Latest Articles Include:
- State of aggregation
- Nat Neurosci 14(4):399 (2011)
Nature Neuroscience | Editorial State of aggregation Journal name:Nature NeuroscienceVolume: 14,Page:399Year published:(2011)DOI:doi:10.1038/nn0411-399Published online28 March 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Neuroscience for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. It is critical that studies examining the functional consequences of aggregated proteins clearly identify the exact source and aggregation state of the protein and critically discuss the implications of their approach. View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Neuroscience for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - Unwrapping HDAC1 and HDAC2 functions in Schwann cell myelination
- Nat Neurosci 14(4):401-403 (2011)
Nature Neuroscience | News and Views Unwrapping HDAC1 and HDAC2 functions in Schwann cell myelination * Robert H Miller1Journal name:Nature NeuroscienceVolume: 14,Pages:401–403Year published:(2011)DOI:doi:10.1038/nn.2788Published online28 March 2011 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Neuroscience for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Two studies now show that epigenetic regulation of gene transcription by histone deacetylases (HDACs) 1 and 2 regulates Schwann cell myelination through activation of a cascade of myelin gene expression. Deletion of HDAC1 and HDAC2 in Schwann cells disrupts interactions with axons, blocks myelination and leads to Schwann cell death. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * The author is at the Center for Translational Neuroscience, Department of Neurosciences, School of Medicine, Case Western Reserve University, Cleveland, Ohio, USA. * Robert H Miller Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Robert H Miller Author Details * Robert H Miller Contact Robert H Miller Search for this author in: * NPG journals * PubMed * Google Scholar Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Neuroscience for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - TDP-43: multiple targets, multiple disease mechanisms?
- Nat Neurosci 14(4):403-405 (2011)
Nature Neuroscience | News and Views TDP-43: multiple targets, multiple disease mechanisms? * Michael Sendtner1Journal name:Nature NeuroscienceVolume: 14,Pages:403–405Year published:(2011)DOI:doi:10.1038/nn.2784Published online28 March 2011 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Neuroscience for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Cytoplasmic inclusions containing TDP-43 are a hallmark of certain neurodegenerative diseases. Two new reports demonstrate that this protein binds a broad range of RNA transcripts, with a preference for UG-rich intronic regions. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Michael Sendtner is at the Institute for Clinical Neurobiology, University of Wuerzburg, Germany. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Michael Sendtner Author Details * Michael Sendtner Contact Michael Sendtner Search for this author in: * NPG journals * PubMed * Google Scholar Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Neuroscience for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - CLC-3 spices up GABAergic synaptic vesicles
- Nat Neurosci 14(4):405-407 (2011)
Nature Neuroscience | News and Views CLC-3 spices up GABAergic synaptic vesicles * Gudrun Ahnert-Hilger1 * Reinhard Jahn2 * Affiliations * Corresponding authorJournal name:Nature NeuroscienceVolume: 14,Pages:405–407Year published:(2011)DOI:doi:10.1038/nn.2786Published online28 March 2011 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Neuroscience for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. How does chloride enter synaptic vesicle, and is it required for neurotransmitter uptake? A new study finds that the chloride transporter CLC-3 is needed for both acidification and transmitter loading of GABAergic synaptic vesicles. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Gudrun Ahnert-Hilger is at AG Functional Cell Biology, Institute for Integrative Neuroanatomy, Charité Universitätsmedizin Berlin, Berlin, Germany. * Reinhard Jahn is in the Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Reinhard Jahn Author Details * Gudrun Ahnert-Hilger Search for this author in: * NPG journals * PubMed * Google Scholar * Reinhard Jahn Contact Reinhard Jahn Search for this author in: * NPG journals * PubMed * Google Scholar Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Neuroscience for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - The multi-laned hippocampus
- Nat Neurosci 14(4):407-408 (2011)
Nature Neuroscience | News and Views The multi-laned hippocampus * Edvard I Moser1Journal name:Nature NeuroscienceVolume: 14,Pages:407–408Year published:(2011)DOI:doi:10.1038/nn.2783Published online28 March 2011 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Neuroscience for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Few brain circuits have generated more research than the mammalian hippocampus, a region of discrete subfields that connect serially to form a 'trisynaptic loop'. A new paper implies that the loop may be made up of parallel subpathways. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Edvard I. Moser is at the Kavli Institute for Systems Neuroscience, Norwegian University of Science and Technology, Trondheim, Norway. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Edvard I Moser Author Details * Edvard I Moser Contact Edvard I Moser Search for this author in: * NPG journals * PubMed * Google Scholar Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Neuroscience for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - Craving, context and the cortex
- Nat Neurosci 14(4):409-410 (2011)
Nature Neuroscience | News and Views Craving, context and the cortex * Olivier George1 * George F Koob1 * Affiliations * Corresponding authorsJournal name:Nature NeuroscienceVolume: 14,Pages:409–410Year published:(2011)DOI:doi:10.1038/nn.2787Published online28 March 2011 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Neuroscience for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. A new study challenges the idea that the ventromedial prefrontal cortex inhibits drug relapse, by selectively inactivating a subpopulation of neurons in this brain area and showing attenuation of context-induced reinstatement of heroin seeking. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Olivier George and George F. Koob are in the Scripps Research Institute, La Jolla, California, USA. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Olivier George or * George F Koob Author Details * Olivier George Contact Olivier George Search for this author in: * NPG journals * PubMed * Google Scholar * George F Koob Contact George F Koob Search for this author in: * NPG journals * PubMed * Google Scholar Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Neuroscience for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - Exocytosis at the hair cell ribbon synapse apparently operates without neuronal SNARE proteins
- Nat Neurosci 14(4):411-413 (2011)
Nature Neuroscience | Brief Communication Exocytosis at the hair cell ribbon synapse apparently operates without neuronal SNARE proteins * Régis Nouvian1, 6 * Jakob Neef1 * Anna V Bulankina1 * Ellen Reisinger2, 3 * Tina Pangršič1, 4 * Thomas Frank1 * Stefan Sikorra5 * Nils Brose3 * Thomas Binz5 * Tobias Moser1 * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 14,Pages:411–413Year published:(2011)DOI:doi:10.1038/nn.2774Received06 December 2010Accepted04 February 2011Published online06 March 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg SNARE proteins mediate membrane fusion. Neurosecretion depends on neuronal soluble NSF attachment protein receptors (SNAREs; SNAP-25, syntaxin-1, and synaptobrevin-1 or synaptobrevin-2) and is blocked by neurotoxin-mediated cleavage or genetic ablation. We found that exocytosis in mouse inner hair cells (IHCs) was insensitive to neurotoxins and genetic ablation of neuronal SNAREs. mRNA, but no synaptically localized protein, of neuronal SNAREs was present in IHCs. Thus, IHC exocytosis is unconventional and may operate independently of neuronal SNAREs. View full text Figures at a glance * Figure 1: Exocytosis in IHCs is resistant to BoNT attack and genetic ablation of neuronal SNAREs. () Representative whole-cell Ca2+ current (ICa2+), membrane capacitance (Cm), membrane conductance (Gm) and series conductance (Gs) traces. Recordings from IHCs were performed as described7. IHCs were repetitively stimulated by 20-ms depolarizations to −27 mV after 10 min treatment with BoNT/E (gray). Control IHCs (without toxins) are shown in black. (–) Average exocytic ΔCm (upper panel, ) and Ca2+ current integrals (QCa2+; lower panel, ), ΔCm per QCa2+ () and ΔCm/QCa2+ ratio normalized to the first response (d) plotted against each 20-ms step depolarization trial in BoNT/E-poisoned (gray) and toxin-free IHCs (black). The number of IHCs is indicated in . () Average ΔCm responses evoked by Ca2+ uncaging in IHCs15 infused with trypsin (25.2 μM, dark gray), BoNT/E (2 μM, light gray) or control solution (without enzyme, black). On average, the ultraviolet flash was delivered 8.2 min after break-in (indicated as time 0) and postflash [Ca2+]i levels were comparable for ! the three conditions. The ΔCm amplitudes of control and trypsin-infused cells, as well as of BoNT/E- and trypsin-infused cells, differed (Mann-Whitney U test, P = 0.003 for both). () Representative perforated patch recordings of ICa2+, Cm,Gm and Gs in lethal-wasting mutant (Lew, gray) and wild-type (WT, black) littermate IHCs in response to 20 ms of maximal Ca2+ current. () Exocytic ΔCm and QCa2+ versus duration of depolarization for Lew (gray) and wild-type (black) IHCs. () Representative ICa2+, Cm, Gm and Gs perforated-patch recordings from IHCs of C57BL/6 (black), synaptobrevin-2/3 double knockout (Syb2/3 dko, dark gray) and SNAP-25 knockout (SNAP-25 ko, light gray) embryonic day 18 organ of Corti cultures in response to 200 ms of maximal Ca2+ current ([Ca2+]e,10 mM). () ΔCm and QCa2+ for 200-ms (top) and ΔCm/QCa2+ for 100–500-ms step depolarization (bottom) of C57BL/6, Syb2/3 dko and SNAP-25 ko. All error bars represent s.e.m. For methods, see Supplementary Method! s. * Figure 2: Neuronal SNARE proteins are abundant in efferent nerve terminals, but not detected in IHCs by immunohistochemistry. (–) IHCs in cochlear cryosections were labeled with a Vglut3 (Synaptic Systems 135203) or otoferlin antibody (Abcam ab53233) and the respective antibodies to neuronal SNAREs (synaptobrevin-1, Synaptic Systems 104002, total number of analyzed cryosections n = 5, ; synaptobrevin-2, Synaptic Systems 104211, n = 8, ; SNAP-25, Covance SMI-81R, n = 12, ; syntaxin-1, Sigma S0664, n = 10, ). The cartoon in the inset illustrates the afferent and efferent connectivity (). Scale bars represent 10 μm. Author information * Author information * Supplementary information Affiliations * InnerEarLab, Department of Otolaryngology and Center for Molecular Physiology of the Brain, University of Göttingen Medical Center, Göttingen, Germany. * Régis Nouvian, * Jakob Neef, * Anna V Bulankina, * Tina Pangršič, * Thomas Frank & * Tobias Moser * Molecular Biology of Cochlear Neurotransmission Group, Department of Otolaryngology, University of Göttingen Medical Center, Göttingen, Germany. * Ellen Reisinger * Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany. * Ellen Reisinger & * Nils Brose * Laboratory of Neuroendocrinology–Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia. * Tina Pangršič * Department of Biochemistry, Medizinische Hochschule Hannover, Hannover, Germany. * Stefan Sikorra & * Thomas Binz * Present address: Inserm U1051, Institute for Neurosciences of Montpellier, Montpellier, France. * Régis Nouvian Contributions The study was designed by R.N. and T.M. The experimental work was performed by R.N., J.N., A.V.B., E.R., T.P., T.F. and S.S. T.M., R.N., J.N., E.R., T.P., N.B. and T.B. prepared the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Tobias Moser Author Details * Régis Nouvian Search for this author in: * NPG journals * PubMed * Google Scholar * Jakob Neef Search for this author in: * NPG journals * PubMed * Google Scholar * Anna V Bulankina Search for this author in: * NPG journals * PubMed * Google Scholar * Ellen Reisinger Search for this author in: * NPG journals * PubMed * Google Scholar * Tina Pangršič Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas Frank Search for this author in: * NPG journals * PubMed * Google Scholar * Stefan Sikorra Search for this author in: * NPG journals * PubMed * Google Scholar * Nils Brose Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas Binz Search for this author in: * NPG journals * PubMed * Google Scholar * Tobias Moser Contact Tobias Moser Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–10, Supplementary Tables 1 and 2, and Supplementary Methods Additional data - Cocaine inverts rules for synaptic plasticity of glutamate transmission in the ventral tegmental area
- Nat Neurosci 14(4):414-416 (2011)
Nature Neuroscience | Brief Communication Cocaine inverts rules for synaptic plasticity of glutamate transmission in the ventral tegmental area * Manuel Mameli1, 2 * Camilla Bellone1 * Matthew T C Brown1 * Christian Lüscher1, 3 * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 14,Pages:414–416Year published:(2011)DOI:doi:10.1038/nn.2763Received18 October 2010Accepted24 January 2011Published online20 February 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The manner in which drug-evoked synaptic plasticity affects reward circuits remains largely elusive. We found that cocaine reduced NMDA receptor excitatory postsynaptic currents and inserted GluA2–lacking AMPA receptors in dopamine neurons of mice. Consequently, a stimulation protocol pairing glutamate release with hyperpolarizing current injections further strengthened synapses after cocaine treatment. Our data suggest that early cocaine-evoked plasticity in the ventral tegmental area inverts the rules for activity-dependent plasticity, eventually leading to addictive behavior. View full text Figures at a glance * Figure 1: Cocaine alters NMDA and AMPA transmission at single synapses onto dopamine neurons of the VTA. () Two-photon laser-scanning microscopy image of a dopamine neuron (top) and a magnified dendrite (inset and bottom) showing two uncaging locations. () 2-PLP evoked a uEPSC at −70 mV at location a, but not at location b. () Summary plot of uEPSC and sEPSCamplitudes obtained in drug-naive mice (uEPSC = 14.7 ± 0.7 pA, sEPSC = 12.2 ± 2 pA, t6 = 1.7, P > 0.5). (,) Sample trace of sEPSC; comparison between sEPSC and uEPSC and blockade by NBQX. () Summary plot of uEPSC and sEPSC decay (90–37%) and rise time (10–90%; decay time, uEPSC = 4.5 ± 0.3 ms, sEPSC = 4.2 ± 0.4 ms, t6 = 1.95, P > 0.5; rise time, uEPSC = 1.8 ± 0.2 ms, sEPSC = 2 ± 0.4 ms, t13 = 0.36, P > 0.5). () 2-PLP–evoked uEPSC at −70, 0 and +40 mV in slices from saline- (gray) and cocaine-treated (red) mice. uNMDA amplitude was taken 10 ms after the EPSC onset (dashed line). (,) Scatter plot and related box plots for uEPSC AMPA and uEPSC NMDA (saline, uNMDA = 25.9 ± 1.6 pA, uAMPA = 14.2 ± 0.6 pA; cocaine! , uNMDA = 15.1 ± 1.2 pA, uAMPA = 18.1 ± 0.6 pA; uAMPA saline versus cocaine, t48 = 3, P < 0.001; uNMDA saline versus cocaine, t74 = 5.8, P < 0.0001). () uAMPA EPSC evoked by 2-PLP at −70, 0, +40 mV in the presence of AP5 (100 μM; note that no current was measured 10 ms after onset; saline, 1.2 ± 0.2 pA; cocaine, 0.9 ± 0.3 pA). (,) Scatter plot obtained for uAMPA values and rectification index (saline, uAMPA = 13.9 ± 0.6 pA, rectification index = 0.8 ± 0.03; cocaine, uAMPA = 17.5 ± 1 pA, rectification index = 1.9 ± 0.1; uAMPA saline versus cocaine, t74 = 4.3, P < 0.0001; rectification index saline versus cocaine, t48 = 5.9, P < 0.0001). () Scatter plot combining the uNMDA, uAMPA and rectification index (color and size coded). The correlation (r2) between AMPAR uEPSC and NMDAR uEPSC was 0.644 (P = 0.001), AMPAR EPSC and rectification index (RI) was −0.649 (P = 0.001) and NMDAR EPSC and rectification index was −0.736 (P = 0.0001). () Averaged AMPA/NMDA ratios; u! AMPA was measured at +40 (left) or −70 (right) mV, whereas u! NMDA was measured at +40 mV (A+40/N+40, saline = 0.26 ± 0.2, cocaine = 0.48 ± 0.1, t20 = 1.9, P < 0.0001; A−70/N+40, saline = 0. 4 ± 0.03, cocaine = 1.8 ± 0.4, t20 = 3.3, P < 0.0001). Compiled data are expressed as mean ± s.e.m. * Figure 2: Cocaine treatment inverts the rules of LTP induction at excitatory inputs onto dopamine neurons of the VTA. () Depolarizing STD protocol (20 bursts at 5-s intervals; burst consisted of five stimuli at 10 Hz). A postsynaptic spike was triggered by injection of positive current (+1.5 nA for 3 ms) 5 ms after the onset of the EPSP. () Hyperpolarizing STD protocol (as above, except injection of a negative current (−1.5 nA for 3 ms)). () Time versus amplitude plots and sample traces of AMPAR EPSPs in slices obtained from saline- (black circles) and cocaine-treated (red circles) mice using the depolarizing STD protocol (saline, 134.5 ± 10%, t5 = 13.22, P < 0.001; cocaine, 98.5 ± 6%, t5 = 1.4, P > 0.05). () Data are presented as in , but the hyperpolarizing STD protocol was used (saline, 93.9 ± 9%, t4 = 0.5, P > 0.05; cocaine, 145.7 ± 17%, t5 = 7.7, P < 0.01). () Time versus amplitude plots and sample traces of STD protocols used in slices from saline- (black circles) and cocaine-treated (red circles) mice when in presence of AP5 (saline, 95.9 ± 7%, t5 = 1.8, P > 0.05; cocaine, 139! .8 ± 15%, t5 = 12, P < 0.0001). () Time versus amplitude plots and sample traces of STD protocols used in slices from saline- (black) and cocaine-treated (red) mice when in presence of NBQX (saline, 118.8 ± 9%, t5 = 2.9, P < 0.05; cocaine, 95.1 ± 8%, t5 = 1.7, P > 0.05). () Time versus amplitude plots and sample traces of the hyperpolarizing STD protocol in slices from cocaine-treated mice in the presence (open circles) or absence (filled circles) of Naspm (control, 153.9 ± 4%; Naspm, 83.2 ± 4%; t7 = 11.42, P < 0.001). () Time versus amplitude plots and sample traces of the hyperpolarizing STD protocol in slices from cocaine-treated mice in the presence (open circles) or absence (filled circles) of intracellular BAPTA (control, 165.3 ± 3%; BAPTA, 80.1 ± 3%; t9 = 21.4, P < 0.001). Compiled data are expressed as mean ± s.e.m. Author information * Author information * Supplementary information Affiliations * Department of Basic Neurosciences, Medical Faculty, University of Geneva, Geneva, Switzerland. * Manuel Mameli, * Camilla Bellone, * Matthew T C Brown & * Christian Lüscher * Institut du Fer à Moulin, UMR-S 839 INSERM/UPMC, Paris, France. * Manuel Mameli * Clinic of Neurology, Department of Clinical Neurosciences, Geneva University Hospital, Geneva, Switzerland. * Christian Lüscher Contributions M.M. carried out all experiments with two–photon laser glutamate uncaging. M.M., M.T.C.B. and C.B. contributed to the long-term plasticity experiments. C.L. designed the study together with M.T.C.B., C.B. and M.M. and wrote the manuscript with the help of all of the authors. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Christian Lüscher Author Details * Manuel Mameli Search for this author in: * NPG journals * PubMed * Google Scholar * Camilla Bellone Search for this author in: * NPG journals * PubMed * Google Scholar * Matthew T C Brown Search for this author in: * NPG journals * PubMed * Google Scholar * Christian Lüscher Contact Christian Lüscher Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1, 2 and Supplementary Methods Additional data - Lasting synaptic changes underlie attention deficits caused by nicotine exposure during adolescence
- Nat Neurosci 14(4):417-419 (2011)
Nature Neuroscience | Brief Communication Lasting synaptic changes underlie attention deficits caused by nicotine exposure during adolescence * Danielle S Counotte1 * Natalia A Goriounova2 * Ka Wan Li1 * Maarten Loos1 * Roel C van der Schors1 * Dustin Schetters3 * Anton N M Schoffelmeer3 * August B Smit1 * Huibert D Mansvelder2, 4 * Tommy Pattij3, 4 * Sabine Spijker1, 4 * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 14,Pages:417–419Year published:(2011)DOI:doi:10.1038/nn.2770Received21 December 2010Accepted20 January 2011Published online20 February 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Tobacco smoking and nicotine exposure during adolescence interfere with prefrontal cortex (PFC) development and lead to cognitive impairments in later life. The molecular and cellular underpinnings of these consequences remain elusive. We found that adolescent nicotine exposure induced lasting attentional disturbances and reduced mGluR2 protein and function on presynaptic terminals of PFC glutamatergic synapses. Restoring mGluR2 activity in vivo by local infusion of a group II mGluR agonist in adult rats that received nicotine as adolescents rescued attentional disturbances. View full text Figures at a glance * Figure 1: Adolescent nicotine exposure affects measures of attentional performance, mGluR2 levels and long-term function on the long term. () Visuospatial divided and sustained attention is (accuracy, left) indicated by the percentage of correct stimulus detections (average of five baseline sessions; Supplementary Methods) and impulsive behavior is indicated by the number of prematurely expressed responses before stimulus onset (right) measured 5 weeks after adolescent (n = 11) or adult (n = 11) nicotine exposure. *P < 0.05, **P < 0.01. () Quantification of immunoblot analysis of synaptic mGluR2 expression (n = 8). #P < 0.1. () Time course of eEPSC amplitude reduction by LY379268 in adolescent nicotine- (black, n = 25) or saline-exposed rats (gray, n = 24); each data point is an average of seven eEPSCs. Insets, example eEPSC traces in control (a) and in the presence of LY379268 (b). Right, average of the last ten responses in the presence of LY379268. () mGluR2/3-dependent inhibition of eEPSC amplitudes was different as a result of adolescent nicotine treatment (saline, n = 39; nicotine, n = 47; F3,128 = 6.2, P! = 0.0006), with no difference in rats treated as adults (saline, n = 22; nicotine, n = 26). Data represent mean ± s.e.m. All experiments were approved by the ethics committee of VU University Amsterdam. * Figure 2: Short-term depression in mPFC is reduced 5 weeks after nicotine exposure during adolescence. (,) Example of short-term plasticity recorded from a layer V pyramidal neuron after extracellular stimulation in layer II/III of adolescent nicotine- or saline-exposed animals. () Summary of short-term depression during ten stimuli (25–200-ms intervals), measured in adolescent-treated (left, F1,137 = 21.6, P < 0.001; saline, n = 10–26; nicotine, n = 10–23) and adult-treated rats (right, F1,255 = 4.57, P = 0.033; saline, n = 24; nicotine, n = 31). Average of the last three responses in the train was normalized to the first one for analysis. () mGluR group II/III antagonist MPPG reduced short-term plasticity in mPFC layer V pyramidal neurons (F2,124 = 7.03, P = 0.012; control, n = 11; 100 μM MPPG, n = 10–11; 200 μM MPPG, n = 6). Data represent mean ± s.e.m. * Figure 3: Intra-mPFC infusion of mGluR2/3 agonist LY379268 reverses long-term attentional disturbances in rats exposed to nicotine as adolescents. () Infusion of the group II antagonist MPPG decreased divided and sustained attention (accuracy) in control animals. *P < 0.05. () LY379268 normalized the nicotine-induced disturbances in divided and sustained attention (accuracy) in rats exposed to nicotine as adolescents (dose, F1,10 = 4.08, P = 0.033), with no effect on rats exposed to saline as adolescents. (,) Impulsive behavior was not affected in control rats by MPPG () or in rats exposed to nicotine as adolescents by LY379268 (), but was increased in saline-exposed rats (dose, F1,10 = 4.98, P = 0.018). Data represent mean ± s.e.m. Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Huibert D Mansvelder, * Tommy Pattij & * Sabine Spijker Affiliations * Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, VU University, Amsterdam, The Netherlands. * Danielle S Counotte, * Ka Wan Li, * Maarten Loos, * Roel C van der Schors, * August B Smit & * Sabine Spijker * Integrative Neurophysiology, Center for Neurogenomics and Cognitive Research, VU University, Amsterdam, The Netherlands. * Natalia A Goriounova & * Huibert D Mansvelder * Anatomy and Neurosciences, VU University Medical Center, Amsterdam, The Netherlands. * Dustin Schetters, * Anton N M Schoffelmeer & * Tommy Pattij Contributions D.S.C. and N.A.G. contributed equally to the experiments in this paper. D.S.C., K.W.L., A.B.S. and S.S. designed the molecular experiments. N.A.G. and H.D.M. designed the physiological experiments. D.S.C., A.N.M.S., S.S. and T.P. designed the behavioral experiments. D.S.C. and R.C.v.d.S. executed the molecular experiments. N.A.G. executed physiological experiments. D.S.C. and D.S. executed behavioral experiments. D.S.C., M.L. and S.S. analyzed molecular experiments. N.A.G. and H.D.M. analyzed physiological experiments. D.S.C. and T.P. analyzed behavioral experiments. D.S.C., H.D.M., T.P. and S.S. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Sabine Spijker Author Details * Danielle S Counotte Search for this author in: * NPG journals * PubMed * Google Scholar * Natalia A Goriounova Search for this author in: * NPG journals * PubMed * Google Scholar * Ka Wan Li Search for this author in: * NPG journals * PubMed * Google Scholar * Maarten Loos Search for this author in: * NPG journals * PubMed * Google Scholar * Roel C van der Schors Search for this author in: * NPG journals * PubMed * Google Scholar * Dustin Schetters Search for this author in: * NPG journals * PubMed * Google Scholar * Anton N M Schoffelmeer Search for this author in: * NPG journals * PubMed * Google Scholar * August B Smit Search for this author in: * NPG journals * PubMed * Google Scholar * Huibert D Mansvelder Search for this author in: * NPG journals * PubMed * Google Scholar * Tommy Pattij Search for this author in: * NPG journals * PubMed * Google Scholar * Sabine Spijker Contact Sabine Spijker Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–10, Supplementary Tables 1–4, Supplementary Methods, Supplementary Results and Supplementary Discussion Additional data - Ventral medial prefrontal cortex neuronal ensembles mediate context-induced relapse to heroin
- Nat Neurosci 14(4):420-422 (2011)
Nature Neuroscience | Brief Communication Ventral medial prefrontal cortex neuronal ensembles mediate context-induced relapse to heroin * Jennifer M Bossert1 * Anna L Stern1 * Florence R M Theberge1 * Carlo Cifani1 * Eisuke Koya1 * Bruce T Hope1 * Yavin Shaham1 * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 14,Pages:420–422Year published:(2011)DOI:doi:10.1038/nn.2758Received01 November 2010Accepted19 January 2011Published online20 February 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg In a rat model of context-induced relapse to heroin, we identified sparsely distributed ventral medial prefrontal cortex (mPFC) neurons that were activated by the heroin-associated context. Selective pharmacogenetic inactivation of these neurons inhibited context-induced drug relapse. A small subset of ventral mPFC neurons formed neuronal ensembles that encode the learned associations between heroin reward and heroin-associated contexts; re-activation of these neuronal ensembles by drug-associated contexts during abstinence provoked drug relapse. View full text Figures at a glance * Figure 1: Context-induced reinstatement of heroin seeking is associated with Fos induction in dorsal and ventral mPFC (dmPFC and vmPFC). () Reinstatement test. Left, total number of active lever and inactive lever presses in rats in which heroin seeking was extinguished in the same (control, AAA) or different (renewal, ABA) context as their heroin self-administration context. ANOVA showed a significant experimental group (control [AAA], renewal [ABA]) × lever (active, inactive) interaction effect (F1,18 = 39.9, P = 0.0001). Right, time course of active lever presses. Data are mean ± s.e.m. *P < 0.05, n = 9–11 per group. () Number of Fos-immunoreactive nuclei per mm2 in dorsal and ventral mPFC (right, area of quantification). ANOVA revealed a significant effect of experimental group (F1,18 = 9.4, P = 0.007), but not mPFC region (dorsal, ventral) or experimental group × mPFC region. n = 9–11 per group. () Fos and NeuN double labeling: representative photomicrographs of Fos and NeuN labeling for dorsal and ventral mPFC (see small squares in the picture in for approximate areas) (n = 4 per group). * Figure 2: Nonselective global inhibition of the majority of ventral, but not dorsal, mPFC neurons by muscimol and baclofen decreased context-induced reinstatement of heroin seeking. (,) Left, total lever presses. The number of active lever presses after bilateral injections of vehicle or muscimol and baclofen (0.03 nmol and 0.3 nmol per side) into dorsal () or ventral () mPFC 5–10 min before exposure to context A or context B are shown. ANOVA revealed a significant drug dose (vehicle or muscimol and baclofen) × mPFC area (dorsal or ventral) × test context (context A or context B) interaction effect (F1,29 = 4.0, P = 0.05). Right, time courses, showing the number of active lever presses. Data are mean ± s.e.m. *P < 0.05 (n = 7–10 per group). () Cannulae placement. Approximate placement and representative pictures of injector tips are shown. Cg1, cingulate area 1; IL, infralimbic cortex; PrL, prelimbic cortex. * Figure 3: Ventral mPFC Daun02 injections after exposure to the heroin-associated context during induction day decreased subsequent context-induced reinstatement of heroin seeking. () Timeline of the experimental procedure: heroin self-administration training (context A), extinction of heroin seeking (context B), induction (context A or B) and context-induced reinstatement test (context A). During the induction day, Daun02 (2 μg per side) or vehicle was injected into the ventral mPFC 90 min after a 30-min exposure to context A or context B. (,) Daun02 injections in the heroin-associated context or the extinction-associated context: total active lever presses and ventral mPFC β-gal expression. ANOVAs of active lever presses and β-gal counts revealed significant Daun02 condition (vehicle, Daun02) × induction day context (context A, context B) interaction effects (F1,53 = 12.6, P = 0.001 and F1,53 = 4.3, P = 0.042, respectively). () Representative images of X-gal staining. Visualization of β-gal–labeled nuclei in ventral mPFC. Dotted red lines indicate approximate area of injector tip. Data are depicted as mean ± s.e.m. * indicates different from ! vehicle, P < 0.05, n = 10–18 per group. Author information * Author information * Supplementary information Affiliations * Behavioral Neuroscience Branch, Intramural Research Program, National Institute on Drug Abuse, US National Institutes of Health/Department of Health and Human Services, Baltimore, Maryland, USA. * Jennifer M Bossert, * Anna L Stern, * Florence R M Theberge, * Carlo Cifani, * Eisuke Koya, * Bruce T Hope & * Yavin Shaham Contributions J.M.B. designed the experiments, ran the experiments and wrote the paper. A.L.S. ran the experiments, managed the data and made the figures. F.R.M.T. and C.C. helped perform the behavioral experiments and the molecular assays. E.K. and B.T.H. provided input on experimental design and the writing of the manuscript, and helped carry out the Daun02 inactivation experiment and the molecular assays. Y.S. supervised the project, designed the experiments and wrote the paper with J.M.B. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Yavin Shaham Author Details * Jennifer M Bossert Search for this author in: * NPG journals * PubMed * Google Scholar * Anna L Stern Search for this author in: * NPG journals * PubMed * Google Scholar * Florence R M Theberge Search for this author in: * NPG journals * PubMed * Google Scholar * Carlo Cifani Search for this author in: * NPG journals * PubMed * Google Scholar * Eisuke Koya Search for this author in: * NPG journals * PubMed * Google Scholar * Bruce T Hope Search for this author in: * NPG journals * PubMed * Google Scholar * Yavin Shaham Contact Yavin Shaham Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (236K) Supplementary Figures 1–3 Additional data - Orthogonal representation of sound dimensions in the primate midbrain
- Nat Neurosci 14(4):423-425 (2011)
Nature Neuroscience | Brief Communication Orthogonal representation of sound dimensions in the primate midbrain * Simon Baumann1 * Timothy D Griffiths1 * Li Sun1 * Christopher I Petkov1 * Alexander Thiele1 * Adrian Rees1 * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 14,Pages:423–425Year published:(2011)DOI:doi:10.1038/nn.2771Received30 November 2010Accepted31 January 2011Published online06 March 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Natural sounds are characterized by their spectral content and the modulation of energy over time. Using functional magnetic resonance imaging in awake macaques, we observed topographical representations of these spectral and temporal dimensions in a single structure, the inferior colliculus, the principal auditory nucleus in the midbrain. These representations are organized as a map with two approximately perpendicular axes: one representing increasing temporal rate and the other increasing spectral frequency. View full text Figures at a glance * Figure 1: BOLD response maps in the inferior colliculus. () Location of the analyzed MRI slices (in red) on a sagittal structural MRI from one animal. V and D indicate the ventral and the dorsal edges of the slice. () t value map of all sound stimuli versus no-sound for one of the slices in (t value shown by scale on right). Robust responses from the inferior colliculi are clearly visible. () Subtraction maps for the boxed area in . BOLD responses are shown to high spectral sound frequencies (hf, left) or temporal rates (hr, right) versus the low spectral frequencies (lf) or temporal rates (lr), respectively, for three animals. Change toward blue indicates increasing dominance of high frequencies or rates. Change toward red indicates increasing dominance of low frequencies or rates. Minimal and maximal response estimate coefficients (beta values) are displayed above and below the color scale, respectively. Letters in upper left corner of left column indicate animal ID. * Figure 2: Estimation of gradient directions by two-dimensional regression analysis. These are shown for the spectral experiment (left) and the temporal experiment (right) in the left inferior colliculus of animal Ws. Top, subtraction maps for the response estimates. Middle, fitting of a plane to the values of the response-estimate coefficients (black dots). Bottom, gradient direction is displayed by the contours of the subtraction maps. Gradient directions are indicated relative to the dorsal-ventral axis. The angle between the axes of the tonotopic and periodotopic gradients is shown below. Author information * Author information * Supplementary information Affiliations * Institute of Neuroscience, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, UK. * Simon Baumann, * Timothy D Griffiths, * Li Sun, * Christopher I Petkov, * Alexander Thiele & * Adrian Rees Contributions S.B., T.D.G. and A.R. designed the experiment. A.T. provided the animals and supervised their handling. L.S. provided the echo planar imaging sequences and optimized them for each animal. S.B. recorded the data. S.B. analyzed the data with help from C.I.P. S.B. and A.R. prepared the manuscript with contributions from T.D.G., A.T., C.I.P. and L.S. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Simon Baumann Author Details * Simon Baumann Contact Simon Baumann Search for this author in: * NPG journals * PubMed * Google Scholar * Timothy D Griffiths Search for this author in: * NPG journals * PubMed * Google Scholar * Li Sun Search for this author in: * NPG journals * PubMed * Google Scholar * Christopher I Petkov Search for this author in: * NPG journals * PubMed * Google Scholar * Alexander Thiele Search for this author in: * NPG journals * PubMed * Google Scholar * Adrian Rees Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–3, Supplementary Table 1 and Supplementary Methods Additional data - Self-related neural response to tailored smoking-cessation messages predicts quitting
- Nat Neurosci 14(4):426-427 (2011)
Nature Neuroscience | Brief Communication Self-related neural response to tailored smoking-cessation messages predicts quitting * Hannah Faye Chua1 * S Shaun Ho2 * Agnes J Jasinska3 * Thad A Polk4 * Robert C Welsh5 * Israel Liberzon2 * Victor J Strecher1 * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 14,Pages:426–427Year published:(2011)DOI:doi:10.1038/nn.2761Received04 October 2010Accepted19 January 2011Published online27 February 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Tailored health interventions can be more effective in eliciting positive behavior change than generic interventions, but the underlying neural mechanisms are not yet understood. Here, 91 smokers participated in a functional magnetic resonance imaging session and a tailored smoking-cessation program. We found that increases in activation in self-related processing regions, particularly dorsomedial prefrontal cortex, to tailored messages predicted quitting during a 4-month follow-up. View full text Author information * Author information * Supplementary information Affiliations * Health Behavior and Health Education, University of Michigan, Ann Arbor, Michigan, USA. * Hannah Faye Chua & * Victor J Strecher * Psychiatry, University of Michigan, Ann Arbor, Michigan, USA. * S Shaun Ho & * Israel Liberzon * Neuroscience Program, University of Michigan, Ann Arbor, Michigan, USA. * Agnes J Jasinska * Psychology, University of Michigan, Ann Arbor, Michigan, USA. * Thad A Polk * Radiology, University of Michigan, Ann Arbor, Michigan, USA. * Robert C Welsh Contributions H.F.C. performed all aspects of this study, including experimental design, data collection, analyses, interpretation and manuscript preparation. S.S.H. and A.J.J. assisted in the data analyses, interpretation and manuscript preparation. T.A.P., R.C.W. and I.L. contributed to the experiment design, data interpretation and manuscript preparation. V.J.S. contributed to the experimental design. Competing financial interests Victor J Strecher is the Chief Visionary Officer and Founder of HealthMedia, a company that develops and licenses computer-tailored health promotion, disease prevention and disease management tools. Corresponding author Correspondence to: * Hannah Faye Chua Author Details * Hannah Faye Chua Contact Hannah Faye Chua Search for this author in: * NPG journals * PubMed * Google Scholar * S Shaun Ho Search for this author in: * NPG journals * PubMed * Google Scholar * Agnes J Jasinska Search for this author in: * NPG journals * PubMed * Google Scholar * Thad A Polk Search for this author in: * NPG journals * PubMed * Google Scholar * Robert C Welsh Search for this author in: * NPG journals * PubMed * Google Scholar * Israel Liberzon Search for this author in: * NPG journals * PubMed * Google Scholar * Victor J Strecher Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (312K) Supplementary Figures 1–4, Supplementary Tables 1–5, Supplementary Results and Supplementary Methods Additional data - HDAC1 and HDAC2 control the transcriptional program of myelination and the survival of Schwann cells
- Nat Neurosci 14(4):429-436 (2011)
Nature Neuroscience | Article HDAC1 and HDAC2 control the transcriptional program of myelination and the survival of Schwann cells * Claire Jacob1 * Carlos N Christen1, 4 * Jorge A Pereira1, 4 * Christian Somandin1, 4 * Arianna Baggiolini1 * Pirmin Lötscher1 * Murat Özçelik1 * Nicolas Tricaud1 * Dies Meijer2 * Teppei Yamaguchi3 * Patrick Matthias3 * Ueli Suter1 * Affiliations * Contributions * Corresponding authorsJournal name:Nature NeuroscienceVolume: 14,Pages:429–436Year published:(2011)DOI:doi:10.1038/nn.2762Received10 September 2010Accepted24 January 2011Published online20 March 2011 Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Histone deacetylases (HDACs) are major epigenetic regulators. We show that HDAC1 and HDAC2 functions are critical for myelination of the peripheral nervous system. Using mouse genetics, we have ablated Hdac1 and Hdac2 specifically in Schwann cells, resulting in massive Schwann cell loss and virtual absence of myelin in mutant sciatic nerves. Expression of Sox10 and Krox20, the main transcriptional regulators of Schwann cell myelination, was greatly reduced. We demonstrate that in Schwann cells, HDAC1 and HDAC2 exert specific primary functions: HDAC2 activates the transcriptional program of myelination in synergy with Sox10, whereas HDAC1 controls Schwann cell survival by regulating the levels of active β-catenin. View full text Figures at a glance * Figure 1: Partial axonal sorting delay, absence of myelination and massive Schwann cell loss in H1/2−/− nerves. (–) Semithin (,) and ultrathin (,) sections of H1/2−/− and DhhCre− control nerves at P1, P5 and P10, and relative area covered by bundles (axon bundle area/total nerve area on cross-sections) quantified in P1 semithin sections of three control and three H1/2−/− sciatic nerves. In , images on the left were taken from semithin sections and images on the right from ultrathin sections. In ,, white arrows highlight one-to-one relationships of axons with Schwann cells and black arrows point to myelinated axons in control nerves. Asterisks indicate axon bundles. In , black arrows highlight Schwann cell cytoplasmic vacuoles and white arrows denote Schwann cell basal lamina remnants. () Percentage of TUNEL-positive cells in sections of P1, P2, P5 and P11 H1/2−/− and control (Co) nerves (three control and three H1/2−/− mice per age group) and staining examples (TUNEL, red; DAPI, blue) at P2. () Immunofluorescence of S100 (red) on sections of P16 H1/2−/− and con! trol nerves. Nuclei are labeled in blue with DAPI. S100 is a Schwann cell marker. Scale bars, 10 μm. Two-tailed Student's t-test; *P < 0.05, ***P < 0.001. Error bars, s.e.m. * Figure 2: In sciatic nerves and long term–transduced RSCs, loss of HDAC1 induces upregulation of HDAC2, and vice versa, whereas in H2+/− or H1+/− sciatic nerves and in short term–transduced cells, expression of the targeted HDAC is reduced without substantial upregulation of the untargeted HDAC. (–) Western blots of HDAC1 and HDAC2 in P5 H1/2−/−, H2−/−, H1−/− and DhhCre− control (Co) nerves (), in P5 H2+/−, H1+/− and control nerves (), and in RSCs transduced for 3 d () or 2 weeks () with nontargeting control (Co), Hdac2 (H2sh) or Hdac1 (H1sh) shRNA lentiviruses. In ,, graphs represent the ratio of HDAC1 or HDAC2 levels normalized to the loading control β-actin or GAPDH. This ratio has been multiplied by an arbitrary factor to set the control value to 100. In ,, graphs represent the percentage of HDAC1 or HDAC2 levels (normalized to β-actin) compared to the control (set to 100%). Nerves of at least three control and three mutant mice were run and analyzed on the same gel, and each lane was loaded with one or two sciatic nerves from a single mouse (,). Three independent experiments were performed for each graph presented in ,. Samples were run on the same gel but not always in consecutive lanes. Full-length blots are presented in Supplementary Fi! gure 10. In ,, significance is indicated by asterisks for HDAC2 and crosses for HDAC1. Two-tailed Student's t-test: *P < 0.05, **P or ++P < 0.01, ***P or +++P < 0.001. Error bars, s.e.m. * Figure 3: HDAC2 is an inducer of Schwann cell differentiation. () Western blots of Sox10, Krox20, P0 and β-actin (loading control) in P5 H1/2−/−, H2+/−, H1+/− and DhhCre− control (Co) nerves. () Semithin sections of P5 control, H2+/− and H1+/− nerves, and g ratio averages (nerves of three mice per group, 50 axons per nerve). () Krox20 immunolabeling (red) and percentage of Krox20-expressing RSCs transduced with nontargeting control, Hdac2 (H2sh) or Hdac1 (H1sh) (green, expressing GFP or DsRed) shRNA lentiviruses in differentiating conditions. For H1sh, Krox20 is false-colored red and DsRed green. Nuclei appear blue. The graph quantifies the percentage of cells expressing detectable Krox20, regardless of the expression level, and thus includes cells with reduced Krox20 expression. White arrows indicate transduced cells; at least 100 transduced cells counted per experiment (three independent experiments). () Western blots of Sox10, P0 and β-actin in RSCs transduced with nontargeting control (Co), H2sh or H1sh lentiviruses.! () Western blots of HDAC2, HDAC1, Krox20, Sox10, P0 and β-actin in differentiated RSCs transfected with GFP (control), HDAC2 (H2 over) or HDAC1 (H1 over) overexpressing constructs. In ,, samples were run on the same gel but not always in consecutive lanes. In , the graph represents the ratio of the proteins normalized to β-actin (ratio multiplied by an arbitrary factor to set the control to 100; at least three nerves of different mice per group). In graphs represent quantification compared to control (set equal to 100%) of three independent experiments. Two-tailed Student's t-test; *P < 0.05, **P < 0.01, ***P < 0.001. Error bars, s.e.m. Full-length blots are presented in Supplementary Figure 10. * Figure 4: Transcriptional regulation of the myelination program by HDACs. () Relative binding of antibodies specific for HDAC2 (anti-HDAC2), anti-HDAC1 or rabbit IgG (negative control) to Sox10 promoter (pro), Sox10 intron 1 (int1), Krox20 pro, Krox20 MSE, P0 pro, P0 int1, control sequence (Cont seq) 1 or Cont seq 2, compared to histone H3 (HH3) antibody (positive control, set equal to 1). () Luciferase fold induction of Sox10 MCS1C, Sox10 MCS1, Krox20 pro, Krox20 MSE, P0 pro and P0 int1 constructs by HDAC2 or HDAC1 overexpression, compared to that seen with GFP expression (set equal to 1), in differentiated RSCs. () Sox10, Krox20 and P0 mRNA fold induction, normalized to Gapdh, in RSCs overexpressing HDAC1 (H1 over) or HDAC2 (H2 over), compared to that in RSCs expressing GFP (set equal to 1). () Immunoprecipitation (IP) of HDAC2, HDAC1, Sox10 or negative control IP (IgG) in proliferating or differentiated RSCs, and western blot (WB) analysis of Sox10, HDAC2 and HDAC1. For reblots of HDAC1 and HDAC2, the membranes were sliced to incubate each IP w! ith the corresponding antibody. () Luciferase fold induction of Sox10 MCS1C, Sox10 MCS1C mutants Δ155–585 and Δ469–470, Krox20 MSE and Krox20 MSE mutant constructs by double overexpression of Sox10/GFP, Sox10/HDAC2 or Sox10/HDAC1, compared to that seen with GFP expression, in differentiated RSCs. At least three independent experiments per graph or IP. Two-tailed Student's t-test, unless stated otherwise in the figure; *P < 0.05, **P < 0.01, ***P < 0.001. Error bars, s.e.m. Full-length blots are presented in Supplementary Figure 10. * Figure 5: Increased activation of the Wnt/β-catenin pathway induces Schwann cell apoptosis and HDAC1 maintains survival by limiting the levels of ABC. (,) Western blots of ABC and β-actin (loading control) in RSCs transduced with nontargeting control (Co), Hdac2 (H2sh) or Hdac1 (H1sh) shRNA lentiviruses (), or in P1 H1/2−/− and DhhCre− control (Co) nerves and P5 H1+/− and control nerves (; three mice per group, both sciatic nerves of each mouse per lane). () Percentage of TUNEL-positive cells in sections of P5 H1+/− and control (Co) nerves (three mice per group) and staining examples (TUNEL, red; DAPI, blue) at P5. (,) Percentage of TUNEL-positive cells in RSC cultures transduced with control (Co), H2sh or H1sh lentiviruses, treated or not with sFRP1 (), or co-transduced with Wnt1-overexpression (over) lentiviruses (). () Western blots of ABC and β-actin in control nerves at birth, P1, P2, P5, P10 and P21 (three sets of two to four mice per age group). In ,,, graphs represent ABC levels normalized to β-actin (, Co set equal to 100%; , for P1 Co and H1/2−/−, percentages calculated compared to ABC at birth i! n control nerves, and for P5 Co and H1+/−, ratios multiplied by an arbitrary factor to set the control to 100; , birth level set equal to 100%). In ,, at least three independent experiments were carried out. Error bars, s.e.m. Two-tailed Student's t-test; *P < 0.05, **P or ++P < 0.01, ***P < 0.001. In , samples were run on the same gel but not always in consecutive lanes. Full-length blots are presented in Supplementary Figure 10. * Figure 6: Sox10 upregulates ABC and ABC promotes the expression of differentiation markers in Schwann cells. () Co-labeling of GFP or Sox10 (green) with ABC (red) in RSCs overexpressing GFP or Sox10. () Luciferase fold induction in differentiated RSCs by ABC overexpression compared to GFP control of regions containing the Sox10 promoter (MCS1C) or intron 1 (MCS1), Krox20 promoter (pro) or MSE, and P0 pro or intron 1 (int1). () Fold increase of Sox10 and Krox20 mRNA, normalized to Gapdh, in RSCs overexpressing ABC compared to GFP (set equal to 1). () ABC, Sox10 and β-actin (loading control) western blots in RSCs overexpressing GFP or ABC and quantification (normalized to β-actin) compared to GFP control (set equal to 100%). () Co-labeling of GFP or ABC (green) and Krox20 (red) in differentiated RSCs overexpressing (over) GFP or ABC. () ABC, Sox10, Krox20 and β-actin western blots in control mouse sciatic nerves treated with Dikkopf-1 (Dkk1) or not treated (NT), and graph representing the ratio of the proteins normalized to β-actin. This ratio has been multiplied by an arbitrary ! factor to set the control to 100. Sciatic nerves of at least three NT and three Dkk1 mice were used (one sciatic nerve of a single mouse per lane). In ,, at least three immunostainings were done and at least 100 cells were analyzed in each case per experiment; white arrows represent some overexpressing cells; nuclei are labeled in blue with DAPI (see text). In –, at least three independent experiments were carried out. Error bars, s.e.m. Two-tailed Student's t-test; *P < 0.05, **P < 0.01. In ,, samples were run on the same gel but not in consecutive lanes. Full-length blots are presented in Supplementary Figure 10. Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Gene Expression Omnibus * GSE27451 Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Carlos N Christen, * Jorge A Pereira & * Christian Somandin Affiliations * Institute of Cell Biology, Department of Biology, ETH Zurich, Zurich, Switzerland. * Claire Jacob, * Carlos N Christen, * Jorge A Pereira, * Christian Somandin, * Arianna Baggiolini, * Pirmin Lötscher, * Murat Özçelik, * Nicolas Tricaud & * Ueli Suter * Department of Cell Biology and Genetics, Erasmus University Medical Center, Rotterdam, The Netherlands. * Dies Meijer * Friedrich Miescher Institute for Biomedical Research, Novartis Research Foundation, Basel, Switzerland. * Teppei Yamaguchi & * Patrick Matthias Contributions C.J. designed the study, analyzed the data and wrote the manuscript; C.J., C.N.C., J.A.P., C.S., A.B, P.L., M.O. and N.T. performed and analyzed the experiments; D.M., T.Y. and P.M. generated and provided crucial mouse lines and support; U.S. conceived and supervised the study and co-wrote the manuscript. All authors commented on the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Claire Jacob or * Ueli Suter Author Details * Claire Jacob Contact Claire Jacob Search for this author in: * NPG journals * PubMed * Google Scholar * Carlos N Christen Search for this author in: * NPG journals * PubMed * Google Scholar * Jorge A Pereira Search for this author in: * NPG journals * PubMed * Google Scholar * Christian Somandin Search for this author in: * NPG journals * PubMed * Google Scholar * Arianna Baggiolini Search for this author in: * NPG journals * PubMed * Google Scholar * Pirmin Lötscher Search for this author in: * NPG journals * PubMed * Google Scholar * Murat Özçelik Search for this author in: * NPG journals * PubMed * Google Scholar * Nicolas Tricaud Search for this author in: * NPG journals * PubMed * Google Scholar * Dies Meijer Search for this author in: * NPG journals * PubMed * Google Scholar * Teppei Yamaguchi Search for this author in: * NPG journals * PubMed * Google Scholar * Patrick Matthias Search for this author in: * NPG journals * PubMed * Google Scholar * Ueli Suter Contact Ueli Suter Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (14M) Supplementary Figures 1–10 and Supplementary Table 1 Additional data - HDAC-mediated deacetylation of NF-κB is critical for Schwann cell myelination
- Nat Neurosci 14(4):437-441 (2011)
Nature Neuroscience | Article HDAC-mediated deacetylation of NF-κB is critical for Schwann cell myelination * Ying Chen1, 2 * Haibo Wang1 * Sung Ok Yoon3 * Xiaomei Xu1 * Michael O Hottiger4 * John Svaren5 * Klaus A Nave6 * Haesun A Kim7 * Eric N Olson2 * Q Richard Lu1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 14,Pages:437–441Year published:(2011)DOI:doi:10.1038/nn.2780Received12 November 2010Accepted18 February 2011Published online20 March 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Schwann cell myelination is tightly regulated by timely expression of key transcriptional regulators that respond to specific environmental cues, but the molecular mechanisms underlying such a process are poorly understood. We found that the acetylation state of NF-κB, which is regulated by histone deacetylases (HDACs) 1 and 2, is critical for orchestrating the myelination program. Mice lacking both HDACs 1 and 2 (HDAC1/2) exhibited severe myelin deficiency with Schwann cell development arrested at the immature stage. NF-κB p65 became heavily acetylated in HDAC1/2 mutants, inhibiting the expression of positive regulators of myelination and inducing the expression of differentiation inhibitors. We observed that the NF-κB protein complex switched from associating with p300 to associating with HDAC1/2 as Schwann cells differentiated. NF-κB and HDAC1/2 acted in a coordinated fashion to regulate the transcriptionally linked chromatin state for Schwann cell myelination. Thus, ! our results reveal an HDAC-mediated developmental switch for controlling myelination in the peripheral nervous system. View full text Figures at a glance * Figure 1: Ablation of HDAC1 and HDAC2 in the Schwann cell lineage results in severe myelination defects in sciatic nerves. () Appearance (upper panel) and electron microscopy analysis (lower panel, cross-section) of sciatic nerves from control and HDAC1cKO (Hdac1loxP/loxP; Dhh-cre), HDAC2cKO (Hdac2loxP/loxP; Dhh-cre) and dCKO mutants at P7. () Electron microscopy analysis of cross sections of control and dCKO sciatic nerves at P0. Inserts are shown for an individual sorted axon (arrows). Arrowheads indicate unsorted axons. () Western blot analysis of HDAC1 and HDAC2 expression using sciatic nerves from control, HDAC1cKO and HDAC2cKO at P4. GAPDH as a loading control. Full-length blots and gels are shown in Supplementary Figure 6. Scale bars represent 1 mm (, top) 5 μm (, bottom, and ). * Figure 2: Effects of HDAC1 and HDAC2 deletion on Schwann cell precursor formation and differentiation. () Cross sections of sciatic nerves of control and dCKO mice at P4 were immunostained with antibodies to S100β and p75. Cell nuclei were counterstained with Topro3. Bottom, quantification of S100β-positive or p75-positive cells per cross-section. () Sciatic nerves of control and dCKO mice at P5 were immunostained with antibodies to myelin components (cross-sections) and transcriptional regulators (longitudinal sections) as indicated. () qRT-PCR analysis of myelin-associated genes, promyelinating transcriptional regulators (top) and negative regulators (bottom) in sciatic nerves of control and dCKO mice at P4 (*P < 0.01). Scale bars represent 60 μm () and 40 μm (). * Figure 3: p65 subunit of NF-κB is an important substrate of HDAC1 and HDAC2 for Schwann cell differentiation. () Lysates of control and dCKO sciatic nerves at P4 were subject to immunoblotting analysis with antibodies to p65, acetyl-p65 K310 (Ac-p65), HDAC1, HDAC2, acetyl-tubulin (Ac-tubulin) and tubulin as indicated. () Sciatic nerves of control and dCKO mice at P4 were immunostained with antibodies to Mbp and acetyl p65. Cell nuclei were counterstained with Topro3. Scale bars represent 50 μm. () Western blot analysis of p65/RelA acetylation in wild-type sciatic nerves at P0, P3, P7 and P14 with antibodies to acetyl-p65 and GAPDH as indicated. () Lysates from primary Schwann cells under the proliferation (Pro) and differentiation (Diff) condition for 4 d were co-immunoprecipitated (IP) with antibody to p65 and blotted with antibodies to HDAC1, HDAC2, p300/CBP and GAPDH, respectively. () Lysates of control and dCKO sciatic nerves at P4 were immunoprecipitated with antibody to p300 and blotted with antibody to acetyl-p65. GAPDH was used as an input control. Full-length blots and gel! s are shown in Supplementary Figure 6. * Figure 4: The acetylation state of NF-κB regulated by HDAC1 and HDAC2 is critical for the Schwann cell differentiation program. (,) Primary rat Schwann cells were transfected with expression vectors for p65, mutant p65 carrying acetylation site mutation(s) and control vector pCMV (−), together with luciferase reporters of Sox10-luc and Mpz-luc () and ID4-luc and Hes5-luc reporters (). (,) Primary rat Schwann cells were transfected with control and expression vectors for HDAC1, HDAC2 or both with luciferase reporters Sox10-luc and Mpz-luc () or ID4-luc (). () The lysates of primary Schwann cells transfected with expression vectors for p65 and/or p300 were immunoblotted with antibodies to acetyl-p65 and GAPDH. Full-length blots and gels are shown in Supplementary Figure 6. (,) Primary Schwann cells were transfected with expression vectors for p65, p300 or both. qRT-PCRs were performed to detect the myelination-associated genes Mbp and Sox10 (), as well as the differentiation inhibitors Id2, Id4, Hes1, Hes5, Jagged1, Delta1 (Dll1) and Delta3 (Dll3) (). Fold changes over controls were measured from thr! ee independent experiments in – and and (n = 3, *P < 0.01). Error bars represent mean ± s.d. * Figure 5: HDAC1 and HDAC2 and NF-κB cooperate to regulate epigenetic marks on the critical genes for Schwann cell differentiation. () Top, the conservation map of the Sox10 promoter among various vertebrates. Bottom, semiquantitative chromatin immunoprecipitation PCR assays for indicated protein/mark recruitment on the Sox10 promoter from control and dCKO sciatic nerves at P5. NA, no antibody control. () Quantification of enrichment of indicated protein/mark association with the Sox10 proximal promoter region by qRT-PCR (n = 3, *P < 0.01). () qRT-PCR analysis of relative enrichment of H3K4me3 and H3K9me3 marks on an active promoter of Gapdh in control and dCKO sciatic nerves at P5. () Top, the conservation map of the Id4 promoter among various vertebrates. Bottom, ChIP PCR assays for protein recruitment on the Id4 promoter in control and dCKO sciatic nerves at P5. () qRT-PCR for enrichment of protein/marks on the Sox10 proximal promoter region in control and dCKO sciatic nerves. Relative fold enrichment in control versus dCKO sciatic nerves (,,) is shown from three independent experiments (n = 3). The b! lue lines in and indicate PCR amplification of promoter elements with or without an NF-κB binding site (red dot) as indicated. (,) Primary Schwann cells isolated from dCKO sciatic nerves at P1 were transfected with control and p65 expression vectors and collected 48 h after transfection. qRT-PCR was performed to detect the myelination-associated genes Mpz, Mbp, Oct6 and Sox10 (), as well as the differentiation inhibitory genes Id2, Id4, Hes1 and Hes5 () from three independent experiments (n = 3). * Figure 6: Activation of canonical Wnt/β-catenin signaling does not inhibit Schwann cell myelination. () Electron microscopy of sciatic nerve cross-sections from control (Ctnnb1lox(ex3)) and Wnt/β-catenin activating mice in the Schwann cell lineage (Ctnnb1lox(ex3)/+; Dhh-cre) at P15. Scale bar represents 5 μm. () The g ratio (ratio of axon diameter to myelinated fiber diameter) of the myelinated axons was measured in sciatic nerves of control and β-catenin activating mice. The histogram shows the percentage of counts for myelinated axons at the different ranges of g ratio as mean ± s.d. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Developmental Biology and Kent Waldrep Foundation Center for Basic Neuroscience Research on Nerve Growth and Regeneration, University of Texas Southwestern Medical Center, Dallas, Texas, USA. * Ying Chen, * Haibo Wang, * Xiaomei Xu & * Q Richard Lu * Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas, USA. * Ying Chen, * Eric N Olson & * Q Richard Lu * Department of Molecular and Cellular Biochemistry, Center for Molecular Neurobiology, Ohio State University, Columbus, Ohio, USA. * Sung Ok Yoon * Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich, Zurich, Switzerland. * Michael O Hottiger * Department of Comparative Biosciences and the Waisman Center, University of Wisconsin, Madison, Wisconsin, USA. * John Svaren * Max Planck Institute of Experimental Medicine, Department of Neurogenetics, Goettingen, Germany. * Klaus A Nave * Department of Biological Sciences, Rutgers University, Newark, New Jersey, USA. * Haesun A Kim Contributions Y.C. conducted the majority of the experiments and analyzed the data. H.W. and X.X. contributed to HDAC mutant generation, phenotype analysis and biochemical assays. S.O.K., J.S. and H.A.S. provided reagents and input. M.H. provided the p65 mutant–expression vectors. K.A.N. provided CNP-Cre mice for initial phenotype observation. E.N.O. provided loxP-flanked HDAC1 and HDAC2 mice and inputs. Q.R.L. supervised the project, analyzed the data and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Q Richard Lu Author Details * Ying Chen Search for this author in: * NPG journals * PubMed * Google Scholar * Haibo Wang Search for this author in: * NPG journals * PubMed * Google Scholar * Sung Ok Yoon Search for this author in: * NPG journals * PubMed * Google Scholar * Xiaomei Xu Search for this author in: * NPG journals * PubMed * Google Scholar * Michael O Hottiger Search for this author in: * NPG journals * PubMed * Google Scholar * John Svaren Search for this author in: * NPG journals * PubMed * Google Scholar * Klaus A Nave Search for this author in: * NPG journals * PubMed * Google Scholar * Haesun A Kim Search for this author in: * NPG journals * PubMed * Google Scholar * Eric N Olson Search for this author in: * NPG journals * PubMed * Google Scholar * Q Richard Lu Contact Q Richard Lu Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–6 Additional data - MHCI negatively regulates synapse density during the establishment of cortical connections
- Nat Neurosci 14(4):442-451 (2011)
Nature Neuroscience | Article MHCI negatively regulates synapse density during the establishment of cortical connections * Marian W Glynn1, 2 * Bradford M Elmer1, 3 * Paula A Garay1, 3 * Xiao-Bo Liu1 * Leigh A Needleman1 * Faten El-Sabeawy1 * A Kimberley McAllister1 * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 14,Pages:442–451Year published:(2011)DOI:doi:10.1038/nn.2764Received17 August 2010Accepted04 January 2011Published online27 February 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Major histocompatibility complex class I (MHCI) molecules modulate activity-dependent refinement and plasticity. We found that MHCI also negatively regulates the density and function of cortical synapses during their initial establishment both in vitro and in vivo. MHCI molecules are expressed on cortical neurons before and during synaptogenesis. In vitro, decreasing surface MHCI (sMHCI) on neurons increased glutamatergic and GABAergic synapse density, whereas overexpression decreased it. In vivo, synapse density was higher throughout development in β2m−/− mice. MHCI also negatively regulated the strength of excitatory, but not inhibitory, synapses and controlled the balance of excitation and inhibition onto cortical neurons. sMHCI levels were modulated by activity and were necessary for activity to negatively regulate glutamatergic synapse density. Finally, acute changes in sMHCI and activity altered synapse density exclusively during early postnatal development. These! results identify a previously unknown function for immune proteins in the negative regulation of the initial establishment and function of cortical connections. View full text Figures at a glance * Figure 1: MHCI is present at the surface of cortical neurons before, during and after synapse formation. (,) Images of dendrites from cortical neurons immunostained for MHCI proteins using the OX-18 antibody at 3, 8 and 14 div are shown proximal to distal from soma (left to right in each image). () MHCI was present in clusters throughout all dendrites at all of the ages that we examined. () sMHCI was also present on dendrites of all of the cells at all of the ages that we examined (n = 540 dendrites, 180 cells). () The density of MHCI detected after permeabilization did not vary over time (normalized to 8 div; 3 div, 1.06 ± 0.05, P = 0.48; 8 div, 1.00 ± 0.05; 14 div, 1.07 ± 0.05, P = 0.45). () sMHCI clusters were present in isolated axons at 3 and 8 div. () sMHCI clusters on dendrites increased in density with age (normalized to 8 div; 3 div, 0.70 ± 0 0.04, P < 0.001; 8 div, 1.00 ± 0.07; 14 div, 1.98 ± 0.09, P < 0.001; * indicates a significant difference from 8 div values). () At 3 div, sMHCI clusters were also present on axonal and dendritic growth cones (n = 57 growth ! cones, 20 neurons). () At 3 and 8 div, sMHCI density was higher on proximal dendrites and lower on distal portions of the same dendrites (normalized to 8 div proximal; 3 div: proximal, 0.58 ± 0.03; distal, 0.38 ± 0.03; P < 0.001; 8 div: proximal, 1.00 ± 0.07; distal, 0.50 ± 0.03; P < 0.001; * indicates a significant difference from 8 div proximal density values; ξ indicates a significant difference between distal and same age proximal densities). At 14 div, this uneven distribution was not observed (sMHCI normalized to 8 div proximal; 14 div: proximal, 1.22 ± 0.08; distal, 1.12 ± 0.06; P = 0.07). () Glutamatergic synapses (excitatory synapses, ES) were also unevenly distributed along dendrites. At 8 div, excitatory synapse density was lower in proximal dendrites and higher in distal dendritic regions (normalized to proximal; proximal, 1.00 ± 0.13; distal, 1.78 ± 0.16; P < 0.001; * indicates a significant difference from same age proximal density values). At 14 div,! synapses were evenly distributed across dendrites (synapse va! lues normalized to 14 div proximal; proximal, 1.00 ± 0.07; distal, 0.83 ± 0.06; P = 0.07). Scale bars represent 5 μm. * Figure 2: Acute β2m knockdown decreases sMHCI and increases glutamatergic and GABAergic synapse density. () β2m siRNA knocked down endogenous β2m and sMHCI protein. Neurons were transfected with β2m siRNA at 5 div, resulting in a significant decrease in β2m cluster density (green, solid line) within 24 h and reaching a maximum of 51% knockdown by 8 div (6 div, 0.83 ± 0.03, P < 0.001; 7 div, 0.51 ± 0.02, P < 0.001; 8 div, 0.49 ± 0.02, P < 0.001). β2m knockdown decreased sMHCI density (blue, dashed line) within 48 h, reaching a maximum knockdown of 46% also by 8 div (6 div, 0.96 ± 0.05, P = 0.8; 7 div, 0.69 ± 0.02, P < 0.001; 8 div, 0.54 ± 0.02, P < 0.001). All values are normalized to age-matched NTS controls. () Images showing the distribution of β2m (top two images) and sMHCI (lower two images) at 8 div. Dendrites are oriented proximal to distal, left to right. (,) Decreasing sMHCI via β2m knockdown increased glutamatergic synapse (excitatory synapse) density (red, solid line) within 48 h of transfection, reaching a maximum 72% increase by 8 div (6 div, 1.08 ± 0.! 07, P = 0.34; 7 div, 1.23 ± 0.06, P < 0.01; 8 div, 1.72 ± 0.08, P < 0.001). () Images of dendrites immunostained for vGlut1 (left) and NR2A/B (middle) from neurons transfected with NTS control (left) or β2m siRNA (right). Yellow in the overlay image indicates synapses. Dendrites are oriented proximal to distal, top to bottom. () The inversely related proximal-distal distributions of sMHCI and glutamatergic synapses were preserved after β2m knockdown (sMHCI density: proximal, 0.60 ± 0.03, P < 0.001; distal, 0.48 ± 0.05, P < 0.001; excitatory synapse (ES) density: proximal, 1.43 ± 0.1, P < 0.01; distal, 2.00 ± 0.14, P < 0.001; * indicates a significant difference from same values in NTS controls, ξ indicates a significant difference from proximal density values). () β2m knockdown increased the percentage of vGlut1 clusters at synapses by almost 30% (26 ± 0.03%, P < 0.001), with no effect on NR2A/B enrichment at synapses (1.07 ± 0.03, P = 0.14). () Addition of exog! enous β2m (eβ2m) did not change the effect of decreasing sMH! CI on excitatory synapse density (normalized to β2m siRNA, 0.93 ± 0.04, P = 0.29). () Images of dendrites immunostained for synapsin (syn, left) and GABA receptor subunits (GABAR, middle) from neurons transfected with NTS control (left) or β2m siRNA (right). Yellow in the overlay image indicates synapses. () Decreasing sMHCI via β2m knockdown increased GABAergic synapse (inhibitory synapse; IS) density by almost 30% (1.27 ± 0.10, P < 0.05, n = 20 dendrites). Scale bars represent 5 μm. * Figure 3: Synapse density is increased between visual cortical neurons from β2m−/− mice both in vitro and in vivo throughout development. (,) Images of neurons from 8 div wild-type () and β2m−/− () mouse cultures immunostained for vGlut1 (green) and PSD-95 (red). Yellow indicates glutamatergic synapses. Scale bar represents 5 μm. () Glutamatergic synapse density was increased by 26% in cultured β2m−/− neurons (normalized to wild type, 1.26 ± 0.08, P < 0.01). * indicates significant difference from wild type. () Total synapse density in β2m−/− mice was greater at all ages examined compared with wild type controls (P8: wild type, 10.35 ± 0.74; β2m−/−, 15.76 ± 1.13; n = 40 sections each, P < 0.001; P11: wild type, 35.42 ± 1.85; β2m−/−, 71.18 ± 2 0.96; n = 48 and 54 sections, respectively, P < 0.001; P23: wild type, 43.80 ± 1.70; β2m−/−, 58.05 ± 2.27; n = 39 sections each, P < 0.001; P60: wild type, 20.65 ± 1.16; β2m−/−, 31.12 ± 1.23; n = 39 sections each, P < 0.001). () Transmission electron micrographs of synapses from P8, P11, P23 and P60 (adult) sections. Sections w! ere blinded and analyzed independently by two different researchers. Only those synapses that were confirmed by both were included in the quantification. Total synapse density was calculated as the number of synapses per 100 μm2 of neuropil. Scale bars represent 0.1 μm. * Figure 4: MHCI overexpression decreases glutamatergic synapse density. () Neurons were transfected with H2-Kb–CFP at 6 div. By 48 h after transfection, MHCI was overexpressed in both proximal and distal dendrites. sMHCI density in dendrites (high-magnification images in middle and right panels) of H2-Kb–CFP–expressing cells (right) was qualitatively greater than in control cells (middle). Dendrites are oriented proximal to distal, top to bottom. Scale bars represent 20 μm (left) and 5 μm (right). () H2-Kb–CFP overexpression increased sMHCI density by 76 ± 0.12% (P < 0.001) and decreased glutamatergic synapse (ES) density by 33 ± 0.03% (P < 0.001). * indicates significant difference from control values. () Neurons transfected with H2-Kb–CFP (right) or their non-transfected neighbors (left) were immunostained for vGlut1 (left) and NR2A/B (NR2, middle). Synapses are yellow in the overlay images (right). Scale bar represents 5 μm. () MHCI overexpression ablated the proximal-distal distribution of both sMHCI (normalized to same region! control; proximal, 1.70 ± 0.14, P < 0.001; distal, 2.13 ± 0.18, P < 0.001; n = 17 dendrites, 8 neurons each) and excitatory synapses (normalized to same region control; proximal, 0.73 ± 0.08, P < 0.05; distal, 0.66 ± 0.03, P < 0.001; n = 19 dendrites, 8 neurons each; # indicates significant difference from same region control values). () Neurons transfected with H2-Kb–CFP (right) or their non-transfected neighbors (left) were immunostained for synapsin (syn, left) and GABAR subunits (middle). Synapses are yellow in the overlay images (right). () H2-Kb–CFP overexpression decreased GABAergic synapse density (IS) by a little over 25% (0.74 ± 0.08, n = 20 dendrites, 10 neurons each, P < 0.05). * Figure 5: MHCI bidirectionally regulates glutamatergic and GABAergic synaptic transmission. () β2m knockdown increased the density of FM1-43–labeled puncta by 55 ± 0.12% (values normalized to NTS; P < 0.01, n = 15 dendrites, 5 cells each; * indicates significant difference from NTS values). () MHCI overexpression decreased the density of FM4-64 staining by 35% (values normalized to GFP-transfected controls; 0.63 ± 0.06, P < 0.05, n = 6 dendrites, 3 neurons each; * indicates significant difference from control values). () Representative traces from whole-cell patch-clamp recordings of mEPSCs from 8–10 div control cultured cortical neurons or neurons transfected with either β2m siRNA or H2-Kb–CFP. Qualitatively, β2m knockdown markedly increased, and H2-Kb overexpression strongly decreased, glutamatergic synaptic transmission. () Representative traces from whole-cell patch-clamp recordings of mIPSCs from 8–10 div control cultured cortical neurons or neurons transfected with either β2m siRNA or H2-Kb–CFP. Qualitatively, β2m knockdown increased, and H2-! Kb overexpression decreased, GABAergic synaptic transmission. () β2m knockdown significantly increased both mEPSC frequency (2.74 ± 0.60, P < 0.001, n = 8) and amplitude (1.63 ± 0.22, P < 0.05, n = 8; * indicates significant difference from NTS values), whereas H2-Kb overexpression decreased both measures (frequency, 0.45 ± 0.04, P < 0.001, n = 8; amplitude, 0.72 ± 0.10, P < 0.05, n = 8; * indicates significant difference from control values). () In contrast, β2m knockdown significantly increased mIPSC frequency (1.77 ± 0.12, P < 0.05, n = 12; * indicates significant difference from NTS values), whereas H2-Kb overexpression decreased it (0.68 ± 0.10, P < 0.05, n = 12; * indicates significant difference from control values), but neither manipulation changed mIPSC amplitude (knockdown, 0.96 ± 0.03, P = 0.78; overexpression, 1.01 ± 0.03, P = 0.36). * Figure 6: Homologous MHCI clusters negatively regulate glutamatergic synapse density. () Exposure of neurons to eβ2m for 36 h decreased the homologous (MHCI alone) to heterologous (MHCI plus β2m) ratio of sMHCI clusters by 45% at 8 div (P < 0.001; * indicates significant difference from control). () ICC staining for vGlut1 (top) and NR2A/B (middle) qualitatively revealed the increase in excitatory synapse density (yellow in overlay, bottom) caused by decreasing homologous sMHCI. () Decreasing the homologous to heterologous ratio of sMHCI clusters increased glutamatergic synapse (ES) density by 53 ± 0.05% (P < 0.001). All values are normalized to the culture-matched controls. Scale bars represent 5 μm. * Figure 7: Changes in MHCI levels are necessary for activity-dependent changes in synapse density. () Exposure to TTX (1 μM, 24 h) decreased sMHCI density at 8 div by 25 ± 0.04% (P < 0.001; * indicates significant difference from control). () TTX treatment increased glutamatergic synapse (ES) density, as defined by colocalized vGlut1 and NR2A/B, by 44 ± 0.07% (P < 0.001). () Images of neurons treated with vehicle (left) or TTX (right) and immunostained for vGlut1 (top) and NR2A/B (middle). Synapses are visible as yellow puncta in the overlay (bottom). Dendrites are oriented proximal to distal, left to right. Scale bar represents 5 μm. () TTX inverted the proximal-distal distribution of sMHCI (normalized to same region control; proximal, 0.63 ± 0.03, P < 0.001; distal, 0.83 ± 0.06, P < 0.05) and glutamatergic synapses (ES; proximal, 1.89 ± 0.15, P < 0.001; distal, 1.22 ± 0.06, P < 0.05), but maintained the inverse relationship between local levels of sMHCI and excitatory synapses (ξ indicates a significant difference of distal density from same age proximal densit! y values). () The TTX-induced increase in excitatory synapse density was completely prevented by MHCI overexpression (OE) (TTX, 1.44 ± 0.05, n = 25 dendrites, P < 0.001; MHCI overexpression, 0.77 ± 0.03, n = 71, P < 0.001; MHCI overexpression + TTX, 0.57 ± 0.03, n = 48, P < 0.001). Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Bradford M Elmer & * Paula A Garay Affiliations * Center for Neuroscience, University of California Davis, Davis, California, USA. * Marian W Glynn, * Bradford M Elmer, * Paula A Garay, * Xiao-Bo Liu, * Leigh A Needleman, * Faten El-Sabeawy & * A Kimberley McAllister * Present address: Dow Pharmaceutical Sciences, Petaluma, California, USA. * Marian W Glynn Contributions M.W.G. initiated the project, conducted most of the experiments using ICC to measure glutamatergic synapse density and wrote a draft of the manuscript. B.M.E. worked with X.-B.L. to generate and quantify the electron microscopy images. P.A.G. established whole-cell patch-clamp recording and performed all of the electrophysiology experiments. L.A.N. performed essential control experiments for antibody specificity. F.E.-S. performed all of the ICC experiments on GABAergic synapses and most of the TTX and MHC experiments. All of the authors edited the manuscript. A.K.M. supported all aspects of this project, designed and helped to analyze all experiments and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * A Kimberley McAllister Author Details * Marian W Glynn Search for this author in: * NPG journals * PubMed * Google Scholar * Bradford M Elmer Search for this author in: * NPG journals * PubMed * Google Scholar * Paula A Garay Search for this author in: * NPG journals * PubMed * Google Scholar * Xiao-Bo Liu Search for this author in: * NPG journals * PubMed * Google Scholar * Leigh A Needleman Search for this author in: * NPG journals * PubMed * Google Scholar * Faten El-Sabeawy Search for this author in: * NPG journals * PubMed * Google Scholar * A Kimberley McAllister Contact A Kimberley McAllister Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (4M) Supplementary Figures 1–5 Additional data - Characterizing the RNA targets and position-dependent splicing regulation by TDP-43
- Nat Neurosci 14(4):452-458 (2011)
Nature Neuroscience | Article Characterizing the RNA targets and position-dependent splicing regulation by TDP-43 * James R Tollervey1, 8 * Tomaž Curk2, 8 * Boris Rogelj3, 8 * Michael Briese1 * Matteo Cereda1, 4 * Melis Kayikci1 * Julian König1 * Tibor Hortobágyi3 * Agnes L Nishimura3 * Vera Župunski3, 5 * Rickie Patani6 * Siddharthan Chandran6, 7 * Gregor Rot2 * Blaž Zupan2 * Christopher E Shaw3 * Jernej Ule1 * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 14,Pages:452–458Year published:(2011)DOI:doi:10.1038/nn.2778Received16 December 2010Accepted02 February 2011Published online27 February 2011 Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg TDP-43 is a predominantly nuclear RNA-binding protein that forms inclusion bodies in frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The mRNA targets of TDP-43 in the human brain and its role in RNA processing are largely unknown. Using individual nucleotide-resolution ultraviolet cross-linking and immunoprecipitation (iCLIP), we found that TDP-43 preferentially bound long clusters of UG-rich sequences in vivo. Analysis of RNA binding by TDP-43 in brains from subjects with FTLD revealed that the greatest increases in binding were to the MALAT1 and NEAT1 noncoding RNAs. We also found that binding of TDP-43 to pre-mRNAs influenced alternative splicing in a similar position-dependent manner to Nova proteins. In addition, we identified unusually long clusters of TDP-43 binding at deep intronic positions downstream of silenced exons. A substantial proportion of alternative mRNA isoforms regulated by TDP-43 encode proteins that regulate neuronal d! evelopment or have been implicated in neurological diseases, highlighting the importance of TDP-43 for the regulation of splicing in the brain. View full text Figures at a glance * Figure 1: Comparison of TDP-43 RNA binding in brain tissue from subjects with and without FTLD-TDP. () To validate the specificity of the antibody to TDP-43 for iCLIP, we isolated the 32P-labeled RNA bound to TDP-43 gel from control (Ctr) or knockdown (KD) HeLa cells in the presence or absence of ultraviolet (UV) cross-linking or antibodies to TDP-43. We used high and low RNase concentrations to confirm the presence of RNA bound to TDP-43. Arrows, positions on gel corresponding to the size of TDP-43 monomer or dimer. TDP-43 western analysis of input extracts confirmed TDP-43 knockdown, and GAPDH was used as a loading control. For full image, see Supplementary Figure 1a. () The proportion of cDNAs (out of all cDNAs that mapped to human genome) from the TDP-43 iCLIP experiments in the four types of samples that mapped to different RNA regions. () The proportion of cDNAs that mapped to different types of ncRNAs. () The proportion of cDNAs that mapped to individual RNAs with at least 10 cDNAs in any experiment. Red, RNAs with the largest significant change between samples from! control subjects and those with FTLD-TDP (difference in proportion of cDNAs > 0.1% and P < 0.05 by Student's t test, one-tailed, unequal variance). The long ncRNA MEG3 (maternally expressed 3) with the largest decrease in TDP-43 binding in FTLD-TDP is also marked. () Real-time PCR analysis of transcripts with largest TDP-43 iCLIP changes in total RNA isolated from control brain samples and samples from subjects with FTLD-TDP. () iCLIP cDNA counts for TDP-43 cross-link positions in NEAT1 in experiments from SH-SY5Y and hES cell lines, and healthy and FTLD-TDP tissue. Blue bars, sequences on the sense strand of the genome; height of bars corresponds to cDNA count. Positions of significant cross-link clusters (XL cluster) are shown on top; RNA sequence underlying the two main clusters is shown below (pink, UG repeats). Data show mean ± s.d. proportion of cDNAs that map to NEAT1 transcript in each experiment (out of all cDNAs mapping to the human genome). () iCLIP cDNA counts! for TDP-43 cross-link positions in EEAT2. Orange bars, sequen! ces on the antisense strand of the genome. Data show mean ± s.d. proportion of cDNAs that map to EEAT2 3′ UTR. The RNA sequence underlying the peak cross-linking sites is shown below; pink, UG repeats. * Figure 2: TDP-43 binding motif analysis. () z-scores of pentamer occurrence within the 61-nt sequence surrounding all cross-link sites (−30 nucleotides (nt) to +30 nt) for iCLIP of healthy samples and samples from subjects with FTLD-TDP. The sequences of the two most enriched pentamers and the Pearson correlation coefficient (r) between the two samples are given. () Enrichment of cross-linking compared to randomized data in UG repeats of different lengths in TDP-43 iCLIP experiments on brain samples from subjects without (blue) and with FTLD-TDP (red), and CELF2 experiments in healthy brain (green). () Enrichment of cross-linking compared to randomized data on UG repeats of different lengths in TDP-43 iCLIP experiments from SH-SY5Y and hES cells. Dark gray, all cross-link sites; light gray, only sites that mapped to cross-link clusters. () Analysis of positions of UGUGU enrichment compared to randomized data around TDP-43 cross-link sites. Color code as in . () Analysis of positions of UGUGU enrichment compared t! o randomized data in TDP-43 iCLIP experiments from SH-SY5Y and hES cells. Color code as in . () TDP-43 cross-linking in its own transcript (TARDBP). Replicate experiments are summed and shown in a single track. The RNA sequence underlying the peak cross-linking site is shown; pink, UG and GU dinucleotides. * Figure 3: The RNA splicing map of TDP-43. Map of cross-link clusters at positions within 500 nt of alternative exons and flanking exons. Cassette exons with ΔIrank > 1 (enhanced exons, red clusters) or ΔIrank < −1 (silenced exons, blue clusters) with at least one cross-link cluster in these regions are shown. The exons are grouped by sequential analysis of cross-link cluster positions in three regions: group S1 is identified by clusters in region 1, 150 nt upstream of the exon and within the exon; group S2 by clusters in region 2, 150–500 nt downstream of the exon; and group E by clusters 0–150 nt downstream of the exon. * Figure 4: TDP-43 regulates splicing of non-coding and protein-coding RNAs. () TDP-43 cross-linking in MIAT ncRNA. Pink bars, UG repeat lengths; black bars, cross-link clusters. Analysis of MIAT exon 10 inclusion (gel electropherogram, left, and quantification, right) in control and TDP-43 knockdown SH-SY5Y cells is shown below. () TDP-43 cross-linking in MEF2D protein-coding transcript. Colours as in . Analysis of MEF2D exon 11 inclusion (gel electropherogram, left, and quantification, right) in control and TDP-43 knockdown SH-SY5Y cells is shown below. () Analysis of BIM exon 3 splicing (gel electropherogram, left, and quantification, right) in control and TDP-43 knockdown SH-SY5Y cells, and in brain samples from subjects without (C23, C25, C30) and with FTLD-TDP (F19, F20, F21). Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions GenBank * E-MTAB-527 * E-MTAB-530 Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * James R Tollervey, * Tomaž Curk & * Boris Rogelj Affiliations * Medical Research Council (MRC) Laboratory of Molecular Biology, Cambridge, UK. * James R Tollervey, * Michael Briese, * Matteo Cereda, * Melis Kayikci, * Julian König & * Jernej Ule * Faculty of Computer and Information Science, University of Ljubljana, Ljubljana, Slovenia. * Tomaž Curk, * Gregor Rot & * Blaž Zupan * MRC Centre for Neurodegeneration Research, King's College London, Institute of Psychiatry, London, UK. * Boris Rogelj, * Tibor Hortobágyi, * Agnes L Nishimura, * Vera Župunski & * Christopher E Shaw * Scientific Institute IRCCS E. Medea, Bosisio Parini (LC), Italy. * Matteo Cereda * Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia. * Vera Župunski * MRC Laboratory for Regenerative Medicine, Department of Clinical Neurosciences, Cambridge, UK. * Rickie Patani & * Siddharthan Chandran * Centre for Clinical Brain Sciences, University of Edinburgh, UK. * Siddharthan Chandran Contributions J.R.T. carried out TDP-43 iCLIP, microarray and PCR experiments. M.B. carried out CELF2 iCLIP. T.C. and G.R. mapped the iCLIP sequence reads to genome, evaluated random barcodes, determined cross-link clusters and annotated the data. T.C. analyzed the reproducibility, sequence and positioning of TDP-43 cross-link sites and performed gene ontology analysis. B.R., A.L.N. and V.Ž. prepared RNA from knockdown cells and brain tissue. T.H. selected, sampled and analyzed the brain samples. M.C. and M.K. analyzed splice-junction microarray data and generated the RNA splicing map. R.P. prepared the embryonic stem cells. S.C., C.E.S., B.Z., J.K. and J.U. supervised the project. J.R.T., T.C., B.R., J.K., C.E.S. and J.U. prepared the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Jernej Ule Author Details * James R Tollervey Search for this author in: * NPG journals * PubMed * Google Scholar * Tomaž Curk Search for this author in: * NPG journals * PubMed * Google Scholar * Boris Rogelj Search for this author in: * NPG journals * PubMed * Google Scholar * Michael Briese Search for this author in: * NPG journals * PubMed * Google Scholar * Matteo Cereda Search for this author in: * NPG journals * PubMed * Google Scholar * Melis Kayikci Search for this author in: * NPG journals * PubMed * Google Scholar * Julian König Search for this author in: * NPG journals * PubMed * Google Scholar * Tibor Hortobágyi Search for this author in: * NPG journals * PubMed * Google Scholar * Agnes L Nishimura Search for this author in: * NPG journals * PubMed * Google Scholar * Vera Župunski Search for this author in: * NPG journals * PubMed * Google Scholar * Rickie Patani Search for this author in: * NPG journals * PubMed * Google Scholar * Siddharthan Chandran Search for this author in: * NPG journals * PubMed * Google Scholar * Gregor Rot Search for this author in: * NPG journals * PubMed * Google Scholar * Blaž Zupan Search for this author in: * NPG journals * PubMed * Google Scholar * Christopher E Shaw Search for this author in: * NPG journals * PubMed * Google Scholar * Jernej Ule Contact Jernej Ule Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (8.3M) Supplementary Figures 1–6 and Tables 1–4 Additional data - Long pre-mRNA depletion and RNA missplicing contribute to neuronal vulnerability from loss of TDP-43
- Nat Neurosci 14(4):459-468 (2011)
Nature Neuroscience | Article Long pre-mRNA depletion and RNA missplicing contribute to neuronal vulnerability from loss of TDP-43 * Magdalini Polymenidou1, 2, 6 * Clotilde Lagier-Tourenne1, 2, 6 * Kasey R Hutt2, 3, 6 * Stephanie C Huelga2, 3 * Jacqueline Moran1, 2 * Tiffany Y Liang2, 3 * Shuo-Chien Ling1, 2 * Eveline Sun1, 2 * Edward Wancewicz4 * Curt Mazur4 * Holly Kordasiewicz1, 2 * Yalda Sedaghat4 * John Paul Donohue5 * Lily Shiue5 * C Frank Bennett4 * Gene W Yeo2, 3 * Don W Cleveland1, 2 * Affiliations * Contributions * Corresponding authorsJournal name:Nature NeuroscienceVolume: 14,Pages:459–468Year published:(2011)DOI:doi:10.1038/nn.2779Received20 December 2010Accepted14 February 2011Published online27 February 2011 Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg We used cross-linking and immunoprecipitation coupled with high-throughput sequencing to identify binding sites in 6,304 genes as the brain RNA targets for TDP-43, an RNA binding protein that, when mutated, causes amyotrophic lateral sclerosis. Massively parallel sequencing and splicing-sensitive junction arrays revealed that levels of 601 mRNAs were changed (including Fus (Tls), progranulin and other transcripts encoding neurodegenerative disease–associated proteins) and 965 altered splicing events were detected (including in sortilin, the receptor for progranulin) following depletion of TDP-43 from mouse adult brain with antisense oligonucleotides. RNAs whose levels were most depleted by reduction in TDP-43 were derived from genes with very long introns and that encode proteins involved in synaptic activity. Lastly, we found that TDP-43 autoregulates its synthesis, in part by directly binding and enhancing splicing of an intron in the 3′ untranslated region of its own ! transcript, thereby triggering nonsense-mediated RNA degradation. View full text Figures at a glance * Figure 1: TDP-43 binds distal introns of pre-mRNA transcripts through UG-rich sites in vivo. () Autoradiograph of TDP-43-RNA complexes trimmed by different concentrations of micrococcal nuclease (MNase, left). Complexes in red box were used for library preparation and sequencing. Immunoblot showing TDP-43 in ~46 kDa and higher molecular weight complexes dependent on ultraviolet (UV) crosslinking (right). () Example of a TDP-43–binding site (CLIP cluster) on Semaphorin 3F defined by overlapping reads from two independent experiments surpassing a gene-specific threshold. () University of California Santa Cruz (UCSC) Genome Browser screenshot of neurexin 3 intron 8 (mm8; chr12:89842000–89847000) displaying three examples of TDP-43–binding modes. The right-most CLIP cluster represents a canonical binding site coinciding GU-rich sequence motifs, whereas the left-most cluster lacks GU-rich sequences and a region containing multiple GU repeats showed no evidence of TDP-43 binding. The second CLIP cluster (middle purple-outlined box) with weak binding was found only w! hen relaxing cluster-finding algorithm parameters. () Flow-chart illustrating the number of reads analyzed from both CLIP-seq experiments. () Histogram of Z scores indicating the enrichment of GU-rich hexamers in CLIP-seq clusters compared with equally sized clusters, randomly distributed in the same pre-mRNAs. Sequences and Z scores of the top eight hexamers are indicated. Pie charts enumerate clusters containing increasing counts of (GU)2 compared with randomly distributed clusters (P≈ 0, χ2 = 21,662). () Pre-mRNAs were divided into annotated regions (top). Distribution of TDP-43 (left) or previously published Argonaute-binding sites21 as a control (right) showed preferential binding of TDP-43 in distal introns. * Figure 2: In vivo depletion of TDP-43 in mouse brain with ASOs. () Strategy for depletion of TDP-43 in mouse striatum. TDP-43–specific or control ASOs were injected into the striatum of adult mice. TDP-43 mRNA was degraded via endogenous RNase H digestion, which specifically recognizes ASO–pre-mRNA (DNA/RNA) hybrids. Mice were killed after 2 weeks and striata were dissected for RNA and protein extraction. () Semi-quantitative immunoblot demonstrating substantial depletion of TDP-43 protein to 20% of controls in TDP-43 ASO–treated mice compared with saline- and control ASO–treated mice. qRT-PCR showed similar TDP-43 depletion at the mRNA level. KD, knockdown. () Flow-chart illustrating the number of reads sequenced and aligned from the RNA-seq experiments. () Differentially regulated genes identified by RNA-seq analysis. Scatter-plot revealed the presence of 362 and 239 genes (diamonds) that were significantly up- (red) or down- (green) regulated after TDP-43 depletion. RNA-seq analysis confirmed that TDP-43 levels were reduced to! 20% of control animals. () Normalized expression (based on RPKM values from RNA-seq) of four noncoding RNAs that were TDP-43 targets and were downregulated after TDP-43 depletion. () Quantitative RT-PCR validation of noncoding RNA Meg3/Gtl2. * Figure 3: Binding of TDP-43 on long transcripts enriched in brain sustains their normal mRNA levels. () Correlation between RNA-seq and CLIP-seq data. Genes were ranked on their degree of regulation after TDP-43 depletion (x axis) and the mean number of intronic CLIP clusters found in the next 100 genes from the ranked list were plotted (y axis, green line). Similarly, the mean total intron length for the next 100 genes was plotted (y axis, red line). The cluster count for each upregulated gene (red dots) and each downregulated gene (green dots) was plotted using the same ordering (inset). () qRT-PCR for selected downregulated genes with long introns (except for Chat) revealed a significant reduction of all transcripts when compared with controls (P < 8 × 10−3). Error bars represent s.d. calculated in each group for 3–5 biological replicates. () Cumulative distribution plots comparing exon length (left) or intron length (middle) across mouse brain tissue–enriched genes (388 genes) and non-brain tissue–enriched genes (15,153 genes). Genes enriched in brain had signi! ficantly longer median intron length compared with genes not enriched in brain (right, solid red line and black lines, P < 6.2 × 10−6 by two-sample Kolmogorov Smirnov goodness-of-fit hypothesis test), whereas a random subset of 388 genes showed no difference in intron length (dashed lines). Similar analysis across human brain tissue–enriched genes (387 genes) and non-brain tissue–enriched genes (17,985 genes) also showed significantly longer introns in brain enriched genes (solid red and black lines, P < 5.3 × 10−6), whereas a random subset of 387 genes showed no difference in intron length (dashed lines). * Figure 4: TDP-43 mediates alternative splicing regulation of its RNA targets. () Schematic representation of different exon classes defined by EST and mRNA libraries, RNA-seq or splicing-sensitive microarray data as indicated (left). Right, bar plot displaying the percentage of exons that contain TDP-43 clusters within 2 kb upstream and downstream of the exon-intron junctions. () Example of alternative splicing change on exon 18 of sortilin 1 analyzed using RNA-seq reads mapping to the exon body (left). Arrows depict increased density of reads in the TDP-43 knockdown samples compared with controls. 76% of the spliced-junction reads in the TDP-43 knockdown samples supported inclusion versus only 19% in the control oligo–treated samples (right). () Comparison of mouse cassette exons detected by splicing-sensitive microarrays to conserved exons in human orthologous genes. 85% and 57% of human exons corresponding to the excluded and included mouse exons, after TDP-43 depletion, respectively (left and middle pie charts), contained EST and mRNA evidence f! or alternative splicing. As a control, the percentage of human exons orthologous to all mouse exons represented on the splicing-sensitive array and that have alternative splicing evidence are shown in the right pie chart. () Semi-quantitative RT-PCR analyses of selected targets confirmed alternative splicing changes in TDP-43 knockdown samples compared with controls. Right, representative acrylamide gel pictures of RT-PCR products from control or knockdown adult brain samples. Quantification of splicing changes from three biological replicates per group (left); error bars represent s.d. * Figure 5: Autoregulation of TDP-43 through binding on the 3′ UTR of its own transcript. () CLIP-seq reads and clusters on TDP-43 transcript showing binding mainly in an alternatively spliced part of the 3′ UTR, lacking long uninterrupted UG repeats. () qRT-PCR showing ~50% reduction of endogenous TDP-43 mRNA in transgenic mice overexpressing human myc–TDP-43 not containing introns and 3′ UTR. () Immunoblots confirming the reduction of endogenous TDP-43 protein (upper panel) to 50% of control levels (by densitometry, lower panel) in response to human myc–TDP-43 overexpression. () Immunoblot showing reduction of endogenous TDP-43 protein in HeLa cells on tetracycline induction of a transgene encoding GFP-myc–TDP-43–HA (annotated GFP–TDP-43). The ~30-kDa product accumulating after 48 h (red arrow) was immunoreactive with four TDP-43–specific antibodies. () qRT-PCR using primers spanning the junctions of TDP-43 isoform 3 (upper panel), showed ~100-fold increase in response to tetracycline induction of GFP–TDP-43. TDP-43 isoform 3 was present at ve! ry low levels before tetracycline induction. () Luciferase assays showing significant reduction of relative fluorescence units (RFUs) in cells expressing renilla luciferase under the control of long TDP-43 3′ UTR when co-transfected with myc–TDP-43–HA. Cells treated with siRNA against UPF1 showed a significant increase of RFUs when expressing Luciferase with the long TDP-43 3′ UTR, but not in controls. () qRT-PCR scoring levels of endogenous TDP-43 isoform 3 in HeLa cells transiently transfected with myc–TDP-43–HA, UPF1 siRNA or both simultaneously. TDP-43 isoform 3 was increased in response to elevated TDP-43 protein levels, blocking of NMD (by UPF1 siRNA) and there was a synergistic effect in the combined condition. * Figure 6: TDP-43 regulates expression of FUS/TLS and progranulin. () CLIP-seq reads and clusters on Fus/Tls transcript showing TDP-43 binding in introns 6 and 7 (also annotated as an alternative 3′ UTR) and the canonical 3′ UTR. RNA-seq reads from control or TDP-43 knockdown samples (equal scales) showed a slight reduction in Fus/Tls mRNA in the TDP-43 knockdown group and expression values from RNA-seq confirmed that Fus/Tls mRNA was reduced to 70% of control on TDP-43 reduction. () qRT-PCR confirmed Fus/Tls mRNA downregulation on TDP-43 depletion. s.d. was calculated within each group for three biological replicas. () Semi-quantitative immunoblot (upper panel) revealed a slight, but consistent, reduction of FUS/TLS protein in ASO-treated mice to ~70% of control levels, as quantified by densitometric analysis (lower panel). () CLIP-seq reads and clusters on progranulin transcript (Grn) showing a sharp binding in the 3′ UTR. RNA-seq reads from control or TDP-43 knockdown samples (equal scales) showed a significant increase in progranu! lin mRNA in the TDP-43 knockdown group compared with controls. () qRT-PCR confirmed the statistically significant increase (P < 3 × 10−4) in progranulin mRNA in samples with reduced TDP-43 levels when compared with controls. s.d. was calculated in each group for three biological replicas. Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions GenBank * GSE27394 Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Magdalini Polymenidou, * Clotilde Lagier-Tourenne & * Kasey R Hutt Affiliations * Ludwig Institute for Cancer Research, University of California at San Diego, La Jolla, California, USA. * Magdalini Polymenidou, * Clotilde Lagier-Tourenne, * Jacqueline Moran, * Shuo-Chien Ling, * Eveline Sun, * Holly Kordasiewicz & * Don W Cleveland * Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, California, USA. * Magdalini Polymenidou, * Clotilde Lagier-Tourenne, * Kasey R Hutt, * Stephanie C Huelga, * Jacqueline Moran, * Tiffany Y Liang, * Shuo-Chien Ling, * Eveline Sun, * Holly Kordasiewicz, * Gene W Yeo & * Don W Cleveland * Stem Cell Program and Institute for Genomic Medicine, University of California at San Diego, La Jolla, California, USA. * Kasey R Hutt, * Stephanie C Huelga, * Tiffany Y Liang & * Gene W Yeo * Isis Pharmaceuticals, Carlsbad, California, USA. * Edward Wancewicz, * Curt Mazur, * Yalda Sedaghat & * C Frank Bennett * RNA Center, Department of Molecular, Cell and Developmental Biology, Sinsheimer Labs, University of California, Santa Cruz, California, USA. * John Paul Donohue & * Lily Shiue Contributions M.P., C.L.-T., J.M. and T.Y.L. performed the experiments. K.R.H., S.C.H. and T.Y.L. conducted the bioinformatics analysis. S.-C.L. developed the monoclonal TDP-43–specific antibody used for CLIP-seq and the tetracycline-inducible GFP–TDP-43–expressing HeLa cells. S.-C.L. and E.S. generated the transgenic myc–TDP-43 mice. J.P.D. and L.S. conducted the preliminary splice-junction microarray analyses. M.P., C.L.-T., E.W., C.M., Y.S., C.F.B. and H.K. conducted the antisense oligonucleotide experiments. M.P., C.L.-T., K.R.H., G.W.Y. and D.W.C. designed the experiments. M.P., C.L.-T., K.R.H., S.C.H., G.W.Y. and D.W.C. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Gene W Yeo or * Don W Cleveland Author Details * Magdalini Polymenidou Search for this author in: * NPG journals * PubMed * Google Scholar * Clotilde Lagier-Tourenne Search for this author in: * NPG journals * PubMed * Google Scholar * Kasey R Hutt Search for this author in: * NPG journals * PubMed * Google Scholar * Stephanie C Huelga Search for this author in: * NPG journals * PubMed * Google Scholar * Jacqueline Moran Search for this author in: * NPG journals * PubMed * Google Scholar * Tiffany Y Liang Search for this author in: * NPG journals * PubMed * Google Scholar * Shuo-Chien Ling Search for this author in: * NPG journals * PubMed * Google Scholar * Eveline Sun Search for this author in: * NPG journals * PubMed * Google Scholar * Edward Wancewicz Search for this author in: * NPG journals * PubMed * Google Scholar * Curt Mazur Search for this author in: * NPG journals * PubMed * Google Scholar * Holly Kordasiewicz Search for this author in: * NPG journals * PubMed * Google Scholar * Yalda Sedaghat Search for this author in: * NPG journals * PubMed * Google Scholar * John Paul Donohue Search for this author in: * NPG journals * PubMed * Google Scholar * Lily Shiue Search for this author in: * NPG journals * PubMed * Google Scholar * C Frank Bennett Search for this author in: * NPG journals * PubMed * Google Scholar * Gene W Yeo Contact Gene W Yeo Search for this author in: * NPG journals * PubMed * Google Scholar * Don W Cleveland Contact Don W Cleveland Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (10.1M) Supplementary Figures 1–14 and Tables 1–6 Additional data - Flotillin-1 is essential for PKC-triggered endocytosis and membrane microdomain localization of DAT
- Nat Neurosci 14(4):469-477 (2011)
Nature Neuroscience | Article Flotillin-1 is essential for PKC-triggered endocytosis and membrane microdomain localization of DAT * M Laura Cremona1, 6 * Heinrich J G Matthies2 * Kelvin Pau1 * Erica Bowton2 * Nicole Speed2 * Brandon J Lute2 * Monique Anderson1 * Namita Sen3 * Sabrina D Robertson2 * Roxanne A Vaughan4 * James E Rothman5 * Aurelio Galli2 * Jonathan A Javitch3 * Ai Yamamoto1 * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 14,Pages:469–477Year published:(2011)DOI:doi:10.1038/nn.2781Received16 August 2010Accepted18 February 2011Published online13 March 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Plasmalemmal neurotransmitter transporters (NTTs) regulate the level of neurotransmitters, such as dopamine (DA) and glutamate, after their release at brain synapses. Stimuli including protein kinase C (PKC) activation can lead to the internalization of some NTTs and a reduction in neurotransmitter clearance capacity. We found that the protein Flotillin-1 (Flot1), also known as Reggie-2, was required for PKC-regulated internalization of members of two different NTT families, the DA transporter (DAT) and the glial glutamate transporter EAAT2, and we identified a conserved serine residue in Flot1 that is essential for transporter internalization. Further analysis revealed that Flot1 was also required to localize DAT within plasma membrane microdomains in stable cell lines, and was essential for amphetamine-induced reverse transport of DA in neurons but not for DA uptake. In sum, our findings provide evidence for a critical role of Flot1-enriched membrane microdomains in PKC-tr! iggered DAT endocytosis and the actions of amphetamine. View full text Figures at a glance * Figure 1: PKC triggers endocytosis of heterologously and endogenously expressed DAT. (,) EM4-YFP-DAT internalizes (white arrows) into EEA1-positive vesicles after exposure to 0.5 μM PMA for 30 min. Cells exposed to PMA were fixed and immunostained for EEA1 as described. Alexa Fluor 633–labeled secondary antibodies were used to prevent overlap with YFP. Scale bars, 10 μm. () Endogenous DAT internalizes in response to PMA in primary dopaminergic neurons. Midbrain cultures were treated with vehicle (Ctrl, n = 32) or 1 μM PMA (+ PMA, n = 35) for 30 min, fixed and immunostained for DAT (intracellular epitope). PMA led to significant internalization of DAT ('Internalization Index,' as described in Online Methods: Ctrl, 0.076 ± 0.013; PMA, 0.309 ± 0.052). One-way analysis of variance (ANOVA); P < 0.001. Scale bars, 20 μm. () Internalization of endogenous DAT in striatal slice preparations (n = 6). Slices were treated with 10 μM PMA or Ctrl for 1 h, then cell surface biotinylated to determine DAT surface levels. Graph indicates the relative amount of DAT at! the cell surface, and calculated as the ratio between the integrated densities of surface DAT after treatment (corrected for total DAT) to surface DAT before treatment (corrected for total DAT). Bars represent mean + s.d. PMA treatment significantly decreased surface DAT (P = 0.0330). Complete blots can be found in Supplementary Figure 9. * Figure 2: Flot1 overexpression attenuates Gö6850-mediated inhibition of PKC-triggered endocytosis. Flot1 overexpression in EM4-YFP-DAT cells attenuated Gö6850 (Gö)-mediated inhibition of PKC-regulated endocytosis. All experiments were performed in this stable cell line unless noted otherwise. (,) Image-based analysis. Percentage of cells with internalization was calculated from cells showing YFP-DAT colocalized to EEA1-positive endosomes (white arrows, ) after 1 μM PMA for 30 min. PKC-triggered internalization of DAT was significantly inhibited by Gö6850 in a dose-dependent manner (open circles, ; P < 0.0001). Data points represent mean + s.e.m. (n = 3). Flot1 overexpression significantly diminished the Gö6850-mediated inhibition (closed circles, P = 0.0078). For each dosage, 150 to 200 cells were analyzed. Scale bar, 10 μm. Complete statistics can be found in Supplementary Statistical Analyses. Flot1 overexpression in the presence of inhibitor (Flot1 + Gö6850) blunted the effect of inhibitor alone, permitting internalization. Scale bar, 10 μm. () Cell surface bio! tinylation measuring YFP-DAT surface availability. DAT surface expression (% vehicle) is calculated as described in Figure 1d. Values are normalized to no PMA. Data are plotted as mean + s.e.m. (n = 5). Ctrl and Gö6850 cells were transfected with mRFP alone as transfection control. Complete blots can be found in Supplementary Figure 10. * Figure 3: Flot1 is required for the PKC-triggered endocytosis of EM4-YFP-DAT and Hela-eGFP-EAAT2. (,) Flot1 is required for PKC-triggered endocytosis of DAT. EM4-YFP-DAT cells transfected with siRNA against Flot1 (siFlot1) failed to internalize DAT despite exposure to 1 μM PMA for 30 min. () Confocal microscopy (n = 4). () Cell surface biotinylation (n = 4). Data are plotted as described in Figure 1d. PMA treatment did not lead to a significant decrease of cell surface DAT after Flot1 depletion (P = 0.226). (,) Flot1 is required for PKC-triggered endocytosis of EAAT2. () GFP-EAAT2 internalized to EAA1-positive structures after 0.5 μM PMA for 30 min (n = 3). () Cell surface biotinylation (n = 3). PMA treatment did not lead to a significant decrease of cell surface EAAT2 after Flot1 depletion (P = 0.104). Cells were treated with 0.5 μM PMA for 30 min. Complete statistics can be found in Supplementary Statistical Analyses. Bars represent mean + s.e.m. () Confocal microscopy. DAT (green) internalized into Flot1-positive vesicles (red) after exposure to 1 μM PMA for 30 mi! n (yellow in overlay). Scale bars, 10 μm. Complete blots can be found in Supplementary Figure 11. * Figure 4: Flot1 must be palmitoylated on residue Cys34. () Schematic representation of Flot1. The conserved cysteine Cys34 is found within the first hydrophobic domain in the PHB homology domain. Four predicted PKC-phosphorylation sites are also noted. () Overexpression of Flot1(C34A) in EM4-YFP-DAT inhibited PKC-triggered internalization of DAT (n = 6 experiments). Cells transiently transfected with Flot1-mRFP, Flot1(C34A)-mRFP or mRFP alone (Ctrl) were exposed to 1 μM PMA for 30 min. Representative images. Scale bar, 10 μm. () Cell surface biotinylation of DAT after PMA treatment in the presence or absence of Gö6850. The C34A mutation inhibited PMA-triggered internalization (left panels, without Gö6850), and also failed to diminish Gö6850 inhibition (right panels, with Gö6850). () Quantification of DAT surface expression of , as described in Figure 1d (n = 3). () Palmitoylation is not required for multimerization of Flot1. EM4-YFP-DAT cells transiently transfected with mRFP, mRFP-Flot1 or mRFP-Flot1(C34A). Flot1 fusion pr! oteins were immunoprecipitated (IP) with an antibody to mRFP and probed (IB) for endogenous Flot1 (left panel). Flot1(C34A)-mRFP still immunoprecipitated with endogenous Flot1 (n = 3). mRFP alone did not immunoprecipitate with Flot1 (right panel). () Palmitoylation is required for proper complex formation of Flot1 and DAT. Flot1(C34A) pulled down significantly less DAT than unmutagenized Flot1 (P < 0.001). PMA (1 μM) for 30 min significantly increased the amount of DAT that immunoprecipitated with WT Flot1 (P = 0.0148). This effect was also abrogated by the C34A mutation (P = 0.1022) (n = 4). Complete statistics can be found in Supplementary Statistical Analyses. Bars represent mean + s.e.m. Int., integrated; A.U., arbitrary units. Complete blots can be found in Supplementary Figure 12. * Figure 5: Flot1 is required for the membrane raft localization of DAT but not for transport of DA. Sucrose gradients revealed that Flot1 was required to maintain DAT in membrane rafts. EM4-YFP-DAT cells were lysed and fractionated across a sucrose gradient. Ten fractions were collected from top (1) to bottom (10) of the gradient. Four different conditions were studied: () Control (Flot1); () Flot1(C34A) transfection; () siFlot1 transfection; and () nystatin (n = 3). () A graphical representation of integrated densities across fractions for each condition for DAT and TfR. () The transport of DA by DAT does not require the presence of Flot1. EM4-YFP-DAT cells were transfected as indicated and exposed to vehicle, then DA uptake assays were performed as described in Online Methods. Data is represented as [3H]DA uptake as a percentage of that in wild type (WT). Neither the palmitoylation-deficiency mutation nor depletion of Flot1 significantly altered DA uptake (n = 4, P = 0.5770). Bars represent mean + s.e.m. Please also refer to Table 1. Complete blots can be found in Supple! mentary Figure 13. * Figure 6: PKC-triggered internalization of DAT requires phosphorylation on Flot1 Ser315. () PMA (1 μM, 30 min) promotes association of Flot1 with DAT. Bars represent the mean ratio between integrated densities of endogenous Flot1 to Flot1-mRFP + s.e.m. (n = 4). PMA significantly increased immunoprecipitation (IP) of mRFP-Flot1 and Flot1 with DAT (ANOVA, *P = 0.0421). Cav1 did not immunoprecipitate with this complex. IB, immunoblot. () Metabolic labeling showing phosphorylation of Flot1 in response to PMA. EM4-YFP-DAT cells were treated with vehicle (Ctrl), 1 μM PMA + vehicle (+ PMA) or 1 μM PMA + 100 nM Gö6850 before incubation (+ Gö) (n = 2). Bars represent the integrated density of 32P incorporation normalized (rel.) to Flot1, + s.e.m. ANOVA showed that PMA led to a significant increase in 32P incorporation (P = 0.0474) that was inhibited by Gö6850 (P = 0.0017). S315A mutation inhibited this PMA-triggered increase (P = 0.1610). Overall 32P incorporation was also significantly less in S315A mutant (P < 0.001). (–) PMA (1 μM, 30 min)-triggered internali! zation of DAT requires Flot1Ser315. Flot1(S315A) overexpression inhibited DAT internalization while Flot1(S54A) did not. () DAT is internalized in non-transfected (white arrows) and Flot1(S54A) transfected cells (yellow arrows), but not in Flot1(S315) transfected cells (yellow arrow). '% Cells with internalization' was determined as described (Fig. 2a) (200 cells/condition) (ANOVA, P < 0.01). Scale bar, 5 μm. () Surface protein biotinylation and quantification of DAT (n = 3) (ANOVA, P < 0.01). () Alignment of region surrounding Flot1 Ser315. Ser315 is largely conserved across species (*, bold). Complete statistics can be found in Supplementary Statistical Analyses. Complete blots can be found in Supplementary Figure 14. * Figure 7: Flot1 is required for PKC-triggered internalization and AMPH-induced reverse transport of DA in primary DA neurons. () Representative DAT-mediated transient currents recorded from DA neurons transduced with lentiviruses carrying control shRNA (shCtrl) or shFlot1 following a voltage jump to −140 mV from a holding potential of −20 mV. The integral of the DAT-mediated transient current is proportional to the number of transporters on the cell surface2. Transient currents were obtained first with vehicle, then in the presence of PMA. Bars represent the mean ratios of PMA transient charge (Q) to pre-PMA responses in the same cell (QCTR). PMA did not reduce DAT-mediated transient currents in shFlot1 neurons, suggesting that Flot1 is necessary for PMA-triggered DAT trafficking. () Loss of Flot1 leads to an inhibition of AMPH-mediated DA efflux. Neurons were loaded through the patch pipette with 2 mM DA and 30 mM Na+. The amperometric current–voltage relationship was obtained by stepping the voltage in 20-mV intervals from 0 mV to 100 mV from a holding potential of −60 mV. DAT-mediated DA! efflux from shFlot1 (red, n = 4) and shCtrl (black, n = 4) is defined as the current recorded in the presence of AMPH, minus the current recorded after the addition of cocaine to the bath with AMPH still present. Mean ± s.e.m. shFlot1 significantly reduced DA efflux as compared to shCtrl at voltages greater than 40 mV (*P < 0.05, **P < 0.01, one-way ANOVA followed by Bonferroni post hoc tests). Author information * Abstract * Author information * Supplementary information Affiliations * Departments of Neurology, and Pathology and Cell Biology, Columbia University, College of Physicians and Surgeons, New York, New York, USA. * M Laura Cremona, * Kelvin Pau, * Monique Anderson & * Ai Yamamoto * Department of Molecular Physiology and Biophysics, Center for Molecular Neuroscience, Kennedy Center, Vanderbilt University, Nashville, Tennessee, USA. * Heinrich J G Matthies, * Erica Bowton, * Nicole Speed, * Brandon J Lute, * Sabrina D Robertson & * Aurelio Galli * Center for Molecular Recognition and Departments of Psychiatry and Pharmacology, Columbia University, College of Physicians and Surgeons, New York, New York, USA. * Namita Sen & * Jonathan A Javitch * University of North Dakota School of Medicine and Health Science, Grand Forks, North Dakota, USA. * Roxanne A Vaughan * Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut, USA. * James E Rothman * Present address: Department of Endocrinology, Columbia University, College of Physicians and Surgeons, New York, New York, USA. * M Laura Cremona Contributions M.L.C., K.P., S.D.R., M.A. and A.Y. contributed toward and performed cell surface biotinylation experiments; M.L.C., H.J.G.M., K.P., M.A. and A.Y. contributed toward and performed image-based internalization experiments; M.L.C., N.S., J.A.J. and A.Y. contributed toward and performed DA and tyramine uptake assays; K.P., E.B., N.S., B.J.L. and A.G. contributed toward and performed electrophysiology experiments; R.A.V. contributed critical reagents and critical reading of manuscript; M.L.C., A.Y. and J.E.R. initiated the study and contributed toward and performed the original functional endocytic screen and membrane microdomain localization study; J.E.R. provided direction in endocytosis studies; A.G. and J.A.J. provided direction in DAT and DA studies; A.Y. and J.A.J. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Ai Yamamoto Author Details * M Laura Cremona Search for this author in: * NPG journals * PubMed * Google Scholar * Heinrich J G Matthies Search for this author in: * NPG journals * PubMed * Google Scholar * Kelvin Pau Search for this author in: * NPG journals * PubMed * Google Scholar * Erica Bowton Search for this author in: * NPG journals * PubMed * Google Scholar * Nicole Speed Search for this author in: * NPG journals * PubMed * Google Scholar * Brandon J Lute Search for this author in: * NPG journals * PubMed * Google Scholar * Monique Anderson Search for this author in: * NPG journals * PubMed * Google Scholar * Namita Sen Search for this author in: * NPG journals * PubMed * Google Scholar * Sabrina D Robertson Search for this author in: * NPG journals * PubMed * Google Scholar * Roxanne A Vaughan Search for this author in: * NPG journals * PubMed * Google Scholar * James E Rothman Search for this author in: * NPG journals * PubMed * Google Scholar * Aurelio Galli Search for this author in: * NPG journals * PubMed * Google Scholar * Jonathan A Javitch Search for this author in: * NPG journals * PubMed * Google Scholar * Ai Yamamoto Contact Ai Yamamoto Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (23M) Supplementary Figures 1–14, Supplementary Table 1 and Supplementary Statistical Analyses Additional data - Presynaptic HCN1 channels regulate CaV3.2 activity and neurotransmission at select cortical synapses
- Nat Neurosci 14(4):478-486 (2011)
Nature Neuroscience | Article Presynaptic HCN1 channels regulate CaV3.2 activity and neurotransmission at select cortical synapses * Zhuo Huang1 * Rafael Lujan2 * Ivan Kadurin3 * Victor N Uebele4 * John J Renger4 * Annette C Dolphin3 * Mala M Shah1 * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 14,Pages:478–486Year published:(2011)DOI:doi:10.1038/nn.2757Received10 November 2010Accepted13 January 2011Published online27 February 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The hyperpolarization-activated cyclic nucleotide–gated (HCN) channels are subthreshold, voltage-gated ion channels that are highly expressed in hippocampal and cortical pyramidal cell dendrites, where they are important for regulating synaptic potential integration and plasticity. We found that HCN1 subunits are also localized to the active zone of mature asymmetric synaptic terminals targeting mouse entorhinal cortical layer III pyramidal neurons. HCN channels inhibited glutamate synaptic release by suppressing the activity of low-threshold voltage-gated T-type (CaV3.2) Ca2+ channels. Consistent with this, electron microscopy revealed colocalization of presynaptic HCN1 and CaV3.2 subunit. This represents a previously unknown mechanism by which HCN channels regulate synaptic strength and thereby neural information processing and network excitability. View full text Figures at a glance * Figure 1: Pharmacological block or deletion of HCN channels enhances mEPSC frequency in entorhinal cortical layer III pyramids. () Morphology of typical mouse entorhinal cortical layer III pyramidal neuron. The scale bar represents 20 μm. (,) Example mEPSC recordings from wild-type (WT) and Hcn1−/− soma and dendrites before and after 15-min bath application of the HCN channel blocker ZD7288 (ZD, 15 μM). The cumulative probability curves for each recording are displayed above the trace and on the right. The average normalized mEPSCs obtained from the recordings are also presented. The scale shown in the upper panel of and applies to all traces in those panels. Graphs depicting the mean (filled squares) and s.e.m. of wild-type and Hcn1−/− soma and dendritic mEPSC frequency in the absence and presence of ZD7288 are also shown. Open squares illustrate the mEPSC frequency from individual experiments. Right, amplitude histograms for the total number of mEPSCs obtained from wild-type soma (n = 7), wild-type dendrites (n = 5), Hcn1−/− soma (n = 6) and Hcn1−/− dendrites (n = 5) with and with! out ZD7288. Superimposed on the histograms are Gaussian fits to demonstrate the peak amplitude of mEPSCs. * Figure 2: HCN channel block or deletion has no effect on mEPSCs in entorhinal cortical layer II and layer V neurons. (,) Typical morphologies of mouse entorhinal cortical layer II stellate cell and layer V neuron. Scale bars represent 20 μm () and 50 μm (). (,) Representative mEPSC recordings from wild-type and Hcn1−/− layer II stellate and layer V pyramid soma before and after application of ZD7288 (15 μM). The values above the traces indicate that outward holding current at −70 mV. The average mEPSC obtained from the recordings are displayed below the traces. The cumulative probability curves for each of the traces are also illustrated on the right of the recordings. The scales shown in the upper trace for and apply to all traces in and , respectively. Right, graphs depicting the individual (open squares) and average (closed squares) mEPSC frequencies recorded from wild-type and Hcn1−/− stellate and layer V pyramid neurons before and after application of ZD7288. * Figure 3: HCN1 subunits are localized to active zones of synaptic terminals. (,) Immunoparticles for HCN1 along the extrasynaptic plasma membrane (for example, arrows) of dendritic spines (s) and shafts (Den) establishing synapses with excitatory terminals () in entorhinal cortical layer III. Presynaptic HCN1 subunits in excitatory terminals (b) were mainly located in the active zone. () Although present at lower proportions, immunoparticles for HCN1 were also observed in putative inhibitory axon terminals (it; for example, arrowheads), recognized by the shape of synaptic vesicles and the lack of a prominent postsynaptic density in the postsynaptic element, establishing synaptic contact with somata. Cyt, cytoplasm. () Immunoreactivity for HCN1 in entorhinal cortical layer III of Hcn1−/− mice as revealed at the electron microscopic level. No labeling could be detected in the Hcn1−/− mice. ax, axon terminal. () Quantitative analysis on the percentage of immunoparticles (n = 2,572) for HCN1 at post- and presynaptic sites in entorhinal cortical l! ayer III revealed that 72% were localized at postsynaptic sites and 28% at presynaptic sites. Scale bars represent 0.5 μm (,,) and 0.2 μm (). * Figure 4: Ca2+ dependence of entorhinal cortical layer III mEPSCs. () Traces from wild-type and Hcn1−/− neurons in the absence, presence and washout of Ca2+-free external solution. The outward holding current values are displayed above the traces. Right, the mean and s.e.m. of nine individual experiments. Scale bars apply to all traces. *P < 0.05. () Time course of the effects of applying Ca2+-free solution. The average frequency per minute during five wild-type and four Hcn1−/− recordings was calculated and normalized. () Example recordings from wild-type neurons under control conditions, following application of Ca2+-free solution and Ca2+-free solution containing ZD7288. The outward holding current values at −70 mV are indicated above the traces. Right, average frequency from four experiments. * Figure 5: T-type Ca2+ channel blockers reduce the increase in mEPSC frequency caused by pharmacological block of Ih or deletion of HCN1 subunits. (,) Recordings obtained in wild-type and Hcn1−/− neurons in the absence and presence of the potent and selective T-type Ca2+ channel blocker TTA-A2 (500 nM). In wild-type neurons, ZD7288 was applied to the neurons after TTA-A2. The outward holding current at a holding potential of −70 mV are indicated above the traces. Right, cumulative probability curves for all the recordings. () Bar graph demonstrating the effects of coapplication of ZD7288 (15 μM) and Ca2+ channel blockers for T-type (TTA-A2, 500 nM; TTA-P2, 1 μM; mibefradil (Mib), 10 μM), P/Q-type (ω-agatoxin IVA, 100 nM), N-type (ω-conotoxin GVIA, 100 nM), L-type (nifedipine, 2 mM) and R-type (SNX-482, 200 nM) Ca2+ channels. The effects of treatment with ZD7288 alone are also shown. *P < 0.05. () Graph illustrating the effects of the T-type (TTA-A2, TTA-P2 and mibefradil) and R-type (SNX-482) Ca2+ channel blockers on mEPSC frequency in Hcn1−/− entorhinal cortical layer III neurons. The numbers of observa! tions for each treatment are indicated above the individual bars (,). * Figure 6: CaV3.2 colocalize with HCN1 at entorhinal cortical synaptic terminals and cause the increase in excitatory synaptic transmission following HCN1 channel inhibition. () Two typical electron micrographs showing immunoreactivity for HCN1 (peroxidase reaction product) and Cav3.2 (immunoparticles) in wild-type sections as detected using pre-embedding double-labeling methods at the electron microscopic level. Immunogold particles for 2 were absent in CaV3.2−/− sections. b, axon terminals; s, dendritic spines; Den, dendrite. Scale bars represent 0.2 μm. () Example recordings showing the effects of 15-min bath application of ZD7288 (15 μM) on mEPSCs recorded from CaV3.2−/− entorhinal cortical layer III neurons and wild-type neurons. The vertical and horizontal scale bars on the traces represent 10 pA and 2 s, respectively. In values above the recordings indicate the outward holding currents at −70 mV. The cumulative probability curves are shown on the right. The average normalized mEPSC traces obtained from the traces before and after ZD7288 application are shown below the recordings. Right, individual (open squares) and mean (fille! d squares) frequency of mEPSCs obtained from wild type (n = 5) and CaV3.2−/− (n = 6) in the absence and presence of ZD7288. *P < 0.05. * Figure 7: Hyperpolarization enhances mEPSC frequency by activating T-type Ca2+ channels. (,) Representative traces and their cumulative probability curves illustrating the effects of lowering the external K+ concentration from 2.5 mM to 1.75 mM in wild-type and Hcn1−/− neurons. The effects of mibefradil (10 μM) on wild-type mEPSCs in 1.75 mM K+ are also shown. The outward holding currents at −70 mV are shown above the traces. Overlays of the average normalized EPSCs from wild-type and Hcn1−/− neurons under control conditions (black), with 1.75 mM K+ (light gray), and in the presence of 1.75 mM K+ and mibefradil (dark gray) are shown. Right, the average mEPSC frequency (filled squares) as well as the individual frequency values obtained from each experiment (open squares) under these conditions in wild-type and Hcn1−/− neurons are shown. *P < 0.05. () Graph showing the time course of the effects of reducing the K+ concentration from 2.5 mM to 1.75 mM in wild-type and Hcn1−/− neurons. The average mEPSC frequency for each minute of the recording ! before and after a 15-min bath application of 1.75 mM KCl is shown. * Figure 8: Effects of pharmacologically blocking HCN channels on evoked synaptic release. () Representative traces in the absence and presence of ZD7288 (15 μM) obtained at the soma are shown above the graph. In between each paired pulse, a single stimulus was used to elicit an EPSC. By subtracting this EPSC from the paired EPSCs, we obtained the amplitude and shape of the individual EPSCs. Insets, overlaid black (second EPSC) and gray (first EPSC) subtracted traces before and after application of ZD7288. The vertical and horizontal scale bars represent 40 pA and 50 ms respectively. () Graph showing the effects of bath applying ZD7288 on the PPR in entorhinal cortical layer III neurons. Pairs of EPSCs were obtained every minute by stimulating the distal dendrites of entorhinal cortical layer III neurons. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Pharmacology, The School of Pharmacy, University of London, London, UK. * Zhuo Huang & * Mala M Shah * Department Ciencias Medicas, Centro de Investigación en Discapacidades Neurológicas, Universidad de Castilla-La Mancha, Albacete, Spain. * Rafael Lujan * Department of Neuroscience, Physiology and Pharmacology, University College, London, UK. * Ivan Kadurin & * Annette C Dolphin * Department of Depression and Circadian Rhythms, Merck Research Laboratories, Pennsylvania, USA. * Victor N Uebele & * John J Renger Contributions Z.H. and M.M.S. performed and analyzed the electrophysiological experiments. Z.H., M.M.S. and R.L. performed the electron microscopy experiments. I.K. and A.C.D. performed western blot experiments and analysis. V.N.U. and J.J.R. provided valuable tools. M.M.S. designed the study and wrote the manuscript with contributions from all of the other authors. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Mala M Shah Author Details * Zhuo Huang Search for this author in: * NPG journals * PubMed * Google Scholar * Rafael Lujan Search for this author in: * NPG journals * PubMed * Google Scholar * Ivan Kadurin Search for this author in: * NPG journals * PubMed * Google Scholar * Victor N Uebele Search for this author in: * NPG journals * PubMed * Google Scholar * John J Renger Search for this author in: * NPG journals * PubMed * Google Scholar * Annette C Dolphin Search for this author in: * NPG journals * PubMed * Google Scholar * Mala M Shah Contact Mala M Shah Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (20M) Supplementary Figures 1–9 and Supplementary Tables 1–4 Additional data - Presynaptic CLC-3 determines quantal size of inhibitory transmission in the hippocampus
- Nat Neurosci 14(4):487-494 (2011)
Nature Neuroscience | Article Presynaptic CLC-3 determines quantal size of inhibitory transmission in the hippocampus * Vladimir Riazanski1, 2 * Ludmila V Deriy1, 2 * Pavel D Shevchenko1 * Brandy Le1 * Erwin A Gomez1 * Deborah J Nelson1 * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 14,Pages:487–494Year published:(2011)DOI:doi:10.1038/nn.2775Received13 September 2010Accepted02 February 2011Published online06 March 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The absence of the chloride channel CLC-3 in Clcn3−/− mice results in hippocampal degeneration with a distinct temporal-spatial sequence that resembles neuronal loss in temporal lobe epilepsy. We examined how the loss of CLC-3 might affect GABAergic synaptic transmission in the hippocampus. An electrophysiological study of synaptic function in hippocampal slices taken from Clcn3−/− mice before the onset of neurodegeneration revealed a substantial decrease in the amplitude and frequency of miniature inhibitory postsynaptic currents compared with those in wild-type slices. We found that CLC-3 colocalized with the vesicular GABA transporter VGAT in the CA1 region of the hippocampus. Acidification of inhibitory synaptic vesicles induced by Cl− showed a marked dependence on CLC-3 expression. The decrease in inhibitory transmission in Clcn3−/− mice suggests that the neurotransmitter loading of synaptic vesicles was reduced, which we attribute to defective vesicular a! cidification. Our observations extend the role of Cl− in inhibitory transmission from that of a postsynaptic permeant species to a presynaptic regulatory element. View full text Figures at a glance * Figure 1: Colocalization of CLC-3 with VGAT. Immunofluorescence labeling of CLC-3 in the CA1 area of hippocampus. () Differential interference contrast image and fluorescent immunostaining for CLC-3 (red dots) show perisomatic localization of CLC-3 around CA1 pyramidal cell bodies (asterisks). Scale bar, 5 μm. (–) Double immunostaining with antibodies to CLC-3730–744 (green) and VGAT (red) in CA1 shows colocalization of CLC-3 with VGAT in the pyramidal cell layer (pcl). () Enlarged image (boxed area in ) shows colocalization to a subset of puncta adjacent to cell bodies of pyramidal neurons. Qualitative analysis of colocalization was done using ImageJ plug-in Jacop. Background subtraction was performed to eliminate noise. Spots analyzed were plotted as the normalized intensity of CLC-3 (green) as a function of VGAT (red). Manders' coefficients (with threshold) were 0.44 for fraction of VGAT overlapping ClC-3 and 0.8 for the fraction of CLC-3 overlapping VGAT with an overlap correlation coefficient of 0.92. Scale b! ar, 10 μm. sr, stratum radiatum; so, stratum oriens. * Figure 2: Immunogold localization of CLC-3 on inhibitory synaptic vesicle fractions. () Electron micrograph of crude CSV fraction (LP2) (see Online Methods). This fraction served as starting material for immunoisolation of subsets of synaptic vesicles. Scale bar, 200 nm. () Electron micrograph of CLC-3 immunodetection on immunoisolated inhibitory synaptic vesicles. Monoclonal VGAT antibody was conjugated to Spherobeads (diameter, 1–1.4 μm) and used for positive selection of inhibitory synaptic vesicles followed by postembedding staining with antibodies to CLC-3 and secondary goat anti-rabbit IgG conjugated to 15-nm gold particles. Scale bar, 200 nm. Lower panel, enlargements of boxed areas above. * Figure 3: Decreased miniature IPSC frequency and amplitudes in Clcn3−/− mice. () Representative miniature IPSCs (mIPSCs) recorded in the presence of tetrodotoxin (TTX; 1 μM) and kynurenic acid (3 mM) from CA1 pyramidal neurons from Clcn3+/+ and Clcn3−/− mice. () Data summary showing a significant decrease in mIPSC frequency in Clcn3−/− neurons (Clcn3+/+ 14 ± 1.3 Hz, n = 13 cells; Clcn3−/− 8.4 ± 1.0 Hz, n = 9 cells; **P < 0.005, one-way ANOVA). () Representative detected and superimposed mIPSC traces recorded from CA1 pyramidal cells in Clcn3+/+ and Clcn3−/− slices. Average current traces are shown by the smooth red line. () Cumulative amplitude distribution of mIPSCs recorded from Clcn3+/+ (filled gray bars) and Clcn3−/− (hatched red bars) neurons; 3,934 events from seven cells were used for analysis for each group. Superimposed cumulative amplitude probability graph of mIPSCs from Clcn3+/+ (black line) and Clcn3−/− (red line) neurons shows that amplitude distribution is shifted to the left for the Clcn3−/− events, indic! ating smaller mIPSCs in the Clcn3−/− mice (P < 0.001, Kolmogorov-Smirnov test). Inset: box plot analysis of average amplitude of mIPSCs from Clcn3+/+ (black) and Clcn3−/− (red) neurons (upper inner fence = 75th percentile + (1.5 × interquartile range), lower inner fence = 25th percentile − (1.5 × interquartile range)). * Figure 4: Buffering with Tris reduces inhibitory synaptic transmission. () Representative evoked IPSC traces recorded before (black line) and during extracellular application of 20 mM (red trace) and 50 mM (blue trace) Tris. () Time course of evoked IPSC peak amplitude changes with bath application of 50 mM Tris (pH 7.4) during 0.2-Hz stimulation. Average of every ten evoked IPSC peaks is shown by the smooth red line. () Representative evoked IPSC traces recorded before (black line) and during extracellular application of 20 mM (red trace) of Tris from representative CA1 pyramidal neurons from Clcn3+/+ and Clcn3−/− mice. () Pooled data showing significantly different (*P < 0.05; **P < 0.005; one-way ANOVA) inhibition of evoked IPSCs with 20 mM Tris in slices from Clcn3+/+ (n = 7) and Clcn3−/− (n = 5) mice and 50 mM (n = 6) Tris in slices from Clcn3+/+ mice. () Representative time course of mIPSC peak amplitude (gray dots) changes with bath application of 50 mM Tris (pH 7.4). Average of every 10,000 mIPSC peaks is shown by the smooth red ! line. () Representative detected and superimposed mIPSC traces recorded from a Clcn3+/+ CA1 pyramidal cell after 10 and 40 min of recording in 50 mM Tris. Average current traces are shown by the smooth red line. () Cumulative amplitude distribution of mIPSCs recorded before (filled gray bars) and 30 min after (hatched red bars) perfusion of Clcn3+/+ slices with 50 mM Tris; 6,000 events from six cells used for analysis for each group. Cumulative amplitude probability graph of mIPSCs peaks during control (black line) and 50 mM Tris (red line) from Clcn3+/+ neurons shows that the amplitude distribution is shifted to the left for events recorded in Tris, indicating smaller mIPSCs (P < 0.001; Kolmogorov-Smirnov test). Inset: box plot analysis of average amplitude of mIPSCs in control period (black) and period with Tris effect (red) on mIPSC (upper inner fence = 75th percentile + (1.5 × interquartile range), lower inner fence = 25th percentile − (1.5 × interquartile range)). ! () Data summary showing changes in mIPSC frequency in control ! period and 30 min after 50 mM Tris application (control period: 16.1 ± 1.8 Hz; 30 min in Tris: 14.6 ± 0.9 Hz; n = 7 cells; gray, individual cells; black, average). * Figure 5: Immunoisolation reveals importance of CLC-3 for acidification of inhibitory synaptic vesicles (SVs). () Averaged traces of [Cl−]-dependent acidification of CSVs from Clcn3+/+ (black, n = 10) and Clcn3−/− (red, n = 9) mice normalized against the original level of acridine orange fluorescence at 2 mM ATP and 0 mM Cl−. () Effect of CLC-3 on the rates of acidification of Clcn3+/+ versus Clcn3−/− CSVs calculated as τdecay from exponential fits of individual traces. Data presented as mean ± s.e.m., Clcn3+/+ (black, n = 10) and Clcn3−/− (red, n = 9); *P < 0.05, one-way ANOVA. Inset: exponential fits of individual acidification traces at a single Cl− concentration as an example of differential effect of CLC-3 on the rate of acidification of Clcn3+/+ versus Clcn3−/− CSVs. () Representative traces of acidification of inhibitory synaptic vesicles (CSV(−)VGLUT1) from Clcn3−/− (red), Clcn3+/+ (black) and Clcn3+/+ CLC-3-depleted (blue) synaptic vesicles. Traces represent preparations each consisting of at least eight animals. (–) Western blot characterizat! ion of immunoisolated fractions of synaptic vesicles from Clcn3+/+ and Clcn3−/− mice. () Upper panel: blot probed with anti-VGLUT1 antibody to show depletion of VGLUT1 (CSV(−)VGLUT1). Middle panel: probed with anti-VGAT antibody to show VGAT enrichment of (CSV(−)VGLUT1) fraction. Lower panel: probed with anti-VAMP antibody as a load control. () Western blot of doubly depleted fraction probed with anti–CLC-3 (upper panel) shows that CSV(−)VGLUT1(−)CLC-3 fraction is devoid of CLC-3. Anti-VAMP detection was used as a load control (lower panel). () Characterization of VGLUT1 depletion (top) and VGAT enrichment (middle) of vesicles immunoisolated from Clcn3−/− mice. Anti-VAMP detection was performed to control for loading. For full-length blots, see Supplementary Figure 5. * Figure 6: Differential degree and rate of acidification of inhibitory versus excitatory synaptic vesicles. () Western blot characterization of rat enriched inhibitory synaptic vesicle fraction immunoisolated by depletion on magnetic beads coated with anti-VGLUT1 antibodies (see Online Methods). Top: blot probed with anti-VGLUT1 antibody to show the depletion of VGLUT1 (CSV(−)VGLUT1). Middle: probed with anti-VGAT antibody to show VGAT enrichment of (CSV(−)VGLUT1) fraction. Bottom: probed with anti-VAMP antibody as a load control. () Representative traces of acidification of inhibitory (CSV(−)VGLUT1) and excitatory (CSV(−)VGAT) synaptic vesicles as a function of various Cl− concentrations and in the presence of 10 mM GABA or glutamate, respectively. FCCP, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone. () Comparison of the degree of [Cl−]-dependent acidification calculated as the fraction of the initial level of acridine orange fluorescence at 2 mM ATP and 0 mM Cl− in inhibitory (blue) versus excitatory (red) synaptic vesicle fractions in the presence of 10 mM! of GABA or glutamate, respectively. () Comparison of the rate of [Cl−]-dependent acidification in inhibitory (blue) and excitatory (red) synaptic vesicle fractions calculated as τdecay determined from exponential fits to individual traces. Data in and are mean ± s.e.m. (n = 3); *P < 0.05, one-way ANOVA. N represents a number of preparations each consisting of at least five animals. For full-length blots, see Supplementary Figure 6. * Figure 7: CLC-3 is important for the degree and the rate of acidification of inhibitory synaptic vesicles. () Western blot characterization of CSVs, inhibitory synaptic vesicle fraction (CSV(−)VGLUT1) and inhibitory synaptic vesicle fraction depleted of CLC-3 on magnetic beads coated with antibodies to CLC-3 (CSV(−)VGLUT1(−)CLC-3). Western blot was probed with antibodies to VGLUT1, VGLUT2, VGAT, CLC-3 and vATPase. Blot probed with anti-VAMP2 served as a load control. () Representative traces of [Cl−]-dependent acidification for inhibitory synaptic vesicles in the presence (black) or absence (red) of CLC-3. Acidification was initiated by addition of 2 mM ATP in the presence of 10 mM GABA. () Comparison of [Cl−]-dependent acidification of inhibitory synaptic vesicles in the presence (black) or absence (red) of CLC-3 as a fraction of the original level at 2 mM ATP and 0 mM Cl−. For full-length blots, see Supplementary Figure 7. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Vladimir Riazanski & * Ludmila V Deriy Affiliations * Department of Neurobiology, Pharmacology and Physiology, University of Chicago, Chicago, Illinois, USA. * Vladimir Riazanski, * Ludmila V Deriy, * Pavel D Shevchenko, * Brandy Le, * Erwin A Gomez & * Deborah J Nelson Contributions V.R., L.V.D. and D.J.N. designed the project, analyzed the data and wrote the manuscript. V.R, L.V.D., P.D.S., B.L. and E.A.G. performed the experiments. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Deborah J Nelson Author Details * Vladimir Riazanski Search for this author in: * NPG journals * PubMed * Google Scholar * Ludmila V Deriy Search for this author in: * NPG journals * PubMed * Google Scholar * Pavel D Shevchenko Search for this author in: * NPG journals * PubMed * Google Scholar * Brandy Le Search for this author in: * NPG journals * PubMed * Google Scholar * Erwin A Gomez Search for this author in: * NPG journals * PubMed * Google Scholar * Deborah J Nelson Contact Deborah J Nelson Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (8M) Supplementary Figures 1–7 and Supplementary Table 1 Additional data - Temporally matched subpopulations of selectively interconnected principal neurons in the hippocampus
- Nat Neurosci 14(4):495-504 (2011)
Nature Neuroscience | Article Temporally matched subpopulations of selectively interconnected principal neurons in the hippocampus * Yuichi Deguchi1, 2 * Flavio Donato1, 2 * Ivan Galimberti1, 2 * Erik Cabuy1 * Pico Caroni1 * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 14,Pages:495–504Year published:(2011)DOI:doi:10.1038/nn.2768Received03 December 2010Accepted18 January 2011Published online27 February 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The extent to which individual neurons are interconnected selectively within brain circuits is an unresolved problem in neuroscience. Neurons can be organized into preferentially interconnected microcircuits, but whether this reflects genetically defined subpopulations is unclear. We found that the principal neurons in the main subdivisions of the hippocampus consist of distinct subpopulations that are generated during distinct time windows and that interconnect selectively across subdivisions. In two mouse lines in which transgene expression was driven by the neuron-specific Thy1 promoter, transgene expression allowed us to visualize distinct populations of principal neurons with unique and matched patterns of gene expression, shared distinct neurogenesis and synaptogenesis time windows, and selective connectivity at dentate gyrus-CA3 and CA3-CA1 synapses. Matched subpopulation marker genes and neuronal subtype markers mapped near clusters of olfactory receptor genes. The n! onoverlapping matched timings of synaptogenesis accounted for the selective connectivities of these neurons in CA3. Therefore, the hippocampus contains parallel connectivity channels assembled from distinct principal neuron subpopulations through matched schedules of synaptogenesis. View full text Figures at a glance * Figure 1: Distinct transcriptomes of Lsi1 and Lsi2 hippocampal principal neurons. () Transcriptome analysis of Lsi1 and Lsi2 granule cells. Left, reproducibility of microarray analysis. Middle, numbers of genes up- or downregulated compared to average (16 weeks data). Right, heat map for 30 differentially regulated genes at 4 ages; note independent clustering in Lsi1 and Lsi2 granule cells (GCs). Error bars, s.e.m. () Differentially expressed genes in Lsi1 and Lsi2 granule cells. Left, columns are average values from three mice. Right, comparable gene expression profiles in granule cells from mice with many (15–20% of total) or few (2–5% of total) GFP-positive neurons; columns are values in one mouse each. () Transcriptomes of Lsi1 and Lsi2 pyramidal neurons. Details as in and . () In silico cell grouping. The unbiased hierarchical tree algorithm grouped cells according to subpopulations of granule cells, and pyramidal neurons in CA3 and CA1. * Figure 2: Detection of Lsi1 and Lsi2 precursors and neurons during hippocampal development. () Transgene expression during hippocampal neurogenesis. Expression in radial glia cells (left) and neuroblast groups (right). Some of the GFP+ neuroblasts are Nestin+ (right, arrow). HN, hippocampal neuroepithelium. () Spatial distribution of GFP+ neuroblasts in hippocampal neuroepithelium of Lsi1 and Lsi2 embryos at E11.5. The heat map (top) visualizes specific differences between Lsi1 and Lsi2 embryos at the same developmental age (see also Supplementary Fig. 12); the quantitative analysis (bottom) represents average values for three embryos each. () Specification of Lsi1 neuroblasts during hippocampal neurogenesis. At E11.5, GFP+ neuroblasts (arrows) co-distribute with regions of proliferating precursors (Tbr2+; top, right). At E13.5, GFP+ neuroblasts (white arrows) accumulate in defined Ki67-depleted non-proliferating regions (n = 6) that had been labeled with EdU at E11.5 (arrow) and are now depleted of Nestin precursors (arrow). Scale bar, 50 μm. * Figure 3: Temporal windows of Lsi1 and Lsi2 neurogenesis during hippocampal development. () Lsi1 and Lsi2 granule cells are generated during early phases of neurogenesis. Top, BrdU labeling experiment; arrows, BrdU+ GFP+ granule cell (yellow), BrdU+ GFP− granule cell (red) and weakly BrdU+ granule cell (white). Bottom left, fractions of total GFP+ granule cells labeled with BrdU at different time intervals (avg., fractions of all granule cells labeled with BrdU). Averages from three mice each; values normalized to troughs between two neurogenesis peaks; vertical line, trough between early and late neurogenesis wave for average granule cells. Statistical analysis: comparisons between Lsi1 and Lsi2 values at individual ages; Mann Whitney test; **P < 0.001; ***P < 0.0001. Error bars, s.e.m. Bottom right, fractions of BrdU+ GFP+ neurons labeled at E12.5 are not affected by total numbers of GFP+ granule cells. () Neurogenesis of Lsi1 and Lsi2 hippocampal pyramidal neurons. Details as in . Scale bar, 20 μm. * Figure 4: Transcripts shared among Lsi1 or Lsi2 subpopulations in dentate gyrus, CA3 and CA1. () Examples of transcripts co-regulated in Lsi1 or Lsi2 principal neurons in dentate gyrus (DG), CA3 and CA1. Error bars, s.e.m. () Marker combinations identifying Lsi1 and Lsi2 principal neurons in the hippocampus. Combined GFP-in situ hybridization detection. Arrows, GFP+ marker+ (yellow) and GFP+ marker− (green). Quantitative analyses: data from eight sections each, covering all anterior-posterior levels of hippocampus; n = 3 mice each. () Genes that are up- or downregulated in all three types of Lsi1 hippocampal principal neurons are closely co-regulated in granule cells, and in pyramidal neurons in CA3 and CA1. Each line represents one gene and its deviation from average values. () High probability that subtype-specific genes map near olfactory receptor (OR) gene clusters. Left: relationship between subtype markers and olfactory receptor gene cluster vicinity. Random: 400 random genes in average granule cells; Maturation: genes enriched in Lsi1 (16 w) over Lsi1 (8 w) ! granule cells; HP(Su1), HP(Su2): genes selectively regulated in all Lsi1 or Lsi2 principal neurons. Right: optimization of OR vicinity for genes up- (4–5 fold) or downregulated (>5 fold) in Lsi1 or Lsi2 principal neurons. * Figure 5: Distinct and matched synaptogenesis schedules by Lsi1 and Lsi2 subpopulations. () Nonoverlapping dendritic maturation and synaptogenesis processes in Lsi1 and Lsi2 pyramidal neurons in CA3 and CA1. Left and center, representative panels and camera lucidas. Mann Whitney test; Error bars, s.e.m. () Immature granule cell transcript contents in Lsi1, Lsi2 and average granule cells. Dcx, doublecortin. () Nonoverlapping dendritic maturation and synaptogenesis processes by Lsi1 and Lsi2 granule cells. Arrows, presence (yellow) or absence (green) of specific contacts between mossy fibers and pyramidal neurons. Scatter plot, presynaptic maturation index values. () Synaptogenesis subgroups in Lmu1 pyramidal neurons and mossy fibers at P7. Panel and camera lucida: representative examples of labeled dendrites (panel, stratum radiatum) and mossy fibers (lucida). Scale bar, 10 μm. *P < 0.05, **P < 0.01, ***P < 0.001. * Figure 6: Selective connectivity between matched granule cells and CA3 pyramidal neuron subpopulations. () Light microscopic analysis of stratum lucidum mossy fiber synapses in Lsi1 mouse at 1 month. Left, overview, and higher magnification view with superimposed camera lucida; yellow arrows, verified contacts. Right, example of verified Lsi1 mossy fiber terminal-pyramidal neuron contact (yellow arrows). High-magnification panels, single confocal planes. Lower row, quantitative analysis of stratum lucidum synaptic contacts in Lsi1 and Lsi2 mice. Left, examples of synaptic contact analysis for six stratum lucidum volumes along CA3, including numbers of GFP+ mossy fibers, numbers of GFP+ pyramidal neurons, and numbers of contacts for each pyramidal neuron. Center, average numbers of Lsi1 (or Lsi2) mossy fiber terminals contacting Lsi1 (or Lsi2) CA3 pyramidal neurons. Normalized to 25 GFP-positive granule cells; 55 μm sections; n = 60 pyramidal neurons, from 3 mice each; random, expected values for random connectivity. Right, cumulative probability values that contacts are chanc! e events for first 12 (100 × 100 × 55 μm) volumes in Lsi1 and Lsi2 mice (averages from 3 mice each). () Immuno-electron microscopic analysis of Lsi1-Lsi1 synaptic contacts in CA3 stratum lucidum. Immunolabeled (yellow) and non-labeled (white) dendrites (dendr.) and large mossy fiber terminals (LMT) at increasing magnification (left to right); yellow arrows, labeled thorny excrescence profiles inside labeled LMTs. Right, example, with three-dimensional reconstruction of labeled pre- and postsynaptic elements. () Connectivity between randomly labeled CA3 pyramidal neurons and Lsi1 granule cells. Left, examples of mCherry-labeled pyramidal neurons (red) and Lsi1 granule cells and pyramidal neurons in stratum lucidum. Center, average distance between Lsi1 mossy fiber synaptic contacts on Lsi1 or mCherry-labeled pyramidal neurons; percentages, fractions of Lsi1-like and non-Lsi1-like pyramidal neurons. Right, average numbers of contacts by Lsi1 mossy fiber terminals with Lsi1! or randomly labeled CA3 pyramidal neurons. Only low random: p! utative Lsi1 CA3 pyramidal neurons with high densities of Lsi1 contacts removed from the dataset. Average dendrite length in stratum lucidum, 184 μm. n = 60 pyramidal neurons, from 3 mice each. Mann-Whitney test; **P < 0.01, ***P < 0.0001. Error bars, s.e.m. Scale bars, 1 (,), 10 (, left) and 2 (, right) μm. * Figure 7: Selective connectivity between matched CA3 and CA1 pyramidal neuron subpopulations. () Examples of labeled CA3 axons (Schaffer collaterals; sc) and CA1 pyramidal neurons in stratum radiatum. Left, Lsi1 Schaffer collateral contacting two Lsi1 CA1 pyramidal neurons. Right, examples of contacts with mCherry-labeled CA1 pyramidal neuron (left) and Lsi1 CA1 pyramidal neuron. Arrows, contacts with (yellow) or without (green) boutons. () Quantitative analysis of Lsi1 connectivity in CA1 stratum radiatum. Left, schematic of how Lsi1 or randomly labeled CA1 pyramidal neurons were analyzed in the same mice for contacts by Lsi1 Schaffer collaterals. Center, fraction of contacts with boutons for Lsi1-Lsi1 and for Lsi1-random CA3-CA1 pairs. n = 90 CA1 pyramidal neurons each, from 3 mice. Error bars, s.e.m. Right, contacts with boutons versus total numbers of contacts for individual Lsi1 and randomly labeled CA1 pyramidal neurons (individual green squares and red dots). Arrows, putative Lsi1 CA1 pyramidal neurons among randomly labeled sample. Gray line, values for 100% ! synaptic connectivity. CA3 Lsi1 axons contacted Lsi1 and randomly labeled pyramidal neurons with undistinguishable frequencies, but only Lsi1-Lsi1 pairs showed high frequencies of boutons. Scale bars, 25 μm (, left), 2 μm (, right). * Figure 8: Influence of synaptogenesis timing and subpopulation identity on selective connectivity. () Schematic representation of heterochronic co-culture experiments. Boxed: age of dentate gyrus (DG) and CA3 fragment at beginning of culture. Mossy fiber synaptogenesis timing is indicated in color (Lsi1: red; Lsi2: blue), and with an arrow pointing from DG to CA3. () Heterochronic co-culture experiments. The panels show a low-magnification view of an heterochronic co-culture (yellow line: DG/CA3 co-culture boundary), and high-magnification views of synaptic and non-synaptic contacts (single confocal planes). Bass: Bassoon. Quantitative analysis: fraction of synaptic (Bassoon+) versus total contacts on Lsi1 or Lsi2 pyramidal neurons in CA3 (see Methods). N = 60, from 3 independent cultures each. Error bars, s.e.m. Scale bars, 50 (, left), 3 (, right) μm. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Yuichi Deguchi, * Flavio Donato & * Ivan Galimberti Affiliations * Friedrich Miescher Institute, Basel, Switzerland. * Yuichi Deguchi, * Flavio Donato, * Ivan Galimberti, * Erik Cabuy & * Pico Caroni Contributions Y.D. conceived and carried out the gene expression and the adult subpopulation mapping analysis. F.D. conceived and carried out the neurogenesis and synaptogenesis analysis and parts of the electron microscopy and connectivity analysis. I.G. conceived and carried out most of the connectivity analysis. E.C. carried out and optimized the cell genomics experiments. P.C. helped devise the experiments and wrote the manuscript. All authors discussed the results and commented on the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Pico Caroni Author Details * Yuichi Deguchi Search for this author in: * NPG journals * PubMed * Google Scholar * Flavio Donato Search for this author in: * NPG journals * PubMed * Google Scholar * Ivan Galimberti Search for this author in: * NPG journals * PubMed * Google Scholar * Erik Cabuy Search for this author in: * NPG journals * PubMed * Google Scholar * Pico Caroni Contact Pico Caroni Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (4M) Supplementary Figures 1–12 Additional data - A neuroprotective role for polyamines in a Xenopus tadpole model of epilepsy
- Nat Neurosci 14(4):505-512 (2011)
Nature Neuroscience | Article A neuroprotective role for polyamines in a Xenopus tadpole model of epilepsy * Mark R Bell1 * James A Belarde1 * Hannah F Johnson1 * Carlos D Aizenman1 * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 14,Pages:505–512Year published:(2011)DOI:doi:10.1038/nn.2777Received19 October 2010Accepted24 January 2011Published online06 March 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Polyamines are endogenous molecules involved in cell damage following neurological insults, although it is unclear whether polyamines reduce or exacerbate this damage. We used a developmental seizure model in which we exposed Xenopus laevis tadpoles to pentylenetetrazole (PTZ), a known convulsant. We found that, after an initial PTZ exposure, seizure onset times were delayed in response to a second PTZ exposure 4 h later. This protective effect was a result of activity-dependent increases in synthesis of putrescine, the simplest polyamine. Unlike more complex polyamines that directly modulate ion channels, putrescine exerted its effect by altering the balance of excitation to inhibition. Tectal neuron recordings, 4 h after the initial seizure, revealed an elevated frequency of GABAergic spontaneous inhibitory postsynaptic currents. Our data suggest that this effect is mediated by an atypical pathway that converts putrescine into GABA, which then activates presynaptic GABAB r! eceptors. Our data suggest that polyamines have a previously unknown neuroprotective role in the developing brain. View full text Figures at a glance * Figure 1: PTZ-induced seizures alter the onset latency of subsequent seizures. () Metabolic pathways of polyamine synthesis. ABAL, 4-aminobutanal; DSAM, decarboxylated s-adenosylmethionine (SAM); SAMDC, S-adenosylmethionine decarboxylase. () Stage 46 Xenopus tadpole under normal conditions and during a seizure. Scale bars represent 2 mm. () Cumulative distribution of seizure onset latencies. Primed group was given a seizure 4 h before testing (see Online Methods). () Average normalized seizure onset latencies in relation to control from (control, 100 ± 4.0%, n = 7; primed, 127.7 ± 4.1%, n = 8). () Seizure onset latency differences between control and primed group at various time-points following initial seizure (0.5 h: control = 657.6 ± 31.9 s, primed = 465.7 ± 60.6 s, n = 9; 1 h: control = 742.1 ± 46.1 s, primed = 597.0 ± 44.5 s, n = 11; 2 h: control = 683.5 ± 41.9 s, primed = 838.3 ± 43.1 s, n = 8; 4 h: control = 723.5 ± 40.9 s, primed = 1,043.5 ± 27.7 s, n = 8). () Protective effects can be obtained by using consultants other than PTZ (con! trol, 100 ± 6.7%, n = 9; PTZ, 137.8 ± 8.7%, n = 9; 4-AP, 234.8% (no seizures occurred in time frame), n = 9; kainate, 117.6 ± 16.6%, n = 9). *P < 0.05, ***P < 0.001, both Mann-Whitney. Error bars are s.e.m. * Figure 2: Increases in putrescine levels delay seizure onset. (,) Cumulative distribution of seizure onset times. Primed group was given a seizure 4 h before testing. DFMO and CGPa groups were given 10 mM DFMO and 10 μM CGPa, respectively, for 6 h before the testing seizure. P+DFMO and P+CGPa groups were treated identically as their DFMO and CGPa counterparts, but with a priming seizure included 4 h before testing. () Average normalized seizure onset times in relation to controls from a and b (control, 100 ± 4.3%, n = 22; primed, 124.3 ± 4.3%, n = 12; DFMO, 90.0 ± 5.3%, n = 8; P+DFMO, 69.7 ± 5.3%, n = 10; CGPa, 134.4 ± 7.6%, n = 8; P+CGPa, 137.2 ± 7.1%, n = 12). (–) Cumulative distribution and average normalized seizure onset times for putrescine (,; control, 100 ± 5.8%, n = 12; putrescine, 163.5 ± 6.0%, n = 10) and spermine (,; control, 100 ± 5.8%, n = 8; spermine, 105.8 ± 6.0%, n = 6). Both putrescine and spermine groups were given 1 μM of their respective polyamine for 2 h before a testing seizure. **P < 0.01, #P < 0.0! 001, both Mann-Whitney. Error bars are s.e.m. * Figure 3: PTZ-induced seizures occur in the optic tectum, but do not alter many of the intrinsic and synaptic properties of tectal neurons. () Example of field potentials taken in control animals and in the presence of 15 mM PTZ. () The retification index, a measure of polyamine block of Ca2+-permiable AMPARs, in control and primed conditions. Top, example traces of AMPA current at +40 and −60 mV. Bottom, retification index average (control, 1.07 ± 0.24, n = 5; primed, 0.98 ± 0.07, n = 5). () Comparison of AMPA to NMDA ratios. Top, example traces of AMPA (−60 mV) and NMDA (+60 mV) in both control and primed conditions. Bottom, AMPA:NMDA average (control, 3.17 ± 0.56, n = 10; primed, 3.36 ± 0.67, n = 8). () Comparison of Na+ and K+I/V curves. Top, example current traces with corresponding voltage step from −60 mV baseline to +30 mV. Bottom, I/V graph of Na+ and K+ currents in both control (n = 13) and primed (n = 9) conditions. () Analysis of Na+ currents after repetitive activation. Top, example current trace. Cells were depolarized from −60 to 0 mV for 15 ms every 35 ms. Bottom, amplitude ratio of t! he fifth peak to the first peak (control, 0.70 ± 0.04, n = 13; primed, 0.81 ± 0.04, n = 9; P = 0.045). Error bars are s.e.m. * Figure 4: Priming seizures and direct putrescine administration both increase GABA spontaneous frequency in tectal neurons. () Examples of whole-cell recordings of spontaneous AMPA- (−45 mV) and GABA-mediated (+5 mV) PSCs. The primed group was given a seizure 4 h before testing and the putrescine group was exposed to 0.1 mM putrescine for 2 h before recording. () Average spontaneous frequency; left and right panels are same data (control, AMPA = 2.46 ± 0.55 events per s, GABA = 2.36 ± 0.37 events per s, n = 14; primed, AMPA = 2.15 ± 0.31 events per s, GABA = 7.1 ± 0.5 events per s, n = 18; putrescine, AMPA = 2.74 ± 0.44 events per s, GABA = 4.06 ± 0.68 events per s, n = 10). () Average spontaneous amplitude; left and right panels are same data (control, AMPA = 7.92 ± 0.35 pA, GABA = 7.24 ± 0.45 pA, n = 14; primed, AMPA = 5.58 ± 0.35 pA, GABA = 6.2 ± 0.53 pA, n = 18; putrescine: AMPA = 6.77 ± 0.57 pA, GABA = 7.15 ± 0.68 pA, n = 10). *P < 0.05, **P < 0.001, #P < 0.0001; Wilcoxon. Error bars are s.e.m. * Figure 5: Putrescine converts to GABA in substantial quantities after a seizure. Treatment with the DAO inhibitor AGBC prevented increased onset times in primed animals by blocking putrescine-to-GABA conversion. () Cumulative distribution of seizures onset times. Primed group was given a seizure 4 h before testing. Primed + AGBC (P+AGBC) group was given 1 mM AGBC for 6 h before the testing seizure in addition to a priming seizure 4 h before testing. () Average normalized seizure onset latencies to control from a (control, 100.0 ± 10.7%, n = 4; primed, 152.9 ± 7.1%, n = 7; P+AGBC, 68.9 ± 11.6%, n = 8). (,) GABA levels measured by ELISA. GABA levels at various time-points after initial seizure, compared to controls, are shown in c (control, 100.0 ± 5.7%, n = 6; 1 h, 237.7 ± 30.2%, n = 6; 4 h, 126.6 ± 17.3%, n = 6). GABA levels compared to controls for AGBC-treated groups are shown in d. AGBC prevented significant increases in GABA levels that occurred 1 h post seizure (control, 100.0 ± 12.2%, n = 6; 1 h + AGBC, 108.7 ± 37.2%, n = 6; AGBC only, 103.! 9 ± 10.7%, n = 3). **P < 0.01, unpaired t test. ***P = 0.0003, Mann-Whitney. Error bars are s.e.m. * Figure 6: Disruption of GABABRs by the GABABR antagonist CGPb in conjunction with priming seizures causes decreases in seizure onset latencies. () Cumulative distribution of seizure onset times. Primed group was given a seizure 4 h before testing. P+CGPb group was given 50 μM of CGPb for 6 h before the testing seizure in addition to a priming seizure 4 h before testing. () Averaged normalized seizure onset latencies to control from a (control, 100.0 ± 6.3%, n = 8; primed, 153.8 ± 6.6%, n = 8; P+CGPb, 48.8 ± 11.6%, n = 8). ***P = 0.0002, Mann-Whitney. Error bars are s.e.m. * Figure 7: GABABRs are a likely target of putrescine-converted GABA. Treatment with the selective GABABR agonist SKF causes increases in seizure onset latencies and corresponding increases in GABA spontaneous frequency. () Cumulative distribution of seizure onset times. SKF group was given 25 μM of SKF for 2 h and then placed in control media for an additional 2 h. () Averaged normalized seizure onset latencies to control from a (control, 100.0 ± 6.4%, n = 26; SKF, 144.7 ± 7.3%, n = 17; ***P = 0.001, unpaired t test). () Data are presented as in a, except SKF acute group was treated with the agonist for 2 h without additional control media exposure. () Averaged normalized seizure onset latencies to control from c (control, 100.0 ± 6.7%, n = 8; SKF acute, 90.7 ± 5.6%, n = 8). (,) Transient treatment with SKF increases GABA spontaneous events. Examples of whole-cell recordings of spontaneous AMPAR- (−45 mV) and GABAR-mediated (+5 mV) currents are shown in . The average spontaneous frequency is shown in f (control, AMPA = 1.64 ± 0.4 even! ts per s, GABA = 1.99 ± 0.32 events per s, n = 12; SKF, AMPA = 1.73 ± 0.33 events per s, GABA = 3.13 ± 0.31 events per s, n = 17; **P < 0.01, Wilcoxon). Error bars are s.e.m. * Figure 8: Blocking putrescine production during a seizure results in long-term damage. () Cumulative distribution of seizure onset times. Primed group was given a seizure 24 h before testing. DFMO and CGPa groups were given 10 mM DFMO and 10 μM of CGPa, respectively, for 2 h before a priming seizure and for 4 h after. Both groups then spent the remaining 20 h in control media before the testing seizure. () Average normalized seizure onset times in relation to control (control, 100.0 ± 5.9%, n = 10; primed, 91.2 ± 6.2%, n = 10; DFMO, 66.6 ± 5.9%, n = 8; CGPa, 100.3 ± 6.4%, n = 10). **P < 0.01, Mann-Whitney. Error bars are s.e.m. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Neuroscience, Brown University, Providence, Rhode Island, USA. * Mark R Bell, * James A Belarde, * Hannah F Johnson & * Carlos D Aizenman Contributions M.R.B. conducted the experiments and wrote the manuscript. J.A.B. contributed to the behavioral testing. H.F.J. contributed to the behavioral testing and the ELISA. C.D.A. supervised the project and edited the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Carlos D Aizenman Author Details * Mark R Bell Search for this author in: * NPG journals * PubMed * Google Scholar * James A Belarde Search for this author in: * NPG journals * PubMed * Google Scholar * Hannah F Johnson Search for this author in: * NPG journals * PubMed * Google Scholar * Carlos D Aizenman Contact Carlos D Aizenman Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information Movies * Supplementary Movie 1 (5M) Xenopus tapole induced PTZ seizures. PDF files * Supplementary Text and Figures (164K) Supplementary Figures 1–3 Additional data - Ultra light-sensitive and fast neuronal activation with the Ca2+-permeable channelrhodopsin CatCh
- Nat Neurosci 14(4):513-518 (2011)
Nature Neuroscience | Technical Report Ultra light-sensitive and fast neuronal activation with the Ca2+-permeable channelrhodopsin CatCh * Sonja Kleinlogel1 * Katrin Feldbauer1, 4 * Robert E Dempski1, 3, 4 * Heike Fotis1 * Phillip G Wood1 * Christian Bamann1 * Ernst Bamberg1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 14,Pages:513–518Year published:(2011)DOI:doi:10.1038/nn.2776Received19 August 2010Accepted02 February 2011Published online13 March 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The light-gated cation channel channelrhodopsin-2 (ChR2) has rapidly become an important tool in neuroscience, and its use is being considered in therapeutic interventions. Although wild-type and known variant ChR2s are able to drive light-activated spike trains, their use in potential clinical applications is limited by either low light sensitivity or slow channel kinetics. We present a new variant, calcium translocating channelrhodopsin (CatCh), which mediates an accelerated response time and a voltage response that is ~70-fold more light sensitive than that of wild-type ChR2. CatCh's superior properties stem from its enhanced Ca2+ permeability. An increase in [Ca2+]i elevates the internal surface potential, facilitating activation of voltage-gated Na+ channels and indirectly increasing light sensitivity. Repolarization following light-stimulation is markedly accelerated by Ca2+-dependent BK channel activation. Our results demonstrate a previously unknown principle: shifti! ng permeability from monovalent to divalent cations to increase sensitivity without compromising fast kinetics of neuronal activation. This paves the way for clinical use of light-gated channels. View full text Figures at a glance * Figure 1: Homology model of ChR2 based on the sensory rhodopsin 2 structure (PDB accession number 1H2S). The target region for the cysteine scanning (Arg115 to Thr139) is located in TM3 and is highlighted in red. The inset shows the presumed location of the mutated L132C, the hydrogen-bonded Cys128 and Asp156, connecting TM3 and TM4 as indicated by the dotted line, and the homolog residues for the proton donor (His134) and proton acceptor (Glu123), respectively. The chromophore is formed by all-trans retinal (ATR) and Lys257 covalently linked by a Schiff base. The cavity formed by the removal of the leucine's methyl groups is depicted as spheres and overlaid on the mutated sulfhydryl group of the cysteine residue (yellow ball). The figure was prepared with Visual Molecular Dynamics27. * Figure 2: Biophysical characterization of CatCh in HEK293 cells and Xenopus oocytes. () Photocurrents in response to a 1-s blue-light pulse. Traces are normalized to the peak photocurrent amplitude to illustrate the increase in the steady state–to–peak current ratio in CatCh compared with the wild type. () Comparison of on kinetics (left) and off kinetics (right) of photocurrents normalized to peak and steady-state currents, respectively. () Typical responses to a 600-ms blue-light pulse of CatCh- and wild type ChR2–expressing Xenopus oocytes in 80 mM extracellular Ca2+ (pH 9) at −120 mV (continuous lower traces). Injection of 1 mM BAPTA (Ca2+ chelator) abolished the superimposed currents of the intrinsic Ca2+-activated chloride channels, while residual channelrhodopsin Ca2+ currents remained (dashed upper traces). Currents were normalized to the wild-type ChR2 peak current. () Ion flux characteristics of CatCh in HEK293 cells at −80 mV (mean ± s.d., n = 6). () Shift of the reversal potential (Er) for wild-type ChR2 (red circles) and CatCh (black ! squares) when exchanging the extracellular solution from 140 mM Na+ (blue triangles, wild-type and CatCh are superimposed) to 90 mM Ca2+. The intracellular solution is kept constant with 140 mM Na+ inside that leads to a negative Er in the presence of Ca2+ (mean ± s.d., n = 5). () Fura-2 measurements of Ca2+ influx in HEK293 cells expressing wild-type ChR2 (red circles) and CatCh (black squares) to 10 s of 460-nm light in the presence of 90 mM extracellular Ca2+ (n = 10). The fluorescence ratios at 340/380 are normalized to the YFP fluorescence (Online Methods). Control untransfected HEK293 cells are also shown (blue triangles). * Figure 3: CatCh expression in hippocampal cultured neurons. () Confocal image of a cultured hippocampal neuron expressing ChR2(L132C)-2A-EGFP under the CAG promoter. Scale bar represents 20 μm. () Typical photocurrents at −60 mV of CatCh and wild-type ChR2 evoked by blue light (J473nm 1 × 1019 photons s−1 cm−2). * Figure 4: Fast and high-sensitivity neural photostimulation. (–) Representative whole-cell current-clamp recordings from a CatCh-expressing hippocampal neuron in response to 2-s light pulses. The blue light intensity required for the wild-type induced a depolarization block (J473nm 2.5 × 1017 photons s−1 cm−2; ). Reducing the light intensity by a factor of 10 re-established firing (J473nm 2.5 × 1016 photons s−1 cm−2; ). Representative light-tuning curve for spike firing (Jmax 9.7 × 1016 photons s−1 cm−2, mean ± s.d.; ). Moderate green illumination also evoked trains of action potentials (J532nm 2.5 × 1017 photons s−1 cm−2; ). () Light pulse-to-spike peak latency throughout light pulse trains consisting of 25 1-ms 473-nm light pulses (J473nm 3 × 1018 photons s−1 cm−2, mean ± s.d. [jitter]) in 2 mM extracellular Ca2+ (squares) and in the absence of Ca2+ (circle), which increased latency to values similar of wild-type ChR2. (,) Spike firing in response to 1-ms 473-nm pulses (gray bars) at a rate of 50 Hz (J47! 3nm 2.8 × 1019 photons s−1 cm−2; ) and in response to 10-ns 473-nm light pulses at 10 Hz (J473nm 1.1 × 1025 photons s−1 cm−2; ). () Incomplete membrane repolarization (double-headed arrow) resulting from inhibition of BK channels by 1 mM TEA applied during a 1-ms 473-nm light pulse train. Overlay of spike before (black) and after first (red) and third (blue) spikes after TEA application (J473nm 1.8 × 1018 photons s−1 cm−2). () Replacement of 2 mM Ca2+ by 3 mM Mg2+ in the extracellular solution caused prolonged depolarization (5 Hz, left) and the formation of multiple spikes at higher 473-nm light pulse frequencies (20 Hz, right; J473nm 8.3 × 1018 photons s−1 cm−2). Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Katrin Feldbauer & * Robert E Dempski Affiliations * Max Planck Institute of Biophysics, Department of Biophysical Chemistry, Frankfurt am Main, Germany. * Sonja Kleinlogel, * Katrin Feldbauer, * Robert E Dempski, * Heike Fotis, * Phillip G Wood, * Christian Bamann & * Ernst Bamberg * Chemical and Pharmaceutical Sciences Department, Johann-Wolfgang-Goethe-University, Frankfurt am Main, Germany. * Ernst Bamberg * Present address: Department of Chemistry and Biochemistry, Worcester Polytechnic Institute, Worcester, Massachusetts, USA. * Robert E Dempski Contributions S.K., R.E.D., P.G.W. and E.B. conceived the experiments. S.K., K.F., H.F. and C.B. carried out the experiments. S.K., C.B. and K.F. performed the data analysis. S.K., C.B. and E.B. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Ernst Bamberg Author Details * Sonja Kleinlogel Search for this author in: * NPG journals * PubMed * Google Scholar * Katrin Feldbauer Search for this author in: * NPG journals * PubMed * Google Scholar * Robert E Dempski Search for this author in: * NPG journals * PubMed * Google Scholar * Heike Fotis Search for this author in: * NPG journals * PubMed * Google Scholar * Phillip G Wood Search for this author in: * NPG journals * PubMed * Google Scholar * Christian Bamann Search for this author in: * NPG journals * PubMed * Google Scholar * Ernst Bamberg Contact Ernst Bamberg Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (252K) Supplementary Figures 1–3, Supplementary Note and Supplementary Discussion Additional data - Optical quantal analysis of synaptic transmission in wild-type and rab3-mutant Drosophila motor axons
- Nat Neurosci 14(4):519-526 (2011)
Nature Neuroscience | Technical Report Optical quantal analysis of synaptic transmission in wild-type and rab3-mutant Drosophila motor axons * Einat S Peled1 * Ehud Y Isacoff1, 2, 3 * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 14,Pages:519–526Year published:(2011)DOI:doi:10.1038/nn.2767Received06 October 2010Accepted21 January 2011Published online06 March 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Synaptic transmission from a neuron to its target cells occurs via neurotransmitter release from dozens to thousands of presynaptic release sites whose strength and plasticity can vary considerably. We report an in vivo imaging method that monitors real-time synaptic transmission simultaneously at many release sites with quantal resolution. We applied this method to the model glutamatergic system of the Drosophila melanogaster larval neuromuscular junction. We find that, under basal conditions, about half of release sites have a very low release probability, but these are interspersed with sites with as much as a 50-fold higher probability. Paired-pulse stimulation depresses high-probability sites, facilitates low-probability sites, and recruits previously silent sites. Mutation of the small GTPase Rab3 substantially increases release probability but still leaves about half of the sites silent. Our findings suggest that basal synaptic strength and short-term plasticity are r! egulated at the level of release probability at individual sites. View full text Figures at a glance * Figure 1: Postsynaptically targeted GCaMP2 (SynapGCaMP2) reports single-action-potential, single-release-site Ca2+ influx. () Single-plane confocal image of basal fluorescence in the NMJ of ventral longitudinal abdominal muscle 4. () Normalized fluorescence changes in the NMJ in , after three separate single-action-potential nerve stimulation trials. Proximal and distal boutons, white arrow and asterisk, respectively. Scale bar, 10 μm (,). () Traces of ΔF due to a single action potential. Left, ΔF in one Ca2+-influx spot. Right, average of 40 ΔF traces, obtained from 26 release sites over 10 nerve stimulation trials. Black arrows, time of nerve stimulation. * Figure 2: SynapGCaMP2 imaging of sucrose-induced spontaneous release events. () mEPSPs () can be associated with spontaneous ΔF spots (numbered panels in ). Inset in bottom of , higher magnification of the third release event (small ΔF spot in yellow square). Proximal and distal boutons, white arrow and asterisk, respectively ( contains two distal boutons). Scale bar, 5 μm (). () ΔF traces (in arbitrary units) corresponding to the three spontaneous events captured in and . Gray vertical lines, time of mEPSP peaks in . Time scale is the same as in . () Correlation of miniature imaging amplitudes with mEPSP amplitudes (n = 89 events, r = 0.57; P < 0.0001, correlation analysis). All data in were obtained from a single NMJ. Similar results were obtained from five additional NMJs (data not shown). * Figure 3: Stimulation-evoked responses have same ΔF/F range of values as spontaneous events. () ΔF/F response map in response to nerve stimulation by a single action potential. () ΔF/F map of a spontaneous event in the same preparation. () Proximal and distal boutons, white arrow and asterisk, respectively. Scale bar, 5 μm. () Two examples of spontaneous ΔF traces in the same preparation as in and . Asterisk, ΔF response to nerve stimulation. () Distributions of evoked and spontaneous ΔF/F values in the same NMJ (n = 461 evoked events; n = 15 spontaneous events). () Representative examples of evoked and spontaneous imaging amplitudes within a single release site (each panel is one site). Left, stimulus-evoked ΔF/F values. Middle and right, spontaneous and evoked ΔF/F values at two other release sites. * Figure 4: Comparison of release probabilities to locations of active zones in the NMJ. () Map of aggregate release probabilities in the NMJ, showing fraction of times each release site participated in response to nerve stimulation by a single action potential (n = 101 trials). Proximal and distal boutons, arrow and asterisk, respectively. () Confocal image of the NMJ from , stained for the presynaptic active-zone protein Brp. () Higher-magnification images showing overlay of probability and Brp images in two boutons. Top panels, distal bouton, surrounded by yellow squares, in and . Bottom panels, bouton from a different branch of the same NMJ. Numbers to right of images, value of the two-dimensional correlation between the probability and Brp patterns. Numbers in parentheses, estimated chance correlation values, obtained after rotation of the Brp patterns by 180°. Scale bars, 10 μm, and ; 2 μm, . * Figure 5: The distal bouton is more active than other boutons along the axonal branch. () Examples of aggregate release probability maps in three NMJs (n = 98, 95 and 89 stimulation trials for top, middle and bottom). Proximal and distal boutons, arrow and asterisk, respectively. Scale bars, 5 μm. () Distribution of single-site release probabilities for all sites in the NMJ (n = 495 release sites, 9 NMJs). The low-release-probability counts are probably underestimated because pixels that responded only once in all stimulation trials would not have been identified as a release site (see Online Methods). () Distribution of release probabilities for sites in distal boutons and all other boutons along the axonal branch (n = 12 branches, 9 NMJs; n = 141 sites in distal boutons and 352 sites in other boutons). () Average number of ΔF spots per bouton (mean ± s.e.m.) versus bouton location from end of branch. Counts normalized by bouton area and normalized to distal bouton (bouton number 1) counts. Numbers in bars are number of boutons averaged (n = 12 branches, 9! NMJs; *P < 0.002, paired t-test, comparing the distal bouton to each of the other boutons). * Figure 6: The distal bouton has more spontaneous (sucrose-induced) ΔF spots than other boutons along the axonal branch. () Two examples of maps of the frequencies of miniature events in the NMJ (n = 80, 53 imaging scans for top and bottom, respectively; see Online Methods). Proximal and distal boutons, arrow and asterisk, respectively (top panel in contains two distal boutons). Scale bars, 5 μm. () Average number of spontaneous ΔF spots per bouton (mean ± s.e.m.) versus bouton location along the branch. Counts are normalized by bouton area and normalized to distal bouton (bouton number 1) counts. Numbers in bars are numbers of boutons averaged (n = 8 branches, 7 NMJs; *P < 0.0002, paired t-test, comparing the distal bouton to each of the other boutons). * Figure 7: Paired-pulse stimulation facilitates low-probability sites and depresses high-probability sites. () ΔF/F single trial response maps for pulses 1 and 2 in paired-pulse stimulation. () Maps of aggregate release probabilities for pulses 1 and 2 (n = 105 paired-pulse stimulation trials). Yellow arrowhead, site with a large increase in probability (see Supplementary Fig. 4 for example of a site with a large decrease in probability). Proximal and distal boutons, white arrow and asterisk, respectively (two distal boutons are present in the NMJ). Scale bar, 5 μm (,). () Change in release probability of individual sites (difference in probabilities between pulses 2 and 1) versus pulse 1 probability. Higher-probability sites tend to depress, whereas lower-probability sites tend to facilitate (n = 458 release sites in 7 NMJs, r = −0.52; P < 0.0001, correlation analysis). () Distributions of ΔF/F values of pulses 1 and 2. n = 4,647, 6,115 and 351 for pulse 1 (blue), pulse 2 only (magenta, pulse 2 ΔF spots at locations that did not respond to pulse 1) and pulse 2 after pulse 1! (green, pulse 2 ΔF spots at same site as pulse 1 ΔF spots), respectively. Median values ± s.e.m. were: pulse 1, 0.157 ± 0.001; pulse 2 only, 0.1548 ± 0.0008 (distribution not significantly different from that of pulse 1; P = 0.18, Kolmogorov-Smirnov test); pulse 2 after pulse 1, 0.146 ± 0.003 (small significant difference from pulse 1 distribution; P = 0.0005, Kolmogorov-Smirnov test). * Figure 8: Transmission properties of rab3-mutant NMJs. () Representative aggregate release probability maps, showing the fraction of times each release site participated in response to nerve stimulation by a single action potential (n = 102, 44 and 96 stimulation trials for top, middle and bottom). () Comparison of release probability pattern (green, determined by SynapGCaMP2 live imaging) to locations of presynaptically enriched active zones (magenta, confocal image of post-hoc staining for the presynaptic active zone protein Brp) in the NMJ of bottom panel in . (,) Proximal and distal boutons, white arrow and asterisk, respectively (middle panel in contains three distal boutons). Scale bars, 5 μm. () Release probability increases with increasing levels of Brp (n = 198 release sites from 2 NMJs; *P < 0.004, independent t-test, comparing medium to low and high Brp levels. See Supplementary Fig. 7 for raw data). () Distribution of release probabilities in wild-type (n = 495 release sites from 9 NMJs) and rab3-mutant (n = 235 rel! ease sites from 5 NMJs) NMJs. () Mean transmission amplitude (ΔF/F value) is larger for sites of higher release probability in both rab3-mutant (n = 35 sites in 4 NMJs, r = 0.68; P < 0.0001, correlation analysis) and wild-type (n = 14 sites in 4 NMJs, r = 0.72; P < 0.004, correlation analysis) NMJs. Only sites that had >9 well-separated ΔF spots were chosen for analysis. () Distribution of transmission amplitudes (ΔF/F values) in wild-type (n = 1,096 evoked transmission events from 5 NMJs) and rab3-mutant (n = 1,786 evoked transmission events from 4 NMJs, n = 12 spontaneous transmission events from 2 NMJs) NMJs. () Average number of ΔF spots per bouton (mean ± s.e.m.) versus bouton location from end of branch in rab3-mutant NMJs. Counts normalized by bouton area and normalized to distal bouton (bouton number 1) counts. Numbers in bars are number of boutons averaged (n = 7 branches, 5 NMJs). Author information * Abstract * Author information * Supplementary information Affiliations * Department of Molecular and Cell Biology, University of California, Berkeley, California, USA. * Einat S Peled & * Ehud Y Isacoff * Helen Wills Neuroscience Institute, University of California, Berkeley, California, USA. * Ehud Y Isacoff * Physical Bioscience Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA. * Ehud Y Isacoff Contributions E.S.P. carried out experiments and data analysis. E.Y.I. supervised the project. E.S.P. and E.Y.I. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Ehud Y Isacoff Author Details * Einat S Peled Search for this author in: * NPG journals * PubMed * Google Scholar * Ehud Y Isacoff Contact Ehud Y Isacoff Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information Movies * Supplementary Movie 1 (72K) Single action-potential evoked responses in the NMJ of Fig. 1. * Supplementary Movie 2 (548K) Spontaneous activity in the NMJ of Fig. 2. * Supplementary Movie 3 (156K) Responses to paired-pulse stimulation (NMJ of Fig. 7). PDF files * Supplementary Text and Figures (640K) Supplementary Figures 1–9, Supplementary Table 1 and Supplementary Text Additional data - Transfection via whole-cell recording in vivo: bridging single-cell physiology, genetics and connectomics
- Nat Neurosci 14(4):527-532 (2011)
Nature Neuroscience | Technical Report Transfection via whole-cell recording in vivo: bridging single-cell physiology, genetics and connectomics * Ede A Rancz1, 2, 6 * Kevin M Franks1, 3, 6 * Martin K Schwarz4 * Bruno Pichler2 * Andreas T Schaefer1, 5 * Troy W Margrie1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 14,Pages:527–532Year published:(2011)DOI:doi:10.1038/nn.2765Received26 October 2011Accepted18 January 2011Published online20 February 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Single-cell genetic manipulation is expected to substantially advance the field of systems neuroscience. However, existing gene delivery techniques do not allow researchers to electrophysiologically characterize cells and to thereby establish an experimental link between physiology and genetics for understanding neuronal function. In the mouse brain in vivo, we found that neurons remained intact after 'blind' whole-cell recording, that DNA vectors could be delivered through the patch-pipette during such recordings and that these vectors drove protein expression in recorded cells for at least 7 d. To illustrate the utility of this approach, we recorded visually evoked synaptic responses in primary visual cortical cells while delivering DNA plasmids that allowed retrograde, monosynaptic tracing of each neuron's presynaptic inputs. By providing a biophysical profile of a cell before its specific genetic perturbation, this combinatorial method captures the synaptic and anatomica! l receptive field of a neuron. View full text Figures at a glance * Figure 1: Recording methodology and biocytin recovery rates. () Seal test current trace used to monitor pipette resistance. () Contact with cell membrane while stepping the electrode indicated by rhythmic changes in pipette resistance associated with heartbeat-related movement. (,) Traces showing seal formation () and whole-cell access (). () Current-voltage relationship (current injections 50-pA steps for 600 ms) recorded from the cell shown in . (–) Current traces in response to voltage steps immediately before () and during pipette retraction () to achieve the outside-out patch configuration (). Inset trace: detail from showing single-channel currents. () Example morphology of a biocytin-labeled layer 2/3 pyramidal cell (mouse terminated 29 h after recording). () Success rates of biocytin-filled cell recovery plotted against series resistance of recording (determined within 30 s of break-in). Open circles represent, on a cell-by-cell basis, recovery success or failure. Gray-filled circles indicate those recordings that did not me! et retraction or series resistance criteria. Large open red circles represent the mean success for each bin (ranges: 20–40, 41–50, 53–70, 71–200 MΩ). All error bars show s.d. Numbers indicate n for each bin. Dashed lines are linear fits to the individual data points. (,) Success rates of cell recovery plotted against duration of recording (; bin ranges: 1–4, 5–6, 7–9, 10–13 min) and the interval between recording and terminating the mouse (; bin ranges: 23–24, 25–29, 31–45, 47–51 h). * Figure 2: Physiological characterization and genetic manipulation. () The firing profile and current-voltage relationship recorded from the layer 5 neuron shown in . Voltage traces shown are in response to 50-pA current steps of 600-ms duration. () Membrane voltage traces showing spontaneous synaptic activity and the kinetics of a resultant action potential. () Synaptic responses (average of ten sweeps) to drifting gratings moving in the direction indicated by the arrows (left). Arrow (top) indicates onset of grating drift. () Polar plot showing integral of the membrane voltage traces (for 1,400 ms from stimulus onset). () Layer 5 pyramidal cell in primary visual cortex 26 h after recording showing GFP expression (left) and biocytin labeling (right). Note the weak, non-specific biocytin signal along the electrode track due to the plume of biocytin expelled while approaching the recorded cell. () GFP signal in dendritic spines and axonal arborizations in the same cell. () Success rates of GFP-labeled cell recovery plotted against series resi! stance of recording (determined within 30 s of break-in). Open circles represent, on a cell-by-cell basis, recovery success or failure. Gray-filled circles are cases that did not meet retraction or series resistance criteria. Open green circles represent the mean success for each bin (ranges: 20–40, 41–50, 53–70, 71–200 MΩ). All error bars show s.d. Numbers indicate n for each bin. Dashed lines are linear fits to the individual data points. (,) Success rates of cell recovery plotted against duration of recording (; bin ranges: 1–4, 5–6, 7–8, 9–13 min) and the interval between recording and terminating each mouse (; bin ranges: 23–25, 26–28, 29–33, 40–47, 48–51 h). * Figure 3: Multiple gene delivery. () Native fluorescence images of a layer 5 neuron in somatosensory cortex 3 d after patching with pCAGGS-Cerulean (top right) and pCAGGS-tdTomato (50 ng μl−1 each, bottom right). Scale bars, 824 μm (left) and 20 μm (right). () Gallery of native fluorescence images for a layer 2/3 cell in somatosensory cortex 5 d after recording with pCAGGS-DsRed2, pCAGGS-Venus and pCAGGS-ChR2-Cerulean. Scale bar, 20 μm. * Figure 4: Synaptic receptive mapping and connectivity of a visual cortical neuron. () Firing profile and current-voltage relationship recorded from the GFP-expressing cell shown in . Voltage traces shown are in response to 50-pA current steps of 600-ms duration. () Synaptic responses (average of ten sweeps) to drifting gratings moving in the direction indicated by the arrows (left). Arrow (top) indicates onset of drift. Current injection (–200 pA) was delivered to isolate evoked synaptic responses from action potential activity. () Polar plot showing integral of the voltage traces (red) recorded during the duration of the drifting grating. The integral of synaptic responses (1,400 ms from stimulus onset) are normalized to the largest response. Below are five example traces recorded for the least and most preferred orientations. For the spiking receptive field (blue), holding current was removed and the stimulus sequence () was repeated five times. () Coronal slice (100 μm thick) showing anti-GFP (green) and anti-RFP (red) fluorescence. The recorded laye! r 5 neuron (green-yellow) in primary visual cortex (V1) and mCherry-labeled cells (red) are clearly identifiable. In this slice we observed ~200 presynaptic cells. Scale bar, 500 μm. Abbreviations: V2ML: secondary visual cortex mediolateral; V2L: secondary visual cortex lateral; CA1–3: hippocampal fields CA1–3; DLG: dorsolateral geniculate nucleus. () High-magnification image showing GFP and mCherry labeled cells in V1. Scale bar, 100 μm. () Eight mCherry-labeled cells in the DLG. Scale bar, 50 μm. () Five maximum intensity projections for consecutive 100-μm-thick slices. The middle panel contains the host cell (green; scale bar, 200 μm). This mouse was killed 7 d after virus injection (9 d after recording). Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Ede A Rancz & * Kevin M Franks Affiliations * Department of Neuroscience, Physiology and Pharmacology, University College London, London, UK. * Ede A Rancz, * Kevin M Franks, * Andreas T Schaefer & * Troy W Margrie * Division of Neurophysiology, The National Institute for Medical Research, Mill Hill, UK. * Ede A Rancz, * Bruno Pichler & * Troy W Margrie * Department of Neuroscience, Columbia University, New York, New York, USA. * Kevin M Franks * Department of Molecular Neurobiology, Max Planck Institute for Medical Research, Heidelberg, Germany. * Martin K Schwarz * SNWG Behavioural Neurophysiology, Max Planck Institute for Medical Research, Heidelberg, Germany. * Andreas T Schaefer Contributions E.A.R., K.M.F., A.T.S. and T.W.M. conceived experiments. K.M.F., A.T.S. and T.W.M. performed original proof-of-principle and multi-plasmid experiments. E.A.R. collected the biocytin, GFP and viral tracing data. M.K.S. co-designed and generated plasmids and provided virus. B.P. provided customized visual stimulation and compiled large-scale imaging data. E.A.R., K.M.F. and T.W.M. wrote the manuscript with input from all other authors. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Troy W Margrie Author Details * Ede A Rancz Search for this author in: * NPG journals * PubMed * Google Scholar * Kevin M Franks Search for this author in: * NPG journals * PubMed * Google Scholar * Martin K Schwarz Search for this author in: * NPG journals * PubMed * Google Scholar * Bruno Pichler Search for this author in: * NPG journals * PubMed * Google Scholar * Andreas T Schaefer Search for this author in: * NPG journals * PubMed * Google Scholar * Troy W Margrie Contact Troy W Margrie Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figure 1 Additional data
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