Latest Articles Include:
- The university student as a model organism
- Nature Neuroscience 13(5):521 (2010)
Studies that use a homogeneous pool of subjects, particularly undergraduates, must be interpreted with caution. - Just a little (lateral prefrontal) patience
Kable JW - Nature Neuroscience 13(5):523-524 (2010)
A new study finds causal evidence that the lateral prefrontal cortex, implicated in executive function, is critical for making decisions in which forgoing a small immediate reward can lead to a better future outcome. These results suggest that this area provides a neural signal that biases behavior in favor of delaying gratification. - Anxious interactions
González-Maeso J - Nature Neuroscience 13(5):524-526 (2010)
The molecular mechanisms responsible for anxiety remain largely unresolved. A study in this issue finds that an interaction between receptors for a hormone and a neurotransmitter regulates anxiety. - Degeneration keeps axons on the straight and narrow
Carter BD - Nature Neuroscience 13(5):526-528 (2010)
Axon degeneration in the adult brain is usually pathological, but a new study finds that mis-sprouting cholinergic axons in the healthy mouse brain are eliminated by a degenerative process that is triggered by myelin via p75NTR. - Neuropeptides strike back
Glauser DA Goodman MB - Nature Neuroscience 13(5):528-529 (2010)
A study identifies a previously unknown neuropeptide-based feedback signaling pathway in C. elegans that modulates the response of primary sensory neurons to chemical stimuli and odorant-evoked behaviors. - Cheesecake-eating rats and the question of food addiction
Epstein DH Shaham Y - Nature Neuroscience 13(5):529-531 (2010)
Rats given extended access to high-fat high-sugar food show behavioral and physiological changes that are similar to those caused by drugs of abuse. However, parallels between drug and food "addiction" should be drawn with caution. - Regulating brain size
Dave KA - Nature Neuroscience 13(5):531 (2010)
In this issue on page 551, Silver and colleagues report a surprising regulator of neural stem cell mitosis and brain size in mice and investigate how its disruption might lead to microcephaly.In a previous mutagenesis screen, the authors had isolated a mutant mouse with a small body size, hypopigmentation and a reduced brain size. - Asymmetric rostro-caudal inhibition in the primary olfactory cortex
Luna VM Pettit DL - Nature Neuroscience 13(5):533-535 (2010)
Nature Neuroscience | Brief Communication Asymmetric rostro-caudal inhibition in the primary olfactory cortex * Victor M Luna1 Search for this author in: * NPG journals * PubMed * Google Scholar * Diana L Pettit1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume:13,Pages:533–535Year published:(2010)DOI:doi:10.1038/nn.2524Received27 January 2010Accepted25 February 2010Published online28 March 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The importance of intracortical inhibitory circuits in setting the feature-selective spatial organization of primary sensory cortices remains controversial. To address this issue, we examined the strength of interneuron-to–pyramidal cell connections across the rat anterior piriform cortex (aPC) and found a pronounced gradient of increasing pyramidal cell inhibition along the aPC rostro-caudal axis. This functional heterogeneity could govern aPC spatial activation in response to varying odor identities and features. View full text Figures at a glance * Figure 1: Asymmetric inhibition along the aPC rostro-caudal axis. () The uncaging beam (white spot) was pulsed at 50-μm lateral intervals from the pyramidal cell soma (Vh = 0 mV). Photolysis of glutamate caused interneurons under the uncaging beam to spike, eliciting IPSCs in connected cells. Left and right, photostimulation-evoked IPSCs recorded in the representative pyramidal cell. Arrows indicate uncaging pulse onset. IPSC charge was measured as the current area above baseline (dashed lines). Note the increase in IPSC charge from rostral to caudal uncaging spots. The pyramidal cell soma was designated as position 0. Scale bar represents 25 μm. () Scatter plot of uncaging spot location versus IPSC charge showing the gradual increase in IPSC charge along the rostro-caudal axis for the cell in . () Scatter plot of uncaging spot location versus mean IPSC charge. On average, caudal spots evoked substantially larger IPSCs than equidistant rostral spots in 26 pyramidal cells. () Left and right, layer 2 inhibitory input onto a bitufted intern! euron and a pyramidal cell. Scale bar represents 20 μm. () Caudal and rostral uncaging spots evoked similar IPSC charge in eight bitufted interneurons. Error bars represent s.e.m. * Figure 2: Caudal pyramidal cells receive stronger inhibitory input than rostral cells. () Mapping inhibitory connections onto a rostral and caudal pyramidal cell separated by ~100 μm in the same slice. Scale bars represent 25 μm. () Comparison of mean IPSC charge (from uncaging spot positions −450 to 450 μm) of rostral versus caudal pyramidal cells that were ~50, 100 and 200 μm apart. *P < 0.02. Error bars represent s.e.m. Author information * Author information * Supplementary information Affiliations * Department of Neuroscience, Albert Einstein College of Medicine, Yeshiva University, New York, New York, USA. * Victor M Luna & * Diana L Pettit Contributions V.M.L. performed and analyzed experiments. V.M.L. and D.L.P. designed the experiments and prepared the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Diana L Pettit (diana.pettit@einstein.yu.edu) Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (280K) Supplementary Figures 1 and 2 and Supplementary Methods Additional data - The amygdala encodes specific sensory features of an aversive reinforcer
Dębiec J Díaz-Mataix L Bush DE Doyère V Ledoux JE - Nature Neuroscience 13(5):536-537 (2010)
Nature Neuroscience | Brief Communication The amygdala encodes specific sensory features of an aversive reinforcer * Jacek Dębiec1, 2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Llorenç Díaz-Mataix1 Search for this author in: * NPG journals * PubMed * Google Scholar * David E A Bush1 Search for this author in: * NPG journals * PubMed * Google Scholar * Valérie Doyère1, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Joseph E LeDoux1, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume:13,Pages:536–537Year published:(2010)DOI:doi:10.1038/nn.2520Received11 December 2009Accepted24 February 2010Published online28 March 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Studies of reconsolidation, in which retrieved memories are altered and restored, offer an approach for exploring the associative structure of fear memory. We found that exposure to the unconditioned stimulus initiates an unconditioned stimulus−specific reconsolidation of learned fear in rats that depended on the amygdala. Thus, specific features of the unconditioned stimulus appear to be encoded in the amygdala as part of fear memories stored there. View full text Figures at a glance * Figure 1: Exposure to the unconditioned stimulus alone triggers memory reconsolidation. () Anisomycin (ANISO) infusions following an exposure to the foot shock unconditioned stimulus (US) alone disrupted the reconsolidation of auditory fear conditioning to both conditioned stimulus a (CSa) and conditioned stimulus b (CSb) (ANOVA, significant main effect of drug, F1,14 = 21.70, P < 0.001, n = 7 and n = 9 for ACSF and ANISO, respectively). LA, lateral amygdala. () The amnesic effects of anisomycin did not reverse in 4 weeks (ANOVA, significant main effect of drug, F1,12 = 36.06, P < 0.0001, n = 7 and n = 7 for ACSF and ANISO, respectively). () Short-term memory was not affected by anisomycin (n = 6 and n = 7 for ACSF and ANISO, respectively; no significant effects of drug (P = 0.7), conditioned stimulus (P = 0.5), or drug × conditioned stimulus (P = 0.5)). Asterisks (*) indicate a significant difference between groups (P < 0.01). Error bars indicate s.e. * Figure 2: Reconsolidation is selective to the reactivated unconditioned stimulus. () Anisomycin infusions following either foot shock (USfoot) or eyelid shock (USeye) selectively disrupted fear memory reconsolidation for the conditioned stimulus associated with the reactivated unconditioned stimulus (drug × CS × reactivation-US interaction, F1,24 = 18.15, P < 0.001). Follow-up of the triple interaction with simple interaction effects indicated that anisomycin, following exposure to the USfoot (n = 8 and n = 6 for ANISO and ACSF, respectively) impaired freezing responding to the USfoot-paired conditioned stimulus (CSfoot), but did not affect freezing to the USeye-paired conditioned stimulus (CSeye) (ANOVA, significant main effects of drug, F1,12 = 14.39, P < 0.01; CSfoot, F1,12 = 6.24, P < 0.05; significant CSfoot × drug interaction, F1,12 = 8.60, P < 0.05). () Anisomycin infusions following an exposure to a USeye (n = 8 and n = 6 for ANISO and ACSF, respectively) impaired freezing responding to the USeye-paired conditioned stimulus (CSeye), but did not! affect freezing to the USfoot-paired conditioned stimulus (CSfoot) (ANOVA, significant main effects of drug, F1,12 = 16.91, P < 0.01; CSeye, F1,12 = 11.70, P < 0.01; significant CSeye × drug interaction, F1,12 = 9.57, P < 0.01). Asterisks (*) indicate a significant difference between groups (P < 0.01). Error bars indicate s.e. Author information * Author information * Supplementary information Affiliations * W.M. Keck Foundation Laboratory of Neurobiology, Center for Neural Science, New York University, New York, New York, USA. * Jacek Dębiec, * Llorenç Díaz-Mataix, * David E A Bush, * Valérie Doyère & * Joseph E LeDoux * Department of Psychiatry, New York University School of Medicine–Bellevue Hospital Center, New York, New York, USA. * Jacek Dębiec * Copernicus Center for Interdisciplinary Studies, Krakow, Poland. * Jacek Dębiec * Centre de Neurosciences Paris-Sud, CNRS UMR8195, Université Paris-Sud, Orsay, France. * Valérie Doyère * Emotional Brain Institute, Nathan S. Kline Institute for Psychiatric Research, Orangeburg, New York, USA. * Joseph E LeDoux Contributions J.D. designed and conducted the experiments, analyzed the data, interpreted the results, wrote the initial manuscript and was involved in the revision process. L.D.-M. was involved in conducting experiments, analyzing the data, interpreting the results and writing and revising the manuscript. D.E.A.B. conducted all the statistical data analysis and was involved in designing the experiments, interpreting the results and writing and revising the manuscript. V.D. was involved in designing and conducting the experiments, interpreting the results and writing and revising the manuscript. J.E.L. was involved in the design of the studies, the interpretation of the results and the preparation and revision of the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Jacek Dębiec (jacek@cns.nyu.edu) Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–6 and Supplementary Methods Additional data - Lateral prefrontal cortex and self-control in intertemporal choice
Figner B Knoch D Johnson EJ Krosch AR Lisanby SH Fehr E Weber EU - Nature Neuroscience 13(5):538-539 (2010)
Nature Neuroscience | Brief Communication Lateral prefrontal cortex and self-control in intertemporal choice * Bernd Figner1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Daria Knoch3 Search for this author in: * NPG journals * PubMed * Google Scholar * Eric J Johnson1, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Amy R Krosch1, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Sarah H Lisanby6 Search for this author in: * NPG journals * PubMed * Google Scholar * Ernst Fehr7 Search for this author in: * NPG journals * PubMed * Google Scholar * Elke U Weber1, 2, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume:13,Pages:538–539Year published:(2010)DOI:doi:10.1038/nn.2516Received14 January 2010Accepted17 February 2010Published online28 March 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Disruption of function of left, but not right, lateral prefrontal cortex (LPFC) with low-frequency repetitive transcranial magnetic stimulation (rTMS) increased choices of immediate rewards over larger delayed rewards. rTMS did not change choices involving only delayed rewards or valuation judgments of immediate and delayed rewards, providing causal evidence for a neural lateral-prefrontal cortex–based self-control mechanism in intertemporal choice. View full text Author information * Author information * Supplementary information Affiliations * Center for Decision Sciences, Columbia University, New York, New York, USA. * Bernd Figner, * Eric J Johnson, * Amy R Krosch & * Elke U Weber * Department of Psychology, Columbia University, New York, New York, USA. * Bernd Figner & * Elke U Weber * Social and Affective Neuroscience, Department of Psychology, University of Basel, Basel, Switzerland. * Daria Knoch * Graduate School of Business, Columbia University, New York, New York, USA. * Eric J Johnson & * Elke U Weber * Department of Psychology, New York University, New York, New York, USA. * Amy R Krosch * Division of Brain Stimulation and Therapeutic Modulation, Columbia University, New York, New York, USA. * Sarah H Lisanby * Institute for Empirical Research in Economics, University of Zurich, Zurich, Switzerland. * Ernst Fehr Contributions All of the authors designed the experiment and edited the manuscript. B.F. and A.R.K. conducted and analyzed the pilot studies. B.F. and D.K. collected the data. B.F., D.K., E.J.J. and E.U.W. analyzed the data and B.F., E.U.W. and E.J.J. prepared the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Bernd Figner (bf2151@columbia.edu) Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–7, Supplementary Table 1, Supplementary Methods, Supplementary Data Analysis, and Supplementary Text Additional data - Chordin-induced lineage plasticity of adult SVZ neuroblasts after demyelination
Jablonska B Aguirre A Raymond M Szabo G Kitabatake Y Sailor KA Ming GL Song H Gallo V - Nature Neuroscience 13(5):541-550 (2010)
Nature Neuroscience | Article Chordin-induced lineage plasticity of adult SVZ neuroblasts after demyelination * Beata Jablonska1, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Adan Aguirre1, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Matthew Raymond1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Gabor Szabo3 Search for this author in: * NPG journals * PubMed * Google Scholar * Yasuji Kitabatake4 Search for this author in: * NPG journals * PubMed * Google Scholar * Kurt A Sailor4 Search for this author in: * NPG journals * PubMed * Google Scholar * Guo-Li Ming4 Search for this author in: * NPG journals * PubMed * Google Scholar * Hongjun Song4 Search for this author in: * NPG journals * PubMed * Google Scholar * Vittorio Gallo1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume:13,Pages:541–550Year published:(2010)DOI:doi:10.1038/nn.2536Received07 January 2010Accepted25 March 2010Published online25 April 2010 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 The mechanisms that regulate the developmental potential of adult neural progenitor populations under physiological and pathological conditions remain poorly defined. Glutamic acid decarboxylase 65 (GAD65)- and Doublecortin (Dcx)-expressing cells constitute major progenitor populations in the adult mouse subventricular zone (SVZ). Under normal physiological conditions, SVZ-derived GAD65-positive and Dcx-positive cells expressed the transcription factor Pax6 and migrated along the rostral migratory stream to the olfactory bulb to generate interneurons. After lysolecithin-induced demyelination of corpus callosum, however, these cells altered their molecular and cellular properties and migratory path. Demyelination upregulated chordin in the SVZ, which redirected GAD65-positive and Dcx-positive progenitors from neuronal to glial fates, generating new oligodendrocytes in the corpus callosum. Our findings suggest that the lineage plasticity of SVZ progenitor cells could be a pote! ntial therapeutic strategy for diseased or injured brain. View full text Figures at a glance * Figure 1: Demyelination increases GAD65-GFP–positive cells expressing glial lineage markers in the SVZ and corpus callosum. (,) GAD65-GFP–positive cells in the anterior subventricular zone (ASVZ) immunolabeled with antibodies to BrdU and Mash1 after NaCl () or LPC () injection at 5 dpl. Dotted lines bound ASVZ and lateral ventricle (LV). Scale bar represents 50 μm. () Total number of GAD65-GFP–positive cells does not change after LPC injections (n = 8 brains). Percentages of double-labeled GAD65-GFP–positive cells stained with antibodies to BrdU, Mash1, Olig2, NG2, Dcx, Pax6 and Dlx2 after LPC injections (n = 4 brains, *P < 0.05, t test). (–) Images of GAD65-GFP–positive cells in NaCl-injected (–) and LPC-injected (–) corpus callosum (CC) at 10 dpl. Cells were colabeled with antibodies to Mash1 (,), Olig2 (,), CC1 (,) or CNP (,). Dotted lines bound corpus callosum. Insets magnify cells indicated by arrows. Scale bar represents 50 μm. () Density of GAD65-GFP–positive cells in LPC- and NaCl-injected corpus callosum. Shown are the percentages of GAD65-GFP–positive and Mash1-posit! ive, GAD65-GFP–positive and Olig2-positive, GAD65-GFP–positive and CC1-positive, and GAD65-GFP–positive and CNP-positive cells in LPC- and in NaCl-injected corpus callosum. ND, not detectable. Bar graphs represent means ± s.e.m. (n = 4–8 brains, ***P < 0.02, t test). (,) Images of GAD65-GFP–positive cells in LPC-injected corpus callosum at 14 dpl. GAD65-GFP–positive, MBP-positive oligodendrocytes were detected only in LPC-injected corpus callosum. Scale bars represent 50 μm. * Figure 2: Demyelination alters the migratory pathway and phenotype of GAD65-GFP–positive cells. () Confocal images of a sagittal brain section showing migratory pathway of GAD65-GFP–positive progenitor cells from the SVZ to the olfactory bulb (OB) along the RMS. Under normal conditions, GAD65-GFP–positive cells were found exclusively in the olfactory bulb; none migrated to the corpus callosum. Scale bar represents 400 μm. () Magnification of RMS GAD65-GFP–positive cells in white boxes, showing their neuronal morphology. Scale bar represents 20 μm. (–) At 14 dpl GAD65-GFP–positive cells in demyelinated corpus callosum coexpressed Mash1, Olig2 and CC1, but not Pax6. White arrowheads indicate GAD65-GFP–positive cells colabeled with different markers. Scale bar represents 20 μm. () Images show GAD65-GFP–positive cells immunolabeled with antibody to MBP in demyelinated corpus callosum at 14 dpl. CTX, cerebral cortex. Scale bar represents 400 μm. () Higher magnification of GAD65-GFP–positive cells colabeled with antibody to MBP in demyelinated corpus call! osum. Scale bar represents 400 μm. * Figure 3: Cellular characterization of the corpus callosum in Dcx-GFP mice after demyelination. (–) Images of Dcx-GFP–positive cells in the ASVZ (,) at 5 dpl and in corpus callosum (–) at 10 dpl after NaCl (,,,,) and LPC (,,,,) injections. Cells were labeled with antibodies to Mash1 and Olig2 (,), NG2 (,), Olig2 (,), CC1 (,) and CNP (,), together with DAPI. Insets magnify cells indicated by arrows. Dotted lines bound ASVZ and corpus callosum. Scale bars represent 50 μm. () The total number of Dcx-GFP–positive cells increased in the corpus callosum after LPC injection at 10 dpl. Bar graphs represent means ± s.e.m. (n = 4 brains, *P < 0.02, t test). () Percentages of Dcx-GFP–positive cells colabeled with antibodies to NG2, Olig2, CC1 and CNP in LPC- and NaCl-injected corpus callosum at 10 dpl. Mature oligodendrocytes stained with antibodies to CC1 and CNP were detected in corpus callosum after demyelination. Bar graphs represent means ± s.e.m. (n = 4 brains, ***P < 0.05, t test). * Figure 4: Cell lineage plasticity of Dcx-expressing progenitors after demyelination. (–) Confocal images of ASVZ (,), RMS (), olfactory bulb (,,), cortex (), white matter (WM, ) and CC (–) from NaCl- (–) and LPC-injected (–) Dcx-CreERT2 brains at 7 dpl. Tissue sections were stained with antibodies to Tuj1 (–), Dcx (), Olig2 (,,,), NG2 () and CC1 (). In the Dcx-CreERT2 mouse, all of the Dcx-CreERT2–GFP–positive cells had neuronal bipolar morphology and expressed the Tuj1 neuronal marker 2 d after tamoxifen injection. In LPC-injected Dcx-CreERT2 brains, Dcx-CreERT2–GFP–positive cells had neuronal morphology in cortex, olfactory bulb and white matter, whereas Dcx-CreERT2–GFP–positive cells had oligodendrocyte morphology and expressed oligodendrocyte markers in corpus callosum. Scale bars represent 200 μm (,), 100 μm (–) and 50 μm (insets). Black and white insets magnify cells indicated by arrows. () Graph represents the percentage of Dcx-CreERT2–GFP–positive cells expressing Olig2, NG2 or CC1 in the olfactory bulb, cortex and corp! us callosum. No oligodendrocyte lineage cells were found in the olfactory bulb and cortex, whereas in corpus callosum all Dcx-CreERT2–GFP–positive cells expressed oligodendrocyte markers. The bar graph shows means ± s.e.m. (n = 3 brains for each, NaCl and LPC injection, ***P < 0.05, t test). * Figure 5: GAD65-GFP–positive cells from the SVZ of LPC-injected brains generate Olig2- and GalC-positive cells in culture. () Bright field (left) and fluorescence (right) images of cultured FACS-purified SVZ GAD65-GFP–positive cells from NaCl- or LPC-injected brains. Arrows indicate GAD65-GFP–positive cells with neuronal (cultures from NaCl-injected brains) or oligodendrocytic (cultures from LPC-injected brains) morphologies. Scale bar represents 50 μm. () Images of FACS-purified SVZ GAD65-GFP–positive cells from NaCl- and LPC-injected brains cultured for 5 d and immunostained with antibodies to MAP2, GalC, Mash1 or Olig2. Scale bar represents 30 μm. () Percentages of GAD65-GFP–positive cells expressing MAP2, Mash1, Olig2 and GalC in 5-d cultures obtained from SVZ cells of NaCl- or LPC-injected brains. Cultures from NaCl-injected brains consisted of 100% MAP2-positive cells; no Olig2- or GalC-positive cells were detected. A percentage of MAP2-positive cells coexpressed Mash1. Cultures from LPC-injected brains consisted of GAD65-GFP–positive, Olig2-positive cells and GAD65-GFP–posit! ive, GalC-positive cells. Bar graphs represent means ± s.e.m. (n = 3 independent cultures, *P < 0.05, **P < 0.03, t test). * Figure 6: Chordin induces cell lineage plasticity in cultured SVZ GAD65-GFP–positive cells. Cultures were processed at 5 d. () FACS-sorted SVZ GAD65-GFP–positive cells cultured in basal medium and with VEGF or chordin. Arrows indicate oligodendrocytes. Scale bar represents 20 μm. () GAD65-GFP–positive cells immunostained with antibodies to Olig2 and MAP2. Scale bar represents 20 μm. () Percentages of GAD65-GFP–positive, MAP2-positive cells and GAD65-GFP–positive, Olig2-positive cells in designated cultures. Bar graphs represent means ± s.e.m. (n = 3 independent cultures,*P < 0.05, t test). () FACS-sorted SVZ GAD65-GFP–positive cells cultured in basal medium with or without chordin. GAD65-GFP–positive cells immunostained with antibodies to Ki67, Mash1, GFAP or Olig2. Scale bar represents 20 μm. () Percentages of total GAD65-GFP–positive cells under different culture conditions. Means ± s.e.m. are shown (n = 3 independent cultures, *P < 0.05, t test). () Percentages of total GAD65-GFP–positive cells that express Ki67, Mash1, GFAP or Olig2 in basa! l medium in the presence of VEGF or chordin. Means ± s.e.m. are shown (n = 3 independent cultures, **P < 0.03, t test). () FACS-purified SVZ GAD65-GFP–positive cells cultured with chordin and either antibody to chordin or with a nonspecific IgG antibody. Scale bar represents 40 μm. () Percentages of GAD65-GFP–positive, Mash1-positive cells and GAD65-GFP–positive, Olig2-positive cells in chordin-treated cultures compared with cells cultured with nonspecific antibody and chordin or chordin alone. Means ± s.e.m. are shown (n = 3 independent cultures, t test). () RT-PCR from FACS-purified SVZ GAD65-GFP–positive cells maintained in culture in the presence of chordin. * Figure 7: Chordin induces cell lineage plasticity in cultured SVZ Dcx-GFP–positive cells. (–) Dcx-GFP–positive cells FACS purified from the adult SVZ were cultured for 5 d in basal medium (,) and in medium with chordin and antibody to chordin (,). Cells were stained with anti-GFAP, anti-GalC and anti-MAP2 antibodies. Scale bars represent 20 μm and 50 μm (inset). Inset magnifies oligodendrocyte indicated by arrow. () Chordin decreased the percentage of Dcx-GFP–positive cells expressing MAP2 and increased the percentage of Dcx-GFP–positive, GalC-positive cells in the cultures. The percentage of Dcx-GFP–positive, GFAP-positive astrocytes remained unchanged. Bar graphs represent means ± s.e.m. (n = 3 independent cultures, *P < 0.05, t test). () RT-PCR from Dcx-GFP–positive cells FACS purified from the adult SVZ and cultured for 5 d. Upregulation of Olig2 and Mbp expression was detected after treatment with chordin. No substantial changes were observed in Gfap expression. Treatment with antibody to chordin downregulates Olig2 and Mbp genes. * Figure 8: Chordin induces oligodendrogenesis in corpus callosum of GAD65-GFP and Dcx-GFP mice after demyelination. (–) LPC- (,) or NaCl-injected (,) corpus callosum of GAD65-GFP mice at 10 dpl. GAD65-GFP–positive cells colabeled with antibody to Olig2, CC1 and GFAP after chordin () or antibody to chordin () infusion. Scale bar represents 50 μm. () GAD65-GFP–positive cell numbers after NaCl, chordin and antibody to chordin infusion in LPC-injected corpus callosum. Bars represent means ± s.e.m. (n = 4 brains per condition, *P < 0.05, ***P < 0.02, t test). () Percentages of GAD65-GFP–positive cells expressing glial markers in LPC- and NaCl-injected corpus callosum. Bars represent means ± s.e.m. (n = 4 brains per condition, **P < 0.03, t test). () GAD65-GFP–positive cell numbers after NaCl, chordin and antibody to chordin infusion in NaCl-injected corpus callosum. Bars represent means ± s.e.m. (n = 4 brains per condition, t test). Dcx–GFP–positive cells colabeled with antibodies to CC1 (), Olig2 (), NG2 () and CNP () in LPC-injected Dcx-GFP brains after chordin or NaCl infu! sion at 10 dpl. Scale bars represent 50 μm. () Dcx-GFP–positive cell number after NaCl and chordin infusion in corpus callosum of LPC- and NaCl-injected brains. Bars represent means ± s.e.m. (n = 4 brains per condition, t-test). () Percentages of Dcx-GFP–positive cells expressing NG2 or glial markers in LPC-injected brains after NaCl or chordin infusion. Bars represent means ± s.e.m. (n = 4 brains per condition, t test). () Percentages of Dcx-GFP–positive cells expressing NG2 or glial markers in NaCl-injected brains after NaCl or chordin infusion. Bars represent means ± s.e.m. (n = 4 brains per condition, t test). Dotted lines define lesion boundaries. The cells indicated arrows are magnified in insets. Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Gene Expression Omnibus * GSE21310 Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Beata Jablonska & * Adan Aguirre Affiliations * Center for Neuroscience Research, Children's National Medical Center, Washington, DC, USA. * Beata Jablonska, * Adan Aguirre, * Matthew Raymond & * Vittorio Gallo * George Washington University, Institute for Biomedical Sciences, Washington, DC, USA. * Matthew Raymond * Department of Gene Technology and Developmental Neurobiology, Institute of Experimental Medicine, Budapest, Hungary. * Gabor Szabo * Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. * Yasuji Kitabatake, * Kurt A Sailor, * Guo-Li Ming & * Hongjun Song Contributions B.J. and A.A. designed, performed and analyzed all of the experiments. M.R. performed some of the in vivo experiments, cell imaging and cell counting. G.S. generated and provided the GAD65-GFP mice. Y.K., K.A.S., G.M. and H.S. generated and provided the Dcx-CreERT2 mice. V.G. designed the experiments with B.J. and A.A., supervised the project and wrote the manuscript with input from B.J. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Vittorio Gallo (vgallo@cnmcresearch.org) Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (4M) Supplementary Figures 1–6 Additional data - The exon junction complex component Magoh controls brain size by regulating neural stem cell division
Silver DL Watkins-Chow DE Schreck KC Pierfelice TJ Larson DM Burnetti AJ Liaw HJ Myung K Walsh CA Gaiano N Pavan WJ - Nature Neuroscience 13(5):551-558 (2010)
Nature Neuroscience | Article The exon junction complex component Magoh controls brain size by regulating neural stem cell division * Debra L Silver1 Search for this author in: * NPG journals * PubMed * Google Scholar * Dawn E Watkins-Chow1 Search for this author in: * NPG journals * PubMed * Google Scholar * Karisa C Schreck2 Search for this author in: * NPG journals * PubMed * Google Scholar * Tarran J Pierfelice2 Search for this author in: * NPG journals * PubMed * Google Scholar * Denise M Larson1 Search for this author in: * NPG journals * PubMed * Google Scholar * Anthony J Burnetti1 Search for this author in: * NPG journals * PubMed * Google Scholar * Hung-Jiun Liaw3 Search for this author in: * NPG journals * PubMed * Google Scholar * Kyungjae Myung3 Search for this author in: * NPG journals * PubMed * Google Scholar * Christopher A Walsh4 Search for this author in: * NPG journals * PubMed * Google Scholar * Nicholas Gaiano2 Search for this author in: * NPG journals * PubMed * Google Scholar * William J Pavan1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume:13,Pages:551–558Year published:(2010)DOI:doi:10.1038/nn.2527Received14 December 2009Accepted03 March 2010Published online04 April 2010Corrected online19 April 2010 Abstract * Abstract * Accession codes * Change history * 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 Brain structure and size require precise division of neural stem cells (NSCs), which self-renew and generate intermediate neural progenitors (INPs) and neurons. The factors that regulate NSCs remain poorly understood, and mechanistic explanations of how aberrant NSC division causes the reduced brain size seen in microcephaly are lacking. Here we show that Magoh, a component of the exon junction complex (EJC) that binds RNA, controls mouse cerebral cortical size by regulating NSC division. Magoh haploinsufficiency causes microcephaly because of INP depletion and neuronal apoptosis. Defective mitosis underlies these phenotypes, as depletion of EJC components disrupts mitotic spindle orientation and integrity, chromosome number and genomic stability. In utero rescue experiments showed that a key function of Magoh is to control levels of the microcephaly-associated protein Lis1 during neurogenesis. Our results uncover requirements for the EJC in brain development, NSC maintenanc! e and mitosis, thereby implicating this complex in the pathogenesis of microcephaly. View full text Figures at a glance * Figure 1: Mutation of Magoh causes microcephaly and reduced body size. () X-ray images of control and MagohMos2/+ adult mice. Average body size, brain size and brain size as a percentage of total body weight are listed beneath each genotype. () Representation of linkage analysis on chromosome 4 with the indicated single-nucleotide polymorphisms and simple sequence length polymorphisms (bold) and the number of recombinants/meioses evaluated. For all markers shown, P < 0.0005. () Sequence chromatogram and corresponding sequences of control (top) and MagohMos2/+ (bottom) genomic DNA. The MagohMos2/+ DNA sequence contains a G deletion (right of the white line), and from this position onward, two peaks, representing the control and Mos2 alleles, are apparent. () The Magoh gene, indicating exons (gray boxes), introns (lines, not drawn to scale) and the locations of three alleles. * Figure 2: E18.5 MagohMos2/+ brains contain disorganized layers and fewer neurons. (–) Whole-mount brains (,), Nissl-stained sagittal sections (,), and hematoxylin and eosin–stained coronal sections (,) from the indicated genotypes. (–) Confocal micrographs of coronal sections from the indicated genotypes stained with antibodies against Cux1 (,,,), Foxp1 (,,,), Foxp2 (,,,) and Tbr1 (,,,). The neuronal layers that they mark are given in parentheses. The boxes in ,,,,,,, indicate the regions shown in ,,,,,,, respectively. Scale bars, 1 mm (,,,); 100 μm (,,–). * Figure 3: Magoh is required for proper numbers of INPs but not NSCs. (–) Confocal micrographs of coronal sections from the indicated genotypes and ages stained with DAPI (blue) and antibodies against Pax6 (green; –), Tbr2 (green; –) and BrdU (red; ,). () Percentage of total DAPI cells that are Pax6-positive in cortices from the indicated genotypes at E13.5, E14.5 and E16.5. () Thickness of the ventricular zone (VZ) as a percentage of total cortical thickness in the indicated genotypes at E13.5 and E14.5. () Percentage of total DAPI cells that are Tbr2-positive in cortices from the indicated genotypes at E13.5, E14.5 and E16.5. () Percentage of Tbr2-positive cells that are BrdU-positive (left) and percentage of BrdU-positive cells that are Tbr2-positive (right). Pregnant females were dissected 1 h after BrdU injection. For –, the average values for all embryos (n = 2–4 per genotype, four to five sections per embryo) is shown, quantified within a 318-μm2 field. *P < 0.05, **P < 0.005; no asterisk indicates no significant differences ! were observed. Error bars, s.d.; scale bars, 50 μm. * Figure 4: Magoh is required to prevent premature neuronal differentiation and apoptosis. (–) Low-magnification (,,,) and high-magnification (,,,,,–) confocal micrographs of coronal sections from the indicated genotypes and ages, stained for Tuj1 (green) and DAPI (blue; –,–), or Calretinin (green) and DAPI (blue; ,,,). () Confocal micrographs of dissociated E12.5 cortical cells from the indicated genotypes stained for Pax6 (green), Tuj1 (red) and DAPI (blue). () Thickness of the cortical plate and intermediate zone together as a percentage of total cortical thickness. The average value for all embryos (n = 3–4 per genotype, four to five sections per embryo) is shown, quantified within a 318-μm2 field. (–) Confocal micrographs of E12.5 coronal sections (–,–) of the indicated genotypes stained for DCX (green; ,), CC3 (red; ,), or DCX (green) and CC3 (red; ,), and E14.5 coronal sections (–,–) of the indicated genotypes stained for Tbr2 (green; ,), TUNEL (red; ,), or Tbr2 (green) and TUNEL (red; ,). **P < 0.005. Error bars, s.d.; scale bars, 50 �! �m. * Figure 5: Magoh and core EJC components regulate the mitotic spindle, ploidy, mitosis and genomic stability. () Confocal micrographs of E11.5 coronal sections stained with DAPI (blue) and rhodamine phalloidin (red), and for PH3 (green). Metaphase cells dividing vertically (arrows) and those with intermediate orientation (arrowheads) are indicated. (,) Percentage of NSCs showing the indicated mitotic cleavage planes at E11.5 (left, ) and E12.5 (right, ), and MEFs showing polyploidy and aneuploidy (). () Representative SKY image from a MagohMos2/+ MEF showing aneuploidy (three copies of chromosomes 8 and 13). (–) Confocal micrographs of HeLa cells treated with scrambled () and Magoh (,) siRNA, and stained with DAPI (blue) for α-tubulin (red) and γ-tubulin (green). Percentages indicate Magoh siRNA-treated monopolar cells containing one () or two () centrosomes. () Percentages of siRNA-treated metaphase cells showing the indicated pole-to-pole distances. Bipolar and monopolar cells are shown independently (left) and together (inset). () Percentages of siRNA-treated cells that are m! onopolar. () Images of E11.5 coronal sections stained for γ-H2AX (brown, arrows). () Number of γ-H2AX–positive cells at the indicated ages. (,) Images of siRNA-treated RPE cells stained with DAPI (blue) and for γ-H2AX-positive foci (green, arrows). () Percentages of siRNA-treated RPE cells showing >5 γ-H2AX–positive and 1–5 γ-H2AX–positive foci. Graphs indicate average values for two to four embryos per genotype (,,), for all siRNA-treated cells (), and for three to six independent experiments (,). *P < 0.05, **P < 0.005, ***P < 0.0005. Error bars, s.d. Scale bars, 5 μm (–), 10 μm (,), 50 μm (). * Figure 6: Magoh acts upstream of the microcephaly-associated protein Lis1 to regulate neurogenesis. () Graphs representing Lis1 gene expression measured by quantitative PCR (left) and Lis1 protein expression measured from immunoblot analyses (right). () Representative cropped immunoblots of cortical lysates from the indicated ages and genotypes, probed with antibodies to Lis1 (46 kDa) and to α-tubulin (55 kDa) as a loading control. Below the lanes are the densitometry values normalized to a loading control set at 1.0 (see Supplementary Fig. 8e for the full-length blots). () Confocal micrographs of coronal sections from E16.5 brains in utero co-electroporated at E13.5 with pCAG-GFP (green) and the following: luciferase shRNA plus empty vector, Magoh shRNA plus empty vector, Magoh shRNA plus Lis1, and luciferase shRNA plus Lis1. In the Magoh shRNA plus empty vector brains, there are fewer GFP-positive cells in the SVZ/ventricular zone (VZ) layers. IZ, intermediate zone; CP, cortical plate. () Percentage of GFP-positive cells in lower SVZ/VZ and upper CP layers of the brain ! for the indicated in utero electroporations. () Confocal micrographs of brains from the indicated in utero electroporations showing GFP (green) and stained for Tbr2 (red) and DAPI (blue). Insets show a higher magnification view of the boxed region, highlighting colocalization (yellow) of Tbr2 (red) and GFP (green). () Percentage of GFP-positive cells expressing Tbr2 for the indicated in utero electroporations. Graphs in and show the average values of all sections. *P < 0.05, **P < 0.005. Error bars, s.d. Scale bars, 100 μm. Accession codes * Abstract * Accession codes * Change history * Author information * Supplementary information Referenced accessions Gene Expression Omnibus * GSE 19168 Change history * Abstract * Accession codes * Change history * Author information * Supplementary informationCorrected online 19 April 2010In the version of this article initially published online, the last sentence of the sixth paragraph in the Discussion section read "Future studies will reveal, for example, whether any of the genes altered in our microarray or proteomics experiments are also essential targets of MAGOH." MAGOH has been corrected to Magoh, denoting the mouse gene. Also, the second sentence of the last paragraph in the Discussion section initially read "Of note, Magoh is found within a 55-gene deletion on chromosome 1p32.3 that is associated with mental retardation and abnormalities in brain size31." Magoh has been corrected to MAGOH, denoting the human gene. These errors have been corrected in the print, HTML and PDF versions of the article. Author information * Abstract * Accession codes * Change history * Author information * Supplementary information Affiliations * Genetic Disease Research Branch, National Human Genome Research Institute (NHGRI), National Institutes of Health (NIH), Bethesda, Maryland, USA. * Debra L Silver, * Dawn E Watkins-Chow, * Denise M Larson, * Anthony J Burnetti & * William J Pavan * Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. * Karisa C Schreck, * Tarran J Pierfelice & * Nicholas Gaiano * Genetics and Molecular Biology Branch, NHGRI, NIH, Bethesda, Maryland, USA. * Hung-Jiun Liaw & * Kyungjae Myung * Division of Genetics, Children's Hospital Boston and Departments of Pediatrics and Neurology, Harvard Medical School, Boston, Massachusetts, USA. * Christopher A Walsh Contributions D.L.S. designed the study, performed all experiments except where noted otherwise and wrote the paper. D.E.W.-C. performed all mRNA analyses and assisted with quantitative analyses. K.C.S. and T.J.P. performed all in utero electroporations and dissections of electroporated brains. D.M.L. performed staining of E18.5 markers. A.J.B. assisted with HeLa cell analyses and quantification of NSCs and INPs. H.L. performed RPE cell analyses. D.L.S., W.J.P., C.A.W., N.G. and K.M. were involved in research design, and all authors were involved in data analysis. D.L.S. and W.J.P. prepared the manuscript. All authors have agreed to the content in the manuscript, including the data as presented. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * William J Pavan (bpavan@mail.nih.gov) Supplementary information * Abstract * Accession codes * Change history * Author information * Supplementary information Excel files * Supplementary Data (84K) Serum chemistries, hematology, and organ weights of control and mutant animals PDF files * Supplementary Text and Figures (12M) Supplementary Figures 1–9 and Supplementary Tables 1–3 Additional data - p75NTR-dependent, myelin-mediated axonal degeneration regulates neural connectivity in the adult brain
Park KJ Grosso CA Aubert I Kaplan DR Miller FD - Nature Neuroscience 13(5):559-566 (2010)
Nature Neuroscience | Article p75NTR-dependent, myelin-mediated axonal degeneration regulates neural connectivity in the adult brain * Katya J Park1, 2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Carlos Ayala Grosso4, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Isabelle Aubert4, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * David R Kaplan2, 3, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Freda D Miller1, 3, 7, 8 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature NeuroscienceVolume:13,Pages:559–566Year published:(2010)DOI:doi:10.1038/nn.2513Received04 December 2009Accepted03 February 2010Published online28 March 2010 Abstract * Abstract * 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 Axonal degeneration is important during development but has not been thought to function in the intact mature nervous system. Here, we provide evidence that degeneration of adult axons occurs in the intact rodent brain through a p75 neurotrophin receptor (p75NTR)- and myelin-dependent mechanism. Specifically, we show that p75NTR-mediated axonal degeneration prevents septal cholinergic axons from aberrantly growing onto myelinated tracts in vivo or on a myelin substrate in culture. Myelin also triggers local degeneration of p75NTR-expressing sympathetic axons that is rescued by increasing TrkA signaling or elevating intracellular cyclic AMP. Myelin-mediated degeneration occurs when neurotrophins bind to p75NTR, and involves p75NTR-dependent sequestration of Rho guanine nucleotide dissociation inhibitor (Rho-GDI). Moreover, degeneration, but not growth inhibition, requires downstream activation of Rho and caspase-6. These data indicate that p75NTR maintains the specificity of ! neural connectivity by preventing inappropriate sprouting onto myelinated tracts and provide a physiological explanation for myelin inhibition after neural injury. View full text Figures at a glance * Figure 1: p75NTR inhibits the growth of septal cholinergic axons on the mature corpus callosum. () Diagram of cholinergic septal neuron projections to the cerebral cortex and hippocampus by way of the supra-callosal pathway in the adult mouse brain. (,) Quantification of the relative number () and length () of ChAT-positive axons projecting vertically into the corpus callosum anterior to the splenium in wild-type (WT) versus Ngfr−/− mice. *P < 0.05, **P < 0.01; n = 3 each. (,) Sagittal sections through the corpus callosum and the supracallosal pathway of adult WT () and Ngfr−/− () mice immunostained for ChAT. Boxed areas in left panels are shown enlarged at right. Arrows, ChAT-positive axons projecting into corpus callosum; scale bar, 50 μm. () High magnification images of sections as in , showing ChAT-positive axons growing on the corpus callosum in WT versus Ngfr−/− mice. Arrows, apparent breaks; scale bar, 10 μm. () Confocal micrographs of a sagittal section through the WT corpus callosum double-labeled for α-tubulin and ChAT. Arrows, axon fragments; ! arrowheads, breaks; scale bar, 20 μm. () Quantification of ChAT positive axons growing into the corpus callosum that show microtubule fragmentation. ***P < 0.001; n = 3 each. In all panels, results are mean ± s.e.m. * Figure 2: Myelin induces degeneration of septal cholinergic axons in low NGF in a p75NTR-dependent fashion. () Mouse septal neurons cultured on BSA or myelin in 100 ng ml−1 NGF for 5 d, switched into 20 ng ml−1 NGF for 2 d and immunostained for βIII-tubulin and p75NTR. Boxed areas in left panels are shown at high magnification at far right. Arrows, degenerating axons; scale bar, 50 μm. () Mouse septal neurons cultured on myelin as in , immunostained for ChAT and p75NTR. Arrows, patchy p75NTR immunostaining in degenerating axons; scale bar, 25 μm. (,) Quantification of cultures similar to those in for degenerating p75NTR-positive axons () and p75NTR-positive neurons with Hoechst-positive condensed, apoptotic nuclei (). **P < 0.01; n = 3. () p75NTR-positive rat septal neurons plated on a control or myelin substrate for 7 d in 100 ng ml−1 NGF, immunostained for βIII-tubulin. Scale bar, 50 μm. () Mouse wild-type and Ngfr−/− ChAT-positive septal neurons cultured as in and immunostained for βIII-tubulin after 2 d in low NGF. Arrows, axon breaks; scale bar, 50 μm. () Qua! ntification of degenerating ChAT-positive axons in cultures similar to those in . *P < 0.05; n = 3. () p75NTR-positive rat septal neurons grown on myelin or a control substrate for 5 d in 100 ng ml−1 NGF, switched for 2 d to 20 ng ml−1 NGF plus anti-BNDF or control IgY, and immunostained for βIII-tubulin. Scale bar, 50 μm. () Quantification of degenerating p75NTR-positive axons in cultures similar to those in . *P < 0.05; n = 3. In all panels, results are mean ± s.e.m. * Figure 3: Myelin induces local degeneration of sympathetic axons, and this can be rescued by high NGF or elevated intracellular cAMP. (–) Mass cultures of sympathetic neurons established on a BSA or myelin substrate in 50 ng ml−1 NGF for 5 d and switched into 10 or 50 ng ml−1 NGF for 2 d more, immunostained for βIII-tubulin (; arrows, degenerating axons). Cultures were quantified for percentage of degenerating axons () and cell death as monitored by Hoechst-positive, condensed, apoptotic nuclei (). ***P < 0.001. Results are from eight cultures in two independent experiments. Scale bar, 50 μm. () Schematic of myelin compartments. Neuronal cell bodies are plated in the center compartment and axons grow into the side compartments, where a drop of myelin has been plated. () Sympathetic axons growing in a myelin compartment as in , immunostained for βIII-tubulin. The white line denotes the border of the myelin drop, with the myelin to the right. Right panels, high magnification of axons growing on or off the myelin; arrows, degenerating axons; scale bar, 125 μm. () Quantification of the percentage of ! degenerating axons in myelin compartments as in . **P < 0.01; n = 3. (,) Video frames of cultures in which neurons were transduced with EGFP adenovirus, switched to low NGF, and axons on () or off () myelin continuously imaged at various time points. Arrows, degenerating EGFP-positive axons; scale bar, 50 μm. (–) βIII-tubulin–immunostained sympathetic axons growing in myelin compartments whose sides were switched into low NGF, with or without dibutyryl cAMP (dbcAMP), for 2 d (). Cultures were quantified for degenerating axons () and axonal growth, the latter shown as a percentage of the off-myelin value (). **P < 0.01; n = 3. Scale bar, 100 μm. In all graphs, results are mean ± s.e.m. * Figure 4: Myelin-induced local sympathetic axon degeneration is p75NTR dependent. () Wild-type (WT) or Ngfr−/− axons growing in myelin compartments in low NGF on myelin or the adjacent control substrate, immunostained for βIII-tubulin. Bottom panels show higher magnification. Arrows indicate degeneration. Scale bar, 50 μm. () Quantification of degenerating axons in experiments similar to those in . ***P < 0.001; n = 3. (,) WT or Ngfr−/− axons growing in myelin compartments on myelin or the adjacent control substrate in low NGF, immunostained for βIII-tubulin () and analyzed for the percentage of axonal growth, shown as a percentage of the off-myelin value (). *P < 0.05; n = 3. Scale bar, 50 μm. () EGFP-positive axons growing on myelin in side compartments of cultures in which Ngfr−/− neurons were adenovirally transduced with EGFP (adEGFP) alone or with p75NTR (adp75). Arrows, degenerating axons; scale bar, 50 μm. () Quantification of the percentage of degenerating, EGFP-positive axons in experiments like those in . ***P < 0.001; n = 3. In! all graphs, data are mean ± s.e.m. * Figure 5: Myelin-induced axonal degeneration requires neurotrophin binding to p75NTR. () Schematic of myelin compartment experiments in which the sides were switched into low NGF plus anti-BDNF for 2 d. () Axons growing in side compartments as in , with anti-BDNF or control chicken IgY, immunostained for βIII-tubulin. In the top left panel, myelin is present to the left of the white line (on myelin), whereas in the top right panel, it is to the right of the line. Boxed areas are shown at higher magnification below. Arrows, degenerating axons; scale bar, 100 μm. () Quantification of the percentage of axonal growth on and off myelin in cultures similar to , shown as a percentage of the off-myelin values. *P < 0.05, **P < 0.01; n = 3. () Axons growing in side compartments as in , showing axon terminals growing on myelin in the presence of anti-BDNF or control IgY. The left two panels are immunostained for βIII-tubulin, and the right panels are double-labeled for βIII-tubulin and p75NTR. Arrows, degenerating axons; scale bar for left panels, 25 μm; scale bar! for right panels, 10 μm. () Quantification of axonal degeneration as in panel . **P < 0.01. n = 3. In all graphs, results are mean ± s.e.m. * Figure 6: p75NTR causes axonal degeneration through a Rho-GDI–Rho–caspase-6 pathway. () Sympathetic axons growing on or off myelin in side compartments that were switched into low NGF plus a control TAT peptide or TAT-Pep5 for 2 d, immunostained for βIII-tubulin and/or p75NTR, shown at low (top) or high (below) magnification. Arrows, degenerating axons; scale bar, 50 μm. (,) Quantification of axonal degeneration () and axonal growth (), with the latter shown as a percentage of the off-myelin value, in experiments similar to those in . *P < 0.05, ***P < 0.001; n = 3. () Quantification of the intensity of phospho-cofilin (p-cofilin) immunostaining in arbitrary units in sympathetic axons growing off or on myelin for 2 d in low NGF, as seen in the micrographs at the bottom of the panel. *P < 0.05; n = 3. Scale bar, 25 μm. (,) Quantification of the percentage of sympathetic axon degeneration () and axon growth () on and off myelin, with the latter shown as a percentage of the off-myelin value, in myelin compartments after the sides were switched for 2 d to low! NGF with (C3) or without (control) of the Rho inhibitor C3 transferase (C3). *P < 0.05; n = 3. () Sympathetic axons growing on myelin in side compartments that were switched for 2 d to low NGF, or to low NGF plus anti-BDNF or plus a control IgY, and immunostained for cleaved caspase-6 (cc-6) and βIII-tubulin. Scale bar, 50 μm. () Sympathetic axons growing on myelin in side compartments that were switched into low NGF with the caspase-6 inhibitor Z-VEID-FMK (Z-VEID) or with vehicle (DMSO). Axons immunostained for βIII-tubulin and/or p75NTR are shown at low (top panels) and high magnification (below). Arrows, degenerating axons; scale bar, 50 μm. (,) Quantification of axonal degeneration () and axonal growth (), with the latter shown as a percentage of the off-myelin value, in experiments similar to those in . ***P < 0.001; n = 3. () p75NTR-positive rat septal neurons grown on myelin or a control substrate for 5 d in 100 ng ml−1 NGF, switched for 2 d to 20 ng ml−1 NG! F containing Z-VEID-FMK or DMSO, and immunostained for βIII-t! ubulin and p75NTR. Arrows, degenerating axons. Scale bar, 50 μm. () Quantification of degenerating p75NTR-positive cholinergic axons in cultures similar to those in . **P < 0.01; n = 3. In all panels, results are mean ± s.e.m. Author information * Abstract * Author information Affiliations * Developmental and Stem Cell Biology, Toronto, Ontario, Canada. * Katya J Park & * Freda D Miller * Cell Biology Programs, Hospital for Sick Children, Toronto, Ontario, Canada. * Katya J Park & * David R Kaplan * Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada. * Katya J Park, * David R Kaplan & * Freda D Miller * Sunnybrook Research Institute, Toronto, Ontario, Canada. * Carlos Ayala Grosso & * Isabelle Aubert * Facultad de Farmacia, Universidad Central de Venezuela, Caracas, Venezuela. * Carlos Ayala Grosso * Departments of Laboratory Medicine and Pathobiology, Toronto, Ontario, Canada. * Isabelle Aubert * Molecular Genetics, Toronto, Ontario, Canada. * David R Kaplan & * Freda D Miller * Physiology, University of Toronto, Toronto, Ontario, Canada. * Freda D Miller Contributions K.J.P. carried out all experiments and co-wrote the paper. C.A.G. participated in the in vivo analysis. I.A. supervised the septal cholinergic experiments. D.R.K. and F.D.M. co-supervised all experiments and co-wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Freda D Miller (fredam@sickkids.ca) or * David R Kaplan (dkaplan@sickkids.ca) Additional data - Cargo recognition failure is responsible for inefficient autophagy in Huntington's disease
Martinez-Vicente M Talloczy Z Wong E Tang G Koga H Kaushik S de Vries R Arias E Harris S Sulzer D Cuervo AM - Nature Neuroscience 13(5):567-576 (2010)
Nature Neuroscience | Article Cargo recognition failure is responsible for inefficient autophagy in Huntington's disease * Marta Martinez-Vicente1, 3, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Zsolt Talloczy2, 3, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Esther Wong1, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Guomei Tang2 Search for this author in: * NPG journals * PubMed * Google Scholar * Hiroshi Koga1 Search for this author in: * NPG journals * PubMed * Google Scholar * Susmita Kaushik1 Search for this author in: * NPG journals * PubMed * Google Scholar * Rosa de Vries2 Search for this author in: * NPG journals * PubMed * Google Scholar * Esperanza Arias1 Search for this author in: * NPG journals * PubMed * Google Scholar * Spike Harris2 Search for this author in: * NPG journals * PubMed * Google Scholar * David Sulzer2 Search for this author in: * NPG journals * PubMed * Google Scholar * Ana Maria Cuervo1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature NeuroscienceVolume:13,Pages:567–576Year published:(2010)DOI:doi:10.1038/nn.2528Received02 December 2009Accepted09 March 2010Published online11 April 2010 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 Continuous turnover of intracellular components by autophagy is necessary to preserve cellular homeostasis in all tissues. Alterations in macroautophagy, the main process responsible for bulk autophagic degradation, have been proposed to contribute to pathogenesis in Huntington's disease (HD), a genetic neurodegenerative disorder caused by an expanded polyglutamine tract in the huntingtin protein. However, the precise mechanism behind macroautophagy malfunction in HD is poorly understood. In this work, using cellular and mouse models of HD and cells from humans with HD, we have identified a primary defect in the ability of autophagic vacuoles to recognize cytosolic cargo in HD cells. Autophagic vacuoles form at normal or even enhanced rates in HD cells and are adequately eliminated by lysosomes, but they fail to efficiently trap cytosolic cargo in their lumen. We propose that inefficient engulfment of cytosolic components by autophagosomes is responsible for their slower tur! nover, functional decay and accumulation inside HD cells. View full text Figures at a glance * Figure 1: Autophagic activity is reduced in HD cells. (–) Degradation of long-lived proteins in MEFs from wild-type (18Q-htt) and mutant huntingtin knock-in (111Q-htt) mice. (,) Rates of protein degradation after serum removal () or treatment with rapamycin (rapam) or thapsigargin (thapsig) (). () Lysosomal degradation calculated as percentage of protein degradation sensitive to NH4Cl. () Contribution of macroautophagy calculated as percentage of protein degradation sensitive to 3-methyladenine (3-MA). () Degradation of long-lived proteins in striatal cells from wild-type (7Q-htt) and mutant huntingtin knock-in mice (111Q-htt) in response to different autophagic stimuli. (,) Rates of protein degradation in wild-type and HD MEFs () and striatal cells () in the absence (control) or presence (Atg7−) of Atg7 RNA interference. () Contribution of macroautophagy (macroaut) to the degradation of long-lived proteins in lymphoblasts from unaffected controls (UC) and people with HD. Mean + s.d. of values from two to four different ind! ividuals and three or four different experiments. *P < 0.05 versus untreated, #P < 0.05 versus control. * Figure 2: Formation and clearance of autophagic vacuoles is normal in HD cells. () Top: LC3 immunostaining of 18Q-htt and 111Q-htt MEFs maintained in the presence (+) or absence (−) of serum and inhibitors of lysosomal proteolysis (inhib+). Bottom: mean number of LC3+ vesicles per cell in cells maintained in the presence (left) or absence (right) of serum. n = 4. () Top: LC3 immunoblot of the same cells after serum (S) removal or thapsigargin (TG) treatment. PI, protease inhibitors. Bottom: LC3-II levels and LC3-II flux (calculated as a multiple of LC3-II value in absence of PI; lateral numbers). n = 4. () LC3-II values and LC3-II flux in neuronal cultures from wild-type (WT) or HD94 mice (HD) grown on wild-type rat astrocyte monolayers, analyzed as in . n = 4. () LC3 staining of lymphoblasts, from three unaffected controls (UC) and people with HD, maintained in the presence or absence of serum. Accession numbers are indicated above panels. Right: mean number of LC3+ vesicles. Extended study in Supplementary Figure 8b,c. () Immunoblots for LC3-II in U! C and HD lymphoblasts treated or not treated with protease inhibitors. Right: LC3-II values and LC3-II flux. Extended study in Supplementary Figure 8a. Mean + s.d.; #P < 0.05. Full-length blots in Supplementary Figure 20. * Figure 3: Abnormalities in autophagic vacuoles in different HD cell types. () Electron micrographs of striatal neurons from control and HD94 mice grown over a wild-type rat astrocyte monolayer. Higher magnification fields show the double membrane and clear content of the cytosolic vesicles. Top right: number of vacuoles per cell profile (13–17 cell profiles in triplicate experiments; *P < 0.05). Arrows, enlarged electron-clear vesicles. () Electron micrographs of striatal cells from mutant huntingtin knock-in (111Q-htt) and wild-type (7Q-htt) mice. () Electron micrographs of lymphoblasts from unaffected controls (UC) and people with HD, maintained in the presence or absence of serum. Arrows, enlarged electron-clear vesicles. () Immunogold for LC3 in striatal cells from mutant huntingtin knock-in mice (111Q-htt), striatal neurons from HD94 mice and lymphoblasts (lymph) from people with HD. Full fields and more details of autophagic vacuoles in Supplementary Figures 10 and 11. * Figure 4: Altered composition of autophagy-related compartments in HD cells. () Electron micrographs of fractions enriched in autophagosomes and autophagolysosomes isolated from liver of 18Q-htt and 111Q-htt mice. Insets: higher magnification of single vesicles. Right: percentage of vesicles with electron-clear (light), vesiculated (multivesicular) or electron-dense (dark) contents. Mean + s.e.m. of three different isolations (> ~1,000 autophagic vacuoles). () Bidimensional electrophoresis and Sypro Ruby staining of these fractions. Left, intact vacuoles; right, vacuole contents. Horizontal dimension, isoelectric point (pH); vertical, molecular mass (kDa). *P < 0.05. * Figure 5: Altered properties of autophagy-related compartments in HD cells. () Immunoblot for the indicated proteins in fractions enriched in APHs and autophagolysosomes (APHL) isolated from livers of 18Q-htt (18Q) and 111Q-htt (111Q) mice. Cyt, cytochrome; Dyn IC, dynein intermediate chain; ADRP, adipose differentiation-related protein; Polyubq, polyubiquitin. Bottom: amount of each protein in 111Q-htt sample, as a multiple of its amount in 18Q-htt sample. Mean + s.d.; n = 4. () Immunoblots for htt and LC3 in homogenates (Homog), cytosol and fractions enriched in APHs and enriched in autophagolysosomes (APHL) isolated from livers of wild-type (18Q-htt) and mutant huntingtin knock-in mice (111Q-htt). Representative one of four experiments with duplicated samples. *P < 0.05. Full-length blots in Supplementary Figure 20. * Figure 6: Distribution of htt and p62 in autophagic vacuoles. () Immunoblot for htt or p62 from total APHs (T) and their corresponding membranes (Mbr) and matrices (Mtx) isolated from livers of wild-type htt (18Q) and 111Q-htt mutant huntingtin knock-in mice (111Q). Left, representative immunoblots. Right, distribution of htt and p62 between Mbr and Mtx calculated by densitometric quantification in six such immunoblots. AV, autophagic vacuoles. Mean + s.d. *P < 0.05 compared to wild-type values. () Filter retardation analysis of the same fractions as in , blotted for htt. Negative signal indicated absence of aggregates retained in the filter. () Immunoblot for ubiquitin in homogenates (Homog), cytosol and the autophagic fractions described in , using 6% (top) and 16% (bottom) gels. Dotted line separates stacking and running parts of gel. () Immunoblot for htt, LC3 and p62, of brain homogenates (Homog) and fractions enriched in APHs from the same mouse groups as in . () Immunoblot for htt and p62 of membranes from autophagic vacuoles sh! own in subjected to co-immunoprecipitation for htt under low stringency. Input for 111Q-htt autophagic vacuoles was one-third of that for 18Q-htt to avoid antibody saturation. Levels of htt (top) and p62 (bottom) in the input, immunoprecipitate (IP) and flow through (FT) are shown. Full-length blots in Supplementary Figure 21. * Figure 7: Consequences of altered recognition of autophagic cargo in HD on cellular lipid content. () Neutral lipids in MEFs from 18Q-htt and 111Q-htt mice stained with BODIPY 493/503. () Electron micrographs of livers from 18Q-htt and 111Q-htt mice. Right: number of lipid droplets (LD) per cell profile (prof.), mean area of LDs and percentage of cellular area occupied by LDs. Mean + s.d.; n = 3. (–) Neutral lipids in striatal cells from 7Q-htt and 111Q-htt mice (), primary striatal neurons from 18Q-htt and 111Q-htt mice grown on a monolayer of their own astrocytes () and lymphoblasts from a control (UC) and HD-affected human () stained with BODIPY 493/503. MAP2 staining highlights neurons. Extended study in Supplementary Figure 15b. () Electron micrographs of a lymphoblast from an individual with HD (with 78 and 15 polyglutamine repeats in the two alleles). Green arrows, LDs. () Fraction of cellular cytosol occupied by LDs quantified in lymphoblasts from three UC and four HD subjects. Numbers of polyglutamine repeats are shown below. Mean + s.d. of >100 cell profiles. ! Dotted red line, mean value of the respective samples. *P < 0.05. () Oil Red O staining of striatal tissue from brain of two different unaffected control individuals (top) and two individuals with HD (bottom). Nuclei are highlighted with hematoxylin. Lipid droplets are visible as red puncta. Subject identifiers are shown in each panel. () The percentage of total cellular area occupied by lipid droplets (left) and the average number of lipid droplets per cell (right) was calculated for each of the samples by quantification of eight or nine different fields. *P < 0.001; analysis of variance, P < 0.01 for each person with HD versus each control individual by Tukey post hoc test. Subject identifiers are as in . * Figure 8: Altered mitochondrial turnover in HD cells. () Top: immunofluorescence for the mitochondrial marker COX IV in 18Q-htt and 111Q-htt MEFs maintained in the presence or absence of serum. Bottom: number of mitochondria per cell. Mean + s.d. of 10–20 cells in three different experiments. () Electron micrographs of striatal neurons from wild-type (Ctr) and HD94 mice grown over a wild-type rat astrocyte monolayer. Arrows, abnormally short (black) or abnormally long (green) mitochondria. Right: number of mitochondria per cell profile. Mean + s.d. of ten cells per group in triplicates. () Left: electron micrographs of lymphoblasts from controls (UC) or people with HD. Arrows as in . Right: percentage of cellular area occupied by mitochondria in different individuals. Line indicates population mean. (,) Striatal cells from 7Q-htt and 111Q-htt mice () and MEFs from 18Q-htt and 111Q-htt mice () stained with Mitotracker and Mito-ROS. Right, merged images. Percentage of colocalization is indicated at the bottom in and is displaye! d in the graph at the bottom in . CCCP was added to control cells in as a positive control for depolarization. *P < 0.05. (,) Striatal cells from 7Q-htt and 111Q-htt mice untreated or treated with vinblastine were co-stained for LC3 and Mitotracker () or BODIPY 493/503 (). Arrows, colocalization events. Extended study in Supplementary Figure 16. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Marta Martinez-Vicente, * Zsolt Talloczy & * Esther Wong Affiliations * Department of Developmental and Molecular Biology, Institute for Aging Studies, Albert Einstein College of Medicine, Bronx, New York, USA. * Marta Martinez-Vicente, * Esther Wong, * Hiroshi Koga, * Susmita Kaushik, * Esperanza Arias & * Ana Maria Cuervo * Departments of Neurology, Psychiatry, Pharmacology, Columbia University Medical School, New York, New York, USA. * Zsolt Talloczy, * Guomei Tang, * Rosa de Vries, * Spike Harris & * David Sulzer * Present addresses: Institute of Neuropathology, IDIBELL–Hospital Universitari de Bellvitge, Hospitalet de Llobregat, Barcelona, Spain (M.M.-Z.) and Novartis Pharmaceuticals Corporation, East Hanover, New Jersey, USA (Z.T.). * Marta Martinez-Vicente & * Zsolt Talloczy Contributions M.M.-V., Z.T. and E.W. performed the experiments that constitute the main body of this work; R.d.V., H.K., S.K., E.A. and G.T. completed the rest of the experiments; S.H. analyzed the electron micrographs of lymphoblasts; A.M.C. and D.S. designed the study and wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Ana Maria Cuervo (ana-maria.cuervo@einstein.yu.edu) or * David Sulzer (ds43@columbia.edu) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (3M) Supplementary Figures 1–21 and Supplementary Text Additional data - Real-time visualization of complexin during single exocytic events
An SJ Grabner CP Zenisek D - Nature Neuroscience 13(5):577-583 (2010)
Nature Neuroscience | Article Real-time visualization of complexin during single exocytic events * Seong J An1 Search for this author in: * NPG journals * PubMed * Google Scholar * Chad P Grabner1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * David Zenisek1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume:13,Pages:577–583Year published:(2010)DOI:doi:10.1038/nn.2532Received21 December 2009Accepted15 March 2010Published online11 April 2010 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 Understanding the fundamental role of soluble NSF attachment protein receptor (SNARE) complexes in membrane fusion requires knowledge of the spatiotemporal dynamics of their assembly. We visualized complexin (cplx), a cytosolic protein that binds assembled SNARE complexes, during single exocytic events in live cells. We found that cplx appeared briefly during full fusion. However, a truncated version of cplx containing only the SNARE-complex binding region persisted at fusion sites for seconds and caused fusion to be transient. Resealing pores with the mutant cplx only partially released transmitter and lipid probes, indicating that the pores are narrow and not purely lipidic in structure. Depletion of cplx similarly caused secretory cargo to be retained. These data suggest that cplx is recruited at a late step in exocytosis and modulates fusion pores composed of SNARE complexes. View full text Figures at a glance * Figure 1: Imaging secretory granules labeled with NPY-mRFP undergoing exocytosis in PC12 cells coexpressing different versions of cplx-GFP. (–) Images (right) are averages of events time-aligned to the moment of fusion: wild type (n = 94 events, 11 cells, ), R59,63E (n = 139 events, 16 cells, ), ΔCT (n = 85 events, 10 cells, ), ΔNT (n = 144 events, 18 cells, ) and short (n = 123 events, 12 cells, ). The fluorescence intensity (left) was calculated by taking the average intensity in a 1.2-μm circle centered on a granule or the corresponding coordinates in the cplx channel and subtracting the average intensity in a concentric 1.3-μm-wide annulus, which served as the local background fluorescence; intensities were plotted against time and then averaged. Exocytosis was stimulated by perfusion with a solution of elevated [K+]. We performed three to six transfections per condition. Scale bar represents 1 μm. Error bars are ± s.e.m. () Schematic diagram of cplx-GFP fusion proteins. () Examples of cplx-GFP signals associated with single fusion events. Cyan circle indicates when fusion occurred. Red line is the a! verage intensity in the trace 3–5 s before fusion. The vertical bar represents 200 fluorescence units and the horizontal bar represents 5 s. * Figure 2: Effect of cplx overexpression on NPY-mRFP exocytosis. () Plot of fusion events (percentage of docked granules) versus cplx expression level. The recording period was 3.33 min. The spatially averaged cplx-GFP fluorescence of the 'footprint' of the cell, where the cell adhered closely to the coverslip (green dashed lines), served as a measure of expression. The cplx expression levels of cells required for detecting SNARE complexes (Fig. 1) fell in the range indicated by the shaded area (~500–1,000 fluorescence units). Representative footprints are shown at the same contrast setting to illustrate the differences in the brightness of transfected cells. Scale bar represents 10 μm. () As in , but for other cplx constructs. All points are the average of a 20-cell bin, except for the rightmost point in each plot, which represented the following number of cells: 17 (wild type), 15 (R59,63E), 15 (ΔCT), 24 (ΔNT) and 18 (short). We performed three to eight transfections per condition. Error bars are ± s.e.m. P values were determined ! using a Student's t test. * Figure 3: Slow and incomplete release of NPY-mRFP when cplx persists during fusion. () Top, images of granules releasing NPY-mRFP completely with wild-type cplx-GFP or incompletely with cplx short–GFP. Scale bar represents 1 μm. Bottom left, background-subtracted fluorescence of same granules, normalized to the intensity during the last 2 s before fusion. Bottom right, average fluorescence in larger, 2.5-μm-diameter circles centered over same granules, normalized to the intensity when fusion occurred. This 'outer-circle' fluorescence analysis is a better measure of the rate of NPY-mRFP release, as it minimizes diffusional loss outside of the region of interest. () Averages of background-subtracted NPY-mRFP fluorescence traces in Figure 1a,e normalized to prefusion intensity. The black trace represents wild-type cplx-GFP and the red trace represents cplx short–GFP. The NPY-mRFP trace with cplx short–GFP is less noisy because it is an average of more events. () Averages of outer-circle NPY-mRFP fluorescence traces of the events in normalized to initia! l-fusion intensity. The black trace represents wild-type cplx-GFP, the red trace represents cplx short–GFP and the blue trace represents the average of five events from wild-type cplx-GFP cells showing fastest loss of NPY-mRFP fluorescence, indicating that NPY-mRFP diffusion is unhindered in the space underneath the cell. () Averaged cplx-GFP signals (from Fig. 1) fitted to single exponential decay from fusion onset. () Plot of cplx-GFP decay times in and rates of NPY-mRFP release measured with the outer circle fluorescence analysis in . The dashed line indicates the rate of NPY-mRFP release (0.93 ± 0.05 s) with the R59,63E-GFP mutant. Error bars in represent s.e. reflecting the goodness of the exponential fits to the average traces in and . * Figure 4: Rapid resealing of granules with persisting cplx. () Images of granules labeled with tPA-phluorin spreading (top) or remaining compact (bottom) during exocytosis. Note that phluorin was completely acid-quenched at −1 s before fusion occurs. () Monitoring granule resealing by alternating the external pH once a second (four frames). Left, plots of background-subtracted fluorescence of granules once they become visible through exocytosis. For clarity, only time points marking the end of a pH pulse are shown (black circles), starting 750 ms after fusion. Events were plotted as rolling averages of three frames to reduce noise. In the bottom three events, cyan circles mark the end of the pulse, when resealing occurred. Right, image sequence of the granules analyzed in the left plots. Scale bar represents 1 μm. () Distribution of resealing times with wild-type cplx-mRFP (17% of 60 granules, median time = 41.5 s), endogenous cplx (22% of 59, 4.25 s) and cplx short–mRFP (40% of 70, 2.5 s). For each condition, more than ten cell! s in two to four transfections were analyzed. () Cumulative frequency distributions of resealing times. The black trace represents wild-type cplx-mRFP, the red trace represents cplx short–mRFP and the blue trace represents endogenous cplx (P < 0.001, Kolmogorov-Smirnov tests). * Figure 5: Restricted lateral diffusion of FM dyes with persisting cplx. () Averaged image sequence of FM4-64 release with wild-type cplx-GFP (n = 37 events, 7 cells) or cplx short–GFP (n = 78 events, 16 cells). The increase in fluorescence on fusion is a result of changes in the proximity of the dye to the glass interface, as well as changes in the dye's orientation in the membrane. Scale bar represents 1 μm. () Plots of background-subtracted FM4-64 fluorescence of granules, normalized to prefusion intensity. The black trace represents wild-type cplx-GFP and the red trace represents cplx short–GFP. (,) Data are presented as in and , but for FM1-84 (wild-type cplx-mRFP, n = 34 events, 8 cells; cplx short-mRFP, n = 105 events, 22 cells). Inset, traces shown at an expanded timescale. () Fluorescence profiles of averaged complete and incomplete FM4-64 release events at selected times relative to fusion (black circles, 30 ms; red circles, 0 ms; orange circles, 30 ms; green circles, 60 ms; blue circles, 90 ms). Images of analyzed events are shown! in Supplementary Figure 11. () Time course of σ2, the square of the width of Gaussian curves (see Online Methods). The slope of each line corresponds to an apparent diffusion coefficient (solid circles: complete release, 0.45 μm2 s−1; open circles: incomplete release, 0.15 μm2 s−1). (,) Data are presented as in and , but for FM1-84 (solid circles: complete release, 0.15 μm2 s−1; open circles: incomplete release, 0.03 μm2 s−1). For comparison, complete and incomplete time courses of σ2 with FM4-64 are replotted as dashed and dotted lines, respectively. Error bars in and are ± s.e.m. Error bars in and are s.e. of the pixel intensity at radial distances from the center. Error bars in and are s.e. reflecting the goodness of fit. * Figure 6: NPY-mRFP exocytosis in cells depleted of cplx. () RNAi-mediated silencing of cplx expression. Cells transfected with GFP and a plasmid encoding short hairpin RNA (shRNA) targeting cplx 2 were isolated by fluorescence-activated cell sorting and analyzed by immunoblotting. () Fluorimetric assay of NPY-mRFP released by cells after a 45-min incubation at low or high [K+] (n = 15). In addition to 0.5 μg of NPY-mRFP plasmid (control), cells were transfected with 1 μg shRNA (RNAi), 0.5 or 2.0 μg cplx-GFP, or shRNA and 0.5 μg cplx-GFP plasmids (rescue). () Immunoblot analysis of the transfection conditions in . (–) TIRFM imaging of NPY-mRFP exocytosis. () Fusion latency of stimulus-evoked events in cells with (red, n = 419 events) or without shRNA (black, n = 480 events). Shading highlights wherever the frequency was lower with shRNA. Time is relative to start of stimulation, which lasted 100 s (dashed line). () Histogram of granule density in cells with (red) or without shRNA (black). The average density was 1.2 ± 0.5 an! d 1.0 ± 0.5 granules per μm2, respectively. () Background-subtracted fluorescence of granules undergoing exocytosis in cells with or without shRNA. Indicated extents of release (%) were based on cell averages calculated by subtracting the fluorescence 4–5 s after fusion from the intensity during the last 1 s before fusion. () Outer-circle fluorescence traces of the same events, averaged and normalized to initial-fusion intensity, in cells with or without shRNA. Rates with wild-type cplx-GFP and cplx short–GFP are replotted from Figure 3c. Error bars are ± s.e.m. * Figure 7: Reduced transmitter release with persisting cplx. () Amperometric recordings of exocytosis from cells expressing wild-type cplx-GFP (upper) or cplx short–GFP (lower). () Averaged current spikes with wild-type cplx-GFP (n = 8 cells) or cplx short–GFP (n = 12 cells). () Histograms of spike amplitudes with wild-type cplx-GFP (black) and cplx short–GFP (red). Dashed lines indicate the 3-pA cutoff. () Distributions in , plotted cumulatively and normalized to the total number of events. Dashed line indicates the 3-pA cutoff. () Averaged current spikes of events above 3 pA from cells with wild-type cplx-GFP (black) or cplx short–GFP (red). () Averaged current spikes of events below 3 pA from cells with wild-type cplx-GFP (black) or cplx short–GFP (red). () Parameters of events above and below 3 pA with wild-type cplx-GFP (black) and cplx short–GFP (red). Error bars are ± s.e.m. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut, USA. * Seong J An, * Chad P Grabner & * David Zenisek * Present address: University of Saarland, Institute for Physiology, Homburg/Saar, Germany. * Chad P Grabner Contributions S.J.A. designed, conducted and analyzed the research and wrote the manuscript. C.P.G. performed and analyzed the amperometry experiments. D.Z. supervised the project. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Seong J An (seong.an@yale.edu) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (3M) Supplementary Figures 1–16 Additional data - Selective induction of astrocytic gliosis generates deficits in neuronal inhibition
Ortinski PI Dong J Mungenast A Yue C Takano H Watson DJ Haydon PG Coulter DA - Nature Neuroscience 13(5):584-591 (2010)
Nature Neuroscience | Article Selective induction of astrocytic gliosis generates deficits in neuronal inhibition * Pavel I Ortinski1, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Jinghui Dong2, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Alison Mungenast2 Search for this author in: * NPG journals * PubMed * Google Scholar * Cuiyong Yue1 Search for this author in: * NPG journals * PubMed * Google Scholar * Hajime Takano1 Search for this author in: * NPG journals * PubMed * Google Scholar * Deborah J Watson3 Search for this author in: * NPG journals * PubMed * Google Scholar * Philip G Haydon2 Search for this author in: * NPG journals * PubMed * Google Scholar * Douglas A Coulter1, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume:13,Pages:584–591Year published:(2010)DOI:doi:10.1038/nn.2535Received07 December 2009Accepted19 March 2010Published online25 April 2010 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 Reactive astrocytosis develops in many neurologic diseases, including epilepsy. Astrocytotic contributions to pathophysiology are poorly understood. Studies examining this are confounded by comorbidities accompanying reactive astrocytosis. We found that high-titer transduction of astrocytes with enhanced green fluorescent protein (eGFP) via adeno-associated virus induced reactive astrocytosis without altering the intrinsic properties or anatomy of neighboring neurons. We examined the consequences of selective astrocytosis induction on synaptic transmission in mouse CA1 pyramidal neurons. Neurons near eGFP-labeled reactive astrocytes had reduced inhibitory, but not excitatory, synaptic currents. This inhibitory postsynaptic current (IPSC) erosion resulted from a failure of the astrocytic glutamate-glutamine cycle. Reactive astrocytes downregulated expression of glutamine synthetase. Blockade of this enzyme normally induces rapid synaptic GABA depletion. In astrocytotic region! s, residual inhibition lost sensitivity to glutamine synthetase blockade, whereas exogenous glutamine administration enhanced IPSCs. Astrocytosis-mediated deficits in inhibition triggered glutamine-reversible hyperexcitability in hippocampal circuits. Thus, reactive astrocytosis could generate local synaptic perturbations, leading to broader functional deficits associated with neurologic disease. View full text Figures at a glance * Figure 1: Astrocyte-specific eGFP expression. (–) Confocal images depicting viral injection–induced eGFP expression (green, ), immunostaining for NeuN (blue, ) and GFAP (red, ) in the hippocampus. () eGFP colocalized with GFAP (astrocyte marker, arrows), but not with NeuN (neuronal marker). (–) High-magnification images of colabeling of eGFP fluorescence with GFAP staining. () Schematic of the AAV2/5–Gfa104-eGFP plasmid. * Figure 2: AAV2/5–Gfa104-eGFP induces a titer-dependent reactivity of astrocytes. (–) Following low viral-titer injection (1010 genome copies (GC) per injection), eGFP-positive cells had normal astrocytic morphology and low levels of GFAP and vimentin staining. (–) In contrast, eGFP-positive cells labeled by high-titer injection (3 × 1010 genome copies per injection) of the virus had a hypertrophic morphology typical of reactive astrocytes () and intense GFAP and vimentin staining (,). (–) Following low-titer injection, eGFP and GFAP double-positive cells had normal astrocytic morphology and were immunoreactive for glutamine synthetase, an astrocyte-specific enzyme. (–) High-titer injection led to a loss of glutamine synthetase immunoreactivity in eGFP and GFAP double-positive cells. Merged images are shown in , , and . (–) Titer dependence of GFAP, vimentin and glutamine synthetase immunostaining in the stratum radiatum. Expression levels were quantified as the area with immunoreactivity exceeding a background threshold intensity and as the av! erage intensity of immunoreactivity. **P < 0.01 and ***P < 0.001 relative to wild-type mice. ##P < 0.01 and ###P < 0.001 relative to saline-injected mice. Error bars represent s.e.m. * Figure 3: Inhibitory neurotransmission is impaired in CA1 pyramidal cells proximal to reactive astrocytes. () Sample traces of eIPSCs from control cells and pyramidal cells located distal (eGFP negative) and proximal (eGFP positive) to AAV2/5-transduced astrocytes. eIPSCs are much smaller in neurons neighboring reactive astrocytes. () Histograms of eIPSC amplitude distribution in CA1 neurons of control and AAV2/5-injected mice. The eIPSC distribution in eGFP-positive neurons was skewed toward smaller values compared with controls. () Cumulative frequency distributions of mIPSC amplitudes from four AAV2/5 and six control cells. Note that the amplitude distribution was shifted to the left in AAV2/5 cells compared with controls and that small-amplitude mIPSCs were maximally shifted (distributions were significantly different, P = 0.001, Kolmogorov-Smirnov test). () mIPSC amplitudes in AAV2/5 and control cells, grouped by quartile, were normalized to the mIPSC average in controls and compared. The degree of mIPSC reduction in AAV2/5 neurons gradually diminished as the quartile increa! sed in the population distribution (**P < 0.001, Kolmogorov-Smirnov test, error bars represent s.e.m.). CFD, cumulative frequency distribution. () Averaged time series of evoked responses before, during and after the train stimulation (four pulses at 50 Hz every 20 s for 15 min) in control and AAV2/5 cells, normalized to the amplitude of the first evoked event. Train stimulation elicits a long-term (persisting for at least the duration of recording, >8 min) depression of eIPSCs in cells from AAV2/5-injected, but not control, mice. Error bars represent s.e.m. () A representative CA1 pyramidal cell filled with biocytin (blue) surrounded by eGFP-positive (green) and GFAP-positive (red) astrocytes. * Figure 4: Preserved excitatory neurotransmission in CA1 pyramidal neurons proximal to reactive astrocytes. () Traces from a CA1 pyramidal cell in a control and an AAV2/5 slice recorded in the absence of glutamate and GABAA receptor antagonists (Vhold = −40 mV). An eIPSC follows an eEPSC in each trace. Note that EPSCs are similar in amplitude, whereas IPSCs are reduced. (,) Histograms of eEPSC and eIPSC amplitude and charge transfer. eEPSC amplitudes and charge transfers were similar (eEPSC amplitude: control, n = 10; AAV2/5, n = 15; P = 0.34; eEPSC charge: control, n = 9; AAV2/5, n = 12; P = 0.59), whereas eIPSC amplitudes and charge transfer were both smaller in AAV2/5 cells (eIPSC amplitude: control, n = 6; AAV2/5, n = 12; *P = 0.014; eIPSC charge: control, n = 6; AAV2/5, n = 12; *P = 0.006). The AAV2/5 eIPSC average excluded two cells that failed to produce an eIPSC at stimulation intensities used to elicit an excitatory response. (,) Time course of eEPSC recovery from RRP (20 Hz, 2 s) and reserve pool (10 Hz, 90 s) stimulations. Amplitudes were normalized to the average (ba! seline) eEPSC during 10–15 single pulses (0.07 Hz) before RRP and reserve pool stimulation. () A histogram of average eEPSC amplitudes. eEPSCs were evoked at 0.07 Hz over a period of 5 min following RRP stimulation and 12 min following the reserve pool stimulation (control: RRP, n = 9; reserve pool, n = 8; AAV2/5: RRP, n = 8; reserve pool, n = 7). Basal eEPSC current amplitudes were not different in the absence and presence of picrotoxin in either the control (P = 0.1) or the AAV2/5 group (P = 0.35). Error bars represent s.e.m. * Figure 5: Glutamate-glutamine cycle deficits reduce the concentration of vesicular GABA. () Left, eIPSCs of MSO-treated cells compared with cells that were not exposed to MSO during and following train stimulation. Right, effect of train stimulation on eIPSC amplitudes following incubation in MSO (1.5 mM) is expressed as the percentage change from eIPSCs recorded in the absence of MSO (Fig. 3e). MSO triggered an activity-dependent decrease of eIPSC amplitude in control cells, but not AAV2/5 cells (**P < 0.001 relative to control). () Left, mIPSC averages in the absence (thin traces) and presence (thick traces) of TPMPA and SR95531. Right, TPMPA significantly reduced mIPSC amplitude in neurons from AAV2/5 eGFP-positive cells (AAV2/5 (+/− TPMPA), 9.5 ± 1.2 pA/7.1 ± 0.9 pA, n = 6, P = 0.005, paired t test; control (+/− TPMPA), 12.5 ± 1.6 pA/12.2 ± 2 pA, n = 6, P = 0.87, paired t test). SR95531 reduced mIPSC amplitude to a similar extent in both groups of cells (AAV2/5 (+/− SR95531), 12.3 ± 1.4 pA/9.9 ± 1.4 pA, n = 5, P = 0.033, paired t test; control (+! /− SR95531), 13.6 ± 2.1 pA/11.1 ± 1.8 pA, P = 0.004, paired t test). *P = 0.027 compared with controls. () Left, current traces before (thin trace) and during (thick trace) application of 10 mM glutamine. Middle, supplementation with glutamine partially reversed eIPSC failure in a subset of AAV2/5 cells (**P < 0.001 relative to control; AAV2/5, n = 7; control, n = 7). Right, bath-applied glutamine restored eIPSC amplitudes to control levels following train stimulation, but failed to prevent eIPSC failure during the train (**P ≤ 0.003 relative to control, #P = 0.04 relative to AAV2/5). Cell numbers (train/post-train) are as follows: control, n = 12/10; AAV2/5, n = 10/6; AAV2/5 and glutamine, n = 6/5. Error bars represent s.e.m. * Figure 6: Reactive gliosis is associated with network hyperexcitability. () Hippocampal preparation. The shaded box represents an area used for snapshots in . DG, dentate gyrus; EC, entorhinal cortex; SC, Schaffer collaterals; stim, stimulation electrode; TA, temporammonic pathway. () VSD signal snapshots at indicated time points following onset of the TAP stimulus. White dotted lines indicate the pixels sampled for Raster plots in SLM, stratum lacunosum moleculare; SR, stratum radiatum; SO, stratum oriens. () Top, amplitude and time course of the fluorescent signal and a Raster plot of hippocampal activity across the CA cell layers. Note the compartmentalization of the EPSP to distal SR in the control, but not the AAV2/5 slice. Bottom, current-clamp traces from CA1 pyramidal neurons. TAP stimulation elicited an EPSP in cells from AAV2/5 eGFP-positive areas and an IPSP in cells from control mice. (,) Amplitude of the fluorescent signal and area activated by TAP stimulation (**P < 0.008, *P = 0.016; control, n = 6; AAV2/5, n = 10). An stratum orie! ns IPSP in the control slices converted to an stratum oriens EPSP in slices from eGFP-positive AAV2/5 slices. () Fluorescence changes triggered by TAP activation before (left) and following (right) a 15-min bath-application of glutamine to an eGFP-positive AAV2/5 slice. (,) Normalized fluorescence change and activated area before and after glutamine application in AAV2/5 eGFP-positive slices (*P = 0.04, paired t test, n = 5). In stratum oriens, the active area was smaller in the AAV2/5 group, but failed to reach significance relative to the control group (P = 0.09, paired t test). Error bars represent s.e.m. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Pavel I Ortinski & * Jinghui Dong Affiliations * Division of Neurology, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA. * Pavel I Ortinski, * Cuiyong Yue, * Hajime Takano & * Douglas A Coulter * Department of Neuroscience, Tufts University School of Medicine, Boston, Massachusetts, USA. * Jinghui Dong, * Alison Mungenast & * Philip G Haydon * Department of Neurosurgery, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA. * Deborah J Watson * Departments of Pediatrics and Neuroscience, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA. * Douglas A Coulter Contributions P.I.O. and J.D. conducted and analyzed all of the experiments. A.M. assisted with viral vector production. C.Y. contributed to VSD data collection and analysis. H.T. assisted with confocal and multiphoton microscope data acquisition and processing. D.J.W. contributed to initial generation of the AAV-injected mice. P.I.O. and D.A.C. wrote the manuscript with help from P.G.H. and J.D. D.A.C. and P.G.H. designed the experiments with P.I.O. and J.D. and supervised the project. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Douglas A Coulter (coulterd@email.chop.edu) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (16M) Supplementary Figures 1–11 and Supplementary Tables 1 and 2 Additional data - Reduction in endocannabinoid tone is a homeostatic mechanism for specific inhibitory synapses
Kim J Alger BE - Nature Neuroscience 13(5):592-600 (2010)
Nature Neuroscience | Article Reduction in endocannabinoid tone is a homeostatic mechanism for specific inhibitory synapses * Jimok Kim1 Search for this author in: * NPG journals * PubMed * Google Scholar * Bradley E Alger1, 2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume:13,Pages:592–600Year published:(2010)DOI:doi:10.1038/nn.2517Received24 December 2009Accepted16 February 2010Published online28 March 2010 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 When chronic alterations in neuronal activity occur, network gain is maintained by global homeostatic scaling of synaptic strength, but the stability of microcircuits can be controlled by unique adaptations that differ from the global changes. It is not understood how specificity of synaptic tuning is achieved. We found that, although a large population of inhibitory synapses was homeostatically scaled down after chronic inactivity, decreased endocannabinoid tone specifically strengthened a subset of GABAergic synapses that express cannabinoid receptors. In rat hippocampal slice cultures, a 3–5-d blockade of neuronal firing facilitated uptake and degradation of anandamide. The consequent reduction in basal stimulation of cannabinoid receptors augmented GABA release probability, fostering rapid depression of synaptic inhibition and on-demand disinhibition. This regulatory mechanism, mediated by activity-dependent changes in tonic endocannabinoid level, permits selective loc! al tuning of inhibitory synapses in hippocampal networks. View full text Figures at a glance * Figure 1: Homeostatic downregulation of inhibitory synapses caused by chronic TTX treatment. () Whole-cell recordings of mIPSCs from CA1 pyramidal neurons in control and TTX-treated (3–5 d) slice cultures of rat hippocampus. Cells were voltage-clamped at −65 mV with a pipette solution containing 142.4 mM Cl−. () Cumulative distributions of mIPSCs (500 events per cell). Chronic TTX treatment reduced mIPSC amplitudes. The distribution of the scaled control data (dashed line), obtained after scaling down individual mIPSCs (see Online Methods) essentially overlapped with the TTX-treated mIPSC distribution (thick gray line) (P > 0.9, Kolmogorov-Smirnov test between TTX and scaled control), suggesting a global scaling of mIPSCs. () uIPSCs were evoked by extracellular minimal stimulation. Stimulus intensity was gradually increased until uIPSCs were elicited in an all-or-none fashion and consistently occurred, as shown in the sample traces. Slice cultures were pre-incubated with 500 nM ω-conotoxin GVIA for 15–30 min before recording to isolate synapses that use P/Q! -type Ca2+ channels for GABA release. () Data are presented as in , but the slice cultures were pre-incubated with 300 nM ω-agatoxin IVA for 15–30 min. () Group data of uIPSC amplitudes. The mean amplitudes were reduced by chronic TTX treatment in both conotoxin- and agatoxin-resistant synapses. *P < 0.02. Error bars represent s.e.m. * Figure 2: Inactivity-induced upregulation of synaptic properties in a subset of inhibitory interneurons. () sIPSCs were recorded at −65 mV with 142.4 mM Cl− in the pipette solution. In each cell, a mixture of sIPSCs and mIPSCs was recorded and mIPSCs alone were then obtained after the addition of 0.5 μM TTX. The sIPSC frequency was found by subtracting the frequency of mIPSCs from the total of sIPSCs and mIPSCs in the same cell. Slice cultures were pre-incubated in 500 nM ω-conotoxin GVIA for 15–30 min. () Data are presented as in , but the cultures were pre-incubated with 300 nM ω-agatoxin IVA for 15–30 min. () Group data of sIPSC frequency. *P < 0.02, **P < 0.0005. () Short-term plasticity of eIPSCs was examined with extracellular stimulation of presynaptic axons at 20 Hz. In this and all subsequent experiments, pipette [Cl−] was lowered to 1.4 mM, which kept eIPSC amplitudes small and preserved adequate voltage clamp control. Slice cultures were pre-incubated with ω-conotoxin GVIA for 15–30 min. The amplitudes of the second and the third eIPSCs were normalize! d to the first eIPSCs. No difference was found between control and TTX-treated cells in short-term depression of eIPSCs from P/Q-type Ca2+ channel–containing terminals (P > 0.2). () Short-term depression of eIPSCs from terminals with N-type Ca2+ channels was measured after pre-incubation with ω-agatoxin IVA for 15–30 min. TTX-treated cells showed more pronounced depression, suggesting an increase in Pr. #P < 0.001. Error bars represent s.e.m. * Figure 3: Tonic activation of CB1R is reduced by activity deprivation. () The mean amplitude of agatoxin-resistant eIPSCs was enhanced the CB1R antagonists SR141716 (2 μM) or AM251 (2 μM). The black traces are averaged eIPSCs before SR141716 treatment and the gray traces are averaged eIPSCs after SR141716 treatment. Right, eIPSC amplitudes were normalized to baseline values. Inset, the effects of SR141716 (S) were not significantly different from those of AM251 (A) in either control or TTX groups (P > 0.5). () SR141716 (2 μM) failed to increase eIPSCs in control slices when a zero-Ca2+ pipette solution was used for recording. () From the data presented in , the ratio of CV−2 in CB1R antagonist to baseline CV−2 (CV−2(antago)/CV−2(base)) was plotted against the ratio of eIPSC amplitudes in CB1R antagonist to baseline values (eIPSC(antago)/eIPSC(base)). Data points in the gray area and along the diagonal line of y = x imply a presynaptic enhancement of transmission caused by either SR147161 or AM251. () The larger increase in eIPSC caus! ed by CB1R antagonists in control cells than in TTX-treated cells was accompanied by a larger increase in PPR3−1, which is proportional to Pr. Data shown in were analyzed. *P < 0.05, **P < 0.01. () The eIPSC increase induced by a CB1R antagonist was plotted against PPR3 before the antagonist. When PPR3 was high (low basal Pr), the eIPSC increase was larger. The straight line is a linear fit with square of correlation coefficient (R2) of 0.71. Error bars represent s.e.m. * Figure 4: Chronic inactivity does not alter the responsiveness of CB1R to WIN55212-2, a CB1R agonist. () Representative traces of agatoxin-resistant eIPSCs from three control and three TTX-treated cells at different concentrations of WIN55212-2 (5, 20 and 200 nM). The black traces are the averages of baseline eIPSCs before WIN55212-2 treatment, and the gray traces are the averages of steady-state eIPSCs in the presence of WIN55212-2. All scale bars represent 100 pA and 20 ms. () eIPSC amplitudes in the presence of WIN55212-2 were normalized to the pre-WIN55212-2 baseline. No significant difference was found between the responses of control and TTX-treated cells at any concentration (P > 0.4). Error bars represent s.e.m. * Figure 5: Basal [Ca2+]in and Ca2+-dependent 2-AG release are not affected by chronic TTX. Slice cultures were pre-incubated with 300 nM ω-agatoxin IVA for 15–30 min. () Somatic [Ca2+]in in postsynaptic cells was imaged at 1 Hz with 200 μM Fura-2 in the pipette solution. Pyramidal cells were depolarized to 0 mV for 500 ms at time 0. Inset, somatic fluorescence was summed in the outlined area. () Basal [Ca2+]in was averaged during the baseline period before depolarization. Average peak [Ca2+]in was measured for 5 s starting from the peak. Neither parameter differed significantly between control and TTX-treated cells (P > 0.2). () Changes in eIPSC amplitudes after 500-ms depolarizations were recorded simultaneously with Ca2+ imaging. The black traces are the averages of eIPSCs before depolarization and the gray traces are the averages of the second and third eIPSCs after depolarization. Bottom, group data of DSI with 500-ms depolarization. The gray area represents integration of eIPSC suppression (the DSI integral) from 6 to 144 s. () DSI with 5-s depolarization! to 0 mV. The DSI integral was obtained from the gray area in the range of 0–240 s. The gray trace is the average of the first three eIPSCs after depolarization. () Group data of DSI integrals showed no difference between control and TTX-treated groups with either 500-ms or 5-s depolarization (P > 0.2). Error bars represent s.e.m. * Figure 6: Uptake and degradation of basal endocannabinoid are enhanced by activity deprivation. All slice cultures were pre-incubated with 300 nM ω-agatoxin IVA for 15–30 min. () AM404-mediated suppression of eIPSC was significantly larger in TTX-treated neurons than in control cells (P < 0.005). Right, representative traces of eIPSCs averaged before (black) and during (gray) application of 20 μM AM404. () In the presence of AM404 (20 μM), the magnitudes of DSI (0 mV, 5 s) in control and TTX-treated cells were similar. Black traces are the averaged eIPSCs before depolarization andthe gray traces are the averages of the first three eIPSCs after depolarization. () DSI integral with 5-s depolarization did not differ between control and TTX-treated cells in the absence (−AM) or presence (+AM) of AM404, whereas AM404 increased DSI in each group. The left two bars are replotted from Figure 5e for comparison. () URB597-mediated suppression of eIPSC was significantly greater in TTX-treated cells than in control cells (P < 0.02). Right, representative traces of averaged ! eIPSCs showing the effect of 1 μM URB597 (gray traces). () From the data shown in and , the ratio of CV−2 in AM404 or URB597 to baseline CV−2 (CV−2(drug)/CV−2(base)) was plotted against the ratio of eIPSCs (eIPSC(drug)/eIPSC(base)). Points in the gray area and along the diagonal line imply presynaptic suppression of eIPSC by either AM404 or URB597. () The larger reduction in eIPSC amplitudes by AM404 or URB597 in TTX-treated cells was accompanied by a larger decrease in PPR3−1, which is proportional to Pr. *P < 0.005. () A cocktail of AM404 (20 μM) and URB597 (1 μM) did not suppress eIPSCs in the presence of 2 μM SR141716, indicating that AM404 and URB597 in and acted via CB1Rs. Inset, representative traces of averaged eIPSCs in the absence (black) or presence (gray) of the cocktail. () Bath-applied anandamide (720 nM) reduced eIPSCs to a lesser extent in TTX-treated cells than in control cells (P < 0.05). Right, averaged traces before and during (AEA) anandam! ide application. Ca2+ was excluded from the pipette solution t! o prevent FAAH from being occupied by endogenous anandamide. Error bars represent s.e.m. * Figure 7: Deafferentation of CA1 decreases tonic CB1R activity and increases basal GABAergic Pr, mimicking chronic TTX treatment. All slice cultures were incubated with ω-agatoxin IVA (300 nM) for 15–30 min before recording. () Micrographs of cultures from which CA3 or subiculum had been removed. CA3 removal eliminated afferent excitation of CA1 neurons, whereas subiculum removal served as a control. Dashed lines indicate the areas removed. () Short-term eIPSC depression (20 Hz) was more pronounced in deafferented slices than in control slices. Recordings were made 5–7 d after the cut. *P < 0.05. () The increase in eIPSCs caused by SR141716 (2 μM) was larger in control cells than in deafferented cells (P < 0.005). The black trace represents baseline and the gray trace represents SR141716 treatment. Amplitude scales, 50 pA for control and 100 pA for deafferented. Time scales, 30 ms. () When basal Pr was lower, as represented by higher PPR3 before SR141716, the SR141716-mediated increase in eIPSCs was larger. The line is a linear regression fit (R2 = 0.84). () The larger enhancement in eIPSC amplit! udes caused by SR141716 in control cells than in deafferented cells was accompanied by a larger increase in PPR3−1. Data in and were analyzed. *P < 0.05. () From the data shown in , the ratio of CV−2 in SR141716 to baseline CV−2 (CV−2(SR)/CV−2(base)) was plotted against the ratio of eIPSCs. Points in the gray area and along the diagonal line suggest presynaptic enhancement of transmission. Error bars represent s.e.m. * Figure 8: Chronic TTX enhances Pr of excitatory synapses independently of tonic endocannabinoid action. () Averaged traces of eEPSCs before and during SR141716 (2 μM) application. SR141716 had no effect on the amplitude of eEPSCs recorded from CA1 pyramidal neurons. () Group data, normalized to pre-SR141716 baseline. SR141716 caused no significant changes ineEPSCs in either treatment group (P > 0.09). () Short-term plasticity of eEPSC was assessed with 20-Hz stimulation. TTX-treated cells displayed more rapid depression. The second and the third eEPSCs were normalized to the first eEPSC amplitude. *P < 0.005. () The ratios of eEPSC3/eEPSC1 were not significantly different (P > 0.8) before (pre-SR) or in the presence of (SR) 2 μM SR141716 in either treatment group, suggesting that the increased glutamatergic Pr (represented by eEPSC3/eEPSC1) was independent of CB1Rs. Each dot indicates individual cell. Error bars represent s.e.m. Author information * Abstract * Author information * Supplementary information Affiliations * Departments of Physiology, University of Maryland School of Medicine, Baltimore, Maryland, USA. * Jimok Kim & * Bradley E Alger * Psychiatry, University of Maryland School of Medicine, Baltimore, Maryland, USA. * Bradley E Alger * Program in Neuroscience, University of Maryland School of Medicine, Baltimore, Maryland, USA. * Bradley E Alger Contributions J.K. and B.E.A. designed the research and wrote the manuscript. J.K. conducted the experiments. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Bradley E Alger (balgerlab@gmail.com) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (432K) Supplementary Figures 1–8 Additional data - Control of submillisecond synaptic timing in binaural coincidence detectors by Kv1 channels
Mathews PJ Jercog PE Rinzel J Scott LL Golding NL - Nature Neuroscience 13(5):601-609 (2010)
Nature Neuroscience | Article Control of submillisecond synaptic timing in binaural coincidence detectors by Kv1 channels * Paul J Mathews1, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Pablo E Jercog2, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * John Rinzel2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Luisa L Scott1 Search for this author in: * NPG journals * PubMed * Google Scholar * Nace L Golding1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume:13,Pages:601–609Year published:(2010)DOI:doi:10.1038/nn.2530Received15 December 2009Accepted08 March 2010Published online04 April 2010 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 Neurons in the medial superior olive process sound-localization cues via binaural coincidence detection, in which excitatory synaptic inputs from each ear are segregated onto different branches of a bipolar dendritic structure and summed at the soma and axon with submillisecond time resolution. Although synaptic timing and dynamics critically shape this computation, synaptic interactions with intrinsic ion channels have received less attention. Using paired somatic and dendritic patch-clamp recordings in gerbil brainstem slices together with compartmental modeling, we found that activation of Kv1 channels by dendritic excitatory postsynaptic potentials (EPSPs) accelerated membrane repolarization in a voltage-dependent manner and actively improved the time resolution of synaptic integration. We found that a somatically biased gradient of Kv1 channels underlies the degree of compensation for passive cable filtering during propagation of EPSPs in dendrites. Thus, both the spati! al distribution and properties of Kv1 channels are important for preserving binaural synaptic timing. View full text Figures at a glance * Figure 1: Propagation of simulated EPSPs from the dendrites to the soma in MSO neurons. () Simultaneous recording from the soma (black) and dendrite (gray) in response to injection of dendritic sEPSCs of three increasing amplitudes (0.2, 0.8 and 2.2 nA). The sEPSPs are shown normalized at the right, revealing a systematic decrease in sEPSP halfwidth with increasing amplitude. () Plot of halfwidth versus sEPSP amplitude for the cell shown in , revealing a steeper VDS of sEPSPs at the soma versus the dendrite. () Population data indicated that the maximum degree of VDS was more prominent at the soma than in the dendrites. There was a trend for stronger VDS at both recording sites with increasing distance from the soma (0.06% per μm dendrite, 0.20% per μm soma, n = 16). () The rate of VDS over the subthreshold voltage range was higher at the soma than at the dendrite (fits: dendrite, 0.02 ± 0.05 μs mV−1 μm−1; soma, 0.26 ± 0.12 μs mV−1 μm−1). (,) Attenuation of backpropagating sEPSPs in the cells shown in and was far less pronounced than during forw! ard propagation from the dendrites to the soma. Note the prominent VDS observed in normalized traces with somatic sEPSC injection (, right). () VDS was insensitive to developmental stage. Despite a significant decline in EPSP duration (minimum halfwidth, measured just below spike threshold, P = 0.03), VDS magnitude and rate in electrophysiologically mature gerbils (P28–34) was not significantly different from that in younger gerbils (P16–19, P = 0.43 and P = 0.21 for magnitude and rate, respectively). * Figure 2: The shape of EPSPs is stable regardless of propagation distance. () sEPSPs from different dual dendritic and somatic recordings elicited with sEPSCs adjusted to be just below action potential threshold. Increasing dendritic depolarizations were required as a function of distance to produce just subthreshold somatic sEPSPs of comparable amplitude and shape (amplitude of current injections: 2.3, 3.2 and 2.8 nA for dendritic recordings; 35, 55 and 73 μm from the soma). () Group data from all paired recordings showing dendritic responses (gray circles) and somatic responses (black circles). Points at 0 μm: sEPSP responses to somatic current injection were obtained using dual somatic recordings to prevent distortions of shape as a result of series resistance. () Rise time and halfwidth of maximal subthreshold sEPSPs (recorded at the soma) as a function of the sEPSC dendritic location. sEPSPs at the soma propagating from different dendritic locations were of comparable rise time and duration as those elicited locally at the soma. Linear fits ! to the data indicate a slight negative trend with increasing distance from the soma (dotted lines). Linear fits have slopes of −0.001 ms/μm and are constrained to pass through the average somatic responses at 0 μm (rise time, 0.39 ms; halfwidth, 0.77 ms). * Figure 3: A DTX-sensitive conductance mediates EPSP sharpening. () Simultaneous dendritic (gray) and somatic (black) voltage responses to sEPSCs recorded in control ACSF and in the presence of 100 nM DTX. sEPSCs were injected into the lateral dendrite 90 μm from the soma (0.2–2.2 nA, 0.2-nA increment). In DTX, sEPSP attenuation was sharply reduced and both the rise time and duration of the response was increased (sEPSC, 0.2–1.2 nA, 0.2-nA increment). () Normalized traces of three selected sEPSPs in showing VDS over the subthreshold voltage range. sEPSPs of identical amplitude exhibited no VDS in DTX (right). () In the neuron shown in , DTX eliminated VDS and increased sEPSP halfwidth by about twofold. Points are the average of five traces. () Group data showing VDS at the soma in response to somatic sEPSCs (local, n = 5) or in the dendrite 55–90 μm away (propagated, n = 4). Local sEPSPs at the soma were generated and measured using different pipettes in dual recordings. Maximum VDS (max VDS) was expressed for EPSP halfwidth in co! ntrol ACSF (gray bars) and in the presence of DTX (black bars). Negative and positive values reflect a relative broadening and sharpening, respectively. Asterisks indicate significant differences between control and DTX conditions (paired two-tailed t test; P = 0.0004 and P = 0.0002 for local and propagated EPSPs, respectively). () DTX slowed the rise time and increased the duration of sEPSPs during propagation from the dendrites to the soma, 90 μm away (same cell shown in –). Control, 2,200 pA; DTX, 1,200 pA. * Figure 4: Characterization of IK-LVA in outside-out patches. (,) Pharmacologically isolated IK-LVA during voltage clamp. Voltage was stepped from −90 mV to potentials between −80 to −20 mV in 10-mV increments. Outward currents resistant to 100 nM DTX (middle) were subtracted from control (top), yielding the DTX-sensitive current (bottom). () Kinetic similarity of DTX-sensitive currents recorded at the soma and dendrites. Voltage steps were the same as in . () Activation and steady-state inactivation of IK-LVA. The peak conductance of DTX-sensitive K+ channels in the soma (blue) and dendrites (brown) elicited by steps between −80 and −20 mV from −90 mV. Steady-state inactivation of somatic IK-LVA: peak conductances from voltage steps to −45 mV from a −90-mV holding potential after 1.5-s prepulses between −120 and −30 mV (n = 6; Supplementary Fig. 1). () Deactivation of IK-LVA. Prepulses (−90 to −40 mV for 10 ms) followed by tail currents elicited between −60 and −120 mV. Fits were made to tails 300 μs to 15! ms after the step (see Online Methods). () Activation and deactivation rates (τw) versus voltage for somatic patches (blue circles and triangles, respectively). Activation rates are shown for dendritic patches (brown). Fits to τw at 25 °C were made according to , also shown adjusted to 35 °C using an experimentally measured Q10 of 3.3 (red). () Distribution of IK-LVA in the soma and dendrites. Inset, normalized peak currents from different locations have similar kinetics. () Average current amplitude at the soma and dendrite (20-μm bins) decreased with a length constant of 42 μm (single exponential fit). Dendritic currents in the 60-μm bin were significantly different from those at the soma (*; P = 0.41, 0.06, 0.03 and 0.06 for bins 20–80 μm, respectively, Wilcoxon signed rank test, unequal sample variances). * Figure 5: Relative timing of IK-LVA and sEPSPs in whole-cell recordings at 35 °C. () IK-LVA elicited by sEPSP voltage commands of amplitudes over the voltage range dominated by DTX-sensitive currents. sEPSP commands were delivered from the presumed resting membrane potential of −60 mV (see Online Methods for leak subtraction). The peak EPSP amplitudes were scaled between 2 and 40 mV with 2- or 5-mV intervals, corresponding to peak voltages between −58 and −20 mV. () Sensitivity of outward currents to 100 nM DTX (n = 6). () The time difference between the sEPSP peak and IK-LVA onset (closed circle) and peak (open circle) is plotted as a function of peak membrane potential. * Figure 6: VDS for dendritic and somatic EPSC injection in a compartmental model of the MSO. () Schematic of dual recordings in the multicompartment model. EPSCs were injected either in the dendrite 67.5 μm from the soma (top) or at the soma itself (bottom). Somatic EPSP halfwidth decreased as depolarization increased for both stimulus locations. () VDS dependence on the spatial distribution of KLVA. VDS at the soma and at 67.5 μm in the dendrites (thin and thick lines) in response to either dendritic or somatic EPSCs (left and right, respectively). Four different IK-LVA density distributions were compared. The green line indicates the exponential-gradient in dendritic IK-LVA density based on experimental data (see Fig. 4f,g), the black line indicates the step-gradient model, the red line indicates the uniform IK-LVA density and the blue line indicates active soma and passive dendrites with GK-LVA in dendrites frozen at its resting value. The step-gradient model and exponential-gradient model best reproduce the experimental data presented in Figure 1. () Step-grad! ient model showing the dependence of VDS on the position of the IK-LVA activation function. The left and right graphs show the dendritic and somatic sites of current injection, respectively. Inset, activation functions, (m∞)4, exhibited the same slopes, but different voltages of half-activation (black, V1/2,m = −58 mV (as in whole-cell data); medium gray, V1/2,m = −63 mV; light gray, V1/2,m = −68 mV (as in outside-outside patch data)). V1/2,m values correspond to single Boltzmann fits of maximal conductance adjusted to give the same resting conductance and potential for soma and dendrite in all three cases (Supplementary Fig. 5). The best match to the VDS data was obtained with V1/2,m = −58 mV. * Figure 7: Spatial effects of VDS in an MSO neuron model. () Effect of IK-LVA on EPSPs propagating through dendrites compared in models with active and passive gK-LVA (step-gradient model and frozen model; black and green traces, respectively). Responses shown from two synaptic locations. () Dendritic and somatic EPSP amplitudes (thick and thin lines) as a function of dendritic EPSC location. EPSP amplitude attenuated from stimulation location to soma as predicted by cable theory. Models are color coded as in . () Duration (halfwidth) of the dendritic and somatic EPSPs in . EPSPs broadened for most of the synaptic locations in both models as predicted by cable theory. IK-LVA generated more attenuation but also decreased EPSP broadening during propagation to the soma. () Comparison of VDS between monolateral and bilateral synaptic inputs 135 μm from the soma. Top, monolateral and bilateral configuration. Conductance in the monolateral case was double that of each bilateral synaptic input. Bottom, time courses for selected locations! for monolateral and bilateral stimulation configurations. () Peak EPSP amplitude during propagation from the synaptic location to the soma. Stimulation occurred at 135 μm from the soma edge for monolateral and bilateral cases. () Peak IK-LVA amplitude induced by EPSPs traveling from the stimulation location to the soma. () EPSP halfwidths for EPSPs recorded locally at the injection site in the dendrites (thick lines) and after propagation to the soma (thin lines). EPSPs broadened during propagation to the soma for most monolateral locations but sharpened for most bilateral locations. * Figure 8: Spatio-temporal dynamics of membrane potential and IK-LVA for bilateral ITD-like distal inputs (750 Hz). () Top, spatial profile of voltage along the soma and dendrites (ordinate) advancing in time for in-phase inputs. Color code indicates voltage. Step-gradient configuration is shown for active (left) and frozen gK-LVA (right). Bottom, spatio-temporal voltage evolution for out-of-phase inputs. Active IK-LVA sharpened voltage time courses, especially proximally. () Somatic voltage and IK-LVA (top and bottom) time courses for in-phase (black) and out-of-phase (red) bilateral inputs. Left, step-gradient model with active gK-LVA. Right, frozen gK-LVA model, with IK-LVA behaving as a leak current. () ITD-tuning curves and sensitivity function for three different spiking models (see Online Methods and Supplementary Fig. 7): step-gradient model (black), frozen gK-LVA in dendrites (blue) and frozen gK-LVA in the whole cell (green). Left, ITD-tuning curve for bilateral inputs at 750 Hz computed as ν (the number of spikes per EPSP pair). For inputs at 750 Hz, the ratio of νin-phase to! νout-of-phase for a model with frozen IK-LVA was small, poorly discriminating different ITDs. The difference in number of spikes between in-phase inputs versus out-of-phase inputs increased as the amount of active IK-LVA in the model increased (from green to blue to black curve). Right, sensitivity functions for different input frequencies in the MSO neurons' physiological range. The step-gradient model with a distribution of IK-LVA similar to that found in vitro showed higher sensitivity for a broader range of input frequencies. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Paul J Mathews & * Pablo E Jercog Affiliations * Section of Neurobiology and Institute for Neuroscience, University of Texas at Austin, Austin, Texas, USA. * Paul J Mathews, * Luisa L Scott & * Nace L Golding * Center for Neural Science, New York University, New York, New York, USA. * Pablo E Jercog & * John Rinzel * Courant Institute of Mathematical Sciences, New York University, New York, New York, USA. * John Rinzel Contributions P.J.M. performed all of the voltage-clamp experiments characterizing IK-LVA in patches and whole cells. N.L.G. conducted dual somatic and dendritic current-clamp recordings. L.L.S. performed some of the experiments from older animals and also made some of the initial observations on voltage-dependent sharpening. P.E.J. performed all of the simulations. N.L.G. and J.R. helped design and supervise the experiments and simulations, respectively. N.L.G. wrote the manuscript, with contributions from P.J.M., P.E.J. and J.R. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Nace L Golding (golding@mail.utexas.edu) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (12M) Supplementary Figures 1–10 Additional data - Hypoxia activates a latent circuit for processing gustatory information in C. elegans
Pocock R Hobert O - Nature Neuroscience 13(5):610-614 (2010)
Nature Neuroscience | Article Hypoxia activates a latent circuit for processing gustatory information in C. elegans * Roger Pocock1 Search for this author in: * NPG journals * PubMed * Google Scholar * Oliver Hobert1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume:13,Pages:610–614Year published:(2010)DOI:doi:10.1038/nn.2537Received09 March 2010Accepted26 March 2010Published online18 April 2010 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 Dedicated neuronal circuits enable animals to engage in specific behavioral responses to environmental stimuli. We found that hypoxic stress enhanced gustatory sensory perception via previously unknown circuitry in Caenorhabditis elegans. The hypoxia-inducible transcription factor HIF-1 upregulated serotonin (5-HT) expression in specific sensory neurons that are not normally required for chemosensation. 5-HT subsequently promoted hypoxia-enhanced sensory perception by signaling through the metabotropic G protein–coupled receptor SER-7 in an unusual peripheral neuron, the M4 motor neuron. M4 relayed this information back into the CNS via the FMRFamide-related neuropeptide FLP-21 and its cognate receptor, NPR-1. Thus, physiological detection of hypoxia results in the activation of an additional, previously unrecognized circuit for processing sensory information that is not required for sensory processing under normoxic conditions. View full text Figures at a glance * Figure 1: 5-HT expression is altered in hypoxia through direct HIF-1 regulation of tph-1. (,) tph-1promgfp expression in normoxia () and after exposure to 1% O2 for 12 h (). Using a reporter strain that expresses tph-1promgfp at low-levels (injected at 2 ng μl−1), we found that tph-1promgfp was also upregulated in the ADF and NSM neurons after hypoxic exposure (Supplementary Fig. 1). (,) 5-HT immunofluorescence of a normoxic animal () and an animal after exposure to 1% O2 for 12 h (). (–) tph-1promgfp was ectopically expressed in ASGL/R under conditions that stabilize hif-1, achieved by either mutating the residue in HIF-1 required for its degradation () or in egl-9 mutants (); the latter effect is genetically dependent on hif-1 (). Red arrowheads indicate ASG chemosensory neurons. Ventral views, anterior is to the left. Scale bar represents 10 μm. () HIF-1–induced tph-1 reporter gene expression (induction achieved through preventing the degradation of HIF-1 in egl-9 mutants) was abolished on deletion of the HRE (6 bp deletion). # = independent transgenic! lines (n = 87–122). * Figure 2: C. elegans shows enhanced sensory perception after hypoxic stress using HIF-1 and 5-HT in a neuron-specific manner. () Hypoxic exposure of C. elegans wild-type adult hermaphrodites enhanced their ability to respond to 500 mM and 250 mM NaCl. () Hypoxic exposure of che-1(ot66) mutant adult animals, which lack functional ASE neurons and therefore do not chemotax well under normoxic conditions, significantly enhanced their ability to chemotax toward 2.5 M NaCl. This behavior was dependent on 5-HT and HIF-1, as it was abolished in che-1(ot66); tph-1(mg280) and che-1(ot66); hif-1(ia4) double mutant animals. Transgenic expression of hif-1 in ADF and ASG, but not the NSM neurons, rescued the loss of HESP in the che-1(ot66); hif-1(ia04) double mutant (# = independent transgenic lines). For cell-specific rescue, we used ops-1 (ASG)37, srh-142 (ADF)38 and ceh-2 (NSM)39 promoters. Statistical significance was assessed using the t test (**P < 0.05, ***P < 0.005, n.s. = not statistically significant from control). * Figure 3: SER-7, a 5-HT7–like metabotropic receptor, acts in the M4 pharyngeal motorneuron to induce HESP. () Loss of ser-7, but not ser-1, ser-2, ser-3 and ser-4, caused defects in HESP to a 250 mM NaCl gradient. () Expression of the ser-7 cDNA exclusively in the M4 neuron with the M4-specific ser-7 and ceh-28 promoter fragments restored HESP in the ser-7(tm1325) mutant. Statistical significance was assessed using the t test (**P < 0.05, ***P < 0.005). * Figure 4: Mutations in flp-21 and npr-1 caused defects in the HESP response. Expression of npr-1 cDNA under the gcy-32 promoter (AQR, PQR and URX expression)40 and not under the ncs-1 promoter (other npr-1–expressing neurons)40 was sufficient to restore the HESP response in npr-1(ad609) animals. Ablation of the oxygen-sensing neurons AQR, PQR and URX (strain qaIs2241)7 also caused defects in HESP. Statistical significance was assessed using the t test (**P < 0.05, ***P < 0.005). Author information * Abstract * Author information * Supplementary information Affiliations * Department of Biochemistry and Molecular Biophysics, Howard Hughes Medical Institute, Columbia University Medical Center, New York, New York, USA. * Roger Pocock & * Oliver Hobert Contributions R.P. initiated this study and conducted all of the experiments. R.P. and O.H. designed and discussed the experiments and wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Roger Pocock (roger.pocock@bric.ku.dk) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–4 Additional data - Neuropeptide feedback modifies odor-evoked dynamics in Caenorhabditis elegans olfactory neurons
Chalasani SH Kato S Albrecht DR Nakagawa T Abbott LF Bargmann CI - Nature Neuroscience 13(5):615-621 (2010)
Nature Neuroscience | Article Neuropeptide feedback modifies odor-evoked dynamics in Caenorhabditis elegans olfactory neurons * Sreekanth H Chalasani1 Search for this author in: * NPG journals * PubMed * Google Scholar * Saul Kato1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Dirk R Albrecht1 Search for this author in: * NPG journals * PubMed * Google Scholar * Takao Nakagawa1 Search for this author in: * NPG journals * PubMed * Google Scholar * L F Abbott2 Search for this author in: * NPG journals * PubMed * Google Scholar * Cornelia I Bargmann1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume:13,Pages:615–621Year published:(2010)DOI:doi:10.1038/nn.2526Received29 October 2009Accepted23 February 2010Published online04 April 2010 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 Many neurons release classical transmitters together with neuropeptide co-transmitters whose functions are incompletely understood. Here we define the relationship between two transmitters in the olfactory system of C. elegans, showing that a neuropeptide-to-neuropeptide feedback loop alters sensory dynamics in primary olfactory neurons. The AWC olfactory neuron is glutamatergic and also expresses the peptide NLP-1. Worms with nlp-1 mutations show increased AWC-dependent behaviors, suggesting that NLP-1 limits the normal response. The receptor for NLP-1 is the G protein-coupled receptor NPR-11, which acts in postsynaptic AIA interneurons. Feedback from AIA interneurons modulates odor-evoked calcium dynamics in AWC olfactory neurons and requires INS-1, a neuropeptide released from AIA. The neuropeptide feedback loop dampens behavioral responses to odors on short and long timescales. Our results point to neuronal dynamics as a site of behavioral regulation and reveal the abili! ty of neuropeptide feedback to remodel sensory networks on multiple timescales. View full text Figures at a glance * Figure 1: AWC releases NLP-1, which acts on NPR-11 in AIA. () AWC sensory neurons, downstream interneurons, and relevant glutamate receptors (from this work (AIA) and ref. 9). (,) Local search behavior 7–12 min after removal from food. RevOmega, coupled reversal-omega behaviors characteristic of local search. Analysis of nlp-1 mutants () and npr-11 mutants (). In all figures, WT indicates control N2 strain, AWCnlp-1 indicates nlp-1 cDNA under AWC-selective odr-3 promoter, AWCnlp-1(OE) indicates the same plasmid injected at high concentrations, AIAnpr-11 indicates npr-11 cDNA under AIA-selective gcy-28.d promoter. Error bars, s.e.m.; *P < 0.05 by t-test or t-test with Bonferroni correction, as appropriate; NS, not significant. Complete behavioral data with all genotypes and time points are in Supplementary Table 1. (,) Response of npr-11- and Gα16Z-, npr-11 or Gα16Z- transfected HEK 293 cells to an NLP-1 peptide and a scrambled NLP-1 peptide (sNLP-1). () Pseudocolor images of fura2-labeled cells indicating fluorescent ratio inten! sities. Scale bar, 100 μm. () Average calcium response of all cells in the window (n = 10 fields for npr-11 and Gα16Z, n = 8 for npr-11 and n = 7 for Gα16Z). Means and s.e.m. are shown. * Figure 2: Calcium responses in AIA interneurons require AWC glutamate and NLP-1. (,) Heat maps showing the ratio of change in fluorescence to total fluorescence in AIA neurons expressing GCaMP2.2b15; addition () and removal () of odor stimulus at t = 10 s in each recording (n = 18). (,) Average G-CaMP fluorescence change in AIA neurons in wild-type (WT; n = 18) and wild-type AWC-ablated worms (n = 12) on addition () and removal () of odor. (–) Mutant AIA responses. () eat-4 (n = 18, WT n = 18). () glc-3 (n = 16, WT n = 16). () nlp-1 (n = 18, WT n = 18) and AWCnlp-1 cell-selective rescue (n = 18). In all imaging figures, odor is a 10−4 dilution of isoamyl alcohol. Light gray shading indicates s.e.m. *Significantly different from wild type; **significantly different from nlp-1 mutant (P < 0.05, t-test with Bonferroni correction). * Figure 3: Altered AWC calcium responses in nlp-1 and npr-11 mutants. (,,,) Heat maps showing ratio change in fluorescence to total fluorescence in AWC neurons expressing G-CaMP1.0. Odor was removed at 10 s in each recording. () Wild type (n = 32); () nlp-1 (n = 32); () wild type (n = 18); () npr-11 (n = 18). (,) Representative AWC calcium responses from individual wild-type worms, nlp-1 () and npr-11 mutants (), and rescued strains. (,) Fourier power analysis of AWC calcium responses in nlp-1 () and npr-11 mutants (). Left, normalized energy density spectrum averaged across all calcium traces of each genotype; arrows indicate range of the middle frequency band (color code on right). Right, the average power ratio of the middle frequency band (0.033–1 Hz) across all calcium traces of each genotype; error bars, s.e.m. *P < 0.05 (t-test with Bonferroni correction). * Figure 4: Worms with mutations in nlp-1 and npr-11 are defective in olfactory adaptation. () Schematic diagram of adaptation assay. () Adaptation in nlp-1 and npr-11 mutants, and cell-selective rescue. Error bars, s.e.m. *P < 0.05 (t-test with Bonferroni correction). (,) AWC calcium responses in wild-type, nlp-1 and AWCnlp-1 transgenic rescued worms () and wild-type, npr-11 and AIAnpr-11 transgenic rescued worms () adapted for 60 min (n = 12 each). Odor pulses are marked. Light gray shading indicates s.e.m. *Significantly different from wild type (P < 0.05, t-test with Bonferroni correction). * Figure 5: ins-1 is a component of the nlp-1-npr-11 pathway. () Local search behavior 7–12 min after removal from food. RevOmega, coupled reversal-omega behaviors characteristic of local search. AIAins-1, ins-1 cDNA expressed under AIA-selective gcy-28.d promoter. Error bars, s.e.m. *P < 0.05, t-test with Bonferroni correction. () Fourier power analysis of AWC calcium responses in ins-1 mutants. Left, the normalized energy density spectrum averaged across all calcium traces of each genotype; arrows indicate range of the middle frequency band (color code on right). Right, the average power ratio of the middle frequency band (0.033–1 Hz) across all calcium traces of each genotype; error bars, s.e.m. *P < 0.05 (t-test with Bonferroni correction). () Adaptation in ins-1 mutants, and cell-selective rescue. *Different from unadapted control (P < 0.05, t-test). Error bars, s.e.m. () AWC calcium responses in wild-type, ins-1 and AIAins-1 rescued transgenic worms adapted for 60 min (n = 12 each). Odor pulses are marked. Light gray shading ! indicates s.e.m. *Different from wild type at P < 0.05, t-test with Bonferroni correction. Author information * Abstract * Author information * Supplementary information Affiliations * Howard Hughes Medical Institute, The Rockefeller University, New York, New York, USA. * Sreekanth H Chalasani, * Saul Kato, * Dirk R Albrecht, * Takao Nakagawa & * Cornelia I Bargmann * Department of Neuroscience, Department of Physiology and Cellular Biophysics, Columbia University College of Physicians and Surgeons, New York, New York, USA. * Saul Kato & * L F Abbott Contributions S.H.C. conceived, conducted and interpreted experiments and co-wrote the paper; S.K., D.R.A. and L.F.A. performed and interpreted data analysis; T.N. performed HEK expression experiments; C.I.B. conceived and interpreted experiments and co-wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Cornelia I Bargmann (cori@rockefeller.edu) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–4, Supplementary Text and Supplementary Table 1 Additional data - CRF receptor 1 regulates anxiety behavior via sensitization of 5-HT2 receptor signaling
Magalhaes AC Holmes KD Dale LB Comps-Agrar L Lee D Yadav PN Drysdale L Poulter MO Roth BL Pin JP Anisman H Ferguson SS - Nature Neuroscience 13(5):622-629 (2010)
Nature Neuroscience | Article CRF receptor 1 regulates anxiety behavior via sensitization of 5-HT2 receptor signaling * Ana C Magalhaes1 Search for this author in: * NPG journals * PubMed * Google Scholar * Kevin D Holmes1 Search for this author in: * NPG journals * PubMed * Google Scholar * Lianne B Dale1 Search for this author in: * NPG journals * PubMed * Google Scholar * Laetitia Comps-Agrar2 Search for this author in: * NPG journals * PubMed * Google Scholar * Dennis Lee1 Search for this author in: * NPG journals * PubMed * Google Scholar * Prem N Yadav3 Search for this author in: * NPG journals * PubMed * Google Scholar * Linsay Drysdale1 Search for this author in: * NPG journals * PubMed * Google Scholar * Michael O Poulter1 Search for this author in: * NPG journals * PubMed * Google Scholar * Bryan L Roth3 Search for this author in: * NPG journals * PubMed * Google Scholar * Jean-Philippe Pin2 Search for this author in: * NPG journals * PubMed * Google Scholar * Hymie Anisman4 Search for this author in: * NPG journals * PubMed * Google Scholar * Stephen S G Ferguson1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume:13,Pages:622–629Year published:(2010)DOI:doi:10.1038/nn.2529Received02 February 2010Accepted04 March 2010Published online11 April 2010 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 Stress and anxiety disorders are risk factors for depression and these behaviors are modulated by corticotrophin-releasing factor receptor 1 (CRFR1) and serotonin receptor (5-HT2R). However, the potential behavioral and cellular interaction between these two receptors is unclear. We found that pre-administration of corticotrophin-releasing factor (CRF) into the prefrontal cortex of mice enhanced 5-HT2R–mediated anxiety behaviors in response to 2,5-dimethoxy-4-iodoamphetamine. In both heterologous cell cultures and mouse cortical neurons, activation of CRFR1 also enhanced 5-HT2 receptor–mediated inositol phosphate formation. CRFR1-mediated increases in 5-HT2R signaling were dependent on receptor internalization and receptor recycling via rapid recycling endosomes, resulting in increased expression of 5-HT2R on the cell surface. Sensitization of 5-HT2R signaling by CRFR1 required intact PDZ domain–binding motifs at the end of the C-terminal tails of both receptor types. ! These data suggest a mechanism by which CRF, a peptide known to be released by stress, enhances anxiety-related behavior via sensitization of 5-HT2R signaling. View full text Figures at a glance * Figure 1: Effect of CRFR1 activation on 5-HT2R signaling. (–) Dose response curves for 5-HT–stimulated inositol phosphate (IP) formation in HEK 293 cells pretreated with and without CRF (500 nM) for 30 min in cells transfected with either FLAG–5-HT2AR and FLAG–5-HT2cR alone (), FLAG–5-HT2AR and HA-CRFR1 () or FLAG–5-HT2cR and HA-CRFR1 (). () Basal and agonist-stimulated inositol formation in cells expressing FLAG–5-HT2cR alone, HA-CRFR1 alone or both FLAG-5-HT2cR and HA-CRFR1. Cells were treated with 500 nM CRF with or without a subsequent exposure to 10 μM 5-HT for 30 min. () Dose response curves for 5-HT–stimulated inositol phosphate formation in HEK 293 cells transfected with FLAG–5-HT2AR and β2AR and pretreated with and without 100 μM isoproterenol (Iso) for 30 min. () Dose response curves for 5-HT–stimulated inositol phosphate formation in HEK 293 cells transfected with FLAG–5-HT2AR and CRFR2 and pretreated with or without 500 nM CRF for 30 min. () Dose response curves for CRF-stimulated cAMP formation! in HEK 293 cells transfected with FLAG–5-HT2AR and HA-CRFR1 and pretreated with or without 10 μM 5-HT for 30 min. The data represent the mean ± s.e.m. for three to six individual experiments. * Figure 2: Effect of CRFR1 activation on 5-HT2R signaling in neurons. (,) Representative laser-scanning confocal micrographs showing the coexpression of endogenous 5-HT2AR (green, ) and CRFR1 (red, ) in a 30-μm neuronal slice derived from prefrontal cortex of a C57/BL6 mouse. () Neurons were also stained with Hoechst. () Colocalization of 5-HT2AR and CRFR1 in a subpopulation of neurons (dashed circles). () Magnified view of 5-HT2AR and CRFR1 colocalization in a subpopulation of neurons (dashed circles) from the dashed box in . Cortical layers are identified by roman numerals. Scale bars represent 50 μm. * Figure 3: Role of endocytosis in CRFR1-dependent augmentation of 5-HT2R signaling. () Dose response curves for 5-HT–stimulated inositol phosphate formation in HEK 293 cells transfected with FLAG–5-HT2AR and HA-CRFR1 and pretreated with or without 500 nM CRF for 30 min in the presence of dominant-negative dynamin I-K44A. The dose response curves represent the mean ± s.e.m. for four independent experiments. (,) Representative laser-scanning confocal micrographs showing the distribution of FLAG-5-HT2AR and HA-CRFR1 () and FLAG-5-HT2CR and HA-CRFR1 () in HEK 293 cells labeled with FLAG and HA antibodies at 4 °C and then warmed to 37 °C for 30 min in the absence of agonist. () Representative laser-scanning confocal micrographs showing the distribution of FLAG–5-HT2AR and HA-CRFR1 labeled with FLAG and HA antibodies at 4 °C and warmed to 37 °C for 30 min in the absence of agonist. () Representative laser-scanning confocal micrographs showing the distribution of FLAG–5-HT2AR and HA-CRFR1 transfected into rat cortical neurons labeled with FLAG and HA ! antibodies at 4 °C and treated with 500 nM CRF and warmed to 37 °C for 30 min. () Representative laser-scanning confocal micrographs showing the distribution of FLAG–5-HT2AR and HA-β2AR transfected into HEK 293 cells labeled with FLAG and HA antibodies at 4 °C and treated with 100 μM isoproterenol and warmed to 37 °C for 30 min. Micrographs are representative images of multiple cells imaged on three independent occasions. Scale bars represent 10 μm. * Figure 4: Role of receptor recycling in CRF-modulated 5-HT2R signaling. () Dose response curves for 5-HT–stimulated inositol phosphate formation in HEK 293 cells transfected with FLAG–5-HT2AR and HA-CRFR1 and pretreated with or without 500 nM CRF for 30 min following the pretreatment of cells with or without 100 μM monensin for 30 min. () Dose response curves for 5-HT–stimulated inositol phosphate formation in HEK 293 cells with transfected FLAG–5-HT2AR, HA-CRFR1 and Rab4-S28N and pretreated with or without 500 nM CRF for 30 min. () Dose response curves for 5-HT–stimulated inositol phosphate formation in HEK 293 cells transfected with FLAG–5-HT2AR, HA-CRFR1 and Rab11-S25N and pretreated with or without 500 nM CRF for 30 min. () Increase in cell surface 5-HT2AR localization following 30 min pretreatment of CRFR1 with 500 nM CRF. The cell surface expression of the 5-HT2AR represents the mean ± s.e.m. for four independent experiments. The full length blot is presented in Supplementary Figure 5. *P < 0.05 versus untreated control. * Figure 5: Receptor determinants of CRF-dependent increases in 5-HT2R signaling. () The change in cell surface 5-HT2AR and 5-HT2AR–ΔSCV localization following 30 min pretreatment of CRFR1 with 500 nM CRF and the change in cell surface 5-HT2AR localization following 30 min pretreatment of CRFR1-ΔTAV with 500 nM CRF are shown. The cell surface expression of the 5-HT2AR represents the mean ± s.e.m. for four independent experiments. *P < 0.05 versus untreated control. () Dose response curves for 5-HT–stimulated inositol phosphate formation in HEK 293 cells transfected with FLAG–5-HT2cR and either HA-CRFR1 or HA-CRFR1 lacking a PDZ domain–binding motif (ΔTAV), pretreated with or without 500 nM CRF for 30 min. () Dose response curves for 5-HT–stimulated inositol phosphate formation in HEK 293 cells transfected with HA-CRFR1 and either FLAG–5-HT2CR or FLAG–5-HT2CR lacking a PDZ domain–binding motif (ΔSSV) pretreated with or without 500 nM CRF for 30 min. () Dose response curves for 5-HT–stimulated inositol phosphate formation in HEK 293 c! ells transfected with HA-CRFR1 and either FLAG–5-HT2AR or FLAG–5-HT2AR lacking a PDZ domain–binding motif (ΔSCV) pretreated with or without 500 nM CRF for 30 min. () Dose response curves for 5-HT–stimulated inositol phosphate formation in HEK 293 cells transfected with HA-CRFR1 and FLAG–5-HT2AR pretreated for 1 h with a Tat-fusion peptide corresponding to the last ten amino acid residues of the CRFR1 carboxyl-terminal tail and then treated with or without 500 nM CRF for 30 min. Dose response curves represent the mean ± s.e.m. for three to five independent experiments. * Figure 6: Analysis of CRF pretreatment on 5-HT2R–mediated anxiety-related behaviors. () Mean latencies for mice to enter the center square in a 5-min open field. () Mean latency to enter the open arms of the elevated plus maze. () The frequency of entries into the open arms of the elevated plus maze. () The frequency of entries into the closed arms of the elevated plus maze. () Time spent in the closed arms of the elevated plus maze. In all experiments, either vehicle or CRF (1.5 μg in 1 μl) was administered to the medial prefrontal cortex via a surgically implanted cannulae for 5 min, and mice were intraperitoneally injected with vehicle or DOI (0.15 mg per kg) 5 min later before behavioral testing. We used nine to ten mice in each test group. Data represents mean ± s.d. *P < 0.01 versus vehicle/vehicle treated control. * Figure 7: Analysis of CRF pretreatment on 5-HT2R–mediated anxiety-related behaviors following M100907 treatment. () Mean latency to enter the open arms of the elevated plus maze in a 5-min test period. () The frequency of entries into the open arms of the elevated plus maze. () Time spent in the open arms of the elevated plus maze. () The frequency of entries into the closed arms of the elevated plus maze. () Time spent in the closed arms of the elevated plus maze. In all experiments, either vehicle or CRF (1.5 μg in 1 μl) was administered to the medial prefrontal cortex via a surgically implanted cannulae for 5 min, mice were intraperitoneally injected with vehicle or DOI (0.15 mg per kg) 5 min later and mice were pretreated intraperitoneally with either vehicle or 0.25 mg per kg of M100907 in a volume of 0.3 ml before DOI administration before behavioral testing. We used six to eight mice in each test group. Data represents mean ± s.d. *P < 0.05 versus respective vehicle control. **P < 0.05 versus respective M100907 treatment. ***P < 0.05 relative to M100907 and CRF treatment. Author information * Abstract * Author information * Supplementary information Affiliations * J. Allyn Taylor Centre for Cell Biology, Molecular Brain Research Group, Robarts Research and the Department of Physiology & Pharmacology, The University of Western Ontario, London, Ontario, Canada. * Ana C Magalhaes, * Kevin D Holmes, * Lianne B Dale, * Dennis Lee, * Linsay Drysdale, * Michael O Poulter & * Stephen S G Ferguson * Institut de Génomique Fonctionnelle, Départment de Pharmacologie Moléculaire, UMR 5203 CNRS, U 661 INSERM, University of Montpellier I & II, Montpellier, France. * Laetitia Comps-Agrar & * Jean-Philippe Pin * Department of Pharmacology, University of North Carolina Chapel Hill, Chapel Hill, North Carolina, USA. * Prem N Yadav & * Bryan L Roth * Institute of Neurosciences, Centre for Research on Stress, Coping & Well-being, Life Sciences Research Centre, Carleton University, Ottawa, Ontario, Canada. * Hymie Anisman Contributions M.O.P., K.D.H., A.C.M., H.A. and S.S.G.F. conceived the experiments. H.A. carried out the behavioral experiments. A.C.M., K.D.H., L.B.D., L.C.-A., J.-P.P., L.D., P.N.Y. and D.L. performed the rest of the experiments. S.S.G.F., A.C.M., B.L.R. and H.A. analyzed the data and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Stephen S G Ferguson (ferguson@robarts.ca) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (584K) Supplementary Figures 1–5 and Table 1 Additional data - PKMζ maintains memories by regulating GluR2-dependent AMPA receptor trafficking
Migues PV Hardt O Wu DC Gamache K Sacktor TC Wang YT Nader K - Nature Neuroscience 13(5):630-634 (2010)
Nature Neuroscience | Article PKMζ maintains memories by regulating GluR2-dependent AMPA receptor trafficking * Paola Virginia Migues1 Search for this author in: * NPG journals * PubMed * Google Scholar * Oliver Hardt1 Search for this author in: * NPG journals * PubMed * Google Scholar * Dong Chuan Wu2 Search for this author in: * NPG journals * PubMed * Google Scholar * Karine Gamache1 Search for this author in: * NPG journals * PubMed * Google Scholar * Todd Charlton Sacktor3 Search for this author in: * NPG journals * PubMed * Google Scholar * Yu Tian Wang2 Search for this author in: * NPG journals * PubMed * Google Scholar * Karim Nader1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume:13,Pages:630–634Year published:(2010)DOI:doi:10.1038/nn.2531Received23 November 2009Accepted01 March 2010Published online11 April 2010 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 maintenance of long-term memory in hippocampus, neocortex and amygdala requires the persistent action of the atypical protein kinase C isoform, protein kinase Mζ (PKMζ). We found that inactivating PKMζ in the amygdala impaired fear memory in rats and that the extent of the impairment was positively correlated with a decrease in postsynaptic GluR2. Blocking the GluR2-dependent removal of postsynaptic AMPA receptors abolished the behavioral impairment caused by PKMζ inhibition and the associated decrease in postsynaptic GluR2 expression, which correlated with performance. Similarly, blocking this pathway for removal of GluR2-containing receptors from postsynaptic sites in amygdala slices prevented the reversal of long-term potentiation caused by inactivating PKMζ. Similar behavioral results were obtained in the hippocampus for unreinforced recognition memory of object location. Together, these findings indicate that PKMζ maintains long-term memory by regulating the t! rafficking of GluR2-containing AMPA receptors, the postsynaptic expression of which directly predicts memory retention. View full text Figures at a glance * Figure 1: Blocking GluR2-dependent AMPA receptor synaptic removal prevents memory impairment induced by PKMζ inactivation. (,) GluR23Y or Scr-GluR23Y was infused into the amygdala 1 d after the training session. ZIP or Scr-ZIP was infused 1 h later. Memory was tested 1 d () or 10 d () after the infusions. Data represent the mean percentage of the freezing time during the tone. Error bars represent s.e.m. ZIP infusion abolished the freezing response (*P < 0.001), but infusions of both GluR23Y and ZIP led to a performance that was similar to that exhibited by the inactive, scrambled peptide–infused controls, as determined by the Kruskal-Wallis analysis of ranks test. * Figure 2: Postsynaptic GluR2 levels are decreased in fear-conditioned rats after PKMζ inactivation and the levels of postsynaptic GluR2 correlate with the magnitude of freezing during memory retention. (,) GluR23Y or Scr-GluR23Y was infused into the amygdala 1 d after the training session. ZIP or Scr-ZIP was infused 1 h later. Memory was tested 1 d after the infusions and rats were killed 1 d later for BLA extraction. Representative western blots (left) and quantification of GluR2 () and GluR1 () protein levels (right) in the indicated subcellular fractions from BLA are shown. The full-length western blots are shown in Supplementary Figure 6. Data were normalized to the Scr-GluR23Y and Scr-ZIP group mean. Bars represent the group means, and error bars represent s.e.m. The levels of postsynaptic GluR2 in the group that received Scr-GluR23Y and ZIP were significantly lower (*P < 0.05) than in the group infused with both scrambled control peptides and in the group that received both GluR23Y and ZIP as determined by the post hoc Tukey's HSD test after significant one-way ANOVA. (,) Relationship between GluR2 () and GluR1 () levels in the postsynaptic or extrasynaptic membrane ! fractions and freezing levels during the test of trained animals. A significant correlation was observed only for the postsynaptic GluR2 fraction (R = 0.65, P < 0.001) by ANOVA overall goodness of fit. * Figure 3: Blocking GluR2-dependent AMPA receptor synaptic removal prevents the LTP impairment induced by PKMζ inactivation in BLA slices. () Bath application of ZIP, but not Scr-ZIP, reversed LTP induced by 200 pulses at 2 Hz paired with postsynaptic depolarization to −5 mV. () The reversal of LTP caused by ZIP was prevented by intracellular perfusion of GluR23Y. () Averaged EPSC amplitude obtained at 40 min after LTP induction. The samples that received both Scr-GluR23Y and ZIP were significantly different from all the other groups (*P < 0.05, post hoc Tukey's HSD test after significant one-way ANOVA). * Figure 4: Blocking GluR2-dependent, postsynaptic AMPA receptor removal prevents the impairment of object location memory induced by PKMζ inactivation in the dorsal hippocampus. GluR23Y or Scr-GluR23Y was infused into the dorsal hippocampus 1 d after the training session. ZIP or Scr-ZIP was infused 1 h later. Memory was tested 1 d after the infusions. () Values represent the mean ratio of the time that the rats spent exploring a familiar object in a novel location over the time they spent exploring a familiar object in the same location as in the training session. ZIP infusions led to a total loss of memory for object location (*P < 0.001), as determined by post hoc Tukey's HSD test after significant one-way ANOVA, such that the rats explored both objects at the level of chance. GluR23Y infusions before ZIP infusions prevented the memory loss. () Values represent the total exploration time. No significant differences were observed among the treatment groups. All error bars represent s.e.m. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Psychology, McGill University, Montreal, Quebec, Canada. * Paola Virginia Migues, * Oliver Hardt, * Karine Gamache & * Karim Nader * Brain Research Center and Department of Medicine, University of British Columbia, Vancouver, British Columbia, Canada. * Dong Chuan Wu & * Yu Tian Wang * Departments of Physiology and Pharmacology, and of Neurology, The Robert Furchgott Center for Neural and Behavioral Science, SUNY Downstate Medical Center, Brooklyn, New York, USA. * Todd Charlton Sacktor Contributions All of the authors contributed to the design of experiments, interpretation of results and editing of the manuscript. P.V.M. and O.H. conducted the behavioral studies. P.V.M. carried out the biochemical studies. D.C.W. conducted the electrophysiological studies. K.G. and O.H. performed the stereotaxic surgeries. P.V.M., O.H., T.C.S. and K.N. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Paola Virginia Migues (virginia.migues@mcgill.ca) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–6 Additional data - Dopamine D2 receptors in addiction-like reward dysfunction and compulsive eating in obese rats
Johnson PM Kenny PJ - Nature Neuroscience 13(5):635-641 (2010)
Nature Neuroscience | Article Dopamine D2 receptors in addiction-like reward dysfunction and compulsive eating in obese rats * Paul M Johnson1 Search for this author in: * NPG journals * PubMed * Google Scholar * Paul J Kenny1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume:13,Pages:635–641Year published:(2010)DOI:doi:10.1038/nn.2519Received29 December 2009Accepted16 February 2010Published online28 March 2010 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 We found that development of obesity was coupled with emergence of a progressively worsening deficit in neural reward responses. Similar changes in reward homeostasis induced by cocaine or heroin are considered to be crucial in triggering the transition from casual to compulsive drug-taking. Accordingly, we detected compulsive-like feeding behavior in obese but not lean rats, measured as palatable food consumption that was resistant to disruption by an aversive conditioned stimulus. Striatal dopamine D2 receptors (D2Rs) were downregulated in obese rats, as has been reported in humans addicted to drugs. Moreover, lentivirus-mediated knockdown of striatal D2Rs rapidly accelerated the development of addiction-like reward deficits and the onset of compulsive-like food seeking in rats with extended access to palatable high-fat food. These data demonstrate that overconsumption of palatable food triggers addiction-like neuroadaptive responses in brain reward circuits and drives the! development of compulsive eating. Common hedonic mechanisms may therefore underlie obesity and drug addiction. View full text Figures at a glance * Figure 1: Weight gain and reward dysfunction in rats with extended access to a cafeteria diet. () Mean (± s.e.m.) weight gain in chow-only, restricted-access and extended-access rats (access × day interaction: F39,702 = 7.9, P < 0.0001; *P < 0.05 compared with chow-only group, post hoc test). () Mean (± s.e.m.) percentage change from baseline reward thresholds (access × time interaction: F78,1092 = 1.7, P < 0.0005; *P < 0.05 compared with chow-only group, post hoc test). * Figure 2: Patterns of consumption in rats with extended access to a cafeteria diet. () Mean (± s.e.m.) daily caloric intake in chow-only, restricted-access and extended-access rats (access: F1,324 = 100.6, P < 0.0001; time: F18,324 = 7.8, P < 0.0001; access × time interaction: F18,324 = 4.6, P < 0.0001; *P < 0.05 compared with chow-only group, post hoc test). () Mean daily caloric intake (± s.e.m.) from chow (access: F2,504 = 349.1, P < 0.0001; time: F18,504 = 5.9, P < 0.0001; access × time interaction: F36,504 = 3.52, P < 0.0001; *P < 0.05 compared with chow-only group, post hoc test). () Mean daily caloric intake (± s.e.m.) from fat (access: F2,486 = 118.7, P < 0.0001; time: F18,486 = 8.8, P < 0.0001; access × time interaction: F36,486 = 6.2, P < 0.0001; *P < 0.05 compared with chow-only group, post hoc test). () Comparison of mean (± s.e.m.) total caloric intake, and calories consumed exclusively from chow, during the entire 40-day period of access (access: F2,54 = 25.0, P < 0.0001; calorie source: F2,54 = 1235.2, P < 0.0001; access × calorie sou! rce interaction: F2,54 = 485.7, P < 0.0001; ***P < 0.001 compared with total calories in chow-only group, ###P < 0.001 compared with total calories in the same group of rats, post hoc test). * Figure 3: Persistent reward dysfunction and hypophagia during abstinence in rats with extended access to a cafeteria diet. () Mean percentage change from baseline reward thresholds (± s.e.m.) during abstinence from a palatable high-fat diet (access: F2,112 = 3.7, P < 0.05; time: F4,112 = 2.3, P > 0.05; *P < 0.05 compared with chow-only group, post hoc test). () Mean caloric intake (± s.e.m.) on the last day of access to the high-fat diet (baseline) and during the 14 d of abstinence when only standard chow was available (access: F2,168 = 41.7, P < 0.0001; time: F6,168 = 65.6, P < 0.0001; access × time interaction: F12,168 = 38.3, P < 0.0001; *P < 0.05 compared with chow-only group, post hoc test). () Change in mean body weight (± s.e.m.) compared with body weight on the last day of access to the high-fat diet (baseline) and during the 14 d of abstinence when only standard chow was available (access: F1,126 = 37.2, P < 0.0001; time: F7,126 = 3.1, P < 0.01; access × time interaction: F7,126 = 40.9, P < 0.0001; *P < 0.05 compared with chow-only group, post hoc test). () Histological reconstruct! ion of the location of BSR stimulating electrodes in the lateral hypothalamus of chow-only (triangles), restricted-access (squares) and extended-access (circles) rats. * Figure 4: Weight gain is inversely related to striatal D2R levels. () Chow-only, restricted-access and extended-access rats were subdivided into two groups per access condition based on a median split of body weights: light (L) or heavy (H). () The entire striatal complex was collected from all rats and D2R levels in each group measured by western blotting. The membrane-associated D2R band was resolved at 70 kDa, and the protein-loading control is displayed below (β-actin, 43 kDa). Full-length immunoblots are shown in Supplementary Figure 12. () Relative amounts of D2R in the striatum of chow-only, restricted-access and extended-access rats were quantified by densitometry (F2,6 = 5.2, P < 0.05, main effect of access; *P < 0.05 and **P < 0.01 compared with chow-only-L group). * Figure 5: Lentivirus-mediated knockdown of striatal D2R expression. () Graphical representation of the striatal areas in which Lenti-D2Rsh was overexpressed. Green circles in the left striatal hemisphere represent the locations at which viral infusions were targeted. Green staining in the right striatal hemisphere is a representative immunochemistry staining for green fluorescent protein (GFP) from the brain of a Lenti-D2Rsh rat. () Representative immunoblot of the decreased D2R expression in the striatum of Lenti-D2Rsh rats. Full-length immunoblots are shown in Supplementary Figure 13. () Relative amounts of D2R in the striatum of Lenti-control and Lenti-D2Rsh rats, quantified by densitometry (*P < 0.05 compared with the Lenti-control group, post hoc test). () Infection of glial cells in the striatum by the Lenti-D2Rsh vector was not detected. Green staining is GFP from virus; red is the astrocyte marker glial fibrillary acidic protein (GFAP); cell nuclei are highlighted by DAPI staining in blue. White arrows indicate a localized area of gl! iosis found only at the site of virus injection in the striatum and not in the surrounding tissues into which the virus has diffused. Even in this area, none of the astrocytes are GFP-positive. The yellow arrows in the magnified image highlight the typical GFP-negative astrocytes that were detected. () High levels of neuronal infection in the striatum by the Lenti-D2Rsh vector. Green staining is GFP from virus; red is the neuronal nuclear marker NeuN; cell nuclei are highlighted by DAPI staining in blue. The yellow arrows in the magnified image highlight GFP-positive and NeuN-positive neurons in the striatum. () A higher-magnification image of a virally infected (GFP-positive) neuron in the striatum of Lenti-D2Rsh rats that shows the typical morphological features of medium spiny neurons. * Figure 6: Knockdown of striatal D2R increases vulnerability to reward dysfunction in rats with extended access to a cafeteria diet. () Mean (± s.e.m.) percentage change from baseline reward thresholds in Lenti-control and Lenti-D2Rsh rats that had extended access to the cafeteria diet for 14 consecutive days (virus: F1,156 = 5.9, P < 0.05; time: F13,156 = 2.2, P < 0.05; virus × time interaction: F13,156 = 2.2, P < 0.05; #P < 0.05, interaction effect). () Mean (± s.e.m.) percentage change from baseline reward thresholds in Lenti-control and Lenti-D2Rsh rats that had chow-only access. () Mean (± s.e.m.) caloric intake of rats during 14 d of chow only or extended access (access: F2,28 = 135.6, ***P < 0.0001). () Mean (± s.e.m.) weight gain during 14 d of chow only or extended access (access: F2,28 = 96.4, P < 0.0001; ***P < 0.001, main effect of access). * Figure 7: Compulsive-like responding for palatable food. () Mean (± s.e.m.) palatable diet consumption in unpunished rats during the 30-min baseline sessions and on the test day when rats were exposed to a neutral conditioned stimulus that was not previously paired with noxious foot shock (access: F2,20 = 5.2, P < 0.05; #P < 0.05 compared with chow-only rats). () Mean (± s.e.m.) palatable diet consumption in punished rats during the 30-min baseline sessions and on the test day when rats were exposed to a conditioned stimulus that was previously paired with noxious foot shock (access: F2,21 = 3.9, P < 0.05; cue: F1,21 = 8.6, P < 0.01; access × cue interaction: F2,21 = 4.7, P < 0.05; *P < 0.05 compared with intake during the baseline session, #P < 0.05 compared with chow-only rats). () Mean (± s.e.m.) palatable diet consumption during the 30-min baseline sessions and on the test day in Lenti-control and Lenti-D2Rsh rats that previously had chow-only or extended access to a cafeteria diet (cue: F1,26 = 29.7, P < 0.0001; *P < 0.05! , **P < 0.01 compared with intake during the baseline sessions, post hoc test). () Mean (± s.e.m.) chow consumption during the 30-min baseline sessions and on the test day in Lenti-control and Lenti-D2Rsh rats that previously had chow only or extended access to a cafeteria diet (cue: F1,26 = 44.9, P < 0.0001; *P < 0.05, **P < 0.01 compared with intake during the baseline sessions, post hoc test). Author information * Abstract * Author information * Supplementary information Affiliations * Laboratory of Behavioral and Molecular Neuroscience, Department of Molecular Therapeutics, The Scripps Research Institute-Scripps Florida, Jupiter, Florida, USA. * Paul M Johnson & * Paul J Kenny Contributions P.M.J. conducted all experiments. P.M.J. and P.J.K. designed the experiments, analyzed the data and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Paul J Kenny (pjkenny@scripps.edu) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (3.2M) Supplementary Figures 1–13 Additional data - Contrast gain control and cortical TrkB signaling shape visual acuity
- Nature Neuroscience 13(5):642-648 (2010)
Nature Neuroscience | Article Contrast gain control and cortical TrkB signaling shape visual acuity * J Alexander Heimel1 Search for this author in: * NPG journals * PubMed * Google Scholar * M Hadi Saiepour1 Search for this author in: * NPG journals * PubMed * Google Scholar * Sridhara Chakravarthy1 Search for this author in: * NPG journals * PubMed * Google Scholar * Josephine M Hermans1 Search for this author in: * NPG journals * PubMed * Google Scholar * Christiaan N Levelt1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume:13,Pages:642–648Year published:(2010)DOI:doi:10.1038/nn.2534Received14 December 2009Accepted22 March 2010Published online18 April 2010 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 During development and aging and in amblyopia, visual acuity is far below the limitations set by the retina. Expression of brain-derived neurotrophic factor (BDNF) in the visual cortex is reduced in these situations. We asked whether neurotrophic tyrosine kinase receptor, type 2 (TrkB) regulates cortical visual acuity in adult mice. We found that genetically interfering with TrkB/BDNF signaling in pyramidal cells in the mature visual cortex reduced synaptic strength and resulted in a loss of neural responses to high spatial-frequency stimuli. Responses to low spatial-frequency stimuli were unaffected. This selective loss was not accompanied by a change in receptive field sizes or plasticity, but apparent contrast was reduced. Our results indicate that a dependence on spatial frequency in the Heeger normalization model explains this selective effect of contrast reduction on high-resolution vision and suggest that it involves contrast gain control operating in the visual corte! x. View full text Figures at a glance * Figure 1: Expression of TrkB.T1-EGFP in pyramidal neurons of the adult visual cortex causes reduced synaptic strength. () Sagittal slice showing expression at postnatal day 68 in cortex and hippocampus. Fluorescence in the thalamus was limited to axons coming from the cortex and hippocampus. () Coronal cross-section of occipital cortex. TrkB.T1-EGFP was mainly expressed in extragranular layers. () Experimental setup for and . We made whole-cell patch-clamp recording in layer 2/3 coupled to electrical stimulation displaced 200 μm in layer 2/3. () Example average response to 50-μA electrical stimulation. () Average layer 2/3 to 2/3 input-output (IO) curves showed a reduced response in TrkB.T1-EGFP mice (T1). () Experimental setup for ,. LFP recording electrode in layer 2/3 of visual cortical slice coupled to electrical stimulation in layer 4. () Example average response to 180-μA stimulation in a wild-type (WT) and a TrkB.T1-EGFP mouse. () Average layer 4 to 2/3 input-output curves showed a reduced response in TrkB.T1-EGFP mice. Error bars denote s.e.m. (*P < 0.05, **P < 0.01, t test). * Figure 2: Acuity is reduced in V1 of TrkB.T1-EGFP mice. (,) Example intrinsic signal responses to a range of spatial frequencies in the visual cortex of a wild-type mouse. Transcranial images of changes in light reflectance are shown in . Scale bar represents 1 mm. Mean reductions in light reflectance in a selected region of the binocular cortex () are shown in . () Average high spatial-frequency cut-offs were reduced in TrkB.T1-EGFP mice compared with controls. () Average intrinsic signal responses for wild-type and TrkB.T1-EGFP mice. The response to the lowest spatial frequency was not substantially reduced. Error bars denote s.e.m. (*P < 0.05, **P < 0.01, t test). * Figure 3: Apparent contrast is reduced in TrkB.T1-EGFP mice. () Example single-unit contrast response curve. C50 was interpolated from a Naka-Rushton fit. () C50 was higher in TrkB.T1-EGFP mice. () Peak latency to the high-contrast gratings was longer in TrkB.T1-EGFP mice. () Average population response curves from single-unit recordings, normalized to wild-type response and fitted with Naka-Rushton curves. () Best fit to TrkB.T1-EGFP data with the wild-type curve (solid line) was achieved by reducing contrast (dashed line), rather than by reducing response strength (dotted line). Error bars denote s.e.m. (*P < 0.05, **P < 0.01, t test). * Figure 4: Apparent contrast reduction in TrkB.T1-EGFP mice explains acuity loss. () Intrinsic signal recordings confirmed the reduction of contrast in TrkB.T1-EGFP mice. Contrast tuning curve of TrkB.T1-EGFP mice was best fitted by scaling the contrast of the wild-type tuning curve rather than the response (least squared error: contrast scaling, 0.003; response scaling, 0.033). (–) Contrast reduction decreases acuity and mimics TrkB.T1-EGFP phenotype. Cortical activity was lower in wild-type mice at 60% compared with 90% contrast, as determined by intrinsic signal measurements (). The loss of response to high spatial-frequency stimuli and the absence of a response reduction at low spatial frequency induced by contrast reduction from 90% to 60% matched the TrkB.T1-EGFP acuity phenotype (,). Lines are linear-threshold fits to wild-type data. Error bars denote s.e.m. (*P < 0.05; **P < 0.01, t test). * Figure 5: Normalization model explains the differential effect of reduced contrast on responses to high and low spatial-frequency stimuli. () V1 normalization model. Neuron i performs an operation on the stimulus that is linear in contrast. This operation is followed by a power law half-rectification. This unnormalized response, Ai, is normalized by division with an activity pool N, which only depends on stimulus contrast and spatial frequency, to give the cell's firing rate Ri. The firing rates of all cells determine the population response P. () Hypothetical examples of unnormalized responses of a cell preferring low spatial frequencies (SFs, top) and one preferring high spatial frequencies (bottom). A reduction in contrast strongly reduces the responses, without altering the tuning preferences or their relative response strengths. () High-contrast, low spatial-frequency stimuli evoke large population response and thus large normalization. (,) Firing rates of hypothetical cells of after normalization by and population response showing that the responses at high spatial frequencies are much more dependent on c! ontrast. * Figure 6: Imaging confirms the model's prediction that population contrast tuning curves at different spatial frequencies are identical when contrast is rescaled. () Contrast responses at 0.1, 0.2 and 0.4 cpd in a single mouse were well fitted by scaling the contrast of the 0.05 cpd contrast tuning curve. (–) Contrast scaling of average 0.05 cpd contrast tuning curve fitted contrast response curves at 0.1, 0.2 and 0.4 cpd better than scaling the response. () Optimally scaled contrast for each spatial frequency of to match the 0.05 cpd response curve. () Optimally scaled response for each spatial frequency of to match the 0.05 cpd response curve. Error bars denote s.e.m. * Figure 7: Model predicts population spatial frequency and contrast relationships in wild-type and TrkB.T1-EGFP mice. (,) From measurements of the contrast tuning at 0.1 cpd () and the spatial frequency tuning at 90% contrast (), the model predicts the response at any combination of contrast and spatial frequency by scaling the contrast of the measured contrast tuning curve in to match the 90% contrast response of the requested spatial frequency in . Horizontal lines show a construction of the predicted response to a 60% contrast grating of 0.2 cpd; the contrast of the contrast tuning curve of 0.1 cpd is scaled such that at 90% the response is identical to the response measured for the high-contrast spatial-frequency curve in . After scaling, the contrast tuning curve response at 60% gives the desired value. (,) The model's predicted spatial-frequency tuning curve at 60% contrast closely matches the 60% contrast measurements in wild-type mice and the 90% contrast measurements in TrkB.T1-EGFP mice. Error bars denote s.e.m. Author information * Abstract * Author information * Supplementary information Affiliations * Molecular Visual Plasticity Group, Netherlands Institute for Neuroscience, Royal Netherlands Academy of Arts and Sciences, Amsterdam, The Netherlands. * J Alexander Heimel, * M Hadi Saiepour, * Sridhara Chakravarthy, * Josephine M Hermans & * Christiaan N Levelt Contributions C.N.L. generated the TrkB.T1-EGFP mice. J.A.H. and C.N.L. devised the experiments and wrote the manuscript. J.A.H. performed the in vivo experiments and implemented the normalization model. M.H.S. carried out the slice experiments. S.C. analyzed parvalbumin puncta. J.M.H. assisted with imaging. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * J Alexander Heimel (heimel@nin.knaw.nl) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (5M) Supplementary Figures 1–9 and Supplementary Equations Additional data - Prefrontal and striatal dopaminergic genes predict individual differences in exploration and exploitation
- Nature Neuroscience 13(5):649 (2010)
Nature Neuroscience | Corrigendum Prefrontal and striatal dopaminergic genes predict individual differences in exploration and exploitation * Michael J Frank Search for this author in: * NPG journals * PubMed * Google Scholar * Bradley B Doll Search for this author in: * NPG journals * PubMed * Google Scholar * Jen Oas-Terpstra Search for this author in: * NPG journals * PubMed * Google Scholar * Francisco Moreno Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature NeuroscienceVolume:13,Page:649Year published:(2010)DOI:doi:10.1038/nn0510-649a Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Nat. Neurosci.12, 1062–1068 (2009); published online 20 July 2009; corrected after print 9 September 2009 In the version of this article initially published, the last sentence of the second paragraph in the right column on page 1065 read "that is, the following term was added to the RT prediction: ρ[σslow (s,t) – σfast (s,t)], where ρ is a free parameter." A variable in the equation contained in this sentence was incorrect. The sentence should read "that is, the following term was added to the RT prediction: ρ[μslow (s,t) – μfast (s,t)], where ρ is a free parameter." The error has been corrected in the HTML and PDF versions of the article. Additional data - Ca2+ and calmodulin initiate all forms of endocytosis during depolarization at a nerve terminal
- Nature Neuroscience 13(5):649 (2010)
Nature Neuroscience | Corrigendum Ca2+ and calmodulin initiate all forms of endocytosis during depolarization at a nerve terminal * Xin-Sheng Wu Search for this author in: * NPG journals * PubMed * Google Scholar * Benjamin D McNeil Search for this author in: * NPG journals * PubMed * Google Scholar * Jianhua Xu Search for this author in: * NPG journals * PubMed * Google Scholar * Junmei Fan Search for this author in: * NPG journals * PubMed * Google Scholar * Lei Xue Search for this author in: * NPG journals * PubMed * Google Scholar * Ernestina Melicoff Search for this author in: * NPG journals * PubMed * Google Scholar * Roberto Adachi Search for this author in: * NPG journals * PubMed * Google Scholar * Li Bai Search for this author in: * NPG journals * PubMed * Google Scholar * Ling-Gang Wu Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature NeuroscienceVolume:13,Page:649Year published:(2010)DOI:doi:10.1038/nn0510-649b Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Nat. Neurosci.12, 1003–1010 (2009); published online 26 July 2009; corrected after print 3 September 2009 In the version of this article initially published, the units for the calcium concentration labels in Figure 2a,d are incorrect. The correct unit should be μM. The error has been corrected in the HTML and PDF versions of the article. Additional data - AP2γ regulates basal progenitor fate in a region- and layer-specific manner in the developing cortex
- Nature Neuroscience 13(5):649 (2010)
Nature Neuroscience | Corrigendum AP2γ regulates basal progenitor fate in a region- and layer-specific manner in the developing cortex * Luisa Pinto Search for this author in: * NPG journals * PubMed * Google Scholar * Daniela Drechsel Search for this author in: * NPG journals * PubMed * Google Scholar * Marie-Theres Schmid Search for this author in: * NPG journals * PubMed * Google Scholar * Jovica Ninkovic Search for this author in: * NPG journals * PubMed * Google Scholar * Martin Irmler Search for this author in: * NPG journals * PubMed * Google Scholar * Monika S Brill Search for this author in: * NPG journals * PubMed * Google Scholar * Laura Restani Search for this author in: * NPG journals * PubMed * Google Scholar * Laura Gianfranceschi Search for this author in: * NPG journals * PubMed * Google Scholar * Chiara Cerri Search for this author in: * NPG journals * PubMed * Google Scholar * Susanne N Weber Search for this author in: * NPG journals * PubMed * Google Scholar * Victor Tarabykin Search for this author in: * NPG journals * PubMed * Google Scholar * Kristin Baer Search for this author in: * NPG journals * PubMed * Google Scholar * François Guillemot Search for this author in: * NPG journals * PubMed * Google Scholar * Johannes Beckers Search for this author in: * NPG journals * PubMed * Google Scholar * Nada Zecevic Search for this author in: * NPG journals * PubMed * Google Scholar * Colette Dehay Search for this author in: * NPG journals * PubMed * Google Scholar * Matteo Caleo Search for this author in: * NPG journals * PubMed * Google Scholar * Hubert Schorle Search for this author in: * NPG journals * PubMed * Google Scholar * Magdalena Götz Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature NeuroscienceVolume:13,Page:649Year published:(2010)DOI:doi:10.1038/nn0510-649c Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Nat. Neurosci.12, 1229–1237 (2009); published online 13 September 2009; corrected after print 25 September 2009 In the version of this article initially published, one of the corresponding authors' email addresses was misspelled. It should be luisapinto@ecsaude.uminho.pt. In addition, errors occurred in some of the numbers listed in the last subsection of the Results section. Instead of "Notably, AP2γ−/− mice also showed alterations in cortical binocularity ( and ) and a tendency toward an increased latency of visual response (wild type = 109.95 ms, AP2γ−/− = 127.19 ms; ). [...] Indeed, monocular deprivation for 3 d caused a significant change in binocularity in adult AP2γ−/− (P = 0.027), but not wild-type (P = 0.365), mice ()," the affected sentences should read, "Notably, AP2γ−/− mice also showed alterations in cortical binocularity ( and ) and a tendency toward an increased latency of visual response (wild type = 110.0 ± 3.8 ms, AP2γ−/− = 127.2 ± 6.4 ms; t-test, P = 0.05; ). [...] Indeed, monocular deprivation for 3 d caused a significant change in ! binocularity in adult AP2γ−/− (P = 0.01), but not wild-type (P = 0.365), mice ()." The errors have been corrected in the HTML and PDF versions of the article. Additional data - Adult generation of glutamatergic olfactory bulb interneurons
- Nature Neuroscience 13(5):649 (2010)
Nature Neuroscience | Erratum Adult generation of glutamatergic olfactory bulb interneurons * Monika S Brill Search for this author in: * NPG journals * PubMed * Google Scholar * Jovica Ninkovic Search for this author in: * NPG journals * PubMed * Google Scholar * Eleanor Winpenny Search for this author in: * NPG journals * PubMed * Google Scholar * Rebecca D Hodge Search for this author in: * NPG journals * PubMed * Google Scholar * Ilknur Ozen Search for this author in: * NPG journals * PubMed * Google Scholar * Roderick Yang Search for this author in: * NPG journals * PubMed * Google Scholar * Alexandra Lepier Search for this author in: * NPG journals * PubMed * Google Scholar * Sergio Gascón Search for this author in: * NPG journals * PubMed * Google Scholar * Ferenc Erdelyi Search for this author in: * NPG journals * PubMed * Google Scholar * Gabor Szabo Search for this author in: * NPG journals * PubMed * Google Scholar * Carlos Parras Search for this author in: * NPG journals * PubMed * Google Scholar * Francois Guillemot Search for this author in: * NPG journals * PubMed * Google Scholar * Michael Frotscher Search for this author in: * NPG journals * PubMed * Google Scholar * Benedikt Berninger Search for this author in: * NPG journals * PubMed * Google Scholar * Robert F Hevner Search for this author in: * NPG journals * PubMed * Google Scholar * Olivier Raineteau Search for this author in: * NPG journals * PubMed * Google Scholar * Magdalena Götz Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature NeuroscienceVolume:13,Page:649Year published:(2010)DOI:doi:10.1038/nn0510-649d Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Nat. Neurosci.12, 1524–1533 (2009); published online 1 November 2009; corrected after print 11 December 2009 In the version of this article initially published, the email address of one of the corresponding authors was misspelled. It should be magdalena.goetz@helmholtz-muenchen.de. The error has been corrected in the HTML and PDF versions of the article. Additional data - Dynamic DNA methylation programs persistent adverse effects of early-life stress
- Nature Neuroscience 13(5):649 (2010)
Nature Neuroscience | Erratum Dynamic DNA methylation programs persistent adverse effects of early-life stress * Chris Murgatroyd Search for this author in: * NPG journals * PubMed * Google Scholar * Alexandre V Patchev Search for this author in: * NPG journals * PubMed * Google Scholar * Yonghe Wu Search for this author in: * NPG journals * PubMed * Google Scholar * Vincenzo Micale Search for this author in: * NPG journals * PubMed * Google Scholar * Yvonne Bockmühl Search for this author in: * NPG journals * PubMed * Google Scholar * Dieter Fischer Search for this author in: * NPG journals * PubMed * Google Scholar * Florian Holsboer Search for this author in: * NPG journals * PubMed * Google Scholar * Carsten T Wotjak Search for this author in: * NPG journals * PubMed * Google Scholar * Osborne F X Almeida Search for this author in: * NPG journals * PubMed * Google Scholar * Dietmar Spengler Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature NeuroscienceVolume:13,Page:649Year published:(2010)DOI:doi:10.1038/nn0510-649e Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Nat. Neurosci.12, 1559–1566 (2009); published online 8 November 2009; corrected after print 3 December 2009 In the version of this article initially published, on page 2, left column, the phrase "...and typically cluster in glucocorticoid-rich regions called CpG islands (CGIs)" should be "...and typically cluster in GC-rich regions called CpG islands (CGIs)". The error has been corrected in the HTML and PDF versions of the article. Additional data
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