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- Shortsighted education reform
- Nat Neurosci 14(1):1 (2011)
Nature Neuroscience | Editorial Shortsighted education reform Journal name:Nature NeuroscienceVolume: 14,Page:1Year published:(2011)DOI:doi:10.1038/nn0111-1Published online27 December 2010 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Neuroscience for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. The UK government decision to remove the cap on undergraduate student fees is likely to have long-lasting negative consequences for science research in the UK. View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Neuroscience for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - An RXR-γ Rx for white-matter damage
- Nat Neurosci 14(1):3-5 (2011)
Nature Neuroscience | News and Views An RXR-γ Rx for white-matter damage * Vittorio Gallo1 Contact Vittorio Gallo Search for this author in: * NPG journals * PubMed * Google Scholar * Li-Jin Chew1 Contact Li-Jin Chew Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Corresponding authorsJournal name:Nature NeuroscienceVolume: 14,Pages:3–5Year published:(2011)DOI:doi:10.1038/nn0111-3Published online27 December 2010 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Neuroscience for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. A new study finds that retinoid X receptor-γ promotes remyelination. Its function in oligodendrocyte progenitor cell maturation sheds light on nuclear receptor signaling in myelin development and paves the way toward therapeutic ligands for myelin repair. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Vittorio Gallo & Li-Jin Chew are at the Center for Neuroscience Research, Children's National Medical Center, Washington, DC, USA. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Vittorio Gallo or * Li-Jin Chew Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Neuroscience for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - Caspase activation without apoptosis: insight into Aβ initiation of neurodegeneration
- Nat Neurosci 14(1):5-6 (2011)
Nature Neuroscience | News and Views Caspase activation without apoptosis: insight into Aβ initiation of neurodegeneration * Bradley T Hyman1 Contact Bradley T Hyman Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature NeuroscienceVolume: 14,Pages:5–6Year published:(2011)DOI:doi:10.1038/nn0111-5Published online27 December 2010 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Neuroscience for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. New work in a mouse model of Alzheimer's disease suggests that early-stage synaptic and memory impairments are caused by abnormal activation of a protease and a phosphatase, both of which could be targeted by inhibitory drugs. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * The author is in the Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts, USA. * Bradley T Hyman Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Bradley T Hyman Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Neuroscience for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - Orphan nuclear receptors control neuronal remodeling during fly metamorphosis
- Nat Neurosci 14(1):6-7 (2011)
Nature Neuroscience | News and Views Orphan nuclear receptors control neuronal remodeling during fly metamorphosis * Takeshi Awasaki1 Contact Takeshi Awasaki Search for this author in: * NPG journals * PubMed * Google Scholar * Tzumin Lee1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Corresponding authorJournal name:Nature NeuroscienceVolume: 14,Pages:6–7Year published:(2011)DOI:doi:10.1038/nn0111-6Published online27 December 2010 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Neuroscience for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Pruning of excess branches is essential for the maturation of developing neuronal circuits. Cross-talk between TGF-β signaling and two antagonistic orphan nuclear receptors governs the pruning of larval γ neurons in the Drosophila pupa. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * The authors are at the Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, Virginia, USA. * Takeshi Awasaki & * Tzumin Lee Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Takeshi Awasaki Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Neuroscience for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - Endocannabinoid signaling in the brain: biosynthetic mechanisms in the limelight
- Nat Neurosci 14(1):9-15 (2011)
Nature Neuroscience | Perspective Endocannabinoid signaling in the brain: biosynthetic mechanisms in the limelight * Vincenzo Di Marzo1 Contact Vincenzo Di Marzo Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature NeuroscienceVolume: 14,Pages:9–15Year published:(2011)DOI:doi:10.1038/nn.2720Published online27 December 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 Studies of the endocannabinoid system in the CNS have been mostly focused on endocannabinoid receptors and inactivating mechanisms. Until recently, very little was known about the role of biosynthetic enzymes in endocannabinoid signaling. New data from the recent development of pharmacological and genetic tools for the study of these enzymes point to their fundamental role in determining where and when endocannabinoids function, and raise the possibility of new intriguing and previously unsuspected concepts in the general strategy of endocannabinoid signaling. However, even with these new tools, the cross-talk between anandamide and 2-arachidonoylglycerol biosynthesis makes it difficult to dissect one from the other, and data will need to be interpreted with this in mind. View full text Author information * Abstract * Author information * Supplementary information Affiliations * Endocannabinoid Research Group, Institute of Biomolecular Chemistry, CNR, Pozzuoli, Italy. * Vincenzo Di Marzo Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Vincenzo Di Marzo Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (200K) Supplementary Figure 1 Additional data - P2X receptor channels show threefold symmetry in ionic charge selectivity and unitary conductance
- Nat Neurosci 14(1):17-18 (2011)
Nature Neuroscience | Brief Communication P2X receptor channels show threefold symmetry in ionic charge selectivity and unitary conductance * Liam E Browne1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Lishuang Cao1, 2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Helen E Broomhead1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Laricia Bragg1 Search for this author in: * NPG journals * PubMed * Google Scholar * William J Wilkinson1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * R Alan North1 Contact R Alan North Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 14,Pages:17–18Year published:(2011)DOI:doi:10.1038/nn.2705Received10 August 2010Accepted20 October 2010Published online19 December 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg In the closed structure of the P2X cation channel, three α-helical transmembrane domains cross the membrane obliquely. In rat P2X2 receptors, these intersect at Thr339. Replacing Thr339 by lysine in one, two or three subunits progressively increased chloride permeability and reduced unitary conductance. This implies that the closed-open transition involves a symmetrical separation of the three subunits and that Thr339 from each subunit contributes symmetrically to the open channel permeation pathway. View full text Figures at a glance * Figure 1: Lysine at 339 progressively increases chloride permeability and outward current. () Current-voltage plots for ATP-induced currents in ten cells expressing concatenated trimeric P2X2 receptors with one, two or three lysines at position 339. Currents are normalized and scale bars apply to all panels (actual currents at −150 mV: wild type (WT), 2,000 pA; T339K, 700 pA; KTT, 3,100 pA; TKT, 2,900 pA; TTK, 2,700 pA; KKT, 230 pA; KTK, 800 pA; TKK, 1,800 pA; TTT, 3,300 pA; KKK, 1,900 pA). ATP concentrations were 10 or 30 μM (close to EC50). () Reversal potential for ATP-evoked currents became dependent on the chloride concentration as lysines were introduced at position 339. Data are mean ± s.e.m. () PCl/PNa (determined from ) increased according to the number of lysines at position 339 and outward rectification increased proportionately (Pearson's r = 0.97). * Figure 2: Lysine at 339 reduces single channel currents. () ATP (0.3 μM or 1 μM) activated single channels in outside-out patches from cells expressing cDNAs encoding wild-type P2X2 (top), P2X2[T339K] (middle) or both (bottom) subunits. The bottom trace shows the intermediate current amplitudes: zero current/closed channel peak is truncated and the arrowhead indicates the position of the third level (<1 pA). Holding potential = −120 mV. () Outside-out recordings of single-channel activity in patches from cells expressing concatenated cDNAs. The amino acid at position 339 in each subunit of the trimer is indicated above each trace. ATP concentrations were 1 to 10 μM. Recordings on the right are all-points histograms used to estimate unitary current amplitudes; zero level peaks are truncated. Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Liam E Browne & * Lishuang Cao Affiliations * Faculty of Medical and Human Sciences, and Faculty of Life Sciences, University of Manchester, Manchester, UK. * Liam E Browne, * Lishuang Cao, * Helen E Broomhead, * Laricia Bragg, * William J Wilkinson & * R Alan North * Present addresses: Pain Research Unit, Pfizer Global Research and Development, Sandwich Laboratories, Sandwich, Kent, UK (L.C.), Department of Biochemistry, University of Cambridge, Cambridge, UK (H.E.B.), School of Biosciences, Cardiff University, Cardiff, UK (W.J.W.). * Lishuang Cao, * Helen E Broomhead & * William J Wilkinson Contributions R.A.N., L.E.B. and L.C. conceived and designed the experiments and analyzed the data. H.E.B., L.B. and W.J.W. generated the constructs and carried out western blotting. L.E.B. and L.C. performed the single-channel and whole-cell electrophysiology. L.E.B. constructed molecular models. R.A.N. wrote the paper with contributions from the other authors. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * R Alan North Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (1012K) Supplementary Figures 1–3, Supplementary Table 1 and Supplementary Methods Additional data - Characterization of the proteome, diseases and evolution of the human postsynaptic density
- Nat Neurosci 14(1):19-21 (2011)
Nature Neuroscience | Brief Communication Characterization of the proteome, diseases and evolution of the human postsynaptic density * Àlex Bayés1, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Louie N van de Lagemaat1, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Mark O Collins2 Search for this author in: * NPG journals * PubMed * Google Scholar * Mike D R Croning1 Search for this author in: * NPG journals * PubMed * Google Scholar * Ian R Whittle3 Search for this author in: * NPG journals * PubMed * Google Scholar * Jyoti S Choudhary2 Search for this author in: * NPG journals * PubMed * Google Scholar * Seth G N Grant1 Contact Seth G N Grant Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 14,Pages:19–21Year published:(2011)DOI:doi:10.1038/nn.2719Received23 June 2010Accepted12 November 2010Published online19 December 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg We isolated the postsynaptic density from human neocortex (hPSD) and identified 1,461 proteins. hPSD mutations cause 133 neurological and psychiatric diseases and were enriched in cognitive, affective and motor phenotypes underpinned by sets of genes. Strong protein sequence conservation in mammalian lineages, particularly in hub proteins, indicates conserved function and organization in primate and rodent models. The hPSD is an important structure for nervous system disease and behavior. View full text Figures at a glance * Figure 1: hPSD diseases and enriched phenotypes. () Distribution of hPSD nervous system diseases in four ICD-10 chapters (left) with chapter VI expanded (right) to show further subclassifications. () Representative human and mouse phenotypes enriched in the hPSD. Categories (bold) of human and mouse phenotypes with numbers of genes (hPSD genes) are shown. Heat map comparing enrichment of these phenotypes in four gene sets relative to the enrichment of the hPSD (shown in red). All other gene sets showed lower enrichment (darker colors with black representing no enrichment). Astrocyte, human astrocyte transcriptome12; brain, whole mouse brain proteome13; genome, human genome; Hs, human; Mm, mouse; neuron, human cortical neuron transcriptome14. HPO and MPO phenotype IDs are shown in brackets after each named phenotype. * Figure 2: hPSD sequence conservation. () Cumulative frequency plot of dN/dS values for hPSD and non-PSD genes expressed in cortical neurons14, and human genome. hPSD neuronal genes are more constrained than non-PSD neuronal genes (P < 10−11). () Median mouse-human dN/dS shown for hPSD and subcellular structures expressed in cortical neurons14. *P < 0.05, ***P < 0.001. (See Supplementary Table 10 and Supplementary Table 12 for a comparison with proteomically derived organelles). () Box plots of dN/dS distribution in hPSD hub proteins (>15 interactions, n = 23), non-hubs (≤15 interactions, n = 725) and the tandem affinity purification of PSD-95 complex11. Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Àlex Bayés & * Louie N van de Lagemaat Affiliations * Genes to Cognition Programme, Wellcome Trust Sanger Institute, Genome Campus, Hinxton, Cambridgeshire, UK. * Àlex Bayés, * Louie N van de Lagemaat, * Mike D R Croning & * Seth G N Grant * Proteomic Mass Spectrometry, Wellcome Trust Sanger Institute, Hinxton, Cambridgeshire, UK. * Mark O Collins & * Jyoti S Choudhary * Division of Clinical Neuroscience, Edinburgh University, Edinburgh, UK. * Ian R Whittle Contributions I.R.W. provided brain samples. A.B., M.O.C. and J.S.C. performed proteomic analysis. A.B. performed OMIM, ICD-10 and evolutionary analyses. L.N.v.L. performed network and enrichment analyses. M.D.R.C. integrated data into G2Cdb. L.N.v.L., M.D.R.C., M.O.C., A.B. and S.G.N.G. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Seth G N Grant Supplementary information * Author information * Supplementary information Excel files * Supplementary Table 1 (28K) Information about Biological Samples. * Supplementary Table 2 (448K) Protein Identifications and Proteomic Data. * Supplementary Table 3 (20K) Summary of OMIM Diseases. * Supplementary Table 4 (144K) OMIM Diseases Identified Among Total hPSD Genes. * Supplementary Table 5 (80K) Human Neural Phenotype gene set enrichment analysis. * Supplementary Table 6 (328K) Brain datasets used in phenotype enrichment analysis. * Supplementary Table 7 (188K) Mammalian Neural Phenotype gene set enrichment Analysis. * Supplementary Table 8 (10M) Comparison of dN/dS between Genome and hPSD. * Supplementary Table 9 (740K) Mouse and Human Brain Datasets dN/dS Analysis. * Supplementary Table 10 (848K) dN/dS Values for genes expressed in human neurons classified by cellular component. * Supplementary Table 11 (136K) Analysis of dN/dS on Hub, non-Hub and TAP-PSD-95 proteins. * Supplementary Table 12 (3M) Mouse to Human dN/dS Values in hPSD and Other Organelle Proteomes. PDF files * Supplementary Text and Figures (744K) Supplementary Figures 1–4 and Supplementary Methods Additional data - Transient neuronal inhibition reveals opposing roles of indirect and direct pathways in sensitization
- Nat Neurosci 14(1):22-24 (2011)
Nature Neuroscience | Brief Communication Transient neuronal inhibition reveals opposing roles of indirect and direct pathways in sensitization * Susan M Ferguson1 Search for this author in: * NPG journals * PubMed * Google Scholar * Daniel Eskenazi1 Search for this author in: * NPG journals * PubMed * Google Scholar * Masago Ishikawa2 Search for this author in: * NPG journals * PubMed * Google Scholar * Matthew J Wanat1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Paul E M Phillips1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Yan Dong2 Search for this author in: * NPG journals * PubMed * Google Scholar * Bryan L Roth4 Search for this author in: * NPG journals * PubMed * Google Scholar * John F Neumaier1, 3 Contact John F Neumaier Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 14,Pages:22–24Year published:(2011)DOI:doi:10.1038/nn.2703Received22 July 2010Accepted12 October 2010Published online05 December 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Dorsal striatum is important for the development of drug addiction; however, a precise understanding of the roles of striatopallidal (indirect) and striatonigral (direct) pathway neurons in regulating behaviors remains elusive. Using viral-mediated expression of an engineered G protein–coupled receptor (hM4D), we found that activation of hM4D receptors with clozapine-N-oxide (CNO) potently reduced striatal neuron excitability. When hM4D receptors were selectively expressed in either direct or indirect pathway neurons, CNO did not change acute locomotor responses to amphetamine, but did alter behavioral plasticity associated with repeated drug treatment. Specifically, transiently disrupting striatopallidal neuronal activity facilitated behavioral sensitization, whereas decreasing excitability of striatonigral neurons impaired its persistence. These findings suggest that acute drug effects can be parsed from the behavioral adaptations associated with repeated drug exposure a! nd highlight the utility of this approach for deconstructing neuronal pathway contributions to behavior. View full text Figures at a glance * Figure 1: Transient and targeted attenuation of striatal cell signaling. () pEnk-hM4D receptors were selectively expressed in striatopallidal MSNs. Green, hemagglutinin (HA); red, ENK (top) and substance P (SP, bottom); yellow, colocalization of neurons. Scale bars represent 10 μm. () pDyn-hM4D receptors were selectively expressed in striatonigral MSNs. Data are presented as in . () Representative voltage trace of CNO-induced hyperpolarization of an hM4D-expressing striatal neuron. (,) CNO decreased input resistance in hM4D-expressing neurons. *P < 0.05 hM4D before versus hM4D after CNO application (n = 4–5). C, CNO treatment; V, vehicle treatment. (,) Representative traces () and summarized data () showed that CNO decreased the number of evoked action potentials in hM4D-expressing neurons. **P < 0.01 hM4D versus hM4D/CNO. () Representative Fos immunohistochemistry sections (red) from pEnk-hM4D infused striatum of vehicle (VEH) and CNO-treated rats. Insets depict single-labeled Fos cells (red), hemagglutinin cells (green) and dual-labeled cell! s (yellow). Scale bars represent 50 μm and 10 μm (insets). () Activation of pEnk-hM4D receptors decreased the number of amphetamine-induced Fos cells (***P = 0.002, n = 5–6 per group). () Amphetamine-evoked c-Fos–positive cells were reduced in both hemagglutinin-positive (*P < 0.05) and hemagglutinin-negative (**P < 0.01) neurons in the pENK-hM4D experiment. () Representative Fos immunohistochemistry sections (red) from pDyn-hM4D infused striatum of vehicle (VEH) and CNO-treated rats. Data are presented as in . () Activation of pDyn-hM4D receptors decreased the number of amphetamine-induced Fos cells (*P < 0.05, n = 5–6 per group). () Amphetamine-evoked c-Fos–positive cells were reduced in hemagglutinin-positive neurons (*P < 0.05) in the pDyn-hM4D experiment. All data represent mean ± s.e.m. * Figure 2: Transiently reducing excitability of striatopallidal or striatonigral neurons had opposing effects on amphetamine sensitization. () Acute locomotor responses to amphetamine following activation of pEnk-hM4D receptors (n = 9–10 per group). ***P < 0.001 versus saline-treated groups. () Activation of pEnk-hM4D receptors during amphetamine treatment enhanced the development of locomotor sensitization. ***P < 0.001 versus session 1 of amphetamine-treated hM4D group, ###P < 0.001 versus amphetamine-treated GFP group. (,) Enhanced sensitization in the amphetamine-pretreated pEnk-hM4D group was maintained during the challenge test. ***P < 0.001 versus saline-pretreated group, ##P < 0.01 versus amphetamine-pretreated GFP group. () Acute locomotor responses to amphetamine following activation of pDyn-hM4D receptors (n = 8–10 per group). () Activation of pDyn-hM4D receptors during amphetamine treatment initially produced locomotor sensitization similar to that of pDyn-GFP controls. **P < 0.01 and *P < 0.05 versus session 1. (,) Sensitization in the amphetamine-pretreated pDyn-hM4D group was no longer evident! on the challenge test. ***P < 0.001 versus saline-pretreated groups, #P < 0.05 versus amphetamine-pretreated GFP group. Data represent mean ± s.e.m. A, amphetamine; S, saline. Squares represent hM4D groups, circles represent GFP groups. Light gray and black symbols represent rats that received amphetamine during the treatment phase, and white and dark gray symbols represent rats that received saline during the treatment phase. All experimental procedures were approved by the University of Washington Institutional Animal Care and Use Committee and were conducted in accordance with US National Institutes of Health guidelines. See Supplementary Methods for additional statistical information. Author information * Author information * Supplementary information Affiliations * Department of Psychiatry and Behavioral Sciences, University of Washington, Seattle, Washington, USA. * Susan M Ferguson, * Daniel Eskenazi, * Matthew J Wanat, * Paul E M Phillips & * John F Neumaier * Program in Neuroscience, Washington State University, Pullman, Washington, USA. * Masago Ishikawa & * Yan Dong * Department of Pharmacology, University of Washington, Seattle, Washington, USA. * Matthew J Wanat, * Paul E M Phillips & * John F Neumaier * Department of Pharmacology, University of North Carolina Medical School, Chapel Hill, North Carolina, USA. * Bryan L Roth Contributions S.M.F. and D.E. generated the viral vector constructs. S.M.F. did the behavioral and immunohistochemical experiments. M.I. and Y.D. did the electrophysiology experiments. M.J.W. and P.E.M.P. did the voltammetry experiments. B.L.R. provided the hM4D plasmids and assisted with experimental design. S.M.F. and J.F.N. designed the overall study and wrote the manuscript. All of the authors contributed to data interpretation and manuscript editing. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * John F Neumaier Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (10M) Supplementary Figures 1–7 and Methods Additional data - Perinatal photoperiod imprints the circadian clock
- Nat Neurosci 14(1):25-27 (2011)
Nature Neuroscience | Brief Communication Perinatal photoperiod imprints the circadian clock * Christopher M Ciarleglio1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * John C Axley2 Search for this author in: * NPG journals * PubMed * Google Scholar * Benjamin R Strauss2 Search for this author in: * NPG journals * PubMed * Google Scholar * Karen L Gamble2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Douglas G McMahon1, 2 Contact Douglas G McMahon Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 14,Pages:25–27Year published:(2011)DOI:doi:10.1038/nn.2699Received24 August 2010Accepted21 October 2010Published online05 December 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Using real-time gene expression imaging and behavioral analysis, we found that the perinatal photoperiod has lasting effects on the circadian rhythms expressed by clock neurons as well as on mouse behavior, and sets the responsiveness of the biological clock to subsequent changes in photoperiod. These developmental gene × environment interactions tune circadian clock responses to subsequent seasonal photoperiods and may contribute to the influence of season on neurobehavioral disorders in humans. View full text Figures at a glance * Figure 1: Persistent effects of perinatal seasonal photoperiod on SCN slice rhythms, on SCN neuronal rhythms, and on behavior. (,) Duration of the SCN peak () and neuronal peak () in Per1GFP expression, measured as the time from 50% maximum on the rising phase to 50% maximum on the falling phase of the molecular circadian rhythm. () Neuronal phase variance calculated from the peak times of individual SCN neurons using Rayleigh circular statistics. () Neuronal period calculated on the first full circadian cycle recorded ex vivo. () SCN period calculated from the first full circadian cycle recorded ex vivo. () Behavioral period calculated from the first 3 d in constant darkness. The black bar indicates the short-day developmental photoperiod, the gray bar indicates the equinox developmental photoperiod and the white bar indicates the long-day developmental photoperiod. *P < 0.05, two-way ANOVA, main effect for perinatal photoperiod. * Figure 2: Interactions of perinatal seasonal photoperiod with subsequent seasonal photoperiod. () Two-way ANOVA interaction plot for peak times of Per1GFP expression in SCN. The developmental photoperiod is shown on the x axis. The continuation photoperiod (PP) for short day (LD 8:16) is represented by black circles, equinox (LD 12:12) by gray inverted triangles and long day (LD 16:8) by open triangles. () Timing of SCN molecular rhythm peaks relative to dusk. The white background represents lights-on and the gray background represents lights-off. The photoperiodic procedure is represented as 'developmental:continuation' (L, long-day photoperiod; E, equinox photoperiod; S, short-day photoperiod). () Two-way ANOVA interaction plot for waveform duration from 50% rise to 50% fall of the first peak ex vivo. () Behavioral duration of activity per circadian cycle in the first 3 d of constant darkness. Error bars represent s.e.m. Significance is indicated by symbols (*, #, †) such that means sharing a symbol are not significantly different (P > 0.05), whereas means with di! fferent symbols are significantly different (P < 0.05). § indicates a significant difference between continuation photoperiods (P < 0.05); indicates a significant main effect of developmental photoperiod (P < 0.05). Author information * Author information * Supplementary information Affiliations * Neuroscience Graduate Program, Vanderbilt University, Nashville, Tennessee, USA. * Christopher M Ciarleglio & * Douglas G McMahon * Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee, USA. * Christopher M Ciarleglio, * John C Axley, * Benjamin R Strauss, * Karen L Gamble & * Douglas G McMahon * Present address: Department of Psychiatry and Behavioral Neurobiology, University of Alabama at Birmingham, Birmingham, Alabama, USA. * Karen L Gamble Contributions C.M.C. and D.G.M. designed the experiments. C.M.C., J.C.A. and B.R.S. performed the experiments and compiled the results. C.M.C. and K.L.G. performed statistical analyses. C.M.C. and D.G.M. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Douglas G McMahon Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (428K) Supplementary Figures 1 and 2, Supplementary Tables 1–3, Supplementary Methods and Supplementary Results Additional data - The surface area of human V1 predicts the subjective experience of object size
- Nat Neurosci 14(1):28-30 (2011)
Nature Neuroscience | Brief Communication The surface area of human V1 predicts the subjective experience of object size * D Samuel Schwarzkopf1, 2 Contact D Samuel Schwarzkopf Search for this author in: * NPG journals * PubMed * Google Scholar * Chen Song1 Search for this author in: * NPG journals * PubMed * Google Scholar * Geraint Rees1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 14,Pages:28–30Year published:(2011)DOI:doi:10.1038/nn.2706Received07 September 2010Accepted21 October 2010Published online05 December 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 surface area of human primary visual cortex (V1) varies substantially between individuals for unknown reasons. We found that this variability was strongly and negatively correlated with the magnitude of two common visual illusions, where two physically identical objects appear different in size as a result of their context. Because such illusions dissociate conscious perception from physical stimulation, our findings indicate that the surface area of V1 predicts variability in conscious experience. View full text Figures at a glance * Figure 1: Size illusions and variability in V1 surface area. () Ebbinghaus illusion13. The two central circles are physically identical, but appear to be different in size because of the presence of the surrounding circles. () A variant of the Ponzo illusion14. The two checkerboard circles are physically identical, but appear to be different in size as a result of the three-dimensional context. () The smaller the V1, the stronger the illusion. Representative maps showing cortical regions V1-V3 on a reconstructed three-dimensional mesh of the left hemisphere gray-white matter surface of three participants (denoted by initials MK, PS and AS). The surface area of the left V1 and Ebbinghaus illusion strength are given for each participant. Red indicates V1, green indicates V2 and blue indicates V3. * Figure 2: Surface area of V1 predicts illusion strength. (,) Scatter plots showing the inter-individual variability of the size of the visual regions V1-V3 plotted as a function of the psychophysically measured strength of the Ebbinghaus () and Ponzo () illusions (Supplementary Methods). Each data point represents a measurement from one participant. The solid black lines indicate the linear regression for each panel. Correlation coefficients and statistical significance are denoted above each panel. The numbers in brackets denote the bootstrapped 95% confidence intervals for the correlation coefficient. Author information * Author information * Supplementary information Affiliations * University College London Institute of Cognitive Neuroscience, University College London, London, UK. * D Samuel Schwarzkopf, * Chen Song & * Geraint Rees * Wellcome Trust Centre for Neuroimaging at University College London, London, UK. * D Samuel Schwarzkopf & * Geraint Rees Contributions D.S.S. conducted the functional magnetic resonance imaging experiment and analyzed the data. C.S. conducted the behavioral experiment. D.S.S., C.S. and G.R. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * D Samuel Schwarzkopf Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (744K) Supplementary Figures 1–3, Table 1 and Results Additional data - An evolving NGF-Hoxd1 signaling pathway mediates development of divergent neural circuits in vertebrates
- Nat Neurosci 14(1):31-36 (2011)
Nature Neuroscience | Article An evolving NGF-Hoxd1 signaling pathway mediates development of divergent neural circuits in vertebrates * Ting Guo1 Search for this author in: * NPG journals * PubMed * Google Scholar * Kenji Mandai1 Search for this author in: * NPG journals * PubMed * Google Scholar * Brian G Condie2 Search for this author in: * NPG journals * PubMed * Google Scholar * S Rasika Wickramasinghe1 Search for this author in: * NPG journals * PubMed * Google Scholar * Mario R Capecchi3 Search for this author in: * NPG journals * PubMed * Google Scholar * David D Ginty1 Contact David D Ginty Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 14,Pages:31–36Year published:(2011)DOI:doi:10.1038/nn.2710Received27 September 2010Accepted08 November 2010Published online12 December 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 Species are endowed with unique sensory capabilities that are encoded by divergent neural circuits. One potential explanation for how divergent circuits have evolved is that conserved extrinsic signals are differentially interpreted by developing neurons of different species to yield unique patterns of axonal connections. Although nerve growth factor (NGF) controls survival, maturation and axonal projections of nociceptors of different vertebrates, whether the NGF signal is differentially transduced in different species to yield unique features of nociceptor circuits is unclear. We identified a species-specific signaling module induced by NGF and mediated by a rapidly evolving Hox transcription factor, Hoxd1. NGF promoted robust expression of Hoxd1 in mice, but not chickens, both in vivo and in vitro. Mice lacking Hoxd1 displayed altered nociceptor circuitry that resembles that normally found in chicks. Conversely, ectopic expression of Hoxd1 in developing chick nociceptors ! promoted a pattern of axonal projections reminiscent of the mouse. Thus, conserved growth factors control divergent neuronal transcriptional events that mediate interspecies differences in neural circuits and the behaviors that they control. View full text Figures at a glance * Figure 1: NGF signaling induces Hoxd1 expression in mammalian, but not avian, embryonic nociceptors. () Diagram of the numbers of unique genes identified by the three microarray screens (at a loose cutoff of probability > 0.5 and fold change > 1.5). () The degree of mRNA level changes of the 14 genes identified by all three independent microarray screens, in log scale. () Application of NGF to cultured mouse DRG explants robustly induced transcription of Hoxd1 within 24 h. () RT-PCR reactions comparing levels of Hoxd1 mRNA in DRGs dissected from E14.5 Bax−/− and Ngf−/−; Bax−/− embryos. () In parallel cultures of mouse and chick DRG explants, NGF induced robust transcription of canonical NGF-dependent genes (CGRP and Ret) in both species, and induced Hoxd1 in mouse, but not in chick, DRGs, as assessed by quantitative PCR. *P < 0.05 by Student's t test (n = 3 independent batches of cultures for each condition). () In situ hybridization for Hoxd1 mRNA in sections of developing DRGs of E14.5 Bax−/− and Ngf−/−; Bax−/− embryos. Scale bar represents 50 μm.! () Comparison of Hoxd1 expression in DRGs and spinal cord of E15.5 mouse and stage 32 (E7.5) chick embryos. TrkA is a general marker for embryonic nociceptors. In chick DRGs, TrkA-positive nociceptors and TrkA-negative non-nociceptors segregate into distinct topographic domains. sc, spinal cord. Scale bar represents 50 μm and 100 μm in low- and high-magnification panels, respectively. () Double fluorescent labeling of Hoxd1 mRNA and TrkA protein in E15.5 mouse and stage 32 chick DRGs. Scale bar represents 20 μm. * Figure 2: Hoxd1 instructs development of mammal-specific features of nociceptor axonal connections in the skin. () Diagram showing the distinct patterns of nociceptor endings in the skin of mammals and birds. () Peripherin staining of mouse hairy skin from proximal limb. Top, transverse sections. Bottom, cross sections. Hair is autofluorescent. Scale bar represents 40 μm and 50 μm in upper and lower panels, respectively. () A marked reduction in the number of nociceptor free nerve endings that penetrate the epidermis of hindlimb footpad glabrous skin of Hoxd1−/− mice was detected by CGRP staining, which labels peptidergic nociceptors. Arrows indicate epidermal nociceptor endings. Scale bar represents 40 μm. () Double fluorescent labeling of Mrgprb4 mRNA and CGRP protein in L3 DRGs of wild-type and Hoxd1−/− mice. () Quantification of the number (± s.e.m.) of transverse lanceolate endings per mm2 skin area in serial sections of hindlimb back thigh hairy skin. *P < 0.001 by Student's t test. () Percentage (± s.e.m.) of Mrgprb4-positive neurons in wild-type and Hoxd1−/− m! ice that coexpressed CGRP or TrkA. *P < 0.001 by Student's t test. () Quantification of the percentage (± s.e.m.) of lumbar DRG neurons that expressed Mrgprb4. *P < 0.001 by Student's t test. () Quantification of the average (± s.e.m.) number of CGRP-positive free nerve endings crossing the dermal-epidermal boundary per unit length (300 μm) of hindlimb footpad glabrous skin. **P < 0.005 by Student's t test. * Figure 3: Hoxd1 controls a species-specific pattern of nociceptor axonal connectivity in the spinal cord. (,) CGRP staining of the dorsal spinal cord of postnatal day 0 (P0) mice and stage 39 (E13) chick. () Deep nociceptor central projections were abnormally increased in the spinal cord of Hoxd1−/− compared with wild-type mice, as seen by staining for CGRP, a peptidergic nociceptor marker. More numerous deep projections were found at all axial levels, including cervical (Cer) and thoracic (Th) segments. V, lamina V; X, lamina X. Arrows point to aberrant deep projections extending into a region near the central canal (lamina X). Some CGRP-positive axons normally reached lamina V similarly in wild-type and Hoxd1−/− mice mostly from ventral projections emanating from the dorsal funiculus. () CGRP staining of nociceptor central projections in stage 39 (E13) chick spinal cord at lower cervical and thoracic levels. Arrows indicate prominent horizontally projecting deep nociceptor axon bundles in the spinal cord of the chick that resemble the abnormally increased fibers in Hox! d1−/− mice. Arrowhead indicates axon bundles that first projected ventrally and then turned medially. Scale bars represent 50 μm (,). () Quantification of , comparing the fraction of 20-μm sections of the spinal cord at different axial levels that contains bundles of deep nociceptor axons in wild-type (n = 5 animals) and Hoxd1−/− (n = 5 animals) mice. Numbers denote the numbers of sections examined. () Quantification of , comparing the average (±s.e.m.) numbers of individual deep CGRP-positive fibers detected per section at different axial levels *P < 0.05 by Student's t test (n = 5 animals in each group). * Figure 4: NGF signaling modulates central axonal projections of mammalian nociceptors in the spinal cord. () Aberrant nociceptor axons, labeled with antibody to peripherin, projected horizontally into deep regions of the spinal cord in Ngf−/−; Bax−/− mice. Bottom, higher magnifications of the images shown in the top panels. Dashed boxes indicate excessive deep nociceptor axons. cc, central canal. Inset, peripherin was expressed at high levels in nociceptors19, 38. Note that in Ngf−/−; Bax−/− mice, in addition to excessive horizontal projections, there was also an abnormally large number of aberrant deep nociceptor axons that projected ventrally from the medial edge of the dorsal horn (arrowhead), some of which extended beyond the central canal. Scale bar represents 60 μm and 30 μm in low- and high-magnification panels, respectively. () Diagram representing nociceptor central projection defects of Ngf−/−; Bax−/− mice in comparison with Bax−/− controls. (,) Quantification of . Fluorescent intensities were measured from areas represented by dashed rect! angular boxes in (). Average (±s.e.m.) fluorescent intensities of peripherin-positive axons in deep ventral and horizontal projections are shown. *P ≤ 0.001 by Student's t test (n ≥ 5 animals in each group). () Fraction (%) of level-matched lumbar spinal cord sections that showed peripherin fluorescent intensity in horizontal projections. * Figure 5: Ectopic expression of Hoxd1 in chick nociceptors impairs their axonal ingrowth into the lateral spinal cord. () DNA constructs used for in ovo electroporation. () Sox10 enhancer–driven in ovo electroporation directs ectopic gene expression in developing chicken DRGs, but not in the spinal cord. Left, a whole-mount lateral view of GFP fluorescence in stage 26 chick embryos 3 d after electroporation. Right, transverse sections of embryos processed as in the left panel, stained with antibody to GFP. sp, spinal cord. () The pattern of central projections of chick nociceptors was altered when Hoxd1 was expressed in the chick DRG. Top, spinal cord section labeled with antibody to TrkA at stage 35 (E9). Bottom, high-magnification views of the boxed areas of the same spinal cord section. The left side is the control; DRGs of the right side of the embryo were electroporated with Hoxd1. Scale bar represents 50 μm and 25 μm in low- and high-magnification panels, respectively. () Quantification of . The ratio between spinal cord areas occupied by TrkA-positive fibers in the electroporated ! side versus the control side is shown. *P = 0.0001 by Student's t test (n > 4 embryos for each group; sections are 25 μm thick and are sampled at least 200 μm apart). cHoxd1, chicken Hoxd1 gene; cΔ, a chicken Hoxd1 gene construct lacking the homeobox motif; mΔ, a mouse Hoxd1 construct lacking the homeobox motif. () Diagram of the different patterns of spinal cord innervation by mammalian and avian nociceptors. * Figure 6: Behavioral responses of Hoxd1−/− mice to somatosensory stimuli. () Hoxd1−/− mice showed a defect in their avoidance response to extreme cold. The paw licking or flinching response latency following exposure of mice to a 0 °C cold plate was significantly increased in Hoxd1−/− mice compared with their wild-type littermate controls (n = 14 for wild type, 13 for Hoxd1−/−). ***P < 0.001 by Student's t test. (–) Hoxd1−/− mice responded normally to acute noxious thermal stimuli. Response latencies in the Hargreaves (n = 12 for wild type, 11 for Hoxd1−/−, ), hot plate (50 °C, n = 9 per genotype, ) and tail-immersion (50 °C, n = 9 per genotype, ) tests did not differ between wild-type and Hoxd1−/− mice. () The paw withdrawal threshold of Hoxd1−/− mice to punctate mechanical stimuli (von Frey test) was comparable to that of wild-type mice (n = 12 per genotype). () Hoxd1−/− mice showed prolonged thermal hyperalgesia 24 h after intraplantar injection of 1% carrageenan as compared with wild-type mice (10 μl, n = ! 9 per genotype). Pre, pre-injection. Data are presented as mean (±s.e.m.). *P < 0.01, two-way ANOVA. Author information * Abstract * Author information * Supplementary information Affiliations * Solomon H. Snyder Department of Neuroscience, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. * Ting Guo, * Kenji Mandai, * S Rasika Wickramasinghe & * David D Ginty * Department of Genetics, University of Georgia, Athens, Georgia, USA. * Brian G Condie * Department of Human Genetics, Howard Hughes Medical Institute, University of Utah School of Medicine, Salt Lake City, Utah, USA. * Mario R Capecchi Contributions K.M. and D.D.G. initiated microarray screens for NGF-dependent genes. B.G.C. and M.R.C. generated the Hoxd1-null mice. T.G., K.M. and S.R.W. performed functional characterization of Hoxd1. T.G., K.M. and D.D.G. analyzed the data. T.G. and D.D.G. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * David D Ginty Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (10M) Supplementary Figures 1–8 and Supplementary Tables 1–3 Additional data - ftz-f1 and Hr39 opposing roles on EcR expression during Drosophila mushroom body neuron remodeling
- Nat Neurosci 14(1):37-44 (2011)
Nature Neuroscience | Article ftz-f1 and Hr39 opposing roles on EcR expression during Drosophila mushroom body neuron remodeling * Ana Boulanger1, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Christelle Clouet-Redt1, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Morgane Farge1 Search for this author in: * NPG journals * PubMed * Google Scholar * Adrien Flandre1 Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas Guignard1 Search for this author in: * NPG journals * PubMed * Google Scholar * Céline Fernando2, 3, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * François Juge2, 3, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Jean-Maurice Dura1 Contact Jean-Maurice Dura Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 14,Pages:37–44Year published:(2011)DOI:doi:10.1038/nn.2700Received03 August 2010Accepted20 October 2010Published online05 December 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 Developmental axon pruning is a general mechanism that is required for maturation of neural circuits. During Drosophila metamorphosis, the larval-specific dendrites and axons of early γ neurons of the mushroom bodies are pruned and replaced by adult-specific processes. We found that the nuclear receptor ftz-f1 is required for this pruning, activates expression of the steroid hormone receptor EcR-B1, whose activity is essential for γ remodeling, and represses expression of Hr39, an ftz-f1 homologous gene. If inappropriately expressed in the γ neurons, HR39 inhibits normal pruning, probably by competing with endogenous FTZ-F1, which results in decreased EcR-B1 expression. EcR-B1 was previously identified as a target of the TGFβ signaling pathway. We found that the ftz-f1 and Hr39 pathway apparently acts independently of TGFβ signaling, suggesting that EcR-B1 is the target of two parallel molecular pathways that act during γ neuron remodeling. View full text Figures at a glance * Figure 1: βFTZ-F1 is required for γ neuron pruning. The expression of GAL4-201Y–driven GFP (green) and FASII (red) is shown in adult γ neurons. (,) Mature axons of wild-type MARCM clones. A wild-type neuroblast clone is shown in in which GFP was expressed in three axon bundles: the large dense lobe of adult γ neurons (arrow) and the weak α and β core bundles (arrowheads). A wild-type single-cell clone is shown in . Arrowhead points to the cell body. Note the presence of only a single medial branch. () Forced expression of βFTZ-F1 did not affect γ neuron pruning. (,) Pruning deficits in ftz-f1−/− mutant MARCM clones. The absence of FTZ-F1 blocked γ neuron pruning (). Arrows point to the larval-type γ neurons surrounding the dorsal and medial αβ lobes defined by FASII labeling. The asterisk indicates the area in which remodeled γ neuron axons should be located. Two-cell clones from ftz-f1−/− mutant flies are shown in . Arrowheads label the cell bodies. Note the presence of two dorsal (arrow) and medial bran! ches. () Forced expression of βFTZ-F1 in γ neurons completely rescued the ftz-f1−/− pruning mutant phenotype. Scale bar represents 20 μm. All of these pictures are composite confocal images except for and , which are deconvoluted z stacks. Genotypes are listed in Supplementary List of Fly Strains. * Figure 2: HR39 ectopic expression blocks γ neuron remodeling. (–) Wild-type adult brain, wild-type neuroblast and wild-type single-cell clone, respectively, labeled with GFP under the control of GAL4–201Y in which all the γ neurons have been remodeled. (–) Lack of larval pruning, in most of the γ neurons, caused by ectopic expression of HR39, respectively, in a brain, a neuroblast and a double two-cell clone. Arrows label unpruned γ neurons. An asterisk labels remodeled γ neurons. Unpruned γ neurons showed higher GFP intensity than the remodeled one, likely as a result of GFP accumulation in these neurons because of the persistence of these axons all along development. Note the presence of two unpruned γ axons and two apparently pruned γ axons in . Scale bar represents 20 μm. Genotypes are listed in Supplementary List of Fly Strains. () Molecular map of the Hr39 locus with the intron/exon structure of the two main categories of mRNAs. The positions of the P element insertions in Hr39C13 and Hr39GS9939 are indicated by a t! riangle. The Hr39C105 remaining regions after imprecise excision of the P[GSV6] element are indicated by lines. () Expression of HR39 detected by western blot of adult heads. Two lanes of control (+/+) and two lanes of mutant (Hr39C105/Df(2L)Exel6048) samples were loaded. The two bands at 87 and 74 kDa, corresponding to the long and the short HR39 proteins, respectively, were absent in mutant samples. Uncropped full-length blots are presented in Supplementary Figure 16. * Figure 3: Forced expression of ECR-B1 rescues ftz-f1−/− and HR39 overexpression γ neuronal remodeling defects. (,) Pruning/remodeling defects observed in ftz-f1−/− mutant neuroblast clones () were rescued by forced ECR-B1 expression (). (,) HR39 overexpression phenotype () was also rescued by ECR-B1 (). Green represents GAL4-201Y–driven GFP and red indicates antibody to FASII. All panels are composite confocal images. Scale bar represents 20 μm. Genotypes: () hs-FLP/X; UAS-mCD8GFP GAL4-201Y/+; ftz-f1ex7FRT2A/tubP-GAL80 FRT2A, () hs-FLP/X; UAS-mCD8GFP GAL4-201Y/+; ftz-f1ex7FRT2A UAS–EcR-B1/tubP-GAL80 FRT2A, () UAS–Hr39 /UAS–mCD8GFP GAL4–201Y, () UAS-Hr39/UAS-mCD8GFP GAL4-201Y; UAS–EcR-B1/+. See full genotypes in Supplementary List of Fly Strains. * Figure 4: Expression of ECR-B1 depends on normal FTZ-F1 and lack of HR39 activity in γ neurons. Composite confocal images of the mushroom body cell body regions in late third instar larval brains are shown. Green represents GAL4-201Y–driven GFP and red represents antibody to ECR-B1. (–) Brains containing wild-type (,) and ftz-f1−/− mutant MARCM clones (,) were immunostained for ECR-B1 expression. The white outline indicates the extent of GFP labeling of the cell bodies belonging to the clone. () Quantification of ECR-B1 signal in arbitrary units. Results are means and s.e.m. (n = 22 for wild type, 23 for mutant; ***P < 0.0001). (–) Brains containing wild-type (,) and HR39-overexpressing γ neurons (,) were immunostained for ECR-B1 expression. The white outline reveals the extent of GFP labeling contained in the cell body of the γ neurons of the brain. Some GFP labeling not enclosed by the outline likely corresponds to neurites (). Note that ECR-B1 was present in the nuclei of γ neurons in wild-type neuroblast clones and brains (,). However, ECR-B1 could bar! ely be detected in either the ftz-f1ex7 mutant neuroblast clones or the HR39-overexpressing γ neurons (,). () Quantification of ECR-B1 signal in arbitrary units. Results are means and s.e.m. (n = 48 for wild type, 47 for mutant). The differences were highly significant in a t test (two-tailed test, P < 0.0001; ,). Scale bars represent 10 μm (–,–). See genotypes in Supplementary List of Fly Strains. * Figure 5: FTZ-F1 represses Hr39 expression to prevent competition. (–) Unpruned γ axons phenotypes. Unpruned γ axons are indicated by arrows and pruned γ axons by an asterisk. The arrowhead points to a wild-type γ neuron that does not belong to the clone. Green represents GAL4-201Y–driven GFP and red represents antibody to FASII. Scale bar represents 20 μm. (–) Hr39 was repressed by FTZ-F1 in γ neurons. Brains containing GAL4-201Y–driven GFP-labeled (green) MARCM control clones (–) and ftz-f1−/− mutant clones (–) were immunostained for HR39 expression (red). HR39 was overexpressed (1.78×) in ftz-f1−/− clones when compared with wild-type clones. Results are means and s.e.m. The differences are significant in a t test (two-tailed test, P < 0.001). Scale bar represents 20 μm. (–) In vivo competition between HR39 and βFTZ-F1 for γ neuron remodeling. MARCM neuroblast clones () overexpressing HR39 were partially () or completely () rescued by βFTZ-F1. The arrow in indicates some unpruned γ neurons. Titration of G! AL4 can be ruled out as having an effect here, as the addition of an UAS-lacZ transgene did not change the Hr39 overexpression phenotype (Table 1). Pictures are composite confocal images. Green represents GAL4-201Y–driven GFP and red represents antibody to FASII. Scale bar represents 20 μm. See genotypes in Supplementary List of Fly Strains. * Figure 6: Hr39 is required for normal αβ neuron development, but not for γ pruning. () Wild-type mushroom body. () Hr39−/− mushroom body in which the γ axons appeared to be wild type (γ), but α axon misguidance was clearly apparent (α). Some axons from the β lobes (β) showed midline crossing defects (MC). Green represents GAL4-201Y–driven GFP and red represents antibody to FASII. () Wild-type neuroblast clone including αβ neurons reflecting Hr39 expression. () Hr39 expression was reduced as shown by the decrease in GAL4-c739–driven GFP expression when FTZ-F1 expression was forced. () Quantification of the GFP signal in arbitrary units. Results are means and s.e.m. with n = 8 in each case. The difference was highly significant in a t test (two-tailed test, ***P < 0.001). When the same experiment was done with UAS-lacZ instead of UAS–βftz-f1, no difference in GFP intensity was detected (WT, 11.1 ± 0.4, n = 8; lacZ, 11.4 ± 0.3, n = 8; P > 0.5) ruling out the possibility of GAL4 titration by an extra UAS sequence. Composite focal images are! shown in –. Scale bars represents 20 μm. Genotypes: () UAS–mCD8GFP GAL4–201Y/+, () Df(2L)Exel6048 UAS–mCD8GFP GAL4–201Y /Hr39C105, () hs–FLP tubP–GAL80 FRT19A/FRT19A; GAL4–c739 UAS–mCD8GFP/+, () hs–FLP tubP–GAL80 FRT19A/FRT19A; GAL4–c739 UAS–mCD8GFP/UAS–βftz-f1. See full genotypes in Supplementary List of Fly Strains. * Figure 7: In vivo binding of FTZ-F1 upstream of the EcR-B1 transcription start site. () Location of FTZ-F1 putative binding sites on the EcR locus is indicated by a corresponding triangle on both strands. () Nine putative FTZ-F1 binding sites are present. () Chromatin immunoprecipitation from L3 brains with antibody to FTZ-F1 was performed for the six sites that are upstream of EcR-B1 transcription start site. FTZ-F1 bound more on the A, B, C and D sites than on a control site in the RP49 locus. The differences were highly significant in a t test (*P = 0.001, ***P < 0.001). The last two sites (E and F) did not show a significant retention of the FTZ-F1 protein when compared with the control RP49 site. (P = 0.13 for E, P = 0.29 for F) Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Ana Boulanger & * Christelle Clouet-Redt Affiliations * Institute of Human Genetics, CNRS UPR1142, Montpellier, France. * Ana Boulanger, * Christelle Clouet-Redt, * Morgane Farge, * Adrien Flandre, * Thomas Guignard & * Jean-Maurice Dura * Institut de Génétique Moléculaire de Montpellier, UMR 5535 CNRS, Montpellier, France. * Céline Fernando & * François Juge * Université Montpellier 2, Montpellier, France. * Céline Fernando & * François Juge * Université Montpellier 1, Montpellier, France. * Céline Fernando & * François Juge Contributions A.B., C.C.-R. and J.-M.D. designed the experiments and analyzed the data. C.F. and F.J. performed the chromatin immunoprecipitation experiments. J.-M.D. carried out the elaboration of the genetic stocks. A.B., C.C.-R., M.F., A.F. and T.G. performed all of the other experiments. The manuscript was written by J.-M.D. and commented on by A.B., C.C.-R. and A.F. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Jean-Maurice Dura Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–16 and List of Fly Strains Additional data - Retinoid X receptor gamma signaling accelerates CNS remyelination
- Nat Neurosci 14(1):45-53 (2011)
Nature Neuroscience | Article Retinoid X receptor gamma signaling accelerates CNS remyelination * Jeffrey K Huang1, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Andrew A Jarjour2, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Brahim Nait Oumesmar3 Search for this author in: * NPG journals * PubMed * Google Scholar * Christophe Kerninon3 Search for this author in: * NPG journals * PubMed * Google Scholar * Anna Williams2 Search for this author in: * NPG journals * PubMed * Google Scholar * Wojciech Krezel4 Search for this author in: * NPG journals * PubMed * Google Scholar * Hiroyuki Kagechika5 Search for this author in: * NPG journals * PubMed * Google Scholar * Julien Bauer6 Search for this author in: * NPG journals * PubMed * Google Scholar * Chao Zhao1 Search for this author in: * NPG journals * PubMed * Google Scholar * Anne Baron-Van Evercooren3 Search for this author in: * NPG journals * PubMed * Google Scholar * Pierre Chambon4 Search for this author in: * NPG journals * PubMed * Google Scholar * Charles ffrench-Constant2 Contact Charles ffrench-Constant Search for this author in: * NPG journals * PubMed * Google Scholar * Robin J M Franklin1 Contact Robin J M Franklin Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature NeuroscienceVolume: 14,Pages:45–53Year published:(2011)DOI:doi:10.1038/nn.2702Received09 July 2010Accepted21 October 2010Published online05 December 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 molecular basis of CNS myelin regeneration (remyelination) is poorly understood. We generated a comprehensive transcriptional profile of the separate stages of spontaneous remyelination that follow focal demyelination in the rat CNS and found that transcripts that encode the retinoid acid receptor RXR-γ were differentially expressed during remyelination. Cells of the oligodendrocyte lineage expressed RXR-γ in rat tissues that were undergoing remyelination and in active and remyelinated multiple sclerosis lesions. Knockdown of RXR-γ by RNA interference or RXR-specific antagonists severely inhibited oligodendrocyte differentiation in culture. In mice that lacked RXR-γ, adult oligodendrocyte precursor cells efficiently repopulated lesions after demyelination, but showed delayed differentiation into mature oligodendrocytes. Administration of the RXR agonist 9-cis-retinoic acid to demyelinated cerebellar slice cultures and to aged rats after demyelination caused an increa! se in remyelinated axons. Our results indicate that RXR-γ is a positive regulator of endogenous oligodendrocyte precursor cell differentiation and remyelination and might be a pharmacological target for regenerative therapy in the CNS. View full text Figures at a glance * Figure 1: Differential expression of Rxrg in CNS remyelination transcriptome. () Hierarchical clustering and graphical analysis of differentially expressed genes at 5, 14 and 28 dpl (P < 0.05). () Ten most upregulated genes at each time point relative to the other time points. () Graphical analysis showing the differential expressions of known genes associated with myelination (P < 0.05). () Top five overall physiological functions in lesions at 5, 14 and 28 dpl using Ingenuity pathway analysis of upregulated genes from each time point. () Volcano plot (x axis = log2 FC at 14 dpl compared to 5 dpl; y axis = log2P) showing highly differentially expressed genes associated with myelination genes. Rxrg (green triangle; x, y = 3.3752, 2.7084) is shown as a highly expressed transcript at 14 dpl compared to 5 dpl. () Real-time qPCR detection of Rxra, Rxrb and Rxrg expression from laser-captured lesions during remyelination (n = 3). Rxrg is barely detectable in non-lesioned CCPs and at 5 dpl, and highly expressed at 14 and 28 dpl. () In situ hybridization sho! ws significant increase of Rxrg+ cells in the CCP at 14 dpl and 28 dpl compared to non-lesioned and 5 dpl CCP. Scale bar, 50 μm. () Quantification of Rxrg+ cells in lesioned CCPs at 5, 14 and 28 dpl (n = 3 per time point). Mean ± s.e.m. are shown. *P < 0.05, ***P < 0.001, one-way ANOVA. * Figure 2: RXR-γ expression by oligodendrocyte lineage cells. (–) Co-immunostaining for RXR-γ (red) and (in green) ED1 (), GFAP (), Nkx2.2 () or CC1 () in lesions at 14 dpl. () Faint, cytosolic detection of RXR-γ+CC1+ oligodendrocytes in non-lesioned white matter. () RXR-γ+ neurons in the striatum. Nuclei were visualized with Hoechst (blue). Scale bar, 25 μm. () Quantification of total Olig2+ RXR-γ+ oligodendrocyte lineage cells relative to the total number of RXR-γ+ cells in CCP lesions at 5, 14 and 28 dpl. () Quantification of RXR-γ+CC1+ oligodendrocytes in CCP lesions at 5, 14 and 28 dpl. (–) Detection of RXR-γ (green) in oligodendrocyte lineage cells from OPC/DRG neuron co-cultures at 2, 6 and 7 d in co-culture. Oligodendrocyte lineage cells were immunolabeled with O4 (red), axons with anti-neurofilament (NF, blue) and nuclei visualized with Hoechst (violet). Scale bar, 60 μm. Mean ± s.e.m. are shown. *P < 0.05, **P < 0.01, ***P < 0.001; one-way ANOVA. * Figure 3: Expression of RXR-γ in multiple sclerosis lesions. (–) Co-immunolabeling for RXR-γ (green) and (in red) MOG (), Sox10 (), Olig1 (), NOG0-A (), MHCII () or GFAP () in active multiple sclerosis lesion areas. Nuclei were visualized with Hoechst (blue). () Luxol fast blue staining followed by anti-MHCII immunoperoxidase labeling showing a typical chronic active multiple sclerosis lesion with active border (A) and chronic inactive core (C), as well as peri-plaque white matter (PPWM). () Quantification of nuclear and cytoplasmic RXR-γ+ cells in multiple sclerosis lesions reveals significantly more nuclear RXR-γ+ cells in active lesions, PPWM and remyelinated shadow plaques (RM) compared to chronic inactive lesions and normal appearing white matter (WM) from non-neurological cases. Scale bars: –, 50 μm; , 2 mm. Mean ± s.e.m. are shown. *P < 0.05, ***P < 0.001; one-way ANOVA. * Figure 4: Loss of RXR-γ function impairs oligodendrocyte differentiation. (–) Purified OPCs transfected with non-targeting siRNAs (), RXRα siRNAs () and RXR-γ siRNAs () and visualized with O4 (red) and antibodies to MBP (green) after 72 h in differentiation medium. Scale bar, 25 μm. () Morphological criteria for the maturation state of differentiating oligodendrocyte defined as simple, complex or membrane morphologies. Cells transfected with RXR-γ siRNAs resulted in increased percentage of O4+ oligodendrocytes with simple morphologies and decreased percentage of complex membrane morphologies compared to mock-treated and non-targeting siRNA–transfected cells. () Western blot shows the specificity of RXR-α or RXR-γ knockdowns. The position of molecular weight standards (in kilodaltons) is shown on the left. Full-length blot presented in Supplementary Figure 4. C, untransfected control; M, mock-transfected; NT, non-targeting siRNA; RXRα, β, γ, RXRα, β or γ siRNA. (,) Ventral spinal cord lesions of Rxrg+/− () and Rxrg−/− () mice ! stained with antibodies to CC1 (green) and Olig2 (red) 15 d after demyelination. Nuclei visualized with Hoechst (blue). () Quantification of oligodendrocyte lineage cells at 15 and 30 dpl shows no difference in the density of total Olig2+ cells between homozygous and heterozygous mutant mice, but a reduction of CC1+ cells and increased Nkx2.2+ cells in lesions of homozygous compared to heterozygous mutant mice. Scale bar, 50 μm. Mean ± s.e.m. are shown. *P < 0.05, **P < 0.01; Student's t test. * Figure 5: Rexinoids influence oligodendrocyte differentiation and myelination. (–) OPC cultures immunolabeled with antibodies to O4 (red) and MBP (green) after RXR antagonist treatment for 72 h. Compared to non-treated cells (), treatment with HX531 (; 2 μM) or PA452 (; 5 μM) resulted in fewer mature oligodendrocytes. Scale bar, 25 μm. () Increasing antagonist concentration resulted in decreasing number of membrane sheet-bearing oligodendrocytes. (–) Oligodendrocyte-DRG co-cultures maintained for 10 d after addition of OPCs immunolabeled with anti-MBP (green), anti-Caspr (red) and anti-NFH (blue). () Control co-culture; () HX531 (2 μM); () PA452 (5 μM); () 9cRA (50 nM). Scale bar, 100 μm. (,) Increasing antagonist concentration resulted in decreased MBP+ oligodendrocytes () and less myelination (). (–) OPC cultures labeled with O4 (red) and anti-MBP (green). () Untreated; () 9cRA alone; () 9cRA and HX531; () 9cRA and PA452. Scale bar, 25 μm. () Quantification showing that 50 nM 9cRA increased mature oligodendrocyte membranes, and low conce! ntrations of HX531 and PA452 were sufficient to abrogate 9cRA-mediated differentiation. () Treatment of cultured OPCs with 9cRA, HX630 or PA024 resulted in increased oligodendrocyte membrane sheets. Mean ± s.e.m. are shown. *P < 0.05 versus control, **P < 0.005 versus control, †P < 0.05 versus 50 nM 9cRA, ††P < 0.005 versus 50 nM 9cRA; Student's t test. * Figure 6: CNS remyelination is enhanced by 9 cis-retinoic acid. () Control cerebellar slices fixed 10 d after demyelination with lysolecithin and immunolabeled with antibodies to NFH (red) and MBP (green). (–) Remyelination was increased by 9cRA (), and decreased by HX531 () or PA452 (). Scale bar, 20 μm. (,) Quantification of NG2+ and MBP+ cells, 48 h () or 14 d () after treatment. *P < 0.05; Student's t test. () Quantification of remyelination after addition of 9cRA, or HX531 or PA452 at high (H, 2 or 5 μM, respectively) or low (L, 0.2 or 0.5 μM) concentrations. *P < 0.05, **P < 0.001; one-way ANOVA. () Treatment with 9cRA increased CC1+ cells in lesions in rats. Student's t test. () Real-time qPCR analysis shows increased Mbp expression in 9cRA-treated mice. Student's t test. () Semi-thin section of a lesioned CCP 27 dpl after 9cRA treatment. Upper left corner shows normal myelinated axons. To the right is a large area of lesion showing axons outlined by thinly remyelinating membranes and dark macrophages. Scale bar, 50 μm. () U! ltrastructural microscopy (1,500×) shows many remyelinated axons (pink) compared to axons that remained demyelinated. (,) Control animal (images as in and , respectively) shows few visible remyelinated axons. () Ranking analysis. Highest rank represents most remyelination. Mann-Whitney U test. () G-ratio is lower in 9cRA-treated mice compared to control mice. Student's t test. () Representative images of myelinated, demyelinated, control remyelinated and 9cRA remyelinated axons. Mean ± s.e.m. are shown. ***P < 0.001. Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Gene Expression Omnibus * GSE24821 Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Jeffrey K Huang & * Andrew A Jarjour Affiliations * MRC Centre for Stem Cell Biology and Regenerative Medicine and Department of Veterinary Medicine, University of Cambridge, Cambridge, UK. * Jeffrey K Huang, * Chao Zhao & * Robin J M Franklin * MRC Centre for Regenerative Medicine and Multiple Sclerosis Society/University of Edinburgh Centre for Translational Research, Centre for Inflammation Research, The Queen's Medical Research Institute, Edinburgh, UK. * Andrew A Jarjour, * Anna Williams & * Charles ffrench-Constant * Centre de Recherche de l'Institut du Cerveau et de la Moelle Epinière, Inserm U.975; Université Pierre et Marie Curie-Paris 6 UMR-S975; Cnrs UMR 7225; and AP-HP Groupe Hospitalier Pitié-Salpêtrière, Fédération de Neurologie, Paris cedex 13, France. * Brahim Nait Oumesmar, * Christophe Kerninon & * Anne Baron-Van Evercooren * Institut de Génétique et de Biologie Moléculaire et Cellulaire, Department of Cell Biology and Development, Illkirch, France. * Wojciech Krezel & * Pierre Chambon * Graduate School of Biomedical Science, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo, Japan. * Hiroyuki Kagechika * Department of Pathology, University of Cambridge, Cambridge, UK. * Julien Bauer Contributions J.K.H. performed in vivo experiments and laser capture microdissections. A.A.J. performed in vitro experiments. C.Z. contributed to in vivo experiments. A.W. performed ex vivo experiments. B.N.O., C.K. and A.B.-V.E. performed multiple sclerosis tissue analysis. H.K. generated RXR antagonists and agonists. W.K. and P.C. generated the RXR-γ mouse mutants. J.B. and J.K.H. performed bioinformatics. C.ff.-C. and R.J.M.F. equally oversaw the project. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Charles ffrench-Constant or * Robin J M Franklin Supplementary information * Abstract * Accession codes * Author information * Supplementary information Excel files * Supplementary Table 1 (5M) Total genes differentially expressed between 5, 14 and 28 days post CCP demyelination. * Supplementary Table 2 (100K) Gene list used for IPA analysis. * Supplementary Table 3 (68K) Active signaling networks found between 5 and 14 dpl. * Supplementary Table 4 (716K) Total genes differentially expressed between 5 and 14 dpl (P < 0.05) used for volcano plot. * Supplementary Table 6 (196K) IPA identified RXR associated pathways from the remyelination transcriptome. PDF files * Supplementary Text and Figures (11M) Supplementary Figures 1–7 and Supplementary Tables 5, 7 Additional data - Sortilin associates with Trk receptors to enhance anterograde transport and neurotrophin signaling
- Nat Neurosci 14(1):54-61 (2011)
Nature Neuroscience | Article Sortilin associates with Trk receptors to enhance anterograde transport and neurotrophin signaling * Christian B Vaegter1 Contact Christian B Vaegter Search for this author in: * NPG journals * PubMed * Google Scholar * Pernille Jansen1 Search for this author in: * NPG journals * PubMed * Google Scholar * Anja W Fjorback2 Search for this author in: * NPG journals * PubMed * Google Scholar * Simon Glerup1 Search for this author in: * NPG journals * PubMed * Google Scholar * Sune Skeldal1 Search for this author in: * NPG journals * PubMed * Google Scholar * Mads Kjolby1 Search for this author in: * NPG journals * PubMed * Google Scholar * Mette Richner1 Search for this author in: * NPG journals * PubMed * Google Scholar * Bettina Erdmann3 Search for this author in: * NPG journals * PubMed * Google Scholar * Jens R Nyengaard2 Search for this author in: * NPG journals * PubMed * Google Scholar * Lino Tessarollo4 Search for this author in: * NPG journals * PubMed * Google Scholar * Gary R Lewin3 Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas E Willnow3 Search for this author in: * NPG journals * PubMed * Google Scholar * Moses V Chao5 Search for this author in: * NPG journals * PubMed * Google Scholar * Anders Nykjaer1 Contact Anders Nykjaer Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature NeuroscienceVolume: 14,Pages:54–61Year published:(2011)DOI:doi:10.1038/nn.2689Received18 August 2010Accepted12 October 2010Published online21 November 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 Binding of target-derived neurotrophins to Trk receptors at nerve terminals is required to stimulate neuronal survival, differentiation, innervation and synaptic plasticity. The distance between the soma and nerve terminal is great, making efficient anterograde Trk transport critical for Trk synaptic translocation and signaling. The mechanism responsible for this trafficking remains poorly understood. Here we show that the sorting receptor sortilin interacts with TrkA, TrkB and TrkC and enables their anterograde axonal transport, thereby enhancing neurotrophin signaling. Cultured DRG neurons lacking sortilin showed blunted MAP kinase signaling and reduced neurite outgrowth upon stimulation with NGF. Moreover, deficiency for sortilin markedly aggravated TrkA, TrkB and TrkC phenotypes present in p75NTR knockouts, and resulted in increased embryonic lethality and sympathetic neuropathy in mice heterozygous for TrkA. Our findings demonstrate a role for sortilin as an anterograde! trafficking receptor for Trk and a positive modulator of neurotrophin-induced neuronal survival. View full text Figures at a glance * Figure 1: Sortilin interacts with Trk receptors. () Colocalization of sortilin with p75NTR (left) and Trk receptors (right) in DRG neurons. () Top: coimmunoprecipitation of sortilin and TrkA in HEK293 cells using pan-anti-Trk or anti-sortilin antibody, respectively (IP). Bottom left: coimmunoprecipitation of sortilin with TrkC in HEK293 using pan-anti-Trk. Bottom right: attempted coimmunoprecipitation of sortilin with Ret. Proteins are visualized by western blotting. () Interaction between sortilin and TrkA in HEK293 cells as measured by reduction in donor fluorophore Alexa 488–sortilin lifetime (FLIM) in the presence of the acceptor fluorophore Alexa 568–TrkA. Distribution of donor lifetime illustrated in color code. () Surface plasmon resonance analysis of receptor interactions. Top: binding of extracellular domains of TrkA and TrkC, but not of Ret and neurturin, to immobilized sortilin ectodomain. Bottom: binding of TrkB to sortilin but not to pro-sortilin (red curve). () Coimmunoprecipitation of TrkB from hippocamp! al neurons by anti-sortilin antibody but not by preimmune IgG. () Immunofluorescence staining of endogenous sortilin and TrkA in cultured SCG neurons. PFRET signal of colocalized receptors was obtained in neurites (bottom right; distribution of PFRET signal illustrated in color code, ranging from 0 (black) to 40 (white)). Panels , contain cropped blots; full-length blots are presented in Supplementary Figure 7. * Figure 2: Sortilin facilitates anterograde neuronal Trk trafficking. () Movement of EGFP-TrkA in neurites of cultured DRG neurons. Arrows indicate anterograde (red) and retrograde (yellow) movement of vesicles. () Kymograph of a neurite as exemplified by panel . Images were captured every 2 s. () Relative frequencies of anterograde, retrograde or no movement of ~350 TrkA-positive vesicles of each genotype measured as described in (mean ± s.e.m.). () Immunofluorescence of sortilin (red) and DAPI staining (blue) of a double-ligated rat sciatic nerve. Accumulation of sortilin proximal and distal to the ligation illustrate anterograde and retrograde sortilin transport, respectively. () Western blot of segments of double-ligated sciatic nerves proximal (P) and/or distal (D) to the ligature in wild-type and sortilin-deficient mice, showing anterograde TrkA transport (top), bidirectional transport of p75NTR (middle) and actin loading control (bottom). () Quantification of anterogradely transported mature and immature TrkA in sortilin knockouts rela! tive to wild-type mice determined by densitometric scanning of blots as illustrated in (n = 3; mean ± s.e.m.). () Western blot of middle cerebral arteries from two representative experiments (left and right) illustrating reduced peripheral TrkA targeting in Sort1−/− mice. () Western blot analysis of TrkB in hippocampal subcellular fractions from two representative experiments (top and bottom) showing reduced TrkB in synaptic fractions (boxed in red) from Sort1−/− mice (n = 4). P1, initial precipitation; S1, initial supernatant; P2, crude synaptosomes; S2, light membrane (see Supplementary Fig. 1). Panels ,, contain cropped blots; full-length blots are presented in Supplementary Figure 7. * Figure 3: Sortilin facilitates Trk signaling in neurons. () Western blot (WB) of phosphorylated ERK1/2 (P-ERK1/2) in primary DRG neuron cultures after stimulation with NGF for 10 min. () Quantification of phospho-ERK1/2 from five experiments as depicted in (mean + s.e.m.). () Staining of DRG neurons with the microtubule marker TuJ1 12 h after plating. (,) Quantification of neurite length (n = 4) after stimulation with NGF () or the Ret ligand neurturin () (mean ± s.e.m.). Panel contains cropped blots; the full-length blots are presented in Supplementary Figure 7. * Figure 4: Aberrant gait and peripheral neuropathy in sortilin and p75NTR double knockout mice. () Abnormal hindlimb posture (arrows) in Sort−/−; Ngfr−/− mice. () Quantification of myelinated axons in the sciatic nerve (left; mean ± s.e.m.) and nerve morphology (right). () Number of neurons in adult L4 and L5 DRGs (n = 4) (mean ± s.e.m.). * Figure 5: Loss of sortilin aggravates TrkC and TrkB phenotypes in p75NTR knockout mice. () Quantification of proprioceptive neurons in L4 and L5 DRGs using markers for NF200 and TrkC (n = 8; mean ± s.e.m.). () Number of muscle spindles in hindlimbs from P1 mice. () Von Frey stimulus-response curves from ten wild-type, eight Sort1−/−, seven Ngfr−/− and six DKO mice. P-values for Sort1−/− and Ngfr−/− are relative to wild-type; for Sort−/−; Ngfr−/−, relative to Ngfr−/− (mean ± s.e.m.). NS, not significant. () Numbers of TrkB-positive DRG neurons in adult mice (n = 8) (mean ± s.e.m.). * Figure 6: Sortilin deficiency aggravates TrkA phenotypes in p75NTR knockout mice. () Thermal response as measured by Hargreaves test (n = 10; mean ± s.e.m.). () Quantification of nociceptive neuron subtypes in L4 and L5 DRGs (n = 8; mean ± s.e.m.). () Electron micrographs showing the morphology of Remak bundle fibers in adult and P15 mice. () Hindlimb degeneration in DKO at 3–4 months of age. * Figure 7: Absent sortilin expression induces TrkA phenotypes in Ntrk1+/− mice. () Schematic representation of possible genotypes of offspring from Sort1+/−; Ntrk1+/− and Sort1−/−; Ntrk1+/+ breeding pairs. () Genotype frequency of offspring at P1. Black bars indicate offspring resulting from male crossover, illustrating excess lethality of Sort1−/−; Ntrk1+/− over Sort1+/−; Ntrk1+/+. () SCG volume in newborns of the indicated genotypes (mean ± s.e.m.). Author information * Abstract * Author information * Supplementary information Affiliations * The Lundbeck Foundation Research Center MIND, Department of Medical Biochemistry, Aarhus University, Aarhus, Denmark. * Christian B Vaegter, * Pernille Jansen, * Simon Glerup, * Sune Skeldal, * Mads Kjolby, * Mette Richner & * Anders Nykjaer * The Lundbeck Foundation Research Center MIND, Stereology and Electron Microscopy Laboratory, Aarhus University, Aarhus, Denmark. * Anja W Fjorback & * Jens R Nyengaard * Max Delbrück Center for Molecular Medicine, Berlin, Germany. * Bettina Erdmann, * Gary R Lewin & * Thomas E Willnow * Center for Cancer Research, National Cancer Institute, Frederick, Maryland, USA. * Lino Tessarollo * Kimmel Center at Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, New York, USA. * Moses V Chao Contributions C.B.V., P.J., A.W.F., S.G., S.S., M.R., M.K. and B.E. conducted the experiments. J.R.N., L.T., G.R.L., T.E.W. and M.V.C. provided reagents and scientific input. C.B.V. and A.N. designed the experiments and evaluated the data, and C.B.V. and A.N. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Anders Nykjaer or * Christian B Vaegter Supplementary information * Abstract * Author information * Supplementary information Movies * Supplementary Video 1 (808K) Live cell imaging of a WT DRG neuron transfected with EGFP-TrkA * Supplementary Video 2 (2M) Live cell imaging of a Sort1−/− DRG neuron transfected with EGFP-TrkA * Supplementary Video 3 (460K) Characteristic waddling gate in sortilin and p75NTR double knockout mouse PDF files * Supplementary Text and Figures (444K) Supplementary Figures 1–7 Additional data - Ca2+-dependent enhancement of release by subthreshold somatic depolarization
- Nat Neurosci 14(1):62-68 (2011)
Nature Neuroscience | Article Ca2+-dependent enhancement of release by subthreshold somatic depolarization * Jason M Christie1 Contact Jason M Christie Search for this author in: * NPG journals * PubMed * Google Scholar * Delia N Chiu1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Craig E Jahr1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 14,Pages:62–68Year published:(2011)DOI:doi:10.1038/nn.2718Received10 October 2010Accepted09 November 2010Published online19 December 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 In many neurons, subthreshold somatic depolarization can spread electrotonically into the axon and modulate subsequent spike-evoked transmission. Although release probability is regulated by intracellular Ca2+, the Ca2+ dependence of this modulatory mechanism has been debated. Using paired recordings from synaptically connected molecular layer interneurons (MLIs) of the rat cerebellum, we observed Ca2+-mediated strengthening of release following brief subthreshold depolarization of the soma. Two-photon microscopy revealed that, at the axon, somatic depolarization evoked Ca2+ influx through voltage-sensitive Ca2+ channels and facilitated spike-evoked Ca2+ entry. Exogenous Ca2+ buffering diminished these Ca2+ transients and eliminated the strengthening of release. Axonal Ca2+ entry elicited by subthreshold somatic depolarization also triggered asynchronous transmission that may deplete vesicle availability and thereby temper release strengthening. In this cerebellar circuit, a! ctivity-dependent presynaptic plasticity depends on Ca2+ elevations resulting from both sub- and suprathreshold electrical activity initiated at the soma. View full text Figures at a glance * Figure 1: Subthreshold somatic depolarization enhances spike-evoked release in MLIs. () In a MLI paired recording, action potentials in the presynaptic neuron evoked GABAAR-mediated IPSCs in the postsynaptic neuron. Subthreshold somatic depolarization (300 ms) elicited before spiking enhanced release. Control action potential responses are in red and test responses preceded by subthreshold depolarization are in black. An amplified view of the first IPSC is shown in the inset. () Paired-pulse IPSCs from the same neuron normalized to the peak of the first response. For clarity, spontaneous IPSCs were blanked before averaging in (inset) and . () Summary data showing that the PPR decreased with somatic depolarization of the presynaptic neuron. *P < 0.05, paired t test. Data are mean values ± s.e.m. () Linear regression analysis revealed that release enhancement elicited by subthreshold depolarization was correlated with the basal PPR. (,) Differences in release enhancement for facilitating and depressing responses measured in 1.5 and 3.0 mM extracellular Ca2+. ! **P < 0.05, ANOVA analysis with post hoc Tukey's multiple comparison tests indicated significance. Data are mean values plotted ± s.e.m. * Figure 2: Subthreshold somatic depolarization evokes and enhances axonal Ca2+ entry. () Two-photon fluorescence image of a representative MLI filled via patch pipette with Alexa 594 (13 μM) and Fluo-5F (200 μM). Right, higher magnification view shows axon varicosities. () Somatic current injection elicited control action potential–evoked responses (red) and test spikes paired with 300-ms subthreshold depolarizations (black). Shown directly below are the resulting Ca2+ transients recorded in an axon varicosity. Integration of the Ca2+ signals accentuates the difference between control and depolarized responses before spike firing. Bottom left, amplified view of Ca2+ transients with dashed lines (100-ms epoch average) demarcating the amplitude of the Ca2+ response evoked by subthreshold depolarization. Bottom right, action potential–evoked Ca2+ transients baselined before spike firing (black bar) isolated depolarization-dependent facilitation of the spike-evoked Ca2+ transient. Exponential fits to the spike-elicited transients were used to determine peak! amplitudes. () Comparison of depolarization-evoked Ca2+ elevation and facilitation of action potential–elicited Ca2+ entry. Line fit of the linear regression is indicated by the dashed line. * Figure 3: Subthreshold depolarization–dependent Ca2+ signaling is EGTA sensitive. () Somatic recording of an action potential (red) and an action potential preceded by a 300-ms subthreshold depolarization (black). The resulting Ca2+ transients in an axon varicosity are shown below in control and following EGTA-AM application (10–20 μM, >10 min). Integrals of the Ca2+ responses are provided for each condition. The inset shows a comparison of action potential–evoked responses, elicited without preceding subthreshold depolarization, in control and with EGTA. Dashed lines indicate the response amplitude before spiking (100-ms epoch) and the exponential fits of action potential–evoked Ca2+ transients used to determine peak amplitudes. () The effect of EGTA on Ca2+ entry evoked by subthreshold depolarization. () EGTA inhibits facilitation of spike-evoked Ca2+ entry caused by subthreshold depolarization. Data are mean ± s.e.m., *P < 0.05, paired t test. * Figure 4: EGTA inhibits enhancement of release elicited by subthreshold depolarization. () Action potential–evoked IPSCs (red) recorded from pairs of connected MLIs in control and following EGTA-AM application. In alternating trials, action potentials were preceded by a 300-ms subthreshold depolarization (black). () Top, EGTA eliminated release strengthening caused by subthreshold depolarization in facilitating cell pairs. Release strengthening was not apparent in cell pairs that depressed in basal conditions. Bottom, linear regression analysis plot of the difference between the percentage change in EGTA and in control relative to the basal PPR. () The effect of EGTA on paired-pulse responses from trials without preceding subthreshold depolarization. Inset, amplified view of the first IPSC. () EGTA altered paired-pulse responses in cell pairs that facilitated in basal conditions. Data are mean ± s.e.m., *P < 0.05, paired t test. Spontaneous IPSCs were blanked before averaging in and . * Figure 5: Subthreshold depolarization evokes asynchronous release. () Paired recording of MLIs showing spontaneous IPSCs in unstimulated control responses (red) and recruitment of asynchronous events with a 300-ms presynaptic subthreshold depolarization (black). Three individual sweeps from the postsynaptic neuron are shown superimposed for each condition with the average response (27 and 28 sweeps for control and depolarized, respectively) at the bottom. () The average charge integration of the postsynaptic response across all cell recordings (±.s.e.m.). *P < 0.05, paired t test. * Figure 6: Depolarization-dependent asynchronous release is triggered by VSCCs. (,) Paired recording from MLIs in tetrodotoxin with representative sweeps from the postsynaptic neuron showing recruitment of asynchronous IPSCs with presynaptic depolarization. The presynaptic holding potential is indicated in gray above each sweep. Responses were recorded in 3 mM extracellular Ca2+ to enhance release. () In the same cell, IPSC frequency was unaltered by presynaptic depolarization after VSCC block (100 μM Cd2+ and Ni2+). () Asynchronous IPSC frequency depended on the presynaptic holding potential. Data are mean ± s.e.m. *P < 0.05, ANOVA analysis (Tukey's multiple comparison test). () Block of VSCCs eliminated the depolarization-dependent increase in asynchronous transmission. Data are mean ± s.e.m., *P < 0.05, ANOVA analysis. * Figure 7: Burst firing reduces the capacity for release enhancement caused by depolarization. () In a recording from connected MLIs, high-frequency bursts of action potentials (ten spikes, 60 Hz) preceded a 300-ms subthreshold stimulation and test spikes in interleaved trials. Amplified views on the right show release enhancement caused by subthreshold depolarization and weakening of transmission when subthreshold excitation was paired with burst firing. () The effect of burst firing on the first IPSC in control trials without subthreshold stimulation. For amplified views (,), spontaneous events were blanked before averaging. () Burst firing eliminated release strengthening induced by subthreshold depolarization. Data are mean ± s.e.m., *P < 0.05, paired t test. Author information * Abstract * Author information * Supplementary information Affiliations * Vollum Institute, Oregon Health & Science University, Portland, Oregon, USA. * Jason M Christie, * Delia N Chiu & * Craig E Jahr * Neuroscience Graduate Program, Oregon Health & Science University, Portland, Oregon, USA. * Delia N Chiu Contributions Each of the authors contributed extensively to the design and implementation of the experiments, interpretation of the data and writing of the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Jason M Christie Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (328K) Supplementary Figure 1 Additional data - Caspase-3 triggers early synaptic dysfunction in a mouse model of Alzheimer's disease
- Nat Neurosci 14(1):69-76 (2011)
Nature Neuroscience | Article Caspase-3 triggers early synaptic dysfunction in a mouse model of Alzheimer's disease * Marcello D'Amelio1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Virve Cavallucci1 Search for this author in: * NPG journals * PubMed * Google Scholar * Silvia Middei3 Search for this author in: * NPG journals * PubMed * Google Scholar * Cristina Marchetti4 Search for this author in: * NPG journals * PubMed * Google Scholar * Simone Pacioni5 Search for this author in: * NPG journals * PubMed * Google Scholar * Alberto Ferri6 Search for this author in: * NPG journals * PubMed * Google Scholar * Adamo Diamantini7 Search for this author in: * NPG journals * PubMed * Google Scholar * Daniela De Zio8 Search for this author in: * NPG journals * PubMed * Google Scholar * Paolo Carrara9 Search for this author in: * NPG journals * PubMed * Google Scholar * Luca Battistini7 Search for this author in: * NPG journals * PubMed * Google Scholar * Sandra Moreno9 Search for this author in: * NPG journals * PubMed * Google Scholar * Alberto Bacci5 Search for this author in: * NPG journals * PubMed * Google Scholar * Martine Ammassari-Teule3 Search for this author in: * NPG journals * PubMed * Google Scholar * Hélène Marie4, 10 Search for this author in: * NPG journals * PubMed * Google Scholar * Francesco Cecconi1, 8 Contact Francesco Cecconi Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 14,Pages:69–76Year published:(2011)DOI:doi:10.1038/nn.2709Received07 July 2010Accepted19 October 2010Published online12 December 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 Synaptic loss is the best pathological correlate of the cognitive decline in Alzheimer's disease; however, the molecular mechanisms underlying synaptic failure are unknown. We found a non-apoptotic baseline caspase-3 activity in hippocampal dendritic spines and an enhancement of this activity at the onset of memory decline in the Tg2576-APPswe mouse model of Alzheimer's disease. In spines, caspase-3 activated calcineurin, which in turn triggered dephosphorylation and removal of the GluR1 subunit of AMPA-type receptor from postsynaptic sites. These molecular modifications led to alterations of glutamatergic synaptic transmission and plasticity and correlated with spine degeneration and a deficit in hippocampal-dependent memory. Notably, pharmacological inhibition of caspase-3 activity in Tg2576 mice rescued the observed Alzheimer-like phenotypes. Our results identify a previously unknown caspase-3–dependent mechanism that drives synaptic failure and contributes to cognitive! dysfunction in Alzheimer's disease. These findings indicate that caspase-3 is a potential target for pharmacological therapy during early disease stages. View full text Figures at a glance * Figure 1: CFC performance and morphology of CA1 neuron dendrites in Tg2576 and wild-type mice. () Percentage of freezing time during CFC test in wild-type (WT) and Tg2576 (Tg) mice at 2 and 3 months of age. Data are expressed as mean ± s.e.m. **P < 0.01 (n = 10 mice for each age and genotype). () Left, representative photomicrograph of a Golgi-stained section of the dorsal hippocampus. Scale bar represents 100 μm. Right, representative segments of basal and apical dendrites of CA1 hippocampal neurons in 3-month-old wild-type and Tg2576 mice. Scale bar represents 10 μm. () Spine density is expressed as the mean spine number per 1-μm dendrite segment ± s.e.m. *P < 0.05, ***P < 0.001 (n = 6 mice for each genotype at 2 months of age, 10 wild-type mice at 3 months of age and 8 Tg2576 mice at 3 months of age; 11 neurons from each mouse). () Cumulative frequencies of spine head diameter values in apical dendrites of pyramidal neurons. A shift of the curve to the left indicates that Tg2576 mice showed a general reduction of spine head diameters. P = 0.039 (n = 5 mice, 1,! 000 spines from each mouse). () Total dendritic length of CA1 hippocampal neurons. Data are presented as mean ± s.e.m. (n = 5 mice, 6 neurons from each mouse). * Figure 2: Altered hippocampal GluR1 distribution in 3-month-old Tg2576 mice. () Representative immunoblots of PSD proteins extracted from wild-type and Tg2576 hippocampi probed with the indicated antibodies and densitometric quantification of changes in gray values expressed as mean ratio (Tg/WT) ± s.d. **P < 0.01 (n = 5 mice from each genotype). () Representative immunoblots of hippocampal total protein extract probed with the indicated antibodies and densitometric quantification of changes in gray values expressed as mean ± s.d. (wild type is indicated as 100%). *P < 0.05 (n = 4 mice). () Representative GluR1 immunoblots of hippocampal synaptic fractionation. Densitometric quantification of changes in gray values expressed as mean ± s.d. (wild type is indicated as 100%) (n = 4). For full-length blots, see Supplementary Figure 22. * Figure 3: Altered basic glutamatergic synaptic transmission and enhanced LTD in 3-month-old Tg2576 mice. () Representative whole-cell traces recorded at −65 mV and +20 mV holding potential for wild-type and Tg2576 mice (open circles indicate the time points at which the AMPA (peak) and the NMDA component were evaluated) and averaged AMPA/NMDA ratio in wild-type (n = 8 cells) and Tg2576 (n = 9) mice. Data are represented as mean ± s.e.m. *P < 0.05. () Representative traces (interstimulus interval, 100 ms) and averaged PPRs in wild-type (n = 8 cells) and Tg2576 (n = 9) mice. () Representative traces for wild-type and Tg2576 mice displaying spontaneous AMPAR-mediated mEPSCs. () Average amplitude and frequency of AMPAR mEPSC for wild-type (n = 16) and Tg2576 (n = 15) mice. Data are represented as mean ± s.e.m. () Representative traces of fEPSPs immediately before (black) and 50 min after (gray) high-frequency stimulation. Averages of ten traces for each example are shown. Bottom, time course of relative changes of fEPSP slopes for wild-type (n = 20 slices) and Tg2576 (n = 14) m! ice. High-frequency stimulation was delivered at time 0. () Representative traces of fEPSPs before (black) and 50 min after (gray) low-frequency stimulation. Averages of ten traces are shown for each example. Bottom, time course of relative changes of fEPSP slopes for wild-type (n = 11 slices) and Tg2576 (n = 12) mice. Low-frequency stimulation was delivered at time 0. * Figure 4: Tg2576 hippocampal dendritic spines accumulate active caspase-3 in the postsynaptic compartment and show apoptotic features. () Caspase-3 activity was revealed by a fluorimetric assay in both total hippocampal homogenate (total) and hippocampal synaptosomes (syn) from wild-type and Tg2576 mice. The histogram shows the caspase-3 activity mean ± s.d. (wild-type is indicated as 100%). **P < 0.01 (n = 6 mice for each age and genotype). () Representative immunoblot showing the presence of cleaved caspase-3 in hippocampal synaptosome preparations, but not in total hippocampal homogenates. Ctrl, positive control for caspase-3 cleavage (proneural cells treated with staurosporine). Right, densitometric quantification of changes in gray values ± s.d. (n = 6). For full-length blots, see Supplementary Figure 22. () Arrows indicate cleaved caspase-3–immunopositive postsynaptic compartments in CA1 dendritic spines. Arrowheads indicate cleaved caspase-3–immunonegative dendritic spines. Scale bar represents 0.5 μm. m, mitochondrion; v, neurotransmitter vesicles. Bottom, proportion of cleaved caspase-3–im! munopositive dendritic spines to the total number found in all of the fields that we examined. *P < 0.05 (n = 3 mice from each genotype, 100 spines from each genotype). () Quadrant analysis from flow cytometry apoptosis assay on synaptosomes. Representative density plot showing calcein AM fluorescence versus annexin V fluorescence for large synaptosomal particles. Mean values ± s.d. (wild type, 22.3 ± 6.8%; Tg2576, 43.4 ± 8.2%). P = 0.021 (n = 6 mice from each genotype, three independent experiments). () Forward scatter signal/propidium iodide fluorescence analysis of hippocampal nuclei. Representative density plot showing propidium iodide incorporation in wild-type and Tg2576 nuclei preparation. Mean values ± s.d. (wild type, 5.9 ± 1.7%; Tg2576, 5.3 ± 1.5%; n = 6 mice, three independent experiments). * Figure 5: Inhibition of caspase-3 activity reduces GluR1 dephosphorylation and its removal from PSD and rescues glutamatergic synaptic transmission in Tg2576 mice. () Caspase-3 activity in Tg2576 hippocampal slices incubated with z-DEVD-fmk or vehicle control; data are expressed as mean ± s.d. (control is indicated as 100%). *P < 0.05, (n = 4 mice for each experimental condition). () PSD proteins from z-DEVD-fmk or vehicle-treated slices were immunoblotted. The densitometric data are expressed as mean ± s.d. (control is indicated as 100%) (n = 4 mice per condition). () Linear regression on the caspase-3 activity ratio (z-DEVD-fmk to vehicle control) and immunoreactivity measures of GluR1 ratio (z-DEVD-fmk to vehicle control). Each point represents a single experiment (n = 8 mice). Correlation coefficient, −0.97 (Pearson correlation test, P = 0.002). () Representative immunoblots of GluR1 and GluR1pSer845 in total protein extract from slices incubated with 5 μM z-DEVD-fmk or with vehicle. The densitometric data are expressed as mean ± s.d. (control is indicated as 100%; n = 4). () Representative whole-cell traces recorded at −65! mV and +20 mV holding potential and averaged ratio from neurons of Tg2576 vehicle-treated (n = 9 cells), Tg2576 z-DEVD-fmk–treated (5 μM z-DEVD-fmk, n = 8) and wild-type untreated (n = 8) slices. The mean ± s.e.m. is shown. () Representative traces (interstimulus interval, 100 ms) and average PPR from neurons of Tg2576 vehicle-treated (n = 9), T2576g z-DEVD-fmk–treated (5 μM z-DEVD-fmk; n = 8) and wild-type untreated (n = 8) slices. For full-length blots, see Supplementary Figure 23. * Figure 6: Inhibition of caspase-3 activity reduces calcineurin cleavage and activity. () Representative immunoblot and densitometric analysis of CN-A full length and its N-terminal fragment in total protein extract from Tg2576 slices incubated with 5 μM z-DEVD-fmk or with vehicle for 2 h. The data shown in the graph are expressed as mean ± s.d. (control is indicated as 100%). *P < 0.05 (n = 4). () The slices were analyzed for calcineurin activity by monitoring 32P release from radiolabeled purified RII substrate. To control for control of calcineurin activity, we added FK506 to protein extracts (+FK506). The data are expressed as mean ± s.d. (control is indicated as 100%) (n = 6). * Figure 7: Caspase-3 inhibition in vivo influences GluR1 distribution and rescues spine head size and memory function in Tg2576 mice. () We analyzed caspase-3 and calcineurin activities 15 h after intra-hippocampal injection of z-DEVD-fmk or vehicle into 3-month-old Tg2576 mice. The data are expressed as mean ± s.d. (control is indicated as 100%). *P < 0.05, (n = 5 mice). () Representative immunoblot and densitometric analysis of CN-A full length and its N-terminal fragment in total protein extract from Tg2576 hippocampus 15 h after z-DEVD-fmk or vehicle injection. The data shown in the graph are expressed as mean ± s.d. (control is indicated as 100%) (n = 4). () Representative immunoblots of hippocampus synaptic fractionation performed 15 h after drug or vehicle injection and GluR1 densitometric quantification of changes in gray values expressed as mean ± s.d. (control is indicated as 100%) (n = 4). For full-length blots, see Supplementary Figure 23. () Left, representative photomicrographs of Golgi-stained apical dendrite segments after injection of z-DEVD-fmk or vehicle. Scale bar represents 10 μm. ! Right, high-magnification micrograph of the area delineated by the box. Scale bar represents 1 μm. Graph shows cumulative frequencies of spine head diameters. P = 0.002 (n = 4 mice, 1,000 spines per group). () Percentage of freezing time during CFC test in vehicle- and z-DEVD-fmk–treated wild-type and Tg2576 mice. Data are expressed as mean ± s.e.m. ***P < 0.001 (n = 7 mice for each experimental group). Author information * Abstract * Author information * Supplementary information Affiliations * Dulbecco Telethon Institute at the Laboratory of Molecular Neuroembryology, Istituto di Ricerca e Cura a Carattere Scientifico (IRCCS) Fondazione Santa Lucia, Rome, Italy. * Marcello D'Amelio, * Virve Cavallucci & * Francesco Cecconi * Laboratory of Molecular Neuroscience, University Campus-Biomedico, Rome, Italy. * Marcello D'Amelio * Consiglio Nazionale delle Ricerche (CNR) Institute of Neuroscience, IRCCS Fondazione Santa Lucia, Rome, Italy. * Silvia Middei & * Martine Ammassari-Teule * Laboratory of Molecular Mechanisms of Synaptic Plasticity, European Brain Research Institute, Rome, Italy. * Cristina Marchetti & * Hélène Marie * Laboratory of Cellular Physiology of Cortical Microcircuits, European Brain Research Institute, Rome, Italy. * Simone Pacioni & * Alberto Bacci * CNR Institute of Neuroscience, Department of Psychobiology and Psychopharmacology, Rome, Italy. * Alberto Ferri * Laboratory of Neuroimmunology, IRCCS Fondazione Santa Lucia, Rome, Italy. * Adamo Diamantini & * Luca Battistini * Department of Biology, University of Rome 'Tor Vergata', Rome, Italy. * Daniela De Zio & * Francesco Cecconi * Department of Biology-Interdepartmental Laboratory of Electron Microscopy (LIME), University Roma Tre, Rome, Italy. * Paolo Carrara & * Sandra Moreno * Present address: Institut de Pharmacologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique/Université Nice-Sophia Antipolis, Valbonne, France. * Hélène Marie Contributions M.D. and V.C. designed and carried out all of the molecular biology experiments and caspase-3 analysis. V.C. helped write the manuscript. S. Middei and M.A.-T. performed behavioral and dendritic spine analysis. S. Middei performed surgery. A.B. and S.P. performed LTP analysis. H.M. and C.M. performed patch-clamp and LTD experiments. A.F. carried out calcineurin activity assays. S. Moreno and P.C. performed immunoelectron microscopy analysis. L.B. and A.D. performed fluorescence-activated cell-sorting analysis. D.D.Z. analyzed the oxidative stress. M.D. and F.C. conceived and designed the study, supervised all of the experiments and wrote the manuscript. All of the authors discussed the results and commented on the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Francesco Cecconi Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (3M) Supplementary Figures 1–23 Additional data - Loss of Cav1.3 (CACNA1D) function in a human channelopathy with bradycardia and congenital deafness
- Nat Neurosci 14(1):77-84 (2011)
Nature Neuroscience | Article Loss of Cav1.3 (CACNA1D) function in a human channelopathy with bradycardia and congenital deafness * Shahid M Baig1, 11 Search for this author in: * NPG journals * PubMed * Google Scholar * Alexandra Koschak2, 11 Search for this author in: * NPG journals * PubMed * Google Scholar * Andreas Lieb2 Search for this author in: * NPG journals * PubMed * Google Scholar * Mathias Gebhart2 Search for this author in: * NPG journals * PubMed * Google Scholar * Claudia Dafinger3 Search for this author in: * NPG journals * PubMed * Google Scholar * Gudrun Nürnberg4 Search for this author in: * NPG journals * PubMed * Google Scholar * Amjad Ali1 Search for this author in: * NPG journals * PubMed * Google Scholar * Ilyas Ahmad1 Search for this author in: * NPG journals * PubMed * Google Scholar * Martina J Sinnegger-Brauns2 Search for this author in: * NPG journals * PubMed * Google Scholar * Niels Brandt5, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Jutta Engel5, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Matteo E Mangoni7 Search for this author in: * NPG journals * PubMed * Google Scholar * Muhammad Farooq1 Search for this author in: * NPG journals * PubMed * Google Scholar * Habib U Khan8 Search for this author in: * NPG journals * PubMed * Google Scholar * Peter Nürnberg4, 9 Search for this author in: * NPG journals * PubMed * Google Scholar * Jörg Striessnig2 Contact Jörg Striessnig Search for this author in: * NPG journals * PubMed * Google Scholar * Hanno J Bolz3, 10 Contact Hanno J Bolz Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature NeuroscienceVolume: 14,Pages:77–84Year published:(2011)DOI:doi:10.1038/nn.2694Received24 May 2010Accepted25 October 2010Published online05 December 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 Deafness is genetically very heterogeneous and forms part of several syndromes. So far, delayed rectifier potassium channels have been linked to human deafness associated with prolongation of the QT interval on electrocardiograms and ventricular arrhythmia in Jervell and Lange-Nielsen syndrome. Cav1.3 voltage-gated L-type calcium channels (LTCCs) translate sound-induced depolarization into neurotransmitter release in auditory hair cells and control diastolic depolarization in the mouse sinoatrial node (SAN). Human deafness has not previously been linked to defects in LTCCs. We used positional cloning to identify a mutation in CACNA1D, which encodes the pore-forming α1 subunit of Cav1.3 LTCCs, in two consanguineous families with deafness. All deaf subjects showed pronounced SAN dysfunction at rest. The insertion of a glycine residue in a highly conserved, alternatively spliced region near the channel pore resulted in nonconducting calcium channels that had abnormal voltage-d! ependent gating. We describe a human channelopathy (termed SANDD syndrome, sinoatrial node dysfunction and deafness) with a cardiac and auditory phenotype that closely resembles that of Cacna1d−/− mice. View full text Figures at a glance * Figure 1: SANDD syndrome mutation c.1208_1209insGGG. () Pedigree of family DEM9. Circles, females; squares, males. Samples from DEM9 individuals I:1, I:2 and II:1–II:4 were subjected to genome-wide linkage analysis. Ages are given next to the pedigree symbols. ECG symbols indicate individuals that were available for ECG recordings and had confirmed SAN dysfunction (red) or normal cardiac rhythm (black). Ins indicates presence of c.1208_1209insGGG (p.403_404insGly); + indicates wild-type sequence. Black symbols indicate congenitally deaf individuals. () Wild-type sequence of the C-terminal part of exon 8B. () Homozygosity for c.1208_1209insGGG as found in affected individuals of families DEM9 and DEM81. () Heterozygosity for the c.1208_1209insGGG mutation as found in carriers. () Pedigree of family DEM81. () Family DEM106 (with heterozygous carriers of the CACNA1D mutation). In families DEM9 and DEM81, affected individuals are homozygous for the CACNA1D mutation. * Figure 2: Haplotype analysis of families with SANDD syndrome. The three families share the same haplotype associated with the c.1208_1209insGGG founder mutation, bounded by SNPs rs720625 and rs1526594 (corresponding to a genomic region of 9.1 Mb). rs723540 (gray background) is an intragenic SNP, indicating the position of the CACNA1D gene. Disease-associated haplotypes are shown in red. Mb: Positions of SNPs on chromosome 3 in megabases. * Figure 3: Audiograms from people with SANDD syndrome and normal controls. Representative pure-tone air conduction audiograms from affected individuals (DEM9: II:3, II:4; DEM81: I:1, I:2) from both families with SANDD. II:5 from the DEM9 family represents a heterozygous CACNA1D mutation carrier. Black, right ear; gray, left ear. * Figure 4: ECG recordings from people with SANDD syndrome and normal controls. (–) ECG recordings from an unaffected person () and three individuals who are homozygous for the CACNA1D mutation (–). In and , asterisks mark P waves that precede QRS complexes; arrows indicate waveforms that suggest P waves coinciding with T waves. In affected individuals, both P-P and R-R intervals are variable (SAN arrhythmia). Numbers indicate heart rate (bpm) calculated from the corresponding beat-to-beat R-R interval. * Figure 5: Cav1.3 α1 subunit containing the p.403_404insGly mutation. () Simplified transmembrane topology of the pore-forming α1 subunit of voltage-gated Ca2+ channels. The approximate position of the insertion mutation p.403_404insGly is indicated by the black dot. () A sequence alignment of exons 8A, 8B and mutant 8B (8Bm) and comparison with other LTCC α1 subunit isoforms (GenBank Accession numbers are EU363339, NM_000720.2, CAA84353, AJ224874 and NP_000060 for human CaV1.3 (exon 8A– and exon 8B–containing splice variants, respectively), CaV1.2, CaV1.4 and CaV1.1, respectively). For CaV1.2, the exon 8A splice variant is shown14. () Immunoblot quantification of CaV1.3 α1 subunits with exons 8A, 8B or 8Bm in membrane protein preparations from transfected tsA-201 cells. Membrane protein isolated in parallel experiments from cells transfected only with accessory subunits but no α1 subunits served as controls (mock, M). Equal loading of lanes was verified by Coomassie staining of immunoblot membranes. One of four experiments is shown. I! ntroduction of the mutation did not reduce expression, which was either similar to wild-type (96 and 100%, two independent experiments) or higher (171 and 184%, two independent experiments). * Figure 6: Biophysical properties of wild-type and mutant Cav1.3 LTCCs. () Current-voltage curves for wild-type (8B, black) and mutant (8Bm, gray) channels and mock-transfected controls (triangles). () Exemplary current traces from experiments shown in (8B: 90502_1; 8Bm: 93001_12; mock: 90402_28). Right panel, enlarged ON-gating currents. () Exemplary ON-gating currents (8B, black: 92601_10; 8Bm, gray 93001_37) at Vrev. () Normalized mean ON-gating currents at Vrev. () Statistical comparison of peak ON-gating current amplitude. () Parameters of gating current kinetics: peak width at 50% of the maximum gating current; TTP, time-to-peak; and τd, mono-exponential decay time constant. Box-plots show median, 25% and 75% percentiles, minimum and maximum; Mann-Whitney test. () Maximal Itail peak amplitude versus QON. Calculated slopes: 6.03 for 8B (different from zero: P < 0.0001) and −0.5 pA fC−1 for 8Bm (P = 0.07). No modulation by 5 μM BayK8644. () Voltage-dependence of integrated ON-gating currents (QON, 8B and 8Bm channels) and of ionic cond! uctance G (Itail, wild-type only). Solutions: intracellular INNMDG (QON-V and G-V) and extracellular EXMg,Cd,La (QON-V) or EXCa (G-V). QON-V, 8B (in mV): Vh: −22.5 ± 2.0, slope: 16.8 ± 1.3; 8Bm(in mV): Vh: −38.7 ± 11.9, slope: 11.9 ± 0.7; G-V, 8B (in mV): Vh: −11.5 ± 2.2, slope: 14.3 ± 0.7. Unpaired Student's t-test: QON-V (8B) versus QON-V (8Bm): Vh, P < 0.0001; slope, P < 0.05; QON-V (8B) versus G-V (8B): Vh, P < 0.002; slope, P < 0.15. Representative ON-gating currents at indicated voltages are shown in the inset (8B, black: 100308_5160; 8Bm, gray: 101208_3240). Number of experiments is indicated in parentheses. Data are means ± s.e.m. **, P < 0.01; *, P < 0.05. * Figure 7: Cav1.3 transcripts containing exon 8A and exon 8B in IHCs and SAN. () Expression of alternatively spliced exon 8A and 8B transcripts in adult mouse total organ of Corti (C) and in two independent RNA preparations of adult mouse cochlear inner hair cells (IHC). () Expression of alternatively spliced exon 8A and 8B RNA transcripts in adult mouse SAN and left ventricle (V). The reactions contained 10 ng of RNA equivalent. The two rightmost lanes show simultaneous amplification of GAPDH (Genbank accession number: AK 144690) and exon 8A or 8B in multiplex reactions. b, adult whole brain; 8A and 8B, specific template controls (~107 molecules per reaction); n, no template. Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions GenBank * EU363339 * NM_028981 * NM_000720.2 * CAA84353 * AJ224874 * NP_000060 * AK 144690 Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Shahid M Baig & * Alexandra Koschak Affiliations * Human Molecular Genetics Laboratory, Health Biotechnology Division, National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan. * Shahid M Baig, * Amjad Ali, * Ilyas Ahmad & * Muhammad Farooq * Pharmacology and Toxicology, Institute of Pharmacy, Center for Molecular Biosciences, University of Innsbruck, Innsbruck, Austria. * Alexandra Koschak, * Andreas Lieb, * Mathias Gebhart, * Martina J Sinnegger-Brauns & * Jörg Striessnig * Institute of Human Genetics, University Hospital of Cologne, Cologne, Germany. * Claudia Dafinger & * Hanno J Bolz * Cologne Center for Genomics and Institute for Genetics, University of Cologne, Cologne, Germany. * Gudrun Nürnberg & * Peter Nürnberg * Institute of Physiology II and Tübingen Hearing Research Centre, University of Tübingen, Tübingen, Germany. * Niels Brandt & * Jutta Engel * Department of Biophysics, Saarland University, Homburg/Saar, Germany. * Niels Brandt & * Jutta Engel * CNRS, UMR 5203, INSERM U661, Université de Montpellier 1 et 2, Institut de Génomique Fonctionnelle, Département de Physiologie, Montpellier, France. * Matteo E Mangoni * Department of Pathology, Khyber Teaching Hospital, Peshawar, Pakistan. * Habib U Khan * Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germany. * Peter Nürnberg * Center for Human Genetics, Bioscientia, Ingelheim, Germany. * Hanno J Bolz Contributions S.M.B. coordinated and conducted the identification of families with deafness and collection of DNA samples and clinical data. A.A., I.A. and M.F. arranged clinical investigations. H.U.K. facilitated and conducted most of the clinical investigations at Khyber Teaching Hospital, Peshawar. G.N. and P.N. performed genetic mapping. C.D. performed molecular genetic analyses. A.K. and A.L. performed electrophysiological analyses. M.G. cloned wild-type and mutant channels and performed PCR and western blot analysis. M.J.S.-B. performed PCR analysis. N.B., J.E. and M.E.M. provided IHC and SAN tissues. A.K. and J.S. coordinated experiments and wrote the manuscript. H.J.B. initiated, planned and coordinated the study and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Hanno J Bolz or * Jörg Striessnig Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–3 Additional data - HCN channelopathy in external globus pallidus neurons in models of Parkinson's disease
- Nat Neurosci 14(1):85-92 (2011)
Nature Neuroscience | Article HCN channelopathy in external globus pallidus neurons in models of Parkinson's disease * C Savio Chan1 Search for this author in: * NPG journals * PubMed * Google Scholar * Kelly E Glajch1 Search for this author in: * NPG journals * PubMed * Google Scholar * Tracy S Gertler1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jaime N Guzman1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jeff N Mercer1, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Alan S Lewis2 Search for this author in: * NPG journals * PubMed * Google Scholar * Alan B Goldberg1 Search for this author in: * NPG journals * PubMed * Google Scholar * Tatiana Tkatch1, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Ryuichi Shigemoto3 Search for this author in: * NPG journals * PubMed * Google Scholar * Sheila M Fleming4 Search for this author in: * NPG journals * PubMed * Google Scholar * Dane M Chetkovich1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Pavel Osten1, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Hitoshi Kita6 Search for this author in: * NPG journals * PubMed * Google Scholar * D James Surmeier1 Contact D James Surmeier Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 14,Pages:85–92Year published:(2011)DOI:doi:10.1038/nn.2692Received30 July 2010Accepted01 October 2010Published online14 November 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 Parkinson's disease is a common neurodegenerative disorder characterized by a profound motor disability that is traceable to the emergence of synchronous, rhythmic spiking in neurons of the external segment of the globus pallidus (GPe). The origins of this pathophysiology are poorly defined for the generation of pacemaking. After the induction of a parkinsonian state in mice, there was a progressive decline in autonomous GPe pacemaking, which normally serves to desynchronize activity. The loss was attributable to the downregulation of an ion channel that is essential in pacemaking, the hyperpolarization and cyclic nucleotide–gated (HCN) channel. Viral delivery of HCN2 subunits restored pacemaking and reduced burst spiking in GPe neurons. However, the motor disability induced by dopamine (DA) depletion was not reversed, suggesting that the loss of pacemaking was a consequence, rather than a cause, of key network pathophysiology, a conclusion that is consistent with the abil! ity of L-type channel antagonists to attenuate silencing after DA depletion. View full text Figures at a glance * Figure 1: Dopamine depletion reduces autonomous activity of GPe neurons. () Representative cell-attached recordings of naive and 6-OHDA–lesioned cells. Right, histogram of discharge rates from naive and various DA-depleting conditions. α-MT, α-methyl-DL-tyrosine methyl ester hydrochloride. () Discharge rate versus limb asymmetry in animal from which neuron was taken; number of observations is denoted by temperature of colors. Right, histogram of the same set of data from 6-OHDA animals. Limb-use symmetry ratio of naive animals was assumed to be 1. () Fraction of active neurons as a function of limb asymmetry after 6-OHDA lesions. * Figure 2: Spontaneous activity of GPe neurons from naive and chronic DA depletion in vivo. () Unit activity recordings of GPe neurons from before and after muscimol injection (0.5 μg μl−1, total injection volume 0.1 μl) into the subthalamic nucleus (STN). In control rats, the firing rate decreased from 44.1 ± 11.8 to 25.2 ± 9.9 (n = 8), compared immediately before injection and 15 min after the injection. () Autocorrelograms of unit activity from various conditions. () Peristimulus time histogram of cortical stimulation–induced responses in the GPe. Cortical stimulation (arrow) evoked combinations of early excitation, inhibition, and late excitation in GPe neurons. * Figure 3: Diminished HCN channel activity in GPe neurons after acute and chronic DA depletion. () Membrane response of GPe neurons from naive and DA-depleted animals. () Summary of membrane response of GPe neurons to a series of current injections (mean ± s.e.m.). Dotted line, discontinuity of the membrane response from −60 mV and below from reserpine-treated group. For clarity, only silent neurons from the reserpine-treated animals were included. () Top, voltage clamp records of HCN currents from control and DA-depleted animals. Bottom, summary of voltage deflection versus current amplitude in control and DA-depleted animals. (P < 0.05, Mann-Whitney). The five reserpine-treated neurons with HCN current amplitudes >180 pA were spontaneously active. () Relationship between voltage deflection and HCN amplitude (correlation coefficient = 0.78, P < 0.001, Spearman rank correlation). () Tail currents from control and reserpine-treated cells. Fitting the current-voltage relationship of the HCN channel with a Boltzmann function reveals no difference (P > 0.05, Mann-Whitne! y) in the voltage dependence of activation (median: naive = −90.2 mV, n = 5; reserpine = −87.4 mV, n = 12). () Records similar to but from 6-OHDA–lesioned animals. () Reduction of HCN current amplitudes in GPe cells from control and 6-OHDA–lesioned mice (P < 0.05, Mann-Whitney). In data presented as box plots, the central line represents the median, the edges of the box represents the interquartile range, and the 'whisker lines' show the extent of the overall distribution. * Figure 4: Western blot and qPCR analysis of HCN channel subunits. () Membrane-bound protein expression of HCN1–HCN4 and TRIP8b in the GPe and the adjacent cortex from naive and DA-depleted mice. Loading control, α-tubulin. Bottom right, quantification of change in the GPe after reserpine treatment. Dotted line, control levels from naive animals. () qPCR expression analysis of HCN1–HCN4 and TRIP8b in GPe relative to whole brain. * Figure 5: Autonomous activity and HCN currents of GPe neurons from mice lacking HCN1 or HCN2. () Representative cell-attached recordings in wild-type mice and mice lacking HCN1 or HCN2. GPe neuron activity from HCN1-null mice (n = 14, P > 0.05, Mann-Whitney) was similar to that seen in wild type (n = 131), whereas that from HCN2-null mice was quiescent (n = 15). () Representative cell-attached recordings and tail current recordings in wild-type mice and mice lacking HCN1 or HCN2. HCN currents from GPe neurons from Hcn1−/− mice (n = 9) were largely similar to those of wild type (n = 50, P > 0.05, Mann-Whitney), whereas those from HCN2-null mice (n = 15, P < 0.05, Mann-Whitney) were markedly reduced. () Summary data of autonomous discharge and HCN current amplitude in GPe neurons from wild-type and subunit-specific mutant mice. * Figure 6: Recombinant HCN2 subunit delivery mediated by adeno-associated virus in GPe. () Schematic of regulated expression constructs and injection site. () Photomicrograph of mouse coronal brain section demonstrating adeno-associated virus infection (enhanced green fluorescent protein (eGFP) fluorescence) with stereotaxic intrapallidal injection. CPu, caudate putamen; Ctx, cortex; hip, hippocampus; Th, thalamus; ac, anterior commisure; ic, internal capsule. () Identification of adeno-associated virus infection in GPe tissue slice (left) and in a GPe neuron (right). Scale bar, 20 μm. () Representative in vitro cell-attached recordings in uninfected (n = 5) and infected neurons. Right, box plot of data. For clarity, we present only active neurons from the infected group. Right, the burst index of neurons were quantified. () Representative in vivo recordings in 6-OHDA–lesioned rats before and after adeno-associated virus infection. Right, box plot of data. * Figure 7: Cellular activity seemed encoded by Ca2+ influx via L-type Ca2+ channels in GPe neurons. (–) Burst firing induced Ca2+ influx in GPe neuron dendrites via Cav1/L-type Ca2+ channel. Antagonism of L-type channels in GPe neurons attenuated Ca2+ transients associated with spike bursts. Top () and bottom () panels, recordings from a pacemaking GPe neuron before and after a depolarizing current (80 pA) step (2 s) while imaging dendritic Ca2+ ~100 μm away from soma (). Current injection induced a robust Ca2+ influx that was sensitive to antagonism of L-type channels (5 μM isradipine). We calculated the extent of the reduction in Ca2+ signal by measuring the change in the G/R ratio before and after bursting. Box plot summarizes four GPe neurons in which the burst response was measured before and after isradipine application; responses were reduced by ~80%. (–) Cav1/L-type Ca2+ channel antagonism attenuates silencing in GPe neurons. Box plot () showing sparing of activity of GPe neurons from isradipine-treated mouse (red). Data are summarized as a cumulative plot (). Author information * Abstract * Author information * Supplementary information Affiliations * Department of Physiology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA. * C Savio Chan, * Kelly E Glajch, * Tracy S Gertler, * Jaime N Guzman, * Jeff N Mercer, * Alan B Goldberg, * Tatiana Tkatch, * Dane M Chetkovich, * Pavel Osten & * D James Surmeier * Department of Neurology and Clinical Neuroscience, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA. * Alan S Lewis & * Dane M Chetkovich * Division of Cerebral Structures, National Institutes for Physiological Sciences, Myodaiji, Okazaki, Japan. * Ryuichi Shigemoto * Department of Psychology, University of Cincinnati, Cincinnati, Ohio, USA. * Sheila M Fleming * Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA. * Pavel Osten * Department of Anatomy and Neurobiology, College of Medicine, The University of Tennessee Memphis, Memphis, Tennessee, USA. * Hitoshi Kita * Present addresses: Department of Orthopaedic Surgery, University of California, Irvine, California, USA (J.N.M.) and Istituto Italiano di Tecnologia, Via Morego, Genova, Italy (T.T.). * Jeff N Mercer & * Tatiana Tkatch Contributions C.S.C. and D.J.S. conceived the study, designed the experiments and directed the project. K.E.G. and P.O. designed the viral expression strategy and generated and validated the HCN2 vectors in vitro and in vivo. C.S.C., T.S.G., J.N.G., J.N.M. and H.K. carried out the recordings. A.S.L., R.S. and D.M.C. provided HCN mutants, generated the antibodies and carried out the western blots. C.S.C., A.B.G. and K.E.G. designed the probes and carried out the qPCRs. T.T. participated in the exploratory phase of the qPCR experiments. K.E.G and S.M.F. carried out the behavioral analyses. C.S.C. and D.J.S. wrote the paper. All authors discussed the results and implications and commented on the manuscript at all stages. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * D James Surmeier Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–7 Additional data - Principles governing recruitment of motoneurons during swimming in zebrafish
- Nat Neurosci 14(1):93-99 (2011)
Nature Neuroscience | Article Principles governing recruitment of motoneurons during swimming in zebrafish * Jens Peter Gabriel1, 2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Jessica Ausborn1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Konstantinos Ampatzis1 Search for this author in: * NPG journals * PubMed * Google Scholar * Riyadh Mahmood1 Search for this author in: * NPG journals * PubMed * Google Scholar * Emma Eklöf-Ljunggren1 Search for this author in: * NPG journals * PubMed * Google Scholar * Abdeljabbar El Manira1 Contact Abdeljabbar El Manira Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 14,Pages:93–99Year published:(2011)DOI:doi:10.1038/nn.2704Received21 September 2010Accepted19 October 2010Published online28 November 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 Locomotor movements are coordinated by a network of neurons that produces sequential muscle activation. Different motoneurons need to be recruited in an orderly manner to generate movement with appropriate speed and force. However, the mechanisms governing recruitment order have not been fully clarified. Using an in vitro juvenile/adult zebrafish brainstem-spinal cord preparation, we found that motoneurons were organized into four pools with specific topographic locations and were incrementally recruited to produce swimming at different frequencies. The threshold of recruitment was not dictated by the input resistance of motoneurons, but was instead set by a combination of specific biophysical properties and the strength of the synaptic currents. Our results provide insights into the cellular and synaptic computations governing recruitment of motoneurons during locomotion. View full text Figures at a glance * Figure 1: Synaptic activity of identified motoneurons. () Schematic drawing of the in vitro brainstem/spinal cord preparation. () Overlay of differential interference contrast (DIC) and fluorescence image showing the location of rhodamine-backfilled motoneurons in the spinal cord. Vertical scale bar indicates how soma position is measured using the normalized distance between the edge of the Mauthner (M) axon and the dorsal edge of the spinal cord. () Transverse section of the spinal cord showing the dorso-ventral and medio-lateral organization of the motor column. () Locomotor episodes were elicited by electrical stimulation (stimulation artifact at beginning of trace) and monitored by recording from the motor nerve. The recorded dorsal sMNs (see position in ) showed only subthreshold membrane potential oscillations. () Ventral sMNs (see position in ) showed large membrane potential oscillations that were correlated with motor nerve bursts and reached the action potential threshold. * Figure 2: Synaptic activity of the four different pools of motoneurons during locomotion. () Position of a recorded pMN in an overlay of DIC and fluorescence images. () Activity of this pMN during a swimming episode. () Expanded recordings from the indicated region in . () Image showing the position of a dorsal sMN. () This dorsal sMN displayed subthreshold membrane potential oscillations during a swimming episode. () Expanded traces from the indicated region in . () Position of a ventromedial sMN close to the edge of the Mauthner axon. () This sMN showed large membrane potential oscillations that reached action potential threshold. () Expanded traces showing that this sMN often fired single action potentials during each locomotor burst. () Ventral sMN with a lateral position in the motor column. () The ventrolateral sMN was firing continuously during the swimming episode. () This type of motoneuron fired bursts of action potentials. Dotted lines indicate the dorsal edge of the Mauthner axon. * Figure 3: Relationship between the motoneuron membrane potential oscillation amplitude and the swimming frequency. (,,,) Quantification of burst frequency (gray) and oscillation amplitude (colored) during a single swimming episode in a typical pMN (cyan), dorsal sMN (purple), ventromedial sMN (green) and ventrolateral sMN (red). (,,,) Graphs showing the change in oscillation amplitude plotted as a function of swimming frequency for the same episodes in pMN (cyan), dorsal sMN (purple), ventromedial sMN (green) and ventrolateral sMN (red). The data in these graphs are from the individual motoneurons shown in Figure 2. * Figure 4: Quantification of parameters important for the recruitment of the different pools of motoneurons. () Incremental recruitment of four individual motoneurons at different swimming frequencies (same data and color code as in Fig. 3). The dotted lines indicate the frequency at which ventrolateral and ventromedial sMNs were recruited. () Averaged slope of the increase in the membrane potential oscillations from all examined motoneurons. The pooled data from all motoneurons are presented as median (middle line) and quartiles (outer lines). () Percentage of motoneurons from each pool that fired action potentials during the swimming episodes and their relative ventro-dorsal position in the spinal cord. () Plot of the membrane potential oscillation amplitude as a function of the relative soma position. All motoneurons in which the mean amplitude of the membrane potential oscillations was above 5 mV (dotted line) fired action potentials during swimming activity. () Plot of the amplitude of membrane potential oscillation as a function of the input resistance of motoneurons. () Plot! of the input resistance as a function of the relative ventro-dorsal position of the different motoneurons. () Correlation between the soma size of motoneurons and their relative ventro-dorsal position in the spinal cord. () Graph of the resting membrane potential and the relative ventro-dorsal position of the different motoneurons. () Principal component analysis using the different variables of all the recorded motoneurons. The motoneurons fell into four pools that were separate from each other. * Figure 5: Excitatory and inhibitory synaptic currents in the four pools of motoneurons. (,,,) Inward EPSCs were measured by holding the membrane potential of motoneurons at −65 mV, which corresponds to the reversal potential of inhibition. (,,,) Outward IPSCs were measured at a holding potential of 0 mV, which corresponds to the reversal potential of excitation. * Figure 6: The amplitude of synaptic currents is correlated with the order of recruitment of motoneurons. (–) Graphs showing the change in the amplitude of EPSCs (filled symbols) and IPSCs (open symbols) as a function of the swimming frequency in the different types of motoneurons (pMN, cyan; dorsal (D) sMN, purple; ventromedial (VM) sMN, green; ventrolateral (VL) sMN, red). () Graph of the average amplitude of EPSCs and IPSCs in individual motoneurons as a function of their position along the ventro-dorsal axis in the spinal cord. () Mean amplitude of excitatory and inhibitory synaptic currents in the different types of motoneurons. Error bars represent mean ± s.e.m. The filled (excitatory current) and open (inhibitory current) circles represent the data points obtained from individual motoneurons recorded in different experiments. () Plot showing the mean amplitude of excitatory synaptic currents from individual motoneurons as a function of their input resistance. () Plot showing the mean amplitude of inhibitory synaptic currents measured in individual motoneurons as a func! tion of their input resistance. Each data point in , and corresponds to a value obtained from an individual motoneuron recorded in separate experiments. * Figure 7: Characteristic intrinsic properties of the four pools of motoneurons. () Primary motoneurons required large currents to reach firing threshold and displayed strong adaptation. () Dorsal secondary motoneurons also required somewhat large currents to reach firing threshold and showed spike frequency adaptation. () Expanded trace from the region indicated with the solid bar in . () Ventromedial secondary motoneurons required less current to reach threshold and fired continuously during current injection. () Ventrolateral secondary motoneurons fired with bursts of action potentials in response to small current injections. () Expanded trace from the region indicated with the solid bar in . () Expanded trace from the region indicated with the solid bar in showing bursting behavior. () Post-inhibitory rebound induced by hyperpolarizing current injection (top). Injection of large hyperpolarizing current induced a sag potential (middle), followed by a burst of action potentials on termination of the current injection (bottom). Author information * Abstract * Author information Primary authors * These authors contributed equally to this work. * Jens Peter Gabriel & * Jessica Ausborn Affiliations * Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden. * Jens Peter Gabriel, * Jessica Ausborn, * Konstantinos Ampatzis, * Riyadh Mahmood, * Emma Eklöf-Ljunggren & * Abdeljabbar El Manira * Present address: Max Planck Institute for Medical Research, Heidelberg, Germany. * Jens Peter Gabriel Contributions J.P.G., J.A., K.A. and A.E.M. conceived the project and planned the experiments. J.P.G., J.A., K.A. and R.M. performed the experiments. All of the authors contributed to the analysis of the data, preparation of the figures and the writing of the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Abdeljabbar El Manira Additional data - The columnar and laminar organization of inhibitory connections to neocortical excitatory cells
- Nat Neurosci 14(1):100-107 (2011)
Nature Neuroscience | Article The columnar and laminar organization of inhibitory connections to neocortical excitatory cells * Dennis Kätzel1 Search for this author in: * NPG journals * PubMed * Google Scholar * Boris V Zemelman2 Search for this author in: * NPG journals * PubMed * Google Scholar * Christina Buetfering1 Search for this author in: * NPG journals * PubMed * Google Scholar * Markus Wölfel2 Search for this author in: * NPG journals * PubMed * Google Scholar * Gero Miesenböck1, 2 Contact Gero Miesenböck Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 14,Pages:100–107Year published:(2011)DOI:doi:10.1038/nn.2687Received18 August 2010Accepted08 October 2010Published online14 November 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 cytoarchitectonic similarities of different neocortical regions have given rise to the idea of 'canonical' connectivity between excitatory neurons of different layers within a column. It is unclear whether similarly general organizational principles also exist for inhibitory neocortical circuits. Here we delineate and compare local inhibitory-to-excitatory wiring patterns in all principal layers of primary motor (M1), somatosensory (S1) and visual (V1) cortex, using genetically targeted photostimulation in a mouse knock-in line that conditionally expresses channelrhodopsin-2 in GABAergic neurons. Inhibitory inputs to excitatory neurons derived largely from the same cortical layer within a three-column diameter. However, subsets of pyramidal cells in layers 2/3 and 5B received extensive translaminar inhibition. These neurons were prominent in V1, where they might correspond to complex cells, less numerous in barrel cortex and absent in M1. Although inhibitory connection p! atterns were stereotypical, the abundance of individual motifs varied between regions and cells, potentially reflecting functional specializations. View full text Figures at a glance * Figure 1: ChR2 expression in GABAergic interneurons. (,) Targeting constructs. Homology sequences, dark gray; promoters, yellow; open reading frames, red; selection markers, light gray. () Construct used to generate the R26ChR2-EGFP allele. Cre-mediated excision of a triple-poly(A) transcriptional STOP cassette (3 × PA, black) flanked by loxP sites enables ChR2-EGFP expression from the CAG promoter. () Construct used to generate the Gad2Cre-ERT2 allele. An internal ribosome entry sequence (IRES, light red) separates the Gad65 and Cre-ERT2 reading frames. The expression of positive and negative selection markers is driven by the promoters of the mouse phosphoglycerate kinase-1 (Pgk1) and RNA polymerase II (RNA Pol2, or Ell2) genes. () Gad2CreT2R26ChR2-EGFP mice after tamoxifen induction express ChR2-GFP in Cre-positive cells (top), which comprise both Gad65- and Gad67-positive interneurons (middle), but not CamKIIα-positive pyramidal cells (bottom). See Supplementary Figure 1 for an analysis of interneuron subtypes and Supple! mentary Table 1 for statistics. Left and center columns, raw confocal images; right column, corresponding colocalization maps produced by multiplying the two fluorescence channels on a pixel-by-pixel basis and normalizing the resulting product image to 8 bits. * Figure 2: Genetically targeted photostimulation of GABAergic interneurons. (,) Responses to photostimulation in cell-attached () and whole-cell current-clamp recordings (); native resting potentials in parentheses. Interneurons followed trains of 20-ms optical pulses at 10 Hz with action potentials; pyramidal neurons were unresponsive to light. () ChR2-mediated photocurrents desensitized during repeated optical stimulation at frequencies >1 Hz (left) but remained stable at stimulation frequencies ≤0.2 Hz (right). () Spiking probabilities as a function of time after stimulus onset, estimated by analyzing 29–198 light-evoked action potentials per cell (n = 4 interneurons in cell-attached recordings; colored traces). Spike times are defined as the times at which the upstroke of an action potential reaches half-maximal amplitude. Average spike latencies (± s.d.) are indicated in matching colors. () Wide-field optical stimulation at 5 Hz (gray bars) evoked IPSCs in pyramidal cells voltage-clamped at 0 mV (gray traces). IPSCs were abolished after ba! th application of 10 μM gabazine (red trace, top) or 0.5 μM TTX (red trace, bottom). * Figure 3: Optogenetic mapping of inhibitory connectivity. () Contour plots depict spiking probabilities (left) and depolarization amplitudes (right) of three interneurons as functions of stimulus location. Blue dots indicate stimulation points; arrowheaded scale bars are 100 μm and point to the pial surface. Perisomatic illumination reliably elicited action potentials (left). Positioning the focus of the stimulating beam near dendritic branches did not cause higher spiking probabilities or larger depolarizations than illumination of dendrite-free neuropil equidistant from the soma (right). () Spiking probabilities of nine interneurons as functions of the distance of the stimulation spot from the soma. Cells were recorded in the cell-attached (n = 4 cells, green traces) or whole-cell configuration (n = 5 cells, blue traces). () Sequential illumination of ten different locations at 2.5 Hz (20 ms, 2 mW; gray tick marks). Illumination of sites marked by blue arrows gave rise to reproducible IPSCs in the recorded pyramidal cell. () Map! s of inhibitory inputs to pyramidal neurons in V1 (top row) or S1 (bottom row), located in L2/3 (left column) or L5B (right column) at comparable depths (±7 μm) from the surface of the slice. Two neurons were recorded simultaneously to test whether cells within the same local network could show different connectivity patterns. The positions of the two cells are marked by triangles; filled triangle denotes the postsynaptic target for each map. White rectangles indicate stimulation grid boundaries. Color symbolizes the average amount of charge flowing during the 100 ms after the onset of the IPSC, at a holding voltage of 0 mV. * Figure 4: Horizontal (columnar) organization of inhibitory connections. (,) Overlay maps of inhibitory inputs to pairs of simultaneously recorded pyramidal neurons in layer 2/3 of S1. The maps depict the locations of inhibitory inputs but not their strength and have been scaled to the size of a standard whisker-related barrel (yellow outlines). Triangles, cell positions; blue, data from the left cell in each pair; red, data from the right cell. () Pairs of pyramidal neurons in the same barrel-related column. () Pairs of pyramidal neurons in adjacent barrel-related columns. () Horizontal profiles of input distributions showing the interpolated number of inhibitory inputs as a function of horizontal distance from the center of the input distribution for each layer, ignoring the laminar (vertical) location of these inputs. Horizontal distances were scaled to the size of a standard somatosensory barrel (yellow outlines) and center-aligned; the number of inhibitory inputs to an excitatory neuron in a given layer at a given distance from the map cente! r was then averaged. The intensity of blue color symbolizes the density of input sources. See Online Methods and Supplementary Table 3 for statistical detail. * Figure 5: Vertical (laminar) organization of inhibitory connections. (–) Average strength of inhibitory input from the indicated source layers (rows) to excitatory neurons located in L2/3 (), L4 (), L5A (), L5B () and L6 (). An example of a neurobiotin-filled excitatory neuron, recovered after recording, is shown to the left of each panel. The figure summarizes data from 30 neurons in M1, 54 neurons in S1 and 53 neurons in V1. The strength of a connection is expressed as the average percentage of inhibitory charge flow arising from identified inputs in a layer. L5 represents the sum of L5A and L5B. Values are represented numerically (s.d. in parentheses) and by the intensity of gray shading. Colored outlines indicate significant differences (P < 0.05), either between two cortical areas (red) or between one area and the other two (blue), as determined by one-way analysis of variance and Tukey honestly significant difference (HSD) post hoc tests (Supplementary Tables 2 and 4). * Figure 6: Area-specific differences in the laminar organization of inhibitory connections. () Discriminant analysis of the laminar source distributions of inhibitory inputs to excitatory neurons in different target layers of M1, S1 and V1 (Supplementary Table 8). Neurons are represented as points in the coordinate system spanned by the discriminant functions Y1 and Y2 (arbitrary units). Borders between colored areas indicate decision boundaries for assigning neurons to M1 (blue), S1 (yellow) and V1 (red). Data points whose fill color matches the background color are classified correctly. Black squares indicate the centroid positions for all cells in each cortical area. () Abundance of interneuron subtypes expressing parvalbumin (P), somatostatin (S) and VIP (V) in the respective cortical layers and areas; pie chart areas represent overall interneuron densities (Supplementary Table 9). () Percentages and absolute numbers of inputs, charge flow and failure rates (means + s.d.) of the four translaminar motifs that showed area-specific differences. Bar colors: data fo! r M1 (blue), S1 (yellow) and V1 (red). Column graphs of data lacking significant differences between cortical areas are shown in faded colors. *P < 0.05, statistically significant differences between cortical areas as determined by analysis of variance and Tukey-HSD post hoc tests (Supplementary Tables 5, 6, 7 and 10). * Figure 7: Cell-specific differences in the laminar organization of inhibitory connections. (,) Hierarchical clustering of pyramidal cells in layers 2/3 () and 5B () of M1, S1 and V1. Classification variables were the strengths of intra- and translaminar inhibitory inputs, quantified as normalized inhibitory charge flow. Neurons in both layers fell into two well separated clusters: a minor population of neurons receiving strong translaminar inhibition (left cluster) and a major population of predominantly home layer–inhibited neurons (right cluster). Bootstrap estimates of cluster distances at the first bifurcation level are 0.64 ± 0.11 and 0.71 ± 0.09 (means ± s.d.) for pyramidal cells in layers 2/3 () and 5B (), respectively. Pie charts indicate the average strengths of inhibitory input from the dominant translaminar layer in black (layer 6 for neurons in layer 5B; layer 5 for neurons in layers 2/3), the home layer in gray and other layers in white. Colored letters denote the cortical area from which each observation is derived. Note the high frequency of V1! neurons and the absence of M1 neurons in the clusters receiving dominant translaminar inhibition. Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions GenBank * AF461397 Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK. * Dennis Kätzel, * Christina Buetfering & * Gero Miesenböck * Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut, USA. * Boris V Zemelman, * Markus Wölfel & * Gero Miesenböck Contributions D.K. and G.M. designed the study, analyzed the results and wrote the paper. B.V.Z. generated the R26ChR2-EGFP and Gad2CreERT2 targeting constructs. C.B. and M.W. helped with the initial characterization of the resulting mouse knock-in lines. D.K. performed all experiments. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Gero Miesenböck Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (3M) Supplementary Figures 1–8 and Supplementary Tables 1–11 Additional data - Auditory cortex spatial sensitivity sharpens during task performance
- Nat Neurosci 14(1):108-114 (2011)
Nature Neuroscience | Article Auditory cortex spatial sensitivity sharpens during task performance * Chen-Chung Lee1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * John C Middlebrooks2 Contact John C Middlebrooks Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Corresponding authorJournal name:Nature NeuroscienceVolume: 14,Pages:108–114Year published:(2011)DOI:doi:10.1038/nn.2713Received22 September 2010Accepted13 November 2010Published online12 December 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 Activity in the primary auditory cortex (A1) is essential for normal sound localization behavior, but previous studies of the spatial sensitivity of neurons in A1 have found broad spatial tuning. We tested the hypothesis that spatial tuning sharpens when an animal engages in an auditory task. Cats performed a task that required evaluation of the locations of sounds and one that required active listening, but in which sound location was irrelevant. Some 26–44% of the units recorded in A1 showed substantially sharpened spatial tuning during the behavioral tasks as compared with idle conditions, with the greatest sharpening occurring during the location-relevant task. Spatial sharpening occurred on a scale of tens of seconds and could be replicated multiple times in ~1.5-h test sessions. Sharpening resulted primarily from increased suppression of responses to sounds at least-preferred locations. That and an observed increase in latencies suggest an important role of inhibitor! y mechanisms. View full text Figures at a glance * Figure 1: Task-dependant modulation of spatial sensitivity. () Poststimulus time histogram (PSTH) showing activity as a function of time (horizontal axis) and head-centered stimulus location (vertical axis) for one example unit in A1 in the right hemisphere during the idle condition (ERRF width = 217°). Colors indicate mean spike activity. The thin white lines at the bottom of the plots indicate the 80-ms stimulus duration. White gaps crossing the plot corresponded to the spatial bins centered at ipsilateral and contralateral 90°, which were omitted from analysis. () PSTH of the same unit during the periodicity detection task (ERRF width = 173°). () PSTH of the same unit during the localization task (ERRF width = 143°). () Average spike rates during the onset response (10–40 ms) as functions of azimuth. Black, blue and red lines plot rate-azimuth functions in the idle condition, the periodicity detection task and the localization task, respectively. The data for three conditions were obtained in a single behavioral session, whi! ch lasted about 100 min. For the computation of the ERRF width, see Supplementary Figure 1. * Figure 2: Modulation of spatial sensitivity in sequential conditions. () PSTH of an example unit from the left hemisphere recorded during the first block of localization trials from the beginning of the recording (0 min) to the first 13 min. () The same unit recorded during a subsequent idle period (13–18 min). () Rate-azimuth functions of the onset responses for the first localization task (red) and the subsequent idle condition (black). () A second block of localization trials (25–34 min). () A second idle period (34–39 min). () Rate-azimuth functions of the onset responses for the second localization task (red) and the subsequent idle condition (black). Data are presented as in Figure 1. * Figure 3: PSTH plots in three task conditions from two units recorded from the left hemisphere that showed offset-dominant responses to 150-ms stimuli. (–) One unit recorded across three conditions (, idle; , periodicity detection; , localization) in a 105-min session. (–) One unit recorded across three conditions (, idle; , periodicity detection; , localization) in a 120-min session. For each unit, the ratio of offset to onset responses increased from idle to periodicity detection to localization conditions. Data are presented as in Figure 1. * Figure 4: Comparisons of ERRF width across conditions for all units that showed excitatory responses in the first 40 ms after stimulus onset. (–) Each symbol represents one unit, with the value in horizontal and vertical axes corresponding to its ERRF width in two different conditions. The symbols lying below the diagonal line represent units for which spatial tuning sharpened (and the ERRF width narrowed) for the condition indicated on the ordinate. o, units that did not show significant sharpening or broadening of ERRF widths; +, units that showed significant sharpening; x, units that showed significant broadening according to the ROC test. () Cumulative distributions of ERRF widths across three conditions. The horizontal dashed line crosses the curves at the median values. The percentages of the distributions to the left of the vertical dashed line had ERRF widths narrower than a hemifield (that is, ≤160°). * Figure 5: Percentage of units that showed significant sharpening or broadening of spatial tuning between condition pairs. Portions of the bars above or below the 0% line represent the percentage of units for which the ERRF width sharpened or broadened significantly for each two-way comparison. The upper portions of the middle and right bars are divided to represent units that sharpened their tuning significantly in the periodicity detection versus idle contrast (dark) or those that did not (light). * Figure 6: First spike latency for preferred locations was longer during behavioral conditions. Distributions of the trial-by-trial medians of first spike latencies for preferred locations are plotted for three conditions. Analysis of variance showed a significant effect of condition for the median first spike latency (P < 0.05). Differences were significant in two-way comparison between the idle condition and the periodicity detection task (t test, P < 0.01) and between the idle condition and the localization task (t test, P < 0.01), but not between the periodicity detection and localization tasks (t test, P = 0.89). Each box shows the upper and lower quartile and median as horizontal lines. + indicate points beyond the quartiles. * Figure 7: Spike rates decreased in the localization task primarily for stimuli at least-preferred locations. () Distributions of spike rates averaged across all stimulus locations for units that showed significant sharpening in the ROC analysis (Fig. 5). Mean spike rates decreased significantly across task conditions (Kruskal-Wallis test, P < 0.01). () Mean spike rates for stimuli at preferred locations showed no significant difference between the localization task and the idle condition (Wilcoxon rank sum test, P = 0.88). () Mean spike rates for least-preferred locations were suppressed significantly for the localization task compared with the idle condition (P < 0.05). * Figure 8: Time course of the task-dependent modulation of the response at single location that showed strongest suppression when the localization task was compared with the idle condition. () Normalized spike rates during four consecutive hit trials (1–4) followed by the four consecutive non-hit trials (5–8). The mean normalized spike rate for each of the four non-hit trials was significantly higher than the mean of all the hit presentations (Wilcoxon rank sum test, P < 0.0001 to P < 0.01, depending on trial). () Four non-hit trials (1–4) followed by four hit trials (5–8). The normalized spike rate for each of the first three non-hit trials was significantly higher than the mean of all the hit presentations that followed (P < 0.001 to P < 0.005). Error bars indicate the s.e.m. Author information * Abstract * Author information * Supplementary information Affiliations * Kresge Hearing Research Institute, Department of Otolaryngology-Head and Neck Surgery, University of Michigan, Ann Arbor, Michigan, USA. * Chen-Chung Lee * Department of Otolaryngology-Head and Neck Surgery and Center for Hearing Research, University of California, Irvine, California, USA. * Chen-Chung Lee & * John C Middlebrooks Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * John C Middlebrooks Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (3M) Supplementary Figure 1 Additional data - Hippocampal brain-network coordination during volitional exploratory behavior enhances learning
- Nat Neurosci 14(1):115-120 (2011)
Nature Neuroscience | Article Hippocampal brain-network coordination during volitional exploratory behavior enhances learning * Joel L Voss1 Contact Joel L Voss Search for this author in: * NPG journals * PubMed * Google Scholar * Brian D Gonsalves1 Search for this author in: * NPG journals * PubMed * Google Scholar * Kara D Federmeier1 Search for this author in: * NPG journals * PubMed * Google Scholar * Daniel Tranel2 Search for this author in: * NPG journals * PubMed * Google Scholar * Neal J Cohen1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 14,Pages:115–120Year published:(2011)DOI:doi:10.1038/nn.2693Received12 July 2010Accepted06 October 2010Published online21 November 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 Exploratory behaviors during learning determine what is studied and when, helping to optimize subsequent memory performance. To elucidate the cognitive and neural determinants of exploratory behaviors, we manipulated the control that human subjects had over the position of a moving window through which they studied objects and their locations. Our behavioral, neuropsychological and neuroimaging data indicate that volitional control benefits memory performance and is linked to a brain network that is centered on the hippocampus. Increases in correlated activity between the hippocampus and other areas were associated with specific aspects of memory, which suggests that volitional control optimizes interactions among specialized neural systems through the hippocampus. Memory is therefore an active process that is intrinsically linked to behavior. Furthermore, brain structures that are typically seen as passive participants in memory encoding (for example, the hippocampus) are a! ctually part of an active network that controls behavior dynamically as it unfolds. View full text Figures at a glance * Figure 1: Volitional control enhances spatial and object-specific memory. () Objects were viewed through a moving window, shown here moving rightward to uncover the topmost row of objects in a 5 × 5 grid. () Window position was under continuous active control for half of the object arrays and was delivered passively for the other half. Passive positions were determined by the active control of the previous experimental subject, so that object viewing was matched across conditions. (,) Results are shown for the spatial recall memory test () and for the object recognition memory test () for experiments (Exp.) 1–3. All experiments involved self-controlled (active) and passive viewing, with the task requirements for each condition varying among experiments (see text). High-confidence responses for the object recognition test are indicated with solid bars and low-confidence responses with shaded bars. The false-alarm rate to new items is also provided for high- and low-confidence levels. Error bars indicate the s.e.m. of the difference between condi! tions represented by adjacent bars and therefore correspond to the within-subjects statistical tests used. *P < 0.05 versus the right-adjacent condition. **P < 0.01 versus the right-adjacent condition. * Figure 2: Volitional control confers no perceptual benefit. Mean identification speed and accuracy are shown for the perceptual identification priming test, in which degraded viewing conditions rendered identification difficult. Error bars indicate the within-subjects s.e.m. of the difference between conditions represented by adjacent bars. Differences between old objects studied volitionally and studied passively were unreliable (P > 0.65). *P < 0.05 for old relative to new objects. * Figure 3: Volitional control caused disproportionate memory enhancement with increasing study durations. (,) Spatial positioning error () and old hit rates () are shown as a function of how long an object was studied (brief versus long based on median split). Average duration ranges for each condition are provided in Supplementary Table 1. Error bars indicate s.e.m. * Figure 4: Volitional control does not benefit memory performance in amnesia subjects with hippocampal amnesia. () Performance on the spatial recall test is quantified for comparison subjects as the proportion of objects placed in precisely the correct location ('bullseye hits'). Performance in subjects with amnesia is quantified as the proportion of objects placed within a perimeter of one object length from the correct location ('near hits') because bullseye hits were at floor levels in these subjects (0.11 volitional and 0.12 passive). () Performance on the object recognition test is quantified as discrimination sensitivity (d′), a normalized measure of hits minus false alarms. Error bars indicate the s.e.m. of the difference between conditions represented by adjacent bars, and therefore correspond to within-subjects volitional/passive difference error. Performance values for each subject are indicated by gray dots connected by gray lines. Performance for each grid size and subject is shown in Supplementary Table 2. * Figure 5: A coordinated brain network for volitional control. Regions that showed significantly greater correlated activity with the hippocampus for the volitional condition than for the passive condition (P < 0.01) are shown overlaid on anatomical MRI images in the transverse plane. The positions of transverse slices are indicated by horizontal lines on the sagittal midline image (right). Five structures for which activity predicted different aspects of memory performance are highlighted (Supplementary Fig. 2). The correlation with the hippocampus averaged across all identified regions is shown for the volitional (blue) and passive (red) conditions, with error bars indicating the s.e.m. of the within-subjects volitional/passive difference. All regions are described in Supplementary Table 3. FG, fusiform gyrus; IPL, inferior parietal lobe. Author information * Abstract * Author information * Supplementary information Affiliations * Beckman Institute for Advanced Science and Technology, University of Illinois Urbana-Champaign, Urbana, Illinois, USA. * Joel L Voss, * Brian D Gonsalves, * Kara D Federmeier & * Neal J Cohen * Department of Neurology, Division of Cognitive Neuroscience, University of Iowa Carver College of Medicine, Iowa City, Iowa, USA. * Daniel Tranel Contributions J.L.V., N.J.C., B.D.G. and K.D.F. conceived the experiments. J.L.V. designed and performed the experiments and analyzed data. D.T. provided access and support for testing subjects with amnesia. All authors co-wrote the paper, discussed results and commented on the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Joel L Voss Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (936K) Supplementary Tables 1–3 and Supplementary Figures 1 and 2 Additional data
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