Latest Articles Include:
- A critical look at connectomics
- Nat Neurosci 13(12):1441 (2010)
There is a public perception that connectomics will translate directly into insights for disease. It is essential that scientists and funding institutions avoid misrepresentation and accurately communicate the scope of their work. - The eye on the needle
- Nat Neurosci 13(12):1443-1444 (2010)
Tiny gaze shifts, or microsaccades, have little function in the eye movement control system and were once thought to be suppressed during fine spatial judgements. A new study suggests that they are important for finely guided visuomotor tasks and may actively contribute to the acquisition of spatial information in the same way as do larger saccades. - How hard is the CNS hardware?
- Nat Neurosci 13(12):1444-1446 (2010)
A study in this issue reveals gene expression differences between neurons that do, and those that do not, show recovery-associated growth after stroke. The differentially expressed genes may provide potential therapeutic targets. - Anandamide serves two masters in the brain
- Nat Neurosci 13(12):1446-1448 (2010)
Two studies in this issue find that postsynaptic TRPV1 receptors affect AMPA receptor endocytosis to mediate anandamide-induced long-term depression in the hippocampus and nucleus accumbens. - Illuminating the locus coeruleus: control of posture and arousal
- Nat Neurosci 13(12):1448-1449 (2010)
Optogenetic stimulation of the locus coeruleus noradrenergic neurons can increase wakefulness, and high-frequency stimulation decreases noradrenaline levels and produces loss of muscle tone similar to that seen in cataplexy. - A common origin of synaptic vesicles undergoing evoked and spontaneous fusion
- Nat Neurosci 13(12):1451-1453 (2010)
Nature Neuroscience | Brief Communication A common origin of synaptic vesicles undergoing evoked and spontaneous fusion * Yunfeng Hua1, 2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Raunak Sinha1, 2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Magalie Martineau2 Search for this author in: * NPG journals * PubMed * Google Scholar * Martin Kahms2 Search for this author in: * NPG journals * PubMed * Google Scholar * Jürgen Klingauf1, 2klingauf@uni-muenster.de Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 13,Pages:1451–1453Year published:(2010)DOI:doi:10.1038/nn.2695Received19 July 2010Accepted25 October 2010Published online21 November 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg There is a longstanding controversy on the identity of synaptic vesicles undergoing spontaneous versus evoked release. A recent study, introducing a new genetic probe, suggested that spontaneous release is driven by a resting pool of synaptic vesicles refractory to stimulation. We found that cross-depletion of spontaneously or actively recycling synaptic vesicle pools occurred on stimulation in rat hippocampal neurons and identified the recycling pool as a major source of spontaneous release. View full text Figures at a glance * Figure 1: Synaptic vesicles labeled by spontaneous or activity-dependent uptake exhibit identical release kinetics on stimulation. () On exocytosis, the biotin tag of biosyn or the ectodomain of Syt1 bearing the epitope were exposed to the extracellular space and bind exogenously added cypHer-labeled streptavidin or antibody. CypHer was quenched at the surface and exhibited maximal fluorescence at acidic pH. () Experimental protocol for labeling selective pools of synaptic vesicles. () Fluorescence images of hippocampal neurons expressing biosyn labeled with fluorescent monovalent streptavidin showed punctuate bouton staining. CypHer-labeled and Alexa488-labeled streptavidin were added in a ratio of 3:1. Average fluorescence responses of boutons labeled with monovalent cypHer-streptavidin spontaneously or by stimulation with 200 action potentials at 20 Hz were nearly identical. Traces were normalized to the size of the total labeled pool uncovered by a pulse of NH4Cl at the end of the experiment (n = 3 for each condition with >30 boutons per experiment). () Fluorescence images of spH-transfected hippoca! mpal neurons labeled with αSyt1-cypHer. Average cypHer fluorescence responses to 200 and 600 action potentials at 20 Hz (n = 4 with >50 boutons each) revealed that both evoked and spontaneously recycled synaptic vesicles exhibited the same release kinetics. Error bars indicate s.e.m. * Figure 2: Synaptic vesicles endocytosed spontaneously and on stimulation recycle equally to the recycling (Rc) and resting (Rs) synaptic vesicle pools. () Fluorescence images of hippocampal boutons labeled with αSyt1-cypHer by activity or spontaneously before (left) and after stimulation (900 action potentials at 20 Hz) in the presence of folimycin (middle), as well as during quenching of nonreleased dye with NH4Cl (right). () Corresponding average normalized fluorescence responses (n = 5 for each condition with >75 boutons per experiment), yielding sizes of the recycling (Rc) and resting (Rs) pool. () Histogram of relative recycling pool sizes (as a fraction of the total pool size) for boutons labeled during stimulation (black) or spontaneously (gray). () Dual-color measurement of spH-transfected neurons colabeled at rest with αSyt1-cypHer following the protocol in (n = 4 with >75 boutons each). Error bars indicate s.e.m. * Figure 3: Synaptic vesicles undergoing spontaneous and activity-dependent recycling originate from the recycling pool. () Schematic of the experimental design. () Fluorescence images of spH-transfected neurons with or without 15–20 min TTX and folimycin pre-incubation. A brief pulse of acid (pH 5.5) and NH4Cl was applied to determine the spontaneously released spH fraction (Fr) and the unreleased vesicular spH fraction (Fv). () Fluorescence amplitudes of Fr and Fv from normalized to the total vesicular fluorescence in control conditions (n = 6 for each condition with >75 boutons per experiment). () Experimental protocol and normalized average spH fluorescence responses to 900 action potentials at 20 Hz and successive NH4Cl perfusion with or without pre-incubation in TTX and folimycin for 15–20 min (n = 6 with >75 boutons). The cross-depletion demonstrates that most spontaneously recycled synaptic vesicles were drawn from the recycling pool. Error bars indicate s.e.m. Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Yunfeng Hua & * Raunak Sinha Affiliations * Department of Membrane Biophysics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany. * Yunfeng Hua, * Raunak Sinha & * Jürgen Klingauf * Department of Cellular Biophysics, Institute for Medical Physics and Biophysics, University of Muenster, Muenster, Germany. * Yunfeng Hua, * Raunak Sinha, * Magalie Martineau, * Martin Kahms & * Jürgen Klingauf Contributions Y.H. and R.S. designed, performed and analyzed the CypHer antibody and spH experiments. M.M. and M.K. designed, performed and analyzed the streptavidin-cypHer experiments. J.K. initialized the project. All of the authors wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Jürgen Klingauf (klingauf@uni-muenster.de) Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (500K) Supplementary Figures 1–5, Supplementary Notes 1–3, Supplementary Discussion and Supplementary Methods Additional data - The same synaptic vesicles drive active and spontaneous release
- Nat Neurosci 13(12):1454-1456 (2010)
Nature Neuroscience | Brief Communication The same synaptic vesicles drive active and spontaneous release * Benjamin G Wilhelm1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Teja W Groemer3 Search for this author in: * NPG journals * PubMed * Google Scholar * Silvio O Rizzoli1s.rizzoli@eni-g.de Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 13,Pages:1454–1456Year published:(2010)DOI:doi:10.1038/nn.2690Received03 June 2010Accepted12 October 2010Published online21 November 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Synaptic vesicles release neurotransmitter both actively (on stimulation) and spontaneously (at rest). It has been assumed that identical vesicles use both modes of release; however, recent evidence has challenged this view. Using several assays (FM dye imaging, pHluorin imaging and antibody-labeling of synaptotagmin) in neuromuscular preparations from Drosophila, frog and mouse, as well as rat cultured neurons, we found that the same vesicles participate in active and spontaneous release. View full text Figures at a glance * Figure 1: Synaptic vesicle recycling was visualized with FM 1-43. () Experimental procedure. Preparations were loaded with FM 1-43 either actively (electrical stimulation) or spontaneously. After a brief wash (10 min, 21–23 °C), the preparations were imaged, followed by unloading either spontaneously or actively. () Typical images of mouse NMJ preparations (see Supplementary Fig. 1 for the other preparations). Scale bar represents 10 μm. All image pairs are scaled identically. () Fraction of fluorescence decrease after unloading, for the four experimental loading/unloading combinations (A-A, active loading–active unloading; A-S, active loading–spontaneous unloading; S-S, spontaneous loading–spontaneous unloading; S-A, spontaneous loading–active unloading). Bars show average ± s.e.m. of 4–10 independent experiments (on average, 15 synapses were analyzed in each experiment). The exact loading/unloading protocols are presented in the Supplementary Methods. No statistically significant differences could be found (P > 0.05, one-w! ay ANOVA tests). As the dye washing time for hippocampal cultures (10 min) was close to the spontaneous release time (15 min), we reproduced these experiments with a shorter washing time (2 min, Supplementary Fig. 4). * Figure 2: Repeated recycling visualized with synaptotagmin antibodies. (–) Single-label marking of synaptic vesicles. () The preparation is incubated with biotinylated antibodies recognizing the lumenal domain of synaptotagmin. This antibody is internalized on active or spontaneous compensatory endocytosis. Antibodies bound to surface synaptotagmin molecules that were not endocytosed were then blocked with unlabeled streptavidin (gray). In a subsequent period of active or spontaneous exocytosis, Cy3-conjugated streptavidin was applied, labeling vesicles that had recycled during both periods of release. () Typical images. Scale bar represents 40 μm. () Average intensity ± s.e.m. for four independent experiments. No significant differences could be found (P > 0.7, one-way ANOVA test). (–) Double-label marking of synaptic vesicles. () The preparation was incubated with unlabeled rabbit antibodies to synaptotagmin (green), which label the vesicles recycling in a first round of active or spontaneous release. Unlabeled mouse synaptotagmin antib! odies (black, thick drawing) were applied to block surface epitopes. During a second round of release (active or spontaneous), fluorescently coupled mouse synaptotagmin antibodies (Atto647N, magenta, thick drawing) were applied. After fixation and permeabilization, the rabbit antibody was detected by conventional immunostaining (secondary antibodies to rabbit fluorescently labeled with Dyomics 480XL, green). Vesicle membranes containing both antibodies (both colors) must have recycled during both release rounds. () Typical isoSTED images. Scale bar represents 0.5 μm. Arrowheads indicate double-labeled vesicles. () Horizontal line scans through the vesicles indicated in . The full width at half maximum, obtained from fitting Gaussian curves to the data, is indicated as a measure of the spot size. () Correlation between Syt1 (green) and Atto647N (magenta) positive vesicles (cumulative histograms). We analyzed 128–174 punctae for each condition from two independent experime! nts. Inset shows the average Pearson's correlation coefficient! (± s.e.m.). No significant differences between the A-A/A-S or S-A/S-S labeling conditions could be found (P > 0.15, t tests). * Figure 3: Synaptic vesicle recycling visualized with spH. () Experimental procedure. spH-expressing neurons were stimulated in the presence of folimycin to inhibit re-acidification of newly endocytosed vesicles, leading to a fluorescence increase (dF1). Application of BPB quenched surface fluorescence, whereas the newly endocytosed, non-acidified vesicles remained fluorescent. Following a 1-min break (to allow for thorough penetration of the BPB into the cultures), a second electrical stimulation was performed (left) or the preparation was incubated for 15 min at rest (right). spH fluorescence of non-acidified vesicles was quenched when they were exocytosed in presence of BPB (dF2). Numbers indicate points of image acquisition. () Typical images of hippocampal cultures stimulated as described in , and then releasing in BPB either actively (top) or spontaneously (bottom). The arrowheads point to representative synapses. Image numbers refer to different experimental steps as indicated in . Scale bar represents 10 μm. () Comparison b! etween the fluorescence increase (amount of endocytosed vesicles) under stimulation in the presence of folimycin (dF1) and the fluorescence loss by exocytosis from this pool in BPB (dF2) either actively (open symbol) or spontaneously (closed symbol). Inset indicates the fraction of the active pool that was re-released in BPB (dF2/dF1, average ± s.e.m.); no significant differences were found (P > 0.7, t test). We analyzed 263 and 164 (A-S) boutons from two independent experiments. Author information * Author information * Supplementary information Affiliations * STED Microscopy Group, European Neuroscience Institute, Deutsche Forschungsgemeinschaft Center for Molecular Physiology of the Brain/Excellence Cluster 171, Göttingen, Germany. * Benjamin G Wilhelm & * Silvio O Rizzoli * International Max Planck Research School Neurosciences, Göttingen, Germany. * Benjamin G Wilhelm * Department of Psychiatry and Psychotherapy, University of Erlangen, Erlangen, Germany. * Teja W Groemer Contributions B.G.W., T.W.G. and S.O.R. designed and performed experiments. B.G.W. and T.W.G. evaluated the manuscript. S.O.R. supervised the project and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Silvio O Rizzoli (s.rizzoli@eni-g.de) Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (992K) Supplementary Figures 1–5 and Supplementary Methods Additional data - 5-HT2CRs expressed by pro-opiomelanocortin neurons regulate insulin sensitivity in liver
- Nat Neurosci 13(12):1457-1459 (2010)
Nature Neuroscience | Brief Communication 5-HT2CRs expressed by pro-opiomelanocortin neurons regulate insulin sensitivity in liver * Yong Xu1, 2, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Eric D Berglund1, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Jong-Woo Sohn1 Search for this author in: * NPG journals * PubMed * Google Scholar * William L Holland3 Search for this author in: * NPG journals * PubMed * Google Scholar * Jen-Chieh Chuang1 Search for this author in: * NPG journals * PubMed * Google Scholar * Makoto Fukuda1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jari Rossi1 Search for this author in: * NPG journals * PubMed * Google Scholar * Kevin W Williams1 Search for this author in: * NPG journals * PubMed * Google Scholar * Juli E Jones4 Search for this author in: * NPG journals * PubMed * Google Scholar * Jeffrey M Zigman1 Search for this author in: * NPG journals * PubMed * Google Scholar * Bradford B Lowell4 Search for this author in: * NPG journals * PubMed * Google Scholar * Philipp E Scherer3 Search for this author in: * NPG journals * PubMed * Google Scholar * Joel K Elmquist1joel.elmquist@utsouthwestern.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 13,Pages:1457–1459Year published:(2010)DOI:doi:10.1038/nn.2664Received21 July 2010Accepted15 September 2010Published online31 October 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Mice lacking 5-HT 2C receptors (5-HT2CRs) displayed hepatic insulin resistance, a phenotype normalized by re-expression of 5-HT2CRs only in pro-opiomelanocortin (POMC) neurons. 5-HT2CR deficiency also abolished the anti-diabetic effects of meta-chlorophenylpiperazine (a 5-HT2CR agonist); these effects were restored when 5-HT2CRs were re-expressed in POMC neurons. Our findings indicate that 5-HT2CRs expressed by POMC neurons are physiologically relevant regulators of insulin sensitivity and glucose homeostasis in the liver. View full text Figures at a glance * Figure 1: mCPP-induced depolarization of POMC neurons is abolished in 2C null mice and restored in 2C/POMC mice. () Whole-cell capacitance and resting membrane potential of POMC neurons. The numbers of cells recorded are indicated in parentheses. WT, wild type. () mCPP-induced changes in membrane potential (ΔmCPP) from all recorded POMC neurons and depolarized POMC neurons only. *P < 0.05; n.s., P > 0.05. () The percentage of POMC neurons that depolarized in response to mCPP. () Representative membrane potential traces. Horizontal bars indicate the period of mCPP treatment. Dotted lines indicate the resting membrane potential. All data are presented as mean ± s.e.m. * Figure 2: Re-expression of 5-HT2CRs in POMC neurons rescues insulin resistance. () ITTs in chow-fed mice (insulin, 1 U kg−1; n = 8 or 9 mice per genotype). *P < 0.05, ***P < 0.001, 2C null versus wild-type mice; #P < 0.05, ###P < 0.001, 2C null versus 2C/POMC mice. () GTTs in chow-fed mice (glucose, 1 g per kg; n = 5–12 mice per genotype). () Serum glucose in the fed or fasted conditions in chow-fed mice (n = 6–12 mice per genotype). () Serum insulin at fed or fasted conditions in chow-fed mice (n = 12–23 mice per genotype). () Serum insulin at 30 min after HFD-fed mice received a glucose bolus injection (0.75 g per kg; n = 6–14 mice per genotype). () Glucose infusion rate during a hyperinsulinemic-euglycemic clamp in chow-fed mice (n = 7–9 mice per genotype). **P < 0.01, 2C null versus wild-type mice; ##P < 0.01, 2C null versus 2C/POMC mice. () Hepatic glucose production at basal (preclamp) or hyperinsulinemic clamp conditions (n = 7–9 mice per genotype). () Glucose disposal rate at basal (pre-clamp) or hyperinsulinemic clamp conditions (! n = 7–9 mice per genotype). () Intraperitoneal injections of saline or mCPP (1.5 mg per kg), followed by GTT (0.75 g per kg) in wild-type, 2C null and 2C/POMC mice. †P < 0.05, ††P < 0.01, saline versus mCPP. () Intraperitoneal injections of saline or mCPP (1.5 mg per kg), followed by ITT (1.5 U kg−1) in wild-type, 2C null and 2C/POMC mice (n = 8–9 mice per group). †††P < 0.001, saline versus mCPP. All data are presented as mean ± s.e.m. Care of all animals and procedures were approved by University of Texas Southwestern Institutional Animal Care and Use Committees. Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Yong Xu & * Eric D Berglund Affiliations * Division of Hypothalamic Research and Departments of Internal Medicine and Pharmacology, UT Southwestern Medical Center, Dallas, Texas, USA. * Yong Xu, * Eric D Berglund, * Jong-Woo Sohn, * Jen-Chieh Chuang, * Makoto Fukuda, * Jari Rossi, * Kevin W Williams, * Jeffrey M Zigman & * Joel K Elmquist * Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA. * Yong Xu * Touchstone Diabetes Center, Department of Internal Medicine, UT Southwestern Medical Center, Dallas, Texas, USA. * William L Holland & * Philipp E Scherer * Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA. * Juli E Jones & * Bradford B Lowell Contributions Y.X. conducted the glucose/insulin experiments and wrote the manuscript. E.D.B. performed clamp studies. J.-W.S. and K.W.W. conducted electrophysiological recordings. J.-C.C., M.F. and J.R. contributed to the islet studies. J.E.J., J.M.Z. and B.B.L. generated mice. W.L.H., P.E.S. and B.B.L. assisted in data interpretation. J.K.E. supervised the project. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Joel K Elmquist (joel.elmquist@utsouthwestern.edu) Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (228K) Supplementary Figure 1, Supplementary Table 1 and Supplementary Methods Additional data - Toll-like receptor 7 mediates pruritus
- Nat Neurosci 13(12):1460-1462 (2010)
Nature Neuroscience | Brief Communication Toll-like receptor 7 mediates pruritus * Tong Liu1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Zhen-Zhong Xu1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Chul-Kyu Park1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Temugin Berta1 Search for this author in: * NPG journals * PubMed * Google Scholar * Ru-Rong Ji1rrji@zeus.bwh.harvard.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 13,Pages:1460–1462Year published:(2010)DOI:doi:10.1038/nn.2683Received30 August 2010Accepted04 October 2010Published online31 October 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Toll-like receptors are typically expressed in immune cells to regulate innate immunity. We found that functional Toll-like receptor 7 (TLR7) was expressed in C-fiber primary sensory neurons and was important for inducing itch (pruritus), but was not necessary for eliciting mechanical, thermal, inflammatory and neuropathic pain in mice. Our results indicate that TLR7 mediates itching and is a potential therapeutic target for anti-itch treatment in skin disease conditions. View full text Figures at a glance * Figure 1: Intact pain, but impaired itch, in Tlr7−/− mice. (,) Acute and persistent inflammatory pain induced by intraplantar formalin (5%, n = 5) and carrageenan (1%, n = 5) (P > 0.05 compared with wild-type control). () Total number of scratches in 30 min following intradermal injection of 50 μl of pruritic agents, including histamine (500 μg), HTMT (100 μg), compound 48/80 (48/80, 100 μg), serotonin (5-HT, 20 μg), endothelin-1 (ET-1, 25 ng), SLIGRL-NH2 (PAR2 agonist, 100 μg) and chloroquine (200 μg) in Tlr7−/− and wild-type (WT) mice (*P < 0.05, versus wild-type control, n = 5–8 mice). All data are means ± s.e.m. * Figure 2: Scratches induced by imiquimod. () Dose-dependent scratches after intradermal imiquimod treatment (n = 5–8). Inset, structure of imiquimod. () Imiquimod-induced scratches in wild-type and Tlr7−/− mice (n = 5). () Imiquimod-induced scratches after RTX and vehicle treatment in Trpv1−/− mice and wild-type control mice, as well as in mast cell–deficient SASH mice. *P < 0.05 versus saline; #P < 0.05; n.s., not significant (n = 5). All data are means ± s.e.m. * Figure 3: Expression of functional TLR7 in DRG neurons. () Immunohistochemistry showing TLR7 expression in DRG neurons. Scale bar represents 50 μm. (,) Single-cell RT-PCR showing colocalization of TLR7 with TPRV1, GRP and MrgprA3. M, molecular weights; N, negative controls from pipettes that did not harvest any cell contents, but were submerged in the bath solution. Asterisk indicates TLR7-positive neurons. Full-length gels are shown in Supplementary Figure 8a. () Inward currents evoked by imiquimod (IMQ, 500 μM) and capsaicin (0.5 μM) in small DRG neurons from wild-type mice; 8 of 17 neurons responded to imiquimod. All ten neurons from Tlr7−/− mice failed to respond to imiquimod. () Amplitude of inward currents evoked by imiquimod (20–500 μM). The numbers over the bars indicate the number of responsive neurons. Error bars represent mean ± s.e.m. () Action potentials evoked by imiquimod (500 μM) and capsaicin (0.5 μM) in small DRG neurons from wild-type mice. Note that imiquimod-induced action potentials were lost in! Tlr7−/− mice (n = 8 neurons). Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Tong Liu, * Zhen-Zhong Xu & * Chul-Kyu Park Affiliations * Sensory Plasticity Laboratory, Pain Research Center, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA. * Tong Liu, * Zhen-Zhong Xu, * Chul-Kyu Park, * Temugin Berta & * Ru-Rong Ji Contributions T.L. conducted behavioral tests for itch and acute pain and participated in experimental design and manuscript preparation. Z.-Z.X. performed immunohistochemistry and behavioral tests of pain. C.-K.P. conducted single-cell PCR and electrophysiology. T.B. performed in situ hybridization. R.-R.J. supervised the project, designed experiments and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Ru-Rong Ji (rrji@zeus.bwh.harvard.edu) Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–10 and Supplementary Methods Additional data - Kinesin 3 and cytoplasmic dynein mediate interkinetic nuclear migration in neural stem cells
- Nat Neurosci 13(12):1463-1471 (2010)
Nature Neuroscience | Article Kinesin 3 and cytoplasmic dynein mediate interkinetic nuclear migration in neural stem cells * Jin-Wu Tsai1, 3, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Wei-Nan Lian1, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Shahrnaz Kemal1 Search for this author in: * NPG journals * PubMed * Google Scholar * Arnold R Kriegstein2 Search for this author in: * NPG journals * PubMed * Google Scholar * Richard B Vallee1rv2025@columbia.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 13,Pages:1463–1471Year published:(2010)DOI:doi:10.1038/nn.2665Received16 July 2010Accepted15 September 2010Published online31 October 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 Radial glial progenitor cells exhibit bidirectional cell cycle–dependent nuclear oscillations. The purpose and underlying mechanism of this unusual 'interkinetic nuclear migration' are poorly understood. We investigated the basis for this behavior by live imaging of nuclei, centrosomes and microtubules in embryonic rat brain slices, coupled with the use of RNA interference (RNAi) and the myosin inhibitor blebbistatin. We found that nuclei migrated independent of centrosomes and unidirectionally away from or toward the ventricular surface along microtubules, which were uniformly oriented from the ventricular surface to the pial surface of the brain. RNAi directed against cytoplasmic dynein specifically inhibited nuclear movement toward the apical surface. An RNAi screen of kinesin genes identified Kif1a, a member of the kinesin-3 family, as the motor for basally directed nuclear movement. These observations provide direct evidence that kinesins are involved in nuclear migra! tion and neurogenesis and suggest that a cell cycle–dependent switch between distinct microtubule motors drives interkinetic nuclear migration. View full text Figures at a glance * Figure 1: Nuclear and centrosomal dynamics in RGPCs throughout the cell cycle. () Schematic diagram of transfection using intraventricular injection and in utero electroporation. RGPCs that expressed GFP expanded through the thickness of the neocortex (arrowheads). Scale bar represents 50 μm. () Live imaging of RGPCs expressing GFP (green) and CFP-histone H1 (magenta; H1 expression is shown for cell body in black and white at bottom) in live rat brain slice from E18. During a 4-h period the RGPC body moved apically toward the ventricular surface (dashed line) and entered mitosis. During mitosis the basal process (arrowheads) persisted, although it was thinner (5:40), and the progeny cells moved basally (Supplementary Video 1). Time shown as h:min. Scale bar represents 5 μm. () Kymograph of the same cell imaged using histone H1 for 18 h showing marked changes in nuclear migration rate in the apical direction, and prolonged, more uniform movement in the basal direction after mitosis. Scale bar represents 5 μm. () Tracings of nuclear movements of the R! GPC shown in and (blue) and two other cells (green and magenta). Each cell generated two progeny nuclei that moved basally at comparable rates. () Velocities of nuclear movements in . Bursts of fast movement up to 1 μm min−1 could be seen during apically directed (−5 h to 0 h) but not during basally directed movement (1 to 10 h). The tracings are aligned to mitotic start time (gray). () Histograms of the velocity during apically and basally directed movements. The velocity distribution of the apical nuclear movements shows a wide distribution of faster movements, whereas the distribution of basal movements is narrow. () Rat brain section fixed at E18, 2 d after in utero electroporation with cDNAs that encode GFP (green) and DsRed-centrin II (magenta). GFP+ cells with DsRed-centrin II–labeled centrosomes are seen in the ventricular zone (VZ; dashed box 1) and subventricular zone (SVZ; dashed box 2) with centrosomes throughout both regions and at the ventricular surfac! e in the endfeet of RGPCs. Scale bars represent 10 μm and 5 �! �m (inset). () Live imaging of a radial glial cell expressing GFP (green) and RFP-centrin II (magenta). Top, during downward (apical) nuclear movement, the centrosome remains at the ventricular surface. At the onset of mitosis (0:40) one centrosome (white arrows) departs from the ventricular surface and, after cytokinesis (2:00), returns to this site (3:00). The other centrosome stays close to the ventricular surface throughout mitosis (black arrows). The cell bodies of the daughter cells then migrate up in a comparably slow and continuous manner (Supplementary Video 2). Bottom, in some cases, one of the centrosomes departs from the ventricular surface (white arrows) while the other returns to the ventricular surface (Supplementary Video 4), as observed in asymmetric cell division20. Scale bar represents 10 μm. * Figure 2: Microtubule organization in RGPCs throughout the cell cycle. () Microtubules of RGPCs expressing GFP-tubulin (green) in E18 rat brain slices. Left, tubulin is distributed throughout interphase cells, with bundles of microtubules parallel to the long axis of the cell (arrows). Right, dividing cell showing mitotic spindle. Basal process remains visible during division (top, arrows), as detected using soluble RFP, whereas tubulin-GFP cannot be found in this region (bottom). () Time-lapse images of plus-ends of growing microtubules labeled with GFP-EB3 (green) in an interphase RGPC at E18. (Centrosome shown in magenta (arrowhead) by DsRed-centrin expression.) Fluorescence images were taken every second. Time series of EB3 images in three regions (basal process (top), soma (middle) and apical process (bottom)) are aligned to form a kymograph. 'Comet tail'-like EB3 streaks move basally (arrowheads). () GFP-EB3 behavior and microtubule organization in radial glial cells at different cell-cycle stages. () Superimposed sequential negative cont! rast images of EB3 streaks (arrowheads) and centrosomes (double arrowheads, red) from 30 s–2 min. () Tracings of the EB3 streaks (multiple colors represent individual microtubules). When the soma is in the top of the ventricular zone (G2), EB3 streaks mostly originate from the centrosomal region in the endfeet, curve around the nucleus and enter the basal process (arrows; Supplementary Videos 5 and 6). During mitosis (M), EB3 streaks radiate from the two spindle poles to form the mitotic spindle. No detectable EB3 streaks enter the basal process (Supplementary Video 7). During cytokinesis (Cyto), the microtubules radiate from the centrosomes in each daughter cell, with many microtubules aimed toward the midbody. EB3 streaks remain absent from the basal processes (Supplementary Video 8). Non-radial glial cells are seen in upper image. G1, left, paired cells after probable symmetric RGPC division with centrosomes at the endfeet of both daughter cells (Supplementary Video 9)! . EB3-tipped microtubules are oriented upward in both cells an! d re-enter the basal fibers (arrows). G1, right, paired cells after probable asymmetric RGPC division. The centrosome of daughter cell at right is shifted away with EB3 streaks emerging radially to form a bidirectional microtubule array (Supplementary Video 10). Scale bars in – represent 5 μm. () Velocity distribution. EB3 movements are comparable to migrating neurons and other cells. Kif1a RNAi does not significantly affect the rate of EB3 movement. () Direction distribution of EB3 movements during interphase: 93% are oriented basally. Kif1a RNAi has no obvious effect on the orientation of EB3 movement. * Figure 3: Effects of dynein functional inhibition on interkinetic nuclear migration in radial glial progenitors. () RGPC in rat brain slice at E21, 5 d after in utero electroporation with cDNA construct expressing cytoplasmic dynein heavy chain shRNA and GFP. The cell had normal bipolar morphology (left), but nuclear motility was limited to gradual drifting toward the ventricular surface (Supplementary Video 11). Scale bar represents 5 μm. () RGPC expressing dynein heavy chain shRNA plus CFP-histone-H1 (magenta) at E19, 3 d after in utero electroporation. The nucleus migrated toward the ventricular surface. but with shorter, more intermittent and slower movements than normal (see text; Supplementary Video 13). () Normal basally directed nuclear movement of paired RGPC nuclei at E19, after 3 d of dynein RNAi (conditions as in ; Supplementary Video 14). () INM was severely inhibited in cells that overexpressed dynamytin for 1.5 d (Supplementary Video 12). () Tracings of the nucleus in cells that expressed dynein shRNA for 5 d (left) and 3 d (right). Most of the nuclei showed neither api! cal nor basal movement after 5 d of dynein RNAi. However, after 3 d of dynein shRNA, apical movements were largely inhibited (dashed lines) but basal movements were normal (Supplementary Table 1). * Figure 4: Myosin IIB is not essential for interkinetic nuclear migration. () Protein blot showing a 65–70% decrease in myosin IIB expression in Rat2 cells transfected with myosin IIB shRNA for 3 d. () E18 rat brain slices monitored by live time-lapse imaging after treatment with myosin II RNAi or blebbistatin. Nuclei were imaged using CFP-histone-H1 and either GFP for RNAi or DsRed with blebbistatin. () GFP (green) and CFP-histone-H1 (magenta) were co-expressed with and without myosin IIB shRNA (Supplementary Videos 15 and 16). Myosin IIB shRNA had no apparent effect on basally directed movement of nuclei. () Cells co-transfected with DsRed (red) and CFP-histone-H1 (blue) were treated with 50 μm blebbistatin for 3–5 h during live cell imaging. Both apically directed () and basally directed () nuclear movement were unaffected. However, mitosis was blocked as expected (). Dashed lines indicate ventricular surface; see Supplementary Videos 17,18,19. Bar, 5 μm. () Duration of mitosis increases about two-fold in cells subjected to myosin IIB RNAi! . **P < 0.01. () Velocities of apically and basally directed movements in cells treated with myosin IIB shRNA and blebbistatin show no significant difference from control. * Figure 5: Kif1a is required for basally directed nuclear movement. () Immunoblotting of PC12 cells exposed to shRNA construct for 2 d shows that Kif1a RNAi reduced Kif1a by 70%. () E16 rat embryonic brains were electroporated with scrambled () or Kif1a shRNAs (), fixed and imaged at E20. Inset from is shown at higher power in . Kif1a RNAi caused clear and potent inhibition of cell redistribution relative to control, with almost complete loss of cells from intermediate zone (IZ) and cortical plate (CP), and accumulation of radial glial somata near ventricular surface. The persistent staining radial glial basal processes in IZ indicates retention of overall cell morphology. Scale bars represent 50 μm. () Quantification of cell distribution in scrambled versus Kif1a shRNA-expressing brain slices showing significant decrease in neural cell redistribution (***P < 0.001; Student's t test, compare with scrambled). () Apically directed nuclear movement recorded by time-lapse imaging of radial glia expressing scrambled shRNA (; Supplementary Video ! 20) and Kif1a shRNA (,). Basally directed nuclear movement was inhibited in both () or one () of the daughter cells. () The centrosome of the mobile daughter moved away from the ventricular surface, consistent with newborn neurons20 (Fig. 1h). Dashed lines indicate ventricular surface; time is hh:min (Supplementary Videos 21 and 22). () Tracings of nuclear migration show no effect of Kif1a RNAi in apical direction. () Kif1a RNAi impairs basal movement of all but one nucleus (blue line), which shows rapid movement characteristic of a newly formed neuron. Note potent inhibition of nuclear migration in sibling of this cell (also in blue). () Final nuclear position after recording of basally directed nuclear movements. Average nuclear positions were 34.096 ± 11.6 μm and 9.6 ± 6.5 μm for control and Kif1a RNAi conditions, respectively. (0 of y axis indicates ventricular surface; P < 0.001; Student's t test, n = 22.) * Figure 6: Effects of Kif1a RNAi on cell cycle progression and cell fate determination. () Brain sections 4 d after electroporation stained with anti-phosphovimentin (4A4) antibody (red) showing mitotic cells. Scale bar represents 50 μm. () Brain sections stained with antibodies to progenitor marker Pax6 (red) and neuronal marker TuJ1 (blue). Right, examples of TuJ1+ (arrows) and Pax6+ (arrowheads) cells in the boxed areas. Scale bar represents 100 μm. () Quantifications of the percentage of Pax6+ or TuJ1+ cells, respectively, among the transfected GFP+ cells in brain sections. *P < 0.05, **P < 0.01; Student's t test. * Figure 7: Rescue of Kif1a RNAi, for INM. () E16 rat brain transfected by in utero electroporation with Kif1a shRNA (green) tested for rescue by co-expression of DsRed (red); DsRed-human KIF1A (red) and myc-human KIF1A and fixed at E20. Co-expression of DsRed-human KIF1A (red) or myc-human KIF1A rescues the neuronal misdistribution phenotype but DsRed alone does not. Bar, 50 μm. () Statistical analysis of rescue experiments shows recovery of cell number within the three brain regions shown (Supplementary Table 3). For rescue by myc-KIF1A we counted all GFP+ cells, whereas for rescue by DsRed-KIF1A, GFP+ DsRed+ cells (~87% of total GFP+ cells) were counted. Significant rescue was produced by DsRed-KIF1A (gray bar, P < 0.001 compared with DsRed; Student's t test) and Myc-KIF1A (white bar, P < 0.001 compared with DsRed, Student's t test). () Live recording of basally directed nuclear movement in Kif1a RNAi cells (green) co-expressing DsRed (red; Supplementary Video 23), or rescued by co-expression of DsRed-KIF1A (red;! Supplementary Video 24). Bar, 5 μm. () Tracings of basally directed nuclear movement in control and DsRed-KIF1A cells. Each color represents progeny of one RGPC division. () Final nuclear position after recording of basally directed nuclear movements. Nuclear localization from apically directed nuclear movements were plotted. Average nuclear position is 34.22 ± 3.31 μm and 9.49 ± 2.61 μm for DsRed-huKIF1A and DsRed rescues of Kif1a RNAi, respectively. (0 μm in y axis indicates ventricular surface; P < 0.001; Student's t test, n = 11). Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Jin-Wu Tsai & * Wei-Nan Lian Affiliations * Department of Pathology and Cell Biology, Columbia University, College of Physicians & Surgeons, New York, New York, USA. * Jin-Wu Tsai, * Wei-Nan Lian, * Shahrnaz Kemal & * Richard B Vallee * Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, California, USA. * Arnold R Kriegstein * Present address: Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, California, USA. * Jin-Wu Tsai Contributions J.-W.T. and R.B.V. conceived and designed the project. J.-W.T. and W.-N.L. performed most of the experiments. S.K. assayed the biochemical effects of RNAi. The study was performed in the lab of R.B.V. and revisions in the lab of A.R.K. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Richard B Vallee (rv2025@columbia.edu) Supplementary information * Abstract * Author information * Supplementary information Movies * Supplementary Movie 1 (2M) Dynamics of normal interkinetic nuclear migration of radial glial progenitors. * Supplementary Movie 2 (3M) Centrosomal dynamics in radial glial prenitors throughout cell cycle. * Supplementary Movie 3 (4M) Centrosomal dynamics in radial glial progenitors throughout cell cycle. * Supplementary Movie 4 (776K) Centrosomal dynamics in radial glial progenitors throughout cell cycle. * Supplementary Movie 5 (364K) EB3 movements in premitotic radial glial progenitors. * Supplementary Movie 6 (468K) EB3 movements in premitotic radial glial progenitors. * Supplementary Movie 7 (1M) EB3 movements in radial glial progenitors during mitosis. * Supplementary Movie 8 (640K) EB3 movements in radial glial progenitors during cytokinesis. * Supplementary Movie 9 (604K) EB3 movements in radial glial progenitors after symmetric cell division. * Supplementary Movie 10 (2M) EB3 movements in radial glial progenitors after asymmetric cell division. * Supplementary Movie 11 (984K) Behavior of a radial glial progenitor after 5 days of dynein HC RNAi. * Supplementary Movie 12 (1M) Behavior of a radial glial progenitor after 1.5 days of dynamytin overepxression. * Supplementary Movie 13 (228K) Behavior of a radial glial progenitor after 3 days of dynein HC RNAi. * Supplementary Movie 14 (268K) Basally directed nuclear movment of a pair of radial glial progenitor progeny after 3 days of dynein HC RNAi. * Supplementary Movie 15 (352K) Basally directed nuclear movement of a radial glial progenitor after 2 days of myosin IIB RNAi. * Supplementary Movie 16 (1M) Basally directed nuclear movement of a radial glial progenitor after 2 days of myosin IIB control RNAi. * Supplementary Movie 17 (320K) Apically directed nuclear movement of a blebbistatin treated radial glial progenitor cell. * Supplementary Movie 18 (164K) Basally directed nuclear movement of a blebbistatin treated radial glial progenitor cell. * Supplementary Movie 19 (284K) Nuclear movement of a blebbistatin treated mitotic radial glial progenitor cell. * Supplementary Movie 20 (292K) Basally directed nuclear movement of a radial glial progenitor cells after 2 days of RNAi using scrambled KIF1A sequence. * Supplementary Movie 21 (124K) Failure in basally directed nuclear movement of a postmitotic radial glial progenitor cell after 2 days of KIF1A RNAi. * Supplementary Movie 22 (392K) Differential effects following 2 days of KIF1A RNAi on postmitotic progeny of asymmetrically divided radial glial progenitor cell. * Supplementary Movie 23 (76K) Failure of basally directed nuclear movement in postmitotic KIF1A shRNA-/DsRed-co-expressing radial glial progenitor cell. * Supplementary Movie 24 (252K) Rescue of basally directed nuclear movement in KIF1A shRNA-/DsRed-human KIF1A-co-expressing radial glial progenitor cell. * Supplementary Movie 25 (268K) Almost normal bidirectional nuclear migration behavior in KIF3A shRNA-expressing radial glial progenitor cell. * Supplementary Movie 26 (924K) Normal basally directed nuclear migration in KIF1B shRNA-expressing radial glial progenitor cell. PDF files * Supplementary Text and Figures (3M) Supplementary Figures 1–4 and Supplementary Tables 1–3 Additional data - Yy1 as a molecular link between neuregulin and transcriptional modulation of peripheral myelination
- Nat Neurosci 13(12):1472-1480 (2010)
Nature Neuroscience | Article Yy1 as a molecular link between neuregulin and transcriptional modulation of peripheral myelination * Ye He1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jin Young Kim1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jeffrey Dupree2 Search for this author in: * NPG journals * PubMed * Google Scholar * Ambika Tewari3 Search for this author in: * NPG journals * PubMed * Google Scholar * Carmen Melendez-Vasquez3 Search for this author in: * NPG journals * PubMed * Google Scholar * John Svaren4 Search for this author in: * NPG journals * PubMed * Google Scholar * Patrizia Casaccia1patrizia.casaccia@mssm.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 13,Pages:1472–1480Year published:(2010)DOI:doi:10.1038/nn.2686Received09 July 2010Accepted27 September 2010Published online07 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 Fast axonal conduction depends on myelin, which is formed by Schwann cells in the PNS. We found that the transcription factor Yin Yang 1 (YY1) is crucial for peripheral myelination. Conditional ablation of Yy1 in the Schwann cell lineage resulted in severe hypomyelination, which occurred independently of altered Schwann cell proliferation or apoptosis. In Yy1 mutant mice, Schwann cells established a 1:1 relationship with axons but were unable to myelinate them. The Schwann cells expressed low levels of myelin proteins and of Egr2 (also called Krox20), which is an important regulator of peripheral myelination. In vitro, Schwann cells that lacked Yy1 did not upregulate Egr2 in response to neuregulin1 and did not express myelin protein zero. This phenotype was rescued by overexpression of Egr2. In addition, neuregulin-induced phosphorylation of YY1 was required for transcriptional activation of Egr2. Thus, YY1 emerges as an important activator of peripheral myelination that lin! ks neuregulin signaling with Egr2 expression. View full text Figures at a glance * Figure 1: Peripheral nerve hypomyelination in mice with conditional ablation of Yy1. () Normal hindlimb postural reflex in a P18 control mouse (Yy1loxP/+;Cnp-cre+/−), characterized by spreading of the limbs, and abnormal reflex in the mutant Yy1loxP/loxPCnp-cre+/− mouse (CKO), characterized by crossing of the hind limbs. () Transcript levels of Yy1 in mouse sciatic nerves during development measured using quantitative reverse transcription PCR (qRT-PCR) n = 3 at each time point). () Teased sciatic nerves from P21 wild-type mice stained for YY1 (red) and myelin basic protein (MBP; green) and processed for confocal analysis. Scale bar, 20 μm. () Immunohistochemistry for YY1 (green) in the sciatic nerve of controls but not in mutant (Yy1loxP/loxP;Cnp-cre+/−) mice at P18. Cell nuclei were counterstained with DAPI (blue). () Gross examination of sciatic nerves dissected from Yy1loxP/loxP;Cnp-cre+/− and control mice at P18 show the white opaque appearance of control nerves and much thinner and more translucent appearance of mutant nerves. () qRT-PCR of RN! A from sciatic nerves of Yy1loxP/loxP;Cnp-cre+/− mice and control siblings at P1, P10 and P21. The bar graphs represent the transcript levels of Mpz and Pmp22 relative to controls. Error bar, s.d.; **P < 0.01, ***P < 0.001 (n = 3). () Longitudinal sections of the sciatic nerves from control and Yy1loxP/loxP; Cnp-cre+/− mice at P10 and P18 were stained for MPZ (green) and nuclei were counterstained with DAPI (blue). Scale bars, 20 μm in , , and 1 mm in . * Figure 2: Ablation of Yy1 impairs the ability of Schwann cells to myelinate. () Electron micrographs of sciatic nerves of controls and Yy1 mutants show severe hypomyelination in Yy1loxP/loxP; Cnp-cre+/− mice at P18. The Schwann cells in the mutant mice established a 1:1 relationship with large-caliber axons but could not myelinate them. () Scatter plot indicating the g ratios of individual fibers as a function of axon diameter (n = 78 axons for Yy1loxP/loxP; Cnp-cre+/− mice; n = 154 axons for controls). () Axons were classified into three categories: myelinated (blue bar), unmyelinated-sorted (red bar) and unmyelinated-Remak bundles (yellow bar) and quantified. () qRT-PCR revealed elevated Pou3f1 transcripts in the sciatic nerves of Yy1 mutant mice compared to controls. () Immunohistochemical validation of Pou3fl expression in longitudinal sections of the sciatic nerves from P18 Yy1loxP/loxP; Cnp-cre+/− mice and control mice. () Electron micrographs of longitudinal sections of sciatic nerves in mutant mice showed heminodes, consisting of unpair! ed paranodal regions, whereas nodal regions were rarely detected. () Longitudinal sections of P18 sciatic nerves from control and Yy1loxP/loxP; Cnp-cre+/− mice were stained for MBP (green) and for the paranodal protein Caspr (red). Note the uniform staining of myelinated MBP+ fibers and paired Caspr expression in control animals and the reduced level of MBP and disorganization of Caspr expression in mutant mice. Scale bars, 2 μm in , 50 μm in and 20 μm in . * Figure 3: Effect of Yy1 ablation on the ability of Schwann cells to myelinate in vitro. () In vitro explant DRG cultures from E13.5 control and Yy1loxP/loxP; Cnp-cre+/− embryos were immunostained for MBP (red) and neurofilament medium chain (NFM, blue). Note the few myelin segments in cultures from the Yy1loxP/loxP; Cnp-cre+/− mice compared to controls. () Bar graph shows the number of myelin segments shown in . () qRT-PCR of mRNA levels of Mpz in myelinating cultures established from Yy1loxP/loxP; Cnp-cre+/− and control DRGs and examined at days 0, 7 and 14 after induction of myelination. () Protein blot analysis of MBP and CNPase in myelinating cocultures derived from Yy1loxP/loxP; Cnp-cre+/− and control DRGs kept in culture for 21 d. Full-length blots are shown in Supplementary Figure 5. (,) Immunofluorescence of cocultures of DRG neurons and Schwann cells from E13.5 transgenic embryos (Yy1loxP/loxP;Plp-creERT) treated with 1 μM Tamoxifen (4OH-TM) for 2 d and then cultured for additional 12 d to induce myelination. MBP (red) and neurofilament (NFM, ! green). () Immunofluorescence of Yy1loxP/loxP; Cnp-cre+/− and control DRG neurons cultured with wild-type rat Schwann cells (WT SC) for 14 d and then stained for MBP (red) and NFM (green). Scale bars, 50 μm. Error bars, s.d.; **P < 0.01. * Figure 4: Ablation of Yy1 modulates Schwann cell S-phase entry during the second week of development. () Longitudinal sections of the sciatic nerves from P18 Yy1loxP/loxP; Cnp-cre+/− and control mice stained with anti-BrdU (red) antibodies after 2 h pulse labeling of BrdU in vivo. Scale bar, 20 μm. () The fraction of BrdU+ cells in the sciatic nerves at multiple developmental time points was quantified and referred to the percentage of DAPI+ cells. () qRT-PCR of genes involved in proliferation (Cdk4, cyclin D1 (Ccnd1) and P21 (Cdkn1a)) in the sciatic nerves of mutant mice and control littermates at P10. () TUNEL assay of the sciatic nerves from mutant mice and control littermates at the indicated time points. The fraction of TUNEL+ cells in the sciatic nerves was quantified and referred to the percentage of DAPI+ cells. () qRT-PCR of genes involved in apoptosis (Jun and Tgfb1) in the sciatic nerves of mutant mice and control littermates at P21. Error bar, s.d.; *P < 0.05, **P < 0.01. n = 3 for each genotype at P2, P10, P18 and P21 and n = 2 at P64. * Figure 5: YY1 regulates Egr2 expression. () Intersection of genes altered in the sciatic nerves of Yy1loxP/loxP; Cnp-cre+/− mice and of Egr2Lo/Lo and Nab1−/−;Nab2−/− mice17. () Comparison of gene expression in Yy1loxP/loxP; Cnp-cre+/−, Egr2Lo/Lo and Nab1−/−;Nab2−/− mice. Similar gene expression signatures in the sciatic nerves of the three mutants suggested that these molecules might share a common signaling pathway. The expression of Nab1, Nab2 () and Egr2 () was measured by qRT-PCR in the sciatic nerves at P21. () Immunohistochemistry showed decreased Egr2 (green) immunoreactivity in teased sciatic nerve fibers of Yy1loxP/loxP; Cnp-cre+/− mice. () Purified mouse Yy1loxP/loxP; Cnp-cre+/− Schwann cells cultured in myelination medium for 3 d showed normal S100 (blue) expression but low immunoreactivity for MPZ (red), and ectopic expression of Egr2 in these cells rescued the phenotype as indicated by the detection of MPZ in GFP+ transfected cells. Bar graph shows quantification of the fraction! of MPZ+ cells among S100+ cells from two experiments. () Protein blot of myelinating cocultures from Yy1loxP/loxP; Cnp-cre+/− and control DRGs maintained for 21 d in vitro showed low Egr2, whereas the NICD was only mildly affected. Full-length protein blots are shown in Supplementary Figure 5. () qRT-PCR of Notch1, Jag1 and CnB1 in the sciatic nerves of Yy1loxP/loxP; Cnp-cre+/− and control mice at distinct developmental time points. Error bar, s.d.; *P < 0.05, **P < 0.01 (n = 3). Scale bars, 20 μm. * Figure 6: YY1 regulates Egr2 expression in response to NRG1. () Purified rat Schwann cells were cultured in minimal medium for 18 h and then exposed to NRG1. The transcript levels of Egr1, Egr2, Nab1, Nab2 and Yy1 at each time point were analyzed by qRT-PCR. Egr2 transcripts increased at 20 min, peaked at 1 h and then gradually returned to basal levels by 2 h, whereas the expression of Yy1 increased only moderately. (−) Transcript levels of Yy1, Egr2, Nab1 and Nab2 in Schwann cells either mock-transfected or transfected with Yy1-specific shRNAs and cultured in the absence or presence of NRG1 for 30 min, 1 h or 2 h. The expression of each gene in control cells at time zero was arbitrarily set as 1. () Luciferase activity measured in NRG1-treated Schwann cells transfected with reporter constructs containing either the Egr2 promoter or the myelinating Schwann cell enhancer (MSE) of Egr2, together with pCX-Yy1 or empty vector. Values were referred to the readings obtained in untreated cells transfected with empty vector. Error bars, s.d! .; *P < 0.05, **P < 0.01. * Figure 7: YY1 binds to chromatin at the Egr2 locus only in Schwann cells treated with NRG1. () Schematic diagram of the promoter and MSE of Egr2. YY1 consensus binding sequences (green ovals) and the regions (R1–R8) analyzed by chromatin immunoprecipitation are indicated. The plot in pink shows highly conserved sequences between rat and human. Scale marks on x axis indicate 50 bp. () Chromatin from Schwann cells cultured in minimal medium (MM, white bar) or treated with NRG1 for 1 h (gray bars) or 12 h (black bars) was immunoprecipitated (ChIP) with antibodies against YY1 and the regions indicated in were amplified. The results are expressed as percent of input. YY1 was recruited to multiple regions of the Egr2 promoter and MSE after 1 h NRG1 treatment. () Protein blot analysis of YY1 in Schwann cells after NRG1 treatment. () Schematic diagram of human YY1 protein including the acidic N terminus, the glycine-lysine–rich central domain (GK) and the C-terminal DNA-binding domain, which is composed of four zinc fingers (ZF). Three highly conserved serine residues ! are marked in red. () Co-immunoprecipitation of protein lysates from rat Schwann cells kept in growth medium (GM) or minimal medium (MM), or treated with NRG1 for 20 min or 1 h. After immunoprecipitation (IP) with anti-YY1 antibodies, the protein blot (WB) was probed with anti-phospho-serine antibodies. Full-length blots are presented in Supplementary Figure 5. * Figure 8: The regulation of Egr2 by phosphorylated YY1 is mediated by NRG1-dependent MEK activation. () Rat Schwann cells were treated with NRG and the PI(3)K inhibitor LY294002, with the MEK inhibitors PD98059 or U0126, or with the calcineurin inhibitor CsA. The transcript levels of Egr2 were assessed by qRT-PCR. () ChIP of samples from Schwann cells either untreated or treated with NRG1 alone or with the MEK inhibitor U0126 or the calcineurin inhibitor CsA for 1 h and immunoprecipitated with antibodies to YY1. Note the decreased binding to region 7 of the Egr2 MSE in cells treated with U0126 but not CsA, in response to NRG stimulation. () Luciferase assay of Schwann cells cotransfected with YY1 SerAla mutation at position 118, 184, 247 and with Yy1 translucent reporter. Note the decreased ability of mutant YY1 to activate a YY1 binding sequence–driven reporter after NRG treatment. () Luciferase assay of Schwann cells treated with NRG1 and cotransfected with the indicated reporter constructs and the mutant Yy1 constructs. Error bar, s.d.; *P < 0.05. **P < 0.01. Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions ArrayExpress * E-MEXP-2919 Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Department of Neuroscience and Genetics and Genomics, Mount Sinai School of Medicine, New York, New York, USA. * Ye He, * Jin Young Kim & * Patrizia Casaccia * Department of Anatomy and Neurobiology, Virginia Commonwealth University, Richmond, Virginia, USA. * Jeffrey Dupree * Department of Cell Biology, Hunter College, New York, New York, USA. * Ambika Tewari & * Carmen Melendez-Vasquez * Department of Comparative Biosciences and Waisman Center, University of Wisconsin, Madison, Wisconsin, USA. * John Svaren Contributions Y.H. conducted the majority of the experiments and analyzed the data. J.Y.K. generated the YY1 point mutation plasmids and performed the immunoprecipitation and western blots. J.D. performed the ultrastructural analysis of the sciatic nerve. C.M.-V. and A.T. helped with the DRG co-culture experiments and conducted part of the in vitro myelination studies. J.S. performed the conservation analysis and contributed to the analysis of gene expression in the three mutants. P.C. supervised the project, analyzed the data, formatted the figures, uploaded microarray data and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Patrizia Casaccia (patrizia.casaccia@mssm.edu) Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–6 and Supplementary Tables 1–4 Additional data - Otx2 controls neuron subtype identity in ventral tegmental area and antagonizes vulnerability to MPTP
- Nat Neurosci 13(12):1481-1488 (2010)
Nature Neuroscience | Article Otx2 controls neuron subtype identity in ventral tegmental area and antagonizes vulnerability to MPTP * Michela Di Salvio1, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Luca Giovanni Di Giovannantonio1, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Dario Acampora1, 2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Raffaele Prosperi4 Search for this author in: * NPG journals * PubMed * Google Scholar * Daniela Omodei1 Search for this author in: * NPG journals * PubMed * Google Scholar * Nilima Prakash5, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Wolfgang Wurst5, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Antonio Simeone1, 2, 3simeone@ceinge.unina.it Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 13,Pages:1481–1488Year published:(2010)DOI:doi:10.1038/nn.2661Received10 August 2010Accepted09 September 2010Published online07 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 Mesencephalic-diencephalic dopaminergic neurons control locomotor activity and emotion and are affected in neurodegenerative and psychiatric diseases. The homeoprotein Otx2 is restricted to ventral tegmental area (VTA) neurons that are prevalently complementary to those expressing Girk2 and glycosylated active form of the dopamine transporter (Dat). High levels of glycosylated Dat mark neurons with efficient dopamine uptake and pronounced vulnerability to Parkinsonian degeneration. We found that Otx2 controls neuron subtype identity by antagonizing molecular and functional features of dorsal-lateral VTA, such as Girk2 and Dat expression. Otx2 limited the number of VTA neurons with efficient dopamine uptake and conferred resistance to the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-HCl (MPTP) neurotoxin. Ectopic Otx2 expression also provided neurons of the substantia nigra with efficient neuroprotection to MPTP. These findings indicate that Otx2 is required to specify neuron! subtype identity in VTA and may antagonize vulnerability to the Parkinsonian toxin MPTP. View full text Figures at a glance * Figure 1: Otx2 postmitotic inactivation and overexpression. (–) Sections from P10 DatICre/+; Otx2GFP/+; R26R-LacZ and DatICre/+; Otx2GFP/loxP; R26R-LacZ mice were stained with antibodies to Otx2 and tyrosine hydroxylase (TH) (,), Otx2 and GFP (,) and β-galactosidase and tyrosine hydroxylase (,). Otx2 was inactivated in tyrosine hydroxylase–positive neurons (,), the residual Otx2 and GFP double-positive neurons were tyrosine hydroxylase negative (,) and β-galactosidase expressed from the R26 locus was ubiquitously activated in tyrosine hydroxylase–positive neurons by Dat-driven ICre recombinase (,). (–) Staining for Otx2 and tyrosine hydroxylase in E18.5 DatICre/+ (,), DatICre/+; R26Otx2/Otx2 (,) and DatICre/+; R26Otx2/Otx2; tOtx2ov/ov (,) mutant mice revealed that, at late gestation, robust Otx2 expression was activated by Dat-driven ICre recombinase in virtually all of the tyrosine hydroxylase–positive neurons of SNpc and VTA. Staining resulting from the activation of extra copies of Otx2 in the SNpc of mutants was compa! rable in intensity to that of the endogenous Otx2 in the VTA of DatICre/+ control mice (compare and to ). Scale bars represent 100 μm () and 200 μm (,). * Figure 2: Otx2 inactivation does not affect the survival of Otx2-positive VTA neurons but induces an increase in the number of GFP and Girk2 double-positive neurons. () Postmitotic inactivation of Otx2 did not affect the internal percentage of GFP-negative and tyrosine hydroxylase–positive, GFP and tyrosine hydroxylase–double-positive, and GFP-positive and tyrosine hydroxylase–negative neurons of DatICre/+; Otx2GFP/loxP mutants, as compared to 14–16-week-old DatICre/+; Otx2GFP/+ mice (*P = 0.1, **P = 0.18; Student's t test). () In the absence of Otx2, the GFP-positive subpopulation exhibited a selective increase in the percentage of GFP and Girk2 double-positive neurons (#P = 0.16, ##P = 0.25, ###P << 0.001; Student's t test). (–) Representative immunostaining of adjacent sections of 14–16-week-old DatICre/+; Otx2GFP/+ and DatICre/+; Otx2GFP/loxP mice with antibodies to Otx2 and tyrosine hydroxylase (,), GFP and tyrosine hydroxylase (,), GFP and Calb (,), GFP and Ahd2 (,), or Girk2 and GFP (,). Girk2 was expressed in more GFP-positive neurons located in the central VTA (arrows in ,) in DatICre/+; Otx2GFP/loxP mice than in con! trols. The dotted lines in – demarcate the central VTA. Magnifications of the boxed areas in and are shown in and . C, central VTA; D, dorsal VTA; V, ventral VTA. Scale bars represent 100 μm (–) and 200 μm (,). * Figure 3: Otx2 activation in the VTA generates a reduction in the number of Girk2-positive neurons. () Compared with 14–16-week-old DatICre/+ control mice, DatICre/+; R26Otx2/Otx2 and DatICre/+; R26Otx2/Otx2; tOtx2ov/ov mutants of the same age exhibited a selective reduction in the percentage of Girk2 and tyrosine hydroxylase double-positive neurons (*P = 0.11, **P = 0.25, ***P = 0.2, #P = 0.24, ##P << 0.001; Student's t test). (–) Representative immunostaining of adjacent sections from DatICre/+, DatICre/+; R26Otx2/Otx2 and DatICre/+; R26Otx2/Otx2; tOtx2ov/ov mice with antibodies to Calb and tyrosine hydroxylase (–), Ahd2 and tyrosine hydroxylase (–), and Girk2 and tyrosine hydroxylase (–). Although no obvious abnormalities were detected for Calb and Ahd2, the number of Girk2-positive neurons and the intensity of their staining were reduced in Otx2-overexpressing mutants (arrows in –). Scale bar represents 100 μm. * Figure 4: The level and expression profile of glyco-Dat are altered in Otx2 mutants. (–) Immunostaining of adult wild-type brain with antibodies to Otx2 and glyco-Dat (,) or Girk2 and glyco-Dat (,) revealed that the majority of Otx2-positive neurons were glyco-Dat negative (,) and that glyco-Dat–positive neurons coexpressed Girk2 (,). (–) Immunohistochemistry in 14–16-week-old DatICre/+; Otx2GFP/+ (,) and DatICre/+; Otx2GFP/loxP (,) showed that the number of GFP-positive neurons coexpressing high levels of glyco-Dat was increased in the central VTA (arrows in ) in mutants lacking Otx2. () Compared with 14–16-week-old DatICre/+; Otx2GFP/+ control mice, the percentage of GFP and glyco-Dat double-positive neurons was significantly increased in DatICre/+; Otx2GFP/loxP mutants of the same age (*P << 0.001; Student's t test). () Western blot revealed that the striatal amount of glyco-Dat was diminished in DatICre/+; R26Otx2/Otx2; tOtx2ov/ov mice. (–) Immunostaining sections from 14–16-week-old DatICre/+ and DatICre/+; R26Otx2/Otx2; tOtx2ov/ov mice wi! th antibodies to tyrosine hydroxylase and glyco-Dat revealed that the amount of expressed glycol-Dat and the number of glyco-Dat–positive neurons were reduced in the SNpc (–) and VTA (–) of mice overexpressing Otx2. () Compared with DatICre/+ control mice, the percentage of tyrosine hydroxylase–positive neurons expressing high levels of glyco-Dat was significantly decreased in the SNpc and VTA of DatICre/+; R26Otx2/Otx2; tOtx2ov/ov mutants (*P << 0.001; Student's t test). Arrows in , and indicate Otx2-positive () or GFP-positive (,) neurons expressing high levels of glyco-Dat. Arrows in indicate Girk2 and glyco-Dat double-positive neurons in the central VTA. Arrows in and indicate tyrosine hydroxylase and glyco-Dat double-positive neurons in the dorsal VTA. The images shown in , , , , , , and are magnifications of the boxed regions in , , , , , , and . Scale bars represent 100 μm (,) and 200 μm (,). The scale bar in refers to the sections shown in , , , , and . Th! e scale bar in refers to the sections shown in , , , , and . T! he scale bars in and refer to the sections shown in and and and , respectively. Full-length western blots are shown in Supplementary Figure 7. * Figure 5: Otx2 negatively controls Dat mRNA expression. () Western blot analysis of extracts from ventral pretectum and ventral mesencephalon of 13-week-old DatICre/+ and DatICre/+; R26Otx2/Otx2; tOtx2ov/ov mice revealed a similar reduction of both glyco-Dat and nonglyco-Dat in mutant mice. () Semiquantitative RT-PCR assays of Dat, tyrosine hydroxylase and Nurr1 in ventral pretectum and ventral mesencephalon revealed a selective reduction of Dat mRNA. RT-PCR assays consisted of 22 (lanes 1 and 3) and 20 (lanes 2 and 4) cycles for Dat and Nurr1 and 20 (lanes 1 and 3) and 18 (lanes 2 and 4) cycles for tyrosine hydroxylase. (–) Glyco-Dat immunohistochemistry (,,,,,) and Dat mRNA in situ hybridization (,,,,,) analysis of DatICre/+; Otx2GFP/+ (,), DatICre/+; Otx2GFP/loxP (,), DatICre/+ (,,,) and DatICre/+; R26Otx2/Otx2; tOtx2ov/ov (,,,) mice revealed that the number of neurons expressing high levels of glyco-Dat and Dat mRNA was increased in the central VTA of DatICre/+; Otx2GFP/loxP mice (compare and with and ), whereas, compared w! ith DatICre/+ control mice, the number of these neurons was decreased in both SNpc (–) and VTA (,) of DatICre/+; R26Otx2/Otx2; tOtx2ov/ov mutants. The arrows in , , and point to the residual neurons expressing high levels of both glyco-Dat and Dat mRNA. Scale bars represent 100 μm () and 200 μm (). The scale bar in refers to the sections shown in – and –. The scale bar in refers to the sections shown in –. Full-length western blots and RT-PCR assays are shown in Supplementary Figure 7. * Figure 6: Otx2 is a neuroprotective factor for the Otx2-positive VTA neurons and this property may be conferred to SNpc neurons. (,) Tyrosine hydroxylase–positive neurons and tyrosine hydroxylase–positive subsets expressing GFP (GFP and tyrosine hydroxylase double positive) or not expressing GFP (GFP negative, tyrosine hydroxylase positive) counted in DatICre/+; Otx2GFP/+ and DatICre/+; Otx2GFP/loxP mice treated with MPTP at 100 mg per kg () and 125 mg per kg (). Compared with control mice, mutants lacking Otx2 exhibited a significant reduction in the number of total tyrosine hydroxylase–positive and GFP and tyrosine hydroxylase double-positive neurons (*P < 0.001, **P = 0.005, ***P = 0.4, #P = 0.3; Student's t test). (,) Ratio (in %) between MPTP-treated and untreated mice of the same genotype. In DatICre/+; Otx2GFP/+ and DatICre/+; Otx2GFP/loxP mice, the GFP-negative, tyrosine hydroxylase–positive VTA neuronal subpopulation exhibited a sensitivity to MPTP similar to that detected in the SNpc of DatICre/+ () or DatICre/+; Otx2GFP/+ () control mice, whereas, compared with DatICre/+; Otx2GFP/+ ! mice, the VTA subpopulation of GFP and tyrosine hydroxylase double-positive neurons in DatICre/+; Otx2GFP/loxP mutants was more severely affected by MPTP both at 100 mg per kg (†P = 0.001, ††P << 0.001, †††P = 0.1; Student's t test; ) and 125 mg per kg (). () Number of tyrosine hydroxylase–positive neurons counted in 14–16-week-old MPTP-treated DatICre/+, DatICre/+; R26Otx2/Otx2 and DatICre/+; R26Otx2/Otx2; tOtx2ov/ov mice. Compared with DatIcre/+ mice, mutant mice showed a relevant increase in the number of tyrosine hydroxylase–positive neurons in both SNpc and VTA. () Ratio (in %) between MPTP-treated and untreated mice of the same genotype. Compared with DatICre/+ control mice, SNpc and VTA tyrosine hydroxylase–positive neurons exhibited higher resistance to MPTP in DatICre/+; R26Otx2/Otx2 mice and were almost insensitive to the neurotoxin in triple mutants (##P = 0.0018; Student's t test). Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Michela Di Salvio & * Luca Giovanni Di Giovannantonio Affiliations * CEINGE Biotecnologie Avanzate, Naples, Italy. * Michela Di Salvio, * Luca Giovanni Di Giovannantonio, * Dario Acampora, * Daniela Omodei & * Antonio Simeone * SEMM European School of Molecular Medicine, Naples site, Naples, Italy. * Dario Acampora & * Antonio Simeone * Institute of Genetics and Biophysics "A. Buzzati-Traverso," CNR, Naples, Italy. * Dario Acampora & * Antonio Simeone * Dipartimento di Matematica e Statistica, Università degli Studi del Sannio, Benevento, Italy. * Raffaele Prosperi * Helmholtz Zentrum München, Technische Universität München, Deutsches Zentrum für Neurodegnerative Erkrankungen München, Institute of Developmental Genetics, Munich/Neuherberg, Germany. * Nilima Prakash & * Wolfgang Wurst * Max Planck Institute of Psychiatry, Molecular Neurogenetics, Munich, Germany. * Nilima Prakash & * Wolfgang Wurst Contributions M.D.S. and L.G.D.G. performed the experiments, D.A. generated the Otx2 mutant mice, R.P. performed the statistical analysis, D.O. and N.P. contributed to the phenotypic analysis, W.W. contributed to the interpretation of results and the writing of the manuscript, and A.S. designed and interpreted the experiments and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Antonio Simeone (simeone@ceinge.unina.it) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–8 and Supplementary Tables 1–3 Additional data - GRLD-1 regulates cell-wide abundance of glutamate receptor through post-transcriptional regulation
- Nat Neurosci 13(12):1489-1495 (2010)
Nature Neuroscience | Article GRLD-1 regulates cell-wide abundance of glutamate receptor through post-transcriptional regulation * George J Wang1 Search for this author in: * NPG journals * PubMed * Google Scholar * Lijun Kang2 Search for this author in: * NPG journals * PubMed * Google Scholar * Julie E Kim1 Search for this author in: * NPG journals * PubMed * Google Scholar * Géraldine S Maro1 Search for this author in: * NPG journals * PubMed * Google Scholar * X Z Shawn Xu2 Search for this author in: * NPG journals * PubMed * Google Scholar * Kang Shen1kangshen@stanford.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 13,Pages:1489–1495Year published:(2010)DOI:doi:10.1038/nn.2667Received16 August 2010Accepted20 September 2010Published online31 October 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 AMPA receptors mediate most of the fast postsynaptic response at glutamatergic synapses. The abundance of AMPA receptors in neurons and at postsynaptic membranes is tightly regulated. It has been suggested that changes in synaptic AMPA receptor levels are an important regulatory event in synaptic plasticity and learning and memory. Although the local, synapse-specific regulation of AMPA receptors has been intensely studied, global, cell-wide control is less well understood. Using a forward genetic approach, we identified glutamate receptor level decreased-1 (GRLD-1), a putative RNA-binding protein that was required for efficient production of GLR-1 in the AVE interneurons in the nematode Caenorhabditis elegans. In grld-1 mutants, GLR-1 levels were markedly reduced. Consistently, glutamate-induced currents in AVE were diminished and glr-1–dependent nose-touch avoidance behavior was defective in grld-1 mutants. We propose that this evolutionarily conserved family of proteins! controls the abundance of GLR-1 by regulating glr-1 transcript splicing. View full text Figures at a glance * Figure 1: grld-1(wy225) mutants have decreased levels of GLR-1 in AVE. () Schematic diagram of AVE. Green represents the postsynaptic segment and red denotes the axonal segment of the AVE process. A, anterior; D, dorsal; P, posterior; V, ventral. () Circuit diagram of the nose-touch avoidance response. The sensory neurons ASH, FLP and OLQ release glutamate to their glr-1–expressing synaptic partners, the interneurons AVE, AVA and AVD. AVE, AVA and AVD synapse onto the A-type motor neurons that stimulate the body wall muscles, resulting in backwards locomotion. (,) Representative confocal image of GLR-1–YFP fluorescence in AVE of L2-stage wild-type () and grld-1(wy225) worms (). The boxed in is shown. Asterisk indicates AVE cell body. Scale bar represents 2 μm. () Comparison of GLR-1–YFP fluorescence intensity (normalized to wild type) between wild-type, grld-1(wy225) and grld-1(wy655) worms (n > 20). Error bars represent s.e.m. ***P < 0.001, n.s. P > 0.05, compared with wild-type worms unless otherwise depicted, t test. * Figure 2: grld-1 mutants are nose-touch defective and exhibit decreased glutamate-gated currents. () Comparison of nose-touch behavioral response between wild-type, glr-1(n2461), grld-1(wy225) and grld-1(wy655) worms. The compared genotypes were assayed on the same days (wild type compared with grld-1(wy225) mutants, n = 177; other comparisons, n > 20). Error bars represent s.e.m. ***P < 0.001, *P < 0.05, t test. (,) In vivo whole-cell patch-clamp of AVE to measure inward currents with application of 1 mM glutamate (black line). Sample inward currents of wild-type () and grld-1(wy225) () AVE are shown. The downward 'spikes' in some traces are typical for many worm neurons that show very high input resistance (~4 GOhm is typical)43. () Comparison of current intensity between wild-type (n = 22) and grld-1(wy225) (n = 8) worms. Error bars represent s.e.m. *P < 0.05, ANOVA. * Figure 3: GRLD-1 is a member of the SPEN family. () Schematic domain structure of the three isoforms of GRLD-1. The arrow indicates the position of the molecular lesion of grld-1(wy225) and the arrowhead indicates that of grld-1(wy655). () Phylogenetic analysis of GRLD-1 and SPEN family members. () grld-1 was expressed in AVE. mCherry (pseudo-colored green) was expressed in AVE by the opt-3 promoter (outlined by white line) and GFP-tagged GRLD-1 (pseudo-colored magenta) was expressed with fosmid recombineering. The nerve ring is anterior to AVE. The image is a single confocal plane (~1 μm) of an L2 stage worm. Scale bar represents 2 μm. (–) GFP–GRLD-1 localized to the nucleus when expressed in AVE. The solid line indicates the cell body and the dashed line indicates the nucleus. Differential interference contrast image of AVE (), pseudo-colored GFP-tagged GRLD-1 () and overlay () at the L4 stage. Scale bar represents 2 μm. * Figure 4: GRLD-1 acts cell autonomously in AVE. () Comparison of GLR-1–YFP fluorescence intensity (normalized to wild type) between wild types, grld-1 mutants and grld-1 mutants expressing grld-1 cDNA under the opt-3 promoter (n > 20). Error bars represent s.e.m. ***P < 0.001 compared with grld-1 mutants, t test. () Analysis of nose-touch behavior with expression of grld-1 cDNA under the opt-3 promoter (wild type compared with grld-1 mutants, n = 177; transgenic worms compared with grld-1 mutants, n > 20). The compared genotypes were assayed on the same days. Error bars represent s.e.m. ***P < 0.001, **P < 0.01, t test. (,) In vivo whole-cell patch-clamp of AVE in grld-1 mutants expressing grld-1 cDNA under the opt-3 promoter. A sample inward current of grld-1(wy225) expressing grld-1 cDNA under the opt-3 promoter is shown (); the black line represents application of 1 mM glutamate. () Comparison of current intensity between wild-type worms (n = 22) and grld-1(wy225) (n = 8) and grld-1(wy225) mutants expressing grld-1 c! DNA under the opt-3 promoter (n = 15). Error bars represent s.e.m. *P < 0.05, n.s. P > 0.05, ANOVA test. * Figure 5: The GRLD-1 RRMs are sufficient to rescue GLR-1 levels in AVE. Comparison of GLR-1–YFP fluorescence intensity (normalized to wild type) between wild type, grld-1 mutants, grld-1 mutants expressing grld-1 RRMs cDNA under the opt-3 promoter and grld-1 mutants expressing grld-1 SPOC cDNA under the opt-3 promoter. * Figure 6: Expression of glr-1 cDNA bypasses the requirement for grld-1. (–) Schematic cartoon of the rescuing constructs. Lines, introns; boxes, exons; pentagons, 3′ UTR. Note that this glr-1 depiction does not contain all of the introns and exons. The glr-1 genomicglr-1 3′ UTR transgene contains all of the endogenous exons, introns and the 3′ UTR (). The glr-1 genomicunc-10 3′ UTR transgene contains all of the endogenous exons, introns and the unc-10 3′ UTR (). The glr-1 cDNAglr-1 3′ UTR transgene contains all of the endogenous exons and the glr-1 3′ UTR, but has no introns (). (–) Representative L2-stage wild-type worms (,) and grld-1(wy225) mutants (,). Asterisk indicates AVE cell body. Scale bar represents 2 μm. () Effectiveness of the glr-1 constructs at rescuing the GLR-1 fluorescent phenotypes. The glrd-1(wy225) intensities were normalized to their respective expression constructs (n ≥ 19). Error bars represent s.e.m. ***P < 0.001, *P < 0.05, t test. () Effectiveness of the glr-1 constructs at rescuing the nose-touch ! behavior defect. The compared genotypes were assayed on the same days (n ≥ 20). Error bars represent s.e.m. **P < 0.01, t test. * Figure 7: Expression of grld-1 after initial development can rescue GLR-1 levels. () Schematic of heat shock experimental timeline. Worms were heat shocked for 2 h at 33 °C during the L2 stage or were not heat shocked (kept at 20 °C). GLR-1–YFP fluorescent intensity was measured 18 h after the heat shock. () Comparison of GLR-1–YFP fluorescence intensity (normalized to wild type) of wild types without heat shock, grld-1 mutants without heat shock, grld-1 mutants with heat shock, grld-1 mutants expressing grld-1 cDNA under the hsp16-2 and hsp16-41 promoters (Phs) without heat shock and grld-1 mutants expressing grld-1 cDNA under the hsp16-2 and hsp16-41 promoters with heat shock (n = 20). Error bars represent s.e.m. ***P < 0.001, t test. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Biology, Howard Hughes Medical Institute, Stanford University, California, USA. * George J Wang, * Julie E Kim, * Géraldine S Maro & * Kang Shen * Life Sciences Institute and Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan, USA. * Lijun Kang & * X Z Shawn Xu Contributions G.J.W. performed most of the experiments. X.Z.S.X. supervised L.K., who carried out the electrophysiological experiments. J.E.K. contributed to the initial screen and transgene development. G.S.M. contributed to the intron experiments. K.S., G.J.W. and G.S.M. analyzed the data and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Kang Shen (kangshen@stanford.edu) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–6 and List of Constructs Additional data - An age-related sprouting transcriptome provides molecular control of axonal sprouting after stroke
- Nat Neurosci 13(12):1496-1504 (2010)
Nature Neuroscience | Article An age-related sprouting transcriptome provides molecular control of axonal sprouting after stroke * Songlin Li1 Search for this author in: * NPG journals * PubMed * Google Scholar * Justine J Overman1 Search for this author in: * NPG journals * PubMed * Google Scholar * Diana Katsman1 Search for this author in: * NPG journals * PubMed * Google Scholar * Serguei V Kozlov2 Search for this author in: * NPG journals * PubMed * Google Scholar * Christopher J Donnelly3 Search for this author in: * NPG journals * PubMed * Google Scholar * Jeffery L Twiss3 Search for this author in: * NPG journals * PubMed * Google Scholar * Roman J Giger4, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Giovanni Coppola1 Search for this author in: * NPG journals * PubMed * Google Scholar * Daniel H Geschwind1 Search for this author in: * NPG journals * PubMed * Google Scholar * S Thomas Carmichael1scarmichael@mednet.ucla.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 13,Pages:1496–1504Year published:(2010)DOI:doi:10.1038/nn.2674Received21 July 2010Accepted21 September 2010Published online07 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 Stroke is an age-related disease. Recovery after stroke is associated with axonal sprouting in cortex adjacent to the infarct. The molecular program that induces a mature cortical neuron to sprout a new connection after stroke is not known. We selectively isolated neurons that sprout a new connection in cortex after stroke and compared their whole-genome expression profile to that of adjacent, non-sprouting neurons. This 'sprouting transcriptome' identified a neuronal growth program that consists of growth factor, cell adhesion, axonal guidance and cytoskeletal modifying molecules that differed by age and time point. Gain and loss of function in three distinct functional classes showed new roles for these proteins in epigenetic regulation of axonal sprouting, growth factor–dependent survival of neurons and, in the aged mouse, paradoxical upregulation of myelin and ephrin receptors in sprouting neurons. This neuronal growth program may provide new therapeutic targets and su! ggest mechanisms for age-related differences in functional recovery. View full text Figures at a glance * Figure 1: Experimental approach and laser capture microdissection of sprouting neurons after stroke. () Stroke was produced by permanently occluding two anterior branches of the distal middle cerebral artery over the parietal cortex and transiently occluding bilateral jugular veins. Two different fluorescent conjugates of the tracer cholera toxin B subunit (CTB) were sequentially injected into the same site of forelimb sensorimotor cortex, the first at the time of stroke (Alexa 488–CTB) and the second at 2/3 the volume either 7 or 21 d after stroke (Alexa 647–CTB). Neurons that project to this forelimb sensorimotor site at the time of stroke are labeled with Alexa 488–CTB; neurons that establish a projection to this site only after stroke are labeled solely with Alexa 647–CTB. () Injection sites from three separate cases. Only mice in which the second tracer injection was entirely within the first were used for further analysis. () Young adult sprouting neurons (top row) and sprouting neurons from an aged brain (bottom row). Left column, neurons labeled by the first! tracer injection; middle column, neurons labeled by the second tracer injection; right column, neurons seen after laser capture of a tracer 2–only neuron. Neurons with double label (non-sprouting neurons; arrowheads) or labeled by CTB-Alexa 647 (tracer 2–only, sprouting neurons; arrows) were separately laser-captured in the peri-infarct cortex. Scale bars, 50 μm () and 10 μm (). * Figure 2: Cellular pattern of ATRX, IGF1 and Lingo1 expression in the brain after stroke. () Nissl-stained photomicrograph of mouse barrel field stroke model. Boxes indicate region of contralateral cortex (left column) and peri-infarct cortex in young adult (middle column) and aged adult (right column). (–) Panels for ATRX (–), IGF1 (–) and Lingo1 (–) show immunoreactivity in each condition. Insets: Colocalized staining of ATRX (red) and NeuN (green) is seen as yellow (). IGF1 staining (red) localizes to astrocytes and not neurons (green) in young adult peri-infarct cortex (). In aged adult peri-infarct cortex, IGF1 (red) co-localizes to NeuN positive neurons (green) (). Lingo1 staining (red) localizes to NeuN positive neurons (green) in aged peri-infarct cortex (). Scale bars, 1 mm (), 50 μm (–) and 20 μm (insets). * Figure 3: Quantitative connectional mapping. () Timeline of experimental design for all in vivo axonal tracer experiments. Mice received a sham surgery or stroke, followed 1 week later by siRNA or drug delivery. Three weeks later biotinylated dextran amine (BDA) was microinjected into forelimb motor cortex. (,) The cortex was then removed, flattened, tangentially cut () and stained for cytochrome oxidase () and BDA in the same sections. (,) The location of BDA-labeled axons was digitally mapped in x,y coordinates relative to the injection site. () The quantitative connectional map of forelimb sensorimotor connections in sham-operated mice (n = 5). () The quantitative connectional map of forelimb sensorimotor cortex connections after stroke (n = 7). () Cytochrome oxidase staining in layer IV identifies the mouse somatosensory body map2 after stroke. () Each connectional map was registered to the somatosensory body map from the same brain to produce a group connectional and functional map of cortex. Axonal sprouting was ! identified when a pattern of cortical connections was precisely mapped and statistically different across treatment conditions. Dark blue indicates areas of dense overlap. Scale bars, 1 mm. * Figure 4: ATRX function in post-stroke axonal sprouting. () Knockdown of ATRX by siRNA reduced axonal outgrowth in vitro compared to scrambled siRNA treatment. ATRX overexpression induced axonal sprouting compared to expression of GFP alone. *P < 0.01 compared to GFP control; **P < 0.001 compared to scrambled siRNA stroke. () Axon numbers in normal control or peri-infarct cortex layer 2/3 with Atrx siRNA or scrambled siRNA. @P ≤ 0.05 compared to saline normal control; **P ≤ 0.01 compared to scrambled siRNA stroke. () Sham-operated, non-stroke forelimb motor cortex connections (blue, n = 10) compared to those from the Atrx siRNA group (red, n = 12). The blue label indicates the position of axons projecting from motor cortex summed from the entire sample of mice in the non-stroke condition. The red label indicates that position of axons from the entire sample of mice treated with Atrx siRNA. Dark blue indicates areas of dense overlap of the two projection systems. There was no significant difference in the pattern of cortical co! nnections. () Quantitative connectional map of forelimb sensorimotor connections in stroke + scrambled siRNA (blue, n = 11) and stroke + Atrx siRNA (red, n = 12), registered to the body map of underlying cortex with stroke. Dark blue shows area of dense overlap of connections in the two conditions. There is a significant increase in motor cortex projections between stroke + scrambled siRNA and stroke + Atrx siRNA. () Polar plots of axonal label from studies in . Shaded regions represent 70th percentile of the distances of labeled axons from the injection site; weighted polar vectors represent the normalized distribution of the number of points in a given segment of the graph. There was a significant difference in distribution of cortical projections between stroke + Atrx siRNA (red) and stroke + scrambled siRNA (blue) and between stroke + scrambled siRNA (blue) and non-stroke vehicle (yellow), and no significant change between stroke + Atrx siRNA (red) and non-stroke vehicl! e (yellow) (n = 10, P > 0.05). Scale bars, 1 mm. Error bars, s! .d. * Figure 5: IGF1 maintains neuronal viability after stroke. () There was no significant difference in the pattern of cortical connections between stroke + saline (n = 5) and stroke + IGF1 (n = 7). () There was no significant change in the polar distribution of connections with IGF1 administration compared to stroke + saline (P > 0.05). () There was a significant loss of cortical connections between stroke + saline (n = 5) and stroke + JB1 (n = 8). () There was a significant decrease in polar distribution of connections with IGF1 blockade (P < 0.001). () Photomicrographs of neurons in peri-infarct cortex, showing no change in number across IGF1 treatment conditions. () Effects of IGF1 and JB1 on neuronal number in layer 5 in normal control or peri-infarct cortex. IGF1 or JB1 were delivered beginning 7 d after stroke. Stroke (n = 6) induced neuronal cell death in peri-infarct cortex (P < 0.01). This neuronal death was not altered by delivery of IGF1 (n = 5, P > 0.05) or vehicle (n = 5, P > 0.05) into peri-infarct cortex. However, IGF1 ! blockade with JB1 hydrogel significantly (n = 5, P < 0.05) increased cell death in peri-infarct cortex when compared to the saline hydrogel (n = 5)–treated mice after stroke. () Photomicrographs of neurons in peri-infarct cortex, showing decrease in number of neurons with JB1-induced IGF1 signaling blockade. Scale bars, 500 μm (,) and 1 mm (,,,). Note that the location of BDA tracer injection was more lateral in ATRX studies (Fig. 4) than in IGF1/JB1 and Lingo1-Fc/NgR1/NgR2 studies, the latter two of which use the same tracer coordinates; this produces slightly different patterns of intracortical connections. Error bars, s.d.; ***P < 0.001 and **P < 0.01 compared to normal control; #P < 0.05 compared to stroke + saline hydrogel. * Figure 6: Lingo1/NgR1 restricts cortical axonal sprouting after stroke. Quantitative connectional maps of forelimb motor connections with NgR1 signaling blockade after hydrogel delivery of Lingo1-Fc + stroke (n = 6) versus hydrogel control-Fc + stroke (n = 7). () Lingo1-Fc release into peri-infarct cortex induced a significantly different pattern of forelimb motor cortex connections. () Registration of motor cortical connections with the underlying cytochrome oxidase body map shows that Lingo1-Fc blocked the connections that form lateral and rostral to mouse somatosensory cortex, in secondary somatosensory cortex and motor cortex. () Polar analysis of forelimb sensorimotor connections from same maps. Lingo1-Fc induced a significant increase in motor cortex connections in the sites seen in the body map registration motor cortex, primary somatosensory cortex and secondary somatosensory cortex (P < 0.001). () Quantitative connectional map of forelimb sensorimotor connections in NgR1 knockout mice with stroke (n = 5) versus control mouse stroke (n =! 5). There was a significant increase in the pattern of cortical connections in NgR1 knockout. () Registering the underlying mouse body map to the motor cortical projections indicates that new projections could be seen in the NgR1 knockout in lateral SI, SII and to a lesser extent in motor cortex. () Polar analysis of forelimb motor connections in stroke + NgR1 knockout versus control. Significantly different connections were seen in motor and lateral somatosensory regions (P < 0.005). Scale bars, 1 mm. Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Gene Expression Omnibus * GSE24442 Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Department of Neurology, David Geffen School of Medicine at University of California, Los Angeles, California, USA. * Songlin Li, * Justine J Overman, * Diana Katsman, * Giovanni Coppola, * Daniel H Geschwind & * S Thomas Carmichael * Cancer and Developmental Biology Laboratory, National Cancer Institute, Frederick, Maryland, USA. * Serguei V Kozlov * Department of Biology, Drexel University, Philadelphia, Pennsylvania, USA. * Christopher J Donnelly & * Jeffery L Twiss * Department of Neurology, University of Michigan, Ann Arbor, Michigan, USA. * Roman J Giger * Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, Michigan, USA. * Roman J Giger Contributions S.L. designed and performed experiments, analyzed data and wrote the paper; J.J.O. developed cortical connectional mapping methodology and analyzed data; D.K. designed and performed experiments and analyzed data; S.V.K. developed a transgenic mouse first reported in this manuscript; C.J.D. and J.L.T. designed and performed the DRG in vitro studies; R.J.G. provided transgenic mice and reagents; G.C. and D.H.G. analyzed the microarray data; S.T.C. designed experiments, analyzed data and wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * S Thomas Carmichael (scarmichael@mednet.ucla.edu) Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (8M) Supplementary Figures 1–13, Supplementary Tables 1–5 and Supplementary Discussion Additional data - Extensive spontaneous plasticity of corticospinal projections after primate spinal cord injury
- Nat Neurosci 13(12):1505-1510 (2010)
Nature Neuroscience | Article Extensive spontaneous plasticity of corticospinal projections after primate spinal cord injury * Ephron S Rosenzweig1, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Gregoire Courtine2, 3, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Devin L Jindrich2 Search for this author in: * NPG journals * PubMed * Google Scholar * John H Brock1 Search for this author in: * NPG journals * PubMed * Google Scholar * Adam R Ferguson4 Search for this author in: * NPG journals * PubMed * Google Scholar * Sarah C Strand5 Search for this author in: * NPG journals * PubMed * Google Scholar * Yvette S Nout4 Search for this author in: * NPG journals * PubMed * Google Scholar * Roland R Roy2 Search for this author in: * NPG journals * PubMed * Google Scholar * Darren M Miller1 Search for this author in: * NPG journals * PubMed * Google Scholar * Michael S Beattie4 Search for this author in: * NPG journals * PubMed * Google Scholar * Leif A Havton2 Search for this author in: * NPG journals * PubMed * Google Scholar * Jacqueline C Bresnahan4 Search for this author in: * NPG journals * PubMed * Google Scholar * V Reggie Edgerton2 Search for this author in: * NPG journals * PubMed * Google Scholar * Mark H Tuszynski1, 6mtuszynski@ucsd.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 13,Pages:1505–1510Year published:(2010)DOI:doi:10.1038/nn.2691Received06 August 2010Accepted27 September 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 Although axonal regeneration after CNS injury is limited, partial injury is frequently accompanied by extensive functional recovery. To investigate mechanisms underlying spontaneous recovery after incomplete spinal cord injury, we administered C7 spinal cord hemisections to adult rhesus monkeys and analyzed behavioral, electrophysiological and anatomical adaptations. We found marked spontaneous plasticity of corticospinal projections, with reconstitution of fully 60% of pre-lesion axon density arising from sprouting of spinal cord midline-crossing axons. This extensive anatomical recovery was associated with improvement in coordinated muscle recruitment, hand function and locomotion. These findings identify what may be the most extensive natural recovery of mammalian axonal projections after nervous system injury observed to date, highlighting an important role for primate models in translational disease research. View full text Figures at a glance * Figure 1: Spontaneous improvement in object retrieval with the hand after C7 lateral hemisection. () Representative EMG activity recorded from forelimb muscles during successful retrievals before and after injury (10–15 trials per time point). Traces are normalized with respect to the timing of forelimb motion, that is, rest, reach and retrieval, as indicated by vertical dotted lines. The reach phase corresponds to the time from the onset of forelimb motion to contact of the hand with the food item. The retrieval phase begins from the end of the reach phase and finishes with contact of the food item to the mouth. Vertical scale bars on the right side of each trace denote 100% of peak muscle activity during locomotion at 1 mph. Time points are shown as a range of weeks to emphasize inter-individual differences in the timing of the recovery. Mean integrated EMG amplitudes (+s.e.m.) for forelimb EMG bursts during successful retrievals are shown. No statistical differences were detected in the amplitude of EMG burst amplitudes between pre- and post-lesion retrievals. () Fi! ne motor control task. From a standardized starting position, monkeys used their affected arm to retrieve a food item resting on a platform. () Mean success in hand use reported as a percent of successful food retrieval trials (±s.e.m.) in five monkeys. #, § and * denote conditions that were significantly different (P < 0.05) from all time points not marked with the same symbol. EDC, extensor digitorum communis; FDS, flexor digitorum superficialis; FPB, flexor pollicis brevis. * Figure 2: Partial recovery in forelimb use during locomotion after C7 hemisection. () Representative stick diagram decompositions (30 ms between sticks) of the right (lesioned) forelimb motion during the swing phase while stepping quadrupedally on the treadmill at 0.45 m s−1 before and at different time points post-injury. The successive (n = 10 steps), color-coded trajectories of the forelimb endpoint (metacarpo-phalangeal joint, MCP) are shown together with the intensity and direction of the forelimb endpoint velocity (arrows) at swing onset. Mean (n = 10 steps) integrated EMG activity of selected forelimb muscles is represented for the different time points. The shaded area indicates the duration of the stance phase. () Mean (+s.e.m.) values for the posterior (negative direction) and anterior (positive direction) positions reached by the forelimb endpoint (MCP) with respect to the shoulder (horizontal distance) during each gait cycle. Dots represent individual values (n = 5 monkeys). () Consistency of forelimb endpoint trajectory measured by PCA. Mean! (+s.e.m.) values of the amount of variance explained by the first principal component are reported. () Mean (+s.e.m.) values for the amplitude of distal joint motion measured during each gait cycle. () Probability density distributions of normalized EMG amplitudes in the FPB and EDC during treadmill stepping. An L-shape pattern observed during stepping pre-lesion indicates reciprocal activation between the antagonist FPB and EDC motor pools. A D-shape during stepping at 4–8 weeks post-lesion indicates coactivation between the FPB and EDC. () Mean EMG amplitude (+s.e.m.) for forelimb EMG bursts during locomotion (n = 3 monkeys). # indicates significantly different from all other time points at P < 0.05; * indicates significantly different from indicated time points at P < 0.05. * Figure 3: Extensive recovery of hindlimb locomotion after C7 hemisection. () Representative stick diagram decompositions (30 ms between sticks) of lesion-side hindlimb movements during the swing phase while stepping quadrupedally on the treadmill at 0.45 m s−1 before and at different time points post-injury. The successive (n = 10 steps), color-coded trajectories (blue, swing; red, drag; gray, stance) of the hindlimb endpoint (MTP) are shown together with the intensity and direction of the hindlimb endpoint velocities (arrows) at swing onset. Mean integrated EMG activity (n = 10 steps) of selected hindlimb muscles (sol, soleus; TA, tibialis anterior) is shown at the bottom of the panel for each time point. Shaded areas indicate the duration of the stance phase and red bars indicate the duration of paw dragging. () Mean (+s.e.m.) duration of paw dragging expressed as a percentage of the total swing phase duration at each time point tested. Dots represent individual values (n = 5). () Mean (+s.e.m.) variability of hip, knee, ankle and MTP joint mo! vements expressed as the sum of the s.d. for each joint over ten successive gait cycles. # indicates significantly different from all other time points at P < 0.05. * indicates significantly different from indicated time points at P < 0.05. * Figure 4: Extensive compensatory plasticity of the lesioned corticospinal tract in primates. (–) Compensatory sprouting of lesioned, D-A488–labeled corticospinal axons in the intermediate zone of the gray matter caudal to the C7 hemisection. Insets demonstrate increased axon caliber in long-term subjects, quantified below. (–) Corticospinal axons in right-side gray matter below the lesion originate from the opposite (left) side of the spinal cord. A long-term lesioned subject is shown in . The lesion is on the right side and the arrow denotes midline. Boxed regions are shown at higher magnification. The path of corticospinal axons as they exit the left dorsolateral fasciculus (), decussate across the spinal cord midline (cc, central canal; ), and terminate in the lateral motor neuron pools () are shown. () Serial reconstruction of a single axon, demonstrating unequivocally that it originated from the left dorsolateral corticospinal tract. () Corticospinal axon density was reduced ~75% 2 weeks after injury and recovered to more than half of pre-lesion axon dens! ity by 24 weeks post-lesion. () Quantification of axon thickness. Long-term lesioned monkeys exhibited a 20% increase in axon caliber below the lesion. There were no significant differences between groups in axon density or caliber above the lesion level; thus, the observed changes were not a result of variability in tracer efficacy. In and , dots denote individual monkeys' data points (*P < 0.05). () Serial reconstructions of axonal arbors in an intact (left) and lesioned, long-term (middle and right) subjects. Axons in lesioned, long-term subjects exhibited high densities of bouton-like swellings (compare left and middle), small, thin processes, ending in swellings that were much smaller than normal boutons (middle inset, arrowheads), and large, abnormal structures that exhibited morphological features resembling growth cones (right inset). (–) Colocalization (arrowheads) of the synaptic marker synaptophysin (red) with D-A488–labeled (green) bouton-like swellings in a! xons sprouting below the C7 lesion site in the long-term group! . A normal-appearing bouton-like swelling is shown in , and examples from atypically large boutons found only in long-term subjects, as shown in , are shown in and . Scale bars represent 100 μm (–), 250 μm (), 86 μm (–), 25 μm () and 2 μm (–). Error bars indicate s.e.m. * Figure 5: Relationship between anatomical plasticity and functional recovery. PCA in the four long-term subjects with both functional and anatomical data revealed a multivariate relationship between corticospinal sprouting density and functional recovery. The first principal component (PC1, circle) reflects a data-driven statistical clustering of the histological and functional outcome variables. Positive loading (analogous to a positive Pearson correlation) of variables onto PC1 is indicated in shades of red and negative loading (analogous to an inverse Pearson correlation) is indicated in shades of blue. The magnitude of the loading is indicated by arrow thickness and below each variable; asterisks indicate statistical significance (|loading| > 0.40). Note that axon density coloads on PC1 with both locomotor function on a treadmill and success in recovering food rewards from a platform. PC1 accounts for 59.2% of the variability in the data. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Ephron S Rosenzweig & * Gregoire Courtine Affiliations * Department of Neurosciences, University of California San Diego, La Jolla, California, USA. * Ephron S Rosenzweig, * John H Brock, * Darren M Miller & * Mark H Tuszynski * Departments of Physiological Science and Neurology, University of California, Los Angeles, California, USA. * Gregoire Courtine, * Devin L Jindrich, * Roland R Roy, * Leif A Havton & * V Reggie Edgerton * Department of Neurology, University of Zurich, Zurich, Switzerland. * Gregoire Courtine * Department of Neurosurgery, University of California, San Francisco, California, USA. * Adam R Ferguson, * Yvette S Nout, * Michael S Beattie & * Jacqueline C Bresnahan * California National Primate Research Center, University of California, Davis, California, USA. * Sarah C Strand * Veterans Administration Medical Center, La Jolla, California, USA. * Mark H Tuszynski Contributions E.S.R., G.C., M.S.B., L.A.H., J.C.B., V.R.E. and M.H.T. designed the study. S.C.S. tested experimental subjects. S.C.S., Y.S.N., G.C., D.L.J. and J.C.B. performed behavioral tests. M.H.T., E.S.R., R.R.R. and Y.S.N. performed surgeries. G.C., E.S.R., D.L.J. and A.R.F. analyzed behavioral, electrophysiological and kinematic data. E.S.R., J.H.B., D.M.M., L.A.H. and M.H.T. analyzed anatomical data. M.H.T., E.S.R. and G.C. 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: * Mark H Tuszynski (mtuszynski@ucsd.edu) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (3M) Supplementary Figures 1–11 and Supplementary Tables 1 and 2 Additional data - TRPV1 activation by endogenous anandamide triggers postsynaptic long-term depression in dentate gyrus
- Nat Neurosci 13(12):1511-1518 (2010)
Nature Neuroscience | Article TRPV1 activation by endogenous anandamide triggers postsynaptic long-term depression in dentate gyrus * Andrés E Chávez1 Search for this author in: * NPG journals * PubMed * Google Scholar * Chiayu Q Chiu1 Search for this author in: * NPG journals * PubMed * Google Scholar * Pablo E Castillo1pablo.castillo@einstein.yu.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 13,Pages:1511–1518Year published:(2010)DOI:doi:10.1038/nn.2684Received23 June 2010Accepted29 September 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 The transient receptor potential TRPV1 is a nonselective cation channel that mediates pain sensations and is commonly activated by a wide variety of exogenous and endogenous, physical and chemical stimuli. Although TRPV1 receptors are mainly found in nociceptive neurons of the peripheral nervous system, these receptors have also been found in the brain, where their role is far less understood. Activation of TRPV1 reportedly regulates neurotransmitter release at several central synapses. However, we found that TRPV1 suppressed excitatory transmission in rat and mouse dentate gyrus by regulating postsynaptic function in an input-specific manner. This suppression was a result of Ca2+-calcineurin and clathrin-dependent internalization of AMPA receptors. Moreover, synaptic activation of TRPV1 triggered a form of long-term depression (TRPV1-LTD) mediated by the endocannabinoid anandamide in a type 1 cannabinoid receptor–independent manner. Thus, our findings reveal a previously ! unknown form of endocannabinoid- and TRPV1-mediated regulation of synaptic strength at central synapses. View full text Figures at a glance * Figure 1: Functional evidence for TRPV1 receptors in the dentate gyrus. () Pharmacological activation of TRPV1 receptors with the agonist capsaicin (CAP, 1 μM) depressed AMPAR-EPSC in an input-specific manner. Top, representative traces of two consecutive AMPAR-EPSCs (100-ms interstimulus interval) before (black) and after (gray) bath application of 1 μM CAP at both medial perforant path (MPP) and mossy cell fibers (MCF). Bottom, summary plot showing the effect of CAP on AMPAR-EPSCs. Application of the group II mGluR agonist DCG-IV (1 μM) selectively depressed MPP-EPSCs, but not MCF-EPSCs, and subsequent application of the AMPAR antagonist NBQX (10 μM) abolished the remaining DCG-IV–insensitive component. () Average traces (top) and summary data (bottom) showing that pretreatment with the TRPV1 antagonist CPZ (10 μM) or loading of DGCs with the antagonist AMG9830 (AMG, 3 μM) eliminated CAP-mediated depression of AMPAR-EPSC in the MPP. () CAP-mediated depression of AMPAR-EPSC was also present in Trpv1+/+, but not Trpv1−/−, mice. The n! umbers of cells (c) or animals (a) are indicated in parentheses. In all panels, averaged sample traces taken at times indicated by numbers are shown next to each summary plot. Summary data consist of mean ± s.e.m. * Figure 2: Postsynaptic TRPV1-mediated suppression of excitatory transmission. () CAP exerted no effect on NMDAR-mediated transmission measured at −40 mV and +40 mV in the presence of 10 μM NBQX. () Representative traces (left) and summary plot (right) showing that 1 μM CAP depressed the amplitude, but not the frequency, of asynchronous AMPA-EPSC. **P < 0.01. () CAP selectively depressed AMPAR-mediated responses evoked by puffing 1 mM glutamate in the MPP, but not the MCF, synaptic field in rat. Representative responses evoked in the MPP synaptic field before (black) and after CAP (gray) application are shown on the left. Vertical arrowheads indicate the time of glutamate puffs. NBQX completely abolished glutamate puff–evoked EPSCs (light gray), indicating that the responses were mediated by AMPARs. The summarized time course of CAP-mediated effect is shown on the right. () Representative traces (left) and summary plot (right) showing that CAP also mediated depression of AMPAR-mediated responses evoked by puffing glutamate in Trpv1+/+, but not in! Trpv1−/−, mice. The numbers of cells (c) or animals (a) are indicated in parentheses. () Representative traces (left) and summary plot (right) showing that transient pharmacological activation of TRPV1 receptors triggered long-lasting depression of AMPAR-mediated transmission that cannot be rescued by subsequent application of the TRPV1 antagonist CPZ. In all panels, averaged sample traces taken at times indicated by numbers are shown next to each summary plot. Summary data consist of mean ± s.e.m. * Figure 3: TRPV1-mediated long-term depression at MPP-DGC synapses. () In the presence of the NMDAR antagonists D-AP5 (50 μM) or MK801 (50 μM), 1-Hz pairing protocol (see Online Methods) induced a robust LTD in rat dentate gyrus, which is blocked by the antagonists CPZ (10 μM) or AMG (3 μM). No substantial changes in either PPR (top right) and 1/CV2 (bottom right) were observed before (pre) and after (post) LTD induction. () The 1-Hz pairing protocol induced robust LTD in Trpv1+/+, but not Trpv1−/−, mice. As seen in rat, TRPV1- LTD in mice was not associated with changes in PPR (top right) or 1/CV2 (bottom right). The numbers of cells (c) or animals (a) are indicated in parentheses. () 1-Hz pairing protocol delivered to MCF inputs did not trigger LTD, strongly suggesting that TRPV1-LTD is input specific (top). TRPV1-LTD also showed associativity (that is, only MPP synapses whose activation was paired with depolarization underwent depression) and specificity (that is, LTD did not spread to unpaired MPP synapses; bottom). () TRPV1-LTD ! could not be induced in the presence of 1 μM CAP (top), whereas CAP had no effect after TRPV1-LTD was established (bottom). Representative responses evoked before (black) and after (gray) 1-Hz protocol are shown on the left (,). Summarized time courses are shown on the right. Summary data consist of mean ± s.e.m. * Figure 4: Role of TRPV1 receptors in long-term potentiation at MPP-DGC synapses. () Under more physiological recording conditions (that is, intact NMDAR transmission), a 1-Hz pairing protocol induced robust MPP-LTP, whose magnitude is enhanced in the presence of CPZ (10 μM) or AMG (3 μM) in the bath. Top, average traces before (black) and after (gray) LTP induction. Bottom, summary plot showing the enhancement of LTP in the presence of CPZ or AMG. () LTP was also enhanced in Trpv1−/− mice compared with Trpv1+/+ mice. The numbers of cells (c) or animals (a) are indicated in parentheses. Summary data consist of mean ± s.e.m. * Figure 5: Activation of mGluR5 is necessary, but not sufficient, to induce TRPV1-LTD. () TRPV1-LTD was blocked by bath application of 4 μM MPEP, but not by 100 μM LY367385, suggesting that activation of mGluR5, but not mGluR1, is required for TRPV1-LTD induction. () Loading DGCs with the G protein antagonist GDPβS (1 mM) or the PLC blocker U73122 (5 μM) blocked TRPV1-LTD, suggesting the involvement of G protein and downstream signaling via PLC in the induction of TRPV1-LTD. () Group I mGluR activation with DHPG (50 μM) for 10 min was not sufficient to trigger long-lasting depression of MPP synaptic transmission in the absence or in the presence of the NMDAR antagonist MK801 (50 μM). () DHPG (50 μM) paired with depolarization triggered robust TRPV1-LTD at MPP-DGC synapses, which was blocked by CPZ (10 μM). Averaged EPSCs before (black) and 25 min after LTD induction (gray) are shown on the left (–). Summary plots (mean ± s.e.m.) showing the time course and drug effects on TRPV1-LTD are shown on the right. * Figure 6: Molecular mechanism underlying TRPV1-mediated depression of synaptic transmission. (,) Both CAP-mediated depression and TRPV1-LTD required Ca2+ rise and Ca2+ release from internal stores. Top, representative averaged traces under control conditions (left) in DGCs loaded with 20 mM BAPTA (center) and in hippocampal slices treated with 30 μM CPA (right). Bottom, summary data. Bar plots show MPP-EPSC amplitude changes calculated 15–20 min following application of CAP () or 25–30 min following the LTD induction protocol (). (,) Representative averaged traces (top) and summary plots (bottom) showing that two different CaN inhibitors, FK506 (50 μM) and Cyclosporin A (CyA; 25 μM), blocked both CAP-mediated depression () and TRPV1-LTD (). The number of cells is indicated in parenthesis. ***P < 0.001, **P < 0.01. All summary data consist of mean ±s.e.m. (,) Intracellular loading of DIP (50 μM) abolished both CAP-mediated depression () and TRPV1-LTD (). Loading DGCs with a scrambled DIP (50 μM) did not affect TRPV1-LTD (). * Figure 7: The endocannabinoid AEA suppresses excitatory synaptic transmission via TRPV1 receptors. () Representative averaged traces (top left) and summary plot (bottom left) showing that, in the presence of the FAAH inhibitor URB597 (1 μM) and the CB1R antagonist AM251 (4 μM), AEA (30 μM) depressed MPP-mediated, but not MCF-mediated, EPSCs, an effect that was not associated with changes in PPR and 1/CV2 (right). () Loading DGCs with AEA (30 μM) also depressed AMPAR-EPSC without changes in PPR and 1/CV2. This AEA-mediated depression was blocked by 10 μM CPZ but not by 4 μM AM251. () Summary plot (bottom left) and representative traces (top left) showing that AEA-mediated depression was abolished in Trpv1−/−, but not Cb1r−/−, mice, and was not associated with PPR or 1/CV2 changes (right). The numbers of cells () or animals () are indicated in parentheses. Summary data consist of mean ± s.e.m. * Figure 8: Anandamide mediates TRPV1-LTD in the dentate gyrus. () Summary plots (bottom) and representative traces (top) showing that a subthreshold 1-Hz pairing protocol (4 stimuli at 100 Hz and paired with 30 mV depolarization, repeated 300 times, rather than 600 times as seen in previous figures), which did not trigger long-term synaptic plasticity under control conditions, triggered robust LTD in the presence of 1 μM URB597 and that this LTD was completely abolished in the presence of 10 μM CPZ. () A subthreshold 1-Hz pairing protocol for LTD under control conditions (as seen in rat, ) induced robust LTD in the presence of 1 μM URB597 in Trpv1+/+ mice (n = 3) but not in Trpv1−/− mice (n = 3). The number of cells is in parentheses. ***P < 0.001. () Loading DGCs with the diacylglycerol lipase inhibitor THL (4 μM) did not affect the induction of TRPV1-LTD (top), but abolished the induction of I-LTD in the CA1 area of the hippocampus (bottom). Averaged EPSCs (top) and IPSCs (bottom) before (black) and 25 min after LTD induction ! (gray) are shown on the left side. Summary plots showing the time course of LTD are shown on the right. Summary data consist of mean ± s.e.m. Author information * Abstract * Author information * Supplementary information Affiliations * Dominick P. Purpura, Department of Neuroscience, Albert Einstein College of Medicine, New York, New York, USA. * Andrés E Chávez, * Chiayu Q Chiu & * Pablo E Castillo Contributions A.E.C. and C.Q.C. performed all of the experiments and analyzed the results. A.E.C., C.Q.C. and P.E.C. designed the experiments, interpreted the results and wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Pablo E Castillo (pablo.castillo@einstein.yu.edu) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (512K) Supplementary Figures 1–7 and Supplementary Tables 1–5 Additional data - Postsynaptic TRPV1 triggers cell type–specific long-term depression in the nucleus accumbens
- Nat Neurosci 13(12):1519-1525 (2010)
Nature Neuroscience | Article Postsynaptic TRPV1 triggers cell type–specific long-term depression in the nucleus accumbens * Brad A Grueter1 Search for this author in: * NPG journals * PubMed * Google Scholar * Gabor Brasnjo1 Search for this author in: * NPG journals * PubMed * Google Scholar * Robert C Malenka1malenka@stanford.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 13,Pages:1519–1525Year published:(2010)DOI:doi:10.1038/nn.2685Received23 June 2010Accepted22 September 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 Synaptic modifications in the nucleus accumbens (NAc) are important for adaptive and pathological reward-dependent learning. Medium spiny neurons (MSNs), the major cell type in the NAc, participate in two parallel circuits that subserve distinct behavioral functions, yet little is known about differences in their electrophysiological and synaptic properties. Using bacterial artificial chromosome transgenic mice, we found that synaptic activation of group I metabotropic glutamate receptors in NAc MSNs in the indirect, but not direct, pathway led to the production of endocannabinoids, which activated presynaptic CB1 receptors to trigger endocannabinoid-mediated long-term depression (eCB-LTD) as well as postsynaptic transient receptor potential vanilloid 1 (TRPV1) channels to trigger a form of LTD resulting from endocytosis of AMPA receptors. These results reveal a previously unknown action of TRPV1 channels and indicate that the postsynaptic generation of endocannabinoids can ! modulate synaptic strength in a cell type–specific fashion by activating distinct pre- and postsynaptic targets. View full text Figures at a glance * Figure 1: Electrophysiological properties of direct (D2+) and indirect (D2−) pathway MSNs in the NAc core. () Sample traces of D2+ and D2− EPSCs at interstimulus intervals (ISI) of 20, 50, 100 and 200 ms. Summary graph of paired-pulse ratios (PPR) from D2+ (filled squares; n = 18) and D2– (open circles; n = 11) MSNs. Calibration bars for evoked EPSCs in this panel and all subsequent figures unless noted are 50 pA and 25 ms. () Sample traces of mEPSCs from D2+ and D2− MSNs. Calibration bars represent 20 pA and 200 ms. Cumulative probability plot of mEPSC frequencies (left) and amplitudes (right) recorded from D2− (open circles; n = 9) and D2+ (filled squares; n = 9) MSNs. Insets show mean ± s.e.m. of frequency and amplitude for the two populations. () Summary of normalized EPSC amplitudes in D-AP5 (50 μM) and with intracellular spermine (0.1 mM) as a function of membrane potential recorded from D2− (open circles; n = 11) and D2+ (filled squares; n = 10) MSNs. () Representative traces of EPSCs recorded at −80 mV and +40 mV from a D2+ MSN and D2− MSN. () Summary grap! h of the ratio of the NMDAR EPSC (measured 50 ms after stimulus at +40 mV) over the peak AMPAR EPSC (peak current recorded at −80 mV) from D2− (n = 11) and D2+ (n = 10) MSNs. () Representative current clamp traces recorded from D2+ and D2− MSNs in response to 200-, 300- and 400-pA, 800-ms current injections. Calibration bars represent 20 mV and 200 ms. () Summary of firing frequency in D2− (open circles: n = 11) and D2+ (filled squares: n = 8) MSNs in response to 800-ms current injections. *P < 0.05. * Figure 2: D2+ MSNs but not D2– NAc MSNs show mGluR5-dependent LTD. (,) Timecourse of EPSC amplitude recorded from a representative D2+ () and D2− () MSN before and after LFS (given at t = 0). () Summary of EPSCs recorded from D2+ (filled squares: n = 25) and D2− (open circles: n = 11) MSNs before and after LFS. () LFS LTD in D2+ MSNs (n = 6) is not blocked by D-AP5 (100 μM). () LTD in D2+ MSNs is blocked by LY341495 at a concentration that blocks all mGluRs (100 μM; open squares, n = 6) but not at a concentration that is specific for group II mGluRs (200 nM; filled squares, n = 6). () The mGluR5 negative allosteric modulator MPEP (10 μM) blocks LTD at D2+ synapses (n = 5). () The group I mGluR agonist RS-DHPG (100 μM) induces LTD in D2+ MSNs (filled squares, n = 14) but not in D2− MSNs (open circles, n = 5). () LTD in D2+ MSNs is occluded 40 min after DHPG application (n = 4). * Figure 3: D2+ MSNs show forms of LTD that are dependent and independent of CB1 receptors and RIM1α. () LTD in D2+ MSNs is reduced but not blocked by the CB1 receptor antagonist AM251 (1–5 μM; n = 12). () The CB1 receptor agonist WIN55,212-2 (WIN; 1 μM) depresses synaptic transmission (filled squares, n = 4), an effect that is blocked by prior application of AM251 (5 μM; open squares, n = 4) but not by AM251 application after WIN application (filled squares). () LTD in D2+ MSNs in Rim1α−/− mice (open squares, n = 8) is reduced compared with LTD in littermate controls (filled squares, n = 6). () DHPG-induced LTD in D2+ MSNs is reduced in Rim1α−/− mice (open squares, n = 4) relative to littermate controls (filled squares, n = 4). * Figure 4: TRPV1 channels trigger LTD at synapses on D2+ NAc MSNs. (,) LFS LTD in D2+ MSNs is reduced by the TRPV1 antagonists capsazepine (, 10 μM; CPZ, open squares, n = 6; controls, filled squares, n = 25; graph from Fig. 2c) and SB366791 (, 20 μM; n = 6). () LTD in D2+ MSNs is blocked by application of both AM251 and CPZ (n = 7). () LTD in D2+ MSNs in Rim1α−/− mice is blocked by CPZ (n = 5). () The TRPV1 agonist capsaicin (3 μM) depresses AMPAR EPSCs in D2+ MSNs (n = 19). () LTD in D2+ MSNs is reduced after capsaicin application (n = 4). () Capsaicin-induced synaptic depression is blocked by the TRPV1 antagonist CPZ (n = 5). () Capsaicin-induced synaptic depression is not blocked by AM251 (n = 7). * Figure 5: Trpv1−/− mice lack CB1 receptor-independent LFS LTD at synapses on D2+ NAc MSNs. () LFS-LTD in D2+ MSNs is reduced in Trpv1−/− mice (open squares, n = 9) compared to Trpv1+/+ littermates (WT; filled squares, n = 5). () The LTD remaining in D2+ MSNs in Trpv1−/− mice is blocked by AM251 (n = 5). () Capsaicin induces a depression of AMPAR EPSCs in D2+ MSNs in Trpv1+/+ mice (filled squares, n = 14; these cells are a subset of those included in Fig. 4e and were selected because they were interleaved with the recordings from the Trpv1−/− slices) but not Trpv1−/− mice (open squares, n = 6). * Figure 6: Postsynaptic TRPV1 channels trigger LTD in D2+ NAc MSNs. () Control LTD (n = 25) and eCB-LTD (n = 23; pharmacologically isolated by TRPV1 antagonists) elicit a decrease in 1/CV2 of AMPAR EPSCs, whereas TRPV1-dependent LTD produces no change in 1/CV2 (n = 12; pharmacologically isolated by AM251). () Application of the CB1 agonist WIN reduces 1/CV2 (n = 4) but application of the TRPV1 agonist capsaicin does not (n = 14). () Representative traces of mEPSCs before and after capsaicin application. Cumulative probability plots of capsaicin-induced changes in mEPSC frequencies (left, n = 6) and amplitudes (right). Insets show mean ± s.e.m. () Representative traces of NMDAR EPSCs before and after capsaicin at +40 mV and −50 mV. Calibration bars represent 25 pA and 25 ms. Summary graph shows that application of capsaicin has no effect on NMDAR EPSCs at either holding potential (n = 4 at −50 mV and n = 6 at +40 mV). () Postsynaptic loading of the TRPV1 antagonist CPZ or SB366791 (open squares; n = 12) reduces LTD in D2+ MSNs compared t! o vehicle-loaded controls (filled squares; n = 7). () Postsynaptic loading of the calcium chelator EGTA (20 mM) prevents capsaicin-induced depression of AMPAR EPSCs (n = 8). () Postsynaptically loading the dynamin inhibitory peptide D15 but not the scrambled peptide S15 in the presence of AM251 prevents LTD in D2+ MSNs (n = 4 and 3, respectively). () LTD is enhanced in slices incubated with the FAAH inhibitor URB597 (1 μM) and AM251 (3 μM; filled squares, n = 6) compared with AM251 alone (open squares, n = 12). () LTD is enhanced in slices from Trpv1−/− mice incubated with the FAAH inhibitor URB597 (1 μM; filled squares, n = 6) compared with those from Trpv1−/− not incubated with URB597 (open squares, n = 9; from the same cells as in Fig. 5a). * Figure 7: Effects of in vivo cocaine administration on LFS LTD in NAc D2+ MSNs and locomotor behavior in Trpv1−/− mice. () LFS LTD in D2+ MSNs is absent 24 h after administration of a single dose of cocaine (open squares, n = 6), whereas it is normal after a single saline injection (filled squares, n = 5). () The depression of AMPAR EPSCs elicited by capsaicin is not affected by prior administration of a single dose of cocaine (open squares, n = 7) or saline (filled squares, n = 9). () Trpv1−/− mice (n = 10, open squares) showed enhanced cocaine-induced locomotor activity relative to Trpv1+/+ mice (n = 12, filled circles). () Timecourse of cocaine-induced changes in stereotypic behaviors in Trpv1−/− and Trpv1+/+ mice. Author information * Abstract * Author information * Supplementary information Affiliations * Nancy Pritzker Laboratory, Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Palo Alto, California, USA. * Brad A Grueter, * Gabor Brasnjo & * Robert C Malenka Contributions B.A.G., G.B. and R.C.M. designed the experiments, interpreted the results and wrote the paper. B.A.G. and G.B. performed all of the experiments and analyzed the results. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Robert C Malenka (malenka@stanford.edu) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (696K) Supplementary Figure 1 Additional data - Tuning arousal with optogenetic modulation of locus coeruleus neurons
- Nat Neurosci 13(12):1526-1533 (2010)
Nature Neuroscience | Article Tuning arousal with optogenetic modulation of locus coeruleus neurons * Matthew E Carter1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Ofer Yizhar3 Search for this author in: * NPG journals * PubMed * Google Scholar * Sachiko Chikahisa4, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Hieu Nguyen2 Search for this author in: * NPG journals * PubMed * Google Scholar * Antoine Adamantidis2 Search for this author in: * NPG journals * PubMed * Google Scholar * Seiji Nishino4 Search for this author in: * NPG journals * PubMed * Google Scholar * Karl Deisseroth1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Luis de Lecea1, 2llecea@stanford.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 13,Pages:1526–1533Year published:(2010)DOI:doi:10.1038/nn.2682Received26 July 2010Accepted27 September 2010Published online31 October 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 Neural activity in the noradrenergic locus coeruleus correlates with periods of wakefulness and arousal. However, it is unclear whether tonic or phasic activity in these neurons is necessary or sufficient to induce transitions between behavioral states and to promote long-term arousal. Using optogenetic tools in mice, we found that there is a frequency-dependent, causal relationship among locus coeruleus firing, cortical activity, sleep-to-wake transitions and general locomotor arousal. We also found that sustained, high-frequency stimulation of the locus coeruleus at frequencies of 5 Hz and above caused reversible behavioral arrests. These results suggest that the locus coeruleus is finely tuned to regulate organismal arousal and that bursts of noradrenergic overexcitation cause behavioral attacks that resemble those seen in people with neuropsychiatric disorders. View full text Figures at a glance * Figure 1: Specific and efficient functional expression of optogenetic transgenes in locus coeruleus neurons. () Representative photomicrographs depicting tyrosine hydroxylase (TH) immunoreactivity (left column, red), viral eYFP expression (center column, green) and merged images (right column) from an animal unilaterally injected with EF1αeYFP rAAV virus into the left locus coeruleus. Top row, global expression in a coronal section counterstained with DAPI (scale bar, 100 μm); middle row, expression in the full locus coeruleus (scale bar, 25 μm); bottom row, individual neurons (scale bar, 5 μm). () Quantification of co-expression of eYFP and TH immunofluorescence from EF1αeYFP transduced mice (n = 4) in 30-μm brain sections from the rostral-to-caudal ends of the locus coeruleus (anteroposterior, −5.20 to −5.80). Cell counts are represented as mean ± s.d. Inset represents the statistics of the total coexpression. () Voltage clamp recording of a neuron expressing eNpHR-eYFP in brainstem slice showing outward current in response to yellow light. () Voltage clamp recording o! f a neuron expressing ChR2-eYFP in brainstem slice showing inward current in response to blue light. () Action potential trains recorded under current clamp conditions from a neuron expressing eNpHR-eYFP in brainstem slice for 5 s (top) or 1 min (bottom). () Blue-light pulse trains (10 ms per pulse) evoked action potential trains at various frequencies in neurons expressing ChR2-eYFP. () Efficiency of action potential trains evoked by blue light pulses in neurons expressing ChR2-eYFP. Data represent mean probability ± s.e.m. from n = 6 neurons. * Figure 2: Photoinhibition of locus coeruleus neurons causes a reduction in the duration of wakefulness. () The percentage of time spent awake, in NREM sleep and in REM sleep during 1 h of photoinhibition in the active (dark) period. Data represent the mean ± s.e.m. of 6 separate 1-h sessions; n = 6 mice throughout. *P < 0.05, two-tailed Student's t test between transduced mice. () The duration of individual wake, NREM and REM episodes during 1 h of photoinhibition during the active period. *P < 0.05, two-tailed Student's t test between transduced mice. () The percentage of sleep state transitions relative to baseline levels during 1 h of photoinhibition during the active period. **P < 0.001, two-tailed Student's t test between transduced mice. () The duration of individual wake episodes in baseline versus photoinhibition conditions (20 episodes per mouse, n = 6 mice). *P < 0.05, two-way ANOVA between stimulation condition and viral transduction followed by Tukey post-hoc test. () Relative EEG power of wakefulness 80–120 s after wake onset in baseline (top) and photoinhibiti! on (bottom) conditions. Data represent the mean ± s.e.m. relative power of 0.5-Hz binned frequencies (20 episodes per mouse, n = 6 mice). * Figure 3: Photostimulation of locus coeruleus neurons causes immediate sleep-to-wake transitions. (–) Data from NREM sleep. (–) Data from REM sleep. (,) Representative traces of EEG/EMG recordings showing an immediate sleep-to-wake transition in NREM () or REM sleep () after acute photostimulation (10-ms pulses at 5 Hz for 5 s) of locus coeruleus neurons during the inactive period in a mouse transduced with ChR2-eYFP (bottom) but not in a mouse transduced with eYFP alone (top). Arrow, onset of sleep-to-wake transition. (,) Cortical EEG traces from ChR2-eYFP mice 5 s before the onset of stimulation (black) and 5 s into stimulation (gray). Quantification based on the average of 15 () or 8 (e) stimulations per mouse, n = 6 mice. (,) Heat maps showing the effects of photostimulation on NREM () or REM () sleep-to-wake transitions in eYFP (n = 6) or ChR2-eYFP (n = 6) transduced mice. Each square represents the mean probability of a sleep-to-wake transition within 10 s of the onset of stimulation. Data analysis is based on an average of 15 () or 8 () stimulations per condit! ion per mouse. * Figure 4: Long-term tonic versus phasic stimulation of the locus coeruleus causes differential promotion of arousal. Tonic (consistent 10-ms pulses at 3 Hz) and phasic (10-ms pulses at 10 Hz for 500 ms every 20 s) stimulation protocols are consistent throughout. (,) The effect of tonic () or phasic () photostimulation for 1 h on sleep architecture in ChR2-eYFP (n = 5) or eYFP (n = 5) transduced mice. Data represent the mean ± s.e.m. percentage time over four trials spent in wake, NREM sleep or REM sleep in baseline conditions or during photostimulation. **P < 0.001, ***P < 0.0001, two-way ANOVA between stimulation condition and viral transduction, followed by Tukey post-hoc test. (,) Sleep recordings in the hour after tonic () or phasic () photostimulation. *P < 0.05, **P < 0.001, Student's t test. (,) Representative traces of locomotor activity in eYFP and ChR2-eYFP transduced mice during tonic () or phasic () photostimulation for 1 h during a 10-min wake period. Quantification (right) shows the mean ± s.e.m. distance traveled by eYFP (n = 5) or ChR2-eYFP (n = 5) transduced mice over th! e 1-h of photostimulation after 5 sessions of stimulation per mouse. *P < 0.05, **P < 0.001, two-way ANOVA between stimulation condition and viral transduction, followed by Tukey post-hoc test. (,) The effect of tonic () or phasic () photostimulation for 5 h on sleep architecture in ChR2-eYFP (n = 5) or eYFP (n = 5) transduced mice. *P < 0.05, two-way ANOVA between stimulation condition and viral transduction, followed by Tukey post-hoc test. * Figure 5: High-frequency photostimulation of the locus coeruleus causes reversible behavioral arrests. () Sequence of events in a behavioral arrest. () Probability of behavioral arrests depends on photostimulation frequency. Data represent mean ± s.e.m. for ChR2-eYFP mice (n = 4 mice, ten trials per frequency per mouse). () The duration of latencies to arrest (time from light onset until behavioral arrest) and durations of arrest (time between the onset and offset of behavioral arrest) in stimulated ChR2-eYFP mice. Data represent the mean ± s.e.m. of 20 trials per mouse, n = 8 mice. *P < 0.05, **P < 0.001 between frequencies, ANOVA followed by Tukey post-hoc test. () Representative EEG and EMG trace of a behavioral arrest after 10-Hz photostimulation. Arrows represent the onset and offset of immobility. () Relative EEG power of the first 10 s of behavioral arrests across multiple mice. Data represent the mean ± s.e.m. relative power of 0.5-Hz binned frequencies (20 episodes per mouse, n = 6 mice). () Extracellular norepinephrine content in prefrontal cortex during 10-Hz st! imulation. Data represent the mean ± s.e.m. of three trials per mouse, n = 4 mice. **P < 0.001, two-way ANOVA between time point and virally-transduced animal followed by Bonferroni post-hoc test. () The duration of latencies to arrest and durations of arrest in ChR2-eYFP stimulated mice upon administration of the norepinephrine reuptake inhibitors atomoxetine and reboxetine. Data represent the mean ± s.e.m. of ten trials per mouse, n = 4 mice. Increased darkness of bars represents increasing pharmacological dose. *P < 0.05, Student's t test between saline and drug-injected mice. Author information * Abstract * Author information * Supplementary information Affiliations * Neurosciences Program, Stanford University, Stanford, California, USA. * Matthew E Carter, * Karl Deisseroth & * Luis de Lecea * Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, California, USA. * Matthew E Carter, * Hieu Nguyen, * Antoine Adamantidis & * Luis de Lecea * Department of Bioengineering, Stanford University, Stanford, California, USA. * Ofer Yizhar & * Karl Deisseroth * Sleep & Circadian Neurobiology Laboratory, Stanford University, Stanford, California, USA. * Sachiko Chikahisa & * Seiji Nishino * Department of Integrative Physiology, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima, Japan. * Sachiko Chikahisa Contributions M.E.C. and L.d.L. designed the study and wrote the manuscript. M.E.C. performed or assisted with all experiments. O.Y. performed and analyzed electrophysiology experiments, S.C. performed HPLC analysis, and H.N. analyzed immunohistochemical co-expression data. A.A. and L.d.L. provided expertise on optogenetic and polysomnographic recording techniques, as well as substantial feedback on the manuscript. S.N., K.D. and L.d.L. provided equipment, reagents and critical feedback. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Luis de Lecea (llecea@stanford.edu) Supplementary information * Abstract * Author information * Supplementary information Movies * Supplementary Movie 1 (904K) Representative sleep-to-wake transition following photostimulation of locus coeruleus neurons during NREM sleep. Photostimulation condition was 10 ms pulses at 5 Hz for 5 s. * Supplementary Movie 2 (3K) Representative behavioral arrest following sustained, high-frequency photostimulation of locus coeruleus neurons with 10 ms pulses at 10 Hz. PDF files * Supplementary Text and Figures (8M) Supplementary Figures 1–11 Additional data - Thalamic synchrony and the adaptive gating of information flow to cortex
- Nat Neurosci 13(12):1534-1541 (2010)
Nature Neuroscience | Article Thalamic synchrony and the adaptive gating of information flow to cortex * Qi Wang1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Roxanna M Webber3 Search for this author in: * NPG journals * PubMed * Google Scholar * Garrett B Stanley1garrett.stanley@bme.gatech.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 13,Pages:1534–1541Year published:(2010)DOI:doi:10.1038/nn.2670Received05 August 2010Accepted21 September 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 Although it has long been posited that sensory adaptation serves to enhance information flow in sensory pathways, the neural basis remains elusive. Simultaneous single-unit recordings in the thalamus and cortex in anesthetized rats showed that adaptation differentially influenced thalamus and cortex in a manner that fundamentally changed the nature of information conveyed about vibrissa motion. Using an ideal observer of cortical activity, we found that performance in detecting vibrissal deflections degraded with adaptation while performance in discriminating among vibrissal deflections of different velocities was enhanced, a trend not observed in thalamus. Analysis of simultaneously recorded thalamic neurons did reveal, however, an analogous adaptive change in thalamic synchrony that mirrored the cortical response. An integrate-and-fire model using experimentally measured thalamic input reproduced the observed transformations. The results here suggest a shift in coding stra! tegy with adaptation that directly controls information relayed to cortex, which could have implications for encoding velocity signatures of textures. View full text Figures at a glance * Figure 1: Statistical properties of cortical response adapt to vibrissa deflections. () Example waveforms from recordings in the thalamic VPm (right) and cortical layer 4 (CTX, left). Single-unit extracellular recordings were made during movement of the identified primary vibrissa in the rostral–caudal plane using a computer-controlled piezoelectric bending actuator. Gray bands denote ± 1 s.d. () Adaptation in the PSTH (2-ms bin size) for a typical cortical regular spiking unit (RSU) in response to a 12-Hz sequence of punctate vibrissa deflections (15 cycles, 800 deg s−1; see Online Methods). () Top: mean spike count (in 30-ms bin following each deflection) across the sample (n = 30 cortical RSUs). Curve is an exponential fit using least squares, excluding the outlier in response to the second pulse. Bottom: corresponding spike count variance (left axis) and Fano factor (variance/mean, right axis), across the cortical sample. () Cortical firing statistics. Each data point represents the response in a 30-ms window following the probe stimulus. Dashed lin! e shows exponential fit of relationship, as compared to the Poisson case for which the variance equals the mean (solid line). In general, response was sub-Poisson, with trial-to-trial spike count variance less than the mean for high spike counts. Error bars are ± 1 s.e.m. * Figure 2: Adaptation degrades stimulus detection for ideal observer of cortical activity. () Observer attributes response to 'signal' or 'noise', in the presence (adapted) and absence (nonadapted) of a preceding adapting stimulus (15 cycles of a 12-Hz sequence of a punctate deflection at 800 deg s−1). () Variations in spike count from trial to trial establish distributions for the signal (black) and the noise (gray) (parametric fits of raw data; Supplementary Note 1), in both the nonadapted (top) and adapted (bottom) states. Above a threshold, the observed spike count was attributed to signal; below, to noise. The mean spontaneous firing rate decreased from 0.68 ± 0.093 Hz in the nonadapted state to 0.42 ± 0.064 Hz in the adapted state (mean ± s.e.m., P < 0.01, Wilcoxon signed-rank test). () The area under the ROC curve was used as a metric for overall performance (AUROC). Diagonal represents chance performance, where probability of false alarm and hit are equal and AUROC is 0.5. () Performance (AUROC) in the detection task in the nonadapted versus adapted s! tates (n = 30 cortical (CTX) RSUs) for the lowest velocity used for the probe. The dashed line is the unity line. () Detection performance for the entire range of probe velocities. Control is the resultant performance when the spike count variance was forced to equal that of the experimentally observed mean spike count in the nonadapted and adapted states (Supplementary Note 2). Performance was significantly better in the nonadapted state than in the adapted (**P < 10−19, **P < 10−20 for control, Wilcoxon signed-rank test). Error bars are ± 1 s.e.m. * Figure 3: Adaptation enhances cortical discriminability. () In the discrimination (discrim) task, observer attributes spike count to one of several possible stimuli (stim), in the presence (adapted) and absence (nonadapted) of a preceding adapting stimulus. () Mean sensitivity of the cortical (CTX) response to the deflection velocity in the nonadapted (dotted) and adapted (solid) states (n = 30 cortical RSUs). The velocities to be discriminated between (s1–s5) are noted on the horizontal axis. () Population spike count distributions for a typical cortical neuron for the velocities (s1–s5) in the nonadapted (top) and adapted (bottom) states. Adaptation attenuated the response but also separated the distributions. Shown in the inset is the performance matrix (see Online Methods). () The overall discriminability performance, quantified as the fraction of correct identifications. The crosses represent cortical neurons that were identified as monosynaptic recipients of VPm inputs (Fig. 5). () Discrimination performance was signific! antly better in the adapted state than in the nonadapted state (n = 30 cortical RSUs, **P < 3 × 10−4 for non-adapted, **P < 1 × 10−4 for control 1 and control 2, Wilcoxon signed-rank test). Control 1 is the case in which the mean spike count in response to each velocity in the nonadapted state was attenuated by deleting 33% of the spikes to match the observed overall spike count reduction with adaptation. Control 2 is the case in which the mean and variance in the nonadapted state were scaled down 33% to match the spike count reduction caused by the adaptation (Supplementary Note 2). In both control cases, there was no difference in performance from the nonadapted state (control 1, P = 0.12; control 2, P = 0.25; Wilcoxon signed-rank test). Error bars are ± 1 s.e.m. * Figure 4: Cortical performance does not trivially mirror activity of thalamic projections. () For each cortical recording, a VPm neuron in the homologous barreloid was recorded simultaneously. The VPm neurons adapted to the persistent, ongoing periodic vibrissa deflection but showed less attenuation in the response (PSTH for a typical example: 1-ms bin, mean spike count across the larger sample, n = 32 VPm units). Curve is an exponential fit using least squares, excluding the outlier in response to the second pulse. The inset shows the adaptation ratio for the cortical (CTX) RSUs (67%) and VPm (80%) neurons. () Discrimination performance in the nonadapted versus adapted states for the recorded VPm units (n = 32). Highlighted with crosses are those that were identified as monosynaptically connected to the RSUs highlighted in Figure 3d. Adaptation did not affect the discrimination performance for an ideal observer of VPm spike count (P = 0.19, n = 32, Wilcoxon signed-rank test). () In contrast to the sensitivity curve in cortex, the sensitivity curve in VPm retained! its shape after adaptation, resulting in little or no change in overall sensitivity and thus no change in performance. Error bars are ± 1 s.e.m. * Figure 5: Shift in discrimination performance is maintained in monosynaptically connected thalamocortical pairs. () An example of the raw (reflected about the horizontal axis) and shuffle-corrected correlograms for a particular VPm–cortex (CTX) pair, along with the corresponding >99% confidence interval (3 s.d.) on an uncorrelated process (dotted line). The inset shows the average shuffle-corrected correlogram for the eight pairs. VPm–CTX pairs were identified as monosynaptically connected on the basis of the presence of a statistically significant, short-latency peak in the spike train cross-correlation function for weak 4-Hz sinusoidal vibrissa stimulation. () The results for the larger sample (Figs. 3 and 4) held for the smaller, monosynaptically connected sample (CTX, **P < 0.007; VPm, P = 0.84; Wilcoxon signed-rank test). () Percent change in discrimination performance from nonadapted to adapted states for each VPm–CTX pair. Gray lines denote pairs identified as likely to be monosynaptically connected. Thick dashed line shows the mean performance change. Error bars are ± 1 ! s.e.m. * Figure 6: Thalamic population synchrony is modulated by adaptation. () Synchrony (synch.) was measured as the central area under the cross-correlogram (± 7.5 ms; see Supplementary Note 4 and Supplementary Fig. 10). Shown also are the measures of synchrony for the five velocities to be discriminated among, in the nonadapted (left) and adapted (right) states. The inset shows the spike cross-correlograms in the nonadapted and adapted states. For VPm pairs recorded simultaneously, the adaptation served to reduce the timing precision across neurons, or desynchronize their firing activity (n = 19 pairs). When the trials were shuffled (dotted line, open symbols), the resulting synchrony in response to the velocity was unchanged. () Synchronous firing across VPm pairs (n = 19 pairs) as a function of deflection velocity, in both the nonadapted (dotted, circle) and adapted states (solid, square). * Figure 7: Thalamocortical network model predictions. () Thalamic spiking that falls within the cortical integration window is relayed to cortex. In the nonadapted state (left), velocity effects on synchrony of VPm activity are small, and signals are strongly relayed to cortex. In the adapted state (right), the integration window widens, and the velocity strongly modulates the VPm synchrony. At lower velocities, VPm spiking falls outside the integration window and does not relay to cortex. () The spiking activity from a population of VPm neurons was used as the input to an integrate-and-fire model of the cortical response. Firing of a VPm input generates an excitatory postsynaptic current (EPSC), the sum of which is integrated in the model to affect the cortical membrane potential. Upon crossing a threshold, the model cortical cell (CTX) fires a spike, then resets. () Velocity sensitivity curves for the simulated cortical response in the nonadapted (dashed) and adapted (solid) states, both normalized to their peak spike count. ! () The change in thalamic synchrony with adaptation served to increase performance in discriminating between different velocities, as judged by cortical spike count response (P < 2 × 10−4 versus non-adapted and control, Wilcoxon signed-rank test). The control shows the case where the adaptation produced changes in the VPm spike count, as observed, but the degree of synchrony was maintained in the nonadapted state, resulting in a loss in performance in the adapted state. Control was not different from non-adapted (P = 0.43, Wilcoxon signed-rank test). Error bars are ± 1 s.e.m. Author information * Abstract * Author information * Supplementary information Affiliations * Coulter Department of Biomedical Engineering, Georgia Institute of Technology & Emory University, Atlanta, Georgia, USA. * Qi Wang & * Garrett B Stanley * School of Engineering & Applied Sciences, Harvard University, Cambridge, Massachusetts, USA. * Qi Wang * Department of Neurobiology, Harvard Medical School, Boston, Massachusetts, USA. * Roxanna M Webber Contributions Q.W. and G.B.S. conceived the study. Q.W., R.M.W. and G.B.S. designed the experiments. Q.W. performed the experiments. Q.W. and G.B.S. analyzed the data, and Q.W., R.M.W. and G.B.S. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Garrett B Stanley (garrett.stanley@bme.gatech.edu) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (3M) Supplementary Figures 1–10 and Supplementary Notes 1–5 Additional data - Functional organization for color and orientation in macaque V4
- Nat Neurosci 13(12):1542-1548 (2010)
Nature Neuroscience | Article Functional organization for color and orientation in macaque V4 * Hisashi Tanigawa1 Search for this author in: * NPG journals * PubMed * Google Scholar * Haidong D Lu1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Anna W Roe1anna.w.roe@vanderbilt.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 13,Pages:1542–1548Year published:(2010)DOI:doi:10.1038/nn.2676Received21 July 2010Accepted28 September 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 Visual area V4 in the macaque monkey is a cortical area that is strongly involved in color and shape perception. However, fundamental questions about V4 are still debated. V4 was initially characterized as a color-processing area, but subsequent studies revealed that it contains a diverse complement of cells, including those with preference for color, orientation, disparity and higher-order feature preferences. This has led to disputes and uncertainty about the role of V4 in vision. Using intrinsic signal optical imaging methods in awake, behaving monkeys, we found that different feature preferences are functionally organized in V4. Optical images revealed that regions with preferential response to color were largely separate from orientation-selective regions. Our results help to resolve long-standing controversies regarding functional diversity and retinotopy in V4 and indicate the presence of spatially biased distribution of featural representation in V4 in the ventral vi! sual pathway. View full text Figures at a glance * Figure 1: Examples of time course of stimulus-evoked reflectance change in V4. () Sampled sites indicated by black crosses on an image of exposed V4 cortical surface taken under 570-nm illumination. Scale bar represents 1 mm. (–) Time courses of average reflectance change (ΔR/R) under 630-nm illumination at the sampled sites (160 μm in diameter) in response to a stimulus patch containing one of four types of gratings (red, 45° R/G; orange, 135° R/G; blue, 45° Lum; blue, 135° Lum; see Online Methods for details). The response values were taken from the single condition map, in which high-pass filtering and blank subtraction were applied. Error bars represent s.e.m. * Figure 2: Functional maps of color and orientation sensitivity in foveal V4. (,) Views of the cortical surface including dorsal V4 through the imaging chamber. The white rectangles indicate the imaged regions (case 1, ; case 3, ). (–) Difference maps in response to R/G versus Lum (,) and 45° versus 135° (,). In these maps, dark versus light spots represent greater response to R/G versus Lum (,, magenta arrowheads) and 45° versus 135° (,, green arrowheads). Such spots were also observed in V2 (,, cyan arrowheads). Gratings above the maps indicate the subtraction pair for each difference map. (–) Statistical maps show significant differences in response to R/G versus Lum (,) and 45° versus 135° (,) (two-tailed t test, n = 414 trials for case 1 and 236 trials for case 3). Colored areas indicate significantly larger response to one of paired conditions, according to the key shown on the right. (–) Statistical maps created using two-way ANOVAs show regions with a significant main effect of stimulus color type (R/G versus Lum) (,, magenta), wit! h a significant main effect of stimulus orientation (,, green) and with a significant interaction (,, white). In –, the brightness of color indicates the significance level, P < 0.05 (dark) and P < 0.0001 (bright), uncorrected, and dark gray regions indicate pixels with large cross-trial variability (Online Methods). The sampled sites in Figure 1 are shown by black crosses in . A, anterior; D, dorsal; io, inferior occipital sulcus; lu, lunate sulcus; st, superior temporal sulcus. Scale bar represents 1 mm (–). * Figure 3: Functional maps of color and orientation sensitivity in parafoveal V4. (–) Data from a parafoveal V4 (~5° eccentricity) of the same animal as case 1 (case 2, n = 400 trials). Data are presented as in Figure 2. () Overlay of the maps of color-sensitive (magenta) and orientation-sensitive (green) regions in the foveal (Fig. 2k) and parafoveal V4 (), aligned using the surface blood vessel pattern. Note the magenta and green regions in V2, consistent with thin and thick/pale stripe organization, respectively12, 14. Scale bar represents 1 mm. * Figure 4: Overall spatial pattern of modulation by the stimulus at different locations in the visual field. (–) Response to shifting the polar angle. Regions with a significant difference in response to R/G versus Lum (–, case 3) and to 45° versus 135° (–), presented at a different polar angle with the same eccentricity as illustrated above (two-tailed t test, n = 161 (,), 159 (,) and 151 trials (,)). The center of stimuli was located at polar angles of 22.5° (,), 45° (,) and 67.5° (,) from the vertical meridian and an eccentricity of 1.5°. As the stimulus location moved away from the vertical meridian along a line of isoeccentricity, the overall activation shifted from posterior to anterior (use vertical red lines as guide). (–) Response to shifting the eccentricity. Regions with a significant difference in response to R/G versus Lum (–) and to 45° versus 135° (–), presented at a different eccentricity along an isopolar axis as illustrated above (two-tailed t test, n = 164 (,), 159 (,) and 159 trials (,)). The center of stimuli was located at a polar angle of ! 45° from the vertical meridian and at eccentricities of 0.5° (,), 1.5° (,) and 2.5° (,). As the stimulus location moved toward larger eccentricities, the overall activation shifted from ventral to dorsal (use horizontal red lines as guide). The size of grating patches was 1° in diameter. Scale bar represents 1 mm. * Figure 5: Functional maps of orientation preference in V4. (,) Difference maps to orthogonally oriented gratings (0°–90°, ; 45°–135°, ; case 1). Dark versus light pixels represent greater response to 0° versus 90° () and 45° versus 135° (). () Color-coded polar map of orientation preference (orientation preference indicated by hue and magnitude of orientation selectivity by brightness. Pure black regions represent pixels without significant selectivity for orientation (ANOVA, 4 orientations, P > 0.05, uncorrected, n = 690 trials). (–) Data from another animal (case 3, n = 468 trials). Data are presented as in –. (–) Angle maps of regions in white frames in and (frame 1, ; frame 2, ; frame 3, ). In these maps, all hues are saturated in brightness to indicate only orientation preference. Scale bars represent 1 mm (–) and 0.5 mm (–). * Figure 6: Functional map of hue preference in V4. () Left, stimulus colors plotted on the CIE 1931-xy chromaticity diagram (dots). M, magenta; R, red; Y, yellow; G, green; C, cyan; B, blue; W, white. Right, key for the hue-preference map. () Time courses of reflectance change at a sampled site (white dot in ) in response to each of six types of colored gratings and one type of achromatic gratings. Error bars represent s.e.m. Data are presented as in Figure 1b. (,) Large field views of color-coded polar maps of hue preference (case 2, ; case 3, ). Pixels with no significant selectivity for hue (one-way ANOVA, six hues, P > 0.05, uncorrected, n = 707 trials for , n = 495 trials for ) and pixels with large cross-trial variability are shaded in black and dark gray, respectively. (,) Difference maps taken from the boxed region in and , respectively. These maps were calculated by subtracting the response to achromatic gratings from the response to each of six colored gratings (top to the second from the bottom). The regions with ! a significantly larger response to one of colored gratings than to achromatic gratings are outlined by that color in the bottom panels (two-tailed t test, P < 0.05 and peak P < 0.0001, uncorrected, n = 231, 230, 227, 229, 231 and 225 trials in , n = 165, 170, 167, 162, 165 and 164 trials in , from magenta to blue, respectively). Scale bars represent 1.0 mm (,) and 0.5 mm (,). * Figure 7: Luminance invariance in responses of hue-preferring region. (–) Difference maps of red/black gratings minus achromatic gratings (Lum) obtained from a color-responsive region of parafoveal V4 (case 4) with different luminance. The luminance of red strips in gratings used was 23.1 cd m−2 in , 15.4 cd m−2 (33.3% darker) in and 7.69 cd m−2 (66.7% darker) in . The luminance of white strips in Lum and background was 23.1 cd m−2 and 11.6 cd m−2, respectively. (–) Statistical maps show regions with a significantly larger response to red/black gratings than to Lum in – (colored areas, two-tailed t test, n = 137, 137 and 138 trials for –, respectively). (–) Difference maps of green/black gratings minus Lum obtained from the same region as in –. The luminance profiles of gratings were the same as in –, respectively. (–) Statistical maps show regions with a significantly larger response to green/black gratings than to Lum in – (two-tailed t test, n = 105, 103 and 104 trials for –, respectively). In the statistical m! aps (–,–), the brightness of color indicates the significance level, P < 0.05 (dark) and P < 0.0001 (bright), uncorrected, and dark gray regions indicate pixels with large cross-trial variability (Online Methods). Scale bar represents 0.5 mm. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Psychology, Vanderbilt University, Nashville, Tennessee, USA. * Hisashi Tanigawa, * Haidong D Lu & * Anna W Roe * Present address: Institute of Neuroscience, Chinese Academy of Sciences, Shanghai, China. * Haidong D Lu Contributions H.T. and A.W.R. designed the experiments. H.T. performed the experiments and analyzed the data. H.L. assisted H.T. with experimental procedures. H.T. and A.W.R. discussed the results and wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Anna W Roe (anna.w.roe@vanderbilt.edu) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–7, Supplementary Table 1 and Supplementary Note Additional data - Microsaccades precisely relocate gaze in a high visual acuity task
- Nat Neurosci 13(12):1549-1553 (2010)
Nature Neuroscience | Article Microsaccades precisely relocate gaze in a high visual acuity task * Hee-kyoung Ko1 Search for this author in: * NPG journals * PubMed * Google Scholar * Martina Poletti1 Search for this author in: * NPG journals * PubMed * Google Scholar * Michele Rucci1, 2, 3mrucci@bu.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 13,Pages:1549–1553Year published:(2010)DOI:doi:10.1038/nn.2663Received28 April 2010Accepted14 September 2010Published online31 October 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The image on the retina is never stationary. Microscopic relocations of gaze, known as microsaccades, occur even during steady fixation. It has long been thought that microsaccades enable exploration of small regions in the scene in the same way saccades are normally used to scan larger regions. This hypothesis, however, has remained controversial, as it is believed that microsaccades are suppressed during fine spatial judgments. We examined the eye movements of human observers in a high-acuity visuomotor task, the threading of a needle in a computer-simulated virtual environment. Using a method for gaze-contingent display that enables accurate localization of the line of sight, we found that microsaccades precisely move the eye to nearby regions of interest and are dynamically modulated by the ongoing demands of the task. These results indicate that microsaccades are part of the oculomotor strategy by which the visual system acquires fine spatial detail. View full text Figures at a glance * Figure 1: Threading a virtual needle. () The arena in which all of the threading experiments were conducted. Subjects used a joypad to align a horizontal bar (the thread) with the gap in a vertical bar (the needle). The gray bars represent the positions of the thread at various times during the trial. The actual stimulus on the display is shown on the right at two different times, t1 and tn. (,) Results from two experiments with conditions similar to those of previous studies10, 11. In , subjects maintained fixation on the needle's eye and had no control over the thread's position. In , subjects were free to move their eyes and fully controlled the thread. The two intervals refer to the initial 4 s (start) and the last 0.5 s (end) of each trial. Both the mean microsaccade rate and the frequency of adjustments in the thread's vertical position are shown in . *P < 0.01, one-tailed t test. Error bars represent s.e.m. * Figure 2: Comparison of saccade characteristics in three different tasks: threading, sustained fixation on a marker and free viewing of natural images. (–) Distributions of saccade amplitudes. The triangles mark the medians of the distributions. The insert in shows the range of small amplitudes. () Mean rates of microsaccades, defined as saccades smaller than 20 arcmin (ANOVA with Scheffe post hoc comparisons, *P = 0.009, **P = 0.004). Error bars represent s.e.m. Individual trials of the threading task are shown in Supplementary Videos 1 and 2. * Figure 3: Modulation of saccade characteristics. (,) Mean instantaneous frequency of microsaccades, defined as saccades smaller than 20 arcmin or saccades smaller than 10 arcmin. () Mean instantaneous saccade amplitude. The two curves in each panel represent data obtained in the presence and absence of background noise. In this latter condition, the background was at a constant gray level and the stimulus was displayed at maximum contrast. Horizontal lines in each panel indicate mean values during sustained fixation (dashed line) and free viewing (dotted line). The asterisk indicates conditions in which measured values were significantly higher during the last 7.5 s of a trial than during the initial 7.5 s (P < 0.04, one-tailed t test in and and Wilcoxon signed-rank test in ). * Figure 4: Analysis of fixation locations. () Each intersaccadic period was classified as a fixation on the eye of the needle, the thread or the background according to the location of its centroid. The distance D between the needle and the thread varied during the course of the trial. () Two examples of spatial distributions of fixations. Each panel corresponds to a different experimental trial. Blue and green circles represent fixations on the thread and on the eye of the needle, respectively. The red crosses mark the trajectory followed by the thread. Inserts, higher magnification of the center of the display. () Mean probabilities of fixation locations. Differences across all conditions were significant (ANOVA with Scheffe post hoc comparisons, P < 0.002). () Fixation probabilities at successive intervals during the course of the trial. * Figure 5: Analysis of microsaccades. () Probabilities of various types of microsaccades during fixation on the needle and on the thread. Microsaccades were subdivided according to where they landed. Data refer to saccades smaller than 20 arcmin. () Influence of microsaccade direction on the direction of the following microsaccade. During the last 2.5 s in each trial, consecutive microsaccades possessed opposite directions on the horizontal axis. In both graphs, all differences within each group were statistically significant (paired z test with Bonferroni corrections, P < 0.001). * Figure 6: Interaction between microsaccades and corrections in the thread-needle alignment. () Probability distributions of adjustments to the thread's position as a function of the location of fixation during which they occurred. () Rates of adjustments. Data points represent the average numbers of changes in the thread's position per fixation. In both and , all differences were statistically significant (paired z test with Bonferroni corrections, P < 0.01). (,) Conditional probabilities of adjustments following different types of microsaccades. The fixation in which the adjustment occurred (x axis) is the target destination of the microsaccade. (,) Conditional probabilities of performing different types of microsaccades following an adjustment. The fixation in which the adjustment occurred (x axis) is the origin of the microsaccade. Data are shown for both microsaccades smaller than 20 arcmin (,) and 10 arcmin (,). In –, microsaccades are arranged according to whether they maintained fixation on the same object (thread or needle) or moved the line of sight from! one to the other. *P < 0.05, paired z-test with Bonferroni corrections. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Psychology, Boston University, Boston, Massachusetts, USA. * Hee-kyoung Ko, * Martina Poletti & * Michele Rucci * Biomedical Engineering, Boston University, Boston, Massachusetts, USA. * Michele Rucci * Program in Neuroscience, Boston University, Boston, Massachusetts, USA. * Michele Rucci Contributions H.-K.K. and M.P. collected data. M.R. supervised the experiments. All of the authors contributed to the design of the experiments, data analysis and the writing of the manuscript. The first two authors contributed equally to this work. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Michele Rucci (mrucci@bu.edu) Supplementary information * Abstract * Author information * Supplementary information Movies * Supplementary Video 1 (840K) Example of experimental trial. Reconstruction of the stimulus experienced by the subject during an experimental trial. The blue cross marks the position of the current location of gaze, which has been superimposed to the stimulus in order to show the eye movements performed by the observer. This cross was not displayed during the actual experiment. Intervals in which the cross turns red represent periods of blink. * Supplementary Video 2 (848K) Example of experimental trial. Reconstruction of the stimulus experienced by the subject during an experimental trial. The blue cross marks the position of the current location of gaze, which has been superimposed to the stimulus in order to show the eye movements performed by the observer. This cross was not displayed during the actual experiment. Intervals in which the cross turns red represent periods of blink. PDF files * Supplementary Text and Figures (96K) Supplementary Figure 1 and Supplementary Results Additional data - When size matters: attention affects performance by contrast or response gain
- Nat Neurosci 13(12):1554-1559 (2010)
Nature Neuroscience | Article When size matters: attention affects performance by contrast or response gain * Katrin Herrmann1 Search for this author in: * NPG journals * PubMed * Google Scholar * Leila Montaser-Kouhsari1 Search for this author in: * NPG journals * PubMed * Google Scholar * Marisa Carrasco1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * David J Heeger1, 2david.heeger@nyu.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature NeuroscienceVolume: 13,Pages:1554–1559Year published:(2010)DOI:doi:10.1038/nn.2669Received09 March 2010Accepted22 September 2010Published online07 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 Covert attention, the selective processing of visual information in the absence of eye movements, improves behavioral performance. We found that attention, both exogenous (involuntary) and endogenous (voluntary), can affect performance by contrast or response gain changes, depending on the stimulus size and the relative size of the attention field. These two variables were manipulated in a cueing task while stimulus contrast was varied. We observed a change in behavioral performance consonant with a change in contrast gain for small stimuli paired with spatial uncertainty and a change in response gain for large stimuli presented at one location (no uncertainty) and surrounded by irrelevant flanking distracters. A complementary neuroimaging experiment revealed that observers' attention fields were wider with than without spatial uncertainty. Our results support important predictions of the normalization model of attention and reconcile previous, seemingly contradictory findin! gs on the effects of visual attention. View full text Figures at a glance * Figure 1: Normalization model of attention exhibits qualitatively different forms of attentional modulation, depending on stimulus size and attention field size (adapted from ref. 1). Each panel shows contrast-response functions for a simulated neuron when attending to a stimulus in the neuron's receptive field and when attending to a stimulus in the opposite hemifield. () Response gain (largest effects at higher contrasts, upward shift of the contrast-response function) for large stimulus size and small attention field. () Contrast gain (largest effect at intermediate contrasts, appears as a leftward shift of the contrast-response function) for small stimulus size and large attention field. Red, simulated responses as a function of contrast when stimuli in the receptive field were attended. Blue, simulated responses when attending to the opposite hemifield. Only stimulus size and attention field size were changed in simulations; all other model parameters were identical in both panels. The solid black circle indicates simulated receptive field size. The dashed red circle indicates simulated attention field size. The vertical black grating indicates stimu! lus size. * Figure 2: Experimental protocols. () Exogenous attention task. ISI, interstimulus interval; ITI, intertrial interval. () Endogenous attention task. () Small stimuli were presented with spatial uncertainty, at one of five predefined isoeccentric locations. Across trials, stimulus locations varied randomly and independently on the left and right sides to encourage observers to employ a larger attention field. The dashed, white circles indicate the possible stimulus locations (not displayed during the experiments). () Large stimuli were presented at fixed stimulus locations with no spatial uncertainty (centered at the middle of the five locations of ). To narrow the size of the attention field for endogenous attention, the two large Gabor stimuli were each surrounded by six irrelevant distracters. * Figure 3: Effects of exogenous and endogenous attention on performance (d′) as a function of contrast. (,) Large stimulus with small attention field. (,) Small stimulus with large attention field. Exogenous attention is shown in and . Endogenous attention is shown in and . Shown are plots of psychometric functions for each attentional condition (valid, neutral and invalid pre-cues) and parameter estimates (c50, contrast yielding half-maximum performance; d′max, asymptotic performance at high contrast). Exponent n (slope) was constrained to have the same value for all pre-cue conditions. Each data point represents the mean across observers. Error bars on data points are ± s.e.m. (n = 4 observers in each experiment, 9,408–14,910 trials per observer, except one observer who completed 21,000 in the exogenous experiment). Error bars on parameter estimates are 68% confidence intervals, obtained by bootstrapping. * Figure 4: Effects of stimulus and attention field size on parameter estimates of individual observers. () Contrast yielding half maximum performance (c50), estimated for valid pre-cues versus invalid pre-cues. () Asymptotic performance at high contrast (d′max). Red symbols indicate large stimulus with small attention field. Blue symbols indicate small stimulus with large attention field. Open symbols indicate individual observers. Filled symbols indicate mean across observers. Squares indicate exogenous attention. Circles indicate endogenous attention. Observer 1 participated in both the exogenous and endogenous attention experiments and is marked with diagonal lines through the symbols. * Figure 5: Attention field size depends on spatial uncertainty. () fMRI responses at a series of locations in left V1 for a typical observer when a small stimulus was presented at the center of the five possible locations during the experiment with spatial uncertainty. The upper abscissa indicates the distance along a 6° isoeccentric arc in the visual field that included the stimulus location. Zero corresponds to the location at which the arc crosses the lower vertical meridian. The lower abscissa indicates the distance in degrees of polar angle. Black symbols represent fMRI response differences (cued minus uncued), plotted at locations corresponding to the centroids of each of several V1 regions of interest, defined based on retinotopic mapping. Open symbol, location corresponding to the center of the stimulus. The gray symbol represents the estimated center of the attention field. The gray line represents the estimated spread of the attention field. () fMRI measurements of the spread of cortical activity in V1, in units of arc degrees! of visual angle in the visual field, with spatial uncertainty versus without spatial uncertainty. Each symbol shape corresponds to one observer. Gray indicates left hemisphere. Black indicates right hemisphere. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Psychology, New York University, New York, New York, USA. * Katrin Herrmann, * Leila Montaser-Kouhsari, * Marisa Carrasco & * David J Heeger * Center for Neural Science, New York University, New York, New York, USA. * Marisa Carrasco & * David J Heeger Contributions K.H. programmed, conducted and analyzed the experiments and co-wrote the manuscript. L.M.-K. conducted and analyzed the psychophysics experiments and assisted in conducting and programming the fMRI experiment. M.C. and D.J.H. conceived and supervised the project and co-wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * David J Heeger (david.heeger@nyu.edu) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (404K) Supplementary Figures 1–3 and Supplementary Tables 1 and 2 Additional data
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