Key Specifications Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|R, A||IH(P), ELISA||Gp||Serum||Polyclonal Antibody|
|Presentation||Purified guinea pig polyclonal with 0.05% sodium azide.|
|Safety Information according to GHS|
|Material Size||50 µL|
|Anti-GABA - 2398912||2398912|
|Anti-GABA - 2166729||2166729|
|Anti-GABA - 2181272||2181272|
|Anti-GABA - 2262684||2262684|
|Anti-GABA - 2332548||2332548|
|Anti-GABA - 2557653||2557653|
|GUINEA PIG ANTI-GABA POLYCLONAL ANTIBODY - 2001218||2001218|
|GUINEA PIG ANTI-GABA POLYCLONAL ANTIBODY - 2034685||2034685|
|GUINEA PIG ANTI-GABA POLYCLONAL ANTIBODY - 2089406||2089406|
References | 30 Available | See All References
|Reference overview||Application||Species||Pub Med ID|
|Mapping synapses by conjugate light-electron array tomography. |
Collman, F; Buchanan, J; Phend, KD; Micheva, KD; Weinberg, RJ; Smith, SJ
The Journal of neuroscience : the official journal of the Society for Neuroscience 35 5792-807 2015
Synapses of the mammalian CNS are diverse in size, structure, molecular composition, and function. Synapses in their myriad variations are fundamental to neural circuit development, homeostasis, plasticity, and memory storage. Unfortunately, quantitative analysis and mapping of the brain's heterogeneous synapse populations has been limited by the lack of adequate single-synapse measurement methods. Electron microscopy (EM) is the definitive means to recognize and measure individual synaptic contacts, but EM has only limited abilities to measure the molecular composition of synapses. This report describes conjugate array tomography (AT), a volumetric imaging method that integrates immunofluorescence and EM imaging modalities in voxel-conjugate fashion. We illustrate the use of conjugate AT to advance the proteometric measurement of EM-validated single-synapse analysis in a study of mouse cortex.
|Inner retinal change in a novel rd1-FTL mouse model of retinal degeneration. |
Greferath, U; Anderson, EE; Jobling, AI; Vessey, KA; Martinez, G; de Iongh, RU; Kalloniatis, M; Fletcher, EL
Frontiers in cellular neuroscience 9 293 2015
While photoreceptor loss is the most devastating result of inherited retinal degenerations such as retinitis pigmentosa, inner retinal neurons also undergo significant alteration. Detailing these changes has become important as many vision restorative therapies target the remaining neurons. In this study, the rd1-Fos-Tau-LacZ (rd1-FTL) mouse model was used to explore inner retinal change at a late stage of retinal degeneration, after the loss of photoreceptor nuclei. The rd1-FTL model carries a mutation in the phosphodiesterase gene, Pde6b, and an axonally targeted transgenic beta galactosidase reporter system under the control of the c-fos promoter. Retinae of transgenic rd1-FTL mice and control FTL animals aged 2-12 months were processed for indirect fluorescence immunocytochemistry. At 2 months of age, a time when the majority of photoreceptor nuclei are lost, there was negligible c-fos reporter (FTL) expression, however, from 4 months, reporter expression was observed to increase within subpopulations of amacrine and ganglion cells within the central retina. These areas of inner retinal FTL expression coincided with regions that contained aberrant Müller cells. Specifically, these cells exhibited reduced glutamine synthetase and Kir4.1 immunolabelling, whilst showing evidence of proliferative gliosis (increased cyclinD1 and glial fibrillary acidic protein expression). These changes were limited to distinct regions where cone photoreceptor terminals were absent. Overall, these results highlight that distinct areas of the rd1-FTL central retina undergo significant glial alterations after cone photoreceptor loss. These areas coincide with up-regulation of the c-fos reporter in the inner retina, which may represent a change in neuronal function/plasticity. The rd1-FTL mouse is a useful model system to probe changes that occur in the inner retina at later stages of retinal degeneration.
|Tonic inhibition in dentate gyrus impairs long-term potentiation and memory in an Alzheimer's [corrected] disease model. |
Wu, Z; Guo, Z; Gearing, M; Chen, G
Nature communications 5 4159 2014
Amyloid plaques and tau tangles are common pathological hallmarks for Alzheimer's disease (AD); however, reducing Aβ production failed to relieve the symptoms of AD patients. Here we report a high GABA (γ-aminobutyric acid) content in reactive astrocytes in the dentate gyrus (DG) of a mouse model for AD (5xFAD) that results in increased tonic inhibition and memory deficit. We also confirm in human AD patient brains that dentate astrocytes have a high GABA content, suggesting that high astrocytic GABA level may be a novel biomarker and a potential diagnostic tool for AD. The excessive GABA in 5xFAD astrocytes is released through an astrocyte-specific GABA transporter GAT3/4, and significantly enhances tonic GABA inhibition in dentate granule cells. Importantly, reducing tonic inhibition in 5xFAD mice rescues the impairment of long-term potentiation (LTP) and memory deficit. Thus, reducing tonic GABA inhibition in the DG may lead to a novel therapy for AD.
|PDE9A is expressed in the inner retina and contributes to the normal shape of the photopic ERG waveform. |
Dhingra, A; Tummala, SR; Lyubarsky, A; Vardi, N
Frontiers in molecular neuroscience 7 60 2014
The ubiquitous second messenger cGMP is synthesized by guanylyl cyclase and hydrolyzed by phosphodiesterase (PDE). cGMP mediates numerous signaling pathways in multiple tissues. In the retina, cGMP regulates signaling in nearly every cell class including photoreceptors, bipolar cells, amacrine cells, and ganglion cells. In order to understand the specific role of cGMP and its regulating enzymes in different cell types, it is first necessary to localize these components and dissect their influence on the circuits. Here we tested the contribution of PDE9A to retinal processing by recording the electroretinograms (ERG) of PDE9A (™/™) (KO) mice and by localizing the enzyme. We found that while the scotopic ERG of KO was the same as that of wild type (WT) in both amplitude and kinetics, the photopic ERG was greatly affected. The greatest effect was on the recovery of the b-wave; the falling phase and the b-wave duration were significantly longer in the KO mice for all photopic stimuli (UV, green, or saturating white flashes). The rising phase was slower in KO than in WT for UV and green stimuli. For certain stimuli, amplitudes of both the a- and b-waves were smaller than in WT. Using Lac-Z expression in KO retinas as a reporter for PDE9A expression pattern, we found that PDE9A is localized to GABA-positive and GABA-negative amacrine cells, and likely also to certain types of ganglion cells. Our results indicate that PDE9A, by controlling the level of cGMP, modulates inhibitory processes within the cone pathway. We speculate that these circuits involve NO/cGMP signaling pathways.
|Ring finger protein 34 (RNF34) interacts with and promotes γ-aminobutyric acid type-A receptor degradation via ubiquitination of the γ2 subunit. |
Jin, H; Chiou, TT; Serwanski, DR; Miralles, CP; Pinal, N; De Blas, AL
The Journal of biological chemistry 289 29420-36 2014
We have found that the large intracellular loop of the γ2 GABAA receptor (R) subunit (γ2IL) interacts with RNF34 (an E3 ubiquitin ligase), as shown by yeast two-hybrid and in vitro pulldown assays. In brain extracts, RNF34 co-immunoprecipitates with assembled GABAARs. In co-transfected HEK293 cells, RNF34 reduces the expression of the γ2 GABAAR subunit by increasing the ratio of ubiquitinated/nonubiquitinated γ2. Mutating several lysines of the γ2IL into arginines makes the γ2 subunit resistant to RNF34-induced degradation. RNF34 also reduces the expression of the γ2 subunit when α1 and β3 subunits are co-assembled with γ2. This effect is partially reversed by leupeptin or MG132, indicating that both the lysosomal and proteasomal degradation pathways are involved. Immunofluorescence of cultured hippocampal neurons shows that RNF34 forms clusters and that a subset of these clusters is associated with GABAergic synapses. This association is also observed in the intact rat brain by electron microscopy immunocytochemistry. RNF34 is not expressed until the 2nd postnatal week of rat brain development, being highly expressed in some interneurons. Overexpression of RNF34 in hippocampal neurons decreases the density of γ2 GABAAR clusters and the number of GABAergic contacts that these neurons receive. Knocking down endogenous RNF34 with shRNA leads to increased γ2 GABAAR cluster density and GABAergic innervation. The results indicate that RNF34 regulates postsynaptic γ2-GABAAR clustering and GABAergic synaptic innervation by interacting with and ubiquitinating the γ2-GABAAR subunit promoting GABAAR degradation.
|Neural circuit interactions between the dorsal raphe nucleus and the lateral hypothalamus: an experimental and computational study. |
Jalewa, J; Joshi, A; McGinnity, TM; Prasad, G; Wong-Lin, K; Hölscher, C
PloS one 9 e88003 2014
Orexinergic/hypocretinergic (Ox) neurotransmission plays an important role in regulating sleep, as well as in anxiety and depression, for which the serotonergic (5-HT) system is also involved in. However, little is known regarding the direct and indirect interactions between 5-HT in the dorsal raphe nucleus (DRN) and Ox neurons in the lateral hypothalamus (LHA). In this study, we report the additional presence of 5-HT1BR, 5-HT2AR, 5-HT2CR and fast ligand-gated 5-HT3AR subtypes on the Ox neurons of transgenic Ox-enhanced green fluorescent protein (Ox-EGFP) and wild type C57Bl/6 mice using single and double immunofluorescence (IF) staining, respectively, and quantify the colocalization for each 5-HT receptor subtype. We further reveal the presence of 5-HT3AR and 5-HT1AR on GABAergic neurons in LHA. We also identify NMDAR1, OX1R and OX2R on Ox neurons, but none on adjacent GABAergic neurons. This suggests a one-way relationship between LHA's GABAergic and Ox neurons, wherein GABAergic neurons exerts an inhibitory effect on Ox neurons under partial DRN's 5-HT control. We also show that Ox axonal projections receive glutamatergic (PSD-95 immunopositive) and GABAergic (Gephyrin immunopositive) inputs in the DRN. We consider these and other available findings into our computational model to explore possible effects of neural circuit connection types and timescales on the DRN-LHA system's dynamics. We find that if the connections from 5-HT to LHA's GABAergic neurons are weakly excitatory or inhibitory, the network exhibits slow oscillations; not observed when the connection is strongly excitatory. Furthermore, if Ox directly excites 5-HT neurons at a fast timescale, phasic Ox activation can lead to an increase in 5-HT activity; no significant effect with slower timescale. Overall, our experimental and computational approaches provide insights towards a more complete understanding of the complex relationship between 5-HT in the DRN and Ox in the LHA.
|Age- and light-dependent development of localised retinal atrophy in CCL2(-/-)CX3CR1(GFP/GFP) mice. |
Chen, M; Hombrebueno, JR; Luo, C; Penalva, R; Zhao, J; Colhoun, L; Pandi, SP; Forrester, JV; Xu, H
PloS one 8 e61381 2013
Previous studies have shown that CCL2/CX3CR1 deficient mice on C57BL/6N background (with rd8 mutation) have an early onset (6 weeks) of spontaneous retinal degeneration. In this study, we generated CCL2(-/-)CX3CR1(GFP/GFP) mice on the C57BL/6J background. Retinal degeneration was not detected in CCL2(-/-)CX3CR1(GFP/GFP) mice younger than 6 months. Patches of whitish/yellowish fundus lesions were observed in 17∼60% of 12-month, and 30∼100% of 18-month CCL2(-/-)CX3CR1(GFP/GFP) mice. Fluorescein angiography revealed no choroidal neovascularisation in these mice. Patches of retinal pigment epithelium (RPE) and photoreceptor damage were detected in 30% and 50% of 12- and 18-month CCL2(-/-)CX3CR1(GFP/GFP) mice respectively, but not in wild-type mice. All CCL2(-/-)CX3CR1(GFP/GFP) mice exposed to extra-light (∼800lux, 6 h/day, 6 months) developed patches of retinal atrophy, and only 20-25% of WT mice which underwent the same light treatment developed atrophic lesions. In addition, synaptophysin expression was detected in the outer nucler layer (ONL) of area related to photoreceptor loss in CCL2(-/-)CX3CR1(GFP/GFP) mice. Markedly increased rhodopsin but reduced cone arrestin expression was observed in retinal outer layers in aged CCL2(-/-)CX3CR1(GFP/GFP) mice. GABA expression was reduced in the inner retina of aged CCL2(-/-)CX3CR1(GFP/GFP) mice. Significantly increased Müller glial and microglial activation was observed in CCL2(-/-)CX3CR1(GFP/GFP) mice compared to age-matched WT mice. Macrophages from CCL2(-/-)CX3CR1(GFP/GFP) mice were less phagocytic, but expressed higher levels of iNOS, IL-1β, IL-12 and TNF-α under hypoxia conditions. Our results suggest that the deletions of CCL2 and CX3CR1 predispose mice to age- and light-mediated retinal damage. The CCL2/CX3CR1 deficient mouse may thus serve as a model for age-related atrophic degeneration of the RPE, including the dry type of macular degeneration, geographic atrophy.
|Survey on amacrine cells coupling to retrograde-identified ganglion cells in the mouse retina. |
Pang, JJ; Paul, DL; Wu, SM
Investigative ophthalmology & visual science 54 5151-62 2013
Retinal amacrine cells (ACs) may make inhibitory chemical synapses and potentially excitatory gap junctions on ganglion cells (GCs). The total number and subtypes of ACs coupled to the entire GC population were investigated in wild-type and three lines of transgenic mice.GCs and GC-coupled ACs were identified by the previously established LY-NB (Lucifer yellow-Neurobiotin) retrograde double-labeling technique, in conjunction with specific antibodies and confocal microscopy.GC-coupled ACs (NB-positive and LY-negative) comprised nearly 11% of displaced ACs and 4% of conventional ACs in wild-type mice, and were 9% and 4% of displaced ACs in Cx45(-/-) and Cx36/45(-/-) mice, respectively. Their somas were small in Cx36/45(-/-) mice, but variable in other strains. They were mostly γ-aminobutyric acid (GABA)-immunoreactive (IR) and located in the GC layer. They comprised only a small portion in the AC subpopulations, including GABA-IR, glycine-IR, calretinin-IR, 5-HT-accumulating, and ON-type choline acetyltransferase (ChAT) ACs in wild-type and ChAT transgenic mice (ChAT- tdTomato). In the distal 80% of the inner plexiform layer (IPL), dense GC dendrites coexisted with rich glycine-IR and GABA-IR. In the inner 20% of the IPL, sparse GC dendrites presented with a major GABA band and sparse glycine-IR.Various subtypes of ACs may couple to GCs. ACs of the same immunoreactivity may either couple or not couple to GCs. Cx36 and Cx45 dominate GC-AC coupling except for small ACs. The overall potency of GC-AC coupling is moderate, especially in the proximal 20% of the IPL, where inhibitory chemical signals are dominated by GABA ACs.
|Anatomical characterization of a rabbit cerebellar eyeblink premotor pathway using pseudorabies and identification of a local modulatory network in anterior interpositus. |
Gonzalez-Joekes, J; Schreurs, BG
The Journal of neuroscience : the official journal of the Society for Neuroscience 32 12472-87 2012
Rabbit eyeblink conditioning is a well characterized model of associative learning. To identify specific neurons that are part of the eyeblink premotor pathway, a retrograde transsynaptic tracer (pseudorabies virus) was injected into the orbicularis oculi muscle. Four time points (3, 4, 4.5, and 5 d) were selected to identify sequential segments of the pathway and a map of labeled structures was generated. At 3 d, labeled first-order motor neurons were found in dorsolateral facial nucleus ipsilaterally. At 4 d, second-order premotor neurons were found in reticular nuclei, and sensory trigeminal, auditory, vestibular, and motor structures, including contralateral red nucleus. At 4.5 d, labeled third-order premotor neurons were found in the pons, midbrain, and cerebellum, including dorsolateral anterior interpositus nucleus and rostral fastigial nucleus. At 5 d, labeling revealed higher-order premotor structures. Labeled fourth-order Purkinje cells were found in ipsilateral cerebellar cortex in cerebellar lobule HVI and in lobule I. The former has been implicated in eyeblink conditioning and the latter in vestibular control. Labeled neurons in anterior interpositus were studied, using neurotransmitter immunoreactivity to classify individual cell types and delineate their interconnectivity. Labeled third-order premotor neurons were immunoreactive for glutamate and corresponded to large excitatory projection neurons. Labeled fourth-order premotor interneurons were immunoreactive for GABA (30%), glycine (18%), or both GABA and glycine (52%) and form a functional network within anterior interpositus involved in modulation of motor commands. These results identify a complete eyeblink premotor pathway, deep cerebellar interconnectivity, and specific neurons responsible for the generation of eyeblink responses.
|Rod and cone pathway signalling is altered in the P2X7 receptor knock out mouse. |
Vessey, KA; Fletcher, EL
PloS one 7 e29990 2012
The P2X7 receptor (P2X7-R) is expressed in the retina and brain and has been implicated in neurodegenerative diseases. However, whether it is expressed by neurons and plays a role as a neurotransmitter receptor has been the subject of controversy. In this study, we first show that the novel vesicular transporter for ATP, VNUT, is expressed in the retina, verifying the presence of the molecular machinery for ATP to act as neurotransmitter at P2X7-Rs. Secondly we show the presence of P2X7-R mRNA and protein in the retina and cortex and absence of the full length variant 1 of the receptor in the P2X7-R knock out (P2X7-KO) mouse. The role of the P2X7-R in neuronal function of the retina was assessed by comparing the electroretinogram response of P2X7-KO with WT mice. The rod photoreceptor response was found to be similar, while both rod and cone pathway post-photoreceptor responses were significantly larger in P2X7-KO mice. This suggests that activation of P2X7-Rs modulates output of second order retinal neurons. In line with this finding, P2X7-Rs were found in the outer plexiform layer and on inner retinal cell classes, including horizontal, amacrine and ganglion cells. The receptor co-localized with conventional synapses in the IPL and was expressed on amacrine cells post-synaptic to rod bipolar ribbon synapses. In view of the changes in visual function in the P2X7-KO mouse and the immunocytochemical location of the receptor in the normal retina, it is likely the P2X7-R provides excitatory input to photoreceptor terminals or to inhibitory cells that shape both the rod and cone pathway response.
|Immunohistochemical identification and synaptic inputs to the diffuse bipolar cell type DB1 in macaque retina. |
Puthussery, T; Gayet-Primo, J; Taylor, WR; Haverkamp, S
The Journal of comparative neurology 519 3640-56 2011
Detailed analysis of the synaptic inputs to the primate DB1 bipolar cell has been precluded by the absence of a suitable immunohistochemical marker. Here we demonstrate that antibodies for the EF-hand calcium-binding protein, secretagogin, strongly label the DB1 bipolar cell as well as a mixed population of GABAergic amacrine cells in the macaque retina. Using secretagogin as a marker, we show that the DB1 bipolar makes synaptic contact with both L/M as well as S-cone photoreceptors and only minimal contact with rod photoreceptors. Electron microscopy showed that the DB1 bipolar makes flat contacts at both triad-associated and nontriad-associated positions on the cone pedicle. Double labeling with various glutamate receptor subunit antibodies failed to conclusively determine the subunit composition of the glutamate receptors on DB1 bipolar cells. In the IPL, DB1 bipolar cell axon terminals expressed the glycine receptor, GlyRα1, at sites of contact with AII amacrine cells, suggesting that these cells receive input from the rod pathway.
|Bcl11b/Ctip2 controls the differentiation of vomeronasal sensory neurons in mice. |
Enomoto, T; Ohmoto, M; Iwata, T; Uno, A; Saitou, M; Yamaguchi, T; Kominami, R; Matsumoto, I; Hirota, J
The Journal of neuroscience : the official journal of the Society for Neuroscience 31 10159-73 2011
The transcription factor Bcl11b/Ctip2 plays critical roles in the development of several systems and organs, including the immune system, CNS, skin, and teeth. Here, we show that Bcl11b/Ctip2 is highly expressed in the developing vomeronasal system in mice and is required for its proper development. Bcl11b/Ctip2 is expressed in postmitotic vomeronasal sensory neurons (VSNs) in the vomeronasal epithelium (VNE) as well as projection neurons and GABAergic interneurons in the accessory olfactory bulb (AOB). In the absence of Bcl11b, these neurons are born in the correct number, but VSNs selectively die by apoptosis. The critical role of Bcl11b in vomeronasal system development is demonstrated by the abnormal phenotypes of Bcl11b-deficient mice: disorganization of layer formation of the AOB, impaired axonal projections of VSNs, a significant reduction in the expression of vomeronasal receptor genes, and defective mature differentiation of VSNs. VSNs can be classified into two major types of neurons, vomeronasal 1 receptor (V1r)/Gα(i2)-positive and vomeronasal 2 receptor (V2r)/Gα(o)-positive VSNs. We found that all Gα(i2)-positive cells coexpressed Gα(o) during embryogenesis. This coexpression is also observed in newly differentiated neurons in the adult VNE. Interestingly, loss of Bcl11b function resulted in an increased number of V1r/Gα(i2)-type VSNs and a decreased number of V2r/Gα(o)-type VSNs, suggesting that Bcl11b regulates the fate choice between these two VSN types. These results indicate that Bcl11b/Ctip2 is an essential regulator of the differentiation and dichotomy of VSNs.
|Morphology and immunoreactivity of retrogradely double-labeled ganglion cells in the mouse retina. |
Pang, JJ; Wu, SM
Investigative ophthalmology & visual science 52 4886-96 2011
To examine the specificity and reliability of a retrograde double-labeling technique that was recently established for identification of retinal ganglion cells (GCs) and to characterize the morphology of displaced (d)GCs (dGs).A mixture of the gap-junction-impermeable dye Lucifer yellow (LY) and the permeable dye neurobiotin (NB) was applied to the optic nerve stump for retrograde labeling of GCs and the cells coupled with them. A confocal microscope was adopted for morphologic observation.GCs were identified by LY labeling, and they were all clearly labeled by NB. Cells coupled to GCs contained a weak NB signal but no LY. LY and NB revealed axon bundles, somas and dendrites of GCs. The retrogradely identified GCs numbered approximately 50,000 per retina, and they constituted 44% of the total neurons in the ganglion cell layer (GCL). Somas of retrogradely identified dGs were usually negative for glycine, ChAT (choline acetyltransferase), bNOS (brain-type nitric oxidase), GAD (glutamate decarboxylase), and glial markers, and occasionally, they were weakly GABA-positive. dGs averaged 760 per retina and composed 1.7% of total GCs. Sixteen morphologic subtypes of dGs were encountered, three of which were distinct from known GCs. dGs sent dendrites to either sublaminas of the IPL, mostly sublamina a.The retrograde labeling is reliable for identification of GCs. dGs participate in ON and OFF light pathways but favor the OFF pathway. ChAT, bNOS, glycine, and GAD remain reliable AC markers in the GCL. GCs may couple to GABAergic ACs, and the gap junctions likely pass NB and GABA.
|Carbonic anhydrase-related protein VIII is expressed in rod bipolar cells and alters signaling at the rod bipolar to AII-amacrine cell synapse in the mammalian retina. |
Puthussery, T; Gayet-Primo, J; Taylor, WR
The European journal of neuroscience 34 1419-31 2011
Mutation of the gene encoding carbonic anhydrase-related protein VIII (CAVIII) results in motor coordination deficits in mice and humans, due to loss of this protein in Purkinje cells of the cerebellum. Recent studies have indicated that the CAVIII gene, Car8, is also expressed in rod bipolar cells (RBCs), a critical glutamatergic neuron for scotopic vision. We investigated the localization of CAVIII in the mouse and macaque retina, and utilized the wdl mouse, which has a null mutation in the Car8 gene, to determine how the loss of CAVIII affects retinal signaling. CAVIII immunoreactivity was observed in RBCs, with particularly high staining intensity in the axon terminals. In addition, weaker staining was observed in a subset of cone bipolar cells and γ-aminobutyric acid (GABA)ergic amacrine cells. Light-evoked current and voltage responses of RBCs were not altered in the wdl mutant. However, light-evoked current responses from the AII-amacrine cell, a postsynaptic partner at the RBC ribbon synapse, were significantly larger, and more prolonged than in control mice. These changes could not be attributed to alterations in calcium current activation or inactivation, or to changes in the density of RBCs. Furthermore, no gross synaptic alterations were evident in the wdl mutant at the light or ultrastructural level. These data provide evidence that the CAVIII protein, which is highly conserved in vertebrates, is selectively expressed within neural circuits, and may be important for modulating retinal neurotransmission.
|Response features of parvalbumin-expressing interneurons suggest precise roles for subtypes of inhibition in visual cortex. |
Runyan, CA; Schummers, J; Van Wart, A; Kuhlman, SJ; Wilson, NR; Huang, ZJ; Sur, M
Neuron 67 847-57 2010
Inhibitory interneurons in the cerebral cortex include a vast array of subtypes, varying in their molecular signatures, electrophysiological properties, and connectivity patterns. This diversity suggests that individual inhibitory classes have unique roles in cortical circuits; however, their characterization to date has been limited to broad classifications including many subtypes. We used the Cre/LoxP system, specifically labeling parvalbumin(PV)-expressing interneurons in visual cortex of PV-Cre mice with red fluorescent protein (RFP), followed by targeted loose-patch recordings and two-photon imaging of calcium responses in vivo to characterize the visual receptive field properties of these cells. Despite their relative molecular and morphological homogeneity, we find that PV+ neurons have a diversity of feature-specific visual responses that include sharp orientation and direction-selectivity, small receptive fields, and band-pass spatial frequency tuning. These results suggest that subsets of parvalbumin interneurons are components of specific cortical networks and that perisomatic inhibition contributes to the generation of precise response properties.
|c-Fos expression during temporal order judgment in mice. |
Wada, M; Higo, N; Moizumi, S; Kitazawa, S
PloS one 5 e10483 2010
The neuronal mechanisms for ordering sensory signals in time still need to be clarified despite a long history of research. To address this issue, we recently developed a behavioral task of temporal order judgment in mice. In the present study, we examined the expression of c-Fos, a marker of neural activation, in mice just after they carried out the temporal order judgment task. The expression of c-Fos was examined in C57BL/6N mice (male, n = 5) that were trained to judge the order of two air-puff stimuli delivered bilaterally to the right and left whiskers with stimulation intervals of 50-750 ms. The mice were rewarded with a food pellet when they responded by orienting their head toward the first stimulus (n = 2) or toward the second stimulus (n = 3) after a visual "go" signal. c-Fos-stained cell densities of these mice (test group) were compared with those of two control groups in coronal brain sections prepared at bregma -2, -1, 0, +1, and +2 mm by applying statistical parametric mapping to the c-Fos immuno-stained sections. The expression of c-Fos was significantly higher in the test group than in the other groups in the bilateral barrel fields of the primary somatosensory cortex, the left secondary somatosensory cortex, the dorsal part of the right secondary auditory cortex. Laminar analyses in the primary somatosensory cortex revealed that c-Fos expression in the test group was most evident in layers II and III, where callosal fibers project. The results suggest that temporal order judgment involves processing bilateral somatosensory signals through the supragranular layers of the primary sensory cortex and in the multimodal sensory areas, including marginal zone between the primary somatosensory cortex and the secondary sensory cortex.Full Text Article
|cJun integrates calcium activity and tlx3 expression to regulate neurotransmitter specification. |
Kurt W Marek,Lisa M Kurtz,Nicholas C Spitzer
Nature neuroscience 13 2010
Neuronal differentiation is accomplished through cascades of intrinsic genetic factors initiated in neuronal progenitors by external gradients of morphogens. Activity has been thought to be important only late in development, but recent evidence suggests that activity also regulates early neuronal differentiation. Activity in post-mitotic neurons before synapse formation can regulate phenotypic specification, including neurotransmitter choice, but the mechanisms are not clear. We identified a mechanism that links endogenous calcium spike activity with an intrinsic genetic pathway to specify neurotransmitter choice in neurons in the dorsal embryonic spinal cord of Xenopus tropicalis. Early activity modulated transcription of the GABAergic/glutamatergic selection gene tlx3 through a variant cAMP response element (CRE) in its promoter. The cJun transcription factor bound to this CRE site, modulated transcription and regulated neurotransmitter phenotype via its transactivation domain. Calcium signaled through cJun N-terminal phosphorylation, which integrated activity-dependent and intrinsic neurotransmitter specification. This mechanism provides a basis for early activity to regulate genetic pathways at critical decision points, switching the phenotype of developing neurons.Full Text Article
|Postsynaptic action of GABA in modulating sensory transmission in co-cultures of rat carotid body via GABA(A) receptors. |
Zhang, M; Clarke, K; Zhong, H; Vollmer, C; Nurse, CA
The Journal of physiology 587 329-44 2009
GABA is expressed in carotid body (CB) chemoreceptor type I cells and has previously been reported to modulate sensory transmission via presynaptic GABA(B) receptors. Because low doses of clinically important GABA(A) receptor (GABA(A)R) agonists, e.g. benzodiazepines, have been reported to depress afferent CB responses to hypoxia, we investigated the potential contribution of GABA(A)R in co-cultures of rat type I cells and sensory petrosal neurones (PNs). During gramicidin perforated-patch recordings (to preserve intracellular Cl-), GABA and/or the GABA(A) agonist muscimol (50 microm) induced a bicuculline-sensitive membrane depolarization in isolated PNs. GABA-induced whole-cell currents reversed at approximately -38 mV and had an EC50 of approximately 10 microm (Hill coefficient = approximately 1) at -60 mV. During simultaneous PN and type I cell recordings at functional chemosensory units in co-culture, bicuculline reversibly potentiated the PN, but not type I cell, depolarizing response to hypoxia. Application of the CB excitatory neurotransmitter ATP (1 microm) over the soma of functional PN induced a spike discharge that was markedly suppressed during co-application with GABA (2 microm), even though GABA alone was excitatory. RT-PCR analysis detected expression of GABAergic markers including mRNA for alpha1, alpha2, beta2, gamma2S, gamma2L and gamma3 GABA(A)R subunits in petrosal ganglia extracts. Also, CB extracts contained mRNAs for GABA biosynthetic markers, i.e. glutamate decarboxylase (GAD) isoforms GAD 67A,E, and GABA transporter isoforms GAT 2,3 and BGT-1. In CB sections, sensory nerve endings apposed to type I cells were immunopositive for the GABA(A)R beta subunit. These data suggest that GABA, released from the CB during hypoxia, inhibits sensory discharge postsynaptically via a shunting mechanism involving GABA(A) receptors.
|Neurokinin-1 receptor immunoreactive neuronal elements in the superficial dorsal horn of the chicken spinal cord: with special reference to their relationship with the tachykinin-containing central axon terminals in synaptic glomeruli. |
Sakamoto, H; Kawate, T; Li, Y; Atsumi, S
Acta histochemica et cytochemica 42 111-9 2009
Synaptic glomeruli that involve tachykinin-containing primary afferent central terminals are numerous in lamina II of the chicken spinal cord. Therefore, a certain amount of noxious information is likely to be modulated in these structures in chickens. In this study, we used immunohistochemistry with confocal and electron microscopy to investigate whether neurokinin-1 receptor (NK-1R)-expressing neuronal elements are in contact with the central primary afferent terminals in synaptic glomeruli of the chicken spinal cord. We also investigated which neuronal elements (axon terminals, dendrites, cell bodies) and which neurons in the spinal cord possess NK-1R, and are possibly influenced by tachykinin in the glomeruli. By confocal microscopy, NK-1R immunoreactivities were seen in a variety of neuronal cell bodies, their dendrites and smaller fibers of unknown origin. Some of the NK-1R immunoreactive profiles also expressed GABA immunoreactivities. A close association was observed between the NK-1R-immunoreactive neurons and tachykinin-immunoreactive axonal varicosities. By electron microscopy, NK-1R immunoreactivity was seen in cell bodies, conventional dendrites and vesicle-containing dendrites in laminae I and II. Among these elements, dendrites and vesicle-containing dendrites made contact with tachykinin-containing central terminals in the synaptic glomeruli. These results indicate that tachykinin-containing central terminals in the chicken spinal cord can modulate second-order neuronal elements in the synaptic glomeruli.
|Combined extrinsic and intrinsic manipulations exert complementary neuronal enrichment in embryonic rat neural precursor cultures: an in vitro and in vivo analysis. |
Furmanski O, Gajavelli S, Lee JW, Collado ME, Jergova S, Sagen J
The Journal of comparative neurology 515 56-71 2009
Numerous central nervous system (CNS) disorders share a common pathology in dysregulation of gamma-aminobutyric acid (GABA) inhibitory signaling. Transplantation of GABA-releasing cells at the site of disinhibition holds promise for alleviating disease symptoms with fewer side effects than traditional drug therapies. We manipulated fibroblast growth factor (FGF)-2 deprivation and mammalian achaete-scute homolog (MASH)1 transcription factor levels in an attempt to amplify the default GABAergic neuronal fate in cultured rat embryonic neural precursor cells (NPCs) for use in transplantation studies. NaÃ¯ve and MASH1 lentivirus-transduced NPCs were maintained in FGF-2 or deprived of FGF-2 for varying lengths of time. Immunostaining and quantitative analysis showed that GABA- and beta-III-tubulin-immunoreactive cells generally decreased through successive passages, suggesting a loss of neurogenic potential in rat neurospheres expanded in vitro. However, FGF-2 deprivation resulted in a small, but significantly increased population of GABAergic cells derived from passaged neurospheres. In contrast to naÃ¯ve and GFP lentivirus-transduced clones, MASH1 transduction resulted in increased bromodeoxyuridine (BrdU) incorporation and clonal colony size. Western blotting showed that MASH1 overexpression and FGF-2 deprivation additively increased beta-III-tubulin and decreased cyclic nucleotide phosphodiesterase (CNPase) expression, whereas FGF-2 deprivation alone attenuated glial fibrillary acidic protein (GFAP) expression. These results suggest that low FGF-2 signaling and MASH1 activity can operate in concert to enrich NPC cultures for a GABA neuronal phenotype. When transplanted into the adult rat spinal cord, this combination also yielded GABAergic neurons. These findings indicate that, even for successful utilization of the default GABAergic neuronal precursor fate, a combination of both extrinsic and intrinsic manipulations will likely be necessary to realize the full potential of NSC grafts in restoring function. Copyright 2009 Wiley-Liss, Inc.Full Text Article
|Co-transmission of dopamine and GABA in periglomerular cells. |
Maher, BJ; Westbrook, GL
Journal of neurophysiology 99 1559-64 2008
Most central neurons package and release a single transmitter. However co-transmission of fast-acting and modulatory transmitters has been observed in vertebrate and invertebrate systems. Here we describe a population of periglomerular cells in mouse brain slices (PND14-21) that co-release dopamine and GABA. We made whole cell recordings from periglomerular cells that expressed enhanced green fluorescent protein (EGFP) under the control of the tyrosine hyrdoxylase (TH) promoter. Immunolabeling confirmed that EGFP+ periglomerular cells synthesized TH as well as glutamic acid decarboxylase (GAD). Stimulation of olfactory receptor neuron (ORN) afferent input evoked excitatory postsynaptic currents (EPSCs) in EGFP+ cells that were inhibited by cocaine, which blocks dopamine transport. These effects were reversed by the D2 receptor antagonist sulpiride. Cocaine also increased the paired-pulse ratio of ORN-evoked EPSCs. These results demonstrate that TH+ periglomerular cells spontaneously release dopamine. In addition to dopamine, TH-EGFP+ cells also released GABA. Brief depolarizing voltage steps in labeled cells evoked a tail current that was completely blocked by the GABA(A) receptor antagonist gabazine and by cadmium, indicative of calcium-dependent self-inhibition in periglomerular cells. However, similar voltage steps were insufficient to cause D2-receptor mediated inhibition of ORN terminals. Our results indicate that TH+ periglomerular cells are directly activated by ORN input and release both dopamine and GABA. We suggest that concerted activation of multiple periglomerular cells may be required to detect dopamine release under normal physiological conditions.
|NKCC1 does not accumulate chloride in developing retinal neurons. |
Zhang, LL; Delpire, E; Vardi, N
Journal of neurophysiology 98 266-77 2007
GABA excites immature neurons due to their relatively high intracellular chloride concentration. This initial high concentration is commonly attributed to the ubiquitous chloride cotransporter NKCC1, which uses a sodium gradient to accumulate chloride. Here we tested this hypothesis in immature retinal amacrine and ganglion cells. Western blotting detected NKCC1 at birth and its expression first increased, then decreased to the adult level. Immunocytochemistry confirmed this early expression of NKCC1 and localized it to all nuclear layers. In the ganglion cell layer, staining peaked at P4 and then decreased with age, becoming undetectable in adult. In comparison, KCC2, the chloride extruder, steadily increased with age localizing primarily to the synaptic layers. For functional tests, we used calcium imaging with fura-2 and chloride imaging with 6-methoxy-N-ethylquinolinium iodide. If NKCC1 accumulates chloride in ganglion and amacrine cells, deleting or blocking it should abolish the GABA-evoked calcium rise. However, at P0-5 GABA consistently evoked a calcium rise that was not abolished in the NKCC1-null retinas, nor by applying high concentrations of bumetanide (NKCC blocker) for long periods. Furthermore, intracellular chloride concentration in amacrine and ganglion cells of the NKCC1-null retinas was approximately 30 mM, same as in wild type at this age. This concentration was not lowered by applying bumetanide or by decreasing extracellular sodium concentration. Costaining for NKCC1 and cellular markers suggested that at P3, NKCC1 is restricted to Müller cells. We conclude that NKCC1 does not serve to accumulate chloride in immature retinal neurons, but it may enable Müller cells to buffer extracellular chloride.
|Synaptic and nonsynaptic localization of GABAA receptors containing the alpha5 subunit in the rat brain. |
Serwanski, DR; Miralles, CP; Christie, SB; Mehta, AK; Li, X; De Blas, AL
The Journal of comparative neurology 499 458-70 2006
The alpha5 subunit of the GABA(A) receptors (GABA(A)Rs) has a restricted expression in the brain. Maximum expression of this subunit occurs in the hippocampus, cerebral cortex, and olfactory bulb. Hippocampal pyramidal cells show high expression of alpha5 subunit-containing GABA(A)Rs (alpha5-GABA(A)Rs) both in culture and in the intact brain. A large pool of alpha5-GABA(A)Rs is extrasynaptic and it has been proposed to be involved in the tonic GABAergic inhibition of the hippocampus. Nevertheless, there are no studies on the localization of the alpha5-GABA(A)Rs at the electron microscope (EM) level. By using both immunofluorescence of cultured hippocampal pyramidal cells and EM postembedding immunogold of the intact hippocampus we show that, in addition to the extrasynaptic pool, there is a pool of alpha5-GABA(A)Rs that concentrates at the GABAergic synapses in dendrites of hippocampal pyramidal cells. The results suggest that the synaptic alpha5-GABA(A)Rs might play a role in the phasic GABAergic inhibition of pyramidal neurons in hippocampus and cerebral cortex.
|Redistribution of synaptic AMPA receptors at glutamatergic synapses in the dorsal cochlear nucleus as an early response to cochlear ablation in rats. |
Rubio, Maria E
Hear. Res., 216-217: 154-67 (2006) 2006
This study investigated whether unilateral deafferentation of the presynaptic neuron is key in the control of morphology and the subunit composition and expression of AMPA type glutamate receptors (GluRs) in neurons of the dorsal cochlear nucleus (DCN). Data showed that there are morphological changes at the postsynaptic sites which precede presynaptic changes at the auditory nerve (AN) synaptic ending in response to peripheral damage, in particular that the postsynaptic densities (PSD) of the AN on fusiform cells (FC) are thicker after denervation. Moreover, GluR2, GluR3 and GluR4 AMPA receptor subunits were redistributed, not only at the synapse of FCs receiving direct contact with the AN, but also at the glutamatergic synapse of the parallel fibers on FC and on cartwheel cells (CwC) which are indirectly innervated by the AN. Interestingly, the same synapses in the DCN contralateral to the lesion and with a normal AN synaptic input also redistributed AMPA receptor subunits at the synapse in respond to deafferentation. In these synapses, there was an increase of immunogold labeling for GluR2/3 subunits but not for GluR2 at 2 days after deafferentation.
|Immunocytochemical analysis of GABA-positive and calretinin-positive horizontal cells in the tiger salamander retina. |
Jian Zhang, Ai-Jun Zhang, Samuel M Wu
The Journal of comparative neurology 499 432-41 2006
By using immunocytochemical techniques, we demonstrate that there are two distinct, nonoverlapping populations of horizontal cells (HCs) in the tiger salamander retina: GABA-positive cells account for about 72% and GABA-negative (calretinin-positive) cells account for 28% of the total HC somas. The calretinin-positive HCs have relatively sparse and thick dendrites: soma diameter of 19.72 +/- 0.29 microm, and soma density of 140 +/- 13 cells/mm(2), morphological features very much like the A-type HCs described in the accompanying article. The GABA-positive HCs have thinner dendritic and coarse axon-terminal-like processes of higher density: soma diameter of 18 +/- 0.18 microm, and soma density of 364 +/- 18 cells/mm(2), features that very much resemble the B-type HCs and B-type HC axon terminals in the accompanying article. By using double and triple immunostaining techniques we found that only 18% of the non-GABAergic HC dendritic clusters contact rods, whereas the remaining 82% of the dendritic clusters contact cones. This is consistent with the physiological finding in the accompanying article that the A-type HCs are cone-dominated. On the other hand, 32% of GABAergic HC dendrites contact rod pedicles and 68% contact cone pedicles, consistent with the physiological finding that B-type HCs and B-type HC axon terminals receive mixed rod/cone inputs. Detailed confocal microscope analysis shows that 4% rods, 6% principal double cones/single cones, and 100% accessory double cones contact calretinin-positive HCs, and 79% rods, 100% principal double cones, 14% accessory double cones, and 82% single cones contact GABAergic HCs. These results suggest that GABAergic and non-GABAergic HC input/output synapses differ and they may mediate different functional pathways in the outer retina.
|Molecular and genetic features of a labeled class of spinal substantia gelatinosa neurons in a transgenic mouse. |
Adam W Hantman, Edward R Perl
The Journal of comparative neurology 492 90-100 2005
Genetic incorporation in a mouse of a transgene containing the prion promoter and the green fluorescent protein (GFP) coding sequence labels a set of substantia gelatinosa (SG) neurons (SG-GFP) homogenous in morphology, electrophysiology, and gamma-amino-butyric acid expression. In the present analysis the SG-GFP neurons are established to have protein kinase C-betaII immunoreactivity and to lack evidence for the presence of calbindin D-28k, parvalbumin, and protein kinase C-gamma. These neurons were hyperpolarized by mediators of descending control, norepinephrine and serotonin. Sequential polymerase chain reactions established the insertion of the transgene to be in the receptor protein tyrosine phosphatase kappa (RPTP-kappa) and the laminin receptor 1 (ribosomal protein SA) pseudogene 1 locus. RPTP-kappa expression in both GFP-labeled dorsal root ganglia and SG neurons raises the possibility that homophilic interactions of RPTP-kappa contribute to establishment of connections between specific classes of primary afferent and SG neurons.
|Differential distribution of synaptic endings containing glutamate, glycine, and GABA in the rat dorsal cochlear nucleus |
Rubio, M. F. and Juiz, J. M.
J. Comp. Neurol., 477(3):253-272 (2004) 2004
|Regional differences in GABA and GAD immunoreactivity in rabbit horizontal cells |
Johnson, M A and Vardi, N
Vis Neurosci, 15:743-53 () 1998
|Coexistence of GABA and peptide immunoreactivity in non-pyramidal neurons of the basolateral amygdala. |
McDonald, A J and Pearson, J C
Neurosci. Lett., 100: 53-8 (1989) 1989
Colocalization of gamma-aminobutyric acid (GABA) immunoreactivity with somatostatin (SOM), neuropeptide Y (NPY), cholecystokinin (CCK), and vasoactive intestinal polypeptide (VIP) immunoreactivity was demonstrated in non-pyramidal neurons of the basolateral amygdala using a two-color immunoperoxidase procedure. Approximately 80-90% of SOM- and NPY-positive neurons in the basolateral amygdala were also immunoreactive for GABA. Virtually all large CCK-positive neurons also exhibited GABA-like immunoreactivity. About one-half of VIP-positive neurons and small CCK-positive cells were also immunoreactive for GABA.
|Identification of gamma-aminobutyric acid-like immunoreactive neurons in the rat cuneate nucleus. |
Roettger, V R, et al.
Neurosci. Lett., 97: 46-50 (1989) 1989
Neurons in the cuneate nucleus of the rat were examined for gamma-aminobutyric acid-like immunoreactivity (GABA-LI) using antiserum raised against GABA-glutaraldehyde-keyhole limpet hemocyanin. GABA-LI neurons were analyzed for size, shape, and distribution and compared to Nissl-stained neurons. GABA-LI cell bodies were located at all rostral-caudal levels and were distributed randomly throughout the nucleus except at the level of the obex, where they were limited to the peripheral region of the cuneate nucleus. GABA-LI cell bodies had a significantly smaller mean cross-sectional area than the total cuneate neuronal population and comprised 21.5% of the total neuronal population as assessed with Nissl-staining. These results are consistent with the hypothesis that GABA is involved in processing somatosensory information in the rat dorsal column nuclei.
|GUINEA PIG ANTI-GABA POLYCLONAL ANTIBODY|