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ABSTRACT: BACKGROUND: GABAergic deficit is one of the major mechanisms underlying epileptic seizures. Previous studies have mainly focused on alterations of synaptic GABAergic inhibition during epileptogenesis. Recent work suggested that tonic inhibition may also play a role in regulating epileptogenesis, but the underlying mechanism is not well understood. RESULTS: We employed molecular and pharmacological tools to investigate the role of tonic inhibition during epileptogenesis both in vitro and in vivo. We overexpressed two distinct subtypes of extrasynaptic GABAA receptors, alpha5beta3gamma2 and alpha6beta3delta receptors, in cultured hippocampal neurons. We demonstrated that overexpression of both alpha5beta3gamma2 and alpha6beta3delta receptors enhanced tonic inhibition and reduced epileptiform activity in vitro. We then showed that injection of THIP (5 muM), a selective agonist for extrasynaptic GABAA receptors at low concentration, into rat brain also suppressed epileptiform burst activity and behavioral seizures in vivo. Mechanistically, we discovered that low concentration of THIP had no effect on GABAergic synaptic transmission and did not affect the basal level of action potentials, but significantly inhibited high frequency neuronal activity induced by epileptogenic agents. CONCLUSIONS: Our studies suggest that extrasynaptic GABAA receptors play an important role in controlling hyperexcitatory activity, such as that during epileptogenesis, but a less prominent role in modulating a low level of basal activity. We propose that tonic inhibition may play a greater role under pathological conditions than in physiological conditions in terms of modulating neural network activity.
Molecular Brain 05/2013; 6(1):21.
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ABSTRACT: Cofilin is an actin-binding protein and a major actin depolymerization factor in the central nervous system (CNS). Cofilin-actin
aggregates are associated with neurodegenerative disorders, but how cofilin-actin aggregation induces pathological effects
in the CNS remains unclear. Here, we demonstrated that cofilin rods disrupted dendritic microtubule integrity in rat hippocampal
cultures. Long term time-lapse imaging revealed that cofilin rods block intracellular trafficking of both mitochondria and
early endosomes. Importantly, cofilin rod formation induced a significant loss of SV2 and PSD-95 puncta as well as dendritic
spines. Cofilin rods also impaired local glutamate receptor responses. We discovered an inverse relationship between the number
of synaptic events and the accumulation of cofilin rods in dendrites. We also detected cofilin rods in aging rat brains in vivo. These results suggest that cofilin aggregation may contribute to neurodegeneration and brain aging by blocking intracellular
trafficking and inducing synaptic loss.
Journal of Biological Chemistry 02/2012; 287(6):3919-3929. · 4.77 Impact Factor
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ABSTRACT: Cofilin is an actin-binding protein and a major actin depolymerization factor in the central nervous system (CNS). Cofilin-actin aggregates are associated with neurodegenerative disorders, but how cofilin-actin aggregation induces pathological effects in the CNS remains unclear. Here, we demonstrated that cofilin rods disrupted dendritic microtubule integrity in rat hippocampal cultures. Long term time-lapse imaging revealed that cofilin rods block intracellular trafficking of both mitochondria and early endosomes. Importantly, cofilin rod formation induced a significant loss of SV2 and PSD-95 puncta as well as dendritic spines. Cofilin rods also impaired local glutamate receptor responses. We discovered an inverse relationship between the number of synaptic events and the accumulation of cofilin rods in dendrites. We also detected cofilin rods in aging rat brains in vivo. These results suggest that cofilin aggregation may contribute to neurodegeneration and brain aging by blocking intracellular trafficking and inducing synaptic loss.
Journal of Biological Chemistry 12/2011; 287(6):3919-29. · 4.77 Impact Factor
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ABSTRACT: Dendritic spines serve as the postsynaptic platform for most excitatory synapses in the mammalian brain, and their shape and size are tightly correlated with synaptic strength. The actin cytoskeleton plays a crucial role in the spine structure and its modifications during synapse development and plasticity, but the underlying regulatory mechanisms remain to be elucidated. Here, we report that actin capping protein (CP), a regulator of actin filament growth, plays an essential role for spine development and synapse formation. We found that CP expression in rat hippocampus is elevated at and after the stage of substantial synapse formation. CP knockdown in hippocampal cultures resulted in a marked decline in spine density accompanied by increased filopodia-like protrusions. Moreover, the spines of CP knockdown neurons exhibited an altered morphology, highlighted by multiple thin filopodia-like protrusions emerging from the spine head. Finally, the number of functional synapses was reduced by CP knockdown as evidenced by a reduction in the density of paired presynaptic and postsynaptic markers and in the frequency of miniature EPSCs. These findings indicate that capping of actin filaments by CP represents an essential step for the remodeling of the actin architecture underlying spine morphogenesis and synaptic formation during development.
Journal of Neuroscience 07/2011; 31(28):10228-33. · 7.11 Impact Factor
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ABSTRACT: Schizophrenia is a severe chronic mental disorder with a high genetic component in its etiology. Several lines of study have suggested that synaptic dysfunction may underlie the pathogenesis of schizophrenia. Neuroligin proteins function as cell-adhesion molecules at post-synaptic membrane and play critical roles in synaptogenesis and synaptic maturation. In this study, we systemically sequenced all the exons and promoter region of neuroligin-2 (NLGN2) gene in a sample of 584 schizophrenia patients and 549 control subjects from Taiwan. In total, we identified 19 genetic variants, including six rare missense mutations such as R215H (one patient), V510M (two patients), R621H (one patient), A637T (two patients), P800L (one patient and one control) and A819S (one patient and one control). In silico analysis predicted that two patient-specific missense mutations, R215H and R621H, had damaging effect, whereas the other missense mutations were benign. Importantly, functional analysis with immunocytochemistry and electrophysiological recordings identified the R215H mutant as a loss-of-function mutant in inducing GABAergic synaptogenesis. Mechanistically, the synaptogenic deficiency of R215H mutant was due to its retention inside the endoplasmic reticulum and inability to be transported to cell membrane. Our study suggests that defects in GABAergic synapse formation in the brain may be an important contributing factor for the onset of schizophrenia. In the family study of this mutation, we found his elder brother also carried this mutation but did not have psychiatric symptoms, indicating that this mutation has incomplete penetrance, and thus the clinical relevance of this mutation should be interpreted with caution.
Human Molecular Genetics 05/2011; 20(15):3042-51. · 7.64 Impact Factor
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ABSTRACT: We have previously demonstrated that cyclothiazide (CTZ) is a potent convulsant drug inducing robust epileptiform activity in hippocampal neurons both in vitro and in vivo. Here we further establish an animal model for CTZ-induced behavioral seizures in freely moving rats. Microinjection of CTZ into the left ventricle dose-dependently induced robust seizure behaviors within 3h after administration. At a dose of 0.75 μmol, CTZ induced Racine score IV-V seizure behaviors in 71% (n=14) of the rats were tested. In addition, CTZ also induced epileptiform EEG activity accompanying behavioral seizures. The convulsant action of CTZ on both behavior and EEG was blocked by pretreatment with clinical anticonvulsant drug diazepam (n=5). In conclusion, our results demonstrate that CTZ is capable of inducing behavioral seizures in intact animals. Since CTZ acts on both GABAergic and glutamatergic systems, this new animal epilepsy model will be useful for anticonvulsant drug testing and general epilepsy research.
Brain research 10/2010; 1355:207-13. · 2.46 Impact Factor
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Jiaping Gu,
Chi Wai Lee,
Yanjie Fan,
Daniel Komlos,
Xin Tang,
Chicheng Sun,
Kuai Yu,
H Criss Hartzell, Gong Chen,
James R Bamburg,
James Q Zheng
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ABSTRACT: Dendritic spines undergo actin-based growth and shrinkage during synaptic plasticity, in which the actin depolymerizing factor (ADF)/cofilin family of actin-associated proteins are important. Elevated ADF/cofilin activities often lead to reduced spine size and immature spine morphology but can also enhance synaptic potentiation in some cases. Thus, ADF/cofilin may have distinct effects on postsynaptic structure and function. We found that ADF/cofilin-mediated actin dynamics regulated AMPA receptor (AMPAR) trafficking during synaptic potentiation, which was distinct from actin's structural role in spine morphology. Specifically, elevated ADF/cofilin activity markedly enhanced surface addition of AMPARs after chemically induced long-term potentiation (LTP), whereas inhibition of ADF/cofilin abolished AMPAR addition. We found that chemically induced LTP elicited a temporal sequence of ADF/cofilin dephosphorylation and phosphorylation that underlies AMPAR trafficking and spine enlargement. These findings suggest that temporally regulated ADF/cofilin activities function in postsynaptic modifications of receptor number and spine size during synaptic plasticity.
Nature Neuroscience 10/2010; 13(10):1208-15. · 15.53 Impact Factor
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ABSTRACT: Astrocytes release ATP and glutamate through vesicular exocytosis to mediate neuron-glial interactions. In contrast to exocytosis, the endocytic pathways in astroglial cells are poorly understood. Here, we identify a constitutive endocytic pathway in cultured astrocytes that is dependent on neither clathrin nor dynamin. This dynamin-independent endocytic pathway is regulated by Rab5, an early endosome protein. The endocytosed vesicles show fast transition from early endosomes to late endosomes and lysosomes within a few minutes. Interestingly, this clathrin- and dynamin-independent endocytosis in astrocytes is potently regulated by intracellular Ca(2+). ATP and glutamate greatly enhance the dynamin-independent endocytosis through elevating the intracellular Ca(2+). In addition, amyloid-beta peptide (A beta) also enhances the dynamin-independent endocytosis by inducing Ca(2+) transients in astrocytes. These results demonstrate a novel endocytic pathway in glial cells that is dynamin independent but tightly regulated by intracellular Ca(2+). The regulation by ATP, glutamate, and A beta suggests an important role of the dynamin-independent endocytosis in both physiological and pathological conditions.
Journal of Neuroscience 07/2009; 29(25):8063-74. · 7.11 Impact Factor
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ABSTRACT: Brain-derived neurotrophic factor (BDNF) and its receptor TrkB play an important function in neuronal development and synaptic plasticity. Recently we have established that cyclothiazide (CTZ) is a novel convulsant drug inducing robust epileptiform activity in hippocampal neurons both in vitro and in vivo. However, the molecular mechanisms underlying such convulsant action of CTZ are unknown. Here, we investigated potential roles of BDNF-TrkB signaling pathway in the CTZ-induction of epileptiform activity. In anaesthetized rats, CTZ dose-dependently induced epileptiform activity characterized by progressing of multiple peaks of population spikes, spontaneous spiking events, and synchronized epileptiform bursts. Pre-injection of a receptor tyrosine kinase inhibitor K252a or a specific antibody for TrkB receptors before intracerebroventricular injection of CTZ significantly suppressed the epileptiform activity induced by CTZ. Similarly, in cultured hippocampal pyramidal neurons, pre-treatment with CTZ together with K252a or TrkB-receptor antibody also inhibited the CTZ-induction of epileptiform activity. Furthermore, we demonstrated that acute application of K252a in hippocampal cultures inhibited epileptiform bursts and action potential firing. We conclude that activation of BDNF-TrkB signaling pathway is fundamentally important during the CTZ-induction of epileptiform activity both in vitro and in vivo.
Neuropharmacology 05/2009; 57(1):49-59. · 4.81 Impact Factor
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ABSTRACT: Accumulation of GABA(A) receptors (GABA(A)Rs) at GABAergic synapses requires the cytoplasmic loop region and C-terminal transmembrane domain of the receptor gamma2 subunit. We here report a novel interaction of gamma2 with Calcium-Modulating cyclophilin Ligand (CAML), an integral membrane protein that regulates this mechanism. Interaction of GABA(A)Rs with CAML depends on both the cytoplasmic region and fourth transmembrane domain of the gamma2 subunit, CAML immunoprecipitates with GABA(A)Rs from transfected cells and brain lysates and colocalizes with gamma2 in ER vesicles in soma and dendrites of neurons. CAML shRNA treatment results in reduced expression of postsynaptic GABA(A)Rs, along with significant reductions in GABA-evoked whole cell currents and GABAergic synaptic function, while glutamatergic transmission is unaffected. Reduced surface expression of GABA(A)Rs in CAML mutant neurons is associated with selective deficits in recycling of endocytosed GABA(A)Rs to the cell surface. Our results indicate a specific role of CAML in functional expression and endocytic recycling of postsynaptic GABA(A)Rs.
Molecular and Cellular Neuroscience 07/2008; 38(2):277-89. · 3.66 Impact Factor
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ABSTRACT: Sequential formation of GABAergic and glutamatergic synapses is thought to be crucial for constructing the stereotypic neural networks during brain development. However, why GABAergic synapses are formed earlier than glutamatergic synapses in the developing brain is not well understood. We used electrophysiology and fluorescence imaging to study GABAergic and glutamatergic synaptogenesis in embryonic hypothalamic cultures, which contain approximately 40% GABAergic and approximately 60% glutamatergic neurons. The newly dissociated embryonic hypothalamic neurons contained a significant pool of functional GABA(A) receptors but a very low level of glutamate receptors. Within the first week of culture, the time course of GABAergic synaptogenesis in embryonic neurons coincided with that of presynaptic vesicle cycling, but both measurements lagged behind the detection of functional GABA(A) receptors. Remarkably, the GABA(A) receptors of newly dissociated embryonic neurons can be rapidly clustered into postsynaptic apparatus and generate functional synaptic currents within 4-6 h when cocultured with mature neurons. Consistent with earlier expression of GABA(A) receptors in immature neurons, synaptic GABAergic events were always detected before the onset of glutamatergic events in both purely embryonic and heterochronic cultures. Interestingly, overexpression of glutamate receptors in embryonic neurons not only increased whole-cell glutamate currents but also significantly increased the frequency of excitatory synaptic events. We conclude that the sequential formation of GABAergic and glutamatergic synapses in immature neurons is likely governed by a sequential expression of GABA(A) and glutamate receptors during neuronal development.
Journal of Neuroscience 11/2007; 27(40):10860-9. · 7.11 Impact Factor
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ABSTRACT: Trans-synaptic cell adhesion molecules neuroligins and neurexins play an important role in promoting central synapse formation. We report here a molecular reconstruction of functional GABAergic synapses in non-neuronal cells with the coexpression of postsynaptic cell adhesion molecule neuroligin-2 (NL-2) and GABA(A) receptors. HEK 293T cells were co-transfected with GABA(A) receptors and NL-2 or its homologue neuroligin-1 (NL-1), and then cocultured with hypothalamic cultures which are enriched with GABAergic neurons. Both spontaneous and action potential-evoked GABAergic events were readily detected in HEK 293T cells coexpressing GABA(A) receptors with NL-2, but not with NL-1. Aggregating NL-2 with specific antibodies in live cells resulted in coaggregation of GABA(A) receptors. Expression of NL-2 in HEK 293T cells also induced stronger GABAergic presynaptic differentiation than that of NL-1 in neuronal cocultures. These results suggest that NL-2 may potentially serve as a central organizer for GABAergic synapse assembly by interacting with both presynaptic neurexins and postsynaptic GABA(A) receptors.
Molecular and Cellular Neuroscience 06/2007; 35(1):14-23. · 3.66 Impact Factor
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ABSTRACT: Deficits in GABAergic inhibitory transmission are a hallmark of temporal lobe epilepsy and have been replicated in animal and tissue culture models of epilepsy. GABAergic inhibition comprises phasic and tonic inhibition that is mediated by synaptic and extrasynaptic GABAA receptors, respectively. We have recently demonstrated that chronic stimulation with cyclothiazide (CTZ) or kainic acid (KA) induces robust epileptiform activity in hippocampal neurons both in vitro and in vivo. Here, we report a downregulation of tonic GABA inhibition after chronic epileptogenic stimulation of rat hippocampal cultures. Chronic pretreatment of hippocampal neurons with CTZ or KA resulted in a marked reduction in GABAergic inhibition, as shown by a significant decrease in whole-cell GABA currents and in the frequency of miniature inhibitory postsynaptic currents (mIPSCs). Interestingly, synaptically localized GABAA receptors remained relatively stable, as evidenced by the unaltered amplitude of mIPSCs, as well as the unchanged punctate immunoreactivity of gamma2 subunit-containing postsynaptic GABAA receptors. In contrast, tonic GABA currents, assessed either by a GABAA receptor antagonist bicuculline or a selective extrasynaptic GABAA receptor agonist THIP, were significantly reduced following epileptogenic stimulation. These results reveal a novel form of neural plasticity, that epileptogenic stimulation can selectively downregulate extrasynaptic GABAA receptors while leaving synaptic GABAA receptors unchanged. Thus, in addition to synaptic alteration of GABAergic transmission, regulation of tonic inhibition may also play an important role during epileptogenesis.
The Journal of Physiology 01/2007; 577(Pt 2):579-90. · 4.72 Impact Factor
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ABSTRACT: Golgi-specific DHHC (Asp-His-His-Cys) zinc finger protein (GODZ) is a DHHC family palmitoyl acyltransferase that is implicated in palmitoylation and regulated trafficking of diverse substrates that function either at inhibitory or excitatory synapses. Of particular interest is the gamma2 subunit of GABA(A) receptors, which is required for targeting these receptors to inhibitory synapses. Here, we report that GODZ and, to a lesser extent, its close paralog sertoli cell gene with a zinc finger domain-beta (SERZ-beta) are the main members of the DHHC family of enzymes that are able to palmitoylate the gamma2 subunit in heterologous cells. Yeast two-hybrid and colocalization assays in human embryonic kidney 293T (HEK293T) cells indicate that GODZ and SERZ-beta show indistinguishable palmitoylation-dependent interaction with the gamma2 subunit. After coexpression in HEK293T cells, they form homomultimers and heteromultimers, as shown by coimmunoprecipitation and in vivo cross-linking experiments. Analyses in neurons transfected with dominant-negative GODZ (GODZ(C157S)) or plasmid-based GODZ-specific RNAi indicate that GODZ is required for normal accumulation of GABA(A) receptors at synapses, for normal whole-cell and synaptic GABAergic inhibitory function and, indirectly, for GABAergic innervation. Unexpectedly, GODZ was found to be dispensable for normal postsynaptic AMPA receptor-mediated glutamatergic transmission. We conclude that GODZ-mediated palmitoylation of GABA(A) receptors and possibly other substrates contributes selectively to the formation and normal function of GABAergic inhibitory synapses.
Journal of Neuroscience 01/2007; 26(49):12758-68. · 7.11 Impact Factor
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ABSTRACT: Developing neurons have greater capacity in experience-dependent plasticity than adult neurons but the molecular mechanism is not well understood. Here we report a developmentally regulated long-term synaptic plasticity through actin-dependent activation of presynaptic silent synapses in cultured hippocampal neurons. Live FM 1-43 imaging and retrospective immunocytochemistry revealed that many presynaptic boutons in immature neurons are functionally silent at resting conditions, but can be converted into active ones after repetitive neuronal stimulation. The activation of presynaptic silent synapses is dependent on L-type calcium channels and protein kinase A (PKA)/PKC signaling pathways. Moreover, blocking actin polymerization with latrunculin A and cytochalasin B abolishes long-term increase of presynaptic functional boutons induced by repetitive stimulation, whereas actin polymerizer jasplakinolide increases the number of active boutons in immature neurons. In mature neurons, however, presynaptic boutons are mostly functional and repetitive stimulation did not induce additional enhancement. Quantitative immunostaining with phalloidin revealed a significant increase in axonal F-actin level after repetitive stimulation in immature but not mature neurons. These results suggest that actin-dependent activation of presynaptic silent synapses contributes significantly to the long-term synaptic plasticity during neuronal development.
Journal of Neuroscience 09/2006; 26(31):8137-47. · 7.11 Impact Factor
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ABSTRACT: Cyclothiazide (CTZ) is a potent blocker of AMPA receptor desensitization. We have recently demonstrated that CTZ also inhibits GABA(A) receptors. Here we report that CTZ induces robust epileptiform activity in hippocampal neurons both in vitro and in vivo. We first found that chronic treatment of hippocampal cultures with CTZ (5 microM, 48 h) results in epileptiform activity in the majority of neurons (80%). The epileptiform activity lasts more than 48 h after washing off CTZ, suggesting a permanent change of the neural network properties after CTZ treatment. We then demonstrated in in vivo recordings that injection of CTZ (5 micromol in 5 microl) into the lateral ventricles of anaesthetized rats also induces spontaneous epileptiform activity in the hippocampal CA1 region. The epileptogenic effect of CTZ is probably due to its enhancing glutamatergic neurotransmission as shown by increasing the frequency and decay time of mEPSCs, and simultaneously inhibiting GABAergic neurotransmission by reducing the frequency of mIPSCs. Comparing to a well-known epileptogenic agent kainic acid (KA), CTZ affects neuronal activity mainly through modulating synaptic transmission without significant change of the intrinsic membrane excitability. Unlike KA, which induces significant cell death in hippocampal cultures, CTZ treatment does not result in any apparent neuronal death. Therefore, the CTZ-induced epilepsy model may provide a novel research tool to elucidate the molecular and cellular mechanisms of epileptogenesis without any complication from drug-induced cell death. The long-lasting epileptiform activity after CTZ washout may also make it a very useful model in screening antiepileptic drugs.
The Journal of Physiology 04/2006; 571(Pt 3):605-18. · 4.72 Impact Factor
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ABSTRACT: This protocol describes a high-efficiency Ca2+-phosphate transfection method with low cell toxicity. The Ca2+-phosphate transfection method is widely used in transfecting neurons because of its low cell toxicity and simplicity in use, but the efficiency is typically low (approximately 1-5%). To solve this problem we have developed a new Ca2+-phosphate transfection protocol that increases the efficiency by 10-fold (< or = 60%), while maintaining low cell toxicity. First, it is critical to have gentle mixing of the DNA-Ca2+ solution with phosphate buffer to form a homogeneous snowlike precipitate (particle size 1-3 microm). Second, the precipitate should be dissolved using a slightly acidic culture medium to reduce cell toxicity. The high efficiency of this new protocol makes it possible to transfect single autaptic neurons as well as mature neurons (15-82 days in vitro) for gene functional analysis. The total time required for the protocol is 2-4 h (including 45 min-3 h incubation time).
Nature Protocol 01/2006; 1(2):695-700. · 8.36 Impact Factor
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ABSTRACT: Modulation of the concentration of postsynaptic GABA(A) receptors contributes to functional plasticity of inhibitory synapses. The gamma2 subunit of GABA(A) receptor is specifically required for clustering of these receptors, for recruitment of the submembrane scaffold protein gephyrin to postsynaptic sites, and for postsynaptic function of GABAergic inhibitory synapses. To elucidate this mechanism, we here have mapped the gamma2 subunit domains required for restoration of postsynaptic clustering and function of GABA(A) receptors in gamma2 subunit mutant neurons. Transfection of gamma2-/- neurons with the gamma2 subunit but not the alpha2 subunit rescues postsynaptic clustering of GABA(A) receptors, results in recruitment of gephyrin to postsynaptic sites, and restores the amplitude and frequency of miniature inhibitory postsynaptic currents to wild-type levels. Analogous analyses of chimeric gamma2/alpha2 subunit constructs indicate, unexpectedly, that the fourth transmembrane domain of the gamma2 subunit is required and sufficient for postsynaptic clustering of GABA(A) receptors, whereas cytoplasmic gamma2 subunit domains are dispensable. In contrast, both the major cytoplasmic loop and the fourth transmembrane domain of the gamma2 subunit contribute to efficient recruitment of gephyrin to postsynaptic receptor clusters and are essential for restoration of miniature IPSCs. Our study points to a novel mechanism involved in targeting of GABA(A) receptors and gephyrin to inhibitory synapses.
Journal of Neuroscience 02/2005; 25(3):594-603. · 7.11 Impact Factor
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ABSTRACT: Ionotropic glutamate and gamma-aminobutyric acid type A (GABAA) receptors mediate critical excitatory and inhibitory actions in the brain. Cyclothiazide (CTZ) is well known for its effect of enhancing glutamatergic transmission and is widely used as a blocker for alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor desensitization. Here, we report that in addition to its action on AMPA receptors, CTZ also exerts a powerful but opposite effect on GABAA receptors. We found that CTZ reversibly inhibited both evoked and spontaneous inhibitory postsynaptic currents, as well as GABA application-induced membrane currents, in a dose-dependent manner. Single-channel analyses revealed further that CTZ greatly reduced the open probability of GABAA receptor channels. These results demonstrate that CTZ interacts with both glutamate and GABAA receptors and shifts the excitation-inhibition balance in the brain by two independent mechanisms. Understanding the molecular mechanism of this double-faceted drug-receptor interaction may help in designing new therapies for neurological diseases.
Proceedings of the National Academy of Sciences 11/2003; 100(22):13025-9. · 9.68 Impact Factor
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ABSTRACT: Accumulation of GABAA receptors (GABAARs) at GABAergic synapses requires the cytoplasmic loop region and C-terminal transmembrane domain of the receptor γ2 subunit. We here report a novel interaction of γ2 with Calcium-Modulating cyclophilin Ligand (CAML), an integral membrane protein that regulates this mechanism. Interaction of GABAARs with CAML depends on both the cytoplasmic region and fourth transmembrane domain of the γ2 subunit, CAML immunoprecipitates with GABAARs from transfected cells and brain lysates and colocalizes with γ2 in ER vesicles in soma and dendrites of neurons. CAML shRNA treatment results in reduced expression of postsynaptic GABAARs, along with significant reductions in GABA-evoked whole cell currents and GABAergic synaptic function, while glutamatergic transmission is unaffected. Reduced surface expression of GABAARs in CAML mutant neurons is associated with selective deficits in recycling of endocytosed GABAARs to the cell surface. Our results indicate a specific role of CAML in functional expression and endocytic recycling of postsynaptic GABAARs.
Molecular and Cellular Neuroscience.
