Sensory-Derived Glutamate Regulates Presynaptic Inhibitory Terminals in Mouse Spinal Cord.
ABSTRACT: Circuit function in the CNS relies on the balanced interplay of excitatory and inhibitory synaptic signaling. How neuronal activity influences synaptic differentiation to maintain such balance remains unclear. In the mouse spinal cord, a population of GABAergic interneurons, GABApre, forms synapses with the terminals of proprioceptive sensory neurons and controls information transfer at sensory-motor connections through presynaptic inhibition. We show that reducing sensory glutamate release results in decreased expression of GABA-synthesizing enzymes GAD65 and GAD67 in GABApre terminals and decreased presynaptic inhibition. Glutamate directs GAD67 expression via the metabotropic glutamate receptor mGluR1? on GABApre terminals and regulates GAD65 expression via autocrine influence on sensory terminal BDNF. We demonstrate that dual retrograde signals from sensory terminals operate hierarchically to direct the molecular differentiation of GABApre terminals and the efficacy of presynaptic inhibition. These retrograde signals comprise a feedback mechanism by which excitatory sensory activity drives GABAergic inhibition to maintain circuit homeostasis.
Project description:Dopaminergic (DAergic) neurons in the ventral tegmental area (VTA) play crucial roles in motivational control of behaviors, and their activity is regulated directly or indirectly via GABAergic neurons by extrinsic afferents from various sources, including the bed nucleus of the stria terminalis (BST). Here, the neurochemical composition of VTA-projecting BST neurons and their outputs to the VTA were studied in adult mouse brains. By combining retrograde tracing with fluorescence in situ hybridization for 67 kDa glutamate decarboxylase (GAD67) and vesicular glutamate transporters (VGluTs), VTA-targeting BST neurons were classified into GAD67-positive (GAD67(+))/VGluT3-negative (VGluT3(-)), GAD67(+)/VGluT3(+), and VGluT2(+) neurons, of which GAD67(+)/VGluT3(-) neurons constituted the majority (?90%) of VTA-projecting BST neurons. GABAergic efferents from the BST formed symmetrical synapses on VTA neurons, which were mostly GABAergic neurons, and expressed GABA(A) receptor ?1 subunit on their synaptic and extrasynaptic membranes. In the VTA, VGluT3 was detected in terminals expressing vesicular inhibitory amino acid transporter (VIAAT), plasmalemmal serotonin transporter, or neither. Of these, VIAAT(+)/VGluT3(+) terminals, which should include those from GAD67(+)/VGluT3(+) BST neurons, formed symmetrical synapses. When single axons from VGluT3(+) BST neurons were examined, almost all terminals were labeled for VIAAT, whereas VGluT3 was often absent from terminals with high VIAAT loads. VGluT2(+) terminals in the VTA exclusively formed asymmetrical synapses, which expressed AMPA receptors on postsynaptic membrane. Therefore, the major mode of the BST-VTA projection is GABAergic, and its activation is predicted to disinhibit VTA DAergic neurons. VGluT2(+) and VGluT3(+) BST neurons further supply additional projections, which may principally convey excitatory or inhibitory inputs, respectively, to the VTA.
Project description:GABAergic interneurons are key elements in neural coding, but the mechanisms that assemble inhibitory circuits remain unclear. In the spinal cord, the transfer of sensory signals to motor neurons is filtered by GABAergic interneurons that act presynaptically to inhibit sensory transmitter release and postsynaptically to inhibit motor neuron excitability. We show here that the connectivity and synaptic differentiation of GABAergic interneurons that mediate presynaptic inhibition is directed by their sensory targets. In the absence of sensory terminals these GABAergic neurons shun other available targets, fail to undergo presynaptic differentiation, and withdraw axons from the ventral spinal cord. A sensory-specific source of brain derived neurotrophic factor induces synaptic expression of the GABA synthetic enzyme GAD65--a defining biochemical feature of this set of interneurons. The organization of a GABAergic circuit that mediates presynaptic inhibition in the mammalian CNS is therefore controlled by a stringent program of sensory recognition and signaling.
Project description:Actions of endocannabinoids in the cerebellum can be demonstrated following distinct stimulation protocols in Purkinje cells. First, depolarization-induced elevations of intracellular Ca2+ lead to the suppression of neurotransmitter release from both inhibitory and excitatory afferents. In another case, postsynaptic group I metabotropic glutamate receptors (mGluRs) trigger a strong inhibition of the glutamatergic inputs from parallel and climbing fibers. Both pathways involve endocannabinoids retrogradely acting on type 1 cannabinoid receptors (CB1Rs) at presynaptic terminals. Here, we show that group I mGluR activation also depresses GABAergic transmission at the synapses between molecular layer interneurons and Purkinje cells. Using paired recordings, we found that application of the group I mGluR agonist (RS)-3,5-dihydroxyphenylglycine reduced the evoked IPSCs in Purkinje cells. This effect was independent of postsynaptic Ca2+ increases and was completely blocked by a CB1R antagonist. Experiments performed with the GTP-analogues GDP-betaS and GTP-gammaS provided evidence that endocannabinoids released after G-protein activation can also inhibit GABAergic inputs onto nearby, unstimulated Purkinje cells. Block of the enzymes DAG lipase or phospholipase C reduced the group I mGluR-dependent inhibition, suggesting that 2-arachidonyl glycerol could act as retrograde messenger. Finally, group I mGluR activation by brief bursts of activity of the parallel fibers induced a short-lived depression of spontaneous IPSCs via presynaptic CB1Rs. Our results reveal a mechanism with potential physiological importance, by which glutamatergic synapses induce an endocannabinoid-mediated inhibition of the GABAergic inputs onto Purkinje cells.
Project description:Stimulation of the postsynaptic metabotropic glutamate receptor mGluR5 triggers retrograde signaling of endocannabinoids that activate presynaptic cannabinoid CB1 receptors on juxtaposing axon terminals. To better understand the synaptic structure that supports mGluR5 mediation of CB1 activation in the prefrontal cortex (PFC) and basolateral amygdala (BLA), we examined electron microscopic dual immunolabeling of these receptors in the prelimbic PFC (prPFC) and BLA of adult male rats. CB1 immunoreactivity was detected in axon terminals that were typically large, complex, and contained dense-core and clear synaptic vesicles. Of terminals forming discernible synaptic specializations, 95% were symmetric inhibitory-type in the prPFC and 90% were inhibitory in the BLA. CB1-immunoreactive terminals frequently contacted dendrites containing mGluR5 adjacent to unlabeled terminals forming excitatory-type synapses. Because most CB1-containing terminals form inhibitory-type synapses, the unlabeled axon terminals forming asymmetric synapses are the likely source of the mGluR5 ligand glutamate. In the prPFC, serial section analysis revealed that GABAergic CB1-containing axon terminals targeted dendrites adjacent to glutamatergic axon terminals, often near dendritic bifurcations. These observations provide ultrastructural evidence that cortical CB1 receptors are strategically positioned for integration of synaptic signaling in response to stimulation of postsynaptic mGluR5 receptors and facilitation of heterosynaptic communication between multiple neurons.
Project description:Euthermia is critical for mammalian homeostasis. Circuits within the preoptic hypothalamus regulate temperature, with fine control exerted via descending GABAergic inhibition of presympathetic motor neurons that control brown adipose tissue (BAT) thermogenesis and cutaneous vascular tone. The thermoregulatory role of hypothalamic excitatory neurons is less clear. Here we report peptidergic regulation of preoptic glutamatergic neurons that contributes to temperature regulation. Tuberoinfundibular peptide of 39 residues (TIP39) is a ligand for the parathyroid hormone 2 receptor (PTH2R). Both peptide and receptor are abundant in the preoptic hypothalamus. Based on PTH2R and vesicular glutamate transporter 2 (VGlut2) immunolabeling in animals with retrograde tracer injection, PTH2R-containing glutamatergic fibers are presynaptic to neurons projecting from the median preoptic nucleus (MnPO) to the dorsomedial hypothalamus. Transneuronal retrograde pathway tracing with pseudorabies virus revealed connectivity between MnPO VGlut2 and PTH2R neurons and BAT. MnPO injection of TIP39 increased body temperature by 2°C for several hours. Mice lacking TIP39 signaling, either because of PTH2R-null mutation or brain delivery of a PTH2R antagonist had impaired heat production upon cold exposure, but no change in basal temperature and no impairment in response to a hot environment. Thus, TIP39 appears to act on PTH2Rs present on MnPO glutamatergic terminals to regulate their activation of projection neurons and subsequent sympathetic BAT activation. This excitatory mechanism of heat production appears to be activated on demand, during cold exposure, and parallels the tonic inhibitory GABAergic control of body temperature.
Project description:The climbing fiber (CF) neurotransmitter not only excites the postsynaptic Purkinje cell (PC) but also suppresses GABA release from inhibitory interneurons converging onto the same PC depending on AMPA-type glutamate receptor (AMPAR) activation. Although the CF-/AMPAR-mediated inhibition of GABA release provides a likely mechanism boosting the CF input-derived excitation, how the CF transmitter reaches target AMPARs to elicit this action remains unknown. Here, we report that the CF transmitter diffused from its release sites directly targets GluR2/GluR3 AMPARs on interneuron terminals to inhibit GABA release. A weak GluR3-AMPAR agonist, bromohomoibotenic acid, produced excitatory currents in the postsynaptic PCs without presynaptic inhibitory effect on GABAergic transmission. Conversely, a specific inhibitor of the GluR2-lacking/Ca2+-permeable AMPARs, philanthotoxin-433, did not affect the CF-induced inhibition but suppressed AMPAR-mediated currents in Bergmann glia. A low-affinity GluR antagonist, gamma-D-glutamylglycine, or retardation of neurotransmitter diffusion by dextran reduced the inhibitory action of CF-stimulation, whereas blockade of glutamate transporters enhanced the CF-induced inhibition. The results suggest that the CF transmitter released after repeated stimulation overwhelms local glutamate uptake and thereby diffuses from the release site to reach GluR2/GluR3 AMPARs on nearby interneuron terminals. Double immunostaining showed that GluR2/3 subunits and glutamate decarboxylase or synaptophysin are colocalized at the perisomatic GABAergic processes surrounding PCs. Finally, electron microscopy detected specific immunoreactivity for GluR2/3 at the presynaptic terminals of symmetric axosomatic synapses on the PC. These findings demonstrate that the CF transmitter directly inhibits GABA release from interneurons to the PC, relying on extrasynaptic diffusion and local heterogeneity in AMPAR subunit compositions.
Project description:The production of gamma-amino butyric acid (GABA) is dependent on glutamate decarboxylases (GAD65 and GAD67), the enzymes that catalyze the decarboxylation of glutamate to GABA. Based on studies suggesting a role of the airway epithelial GABAergic system in asthma-related mucus overproduction, we hypothesized that cigarette smoking, another disorder associated with increased mucus production, may modulate GABAergic system-related gene expression levels in the airway epithelium.We assessed expression of the GABAergic system in human airway epithelium obtained using bronchoscopy to sample the epithelium and microarrays to evaluate gene expression. RT-PCR was used to confirm gene expression of GABAergic system gene in large and small airway epithelium from heathy nonsmokers and healthy smokers. The differences in the GABAergic system gene was further confirmed by TaqMan, immunohistochemistry and Western analysis.The data demonstrate there is a complete GABAergic system expressed in the large and small human airway epithelium, including glutamate decarboxylase, GABA receptors, transporters and catabolism enzymes. Interestingly, of the entire GABAergic system, smoking modified only the expression of GAD67, with marked up-regulation of GAD67 gene expression in both large (4.1-fold increase, p < 0.01) and small airway epithelium of healthy smokers (6.3-fold increase, p < 0.01). At the protein level, Western analysis confirmed the increased expression of GAD67 in airway epithelium of healthy smokers compared to healthy nonsmokers (p < 0.05). There was a significant positive correlation between GAD67 and MUC5AC gene expression in both large and small airway epithelium (p < 0.01), implying a link between GAD67 and mucin overproduction in association with smoking.In the context that GAD67 is the rate limiting enzyme in GABA synthesis, the correlation of GAD67 gene expression with MUC5AC expressions suggests that the up-regulation of airway epithelium expression of GAD67 may contribute to the increase in mucus production observed in association with cigarette smoking.NCT00224198; NCT00224185.
Project description:UNLABELLED:During infections with the protozoan parasite Toxoplasma gondii, gamma-aminobutyric acid (GABA) is utilized as a carbon source for parasite metabolism and also to facilitate parasite dissemination by stimulating dendritic-cell motility. The best-recognized function for GABA, however, is its role in the nervous system as an inhibitory neurotransmitter that regulates the flow and timing of excitatory neurotransmission. When this pathway is altered, seizures develop. Human toxoplasmosis patients suffer from seizures, suggesting that Toxoplasma interferes with GABA signaling in the brain. Here, we show that while excitatory glutamatergic presynaptic proteins appeared normal, infection with type II ME49 Toxoplasma tissue cysts led to global changes in the distribution of glutamic acid decarboxylase 67 (GAD67), a key enzyme that catalyzes GABA synthesis in the brain. Alterations in GAD67 staining were not due to decreased expression but rather to a change from GAD67 clustering at presynaptic termini to a more diffuse localization throughout the neuropil. Consistent with a loss of GAD67 from the synaptic terminals, Toxoplasma-infected mice develop spontaneous seizures and are more susceptible to drugs that induce seizures by antagonizing GABA receptors. Interestingly, GABAergic protein mislocalization and the response to seizure-inducing drugs were observed in mice infected with type II ME49 but not type III CEP strain parasites, indicating a role for a polymorphic parasite factor(s) in regulating GABAergic synapses. Taken together, these data support a model in which seizures and other neurological complications seen in Toxoplasma-infected individuals are due, at least in part, to changes in GABAergic signaling. IMPORTANCE:Infections of the central nervous system can cause seizures. While inflammation in the brain has been proposed to initiate the onset of the seizures, relatively little is known about how inflammation impacts the structure and function of the neurons. Here we used a parasite called Toxoplasma gondii that infects the brain and showed that seizures arise due to a defect in signaling of GABA, which is the neurotransmitter primarily responsible for preventing the onset of seizures.
Project description:In human prefrontal cortex (PFC), ~85% of ?-aminobutyric acid (GABA)-expressing neurons can be subdivided into non-overlapping groups by the presence of calbindin (CB), calretinin (CR) or parvalbumin (PV). Substantial research has focused on the differences in the laminar locations of the cells bodies of these neurons, with limited attention to the distribution of their axon terminals, their sites of action. We previously reported that in non-human primates subtypes of these cells are distinguishable by differences in terminal protein levels of the GABA synthesizing enzymes glutamic acid decarboxylase 65 (GAD65) and GAD67. Here we used multi-label fluorescence microscopy in human PFC to assess: (1) the laminar distributions of axon terminals containing CB, CR, or PV; and (2) the relative protein levels of GAD65, GAD67 and vesicular GABA transporter (vGAT) in CB, CR and PV terminals. The densities of the different CB, CR and PV terminal subpopulations differed across layers of the PFC. PV terminals comprised two subsets based on the presence of only GAD67 (GAD67+) or both GADs (GAD65/GAD67+), whereas CB and CR terminals comprised three subsets (GAD65+, GAD67+, or GAD65/GAD67+). The densities of the different CB, CR and PV GAD terminal subpopulations also differed across layers. Finally, within each of the three calcium-binding protein subpopulations intra-terminal protein levels of GAD and vGAT differed by GAD subpopulation. These findings are discussed in the context of the laminar distributions of CB, CR and PV cell bodies and the synaptic targets of their axons.
Project description:The precision of skilled movement depends on sensory feedback and its refinement by local inhibitory microcircuits. One specialized set of spinal GABAergic interneurons forms axo-axonic contacts with the central terminals of sensory afferents, exerting presynaptic inhibitory control over sensory-motor transmission. The inability to achieve selective access to the GABAergic neurons responsible for this unorthodox inhibitory mechanism has left unresolved the contribution of presynaptic inhibition to motor behaviour. We used Gad2 as a genetic entry point to manipulate the interneurons that contact sensory terminals, and show that activation of these interneurons in mice elicits the defining physiological characteristics of presynaptic inhibition. Selective genetic ablation of Gad2-expressing interneurons severely perturbs goal-directed reaching movements, uncovering a pronounced and stereotypic forelimb motor oscillation, the core features of which are captured by modelling the consequences of sensory feedback at high gain. Our findings define the neural substrate of a genetically hardwired gain control system crucial for the smooth execution of movement.