Search

The axon initial segment (AIS) of cerebellar Purkinje cells (PCs) is embraced by ramified axons of GABAergic basket cells (BCs) called the pinceau formation. This unique structure has been assumed to be a device for the modulation of PC outputs through electrical and/or GABAergic inhibition. Electrical inhibition is supported by enriched potassium channels, absence of sodium channels, and developed septate-like junctions between BC axons. The neurochemical basis for GABAergic inhibition, however, has not been well investigated. Here we addressed this issue using C56BL/6 mice. First, we confirmed previous observations that typical synaptic contacts were rare and confined to proximal axonal portions, with the remaining portions being mostly covered by astrocytic processes. Then we examined the expression of molecules involved in GABAergic signaling, including GABA synthetic enzyme glutamic acid decarboxylase (GAD), vesicular GABA transporter vesicular inhibitory amino acid transporter (VIAAT), cytomatrix active zone protein bassoon, GABA receptor GABAAR&agr;1, and cell adhesion molecule neuroligin-2. These molecules were recruited to form a functional assembly at perisomatic BC-PC synapses and along the AIS of hippocampal and neocortical pyramidal cells.GADand VIAAT immunogold labeling was five times lower in the pinceau formation compared with perisomatic BC terminals and showed no accumulation toward the AIS. Moreover, bassoon, neuroligin-2, and GABAAR&agr;1 formed no detectable clusters along the ankyrin-G-positive AIS proper. These findings indicate that GABAergic signaling machinery is organized loosely and even incompletely in the pinceau formation. Together, BCs do not appear to exert GABAergic synaptic inhibition on the AIS, although the mode of action of the pinceau formation remains to be explored. [ABSTRACT FROM AUTHOR]

The remodeling of neural circuitry and changes in synaptic efficacy after peripheral sensory nerve injury are considered the basis for functional reorganization in the brain, including changes in receptive fields. However, when or how the remodeling occurs is largely unknown. Here we show the rapid rewiring of afferent fibers in the mature ventral posteromedial thalamic nucleus of mice after transection of the peripheral whisker sensory nerve, using the whole-cell voltage-clamp technique. Transection induced the recruitment of afferent fibers to a thalamic relay neuron within 5- 6 d of injury. The rewiring was pathway specific, but not sensory experience dependent or peripheral nerve activity dependent. The newly recruited fibers mediated small EPSCs, and postsynaptic GluA2-containing AMPA receptors were selectively upregulated at the new synapses. This rapid and pathway-specific remodeling of thalamic circuitry may be an initial step in the massive axonal reorganization at supraspinal levels, which occurs months or years after peripheral sensory nerve injury. [ABSTRACT FROM AUTHOR]

Acetylcholine (ACh) plays important roles for higher brain functions, including arousal, attention, and cognition. These effects are mediated largely by muscarinic acetylcholine receptors (mAChRs). However, it remains inconclusive whether the mode of ACh-mAChR signaling is synaptic, so-called "wired," transmission mediated by ACh released into the synaptic cleft, or nonsynaptic, so-called "volume," transmission by ambient ACh. To address this issue, we examined cellular and subcellular distribution ofM1 , the most predominant mAChR subtype in the cerebral cortex and hippocampus, and pursued its anatomical relationship with cholinergic varicosities in these regions of adult mice. M1 was highly expressed in glutamatergic pyramidal neurons, whereas it was low or undetectable in various GABAergic interneuron subtypes. M1 was preferentially distributed on the extrasynaptic membrane of pyramidal cell dendrites and spines. Cholinergic varicosities often made direct contact to pyramidal cell dendrites and synapses. At such contact sites, however, synapse-like specialization was infrequent, and no particular accumulation was found at around contact sites for both M1 and presynpatic active zone protein Bassoon. These features contrasted with those of the glutamatergic system, in which AMPA receptor GluA2 and metabotropic receptor mGluR5 were recruited to the synaptic or perisynaptic membrane, respectively, and Bassoon was highly accumulated in the presynaptic terminals. These results suggest that M1 is so positioned to sense ambient ACh released from cholinergic varicosities at variable distances, and to enhance the synaptic efficacy and excitability of pyramidal cells. These molecular-anatomical arrangements will provide the evidence for volume transmission, at least in M1-mediated cortical cholinergic signaling. [ABSTRACT FROM AUTHOR]

GluN2B (GluRϵ2/NR2B) subunit is involved in synapse development, synaptic plasticity, and cognitive function. However, its roles in synaptic expression and function of NMDA receptors (NMDARs) in the brain remain mostly unknown because of the neonatal lethality of global knock-out mice. To address this, we generated conditional knock-out mice, in which GluN2B was ablated exclusively in hippocampal CA3 pyramidal cells. By immunohistochemistry, GluN2B disappeared and GluN1 (GluRζ1/NR1) was moderately reduced, whereas GluN2A (GluRϵ1/NR2A) and postsynaptic density-95 (PSD-95) were unaltered in the mutant CA3. This was consistent with protein contents in the CA3 crude fraction: 9.6% of control level for GluN2B, 47.7% for GluN1, 90.6% for GluN2A, and 98.0% for PSD-95. Despite the remaining NMDARs, NMDAR-mediated currents and long-term potentiation were virtually lost at various CA3 synapses. Then, we compared synaptic NMDARs by postembedding immunogold electron microscopy and immunoblot using the PSD fraction. In the mutant CA3, GluN1 was severely reduced in both immunogold (20.6-23.6%) and immunoblot (24.6%), whereas GluN2A and PSD-95 were unchanged in immunogold but markedly reduced in the PSD fraction (51.4 and 36.5%, respectively), indicating increased detergent solubility of PSD molecules. No such increased solubility was observed for GluN2B in the CA3 of GluN2A-knock-out mice. Furthermore, significant decreases were found in the ratio of filamentous to globular actin (49.5%) and in the density of dendritic spines (76.2%). These findings suggest that GluN2B is critically involved in NMDAR channel function, organization of postsynaptic macromolecular complexes, formation or maintenance of dendritic spines, and regulation of the actin cytoskeleton. [ABSTRACT FROM AUTHOR]

Activation of cannabinoid receptors suppresses neurotransmitter release in various brain regions. In cerebellar Purkinje cells (PCs), cannabinoid agonists suppress both EPSC and IPSC evoked by stimulating the corresponding inputs. However, cannabinoid agonists suppress miniature IPSC (mIPSC) but not miniature EPSC (mEPSC) at normal external Ca2+ concentration ([Ca2+]o). Therefore, cannabinoid agonists are thought to suppress release machinery for IPSCs but not that for EPSCs. Here we investigated the possible cause of this difference and found that cannabinoid agonists selectively suppressed Ca2+-enhanced miniature events. A cannabinoid agonist, WIN55,212-2 (5 µM), did not affect mEPSC frequency with 2 mM extracellular Ca2+ (Ca2+o). However, WIN55,212-2 became effective when mEPSC frequency was enhanced by elevation of presynaptic Ca2+level by perfusion with 5mM Ca2+o or bath application of A23187, a Ca2+ ionophore. In contrast, WIN55,212-2 suppressed mIPSC frequency with 2mM Ca2+o, but it became ineffective when the presynaptic Ca2+ level was lowered by perfusion with a Ca2+-free solution containing BAPTA-AM. Experiments with systematic [Ca2+]o changes revealed that mIPSC but not mEPSC regularly involved events elicited by presynaptic Ca2+ rise with 2mM Ca2+o. Importantly, Ca2+-enhancement of mEPSC and mIPSC was not attributable to activation of voltage-dependent Ca2+ channels. Activation of GABAB receptor or group III metabotropic glutamate receptor, which couple to Gi/o-protein, also preferentially suppressed Ca2+-enhanced miniature events in PCs. These results suggest that the occurrence of Ca2+-enhanced miniature events at normal [Ca2+]o determines the sensitivity to the presynaptic depression mediated by cannabinoid receptors and other Gi/o-coupled receptors in PCs. [ABSTRACT FROM AUTHOR]

Glutamate receptor (GluR) δ2 selectively expressed in cerebellar Purkinje cells (PCs) plays key roles in long-term depression (LTD) induction at parallel fiber (PF)-PC synapses, motor learning, the matching and connection of PF-PC synapses in developing and adult cerebella, the elimination of multiple climbing fibers (CFs) during development, and the regulation of CF territory on PCs. However, it remains unsolved how GluRδ2 regulates cerebellar synaptic plasticity, PF-PC synapse formation, and CF wiring. One possible signaling mechanism through GluRδ2 is signaling by protein-protein interactions. The C-terminal region of GluRδ2 contains at least three domains for protein-protein interactions. The PDZ (postsynaptic density-95/Discs large/zona occludens 1)-binding domain at the C terminal, named as the T site, interacts with several postsynaptic density proteins. Here, we generated GluRδ2ΔT mice carrying mutant GluRδ2 lacking the T site. There were no significant differences in the amount of receptor proteins at synapses, histological features, and the fine structures of PF-PC synapses between wild-type and GluRδ2ΔT mice. However, LTD induction at PF-PC synapses and improvement in the accelerating rotarod test were impaired in GluRδ2ΔT mice. Furthermore, CF territory expanded distally and ectopic innervation of CFs occurred at distal dendrites in GluRδ2ΔT mice, but the elimination of surplus CF innervation at proximal dendrites appeared to proceed normally. These results suggest that the C-terminal T site of GluRδ2 is essential for LTD induction and the regulation of CF territory but is dispensable for PF-PC synapse formation and the elimination of surplus CFs at proximal dendrites during development. [ABSTRACT FROM AUTHOR]

Dopamine neurons play crucial roles in pleasure, reward, memory, learning, and fine motor skills and their dysfunction is associated with various neuropsychiatric diseases. Dopamine receptors are the main target of treatment for neurologic and psychiatric disorders. Antipsychotics that antagonize the dopamine D2 receptor (DRD2) are used to alleviate the symptoms of these disorders but may also sometimes cause disabling side effects such as parkinsonism (catalepsy in rodents). Here we show that GPR143, a G-protein-coupled receptor for L-3,4-dihydroxyphenylalanine (L-DOPA), expressed in striatal cholinergic interneurons enhances the DRD2-mediated side effects of haloperidol, an antipsychotic agent. Haloperidol-induced catalepsy was attenuated in male Gpr143 gene -deficient ( Gpr143 -/y ) mice compared with wild-type (Wt) mice. Reducing the endogenous release of L-DOPA and preventing interactions between GPR143 and DRD2 suppressed the haloperidol-induced catalepsy in Wt mice but not Gpr143 -/y mice. The phenotypic defect in Gpr143 -/y mice was mimicked in cholinergic interneuron-specific Gpr143 -/y ( Chat-cre;Gpr143 flox/y ) mice. Administration of haloperidol increased the phosphorylation of ribosomal protein S6 at Ser 240/244 in the dorsolateral striatum of Wt mice but not Chat-cre;Gpr143 flox/y mice. In Chinese hamster ovary cells stably expressing DRD2, co-expression of GPR143 increased cell surface expression level of DRD2, and L-DOPA application further enhanced the DRD2 surface expression. Shorter pauses in cholinergic interneuron firing activity were observed after intrastriatal stimulation in striatal slice preparations from Chat-cre;Gpr143 flox/y mice compared with those from Wt mice. Together, these findings provide evidence that GPR143 regulates DRD2 function in cholinergic interneurons and may be involved in parkinsonism induced by antipsychotic drugs., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 the authors.)

Unlabelled: The formation and refinement of thalamocortical axons (TCAs) is an activity-dependent process (Katz and Shatz, 1996), but its mechanism and nature of activity are elusive. We studied the role of spike timing-dependent plasticity (STDP) in TCA formation and refinement in mice. At birth (postnatal day 0, P0), TCAs invade the cortical plate, from which layers 4 (L4) and L2/3 differentiate at P3-P4. A portion of TCAs transiently reach toward the pia surface around P2-P4 (Senft and Woolsey, 1991; Rebsam et al., 2002) but are eventually confined below the border between L2/3 and L4. We previously showed that L4-L2/3 synapses exhibit STDP with only potentiation (timing-dependent long-term potentiation [t-LTP]) during synapse formation, then switch to a Hebbian form of STDP. Here we show that TCA-cortical plate synapses exhibit robust t-LTP in neonates, whose magnitude decreased gradually after P4-P5. After L2/3 is differentiated, TCA-L2/3 gradually switched to STDP with only depression (t-LTD) after P7-P8, whereas TCA-L4 lost STDP. t-LTP was dependent on NMDA receptor and PKA, whereas t-LTD was mediated by Type 1 cannabinoid receptors (CB1Rs) probably located at TCA terminals, revealed by global and cortical excitatory cell-specific knock-out of CB1R. Moreover, we found that administration of CB1R agonists, including Δ(9)-tetrahydrocannabinol, caused substantial retraction of TCAs. Consistent with this, individual thalamocortical axons exuberantly innervated L2/3 at P12 in CB1R knock-outs, indicating that endogenous cannabinoid signaling shapes TCA projection. These results suggest that the developmental switch in STDP and associated appearance of CB1R play important roles in the formation and refinement of TCAs., Significance Statement: It has been shown that neural activity is required for initial synapse formation of thalamocortical axons with cortical cells, but precisely what sort of activities in presynaptic and postsynaptic cells are required is not yet clear. In addition, how activity is further translated into structural changes is unclear. We show here that the period during which spike timing-dependent long-term potentiation and depression (t-LTP, t-LTD) can be induced closely matches the time course of synapse formation and retraction, respectively, at the thalamocortical synapse. Moreover, administration of cannabinoid agonists, which mimic t-LTD, caused TCA retraction, suggesting that cannabinoids translate physiological changes into morphological consequences., (Copyright © 2016 the authors 0270-6474/16/367040-16$15.00/0.)

Invaginating synapses in the basal amygdala are a unique type of GABAergic synapses equipped with molecular-anatomical organization specialized for 2-arachidonoylglycerol (2-AG)-mediated endocannabinoid signaling. Cholecystokinin (CCK)-positive basket cell terminals protrude into pyramidal cell somata and form invaginating synapses, where apposing presynaptic and postsynaptic elements are highly loaded with cannabinoid receptor CB₁ or 2-AG synthetic enzyme diacylglycerol lipase-α (DGLα), respectively. The present study scrutinized their neurochemical and neuroanatomical phenotypes in adult mouse telencephalon. In the basal amygdala, vesicular glutamate transporter-3 (VGluT3) was transcribed in one-fourth of CB₁-expressing GABAergic interneurons. The majority of VGluT3-positive CB₁-expressing basket cell terminals apposed DGLα clusters, whereas the majority of VGluT3-negative ones did not. Importantly, VGluT3-positive basket cell terminals selectively constructed invaginating synapses. GABAA receptors accumulated on the postsynaptic membrane of invaginating synapses, whereas metabotropic glutamate receptor-5 (mGluR₅) was widely distributed on the somatodendritic surface of pyramidal cells. Moreover, CCK₂ receptor (CCK₂R) was highly transcribed in pyramidal cells. In cortical regions, pyramidal cells equipped with such VGluT3/CB₁/DGLα-accumulated invaginating synapses were found at variable frequencies depending on the subregions. Therefore, in addition to extreme proximity of CB₁- and DGLα-loaded presynaptic and postsynaptic elements, tripartite transmitter phenotype of GABA/glutamate/CCK is the common neurochemical feature of invaginating synapses, suggesting that glutamate, CCK, or both can promote 2-AG synthesis through activating Gαq/₁₁ protein-coupled mGluR₅ and CCK₂R. These molecular configurations led us to hypothesize that invaginating synapses might be evolved to provide some specific mechanisms of induction, regulation, and cooperativity for 2-AG-mediated retrograde signaling in particular cortical and cortex-like amygdaloid regions., (Copyright © 2015 the authors 0270-6474/15/354215-14$15.00/0.)

More than 30 mutations in LGI1, a secreted neuronal protein, have been reported with autosomal dominant lateral temporal lobe epilepsy (ADLTE). Although LGI1 haploinsufficiency is thought to cause ADLTE, the underlying molecular mechanism that results in abnormal brain excitability remains mysterious. Here, we focused on a mode of action of LGI1 autoantibodies associated with limbic encephalitis (LE), which is one of acquired epileptic disorders characterized by subacute onset of amnesia and seizures. We comprehensively screened human sera from patients with immune-mediated neurological disorders for LGI1 autoantibodies, which also uncovered novel autoantibodies against six cell surface antigens including DCC, DPP10, and ADAM23. Our developed ELISA arrays revealed a specific role for LGI1 antibodies in LE and concomitant involvement of multiple antibodies, including LGI1 antibodies in neuromyotonia, a peripheral nerve disorder. LGI1 antibodies associated with LE specifically inhibited the ligand-receptor interaction between LGI1 and ADAM22/23 by targeting the EPTP repeat domain of LGI1 and reversibly reduced synaptic AMPA receptor clusters in rat hippocampal neurons. Furthermore, we found that disruption of LGI1-ADAM22 interaction by soluble extracellular domain of ADAM22 was sufficient to reduce synaptic AMPA receptors in rat hippocampal neurons and that levels of AMPA receptor were greatly reduced in the hippocampal dentate gyrus in the epileptic LGI1 knock-out mouse. Therefore, either genetic or acquired loss of the LGI1-ADAM22 interaction reduces the AMPA receptor function, causing epileptic disorders. These results suggest that by finely regulating the synaptic AMPA receptors, the LGI1-ADAM22 interaction maintains physiological brain excitability throughout life.

In the adult cerebellum, each Purkinje cell (PC) is innervated by a single climbing fiber (CF) in proximal dendrites and 10(5)-10(6) parallel fibers (PFs) in distal dendrites. This organized wiring is established postnatally through heterosynaptic competition between PFs and CFs and homosynaptic competition among multiple CFs. Using PC-specific Cav2.1 knock-out mice (PC-Cav2.1 KO mice), we have demonstrated recently that postsynaptic Cav2.1 plays a key role in the homosynaptic competition by promoting functional strengthening and dendritic translocation of single "winner" CFs. Here, we report that Cav2.1 in PCs, but not in granule cells, is also essential for the heterosynaptic competition. In PC-Cav2.1 KO mice, the extent of CF territory was limited to the soma and basal dendrites, whereas PF territory was expanded reciprocally. Consequently, the proximal somatodendritic domain of PCs displayed hyperspiny transformation and fell into chaotic innervation by multiple CFs and numerous PFs. PC-Cav2.1 KO mice also displayed patterned degeneration of PCs, which occurred preferentially in aldolase C/zebrin II-negative cerebellar compartments. Furthermore, the mutually complementary expression of phospholipase Cβ3 (PLCβ3) and PLCβ4 was altered such that their normally sharp boundary was blurred in the PCs of PC-Cav2.1 KO mice. This blurring was caused by an impaired posttranscriptional downregulation of PLCβ3 in PLCβ4-dominant PCs during the early postnatal period. A similar alteration was noted in the banded expression of the glutamate transporter EAAT4 in PC-Cav2.1 KO mice. Therefore, Cav2.1 in PCs is essential for competitive synaptic wiring, cell survival, and the establishment of precise boundaries and reciprocity of biochemical compartments in PCs.