Serine/Threonine Phosphatases in LTP: Two B or Not to Be the Protein Synthesis Blocker-Induced Impairment of Early Phase.

Dephosphorylation of target proteins at serine/threonine residues is one of the most crucial mechanisms regulating their activity and, consequently, the cellular functions. The role of phosphatases in synaptic plasticity, especially in long-term depression or depotentiation, has been reported. We studied serine/threonine phosphatase activity during the protein synthesis blocker (PSB)-induced impairment of long-term potentiation (LTP). Established protein phosphatase 2B (PP2B, calcineurin) inhibitor cyclosporin A prevented the LTP early phase (E-LTP) decline produced by pretreatment of hippocampal slices with cycloheximide or anisomycin. For the first time, we directly measured serine/threonine phosphatase activity during E-LTP, and its significant increase in PSB-treated slices was demonstrated. Nitric oxide (NO) donor SNAP also heightened phosphatase activity in the same manner as PSB, and simultaneous application of anisomycin + SNAP had no synergistic effect. Direct measurement of the NO production in hippocampal slices by the NO-specific fluorescent probe DAF-FM revealed that PSBs strongly stimulate the NO concentration in all studied brain areas: CA1, CA3, and dentate gyrus (DG). Cyclosporin A fully abolished the PSB-induced NO production in the hippocampus, suggesting a close relationship between nNOS and PP2B activity. Surprisingly, cyclosporin A alone impaired short-term plasticity in CA1 by decreasing paired-pulse facilitation, which suggests bi-directionality of the influences of PP2B in the hippocampus. In conclusion, we proposed a minimal model of signaling events that occur during LTP induction in normal conditions and the PSB-treated slices.

Topoisomerase 3ß knockout mice show transcriptional and behavioural impairments associated with neurogenesis and synaptic plasticity.

Topoisomerase 3ß (Top3ß) is the only dual-activity topoisomerase in animals that can change topology for both DNA and RNA, and facilitate transcription on DNA and translation on mRNAs. Top3ß mutations have been linked to schizophrenia, autism, epilepsy, and cognitive impairment. Here we show that Top3ß knockout mice exhibit behavioural phenotypes related to psychiatric disorders and cognitive impairment. The mice also display impairments in hippocampal neurogenesis and synaptic plasticity. Notably, the brains of the mutant mice exhibit impaired global neuronal activity-dependent transcription in response to fear conditioning stress, and the affected genes include many with known neuronal functions. Our data suggest that Top3ß is essential for normal brain function, and that defective neuronal activity-dependent transcription may be a mechanism by which Top3ß deletion causes cognitive impairment and psychiatric disorders.

The Familial dementia gene ITM2b/BRI2 facilitates glutamate transmission via both presynaptic and postsynaptic mechanisms.

Mutations in the Integral membrane protein 2B (ITM2b/BRI2) gene, which codes for a protein called BRI2, cause familial British and Danish dementia (FBD and FDD). Loss of BRI2 function and/or accumulation of amyloidogenic mutant BRI2-derived peptides have been proposed to mediate FDD and FBD pathogenesis by impairing synaptic Long-term potentiation (LTP). However, the precise site and nature of the synaptic dysfunction remain unknown. Here we use a genetic approach to inactivate Itm2b in either presynaptic (CA3), postsynaptic (CA1) or both (CA3 + CA1) neurons of the hippocampal Schaeffer-collateral pathway in both female and male mice. We show that after CA3 + CA1 Itm2b inactivation, spontaneous glutamate release and AMPAR-mediated responses are decreased, while short-term synaptic facilitation is increased. Moreover, AMPAR-mediated responses are decreased after postsynaptic but not presynaptic deletion of Itm2b. In contrast, the probability of spontaneous glutamate release is decreased, while short-term synaptic facilitation is increased, primarily after presynaptic deletion of Itm2b. Collectively, these results indicate a dual physiological role of Itm2b in the regulation of excitatory synaptic transmission at both presynaptic termini and postsynaptic termini and suggest that presynaptic and postsynaptic dysfunctions may be a pathogenic event leading to dementia and neurodegeneration in FDD and FBD.

Dystrobrevin is required postsynaptically for homeostatic potentiation at the Drosophila NMJ.

Evolutionarily conserved homeostatic systems have been shown to modulate synaptic efficiency at the neuromuscular junctions of organisms. While advances have been made in identifying molecules that function presynaptically during homeostasis, limited information is currently available on how postsynaptic alterations affect presynaptic function. We previously identified a role for postsynaptic Dystrophin in the maintenance of evoked neurotransmitter release. We herein demonstrated that Dystrobrevin, a member of the Dystrophin Glycoprotein Complex, was delocalized from the postsynaptic region in the absence of Dystrophin. A newly-generated Dystrobrevin mutant showed elevated evoked neurotransmitter release, increased bouton numbers, and a readily releasable pool of synaptic vesicles without changes in the function or numbers of postsynaptic glutamate receptors. In addition, we provide evidence to show that the highly conserved Cdc42 Rho GTPase plays a key role in the postsynaptic Dystrophin/Dystrobrevin pathway for synaptic homeostasis. The present results give novel insights into the synaptic deficits underlying Duchenne Muscular Dystrophy affected by a dysfunctional Dystrophin Glycoprotein complex.

Synaptotagmin-11 mediates a vesicle trafficking pathway that is essential for development and synaptic plasticity.

Synaptotagmin-11 (Syt11) is a Synaptotagmin isoform that lacks an apparent ability to bind calcium, phospholipids, or SNARE proteins. While human genetic studies have linked mutations in the Syt11 gene to schizophrenia and Parkinson's disease, the localization or physiological role of Syt11 remain unclear. We found that in neurons, Syt11 resides on abundant vesicles that differ from synaptic vesicles and resemble trafficking endosomes. These vesicles recycle via the plasma membrane in an activity-dependent manner, but their exocytosis is slow and desynchronized. Constitutive knockout mice lacking Syt11 died shortly after birth, suggesting Syt11-mediated membrane transport is required for survival. In contrast, selective ablation of Syt11 in excitatory forebrain neurons using a conditional knockout did not affect life span but impaired synaptic plasticity and memory. Syt11-deficient neurons displayed normal secretion of fast neurotransmitters and peptides but exhibited a reduction of long-term synaptic potentiation. Hence, Syt11 is an essential component of a neuronal vesicular trafficking pathway that differs from the well-characterized synaptic vesicle trafficking pathway but is also essential for life.

Presynaptic SNAP-25 regulates retinal waves and retinogeniculate projection via phosphorylation.

Patterned spontaneous activity periodically displays in developing retinas termed retinal waves, essential for visual circuit refinement. In neonatal rodents, retinal waves initiate in starburst amacrine cells (SACs), propagating across retinal ganglion cells (RGCs), further through visual centers. Although these waves are shown temporally synchronized with transiently high PKA activity, the downstream PKA target important for regulating the transmission from SACs remains unidentified. A t-SNARE, synaptosome-associated protein of 25 kDa (SNAP-25/SN25), serves as a PKA substrate, implying a potential role of SN25 in regulating retinal development. Here, we examined whether SN25 in SACs could regulate wave properties and retinogeniculate projection during development. In developing SACs, overexpression of wild-type SN25b, but not the PKA-phosphodeficient mutant (SN25b-T138A), decreased the frequency and spatial correlation of wave-associated calcium transients. Overexpressing SN25b, but not SN25b-T138A, in SACs dampened spontaneous, wave-associated, postsynaptic currents in RGCs and decreased the SAC release upon augmenting the cAMP-PKA signaling. These results suggest that SN25b overexpression may inhibit the strength of transmission from SACs via PKA-mediated phosphorylation at T138. Moreover, knockdown of endogenous SN25b increased the frequency of wave-associated calcium transients, supporting the role of SN25 in restraining wave periodicity. Finally, the eye-specific segregation of retinogeniculate projection was impaired by in vivo overexpression of SN25b, but not SN25b-T138A, in SACs. These results suggest that SN25 in developing SACs dampens the spatiotemporal properties of retinal waves and limits visual circuit refinement by phosphorylation at T138. Therefore, SN25 in SACs plays a profound role in regulating visual circuit refinement.

Genetic Dissection of Presynaptic and Postsynaptic BDNF-TrkB Signaling in Synaptic Efficacy of CA3-CA1 Synapses.

Brain-derived neurotrophic factor (BDNF) and its high-affinity receptor, tropomyosin receptor kinase B (TrkB), regulate long-term potentiation (LTP) in the hippocampus, although the sites of BDNF-TrkB receptors in this process are controversial. We used a viral-mediated approach to delete BDNF or TrkB specifically in CA1 and CA3 regions of the Schaffer collateral pathway. Deletion of BDNF in CA3 or CA1 revealed that presynaptic BDNF is involved in LTP induction, while postsynaptic BDNF contributes to LTP maintenance. Similarly, loss of presynaptic or postsynaptic TrkB receptors leads to distinct LTP deficits, with presynaptic TrkB required to maintain LTP, while postsynaptic TrkB is essential for LTP formation. In addition, loss of TrkB in CA3 significantly diminishes release probability, uncovering a role for presynaptic TrkB receptors in basal neurotransmission. Taken together, this direct comparison of presynaptic and postsynaptic BDNF-TrkB reveals insight into BDNF release and TrkB activation sites in hippocampal LTP.

The Low-Threshold Calcium Channel Cav3.2 Mediates Burst Firing of Mature Dentate Granule Cells.

Mature granule cells are poorly excitable neurons that were recently shown to fire action potentials, preferentially in bursts. It is believed that the particularly pronounced short-term facilitation of mossy fiber synapses makes granule cell bursting a very effective means of properly transferring information to CA3. However, the mechanism underlying the unique bursting behavior of mature granule cells is currently unknown. Here, we show that Cav3.2 T-type channels at the axon initial segment are responsible for burst firing of mature granule cells in rats and mice. Accordingly, Cav3.2 knockout mice fire tonic spikes and exhibit impaired bursting, synaptic plasticity and dentate-to-CA3 communication. The data show that Cav3.2 channels are strong modulators of bursting and can be considered a critical molecular switch that enables effective information transfer from mature granule cells to the CA3 pyramids.

Social deficits in Shank3-deficient mouse models of autism are rescued by histone deacetylase (HDAC) inhibition.

Haploinsufficiency of the SHANK3 gene is causally linked to autism spectrum disorder (ASD), and ASD-associated genes are also enriched for chromatin remodelers. Here we found that brief treatment with romidepsin, a highly potent class I histone deacetylase (HDAC) inhibitor, alleviated social deficits in Shank3-deficient mice, which persisted for ~3 weeks. HDAC2 transcription was upregulated in these mice, and knockdown of HDAC2 in prefrontal cortex also rescued their social deficits. Nuclear localization of ß-catenin, a Shank3-binding protein that regulates cell adhesion and transcription, was increased in Shank3-deficient mice, which induced HDAC2 upregulation and social deficits. At the downstream molecular level, romidepsin treatment elevated the expression and histone acetylation of Grin2a and actin-regulatory genes and restored NMDA-receptor function and actin filaments in Shank3-deficient mice. Taken together, these findings highlight an epigenetic mechanism underlying social deficits linked to Shank3 deficiency, which may suggest potential therapeutic strategies for ASD patients bearing SHANK3 mutations.

Regulation of Dendritic Spine Morphology in Hippocampal Neurons by Copine-6.

Dendritic spines compartmentalize information in the brain, and their morphological characteristics are thought to underly synaptic plasticity. Here we identify copine-6 as a novel modulator of dendritic spine morphology. We found that brain-derived neurotrophic factor (BDNF) - a molecule essential for long-term potentiation of synaptic strength - upregulated and recruited copine-6 to dendritic spines in hippocampal neurons. Overexpression of copine-6 increased mushroom spine number and decreased filopodia number, while copine-6 knockdown had the opposite effect and dramatically increased the number of filopodia, which lacked PSD95. Functionally, manipulation of post-synaptic copine-6 levels affected miniature excitatory post-synaptic current (mEPSC) kinetics and evoked synaptic vesicle recycling in contacting boutons, and post-synaptic knockdown of copine-6 reduced hippocampal LTP and increased LTD. Mechanistically, copine-6 promotes BDNF-TrkB signaling and recycling of activated TrkB receptors back to the plasma membrane surface, and is necessary for BDNF-induced increases in mushroom spines in hippocampal neurons. Thus copine-6 regulates BDNF-dependent changes in dendritic spine morphology to promote synaptic plasticity.

ß-Catenin in the Adult Visual Cortex Regulates NMDA-Receptor Function and Visual Responses.

The formation, plasticity and maintenance of synaptic connections is regulated by molecular and electrical signals. ß-Catenin is an important protein in these events and regulates cadherin-mediated cell adhesion and the recruitment of pre- and postsynaptic proteins in an activity-dependent fashion. Mutations in the ß-catenin gene can cause cognitive disability and autism, with life-long consequences. Understanding its synaptic function may thus be relevant for the treatment of these disorders. So far, ß-catenin's function has been studied predominantly in cell culture and during development but knowledge on its function in adulthood is limited. Here, we show that ablating ß-catenin in excitatory neurons of the adult visual cortex does not cause the same synaptic deficits previously observed during development. Instead, it reduces NMDA-receptor currents and impairs visual processing. We conclude that ß-catenin remains important for adult cortical function but through different mechanisms than during development.

The L-type voltage-gated calcium channel CaV1.2 mediates fear extinction and modulates synaptic tone in the lateral amygdala.

L-type voltage-gated calcium channels (LVGCCs) have been implicated in both the formation and the reduction of fear through Pavlovian fear conditioning and extinction. Despite the implication of LVGCCs in fear learning and extinction, studies of the individual LVGCC subtypes, CaV1.2 and CaV1.3, using transgenic mice have failed to find a role of either subtype in fear extinction. This discontinuity between the pharmacological studies of LVGCCs and the studies investigating individual subtype contributions could be due to the limited neuronal deletion pattern of the CaV1.2 conditional knockout mice previously studied to excitatory neurons in the forebrain. To investigate the effects of deletion of CaV1.2 in all neuronal populations, we generated CaV1.2 conditional knockout mice using the synapsin1 promoter to drive Cre recombinase expression. Pan-neuronal deletion of CaV1.2 did not alter basal anxiety or fear learning. However, pan-neuronal deletion of CaV1.2 resulted in a significant deficit in extinction of contextual fear, implicating LVGCCs, specifically CaV1.2, in extinction learning. Further exploration on the effects of deletion of CaV1.2 on inhibitory and excitatory input onto the principle neurons of the lateral amygdala revealed a significant shift in inhibitory/excitatory balance. Together these data illustrate an important role of CaV1.2 in fear extinction and the synaptic regulation of activity within the amygdala.

Activation of RAGE/STAT3 pathway by methylglyoxal contributes to spinal central sensitization and persistent pain induced by bortezomib.

Bortezomib is a first-line chemotherapeutic drug widely used for multiple myeloma and other nonsolid malignancies. Although bortezomib-induced persistent pain is easily diagnosed in clinic, the pathogenic mechanism remains unclear. Here, we studied this issue with use of a rat model of systemic intraperitoneal administration of bortezomib for consecutive 5days. Consisted with our previous study, we found that bortezomib treatment markedly induced mechanical allodynia in rats. Furthermore, we first found that bortezomib treatment significantly induced the upregulation of methylglyoxal in spinal dorsal horn of rats. Spinal local application of methylglyoxal also induced mechanical allodynia and central sensitization in normal rats. Moreover, administration of bortezomib upregulated the expression of receptors for advanced glycation end products (RAGE) and phosphorylated STAT3 (p-STAT3) in dorsal horn. Importantly, intrathecal injection of metformin, a known scavenger of methylglyoxal, significantly attenuated the upregulation of methylglyoxal and RAGE in dorsal horn, central sensitization and mechanical allodynia induced by bortezomib treatment, and blockage of RAGE also prevented the upregulation of p-STAT3, central sensitization and mechanical allodynia induced by bortezomib treatment. In addition, inhibition of STAT3 activity by S3I-201 attenuated bortezomib-induced mechanical allodynia and central sensitization. Local knockdown of STAT3 also ameliorated the mechanical allodynia induced by bortezomib administration. Our results suggest that accumulation of methylglyoxal may activate the RAGE/STAT3 signaling pathway in dorsal horn, and contributes to the spinal central sensitization and persistent pain induced by bortezomib treatment.

Synaptotagmin-7-Mediated Asynchronous Release Boosts High-Fidelity Synchronous Transmission at a Central Synapse.

Synchronous release triggered by Ca2+ binding to synaptotagmin-1, -2, or -9 is thought to drive fast synaptic transmission, whereas asynchronous release induced by Ca2+ binding to synaptotagmin-7 is thought to produce delayed synaptic signaling, enabling prolonged synaptic computations. However, it is unknown whether synaptotagmin-7-dependent asynchronous release performs a physiological function at fast synapses lacking a prolonged signaling mode, such as the calyx of Held synapse. Here, we show at the calyx synapse that synaptotagmin-7-dependent asynchronous release indeed does not produce a prolonged synaptic signal after a stimulus train and does not contribute to short-term plasticity, but induces a steady-state, asynchronous postsynaptic current during stimulus trains. This steady-state postsynaptic current does not increase overall synaptic transmission but instead sustains reliable generation of postsynaptic spikes that are precisely time locked to presynaptic spikes. Thus, asynchronous release surprisingly functions, at least at some synapses, to sustain high-fidelity neurotransmission driven by synchronous release during high-frequency stimulus trains.

Neuron-astrocyte signaling is preserved in the aging brain.

Astrocytes play crucial roles in brain homeostasis and are emerging as regulatory elements of neuronal and synaptic physiology by responding to neurotransmitters with Ca2+ elevations and releasing gliotransmitters that activate neuronal receptors. Aging involves neuronal and astrocytic alterations, being considered risk factor for neurodegenerative diseases. Most evidence of the astrocyte-neuron signaling is derived from studies with young animals; however, the features of astrocyte-neuron signaling in adult and aging brain remain largely unknown. We have investigated the existence and properties of astrocyte-neuron signaling in physiologically and pathologically aging mouse hippocampal and cortical slices at different lifetime points (0.5 to 20 month-old animals). We found that astrocytes preserved their ability to express spontaneous and neurotransmitter-dependent intracellular Ca2+ signals from juvenile to aging brains. Likewise, resting levels of gliotransmission, assessed by neuronal NMDAR activation by glutamate released from astrocytes, were largely preserved with similar properties in all tested age groups, but DHPG-induced gliotransmission was reduced in aged mice. In contrast, gliotransmission was enhanced in the APP/PS1 mouse model of Alzheimer's disease, indicating a dysregulation of astrocyte-neuron signaling in pathological conditions. Disruption of the astrocytic IP3 R2 mediated-signaling, which is required for neurotransmitter-induced astrocyte Ca2+ signals and gliotransmission, boosted the progression of amyloid plaque deposits and synaptic plasticity impairments in APP/PS1 mice at early stages of the disease. Therefore, astrocyte-neuron interaction is a fundamental signaling, largely conserved in the adult and aging brain of healthy animals, but it is altered in Alzheimer's disease, suggesting that dysfunctions of astrocyte Ca2+ physiology may contribute to this neurodegenerative disease. GLIA 2017 GLIA 2017;65:569-580.

Presynaptic gain control by endogenous cotransmission of dopamine and GABA in the olfactory bulb.

In the olfactory bulb, lateral inhibition mediated by local juxtaglomerular interneurons has been proposed as a gain control mechanism, important for decorrelating odorant responses. Among juxtaglomerular interneurons, short axon cells are unique as dual-transmitter neurons that release dopamine and GABA. To examine their intraglomerular function, we expressed channelrhodopsin under control of the DAT-cre promoter and activated olfactory afferents within individual glomeruli. Optical stimulation of labeled cells triggered endogenous dopamine release as measured by cyclic voltammetry and GABA release as measured by whole cell GABAA receptor currents. Activation of short axon cells reduced the afferent presynaptic release probability via D2 and GABAB receptor activation, resulting in reduced spiking in both mitral and external tufted cells. Our results suggest that short axon cells influence glomerular activity not only by direct inhibition of external tufted cells but also by inhibition of afferent inputs to external tufted and mitral cells.NEW & NOTEWORTHY Sensory systems, including the olfactory system, encode information across a large dynamic range, making synaptic mechanisms of gain control critical to proper function. Here we demonstrate that a dual-transmitter interneuron in the olfactory bulb controls the gain of intraglomerular afferent input via two distinct mechanisms, presynaptic inhibition as well as inhibition of a principal neuron subtype, and thereby potently controls the synaptic gain of afferent inputs.

Alterations in hypoglossal motor neurons due to GAD67 and VGAT deficiency in mice.

There is an emerging body of evidence that glycinergic and GABAergic synaptic inputs onto motor neurons (MNs) help regulate the final number of MNs and axonal muscle innervation patterns. Using mutant glutamate decarboxylase 67 (GAD67) and vesicular inhibitory amino acid transporter (VGAT) deficient mice, we describe the effect that deficiencies of presynaptic GABAergic and/or glycinergic release have on the post-synaptic somato-dendritic structure of motor neurons, and the development of excitatory and inhibitory synaptic inputs to MNs. We use whole-cell patch clamp recording of synaptic currents in E18.5 hypoglossal MNs from brainstem slices, combined with dye-filling of these recorded cells with Neurobiotin™, high-resolution confocal imaging and 3-dimensional reconstructions. Hypoglossal MNs from GAD67- and VGAT-deficient mice display decreased inhibitory neurotransmission and increased excitatory synaptic inputs. These changes are associated with increased dendritic arbor length, increased complexity of dendritic branching, and increased density of spiny processes. Our results show that presynaptic release of inhibitory amino acid neurotransmitters are potent regulators of hypoglossal MN morphology and key regulators of synaptic inputs during this critical developmental time point.

Inhibitory Gating of Basolateral Amygdala Inputs to the Prefrontal Cortex.

UNLABELLED: Interactions between the prefrontal cortex (PFC) and basolateral amygdala (BLA) regulate emotional behaviors. However, a circuit-level understanding of functional connections between these brain regions remains incomplete. The BLA sends prominent glutamatergic projections to the PFC, but the overall influence of these inputs is predominantly inhibitory. Here we combine targeted recordings and optogenetics to examine the synaptic underpinnings of this inhibition in the mouse infralimbic PFC. We find that BLA inputs preferentially target layer 2 corticoamygdala over neighboring corticostriatal neurons. However, these inputs make even stronger connections onto neighboring parvalbumin and somatostatin expressing interneurons. Inhibitory connections from these two populations of interneurons are also much stronger onto corticoamygdala neurons. Consequently, BLA inputs are able to drive robust feedforward inhibition via two parallel interneuron pathways. Moreover, the contributions of these interneurons shift during repetitive activity, due to differences in short-term synaptic dynamics. Thus, parvalbumin interneurons are activated at the start of stimulus trains, whereas somatostatin interneuron activation builds during these trains. Together, these results reveal how the BLA impacts the PFC through a complex interplay of direct excitation and feedforward inhibition. They also highlight the roles of targeted connections onto multiple projection neurons and interneurons in this cortical circuit. Our findings provide a mechanistic understanding for how the BLA can influence the PFC circuit, with important implications for how this circuit participates in the regulation of emotion. SIGNIFICANCE STATEMENT: The prefrontal cortex (PFC) and basolateral amygdala (BLA) interact to control emotional behaviors. Here we show that BLA inputs elicit direct excitation and feedforward inhibition of layer 2 projection neurons in infralimbic PFC. BLA inputs are much stronger at corticoamygdala neurons compared with nearby corticostriatal neurons. However, these inputs are even more powerful at parvalbumin and somatostatin expressing interneurons. BLA inputs thus activate two parallel inhibitory networks, whose contributions change during repetitive activity. Finally, connections from these interneurons are also more powerful at corticoamygdala neurons compared with corticostriatal neurons. Together, our results demonstrate how the BLA predominantly inhibits the PFC via a complex sequence involving multiple cell-type and input-specific connections.

Neuroligins Are Selectively Essential for NMDAR Signaling in Cerebellar Stellate Interneurons.

UNLABELLED: Neuroligins are postsynaptic cell-adhesion molecules that contribute to synapse specification. However, many other postsynaptic cell-adhesion molecules are known and the relative contributions of neuroligins versus other such molecules in different types of synapses and neurons remains largely unknown. Here, we have studied the role of neuroligins in cerebellar stellate interneurons that participate in a well defined circuit that converges on Purkinje cells as the major output neurons of cerebellar cortex. By crossing triple conditional knock-out (cKO) mice targeting all three major neuroligins [neuroligin-1 to neuroligin-3 (NL123)] with parvalbumin-Cre (PV-Cre) transgenic mice, we deleted neuroligins from inhibitory cerebellar interneurons and Purkinje cells, allowing us to study the effects of neuroligin deletions on cerebellar stellate cell synapses by electrophysiology in acute slices. PV-Cre/NL123 cKO mice did not exhibit gross alterations of cerebellar structure or cerebellar interneuron morphology. Strikingly, electrophysiological recordings in stellate cells from these PV-Cre/NL123 cKO mice revealed a large decrease in NMDAR-mediated excitatory synaptic responses, which, in stellate cells, are largely extrasynaptic, without a change in AMPA-receptor-mediated responses. Parallel analyses in PV-Cre/NL1 mice that are single NL1 cKO mice uncovered the same phenotype, demonstrating that NL1 is responsible for recruiting extrasynaptic NMDARs. Moreover, we observed only a modest impairment in inhibitory synaptic responses in stellate cells lacking NL123 despite a nearly complete suppression of inhibitory synaptic transmission in Purkinje cells by the same genetic manipulation. Our results suggest that, unlike other types of neurons investigated, neuroligins are selectively essential in cerebellar stellate interneurons for enabling the function of extrasynaptic NMDARs. SIGNIFICANCE STATEMENT: Neuroligins are postsynaptic cell-adhesion molecules genetically linked to autism. However, the contributions of neuroligins to interneuron functions remain largely unknown. Here, we analyzed the role of neuroligins in cerebellar stellate interneurons. We deleted neuroligin-1, neuroligin-2, and neuroligin-3, the major cerebellar neuroligin isoforms, from stellate cells in triple NL123 conditional knock-out mice and analyzed synaptic responses by acute slice electrophysiology. We find that neuroligins are selectively essential for extrasynaptic NMDAR-mediated signaling, but dispensable for both AMPAR-mediated and inhibitory synaptic transmission. Our results reveal a critical and selective role for neuroligins in the regulation of NMDAR responses in cerebellar stellate interneurons.

Effects of hypotaurine on substantia gelatinosa neurons of the trigeminal subnucleus caudalis in immature mice.

To understand the action and mechanism of hypotaurine, an immediate precursor of taurine, on orofacial nociceptive processing, we examined the direct effects and receptor types involved in hypotaurine-induced responses using the whole-cell patch clamp technique in the substantia gelatinosa (SG) neurons of the trigeminal subnucleus caudalis (Vc) of immature mice. Under the condition of high-chloride pipette solution, hypotaurine elicited inward currents or upward deflections of membrane potential, which increased in a concentration-dependent manner (30-3000 µM) with the EC50 of 663.8 and 337.6 µM, respectively. The responses to 300 µM hypotaurine were reproducible and recovered upon washout. The 300 µM hypotaurine-induced currents were maintained in the presence of TTX, CNQX, and AP5, indicating direct postsynaptic action of hypotaurine on SG neurons. Responses to both low (300 µM) and high (1 or 3 mM) concentrations of hypotaurine were completely and reversibly blocked by the glycine receptor antagonist strychnine (2 µM), but unaffected by the GABAA receptor antagonist gabazine (3 µM) which blocks synaptic GABAA receptors at low concentration. Furthermore, responses to 300 µM hypotaurine and a maximal concentration of glycine (3 mM) were not additive, indicating that hypotaurine and glycine act on the same receptor. Hypotaurine-induced currents were partially antagonized by picrotoxin (50 µM) which blocks homomeric glycine receptors and by bicuculline (10 µM) which is an antagonist of α2 subunit-containing glycine receptors. These results suggest that hypotaurine-induced responses were mediated by glycine receptor activation in the SG neurons and hypotaurine might be used as an effective therapeutics for orofacial pain.