Early correction of synaptic long-term depression improves abnormal anxiety-like behavior in adult GluN2B-C456Y-mutant mice.

Extensive evidence links Glutamate receptor, ionotropic, NMDA2B (GRIN2B), encoding the GluN2B/NR2B subunit of N-methyl-D-aspartate receptors (NMDARs), with various neurodevelopmental disorders, including autism spectrum disorders (ASDs), but the underlying mechanisms remain unclear. In addition, it remains unknown whether mutations in GluN2B, which starts to be expressed early in development, induces early pathophysiology that can be corrected by early treatments for long-lasting effects. We generated and characterized Grin2b-mutant mice that carry a heterozygous, ASD-risk C456Y mutation (Grin2b+/C456Y). In Grin2b+/C456Y mice, GluN2B protein levels were strongly reduced in association with decreased hippocampal NMDAR currents and NMDAR-dependent long-term depression (LTD) but unaltered long-term potentiation, indicative of mutation-induced protein degradation and LTD sensitivity. Behaviorally, Grin2b+/C456Y mice showed normal social interaction but exhibited abnormal anxiolytic-like behavior. Importantly, early, but not late, treatment of young Grin2b+/C456Y mice with the NMDAR agonist D-cycloserine rescued NMDAR currents and LTD in juvenile mice and improved anxiolytic-like behavior in adult mice. Therefore, GluN2B-C456Y haploinsufficiency decreases GluN2B protein levels, NMDAR-dependent LTD, and anxiety-like behavior, and early activation of NMDAR function has long-lasting effects on adult mouse behavior.

Lrfn2-Mutant Mice Display Suppressed Synaptic Plasticity and Inhibitory Synapse Development and Abnormal Social Communication and Startle Response.

SALM1 (SALM (synaptic adhesion-like molecule), also known as LRFN2 (leucine rich repeat and fibronectin type III domain containing), is a postsynaptic density (PSD)-95-interacting synaptic adhesion molecule implicated in the regulation of NMDA receptor (NMDAR) clustering largely based on in vitro data, although its in vivo functions remain unclear. Here, we found that mice lacking SALM1/LRFN2 (Lrfn2-/- mice) show a normal density of excitatory synapses but altered excitatory synaptic function, including enhanced NMDAR-dependent synaptic transmission but suppressed NMDAR-dependent synaptic plasticity in the hippocampal CA1 region. Unexpectedly, SALM1 expression was detected in both glutamatergic and GABAergic neurons and Lrfn2-/- CA1 pyramidal neurons showed decreases in the density of inhibitory synapses and the frequency of spontaneous inhibitory synaptic transmission. Behaviorally, ultrasonic vocalization was suppressed in Lrfn2-/- pups separated from their mothers and acoustic startle was enhanced, but locomotion, anxiety-like behavior, social interaction, repetitive behaviors, and learning and memory were largely normal in adult male Lrfn2-/- mice. These results suggest that SALM1/LRFN2 regulates excitatory synapse function, inhibitory synapse development, and social communication and startle behaviors in mice.SIGNIFICANCE STATEMENT Synaptic adhesion molecules regulate synapse development and function, which govern neural circuit and brain functions. The SALM/LRFN (synaptic adhesion-like molecule/leucine rich repeat and fibronectin type III domain containing) family of synaptic adhesion proteins consists of five known members for which the in vivo functions are largely unknown. Here, we characterized mice lacking SALM1/LRFN2 (SALM1 KO) known to associate with NMDA receptors (NMDARs) and found that these mice showed altered NMDAR-dependent synaptic transmission and plasticity, as expected, but unexpectedly also exhibited suppressed inhibitory synapse development and synaptic transmission. Behaviorally, SALM1 KO pups showed suppressed ultrasonic vocalization upon separation from their mothers and SALM1 KO adults showed enhanced responses to loud acoustic stimuli. These results suggest that SALM1/LRFN2 regulates excitatory synapse function, inhibitory synapse development, social communication, and acoustic startle behavior.

Autistic-like social behaviour in Shank2-mutant mice improved by restoring NMDA receptor function.

Autism spectrum disorder (ASD) is a group of conditions characterized by impaired social interaction and communication, and restricted and repetitive behaviours. ASD is a highly heritable disorder involving various genetic determinants. Shank2 (also known as ProSAP1) is a multi-domain scaffolding protein and signalling adaptor enriched at excitatory neuronal synapses, and mutations in the human SHANK2 gene have recently been associated with ASD and intellectual disability. Although ASD-associated genes are being increasingly identified and studied using various approaches, including mouse genetics, further efforts are required to delineate important causal mechanisms with the potential for therapeutic application. Here we show that Shank2-mutant (Shank2(-/-)) mice carrying a mutation identical to the ASD-associated microdeletion in the human SHANK2 gene exhibit ASD-like behaviours including reduced social interaction, reduced social communication by ultrasonic vocalizations, and repetitive jumping. These mice show a marked decrease in NMDA (N-methyl-D-aspartate) glutamate receptor (NMDAR) function. Direct stimulation of NMDARs with D-cycloserine, a partial agonist of NMDARs, normalizes NMDAR function and improves social interaction in Shank2(-/-) mice. Furthermore, treatment of Shank2(-/-) mice with a positive allosteric modulator of metabotropic glutamate receptor 5 (mGluR5), which enhances NMDAR function via mGluR5 activation, also normalizes NMDAR function and markedly enhances social interaction. These results suggest that reduced NMDAR function may contribute to the development of ASD-like phenotypes in Shank2(-/-) mice, and mGluR modulation of NMDARs offers a potential strategy to treat ASD.

Cerebellar Shank2 Regulates Excitatory Synapse Density, Motor Coordination, and Specific Repetitive and Anxiety-Like Behaviors.

Shank2 is a multidomain scaffolding protein implicated in the structural and functional coordination of multiprotein complexes at excitatory postsynaptic sites as well as in psychiatric disorders, including autism spectrum disorders. While Shank2 is strongly expressed in the cerebellum, whether Shank2 regulates cerebellar excitatory synapses, or contributes to the behavioral abnormalities observed in Shank2-/- mice, remains unexplored. Here we show that Shank2-/- mice show reduced excitatory synapse density in cerebellar Purkinje cells in association with reduced levels of excitatory postsynaptic proteins, including GluD2 and PSD-93, and impaired motor coordination in the Erasmus test. Shank2 deletion restricted to Purkinje cells (Pcp2-Cre;Shank2fl/fl mice) leads to similar reductions in excitatory synapse density, synaptic protein levels, and motor coordination. Pcp2-Cre;Shank2fl/fl mice do not recapitulate autistic-like behaviors observed in Shank2-/- mice, such as social interaction deficits, altered ultrasonic vocalizations, repetitive behaviors, and hyperactivity. However, Pcp2-Cre;Shank2fl/fl mice display enhanced repetitive behavior in the hole-board test and anxiety-like behavior in the light-dark test, which are not observed in Shank2-/- mice. These results implicate Shank2 in the regulation of cerebellar excitatory synapse density, motor coordination, and specific repetitive and anxiety-like behaviors. SIGNIFICANCE STATEMENT: The postsynaptic side of excitatory synapses contains multiprotein complexes, termed the postsynaptic density, which contains receptors, scaffolding/adaptor proteins, and signaling molecules. Shank2 is an excitatory postsynaptic scaffolding protein implicated in the formation and functional coordination of the postsynaptic density and has been linked to autism spectrum disorders. Using Shank2-null mice and Shank2-conditional knock-out mice with a gene deletion restricted to cerebellar Purkinje cells, we explored functions of Shank2 in the cerebellum. We found that Shank2 regulates excitatory synapse density, motor coordination, and specific repetitive and anxiety-like behaviors, but is not associated with autistic-like social deficits or repetitive behaviors.

Synaptic connectivity of the TRPV1-positive trigeminal afferents in the rat lateral parabrachial nucleus.

Recent studies have shown a direct projection of nociceptive trigeminal afferents into the lateral parabrachial nucleus (LPBN). Information about the synaptic connectivity of these afferents may help understand how orofacial nociception is processed in the LPBN, which is known to be involved primarily in the affective aspect of pain. To address this issue, we investigated the synapses of the transient receptor potential vanilloid 1-positive (TRPV1+) trigeminal afferent terminals in the LPBN by immunostaining and serial section electron microscopy. TRPV1 + afferents arising from the ascending trigeminal tract issued axons and terminals (boutons) in the LPBN. TRPV1+ boutons formed synapses of asymmetric type with dendritic shafts and spines. Almost all (98.3%) TRPV1+ boutons formed synapses with one (82.6%) or two postsynaptic dendrites, suggesting that, at a single bouton level, the orofacial nociceptive information is predominantly transmitted to a single postsynaptic neuron with a small degree of synaptic divergence. A small fraction (14.9%) of the TRPV1+ boutons formed synapses with dendritic spines. None of the TRPV1+ boutons were involved in axoaxonic synapses. Conversely, in the trigeminal caudal nucleus (Vc), TRPV1+ boutons often formed synapses with multiple postsynaptic dendrites and were involved in axoaxonic synapses. Number of dendritic spine and total number of postsynaptic dendrites per TRPV1+ bouton were significantly fewer in the LPBN than Vc. Thus, the synaptic connectivity of the TRPV1+ boutons in the LPBN differed significantly from that in the Vc, suggesting that the TRPV1-mediated orofacial nociception is relayed to the LPBN in a distinctively different manner than in the Vc.

Increase of glutamate in satellite glial cells of the trigeminal ganglion in a rat model of craniofacial neuropathic pain.

Introduction: Satellite glial cells (SGCs) that envelop the cell bodies of neurons in sensory ganglia have been shown to both release glutamate, and be activated by glutamate in the context of nociceptive signaling. However, little is known about the subpopulations of SGCs that are activated following nerve injury and whether glutamate mechanisms in the SGCs are involved in the pathologic pain. Methods: To address this issue, we used light and electron microscopic immunohistochemistry to examine the change in the glutamate levels in the SGCs and the structural relationship between neighboring neurons in the trigeminal ganglion (TG) in a rat model of craniofacial neuropathic pain, CCI-ION. Results: Administration of ionomycin, ATP and Bz-ATP induced an increase of extracellular glutamate concentration in cultured trigeminal SGCs, indicating a release of glutamate from SGCs. The level of glutamate immunostaining in the SGCs that envelop neurons of all sizes in the TG was significantly higher in rats with CCI-ION than in control rats, suggesting that SGCs enveloping nociceptive as well as non-nociceptive mechanosensitive neurons are activated following nerve injury, and that the glutamate release from SGCs increases in pathologic pain state. Close appositions between substance-P (SP)-immunopositive (+) or calcitonin gene-related peptide (CGRP)+, likely nociceptive neurons, between Piezo1+, likely non-nociceptive, mechanosensitive neurons and SP+ or CGRP+ neurons, and between SGCs of neighboring neurons were frequently observed. Discussion: These findings suggest that glutamate in the trigeminal SGCs that envelop all types of neurons may play a role in the mechanisms of neuropathic pain, possibly via paracrine signaling.

Ultrastructure of Rat Rostral Nucleus of the Solitary Tract Terminals in the Parabrachial Nucleus and Medullary Reticular Formation.

Neurons in the rostral nucleus of the solitary tract (rNST) receive taste information from the tongue and relay it mainly to the parabrachial nucleus (PBN) and the medullary reticular formation (RF) through two functionally different neural circuits. To help understand how the information from the rNST neurons is transmitted within these brainstem relay nuclei in the taste pathway, we examined the terminals of the rNST neurons in the PBN and RF by use of anterograde horseradish peroxidase (HRP) labeling, postembedding immunogold staining for glutamate, serial section electron microscopy, and quantitative analysis. Most of the anterogradely labeled, glutamate-immunopositive axon terminals made a synaptic contact with only a single postsynaptic element in PBN and RF, suggesting that the sensory information from rNST neurons, at the individual terminal level, is not passed to multiple target cells. Labeled terminals were usually presynaptic to distal dendritic shafts in both target nuclei. However, the frequency of labeled terminals that contacted dendritic spines was significantly higher in the PBN than in the RF, and the frequency of labeled terminals that contacted somata or proximal dendrites was significantly higher in the RF than in the PBN. Labeled terminals receiving axoaxonic synapses, which are a morphological substrate for presynaptic modulation frequently found in primary sensory afferents, were not observed. These findings suggest that the sensory information from rNST neurons is processed in a relatively simple manner in both PBN and RF, but in a distinctly different manner in the PBN as opposed to the RF.

Expression of Piezo2 in the Dental Pulp, Sensory Root, and Trigeminal Ganglion and Its Coexpression with Vesicular Glutamate Transporters.

INTRODUCTION: Information on the type of vesicular glutamate transporter (VGLUT) that is expressed in the Piezo2-positive (Piezo2+) neurons in the trigeminal ganglion (TG) and on the type of Piezo2+ axons and their distribution in the dental pulp is important for understanding dental pain elicited by mechanical stimuli and developing new therapeutic strategies. METHODS: We examined the expression of Piezo2 and its coexpression with VGLUT1 and VGLUT2 in rat TG, the sensory root, and human dental pulp using light and electron microscopic immunohistochemistry and quantitative analysis. RESULTS: VGLUT1 and VGLUT2 were expressed in the TG neurons. Piezo2 was expressed in axons of all types but primarily in small myelinated (Aδ) axons in the sensory root. In the dental pulp, Piezo2 was expressed densely in the numerous axons that form a plexus in the peripheral pulp. Piezo2+ axons in the peripheral pulp were mostly unmyelinated, and Piezo2 immunoreactivity was often concentrated near the axolemma, suggesting that it may represent functional receptors. CONCLUSIONS: These findings suggest that VGLUT1 and VGLUT2 are involved in the glutamate signaling in Piezo2+ neurons, Piezo2 may be primarily activated by noxious mechanical stimuli, and Piezo2-mediated dental mechanotransduction may be primarily elicited in the peripheral pulp.

Expression of Piezo1 in the Trigeminal Neurons and in the Axons That Innervate the Dental Pulp.

Information on the neurons and axons that express the mechanosensitive channel Piezo1 and its expression in axons innervating the dental pulp may help understand the nature of the Piezo1-mediated mechanosensation and the underlying mechanism of dentin sensitivity elicited by mechanical stimuli. For this, we here investigated the neurochemical properties of the neurons in the rat trigeminal ganglion (TG) and their axons in its sensory root that express Piezo1 and the expression of Piezo1 in the rat and human dental pulp by light and electron microscopic immunohistochemistry and quantitative analysis. Piezo1 was expressed mainly in medium-sized and large TG neurons. Piezo1-immunopositive (+) neurons frequently coexpressed the marker for neurons with myelinated axons, NF200, but rarely the markers for neurons with unmyelinated axons, CGRP or IB4. In the sensory root of TG, Piezo1 was expressed primarily in small myelinated axons (Aδ, 60.2%) but also in large myelinated (Aß, 24.3%) and unmyelinated (C, 15.5%) axons. In the human dental pulp, Piezo1 was expressed in numerous NF200+ axons, which formed a network in the peripheral pulp and often "ascended" toward the dentin. Most Piezo1+ myelinated axons in the radicular pulp became unmyelinated in the peripheral pulp, where Piezo1 immunoreaction product was associated with the axonal plasma membrane, suggesting a functional role of Piezo1 in the peripheral pulp. These findings suggest that Piezo1 is involved primarily in mediating the acute pain elicited by high-threshold mechanical stimuli, and that the Piezo1-mediated dental mechanotransduction occurs primarily in the axons in the peripheral pulp.

A Deficiency of the Psychiatric Risk Gene DLG2/PSD-93 Causes Excitatory Synaptic Deficits in the Dorsolateral Striatum.

Genetic variations resulting in the loss of function of the discs large homologs (DLG2)/postsynaptic density protein-93 (PSD-93) gene have been implicated in the increased risk for schizophrenia, intellectual disability, and autism spectrum disorders (ASDs). Previously, we have reported that mice lacking exon 14 of the Dlg2 gene (Dlg2 -/- mice) display autistic-like behaviors, including social deficits and increased repetitive behaviors, as well as suppressed spontaneous excitatory postsynaptic currents in the striatum. However, the neural substrate underpinning such aberrant synaptic network activity remains unclear. Here, we found that the corticostriatal synaptic transmission was significantly impaired in Dlg2 -/- mice, which did not seem attributed to defects in presynaptic releases of cortical neurons, but to the reduced number of functional synapses in the striatum, as manifested in the suppressed frequency of miniature excitatory postsynaptic currents in spiny projection neurons (SPNs). Using transmission electron microscopy, we found that both the density of postsynaptic densities and the fraction of perforated synapses were significantly decreased in the Dlg2 -/- dorsolateral striatum. The density of dendritic spines was significantly reduced in striatal SPNs, but notably, not in the cortical pyramidal neurons of Dlg2 -/- mice. Furthermore, a DLG2/PSD-93 deficiency resulted in the compensatory increases of DLG4/PSD-95 and decreases in the expression of TrkA in the striatum, but not particularly in the cortex. These results suggest that striatal dysfunction might play a role in the pathology of psychiatric disorders that are associated with a disruption of the Dlg2 gene.

Regulation of dendritic spines, spatial memory, and embryonic development by the TANC family of PSD-95-interacting proteins.

PSD-95 (postsynaptic density-95) is thought to play important roles in the regulation of dendritic spines and excitatory synapses, but the underlying mechanisms have not been fully elucidated. TANC1 is a PSD-95-interacting synaptic protein that contains multiple domains for protein-protein interactions but whose function is not well understood. In the present study, we provide evidence that TANC1 and its close relative TANC2 regulate dendritic spines and excitatory synapses. Overexpression of TANC1 and TANC2 in cultured neurons increases the density of dendritic spines and excitatory synapses in a manner that requires the PDZ (PSD-95/Dlg/ZO-1)-binding C termini of TANC proteins. TANC1-deficient mice exhibit reduced spine density in the CA3 region of the hippocampus, but not in the CA1 or dentate gyrus regions, and show impaired spatial memory. TANC2 deficiency, however, causes embryonic lethality. These results suggest that TANC1 is important for dendritic spine maintenance and spatial memory, and implicate TANC2 in embryonic development.

Regulation of synaptic Rac1 activity, long-term potentiation maintenance, and learning and memory by BCR and ABR Rac GTPase-activating proteins.

Rho family small GTPases are important regulators of neuronal development. Defective Rho regulation causes nervous system dysfunctions including mental retardation and Alzheimer's disease. Rac1, a member of the Rho family, regulates dendritic spines and excitatory synapses, but relatively little is known about how synaptic Rac1 is negatively regulated. Breakpoint cluster region (BCR) is a Rac GTPase-activating protein known to form a fusion protein with the c-Abl tyrosine kinase in Philadelphia chromosome-positive chronic myelogenous leukemia. Despite the fact that BCR mRNAs are abundantly expressed in the brain, the neural functions of BCR protein have remained obscure. We report here that BCR and its close relative active BCR-related (ABR) localize at excitatory synapses and directly interact with PSD-95, an abundant postsynaptic scaffolding protein. Mice deficient for BCR or ABR show enhanced basal Rac1 activity but only a small increase in spine density. Importantly, mice lacking BCR or ABR exhibit a marked decrease in the maintenance, but not induction, of long-term potentiation, and show impaired spatial and object recognition memory. These results suggest that BCR and ABR have novel roles in the regulation of synaptic Rac1 signaling, synaptic plasticity, and learning and memory, and that excessive Rac1 activity negatively affects synaptic and cognitive functions.

Ultrastructural analysis of glutamate-, GABA-, and glycine-immunopositive boutons from supratrigeminal premotoneurons in the rat trigeminal motor nucleus.

The supratrigeminal region (Vsup) is important for coordination of smooth jaw movement. However, little is known about the synaptic connections of the Vsup premotoneurons with the trigeminal motor neurons. In the present study, we examined axon terminals of Vsup premotoneurons in the contralateral trigeminal motor nucleus (Vmo) by a combination of anterograde tracing with cholera toxin B-horseradish peroxidase (CTB-HRP), postembedding immunohistochemistry for the amino acid transmitters glutamate, GABA, and glycine, and electron microscopy. Tracer injections resulted in anterograde labeling of axon terminals of the Vsup premotoneurons in the motor trigeminal nucleus (Vmo). The labeled boutons in Vmo exhibited immunoreactivity for glutamate, GABA, or glycine: glutamate-immunopositive boutons (69%) were more frequently observed than GABA- or glycine-immunopositive boutons (19% and 12%, respectively). Although most labeled boutons (97%) made synaptic contacts with a single postsynaptic dendrite, a few glutamate-immunopositive boutons (3%) showed synaptic contact with two dendrites. No labeled boutons participated in axoaxonic synaptic contacts. Most labeled boutons (78%) were presynaptic to dendritic shafts, and the remaining 22% were presynaptic to somata or primary dendrites. A large proportion of GABA- or glycine-immunopositive boutons (40%) were presynaptic to somata or primary dendrites, whereas most glutamate-immunopositive boutons (86%) were presynaptic to dendritic shafts. These results indicate that axon terminals of Vsup premotoneurons show simple synaptic connection with Vmo neurons. This may provide the anatomical basis for the neural information processing responsible for jaw movement control.

Synaptic connectivity of urinary bladder afferents in the rat superficial dorsal horn and spinal parasympathetic nucleus.

That visceral sensory afferents are functionally distinct from their somatic analogues has been known for a long time but the detailed knowledge of their synaptic connections and neurotransmitters at the first relay nucleus in the spinal cord has been limited. To provide information on these topics, we investigated the synapses and neurotransmitters of identified afferents from the urinary bladder to the superficial laminae of the rat spinal dorsal horn (DH) and the spinal parasympathetic nucleus (SPN) by tracing with horseradish peroxidase, quantitative electron microscopical analysis, and immunogold staining for GABA and glycine. In the DH, most bladder afferent boutons formed synapses with 1-2 postsynaptic dendrites, whereas in the SPN, close to a half of them formed synapses with 3-8 postsynaptic dendrites. The number of postsynaptic dendrites and dendritic spines per bladder afferent bouton, both measures of synaptic divergence and of potential for synaptic plasticity at a single bouton level, were significantly higher in the SPN than in the DH. Bladder afferent boutons frequently received inhibitory axoaxonic synapses from presynaptic endings in the DH but rarely in the SPN. The presynaptic endings were GABA- and/or glycine-immunopositive. The bouton volume, mitochondrial volume, and active zone area, all determinants of synaptic strength, of the bladder afferent boutons were positively correlated with the number of postsynaptic dendrites. These findings suggest that visceral sensory information conveyed via the urinary bladder afferents is processed differently in the DH than in the SPN, and differently from the way somatosensory information is processed in the spinal cord.

Expression of P2X3 receptor in the trigeminal sensory nuclei of the rat.

Trigeminal primary afferents expressing P2X(3) receptor are involved in the transmission of orofacial nociceptive information. However, little is known about their central projection pattern and ultrastructural features within the trigeminal brainstem sensory nuclei (TBSN). Here we use multiple immunofluorescence and electron microscopy to characterize the P2X(3)-immunopositive (+) neurons in the trigeminal ganglion and describe the distribution and synaptic organization of their central terminals within the rat TBSN, including nuclei principalis (Vp), oralis (Vo), interpolaris (Vi), and caudalis (Vc). In the trigeminal ganglion, P2X(3) immunoreactivity was mainly in small and medium-sized somata, but also frequently in large somata. Although most P2X(3) (+) somata costained for the nonpeptidergic marker IB4, few costained for the peptidergic marker substance P. Most P2X(3) (+) fibers in the sensory root of trigeminal ganglion (92.9%) were unmyelinated, whereas the rest were small myelinated. In the TBSN, P2X(3) immunoreactivity was dispersed in the rostral TBSN but was dense in the superficial laminae of Vc, especially in the inner lamina II. The P2X(3) (+) terminals contained numerous clear, round vesicles and sparse large, dense-core vesicles. Typically, they were presynaptic to one or two dendritic shafts and also frequently postsynaptic to axonal endings, containing pleomorphic vesicles. Such P2X(3) (+) terminals, showing glomerular shape and complex synaptic relationships, and those exhibiting axoaxonic contacts, were more frequently seen in Vp than in any other TBSN. These results suggest that orofacial nociceptive information may be transmitted via P2X(3) (+) afferents to all TBSN and that it may be processed differently in different TBSN.

Extrasynaptic homomeric glycine receptors in neurons of the rat trigeminal mesencephalic nucleus.

The neurons in the trigeminal mesencephalic nucleus (Vmes) innervate jaw-closing muscle spindles and periodontal ligaments, and play a crucial role in the regulation of jaw movements. Recently, it was shown that many boutons that form synapses on them are immunopositive for glycine (Gly+), suggesting that these neurons receive glycinergic input. Information about the glycine receptors that mediate this input is needed to help understand the role of glycine in controlling Vmes neuron excitability. For this, we investigated the expression of glycine receptor subunit alpha 3 (GlyRα3) and gephyrin in neurons in Vmes and the trigeminal motor nucleus (Vmo), and the Gly+ boutons that contact them by light- and electron-microscopic immunocytochemistry and quantitative ultrastructural analysis. The somata of the Vmes neurons were immunostained for GlyRα3, but not gephyrin, indicating expression of homomeric GlyR. The immunostaining for GlyRα3 was localized away from the synapses in the Vmes neuron somata, in contrast to the Vmo neurons, where the staining for GlyRα3 and gephyrin were localized at the subsynaptic zones in somata and dendrites. Additionally, the ultrastructural determinants of synaptic strength, bouton volume, mitochondrial volume, and active zone area, were significantly smaller in Gly+ boutons on the Vmes neurons than in those on the Vmo neurons. These findings support the notion that the Vmes neurons receive glycinergic input via putative extrasynaptic homomeric glycine receptors, likely mediating a slow, tonic modulation of the Vmes neuron excitability.

NGL-2 Deletion Leads to Autistic-like Behaviors Responsive to NMDAR Modulation.

Netrin-G ligand 2 (NGL-2)/LRRC4, implicated in autism spectrum disorders and schizophrenia, is a leucine-rich repeat-containing postsynaptic adhesion molecule that interacts intracellularly with the excitatory postsynaptic scaffolding protein PSD-95 and trans-synaptically with the presynaptic adhesion molecule netrin-G2. Functionally, NGL-2 regulates excitatory synapse development and synaptic transmission. However, whether it regulates synaptic plasticity and disease-related specific behaviors is not known. Here, we report that mice lacking NGL-2 (Lrrc4-/- mice) show suppressed N-Methyl-D-aspartate receptor (NMDAR)-dependent synaptic plasticity in the hippocampus. NGL-2 associates with NMDARs through both PSD-95-dependent and -independent mechanisms. Moreover, Lrrc4-/- mice display mild social interaction deficits and repetitive behaviors that are rapidly improved by pharmacological NMDAR activation. These results suggest that NGL-2 promotes synaptic stabilization of NMDARs, regulates NMDAR-dependent synaptic plasticity, and prevents autistic-like behaviors from developing in mice, supporting the hypothesis that NMDAR dysfunction contributes to autism spectrum disorders.

Increased Excitatory Synaptic Transmission of Dentate Granule Neurons in Mice Lacking PSD-95-Interacting Adhesion Molecule Neph2/Kirrel3 during the Early Postnatal Period.

Copy number variants and point mutations of NEPH2 (also called KIRREL3) gene encoding an immunoglobulin (Ig) superfamily adhesion molecule have been linked to autism spectrum disorders, intellectual disability and neurocognitive delay associated with Jacobsen syndrome, but the physiological roles of Neph2 in the mammalian brain remain largely unknown. Neph2 is highly expressed in the dentate granule (DG) neurons of the hippocampus and is localized in both dendrites and axons. It was recently shown that Neph2 is required for the formation of mossy fiber filopodia, the axon terminal structure of DG neurons forming synapses with GABAergic neurons of CA3. In contrast, however, it is unknown whether Neph2 also has any roles in the postsynaptic compartments of DG neurons. We here report that, through its C-terminal PDZ domain-binding motif, Neph2 directly interacts with postsynaptic density (PSD)-95, an abundant excitatory postsynaptic scaffolding protein. Moreover, Neph2 protein is detected in the brain PSD fraction and interacts with PSD-95 in synaptosomal lysates. Functionally, loss of Neph2 in mice leads to age-specific defects in the synaptic connectivity of DG neurons. Specifically, Neph2-/- mice show significantly increased spontaneous excitatory synaptic events in DG neurons at postnatal week 2 when the endogenous Neph2 protein expression peaks, but show normal excitatory synaptic transmission at postnatal week 3. The evoked excitatory synaptic transmission and synaptic plasticity of medial perforant pathway (MPP)-DG synapses are also normal in Neph2-/- mice at postnatal week 3, further confirming the age-specific synaptic defects. Together, our results provide some evidence for the postsynaptic function of Neph2 in DG neurons during the early postnatal period, which might be implicated in neurodevelopmental and cognitive disorders caused by NEPH2 mutations.

Rat odontoblasts may use glutamate to signal dentin injury.

Accumulating evidence indicates that odontoblasts act as sensor cells, capable of triggering action potentials in adjacent pulpal nociceptive axons, suggesting a paracrine signaling via a currently unknown mediator. Since glutamate can mediate signaling by non-neuronal cells, and peripheral axons may express glutamate receptors (GluR), we hypothesized that the expression of high levels of glutamate, and of sensory receptors in odontoblasts, combined with an expression of GluR in adjacent pulpal axons, is the morphological basis for odontoblastic sensory signaling. To test this hypothesis, we investigated the expression of glutamate, the thermo- and mechanosensitive ion channels transient receptor potential vanilloid 1 (TRPV1), transient receptor potential ankyrin 1 (TRPA1), and TWIK-1-related K+channel (TREK-1), and the glutamate receptor mGluR5, in a normal rat dental pulp, and following dentin injury. We also examined the glutamate release from odontoblast in cell culture. Odontoblasts were enriched with glutamate, at the level as high as in adjacent pulpal axons, and showed immunoreactivity for TRPV1, TRPA1, and TREK-1. Pulpal sensory axons adjacent to odontoblasts expressed mGluR5. Both the levels of glutamate in odontoblasts, and the expression of mGluR5 in nearby axons, were upregulated following dentin injury. The extracellular glutamate concentration was increased significantly after treating of odontoblast cell line with calcium permeable ionophore, suggesting glutamate release from odontoblasts. These findings lend morphological support to the hypothesis that odontoblasts contain glutamate as a potential neuroactive substance that may activate adjacent pulpal axons, and thus contribute to dental pain and hypersensitivity.

LRRTM3 Regulates Excitatory Synapse Development through Alternative Splicing and Neurexin Binding.

The four members of the LRRTM family (LRRTM1-4) are postsynaptic adhesion molecules essential for excitatory synapse development. They have also been implicated in neuropsychiatric diseases. Here, we focus on LRRTM3, showing that two distinct LRRTM3 variants generated by alternative splicing regulate LRRTM3 interaction with PSD-95, but not its excitatory synapse-promoting activity. Overexpression of either LRRTM3 variant increased excitatory synapse density in dentate gyrus (DG) granule neurons, whereas LRRTM3 knockdown decreased it. LRRTM3 also controlled activity-regulated AMPA receptor surface expression in an alternative splicing-dependent manner. Furthermore, Lrrtm3-knockout mice displayed specific alterations in excitatory synapse density, excitatory synaptic transmission and excitability in DG granule neurons but not in CA1 pyramidal neurons. Lastly, LRRTM3 required only specific splice variants of presynaptic neurexins for their synaptogenic activity. Collectively, our data highlight alternative splicing and differential presynaptic ligand utilization in the regulation of LRRTMs, revealing key regulatory mechanisms for excitatory synapse development.