CalDAG-GEFI mediates striatal cholinergic modulation of dendritic excitability, synaptic plasticity and psychomotor behaviors.

CalDAG-GEFI (CDGI) is a protein highly enriched in the striatum, particularly in the principal spiny projection neurons (SPNs). CDGI is strongly down-regulated in two hyperkinetic conditions related to striatal dysfunction: Huntington's disease and levodopa-induced dyskinesia in Parkinson's disease. We demonstrate that genetic deletion of CDGI in mice disrupts dendritic, but not somatic, M1 muscarinic receptors (M1Rs) signaling in indirect pathway SPNs. Loss of CDGI reduced temporal integration of excitatory postsynaptic potentials at dendritic glutamatergic synapses and impaired the induction of activity-dependent long-term potentiation. CDGI deletion selectively increased psychostimulant-induced repetitive behaviors, disrupted sequence learning, and eliminated M1R blockade of cocaine self-administration. These findings place CDGI as a major, but previously unrecognized, mediator of cholinergic signaling in the striatum. The effects of CDGI deletion on the self-administration of drugs of abuse and its marked alterations in hyperkinetic extrapyramidal disorders highlight CDGI's therapeutic potential.

Dysregulated clock gene expression and abnormal diurnal regulation of hippocampal inhibitory transmission and spatial memory in amyloid precursor protein transgenic mice.

Patients with Alzheimer's disease (AD) often have fragmentation of sleep/wake cycles and disrupted 24-h (circadian) activity. Despite this, little work has investigated the potential underlying day/night disruptions in cognition and neuronal physiology in the hippocampus. The molecular clock, an intrinsic transcription-translation feedback loop that regulates circadian behavior, may also regulate hippocampal neurophysiological activity. We hypothesized that disrupted diurnal variation in clock gene expression in the hippocampus corresponds with loss of normal day/night differences in membrane excitability, synaptic physiology, and cognition. We previously reported disrupted circadian locomotor rhythms and neurophysiological output of the suprachiasmatic nucleus (the primary circadian clock) in Tg-SwDI mice with human amyloid-beta precursor protein mutations. Here, we report that Tg-SwDI mice failed to show day/night differences in a spatial working memory task, unlike wild-type controls that exhibited enhanced spatial working memory at night. Moreover, Tg-SwDI mice had lower levels of Per2, one of the core components of the molecular clock, at both mRNA and protein levels when compared to age-matched controls. Interestingly, we discovered neurophysiological impairments in area CA1 of the Tg-SwDI hippocampus. In controls, spontaneous inhibitory post-synaptic currents (sIPSCs) in pyramidal cells showed greater amplitude and lower inter-event interval during the day than the night. However, the normal day/night differences in sIPSCs were absent (amplitude) or reversed (inter-event interval) in pyramidal cells from Tg-SwDI mice. In control mice, current injection into CA1 pyramidal cells produced more firing during the night than during the day, but no day/night difference in excitability was observed in Tg-SwDI mice. The normal day/night difference in excitability in controls was blocked by GABA receptor inhibition. Together, these results demonstrate that the normal diurnal regulation of inhibitory transmission in the hippocampus is diminished in a mouse model of AD, leading to decreased daytime inhibition onto hippocampal CA1 pyramidal cells. Uncovering disrupted day/night differences in circadian gene regulation, hippocampal physiology, and memory in AD mouse models may provide insight into possible chronotherapeutic strategies to ameliorate Alzheimer's disease symptoms or delay pathological onset.

Regulation and dysregulation of neuronal circuits by KARs.

Kainate receptors (KARs) constitute a family of ionotropic glutamate receptors (iGluRs) with distinct physiological roles in synapses and neuronal circuits. Despite structural and biophysical commonalities with the other iGluRs, AMPA receptors and NMDA receptors, their role as post-synaptic receptors involved in shaping EPSCs to transmit signals across synapses is limited to a small number of synapses. On the other hand KARs regulate presynaptic release mechanisms and control ion channels and signaling pathways through non-canonical metabotropic actions. We review how these different KAR-dependent mechanisms concur to regulate the activity and plasticity of neuronal circuits in physiological conditions of activation of KARs by endogenous glutamate (as opposed to pharmacological activation by exogenous agonists). KARs have been implicated in neurological disorders, based on genetic association and on physiopathological studies. A well described example relates to temporal lobe epilepsy for which the aberrant recruitment of KARs at recurrent mossy fiber synapses takes part in epileptogenic neuronal activity. In conclusion, KARs certainly represent an underestimated actor in the regulation of neuronal circuits, and a potential therapeutic target awaiting more selective and efficient genetic tools and/or ligands. This article is part of the special Issue on 'Glutamate Receptors - Kainate receptors'.

Decreased excitatory drive onto hilar neuronal nitric oxide synthase expressing interneurons in chronic models of epilepsy.

Excitation-inhibition imbalance of GABAergic interneurons is predisposed to develop chronic temporal lobe epilepsy (TLE). We have previously shown that virtually every neuronal nitric oxide synthase (nNOS)-positive cell is a GABAergic inhibitory interneuron in the denate gyrus. The present study was designed to quantify the number of nNOS-containing hilar interneurons using stereology in pilocapine- and kainic acid (KA)-exposed transgenic adult mice that expressed GFP under the nNOS promoter. In addition, we studied the properties of miniature excitatory postsynaptic current (mEPSC) and paired-pulse response ratio (PPR) of evoked EPSC in nNOS interneurons using whole cell recording techniques. Results showed that there were fewer nNOS-immunoreactive interneurons of chronically epileptic animals. Importantly, patch-clamp recordings revealed reduction in mEPSC frequency, indicating diminished global excitatory input. In contrast, PPR of evoked EPSC following the granule cell layer stimulation was increased in epileptic animals suggesting reduced neurotransmitter release from granule cell input. In summary, we propose that impaired excitatory drive onto hippocampal nNOS interneurons may be implicated in the development of refractory epilepsy.

Decreased neuronal synaptosome associated protein 29 contributes to poststroke cognitive impairment by disrupting presynaptic maintenance.

Background: Poststroke cognitive impairments are common in stroke survivors, and pose a high risk of incident dementia. However, the cause of these cognitive impairments is obscure and required an investigation. Methods: Oxygen-glucose deprivation (OGD) model and middle cerebral artery occlusion (MCAO) model were used to imitate in vitro or in vivo acute cerebral ischemia, respectively. The differentially expressed synaptosome associated protein 29 (SNAP29)-interacting proteins upon ischemia and reperfusion were analyzed with bioinformatics analysis and the results indicated that the changes of SNAP29 after acute ischemia were mainly involved in the synaptic functions. The outcomes of SNAP29 reduction were assessed with SNAP29 knockdown, which mimicked the distribution of SNAP29 along neuronal processes after acute ischemia. Using the whole-cell patch clamp recording method and transmission electron microscope, the pre-synaptic function and readily releasable pool (RRP) were observed after SNAP29 knock down. Using photogenetic manipulations and behavioral tests, the neuronal projection and cognitive functions of mice with SNAP29 knock down in hippocampus CA1 region were evaluated. Results: It was found that SNAP29 protein levels decreased in both in vitro and in vivo ischemic models. Further, the SNAP29 reduction wasn't associated with impaired autophagy flux and neuronal survival. When SNAP29 was knocked down in primary cortical neurons, the frequency of AMPARs-mediated mEPSCs, but not the amplitude, significantly decreased. Meanwhile, the mice with SNAP29 knockdown at CA1 region of hippocampus developed an impairment in hippocampus-mPFC (middle prefrontal cortex) circuit and behavioral dysfunctions. Moreover, the size of RRP at presynaptic sites was diminished. Conclusion: Since SNAP29 protein levels didn't significantly influence the neuronal survival and its decrease was sufficient to disturb the neural circuit via a presynaptic manner, the SNAP29-associated strategies may be an efficient target against poststroke synaptic dysfunction and cognitive deficits.

The amino-terminal domain of GluA1 mediates LTP maintenance via interaction with neuroplastin-65.

Long-term potentiation (LTP) has long been considered as an important cellular mechanism for learning and memory. LTP expression involves NMDA receptor-dependent synaptic insertion of AMPA receptors (AMPARs). However, how AMPARs are recruited and anchored at the postsynaptic membrane during LTP remains largely unknown. In this study, using CRISPR/Cas9 to delete the endogenous AMPARs and replace them with the mutant forms in single neurons, we have found that the amino-terminal domain (ATD) of GluA1 is required for LTP maintenance. Moreover, we show that GluA1 ATD directly interacts with the cell adhesion molecule neuroplastin-65 (Np65). Neurons lacking Np65 exhibit severely impaired LTP maintenance, and Np65 deletion prevents GluA1 from rescuing LTP in AMPARs-deleted neurons. Thus, our study reveals an essential role for GluA1/Np65 binding in anchoring AMPARs at the postsynaptic membrane during LTP.

Orexin-A differentially modulates inhibitory and excitatory synaptic transmission in rat inner retina.

In this work, modulation by orexin-A of the release of glutamate and GABA from bipolar and amacrine cells respectively was studied by examining the effects of the neuropeptide on miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs) of rat retinal ganglion cells (GCs). Using RNAscope in situ hybridization in combination with immunohistochemistry, we showed positive signals for orexin receptor-1 (OX1R) mRNA in the bipolar cell terminals and those for orexin receptor-2 (OX2R) mRNA in the amacrine cell terminals. With whole-cell patch-clamp recordings in rat retinal slices, we demonstrated that application of orexin-A reduced the interevent interval of mEPSCs of GCs through OX1R. However, it increased the interevent interval of mIPSCs, mediated by GABAA receptors, through OX2R. Furthermore, orexin-A-induced reduction of mEPSC interevent interval was abolished by the application of PI-PLC inhibitors or PKC inhibitors. In contrast, orexin-A-induced increase of GABAergic mIPSC interevent interval was mimicked by 8-Br-cAMP or an adenylyl cyclase activator, but was eliminated by PKA antagonists. Finally, application of nimodipine, an L-type Ca2+ channel blocker, increased both mEPSC and mIPSC interevent interval, and co-application of orexin-A no longer changed the mEPSCs and mIPSCs. We conclude that orexin-A increases presynaptic glutamate release onto GCs by activating L-type Ca2+ channels in bipolar cells, a process that is mediated by an OX1R/PI-PLC/PKC signaling pathway. However, orexin-A decreases presynaptic GABA release onto GCs by inhibiting L-type Ca2+ channels in amacrine cells, a process that is mediated by an OX2R/cAMP-PKA signaling pathway.

Structural and Functional Synaptic Plasticity Induced by Convergent Synapse Loss in the Drosophila Neuromuscular Circuit.

Throughout the nervous system, the convergence of two or more presynaptic inputs on a target cell is commonly observed. The question we ask here is to what extent converging inputs influence each other's structural and functional synaptic plasticity. In complex circuits, isolating individual inputs is difficult because postsynaptic cells can receive thousands of inputs. An ideal model to address this question is the Drosophila larval neuromuscular junction (NMJ) where each postsynaptic muscle cell receives inputs from two glutamatergic types of motor neurons (MNs), known as 1b and 1s MNs. Notably, each muscle is unique and receives input from a different combination of 1b and 1s MNs; we surveyed multiple muscles for this reason. Here, we identified a cell-specific promoter that allows ablation of 1s MNs postinnervation and measured structural and functional responses of convergent 1b NMJs using microscopy and electrophysiology. For all muscles examined in both sexes, ablation of 1s MNs resulted in NMJ expansion and increased spontaneous neurotransmitter release at corresponding 1b NMJs. This demonstrates that 1b NMJs can compensate for the loss of convergent 1s MNs. However, only a subset of 1b NMJs showed compensatory evoked neurotransmission, suggesting target-specific plasticity. Silencing 1s MNs led to similar plasticity at 1b NMJs, suggesting that evoked neurotransmission from 1s MNs contributes to 1b synaptic plasticity. Finally, we genetically blocked 1s innervation in male larvae and robust 1b synaptic plasticity was eliminated, raising the possibility that 1s NMJ formation is required to set up a reference for subsequent synaptic perturbations.SIGNIFICANCE STATEMENT In complex neural circuits, multiple convergent inputs contribute to the activity of the target cell, but whether synaptic plasticity exists among these inputs has not been thoroughly explored. In this study, we examined synaptic plasticity in the structurally and functionally tractable Drosophila larval neuromuscular system. In this convergent circuit, each muscle is innervated by a unique pair of motor neurons. Removal of one neuron after innervation causes the adjacent neuron to increase neuromuscular junction outgrowth and functional output. However, this is not a general feature as each motor neuron differentially compensates. Further, robust compensation requires initial coinnervation by both neurons. Understanding how neurons respond to perturbations in adjacent neurons will provide insight into nervous system plasticity in both healthy and disease states.

SYNGAP1 Controls the Maturation of Dendrites, Synaptic Function, and Network Activity in Developing Human Neurons.

SYNGAP1 is a major genetic risk factor for global developmental delay, autism spectrum disorder, and epileptic encephalopathy. De novo loss-of-function variants in this gene cause a neurodevelopmental disorder defined by cognitive impairment, social-communication disorder, and early-onset seizures. Cell biological studies in mouse and rat neurons have shown that Syngap1 regulates developing excitatory synapse structure and function, with loss-of-function variants driving formation of larger dendritic spines and stronger glutamatergic transmission. However, studies to date have been limited to mouse and rat neurons. Therefore, it remains unknown how SYNGAP1 loss of function impacts the development and function of human neurons. To address this, we used CRISPR/Cas9 technology to ablate SYNGAP1 protein expression in neurons derived from a commercially available induced pluripotent stem cell line (hiPSC) obtained from a human female donor. Reducing SynGAP protein expression in developing hiPSC-derived neurons enhanced dendritic morphogenesis, leading to larger neurons compared with those derived from isogenic controls. Consistent with larger dendritic fields, we also observed a greater number of morphologically defined excitatory synapses in cultures containing these neurons. Moreover, neurons with reduced SynGAP protein had stronger excitatory synapses and expressed synaptic activity earlier in development. Finally, distributed network spiking activity appeared earlier, was substantially elevated, and exhibited greater bursting behavior in SYNGAP1 null neurons. We conclude that SYNGAP1 regulates the postmitotic maturation of human neurons made from hiPSCs, which influences how activity develops within nascent neural networks. Alterations to this fundamental neurodevelopmental process may contribute to the etiology of SYNGAP1-related disorders.SIGNIFICANCE STATEMENTSYNGAP1 is a major genetic risk factor for global developmental delay, autism spectrum disorder, and epileptic encephalopathy. While this gene is well studied in rodent neurons, its function in human neurons remains unknown. We used CRISPR/Cas9 technology to disrupt SYNGAP1 protein expression in neurons derived from an induced pluripotent stem cell line. We found that induced neurons lacking SynGAP expression exhibited accelerated dendritic morphogenesis, increased accumulation of postsynaptic markers, early expression of synapse activity, enhanced excitatory synaptic strength, and early onset of neural network activity. We conclude that SYNGAP1 regulates the postmitotic differentiation rate of developing human neurons and disrupting this process impacts the function of nascent neural networks. These altered developmental processes may contribute to the etiology of SYNGAP1 disorders.

Exposure to the predator odor TMT induces early and late differential gene expression related to stress and excitatory synaptic function throughout the brain in male rats.

Persistent changes in brain stress and glutamatergic function are associated with post-traumatic stress disorder (PTSD). Rodent exposure to the predator odor trimethylthiazoline (TMT) is an innate stressor that produces lasting behavioral consequences relevant to PTSD. As such, the goal of the present study was to assess early (6 hours and 2 days-Experiment 1) and late (4 weeks-Experiment 2) changes to gene expression (RT-PCR) related to stress and excitatory function following TMT exposure in male, Long-Evans rats. During TMT exposure, rats engaged in stress reactive behaviors, including digging and immobility. Further, the TMT group displayed enhanced exploration and mobility in the TMT-paired context 1 week after exposure, suggesting a lasting contextual reactivity. Gene expression analyses revealed upregulated FKBP5 6 hours post-TMT in the hypothalamus and dorsal hippocampus. Two days after TMT, GRM3 was downregulated in the prelimbic cortex and dorsal hippocampus, but upregulated in the nucleus accumbens. This may reflect an early stress response (FKBP5) that resulted in later glutamatergic adaptation (GRM3). Finally, another experiment 4 weeks after TMT exposure showed several differentially expressed genes known to mediate excitatory tripartite synaptic function in the prelimbic cortex (GRM5, DLG4 and SLC1A3 upregulated), infralimbic cortex (GRM2 downregulated, Homer1 upregulated), nucleus accumbens (GRM7 and SLC1A3 downregulated), dorsal hippocampus (FKBP5 and NR3C2 upregulated, SHANK3 downregulated) and ventral hippocampus (CNR1, GRM7, GRM5, SHANK3 and Homer1 downregulated). These data show that TMT exposure induces stress and excitatory molecular adaptations, which could help us understand the persistent glutamatergic dysfunction observed in PTSD.

Molecular Correlates of Topiramate and GRIK1 rs2832407 Genotype in Pluripotent Stem Cell-Derived Neural Cultures.

BACKGROUND: There is growing evidence that the anticonvulsant topiramate is efficacious in reducing alcohol consumption. Further, an intronic single nucleotide polymorphism (rs2832407, C A) in the GRIK1 gene, which encodes the GluK1 subunit of the excitatory kainate receptor, predicted topiramate's effectiveness in reducing heavy drinking in a clinical trial. The molecular correlates of GRIK1 genotype that may relate to topiramate's ability to reduce drinking remain unknown. METHODS: We differentiated induced pluripotent stem cells (iPSCs) characterized by GRIK1 rs2832407 genotype from 8 A/A and 8 C/C donors into forebrain-lineage neural cultures. Our differentiation protocol yielded mixed neural cultures enriched for glutamatergic neurons. Basal mRNA expression of the GRIK1 locus was examined via quantitative polymerase chain reaction (qPCR). The effects of acute topiramate exposure on excitatory spontaneous synaptic activity were examined via whole-cell patch-clamp electrophysiology. Results were compared and contrasted between iPSC donor genotypes. RESULTS: Although characterization of the GRIK1 locus revealed no effect of rs2832407 genotype on GRIK1 isoform mRNA expression, a significant difference was observed on GRIK1 antisense-2 expression, which was greater in C/C neural cultures. Differential effects of acute exposure to 5 µM topiramate were observed on spontaneous synaptic activity in A/A versus C/C neurons, with a smaller reduction in excitatory event frequency observed in C/C donor neurons. CONCLUSIONS: This work highlights the use of iPSC technologies to study pharmacogenetic treatment effects in psychiatric disorders and furthers our understanding of the molecular effects of topiramate exposure in human neural cells.

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.

Autism-Associated Shank3 Is Essential for Homeostatic Compensation in Rodent V1.

Mutations in Shank3 are strongly associated with autism spectrum disorders and neural circuit changes in several brain areas, but the cellular mechanisms that underlie these defects are not understood. Homeostatic forms of plasticity allow central circuits to maintain stable function during experience-dependent development, leading us to ask whether loss of Shank3 might impair homeostatic plasticity and circuit-level compensation to perturbations. We found that Shank3 loss in vitro abolished synaptic scaling and intrinsic homeostatic plasticity, deficits that could be rescued by treatment with lithium. Further, Shank3 knockout severely compromised the in vivo ability of visual cortical circuits to recover from perturbations to sensory drive. Finally, lithium treatment ameliorated a repetitive self-grooming phenotype in Shank3 knockout mice. These findings demonstrate that Shank3 loss severely impairs the ability of central circuits to harness homeostatic mechanisms to compensate for perturbations in drive, which, in turn, may render them more vulnerable to such perturbations.

Adult loss of Cacna1a in mice recapitulates childhood absence epilepsy by distinct thalamic bursting mechanisms.

Inborn errors of CACNA1A-encoded P/Q-type calcium channels impair synaptic transmission, producing early and lifelong neurological deficits, including childhood absence epilepsy, ataxia and dystonia. Whether these impairments owe their pathologies to defective channel function during the critical period for thalamic network stabilization in immature brain remains unclear. Here we show that mice with tamoxifen-induced adult-onset ablation of P/Q channel alpha subunit (iKOp/q) display identical patterns of dysfunction, replicating the inborn loss-of-function phenotypes and, therefore demonstrate that these neurological defects do not rely upon developmental abnormality. Unexpectedly, unlike the inborn model, the adult-onset pattern of excitability changes believed to be pathogenic within the thalamic network is non-canonical. Specifically, adult ablation of P/Q channels does not promote Cacna1g-mediated burst firing or T-type calcium current (IT) in the thalamocortical relay neurons; however, burst firing in thalamocortical relay neurons remains essential as iKOp/q mice generated on a Cacna1g deleted background show substantially diminished seizure generation. Moreover, in thalamic reticular nucleus neurons, burst firing is impaired accompanied by attenuated IT. Interestingly, inborn deletion of thalamic reticular nucleus-enriched, human childhood absence epilepsy-linked gene Cacna1h in iKOp/q mice reduces thalamic reticular nucleus burst firing and promotes rather than reduces seizure, indicating an epileptogenic role for loss-of-function Cacna1h gene variants reported in human childhood absence epilepsy cases. Together, our results demonstrate that P/Q channels remain critical for maintaining normal thalamocortical oscillations and motor control in the adult brain, and suggest that the developmental plasticity of membrane currents regulating pathological rhythmicity is both degenerate and age-dependent.

TREK-1 Null Impairs Neuronal Excitability, Synaptic Plasticity, and Cognitive Function.

TREK-1, a two-pore-domain K+ channel, is highly expressed in the central nervous system. Although aberrant expression of TREK-1 is implicated in cognitive impairment, the cellular and functional mechanism underlying this channelopathy is poorly understood. Here we examined TREK-1 contribution to neuronal morphology, excitability, synaptic plasticity, and cognitive function in mice deficient in TREK-1 expression. TREK-1 immunostaining signal mainly appeared in hippocampal pyramidal neurons, but not in astrocytes. TREK-1 gene knockout (TREK-1 KO) increases dendritic sprouting and the number of immature spines in hippocampal CA1 pyramidal neurons. Functionally, TREK-1 KO increases neuronal excitability and enhances excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs). The increased EPSCs appear to be attributed to an increased release probability of presynaptic glutamate and functional expression of postsynaptic AMPA receptors. TREK-1 KO decreased the paired-pulse ratio and severely occluded the long-term potentiation (LTP) in the CA1 region. These altered synaptic transmission and plasticity are associated with recognition memory deficit in TREK-1 KO mice. Although astrocytic expression of TREK-1 has been reported in previous studies, TREK-1 KO does not alter astrocyte membrane K+ conductance or the syncytial network function in terms of syncytial isopotentiality. Altogether, TREK-1 KO profoundly affects the cellular structure and function of hippocampal pyramidal neurons. Thus, the impaired cognitive function in diseases associated with aberrant expression of TREK-1 should be attributed to the failure of this K+ channel in regulating neuronal morphology, excitability, synaptic transmission, and plasticity.

The Nebulin Family LIM and SH3 Proteins Regulate Postsynaptic Development and Function.

Neuronal dendrites have specialized actin-rich structures called dendritic spines that receive and integrate most excitatory synaptic inputs. The stabilization of dendrites and spines during neuronal maturation is essential for proper neural circuit formation. Changes in dendritic morphology and stability are largely mediated by regulation of the actin cytoskeleton; however, the underlying mechanisms remain to be fully elucidated. Here, we present evidence that the nebulin family members LASP1 and LASP2 play an important role in the postsynaptic development of rat hippocampal neurons from both sexes. We find that both LASP1 and LASP2 are enriched in dendritic spines, and their knockdown impairs spine development and synapse formation. Furthermore, LASP2 exerts a distinct role in dendritic arbor and dendritic spine stabilization. Importantly, the actin-binding N-terminal LIM domain and nebulin repeats of LASP2 are required for spine stability and dendritic arbor complexity. These findings identify LASP1 and LASP2 as novel regulators of neuronal circuitry.SIGNIFICANCE STATEMENT Proper regulation of the actin cytoskeleton is essential for the structural stability of dendrites and dendritic spines. Consequently, the malformation of dendritic structures accompanies numerous neurologic disorders, such as schizophrenia and autism. Nebulin family members are best known for their role in regulating the stabilization and function of actin thin filaments in muscle. The two smallest family members, LASP1 and LASP2, are more structurally diverse and are expressed in a broader array of tissues. While both LASP1 and LASP2 are highly expressed in the brain, little is currently known about their function in the nervous system. In this study, we demonstrate the first evidence that LASP1 and LASP2 are involved in the formation and long-term maintenance of dendrites and dendritic spines.

Synaptic Inhibition of Medial Olivocochlear Efferent Neurons by Neurons of the Medial Nucleus of the Trapezoid Body.

Medial olivocochlear (MOC) efferent neurons in the brainstem comprise the final stage of descending control of the mammalian peripheral auditory system through axon projections to the cochlea. MOC activity adjusts cochlear gain and frequency tuning, and protects the ear from acoustic trauma. The neuronal pathways that activate and modulate the MOC somata in the brainstem to drive these cochlear effects are poorly understood. Evidence suggests that MOC neurons are primarily excited by sound stimuli in a three-neuron activation loop from the auditory nerve via an intermediate neuron in the cochlear nucleus. Anatomical studies suggest that MOC neurons receive diverse synaptic inputs, but the functional effect of additional synaptic influences on MOC neuron responses is unknown. Here we use patch-clamp electrophysiological recordings from identified MOC neurons in brainstem slices from mice of either sex to demonstrate that in addition to excitatory glutamatergic synapses, MOC neurons receive inhibitory GABAergic and glycinergic synaptic inputs. These synapses are activated by electrical stimulation of axons near the medial nucleus of the trapezoid body (MNTB). Focal glutamate uncaging confirms MNTB neurons as a source of inhibitory synapses onto MOC neurons. MNTB neurons inhibit MOC action potentials, but this effect depresses with repeat activation. This work identifies a new pathway of connectivity between brainstem auditory neurons and indicates that MOC neurons are both excited and inhibited by sound stimuli received at the same ear. The pathway depression suggests that the effect of MNTB inhibition of MOC neurons diminishes over the course of a sustained sound.SIGNIFICANCE STATEMENT Medial olivocochlear (MOC) neurons are the final stage of descending control of the mammalian auditory system and exert influence on cochlear mechanics to modulate perception of acoustic stimuli. The brainstem pathways that drive MOC function are poorly understood. Here we show for the first time that MOC neurons are inhibited by neurons of the MNTB, which may suppress the effects of MOC activity on the cochlea.

Ceftriaxone Treatment Affects EAAT2 Expression and Glutamatergic Neurotransmission and Exerts a Weak Anticonvulsant Effect in Young Rats.

Epilepsy is a common neurological disorder. Despite the availability of a wide range of antiepileptic drugs, these are unsuccessful in preventing seizures in 20-30% of patients. Therefore, new pharmacological strategies are urgently required to control seizures. Modulation of glutamate uptake may have potential in the treatment of pharmacoresistant forms of epilepsy. Previous research showed that the antibiotic ceftriaxone (CTX) increased the expression and functional activity of excitatory amino acid transporter 2 (EAAT2) and exerted considerable anticonvulsant effects. However, other studies did not confirm a significant anticonvulsant effect of CTX administration. We investigated the impacts of CTX treatment on EAAT expression and glutamatergic neurotransmission, as well its anticonvulsant action, in young male Wistar rats. As shown by a quantitative real-time polymerase chain reaction (qPCR) assay and a Western blot analysis, the mRNA but not the protein level of EAAT2 increased in the hippocampus following CTX treatment. Repetitive CTX administration had only a mild anticonvulsant effect on pentylenetetrazol (PTZ)-induced convulsions in a maximal electroshock threshold test (MEST). CTX treatment did not affect the glutamatergic neurotransmission, including synaptic efficacy, short-term facilitation, or the summation of excitatory postsynaptic potentials (EPSPs) in the hippocampus and temporal cortex. However, it decreased the field EPSP (fEPSP) amplitudes evoked by intense electrical stimulation. In conclusion, in young rats, CTX treatment did not induce overexpression of EAAT2, therefore exerting only a weak antiseizure effect. Our data provide new insight into the effects of modulation of EAAT2 expression on brain functioning.

N-terminal alternative splicing of GluN1 regulates the maturation of excitatory synapses and seizure susceptibility.

The majority of NMDA receptors (NMDARs) in the brain are composed of 2 GluN1 and 2 GluN2 subunits. The inclusion or exclusion of 1 N-terminal and 2 C-terminal domains of GluN1 results in 8 splicing variants that exhibit distinct temporal and spatial patterns of expression and functional properties. However, previous functional analyses of Grin1 variants have been done using heterologous expression and the in vivo function of Grin1 splicing is unknown. Here we show that N-terminal splicing of GluN1 has important functions in the maturation of excitatory synapses. The inclusion of exon 5 of Grin1 is up-regulated in several brain regions such as the thalamus and neocortex. We find that deletion of Grin1 exon 5 disrupts the developmental remodeling of NMDARs in thalamic neurons and the effect is distinct from that of Grin2a (GluN2A) deletion. Deletion of Grin2a or exon 5 of Grin1 alone partially attenuates the shortening of NMDAR-mediated excitatory postsynaptic currents (NMDAR-EPSCs) during early life, whereas deletion of both Grin2a and exon 5 of Grin1 completely abolishes the developmental change in NMDAR-EPSC decay time. Deletion of exon 5 of Grin1 leads to an overproduction of excitatory synapses in layer 5 pyramidal neurons in the cortex and increases seizure susceptibility in adult mice. Our findings demonstrate that N-terminal splicing of GluN1 has important functions in synaptic maturation and neuronal network excitability.

Gene-environment interaction counterbalances social impairment in mouse models of autism.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by social communication deficits and repetitive/restricted behaviors. Although gene-environment interactions may explain the heterogeneous etiology of ASD, it is still largely unknown how the gene-environment interaction affects behavioral symptoms and pathophysiology in ASD. To address these questions, we used Cntnap2 knockout mice (genetic factor, G) exposed to valproic acid during embryonic development (environmental factor, E) as a gene-environment interaction (G × E) model. Paradoxically, the social deficits observed in the respective G and E models were improved in the G × E model; however, the high seizure susceptibility was more severe in the G × E -model than in the G and E models. Repetitive self-grooming and hyperactivity did not differ among the three models. The amplitudes of miniature excitatory postsynaptic currents in layer 2/3 pyramidal neurons of the medial prefrontal cortex were aberrant and similar in the G × E model when compared to the control group. Our findings suggest that the interaction of two risk factors does not always aggravate ASD symptoms but can also alleviate them, which may be key to understanding individual differences in behavioral phenotypes and symptom intensity.