Density Of Excitatory Synapses Research Articles

Altered offspring neurodevelopment in an arginine vasopressin preeclampsia model

Preeclampsia is a severe gestational hypertensive condition linked to child neuropsychiatric disorders, although underlying mechanisms are unclear. We used a recently developed, clinically relevant animal model of preeclampsia to assess offspring. C57BL/6J mouse dams were chronically infused with arginine vasopressin (AVP) or saline (24 ng/h) throughout pregnancy. Adult offspring were behaviorally tested (Y-maze, open field, rotarod, social approach, and elevated plus maze). Offspring brain was assessed histologically and by RNA sequencing. Preeclampsia-exposed adult males exhibited increased anxiety-like behavior and social approach while adult females exhibited impaired procedural learning. Adult AVP-exposed males had reduced total neocortical volume. Adult AVP-exposed females had increased caudate–putamen volume, increased caudate–putamen cell number, and decreased excitatory synapse density in hippocampal dentate gyrus (DG), CA1, and CA3. At postnatal day 7 (P7), AVP-exposed male and female offspring both had smaller neocortex. At P7, AVP-exposed males also had smaller caudate–putamen volume, while females had increased caudate–putamen volume relative to neocortical size. Similar to P7, E18 AVP-exposed offspring had smaller dorsal forebrain, mainly in reduced intermediate, subventricular, and ventricular zone volume, particularly in males. Decreased volume was not accounted for by cell size or cerebrovascular vessel diameter changes. E18 cortical RNAseq revealed 49 differentially-expressed genes in male AVP-exposed offspring, over-representing cytoplasmic translation processes. In females, 31 genes were differentially-expressed, over-representing collagen-related and epithelial regulation pathways. Gene expression changes in E18 AVP-exposed placenta indicated potential underlying mechanisms. Deficits in behavior and forebrain development in this AVP-based preeclampsia model were distinctly different in males and females, implicating different neurobiological bases.

Astrocytes as a target for Nogo-A and implications for synapse formation in vitro and in a model of acute demyelination.

Central nervous system (CNS) function depends on precise synaptogenesis, which is shaped by environmental cues and cellular interactions. Astrocytes are outstanding regulators of synapse development and plasticity through contact-dependent signals and through the release of pro- and antisynaptogenic factors. Conversely, myelin and its associated proteins, including Nogo-A, affect synapses in a inhibitory fashion and contribute to neural circuitry stabilization. However, the roles of Nogo-A-astrocyte interactions and their implications in synapse development and plasticity have not been characterized. Therefore, we aimed to investigate whether Nogo-A affects the capacity of astrocytes to induce synaptogenesis. Additionally, we assessed whether downregulation of Nogo-A signaling in an in vivo demyelination model impacts the synaptogenic potential of astrocytes. Our in vitro data show that cortical astrocytes respond to Nogo-A through RhoA pathway activation, exhibiting stress fiber formation and decreased ramified morphology. This phenotype was associated with reduced levels of GLAST protein and aspartate uptake, decreased mRNA levels of the synaptogenesis-associated genes Hevin, glypican-4, TGF-β1 and BDNF, and decreased and increased protein levels of Hevin and SPARC, respectively. Corroborating these findings, conditioned medium from Nogo-A-treated astrocytes suppressed the formation of structurally and functionally mature synapses in cortical neuronal cultures. After cuprizone-induced acute demyelination, we observed reduced immunostaining for Nogo-A in the visual cortex accompanied by higher levels of Hevin expression in astrocytes and an increase in excitatory synapse density. Hence, we suggest that interactions between Nogo-A and astrocytes might represent an important pathway of plasticity regulation and could be a target for therapeutic intervention in demyelinating diseases in the future.

Glioma-Derived TSP2 Promotes Excitatory Synapse Formation and Results in Hyperexcitability in the Peritumoral Cortex of Glioma.

Seizures are common in patients with glioma, especially low-grade glioma (LGG). However, the epileptogenic mechanisms are poorly understood. Recent evidence has indicated that abnormal excitatory synaptogenesis plays an important role in epileptogenesis. The thrombospondin (TSP) family is a key regulator of synaptogenesis. Thus, this study aimed to elucidate the role of TSP2 in epileptogenesis in glioma-related epilepsy. The expression of TSP2 was increased in tumor tissue specimens from LGG patients, and this increase may have contributed to an increase in the density of spines and excitatory synapses in the peritumoral area. A glioma cell-implanted rat model was established by stereotactic implantation of wild-type TSP2-expressing, TSP2-overexpressing or TSP2-knockout C6 cells into the neocortex. Similarly, an increase in the density of excitatory synapses was also observed in the peritumoral area of the implanted tumor. In addition, epileptiform discharges occurred in the peritumoral cortex and were positively correlated with the TSP2 level in glioma tissues. Moreover, α2δ1/Rac1 signaling was enhanced in the peritumoral region, and treatment with the α2δ1 antagonist gabapentin inhibited epileptiform discharges in the peritumoral cortex. In conclusion, glioma-derived TSP2 promotes excitatory synapse formation, probably via the α2δ1/Rac1 signaling pathway, resulting in hyperexcitability in the peritumoral cortical networks, which may provide new insight into the epileptogenic mechanisms underlying glioma-related epilepsy.

The Interaction of TRAF6 With Neuroplastin Promotes Spinogenesis During Early Neuronal Development.

Correct brain wiring depends on reliable synapse formation. Nevertheless, signaling codes promoting synaptogenesis are not fully understood. Here, we report a spinogenic mechanism that operates during neuronal development and is based on the interaction of tumor necrosis factor receptor-associated factor 6 (TRAF6) with the synaptic cell adhesion molecule neuroplastin. The interaction between these proteins was predicted in silico and verified by co-immunoprecipitation in extracts from rat brain and co-transfected HEK cells. Binding assays show physical interaction between neuroplastin’s C-terminus and the TRAF-C domain of TRAF6 with a Kd value of 88 μM. As the two proteins co-localize in primordial dendritic protrusions, we used young cultures of rat and mouse as well as neuroplastin-deficient mouse neurons and showed with mutagenesis, knock-down, and pharmacological blockade that TRAF6 is required by neuroplastin to promote early spinogenesis during in vitro days 6-9, but not later. Time-framed TRAF6 blockade during days 6–9 reduced mEPSC amplitude, number of postsynaptic sites, synapse density and neuronal activity as neurons mature. Our data unravel a new molecular liaison that may emerge during a specific window of the neuronal development to determine excitatory synapse density in the rodent brain.

Sex Differences of Microglia and Synapses in the Hippocampal Dentate Gyrus of Adult Mouse Offspring Exposed to Maternal Immune Activation.

Schizophrenia is a psychiatric disorder affecting ∼1% of humans worldwide. It is earlier and more frequently diagnosed in men than woman, and men display more pronounced negative symptoms together with greater gray matter reductions. Our previous findings utilizing a maternal immune activation (mIA) mouse model of schizophrenia revealed exacerbated anxiety-like behavior and sensorimotor gating deficits in adult male offspring that were associated with increased microglial reactivity and inflammation in the hippocampal dentate gyrus (DG). However, both male and female adult offspring displayed stereotypy and impairment of sociability. We hypothesized that mIA may lead to sex-specific alterations in microglial pruning activity, resulting in abnormal synaptic connectivity in the DG. Using the same mIA model, we show in the current study sex-specific differences in microglia and synapses within the DG of adult offspring. Specifically, microglial levels of cluster of differentiation (CD)68 and CD11b were increased in mIA-exposed females. Sex-specific differences in excitatory and inhibitory synapse densities were also observed following mIA. Additionally, inhibitory synaptic tone was increased in DG granule cells of both males and females, while changes in excitatory synaptic transmission occurred only in females with mIA. These findings suggest that phagocytic and complement pathways may together contribute to a sexual dimorphism in synaptic pruning and neuronal dysfunction in mIA, and may propose sex-specific therapeutic targets to prevent schizophrenia-like behaviors.

Ultrastructural evidence for glutamatergic dysregulation in schizophrenia

The aim of this paper is to summarize ultrastructural evidence for glutamatergic dysregulation in several linked regions in postmortem schizophrenia brain. Following a brief summary of glutamate circuitry and how synapses are identified at the electron microscopic (EM) level, we will review EM pathology in the cortex and basal ganglia. We will include the effects of antipsychotic drugs and the relation of treatment response. We will discuss how these findings support or confirm other postmortem findings as well as imaging results. Briefly, synaptic and mitochondrial density in anterior cingulate cortex was decreased in schizophrenia, versus normal controls (NCs), in a selective layer specific pattern. In dorsal striatum, increases in excitatory synaptic density were detected in caudate matrix, a compartment associated with cognitive and motor function, and in the putamen patches, a region associated with limbic function and in the core of the nucleus accumbens. Patients who were treatment resistant or untreated had significantly elevated numbers of excitatory synapses in limbic striatal areas in comparison to NCs and responders. Protein levels of vGLUT2, found in subcortical glutamatergic neurons, were increased in the nucleus accumbens in schizophrenia. At the EM level, schizophrenia subjects had an increase in density of excitatory synapses in several areas of the basal ganglia. In the substantia nigra, the protein levels of vGLUT2 were elevated in untreated patients compared to NCs. The density of inhibitory synapses was decreased in schizophrenia versus NCs. In schizophrenia, glutamatergic synapses are differentially affected depending on the brain region, treatment status, and treatment response.

17\u03b1 Estradiol promotes plasticity of spared inputs in the adult amblyopic visual cortex

The promotion of structural and functional plasticity by estrogens is a promising approach to enhance central nervous system function in the aged. However, how the sensitivity to estrogens is regulated across brain regions, age and experience is poorly understood. To ask if estradiol treatment impacts structural and functional plasticity in sensory cortices, we examined the acute effect of 17α-Estradiol in adult Long Evans rats following chronic monocular deprivation, a manipulation that reduces the strength and selectivity of deprived eye vision. Chronic monocular deprivation decreased thalamic input from the deprived eye to the binocular visual cortex and accelerated short-term depression of the deprived eye pathway, but did not change the density of excitatory synapses in primary visual cortex. Importantly, we found that the classical estrogen receptors ERα and ERβ were robustly expressed in the adult visual cortex, and that a single dose of 17α-Estradiol reduced the expression of the calcium-binding protein parvalbumin, decreased the integrity of the extracellular matrix and increased the size of excitatory postsynaptic densities. Furthermore, 17α-Estradiol enhanced experience-dependent plasticity in the amblyopic visual cortex, by promoting response potentiation of the pathway served by the non-deprived eye. The promotion of plasticity at synapses serving the non-deprived eye may reflect selectivity for synapses with an initially low probability of neurotransmitter release, and may inform strategies to remap spared inputs around a scotoma or a cortical infarct.

βPix heterozygous mice have defects in neuronal morphology and social interaction

βPix activates Rho family small GTPases, Rac1 and Cdc42 as a guanine nucleotide exchange factor. Although overexpression of βPix in cultured neurons indicates that βPix is involved in spine morphogenesis and synapse formation in vitro, the in vivo role of βPix in the neuron is not well understood. Recently, we generated βPix knockout mice that showed lethality at embryonic day 9.5. Here, we investigate the neuronal role of βPix using βPix heterozygous mice that are viable and fertile. βPix heterozygous mice show decreased expression levels of βPix proteins in various tissues including the brain. Cultured hippocampal neurons from βPix heterozygous mice show a decrease in neurite length and complexity as well as synaptic density. Both excitatory and inhibitory synapse densities are decreased in these neurons. Golgi-staining of hippocampal tissues from the brain of these mice show reduced dendritic complexity and spine density in the hippocampal neurons. Expression levels of NMDA- and AMPA-receptor subunits and Git1 protein in hippocampal tissues are also decreased in these mice. Behaviorally, βPix heterozygous mice exhibit impaired social interaction. Altogether, these results indicate that βPix is required for neurite morphogenesis and synapse formation, and the reduced expression of βPix proteins results in a defect in social behavior.

Regulation of dendrite morphology and excitatory synapse formation by zDHHC15.

ABSTRACTProtein palmitoylation is the most common post-translational lipid modification in the brain and is mediated by a family of 24 zDHHC enzymes. There has been growing interest in zDHHCs due to mounting evidence that these enzymes play key roles in the development and function of neuronal connections, and the fact that a number of zDHHCs have been associated with neurodevelopmental and neurodegenerative diseases. Loss-of-function variants in several zDHHCs, including zDHHC15, have been identified in patients with intellectual disabilities; however, the function of zDHHC15 in the brain has not been well studied. Here, we demonstrate that knocking down zDHHC15 in primary rat hippocampal cultures reduces dendritic outgrowth and arborization, as well as spine maturation. Moreover, knockdown of zDHHC15 reduces palmitoylation of PSD-95 and its trafficking into dendrites, resulting in an overall decrease in the density of excitatory synapses being formed onto mutant cells.

α-synuclein oligomers enhance astrocyte-induced synapse formation through TGF-β1 signaling in a Parkinson's disease model.

Parkinson's disease (PD) is characterized by selective death of dopaminergic neurons in the substantia nigra, degeneration of the nigrostriatal pathway, increases in glutamatergic synapses in the striatum and aggregation of α-synuclein. Evidence suggests that oligomeric species of α-synuclein (αSO) are the genuine neurotoxins of PD. Although several studies have supported the direct neurotoxic effects of αSO on neurons, their effects on astrocytes have not been directly addressed. Astrocytes are essential to several steps of synapse formation and function, including secretion of synaptogenic factors, control of synaptic elimination and stabilization, secretion of neural/glial modulators, and modulation of extracellular ions, and neurotransmitter levels in the synaptic cleft. Here, we show that αSO induced the astrocyte reactivity and enhanced the synaptogenic capacity of human and murine astrocytes by increasing the levels of the known synaptogenic molecule transforming growth factor beta 1 (TGF-β1). Moreover, intracerebroventricular injection of αSO in mice increased the number of astrocytes, the density of excitatory synapses, and the levels of TGF-β1 in the striatum of injected animals. Inhibition of TGF-β1 signaling impaired the effect of the astrocyte-conditioned medium on glutamatergic synapse formation in vitro and on striatal synapse formation in vivo, whereas addition of TGF-β1 protected mesencephalic neurons against synapse loss triggered by αSO. Together, our data suggest that αSO have important effects on astrocytic functions and describe TGF-β1 as a new endogenous astrocyte-derived molecule involved in the increase in striatal glutamatergic synaptic density present in early stages of PD. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. Cover Image for this issue: doi: 10.1111/jnc.14514.

Close Homolog of L1 Regulates Dendritic Spine Density in the Mouse Cerebral Cortex Through Semaphorin 3B.

Dendritic spines in the developing mammalian neocortex are initially overproduced and then eliminated during adolescence to achieve appropriate levels of excitation in mature networks. We show here that the L1 family cell adhesion molecule Close Homolog of L1 (CHL1) and secreted repellent ligand Semaphorin 3B (Sema3B) function together to induce dendritic spine pruning in developing cortical pyramidal neurons. Loss of CHL1 in null mutant mice in both genders resulted in increased spine density and a greater proportion of immature spines on apical dendrites in the prefrontal and visual cortex. Electron microscopy showed that excitatory spine synapses with postsynaptic densities were increased in the CHL1-null cortex, and electrophysiological recording in prefrontal slices from mutant mice revealed deficiencies in excitatory synaptic transmission. Mechanistically, Sema3B protein induced elimination of spines on apical dendrites of cortical neurons cultured from wild-type but not CHL1-null embryos. Sema3B was secreted by the cortical neuron cultures, and its levels increased when cells were treated with the GABA antagonist gabazine. In vivo CHL1 was coexpressed with Sema3B in pyramidal neuron subpopulations and formed a complex with Sema3B receptor subunits Neuropilin-2 and PlexinA4. CHL1 and NrCAM, a closely related L1 adhesion molecule, localized primarily to distinct spines and promoted spine elimination to Sema3B or Sema3F, respectively. These results support a new concept in which selective spine elimination is achieved through different secreted semaphorins and L1 family adhesion molecules to sculpt functional neural circuits during postnatal maturation.SIGNIFICANCE STATEMENT Dendritic spines in the mammalian neocortex are initially overproduced and then pruned in adolescent life through unclear mechanisms to sculpt maturing cortical circuits. Here, we show that spine and excitatory synapse density of pyramidal neurons in the developing neocortex is regulated by the L1 adhesion molecule, Close Homolog of L1 (CHL1). CHL1 mediated spine pruning in response to the secreted repellent ligand Semaphorin 3B and associated with receptor subunits Neuropilin-2 and PlexinA4. CHL1 and related L1 adhesion molecule NrCAM localized to distinct spines, and promoted spine elimination to Semaphorin 3B and -3F, respectively. These results support a new concept in which selective elimination of individual spines and nascent synapses can be achieved through the action of distinct secreted semaphorins and L1 adhesion molecules.

Antagonizing Increased miR-135a Levels at the Chronic Stage of Experimental TLE Reduces Spontaneous Recurrent Seizures.

Mesial temporal lobe epilepsy (mTLE) is a chronic neurological disease characterized by recurrent seizures. The antiepileptic drugs currently available to treat mTLE are ineffective in one-third of patients and lack disease-modifying effects. miRNAs, a class of small noncoding RNAs which control gene expression at the post-transcriptional level, play a key role in the pathogenesis of mTLE and other epilepsies. Although manipulation of miRNAs at acute stages has been reported to reduce subsequent spontaneous seizures, it is uncertain whether targeting miRNAs at chronic stages of mTLE can also reduce seizures. Furthermore, the functional role and downstream targets of most epilepsy-associated miRNAs remain poorly understood. Here, we show that miR-135a is selectively upregulated within neurons in epileptic brain and report that targeting miR-135a in vivo using antagomirs after onset of spontaneous recurrent seizures can reduce seizure activity at the chronic stage of experimental mTLE in male mice. Further, by using an unbiased approach combining immunoprecipitation and RNA sequencing, we identify several novel neuronal targets of miR-135a, including Mef2a Mef2 proteins are key regulators of excitatory synapse density. Mef2a and miR-135a show reciprocal expression regulation in human (of both sexes) and experimental TLE, and miR-135a regulates dendritic spine number and type through Mef2. Together, our data show that miR-135a is target for reducing seizure activity in chronic epilepsy, and that deregulation of miR-135a in epilepsy may alter Mef2a expression and thereby affect synaptic function and plasticity.SIGNIFICANCE STATEMENT miRNAs are post-transcriptional regulators of gene expression with roles in the pathogenesis of epilepsy. However, the precise mechanism of action and therapeutic potential of most epilepsy-associated miRNAs remain poorly understood. Our study reveals dramatic upregulation of the key neuronal miRNA miR-135a in both experimental and human mesial temporal lobe epilepsy. Silencing miR-135a in experimental temporal lobe epilepsy reduces seizure activity at the spontaneous recurrent seizure stage. These data support the exciting possibility that miRNAs can be targeted to combat seizures after spontaneous seizure activity has been established. Further, by using unbiased approaches novel neuronal targets of miR-135a, including members of the Mef2 protein family, are identified that begin to explain how deregulation of miR-135a may contribute to epilepsy.

Ephrin-B3 controls excitatory synapse density through cell-cell competition for EphBs.

Cortical networks are characterized by sparse connectivity, with synapses found at only a subset of axo-dendritic contacts. Yet within these networks, neurons can exhibit high connection probabilities, suggesting that cell-intrinsic factors, not proximity, determine connectivity. Here, we identify ephrin-B3 (eB3) as a factor that determines synapse density by mediating a cell-cell competition that requires ephrin-B-EphB signaling. In a microisland culture system designed to isolate cell-cell competition, we find that eB3 determines winning and losing neurons in a contest for synapses. In a Mosaic Analysis with Double Markers (MADM) genetic mouse model system in vivo the relative levels of eB3 control spine density in layer 5 and 6 neurons. MADM cortical neurons in vitro reveal that eB3 controls synapse density independently of action potential-driven activity. Our findings illustrate a new class of competitive mechanism mediated by trans-synaptic organizing proteins which control the number of synapses neurons receive relative to neighboring neurons.

Neuroligins Differentially Mediate Subtype-Specific Synapse Formation in Pyramidal Neurons and Interneurons.

Neuroligins (NLs) are postsynaptic cell-adhesion proteins that play important roles in synapse formation and the excitatory-inhibitory balance. They have been associated with autism in both human genetic and animal model studies, and affect synaptic connections and synaptic plasticity in several brain regions. Yet current research mainly focuses on pyramidal neurons, while the function of NLs in interneurons remains to be understood. To explore the functional difference among NLs in the subtype-specific synapse formation of both pyramidal neurons and interneurons, we performed viral-mediated shRNA knockdown of NLs in cultured rat cortical neurons and examined the synapses in the two major types of neurons. Our results showed that in both types of neurons, NL1 and NL3 were involved in excitatory synapse formation, and NL2 in GABAergic synapse formation. Interestingly, NL1 affected GABAergic synapse formation more specifically than NL3, and NL2 affected excitatory synapse density preferentially in pyramidal neurons. In summary, our results demonstrated that different NLs play distinct roles in regulating the development and balance of excitatory and inhibitory synapses in pyramidal neurons and interneurons.

Endothelium-Derived Semaphorin 3G Regulates Hippocampal Synaptic Structure and Plasticity via Neuropilin-2/PlexinA4

The proper interactions between blood vessels and neurons are critical for maintaining the strength of neural circuits and cognitive function. However, the precise molecular events underlying these interactions remain largely unknown. Here, we report that the selective knockout of semaphorin 3G (Sema3G) in endothelial cells impaired hippocampal-dependent memory and reduced dendritic spine density in CA1 neurons in mice; these effects were reversed after restoration of Sema3G levels in the hippocampus by AAV transfection. We further show that Sema3G increased excitatory synapse density via neuropilin-2/PlexinA4 signaling and through activation of Rac1. These results provide the first evidence that, in the central nervous system, endothelial Sema3G serves as a vascular-derived synaptic organizer that regulates synaptic plasticity and hippocampal-dependent memory. Our findings highlight the role of vascular endothelial cells in regulating cognitive function through intercellular communication with neurons in the hippocampus.

LiCl Pretreatment Ameliorates Adolescent Methamphetamine Exposure-Induced Long-Term Alterations in Behavior and Hippocampal Ultrastructure in Adulthood in Mice.

BackgroundAdolescent methamphetamine exposure causes a broad range of neurobiological deficits in adulthood. Glycogen synthase kinase-3β is involved in various cognitive and behavioral processes associated with methamphetamine exposure. This study aims to investigate the protective effects of the glycogen synthase kinase-3β inhibitor lithium chloride on adolescent methamphetamine exposure-induced long-term alterations in emotion, cognition, behavior, and molecule and hippocampal ultrastructure in adulthood.MethodsA behavioral test battery was used to investigate the protective effects of lithium chloride on adolescent methamphetamine exposure-induced long-term emotional, cognitive, and behavioral impairments in mice. Western blotting and immunohistochemistry were used to detect glycogen synthase kinase-3β activity levels in the medial prefrontal cortex and dorsal hippocampus. Electron microscopy was used to analyze changes in synaptic ultrastructure in the dorsal hippocampus. Locomotor sensitization with a methamphetamine (1 mg/kg) challenge was examined 80 days after adolescent methamphetamine exposure.ResultsAdolescent methamphetamine exposure induced long-term alterations in locomotor activity, novel spatial exploration, and social recognition memory; increases in glycogen synthase kinase-3β activity in dorsal hippocampus; and decreases in excitatory synapse density and postsynaptic density thickness in CA1. These changes were ameliorated by lithium chloride pretreatment. Adolescent methamphetamine exposure-induced working memory deficits in Y-maze spontaneous alternation test and anxiety-like behavior in elevated-plus maze test spontaneously recovered after long-term methamphetamine abstinence. No significant locomotor sensitization was observed after long-term methamphetamine abstinence.ConclusionsHyperactive glycogen synthase kinase-3β contributes to adolescent chronic methamphetamine exposure-induced behavioral and hippocampal impairments in adulthood. Our results suggest glycogen synthase kinase-3β may be a potential target for the treatment of deficits in adulthood associated with adolescent methamphetamine abuse.

Sociability and synapse subtype-specific defects in mice lacking SRPX2, a language-associated gene.

The FoxP2 transcription factor and its target genes have been implicated in developmental brain diseases with a prominent language component, such as developmental verbal dyspraxia and specific language impairment. How FoxP2 affects neural circuitry development remains poorly understood. The sushi domain protein SRPX2 is a target of FoxP2, and mutations in SRPX2 are associated with language defects in humans. We have previously shown that SRPX2 is a synaptogenic protein that increases excitatory synapse density. Here we provide the first characterization of mice lacking the SRPX2 gene, and show that these mice exhibit defects in both neural circuitry and communication and social behaviors. Specifically, we show that mice lacking SRPX2 show a specific reduction in excitatory VGlut2 synapses in the cerebral cortex, while VGlut1 and inhibitory synapses were largely unaffected. SRPX2 KO mice also exhibit an abnormal ultrasonic vocalization ontogenetic profile in neonatal pups, and reduced preference for social novelty. These data demonstrate a functional role for SRPX2 during brain development, and further implicate FoxP2 and its targets in regulating the development of vocalization and social circuits.

Genetic Ablation of All Cerebellins Reveals Synapse Organizer Functions in Multiple Regions Throughout the Brain.

Cerebellins are synaptic organizer molecules that bind to presynaptic neurexins and postsynaptic receptors. They are well studied in the cerebellum, but three of the four cerebellins (Cbln1, Cbln2, and Cbln4) are also broadly expressed outside of the cerebellum, suggesting that they perform general functions throughout the brain. Here, we generated male and female constitutive single (KO), double KO (dKO), and triple KO (tKO) mice of Cbln1, Cbln2, and Cbln4. We found that all constitutive cerebellin-deficient mice were viable and fertile, suggesting that cerebellins are not essential for survival. Cbln1/2 dKO mice exhibited salience-induced seizures that were aggravated in Cbln1/2/4 tKO mice, suggesting that all cerebellins contribute to brain function. As described previously, Cbln1 KO mice displayed major motor impairments that were aggravated by additional KO of Cbln2. Strikingly, the Cbln1/2 dKO did not cause alterations in synapse density in the hippocampus of young adult (1- and 2-month-old) mice, but produced a selective ∼50% decrease in hippocampal synapse density in the stratum lacunosum moleculare of the CA1 region and in the dentate gyrus of aging, 6-month-old mice. A similar decrease in excitatory synapse density was observed in the striatum and retrosplenial cortex. Behaviorally, the Cbln1 KO produced dramatic changes in motor behaviors that were partly aggravated by additional deletion of Cbln2 and/or Cbln4. Our results show that cerebellins are not essential for survival and do not contribute to initial synapse formation, but perform multiple functions throughout the brain; as a consequence, their ablation results in a delayed loss of synapses and in behavioral impairments.SIGNIFICANCE STATEMENT Cerebellins (Cbln1-4) are trans-synaptic cell adhesion molecules. In the cerebellum, Cbln1 functions as a bidirectional organizer of parallel fiber-Purkinje cell synapses by binding to presynaptic neurexins and postsynaptic GluRδ2. Little is known about the function of cerebellins outside of the cerebellum; therefore, the present study used single, double, and triple constitutive KO mice of Cbln1, Cbln2, and Cbln4 to analyze the overall function of cerebellins. We show that cerebellins act as important synaptic organizers in specific subsets of neurons and likely contribute to many different brain functions. We also show that cerebellins are not initially required for synapse formation, but rather for specification and long-term synapse maintenance and demonstrate that all cerebellins, not just Cbln1, contribute to brain function.

Comprehensive Study on the Potential Therapeutic Use of Brevetoxin‐2 to Aid Post‐stroke Recovery

OBJECTIVEIschemic stroke is a leading cause of long‐term disability that triggers structural and physiological changes in neuronal circuits adjacent to the infarct region. This region, peri‐infarct area, plays a key role in recovery and represents a potential therapeutic target. Brevetoxin‐2 (PbTx‐2) interacts with site 5 of the α‐subunit of the voltage gated sodium channels (VGSC) and augments Na+ influx and Ca2+ dependent downstream signaling pathways. Previous studies in murine neocortical neurons found that PbTx‐2 influences the activation of key signaling proteins that alter physiologic events involved in survival of neurons as well as synaptic plasticity. We therefore assessed the effects of PbTx‐2 on parameters such as motor function, cerebralcortical neuron dendritic arborization and synapse density.HYPOTHESESWe hypothesized that PbTx‐2 would aid in functional recovery post‐stroke by increasing dendritic arbor complexity of the layer V pyramidal neurons and excitatory synapse density in the peri‐infarct regionMETHODSA photothrombotic stroke was produced by administration of an intraperitoneal injection of Rose Bengal solution followed by a 12.5 min light illumination (150 mW) of the motor cortex region resulting in clotting in 2 to 4 months old YFP mice. Epicortical application of hydrogel (controls) or hydrogel + PbTx‐2 was applied over the infarct area on day 5 post‐stroke using a Hamilton syringe. A pasta matrix reach task (PMRT) was used to assess the ability of food restricted mice to grasp pasta pieces so as to determine the number of pieces of pasta retrieved post‐stroke and post‐hydrogel treatment from a 5x5 matrix of pasta. A second motor test, the foot fault test (FFT), was performed where the mouse could freely walk on an elevated wire grid for five minutes, was recorded by video camera, and manually scored for foot faults. Mice were sacrificed by 24 hours on day 6 and the brains were collected to obtain 100 μm sections. These sections were imaged using a Leica confocal microscope which were then analyzed with Imaris software to measure dendritic arbor complexity by Sholl analysis. Immunohistochemistry (IHC) was performed using VGlut1 and PSD95 as synaptic markers to measure excitatory synapse density that was analyzed by Volocity Software.RESULTSThe number of pasta pieces retrieved was significantly impaired on day 1 post‐stroke in all animals with confirmed evidence of stroke. There was subsequent improvement of this task following treatment with PbTx‐2 as compared to the controls on day 6. In addition, the PbTx‐2 treated mice had a decrease in the number of foot faults in comparison to the controls on day 6. PbTx‐2 treated layer V pyramidal neurons had a two‐fold increase in dendritic arbor complexity as compared to controls. Preliminary IHC data suggest that the excitatory synapse density in the peri‐infarct region was increased in PbTx‐2 treated mice compared to the control mice.CONCLUSIONPreclinical research in a murine stroke model provided behavioral assessments of neural insult‐induced functional deficits and PbTx‐2 induced motor recovery. The PMRT and FFT proved to be sensitive for the study of motor impairment and recovery. Increased dendritic arbor complexity and excitatory synapse density indicates that PbTx‐2 can potentially augment neuroplasticity in the peri‐infarct region. Identifying the appropriate dosage range of PbTx‐2 in future studies will establish the dose‐response relationships for these effects.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

32.2 ABNORMALITIES OF SYNAPTIC PROTEOMES IN SCHIZOPHRENIA

BackgroundThe human brain is comprised of billions of neurons that form networks of connections within and between brain regions. These connections facilitate neuroplastic events that underlie learning and memory, critical aspects of cognitive function often perturbed in neuropsychiatric illnesses. Neuronal signaling is mediated by fast and slow transmission events, encompassing receptors, ligands, ions, enzymes, and other substrates. These elements are spatially arranged in subcellular microdomains, facilitating juxtaposition of proteins that coordinate various biological processes. For example, synaptic transmission is modulated via release of neurotransmitter into the synaptic cleft, where receptors are activated and the postsynaptic cell modulated via electrical and chemical signals. The pre- and postsynaptic compartments include highly specialized protein clusters, with elegant and complex regulatory mechanisms that traffic proteins to and from these zones. In particular, postsynaptic densities are microdomains comprised of about 1000 unique proteins that are interacting with one another via specialized multipotent scaffolding molecule. Postsynaptic density-95 (PSD-95) is a multipotent scaffolding, trafficking, and clustering protein that links glutamate receptors, signaling molecules, and other structural proteins at postsynaptic sites. More than 95% of PSD-95 expression is localized to excitatory synapses, and it is the most abundant scaffolding protein within the postsynaptic density. Mounting genetic, proteomic, and pharmacological evidence converges on alterations in the postsynaptic density of excitatory synapses in subjects with schizophrenia. Cognitive and negative symptoms associated with dysfunction of limbic circuitry, including working memory and motivation, are particularly implicated by this mechanism. To investigate excitatory postsynaptic protein hubs in schizophrenia, we assessed the PSD-95 protein interactome from brain tissue of subjects with schizophrenia and controls.MethodsHuman brain tissue from fifteen subjects with schizophrenia and fifteen control subjects from the DLPFC was processed for affinity purification of PSD-95 protein complexes. We confirmed PSD-95 capture and enrichment from each sample using Western blot analyses. Samples were then pooled by region and assessed by mass spectrometry for a quality control step. Pooled samples were run in triplicate. Go versus nogo was based on finding more than 500 unique peptides in each pooled sample from each region. Next, individual samples were run through our mass spectrometry protocol in triplicate. We then subtracted any non-specifically captured peptides identified by our IgG control studies that were performed in parallel to PSD-95 affinity purification. Data were normalized within each of the mass spec runs to the most intense PSD-95 peptide. This PSD-95 peptide used for normalization was the same in every sample. Peptides that were present in at least 2 of 3 technical replicates were carried forward and subjected to quantile normalization. Peptides missing in a technical replicate were replaced by imputation, and the dataset subjected to unsupervised clustering. We also performed a semisupervised clustering protocol, non negative matrix factoring (NMF). Consensus signatures of 200 peptides were identified for each brain region, and subjected to traditional and alternative bioinformatics analyses to identify pathways, processes and compounds associated with the signatures for each brain region.ResultsPreliminary analyses indicate changes in the PSD-95 interactome consistent with diminished NMDA receptor signaling complex activity in schizophrenia, with lower levels of NMDA-subtype glutamate receptor subunits, as well as protein kinases associated with postsynaptic signaling in this complex. Specific biological pathways and processes identified include metabolic and inflammatory pathways. We will also present proteomic signatures generated from this dataset, and interrogate the iLINCS perturbagen database to identify drugs and genes that simulate or reverse this signature.DiscussionOur preliminary data suggest that NMDA receptor function is compromised in schizophrenia. Additional work is needed to see if this is an effect of antipsychotic medications. Our proteomic findings extend the NMDA receptor hypothesis beyond the transcriptome, highlighting an important new approach for assessing abnormalities of synapses in postmortem brain.