Sex-biased effects on hippocampal circuit development by perinatal SERT expression in CA3 pyramidal neurons.

Neurodevelopmental disorders ranging from autism to intellectual disability display sex-biased prevalence and phenotypical presentations. Despite increasing knowledge about temporospatial cortical map development and genetic variants linked to neurodevelopmental disorders, when and how sex-biased neural circuit derailment may arise in diseased brain remain unknown. Here, we identify in mice that serotonin uptake transporter (SERT) in non-serotonergic neurons - hippocampal and prefrontal pyramidal neurons - confers sex-biased effects specifically during neural circuit development. A set of gradient-patterned CA3 pyramidal neurons transiently express SERT to clear extracellular serotonin, coinciding with hippocampal synaptic circuit establishment. Ablating pyramidal neuron SERT (SERTPyramidΔ) alters dendritic spine developmental trajectory in the hippocampus, and precipitates sex-biased impairments in long-term activity-dependent hippocampal synaptic plasticity and cognitive behaviors. Transcriptomic analyses identify sex-biased alterations in gene sets associated with autism, dendritic spine structure, synaptic function and male-specific enrichment of dysregulated genes in glial cells in early postnatal SERTPyramidΔ hippocampus. Our data suggest that SERT function in these pyramidal neurons underscores a temporal- and brain region-specific regulation of normal sex-dimorphic circuit development and a source for sex-biased vulnerability to cognitive and behavioral impairments. This article has an associated 'The people behind the papers' interview.

Elevated insulin growth factor-1 in dentate gyrus induces cognitive deficits in pre-term newborns.

Prematurely born infants are deprived of maternal hormones and cared for in the stressful environment of Neonatal Intensive Care Units (NICUs). They suffer from long-lasting deficits in learning and memory. Here, we show that prematurity and associated neonatal stress disrupt dentate gyrus (DG) development and induce long-term cognitive deficits and that these effects are mediated by insulin growth factor-1 (IGF1). Nonmaternal care of premature rabbits increased the number of granule cells and interneurons and reduced neurogenesis, suggesting accelerated premature maturation of DG. However, the density of glutamatergic synapses, mature dendritic spines, and synaptic transmission were reduced in preterm kits compared with full-term controls, indicating that premature synaptic maturation was abnormal. These findings were consistent with cognitive deficits observed in premature rabbits and appeared to be driven by transcriptomic changes in the granule cells. Preterm kits displayed reduced weight, elevated serum cortisol and growth hormone, and higher IGF1 expression in the liver and DG relative to full-term controls. Importantly, blocking IGF-1 receptor in premature kits restored cognitive deficits, increased the density of glutamatergic puncta, and rescued NR2B and PSD95 levels in the DG. Hence, IGF1 inhibition alleviates prematurity-induced cognitive dysfunction and synaptic changes in the DG through modulation of NR2B and PSD95. The study identifies a novel strategy to potentially rescue DG maldevelopment and cognitive dysfunction in premature infants under stress in NICUs.

Impact of schizophrenia GWAS loci converge onto distinct pathways in cortical interneurons vs glutamatergic neurons during development.

Remarkable advances have been made in schizophrenia (SCZ) GWAS, but gleaning biological insight from these loci is challenging. Genetic influences on gene expression (e.g., eQTLs) are cell type-specific, but most studies that attempt to clarify GWAS loci's influence on gene expression have employed tissues with mixed cell compositions that can obscure cell-specific effects. Furthermore, enriched SCZ heritability in the fetal brain underscores the need to study the impact of SCZ risk loci in specific developing neurons. MGE-derived cortical interneurons (cINs) are consistently affected in SCZ brains and show enriched SCZ heritability in human fetal brains. We identified SCZ GWAS risk genes that are dysregulated in iPSC-derived homogeneous populations of developing SCZ cINs. These SCZ GWAS loci differential expression (DE) genes converge on the PKC pathway. Their disruption results in PKC hyperactivity in developing cINs, leading to arborization deficits. We show that the fine-mapped GWAS locus in the ATP2A2 gene of the PKC pathway harbors enhancer marks by ATACseq and ChIPseq, and regulates ATP2A2 expression. We also generated developing glutamatergic neurons (GNs), another population with enriched SCZ heritability, and confirmed their functionality after transplantation into the mouse brain. Then, we identified SCZ GWAS risk genes that are dysregulated in developing SCZ GNs. GN-specific SCZ GWAS loci DE genes converge on the ion transporter pathway, distinct from those for cINs. Disruption of the pathway gene CACNA1D resulted in deficits of Ca2+ currents in developing GNs, suggesting compromised neuronal function by GWAS loci pathway deficits during development. This study allows us to identify cell type-specific and developmental stage-specific mechanisms of SCZ risk gene function, and may aid in identifying mechanism-based novel therapeutic targets.

Zelquistinel Is an Orally Bioavailable Novel NMDA Receptor Allosteric Modulator That Exhibits Rapid and Sustained Antidepressant-Like Effects.

BACKGROUND: The role of glutamatergic receptors in major depressive disorder continues to be of great interest for therapeutic development. Recent studies suggest that both negative and positive modulation of N-methyl-D-aspartate receptors (NMDAR) can produce rapid antidepressant effects. Here we report that zelquistinel, a novel NMDAR allosteric modulator, exhibits high oral bioavailability and dose-proportional exposures in plasma and the central nervous system and produces rapid and sustained antidepressant-like effects in rodents by enhancing activity-dependent, long-term synaptic plasticity. METHODS: NMDAR-mediated functional activity was measured in cultured rat brain cortical neurons (calcium imaging), hNR2A or B subtype-expressing HEK cells, and synaptic plasticity in rat hippocampal and medial prefrontal cortex slices in vitro. Pharmacokinetics were evaluated in rats following oral administration. Antidepressant-like effects were assessed in the rat forced swim test and the chronic social deficit mouse model. Target engagement and the safety/tolerability profile was assessed using phencyclidine-induced hyperlocomotion and rotarod rodent models. RESULTS: Following a single oral dose, zelquistinel (0.1-100 µg/kg) produced rapid and sustained antidepressant-like effects in the rodent depression models. Brain/ cerebrospinal fluid concentrations associated with zelquistinel antidepressant-like activity also increased NMDAR function and rapidly and persistently enhanced activity-dependent synaptic plasticity (long-term potentiation), suggesting that zelquistinel produces antidepressant-like effects by enhancing NMDAR function and synaptic plasticity. Furthermore, Zelquistinel inhibited phencyclidine (an NMDAR antagonist)-induced hyperlocomotion and did not impact rotarod performance. CONCLUSIONS: Zelquistinel produces rapid and sustained antidepressant effects by positively modulating the NMDARs, thereby enhancing long-term potentiation of synaptic transmission.

Developmental Time Course of SNAP-25 Isoforms Regulate Hippocampal Long-Term Synaptic Plasticity and Hippocampus-Dependent Learning.

SNAP-25 is essential to activity-dependent vesicle fusion and neurotransmitter release in the nervous system. During early development and adulthood, SNAP-25 appears to have differential influences on short- and long-term synaptic plasticity. The involvement of SNAP-25 in these processes may be different at hippocampal and neocortical synapses because of the presence of two different splice variants, which are developmentally regulated. We show here that the isoform SNAP-25a, which is expressed first developmentally in rodent brain, contributes to developmental regulation of the expression of both long-term depression (LTD) and long-term potentiation (LTP) at Schaffer collateral-CA1 synapses in the hippocampus. In one month old mice lacking the developmentally later expressed isoform SNAP-25b, Schaffer collateral-CA1 synapses showed faster release kinetics, decreased LTP and enhanced LTD. By four months of age, SNAP-25b-deficient mice appeared to have compensated for the lack of the adult SNAP-25b isoform, now exhibiting larger LTP and no differences in LTD compared to wild type mice. Interestingly, learning a hippocampus-dependent task reversed the reductions in LTP, but not LTD, seen at one month of age. In four month old adult mice, learning prevented the compensatory up-regulation of LTD that we observed prior to training. These findings support the hypothesis that SNAP-25b promotes stronger LTP and weakens LTD at Schaffer collateral-CA1 synapses in young mice, and suggest that compensatory mechanisms can reverse alterations in synaptic plasticity associated with a lack of SNAP-25b, once mice reach adulthood.

Positive N-Methyl-D-Aspartate Receptor Modulation by Rapastinel Promotes Rapid and Sustained Antidepressant-Like Effects.

BACKGROUND: Modulation of glutamatergic synaptic transmission by N-methyl-D-aspartate receptors can produce rapid and sustained antidepressant effects. Rapastinel (GLYX-13), initially described as a N-methyl-D-aspartate receptor partial glycine site agonist, exhibits rapid antidepressant effect in rodents without the accompanying dissociative effects of N-methyl-D-aspartate receptor antagonists. METHODS: The relationship between rapastinel's in vitro N-methyl-D-aspartate receptor pharmacology and antidepressant efficacy was determined by brain microdialysis and subsequent pharmacological characterization of therapeutic rapastinel concentrations in N-methyl-D-aspartate receptor-specific radioligand displacement, calcium mobilization, and medial prefrontal cortex electrophysiology assays. RESULTS: Brain rapastinel concentrations of 30 to 100 nM were associated with its antidepressant-like efficacy and enhancement of N-methyl-D-aspartate receptor-dependent neuronal intracellular calcium mobilization. Modulation of N-methyl-D-aspartate receptors by rapastinel was independent of D-serine concentrations, and glycine site antagonists did not block rapastinel's effect. In rat medial prefrontal cortex slices, 100 nM rapastinel increased N-methyl-D-aspartate receptor-mediated excitatory postsynaptic currents and enhanced the magnitude of long-term potentiation without any effect on miniature EPSCs or paired-pulse facilitation responses, indicating postsynaptic action of rapastinel. A critical amino acid within the NR2 subunit was identified as necessary for rapastinel's modulatory effect. CONCLUSION: Rapastinel brain concentrations associated with antidepressant-like activity directly enhance medial prefrontal cortex N-methyl-D-aspartate receptor activity and N-methyl-D-aspartate receptor-mediated synaptic plasticity in vitro. At therapeutic concentrations, rapastinel directly enhances N-methyl-D-aspartate receptor activity through a novel site independent of the glycine coagonist site. While both rapastinel and ketamine physically target N-methyl-D-aspartate receptors, the 2 molecules have opposing actions on N-methyl-D-aspartate receptors. Modest positive modulation of N-methyl-D-aspartate receptors by rapastinel represents a novel pharmacological approach to promote well-tolerated, rapid, and sustained improvements in mood disorders.

Concussion, microvascular injury, and early tauopathy in young athletes after impact head injury and an impact concussion mouse model.

The mechanisms underpinning concussion, traumatic brain injury, and chronic traumatic encephalopathy, and the relationships between these disorders, are poorly understood. We examined post-mortem brains from teenage athletes in the acute-subacute period after mild closed-head impact injury and found astrocytosis, myelinated axonopathy, microvascular injury, perivascular neuroinflammation, and phosphorylated tau protein pathology. To investigate causal mechanisms, we developed a mouse model of lateral closed-head impact injury that uses momentum transfer to induce traumatic head acceleration. Unanaesthetized mice subjected to unilateral impact exhibited abrupt onset, transient course, and rapid resolution of a concussion-like syndrome characterized by altered arousal, contralateral hemiparesis, truncal ataxia, locomotor and balance impairments, and neurobehavioural deficits. Experimental impact injury was associated with axonopathy, blood-brain barrier disruption, astrocytosis, microgliosis (with activation of triggering receptor expressed on myeloid cells, TREM2), monocyte infiltration, and phosphorylated tauopathy in cerebral cortex ipsilateral and subjacent to impact. Phosphorylated tauopathy was detected in ipsilateral axons by 24 h, bilateral axons and soma by 2 weeks, and distant cortex bilaterally at 5.5 months post-injury. Impact pathologies co-localized with serum albumin extravasation in the brain that was diagnostically detectable in living mice by dynamic contrast-enhanced MRI. These pathologies were also accompanied by early, persistent, and bilateral impairment in axonal conduction velocity in the hippocampus and defective long-term potentiation of synaptic neurotransmission in the medial prefrontal cortex, brain regions distant from acute brain injury. Surprisingly, acute neurobehavioural deficits at the time of injury did not correlate with blood-brain barrier disruption, microgliosis, neuroinflammation, phosphorylated tauopathy, or electrophysiological dysfunction. Furthermore, concussion-like deficits were observed after impact injury, but not after blast exposure under experimental conditions matched for head kinematics. Computational modelling showed that impact injury generated focal point loading on the head and seven-fold greater peak shear stress in the brain compared to blast exposure. Moreover, intracerebral shear stress peaked before onset of gross head motion. By comparison, blast induced distributed force loading on the head and diffuse, lower magnitude shear stress in the brain. We conclude that force loading mechanics at the time of injury shape acute neurobehavioural responses, structural brain damage, and neuropathological sequelae triggered by neurotrauma. These results indicate that closed-head impact injuries, independent of concussive signs, can induce traumatic brain injury as well as early pathologies and functional sequelae associated with chronic traumatic encephalopathy. These results also shed light on the origins of concussion and relationship to traumatic brain injury and its aftermath.awx350media15713427811001.

NYX-2925 Is a Novel NMDA Receptor-Specific Spirocyclic-ß-Lactam That Modulates Synaptic Plasticity Processes Associated with Learning and Memory.

Background: N-methyl-D-aspartate receptors are one member of a family of ionotropic glutamate receptors that play a pivotal role in synaptic plasticity processes associated with learning and have become attractive therapeutic targets for diseases such as depression, anxiety, schizophrenia, and neuropathic pain. NYX-2925 ((2S, 3R)-3-hydroxy-2-((R)-5-isobutyryl-1-oxo-2,5-diazaspiro[3.4]octan-2-yl)butanamide) is one member of a spiro-ß-lactam-based chemical platform that mimics some of the dipyrrolidine structural features of rapastinel (formerly GLYX-13: threonine-proline-proline-threonine) and is distinct from known N-methyl-D-aspartate receptor agonists or antagonists such as D-cycloserine, ketamine, MK-801, kynurenic acid, or ifenprodil. Methods: The in vitro and in vivo pharmacological properties of NYX-2925 were examined. Results: NYX-2925 has a low potential for "off-target" activity, as it did not exhibit any significant affinity for a large panel of neuroactive receptors, including hERG receptors. NYX-2925 increased MK-801 binding to human N-methyl-D-aspartate receptor NR2A-D subtypes expressed in HEK cells and enhanced N-methyl-D-aspartate receptor current and long-term potentiation (LTP) in rat hippocampal slices (100-500 nM). Single dose ex vivo studies showed increased metaplasticity in a hippocampal LTP paradigm and structural plasticity 24 hours after administration (1 mg/kg p.o.). Significant learning enhancement in both novel object recognition and positive emotional learning paradigms were observed (0.01-1 mg/kg p.o.), and these effects were blocked by the N-methyl-D-aspartate receptor antagonist CPP. NYX-2925 does not show any addictive or sedative/ataxic side effects and has a therapeutic index of >1000. NYX-2925 (1 mg/kg p.o.) has a cerebrospinal fluid half-life of 1.2 hours with a Cmax of 44 nM at 1 hour. Conclusions: NYX-2925, like rapastinel, activates an NMDA receptor-mediated synaptic plasticity process and may have therapeutic potential for a variety of NMDA receptor-mediated central nervous system disorders.

IGFBP2 Produces Rapid-Acting and Long-Lasting Effects in Rat Models of Posttraumatic Stress Disorder via a Novel Mechanism Associated with Structural Plasticity.

Background: Posttraumatic stress disorder is an anxiety disorder characterized by deficits in the extinction of aversive memories. Insulin-like growth factor 1 (IGF1) is the only growth factor that has shown anxiolytic and antidepressant properties in human clinical trials. In animal studies, insulin-like growth factor binding protein 2 (IGFBP2) shows both IGF1-dependent and IGF1-independent pharmacological effects, and IGFBP2 expression is upregulated by rough-and-tumble play that induces resilience to stress. Methods: IGFBP2 was evaluated in Porsolt, contextual fear conditioning, and chronic unpredictable stress models of posttraumatic stress disorder. The dependence of IGFBP2 effects on IGF1- and AMPA-receptor activation was tested using selective receptor antagonists. Dendritic spine morphology was measured in the dentate gyrus and the medial prefrontal cortex 24 hours after in vivo dosing. Results: IGFBP2 was 100 times more potent than IGF1 in the Porsolt test. Unlike IGF1, effects of IGFBP2 were not blocked by the IGF1-receptor antagonist JB1, or by the AMPA-receptor antagonist 2,3-Dioxo-6-nitro-1,2,3,4 tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) in the Porsolt test. IGFBP2 (1 µg/kg) and IGF1 (100 µg/kg i.v.) each facilitated contextual fear extinction and consolidation. Using a chronic unpredictable stress paradigm, IGFBP2 reversed stress-induced effects in the Porsolt, novelty-induced hypophagia, sucrose preference, and ultrasonic vocalization assays. IGFBP2 also increased mature dendritic spine densities in the medial prefrontal cortex and hippocampus 24 hours postdosing. Conclusions: These data suggest that IGFBP2 has therapeutic-like effects in multiple rat models of posttraumatic stress disorder via a novel IGF1 receptor-independent mechanism. These data also suggest that the long-lasting effects of IGFBP2 may be due to facilitation of structural plasticity at the dendritic spine level. IGFBP2 and mimetics may have therapeutic potential for the treatment of posttraumatic stress disorder.

Insulin-Like Growth Factor I Produces an Antidepressant-Like Effect and Elicits N-Methyl-D-Aspartate Receptor Independent Long-Term Potentiation of Synaptic Transmission in Medial Prefrontal Cortex and Hippocampus.

BACKGROUND: Growth factors play an important role in regulating neurogenesis and synapse formation and may be involved in regulating the antidepressant response to conventional antidepressants. To date, Insulin-like growth factor I (IGFI) is the only growth factor that has shown antidepressant properties in human clinical trials. However, its mechanism of action remains unclear. METHODS: The antidepressant-like effect of a single IV dose of IGFI was determined using a chronic unpredictable stress paradigm in the rat Porsolt, sucrose preference, novelty-induced hypophagia, and ultrasonic vocalization models. The dependence of the medial prefrontal cortex for these effects was determined by direct medial prefrontal cortex injection followed by Porsolt testing as well as IGFI receptor activation in the medial prefrontal cortex following an optimal IV antidepressant-like dose of IGFI. The effect of IGFI on synaptic transmission and long-term potentiation (LTP) of synaptic strength was assessed in the hippocampus and medial prefrontal cortex. The dependence of these effects on IGFI and AMPA receptor activation and protein synthesis were also determined. RESULTS: IGFI produced a rapid-acting and long-lasting antidepressant-like effect in each of the depression models. These effects were blocked by IGFI and AMPA receptor antagonists, and medial prefrontal cortex was localized. IGFI robustly increased synaptic strength in the hippocampus and medial prefrontal cortex and these effects were IGFI receptor and protein synthesis-dependent but N-methyl-d-aspartate receptor independent. IGFI also robustly facilitated hippocampal metaplasticity 24 hours postdosing. CONCLUSIONS: These data support the conclusion that the antidepressant-like effects of IGFI are mediated by a persistent, LTP-like enhancement of synaptic strength requiring both IGFIR activation and ongoing protein synthesis.

Essential role for synaptopodin in dendritic spine plasticity of the developing hippocampus.

Dendritic spines are a major substrate of brain plasticity. Although many studies have focused on Ca(2+)/calmodulin-dependent protein kinase II (CaMKII)-mediated regulation of spine dynamics and synaptic function in adult brain, much less is know about protein kinase A (PKA)-dependent regulation of spine shape dynamics during postnatal brain development. Synaptopodin is a dendritic spine associated modulator of actin dynamics and a substrate of PKA. Here we show that NMDA and cAMP-induced dendritic spine expansion is impaired in hippocampal slices from 15- and 21-d-old synaptopodin-deficient mice. We further show that synaptopodin is required for full expression of PKA-dependent hippocampal long-term potentiation in 15- and 21-d-old, but not adult, mice. PKA-induced cAMP response element-binding phosphorylation is normal in the hippocampus of synaptopodin-deficient mice, suggesting that synaptopodin functions independently of cAMP response element-binding. Our results identify synaptopodin as a substrate of PKA in hippocampal neurons and point to an essential role for synaptopodin in activity-dependent regulation of dendritic spine dynamics and synaptic plasticity in postnatal brain development.

Two distinct modes of exocytotic fusion pore expansion in large astrocytic vesicles.

Formation of the fusion pore is a central question for regulated exocytosis by which secretory cells release neurotransmitters or hormones. Here, by dynamically monitoring exocytosis of large vesicles (2-7 µM) in astrocytes with two-photon microscopy imaging, we found that the exocytotic fusion pore was generated from the SNARE-dependent fusion at a ring shape of the docked plasma-vesicular membrane and the movement of a fusion-produced membrane fragment. We observed two modes of fragment movements, 1) a shift fragment that shifted to expand the fusion pore and 2) a fall-in fragment that fell into the collapsed vesicle to expand the fusion pore. Shift and fall-in modes are associated with full and partial collapses of large vesicles, respectively. The astrocytic marker, sulforhodamine 101, stained the fusion-produced membrane fragment more brightly than FM 1-43. Sulforhodamine 101 imaging showed that double fusion pores could simultaneously occur in a single vesicle (16% of large vesicles) to accelerate discharge of vesicular contents. Electron microscopy of large astrocytic vesicles showed shift and fall-in membrane fragments. Two modes of fusion pore formation demonstrate a novel mechanism underlying fusion pore expansion and provide a new explanation for full and partial collapses of large secretory vesicles.

Altered neurotransmitter release, vesicle recycling and presynaptic structure in the pilocarpine model of temporal lobe epilepsy.

In searching for persistent seizure-induced alterations in brain function that might be causally related to epilepsy, presynaptic transmitter release has relatively been neglected. To measure directly the long-term effects of pilocarpine-induced status epilepticus on vesicular release and recycling in hippocampal mossy fibre presynaptic boutons, we used (i) two-photon imaging of FM1-43 vesicular release in rat hippocampal slices; and (ii) transgenic mice expressing the genetically encoded pH-sensitive fluorescent reporter synaptopHluorin preferentially at glutamatergic synapses. In this study we found that, 1-2 months after pilocarpine-induced status epilepticus, there were significant increases in mossy fibre bouton size, faster rates of action potential-driven vesicular release and endocytosis. We also analysed the ultrastructure of rat mossy fibre boutons using transmission electron microscopy. Pilocarpine-induced status epilepticus led to a significant increase in the number of release sites, active zone length, postsynaptic density area and number of vesicles in the readily releasable and recycling pools, all correlated with increased release probability. Our data show that presynaptic release machinery is persistently altered in structure and function by status epilepticus, which could contribute to the development of the chronic epileptic state and may represent a potential new target for antiepileptic therapies.

Astrocyte and neuronal Panx1 support long-term reference memory in mice.

Pannexin 1 (Panx1) are ubiquitously expressed proteins that form plasma membrane channels permeable to anions and moderate sized signaling molecules (e.g., ATP, glutamate). In the nervous system, activation of Panx1 channels have been extensively shown to contribute to distinct neurological disorders (epilepsy, chronic pain, migraine, neuroAIDS, etc.) but knowledge of extent to which these channels have a physiological role remains restricted to three studies supporting their involvement in hippocampus dependent learning. Given that Panx1 channels may provide an important mechanism for activity-dependent neuron-glia interaction, we used Panx1 transgenic mice with global and cell-type specific deletions of Panx1 to interrogate their participation in working and reference memory. Using the 8-arm radial maze, we show that long-term spatial reference memory, but not spatial working memory, is deficient in Panx1-null mice and that both astrocyte and neuronal Panx1 contribute to the consolidation of long-term spatial memory. Field potential recordings in hippocampal slices of Panx1-null mice revealed an attenuation of both long-term potentiation (LTP) of synaptic strength and long-term depression (LTD) at Schaffer collateral - CA1 synapses without alterations basal synaptic transmission or pre-synaptic paired-pulse facilitation. Our results implicate both neuronal and astrocyte Panx1 channels as critical players for the development and maintenance of long-term spatial reference memory in mice.

Astrocyte and Neuronal Panx1 Support Long-Term Reference Memory in Mice.

Pannexin 1 (Panx1) is an ubiquitously expressed protein that forms plasma membrane channels permeable to anions and moderate-sized signaling molecules (e.g., ATP, glutamate). In the nervous system, activation of Panx1 channels has been extensively shown to contribute to distinct neurological disorders (epilepsy, chronic pain, migraine, neuroAIDS, etc.), but knowledge of the extent to which these channels have a physiological role remains restricted to three studies supporting their involvement in hippocampus dependent learning. Given that Panx1 channels may provide an important mechanism for activity-dependent neuron-glia interaction, we used Panx1 transgenic mice with global and cell-type specific deletions of Panx1 to interrogate their participation in working and reference memory. Using the eight-arm radial maze, we show that long-term spatial reference memory, but not spatial working memory, is deficient in Panx1-null mice and that both astrocyte and neuronal Panx1 contribute to the consolidation of long-term spatial memory. Field potential recordings in hippocampal slices of Panx1-null mice revealed an attenuation of both long-term potentiation (LTP) of synaptic strength and long-term depression (LTD) at Schaffer collateral-CA1 synapses without alterations of basal synaptic transmission or pre-synaptic paired-pulse facilitation. Our results implicate both neuronal and astrocyte Panx1 channels as critical players for the development and maintenance of long-term spatial reference memory in mice.

Myeloid cell deficiency of the inflammatory transcription factor Stat4 protects long-term synaptic plasticity from the effects of a high-fat, high-cholesterol diet.

Neuroinflammation is associated with neurodegenerative diseases, including Alzheimer's and Parkinson's. The cytokine interleukin-12 activates signal transducer and activator of transcription 4 (Stat4), and consumption of a high-fat, high-cholesterol diet (HFD-C) and Stat4 activity are associated with inflammation, atherosclerosis, and a diabetic metabolic phenotype. In studies of in vitro hippocampal slices from control Stat4fl/flLdlr-/- mice fed a HFD-C diabetogenic diet, we show that Schaffer collateral-CA1 synapses exhibited larger reductions in activity-dependent, long-term potentiation (LTP) of synaptic transmission, compared to mice fed a standard diet. Glucose tolerance and insulin sensitivity shifts produced by HFD-C diet were reduced in Stat4ΔLysMLdlr-/- mice compared to Stat4fl/flLdlr-/- controls. Stat4ΔLysMLdlr-/- mice, which lack Stat4 under control of the LysMCre promoter, were resistant to HFD-C induced impairments in LTP. In contrast, Schaffer collateral-CA1 synapses in Stat4ΔLysMLdlr-/- mice fed the HFD-C diet showed larger LTP than control Stat4fl/flLdlr-/- mice. Expression of a number of neuroinflammatory and synaptic plasticity genes was reduced by HFD-C diet in control mice, and less affected by HFD-C diet in Stat4ΔLysMLdlr-/- mice. These data suggest that suppression of Stat4 activation may protect against effects of Western diet on cognition, type 2 diabetes, and reduce risk of Alzheimer's disease and other neurodegenerative disorders associated with neuroinflammation.

Human cortical interneurons optimized for grafting specifically integrate, abort seizures, and display prolonged efficacy without over-inhibition.

Previously, we demonstrated the efficacy of human pluripotent stem cell (hPSC)-derived GABAergic cortical interneuron (cIN) grafts in ameliorating seizures. However, a safe and reliable clinical translation requires a mechanistic understanding of graft function, as well as the assurance of long-term efficacy and safety. By employing hPSC-derived chemically matured migratory cINs in two models of epilepsy, we demonstrate lasting efficacy in treating seizures and comorbid deficits, as well as safety without uncontrolled growth. Host inhibition does not increase with increasing grafted cIN densities, assuring their safety without the risk of over-inhibition. Furthermore, their closed-loop optogenetic activation aborted seizure activity, revealing mechanisms of graft-mediated seizure control and allowing graft modulation for optimal translation. Monosynaptic tracing shows their extensive and specific synaptic connections with host neurons, resembling developmental connection specificity. These results offer confidence in stem cell-based therapy for epilepsy as a safe and reliable treatment for patients suffering from intractable epilepsy.

Novel organotypic in vitro slice culture model for intraventricular hemorrhage of premature infants.

Mechanisms of brain injury in intraventricular hemorrhage (IVH) of premature infants are elusive, and no therapeutic strategy exists to prevent brain damage in these infants. Therefore, we developed an in vitro organotypic forebrain slice culture model to advance mechanistic studies and therapeutic developments for this disorder. We cultured forebrain slices from E29 rabbit pups and treated the cultured slices (CS) with moderate (50 µl) or large (100 µl) amounts of autologous blood to mimic moderate and severe IVH. Blood-induced damage to CS was evaluated by propidium iodide staining, lactate dehydrogenase (LDH) levels, microglial density, neuronal degeneration, myelination, and gliosis over 2 weeks after the initiation of culture. CS were viable for at least 14 days in vitro (DIV). The application of blood induced significant neural cell degeneration. Degenerating cells were more abundant and LDH levels were elevated in a dose-dependent manner in CS treated with 50 versus 100 µl of blood compared with untreated controls. Microglial density was higher in blood-treated CS compared with controls at both 7 and 14 days posttreatment, and myelination was reduced and gliosis enhanced. Selective application of blood fractions revealed that CS treated with plasma displayed more hypomyelination and gliosis compared with erythrocyte-treated slices. This study develops and characterizes a novel rabbit forebrain slice culture model of IVH that exhibits neuropatholgical changes similar to those in human infants with IVH. Importantly, plasma appears to induce greater white matter damage than erythrocytes in IVH,indicating plasma as a source of neurotoxic components.

Differential antagonism of tetramethylenedisulfotetramine-induced seizures by agents acting at NMDA and GABA(A) receptors.

Tetramethylenedisulfotetramine (TMDT) is a highly lethal neuroactive rodenticide responsible for many accidental and intentional poisonings in mainland China. Ease of synthesis, water solubility, potency, and difficulty to treat make TMDT a potential weapon for terrorist activity. We characterized TMDT-induced convulsions and mortality in male C57BL/6 mice. TMDT (ip) produced a continuum of twitches, clonic, and tonic-clonic seizures decreasing in onset latency and increasing in severity with increasing dose; 0.4mg/kg was 100% lethal. The NMDA antagonist, ketamine (35mg/kg) injected ip immediately after the first TMDT-induced seizure, did not change number of tonic-clonic seizures or lethality, but increased the number of clonic seizures. Doubling the ketamine dose decreased tonic-clonic seizures and eliminated lethality through a 60min observation period. Treating mice with another NMDA antagonist, MK-801, 0.5 or 1mg/kg ip, showed similar effects as low and high doses of ketamine, respectively, and prevented lethality, converting status epilepticus EEG activity to isolated interictal discharges. Treatment with these agents 15min prior to TMDT administration did not increase their effectiveness. Post-treatment with the GABA(A) receptor allosteric enhancer diazepam (5mg/kg) greatly reduced seizure manifestations and prevented lethality 60min post-TMDT, but ictal events were evident in EEG recordings and, hours post-treatment, mice experienced status epilepticus and died. Thus, TMDT is a highly potent and lethal convulsant for which single-dose benzodiazepine treatment is inadequate in managing electrographic seizures or lethality. Repeated benzodiazepine dosing or combined application of benzodiazepines and NMDA receptor antagonists is more likely to be effective in treating TMDT poisoning.

Extracellular application of the N-methyl-D-aspartate receptor allosteric modulator rapastinel acts remotely to regulate Ca2+ inactivation at an intracellular locus.

BACKGROUND: A novel N-methyl-D-aspartate receptor (NMDAR) allosteric modulator, rapastinel (RAP, formerly GLYX-13), elicits long-lasting antidepressant-like effects by enhancing long-term potentiation (LTP) of synaptic transmission. RAP elicits these effects by binding to a unique site in the extracellular region of the NMDAR complex, transiently enhancing NMDAR-gated current in pyramidal neurons of both hippocampus and medial prefrontal cortex. METHODS: We compared efficacy of RAP in modulating Schaffer collateral-evoked NMDAR-currents as a function of kinetics of the Ca2+ chelator in the intracellular solution, using whole-cell patch-clamp recordings. The intracellular solution contained either the slow Ca2+ chelator EGTA [3,12-bis(carboxymethyl)-6,9-dioxa-3,12-diazatetradecane-1,14-dioic acid, 0.5 mmol/l] or the 40-500-fold kinetically faster, more selective Ca2+ chelator BAPTA {2,2',2″,2‴-[ethane-1,2-diylbis(oxy-2,1-phenylenenitrilo)] tetraacetic acid, 5 mmol/l}. NMDAR-gated currents were pharmacologically isolated by bath application of the 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid receptor antagonist 6-nitro-2,3-dioxo-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (10 µmol/l) plus the GABA receptor blocker bicuculline (20 µmol/l). RESULTS: When the slow Ca2+ chelator EGTA was in the intracellular solution, RAP elicited significant enhancement of NMDAR-gated current at a 1 µmol/l concentration, and significantly reduced current at 10 µmol/l. In contrast, when recording with the 40-500-fold kinetically faster, more selective Ca2+ chelator BAPTA, NMDAR current increased in magnitude by 84% as BAPTA washed into the cell, and the enhancement of NMDAR current by 1 µmol/l RAP was completely blocked. Interestingly, the reduction in NMDAR current from 10 µmol/l RAP was not affected by the presence of BAPTA in the recording pipette, indicating that this effect is mediated by a different mechanism. CONCLUSION: Extracellular binding of RAP to the NMDAR produces a novel, long-range reduction in affinity of the Ca2+ inactivation site on the NMDAR C-terminus accessible to the intracellular space. This action underlies enhancement in NMDAR-gated conductance elicited by RAP.