X-linked cellular mosaicism underlies age-dependent occurrence of seizure-like events in mouse models of CDKL5 deficiency disorder.

CDKL5 deficiency disorder (CDD) is an infantile, epileptic encephalopathy presenting with early-onset seizures, intellectual disability, motor impairment, and autistic features. The disorder has been linked to mutations in the X-linked CDKL5, and mouse models of the disease recapitulate several aspects of CDD symptomology, including learning and memory impairments, motor deficits, and autistic-like features. Although early-onset epilepsy is one of the hallmark features of CDD, evidence of spontaneous seizure activity has only recently been described in Cdkl5-deficient heterozygous female mice, but the etiology, prevalence, and sex-specificity of this phenotype remain unknown. Here, we report the first observation of disturbance-associated seizure-like events in heterozygous female mice across two independent mouse models of CDD: Cdkl5 knockout mice and CDKL5 R59X knock-in mice. We find that both the prevalence and severity of this phenotype increase with aging, with a median onset around 28 weeks of age. Similar seizure-like events are not observed in hemizygous knockout male or homozygous knockout female littermates, suggesting that X-linked cellular mosaicism is a driving factor underlying these seizure-like events. Together, these findings not only contribute to our understanding of the effects of CDKL5 loss on seizure susceptibility, but also document a novel, pre-clinical phenotype for future therapeutic investigation.

α-Synuclein (αSyn) Preformed Fibrils Induce Endogenous αSyn Aggregation, Compromise Synaptic Activity and Enhance Synapse Loss in Cultured Excitatory Hippocampal Neurons.

Synucleinopathies are characterized by the accumulation of insoluble α-synuclein (αSyn). To test whether αSyn aggregates modulate synaptic activity, we used a recently developed model in primary neurons for inducing αSyn pathology. We demonstrated that preformed fibrils (PFFs) generated with recombinant human αSyn compromised synaptic activity in a time- and dose-dependent manner and that the magnitude of these deficits correlated with the formation of αSyn pathology in cultured excitatory hippocampal neurons from both sexes of mice. Remarkably, acute passive infusion of αSyn PFFs from whole-cell patch-clamp pipette decreased mEPSC frequency within 10 min followed by induction of αSyn pathology within 1 d. Moreover, by direct addition of αSyn PFFs into culture medium, the formation of misfolded αSyn inclusions dramatically compromised the colocalization of synaptic markers and altered dynamic changes of dendritic spines, but the viability of neurons was not affected up to 7 d post-treatment with αSyn PFFs. Our data indicate that intraneuronal αSyn fibrils impaired the initiation of synaptogenesis and their physiological functions, thereby suggesting that targeting synaptic dysfunction in synucleinopathies may provide a promising therapeutic direction.SIGNIFICANCE STATEMENT Under pathological conditions, the presynaptic protein α-synuclein (αSyn) aggregates to form intraneuronal inclusions. To understand how and to what extent αSyn aggregates modulate synaptic activity before neuron loss, we demonstrate that αSyn preformed fibrils (PFFs) reduced synaptic activity in a dose- and time-dependent manner. The magnitude of these deficits correlated with the deposition of αSyn pathology, which dramatically compromised the colocalization of synaptic markers and altered the dendritic spine dynamics. The present work further highlights the impact of αSyn PFFs on synaptogenesis and physiological function, which may be applicable to other types of synucleinopathies.

Circuit-based interventions in the dentate gyrus rescue epilepsy-associated cognitive dysfunction.

Temporal lobe epilepsy is associated with significant structural pathology in the hippocampus. In the dentate gyrus, the summative effect of these pathologies is massive hyperexcitability in the granule cells, generating both increased seizure susceptibility and cognitive deficits. To date, therapeutic approaches have failed to improve the cognitive symptoms in fully developed, chronic epilepsy. As the dentate's principal signalling population, the granule cells' aggregate excitability has the potential to provide a mechanistically-independent downstream target. We examined whether normalizing epilepsy-associated granule cell hyperexcitability-without correcting the underlying structural circuit disruptions-would constitute an effective therapeutic approach for cognitive dysfunction. In the systemic pilocarpine mouse model of temporal lobe epilepsy, the epileptic dentate gyrus excessively recruits granule cells in behavioural contexts, not just during seizure events, and these mice fail to perform on a dentate-mediated spatial discrimination task. Acutely reducing dorsal granule cell hyperactivity in chronically epileptic mice via either of two distinct inhibitory chemogenetic receptors rescued behavioural performance such that they responded comparably to wild type mice. Furthermore, recreating granule cell hyperexcitability in control mice via excitatory chemogenetic receptors, without altering normal circuit anatomy, recapitulated spatial memory deficits observed in epileptic mice. However, making the granule cells overly quiescent in both epileptic and control mice again disrupted behavioural performance. These bidirectional manipulations reveal that there is a permissive excitability window for granule cells that is necessary to support successful behavioural performance. Chemogenetic effects were specific to the targeted dorsal hippocampus, as hippocampal-independent and ventral hippocampal-dependent behaviours remained unaffected. Fos expression demonstrated that chemogenetics can modulate granule cell recruitment via behaviourally relevant inputs. Rather than driving cell activity deterministically or spontaneously, chemogenetic intervention merely modulates the behaviourally permissive activity window in which the circuit operates. We conclude that restoring appropriate principal cell tuning via circuit-based therapies, irrespective of the mechanisms generating the disease-related hyperactivity, is a promising translational approach.

Loss of CDKL5 in Glutamatergic Neurons Disrupts Hippocampal Microcircuitry and Leads to Memory Impairment in Mice.

Cyclin-dependent kinase-like 5 (CDKL5) deficiency is a neurodevelopmental disorder characterized by epileptic seizures, severe intellectual disability, and autistic features. Mice lacking CDKL5 display multiple behavioral abnormalities reminiscent of the disorder, but the cellular origins of these phenotypes remain unclear. Here, we find that ablating CDKL5 expression specifically from forebrain glutamatergic neurons impairs hippocampal-dependent memory in male conditional knock-out mice. Hippocampal pyramidal neurons lacking CDKL5 show decreased dendritic complexity but a trend toward increased spine density. This morphological change is accompanied by an increase in the frequency of spontaneous miniature EPSCs and interestingly, miniature IPSCs. Using voltage-sensitive dye imaging to interrogate the evoked response of the CA1 microcircuit, we find that CA1 pyramidal neurons lacking CDKL5 show hyperexcitability in their dendritic domain that is constrained by elevated inhibition in a spatially and temporally distinct manner. These results suggest a novel role for CDKL5 in the regulation of synaptic function and uncover an intriguing microcircuit mechanism underlying impaired learning and memory.SIGNIFICANCE STATEMENT Cyclin-dependent kinase-like 5 (CDKL5) deficiency is a severe neurodevelopmental disorder caused by mutations in the CDKL5 gene. Although Cdkl5 constitutive knock-out mice have recapitulated key aspects of human symptomatology, the cellular origins of CDKL5 deficiency-related phenotypes are unknown. Here, using conditional knock-out mice, we show that hippocampal-dependent learning and memory deficits in CDKL5 deficiency have origins in glutamatergic neurons of the forebrain and that loss of CDKL5 results in the enhancement of synaptic transmission and disruptions in neural circuit dynamics in a spatially and temporally specific manner. Our findings demonstrate that CDKL5 is an important regulator of synaptic function in glutamatergic neurons and serves a critical role in learning and memory.

Mechanisms of epileptogenesis: a convergence on neural circuit dysfunction.

Epilepsy is a prevalent neurological disorder associated with significant morbidity and mortality, but the only available drug therapies target its symptoms rather than the underlying cause. The process that links brain injury or other predisposing factors to the subsequent emergence of epilepsy is termed epileptogenesis. Substantial research has focused on elucidating the mechanisms of epileptogenesis so as to identify more specific targets for intervention, with the hope of preventing epilepsy before seizures emerge. Recent work has yielded important conceptual advances in this field. We suggest that such insights into the mechanisms of epileptogenesis converge at the level of cortical circuit dysfunction.

Respiratory failure, cleft palate and epilepsy in the mouse model of human Xq22.1 deletion syndrome.

Chromosomal segmental deletion is a frequent cause of human diseases. A familial 1.1 Mb deletion of human chromosome Xq22.1 associates with epilepsy, cleft palate and developmental defects in heterozygous female patients. Here, we describe a mouse mutant with a targeted deletion of the syntenic segment of the mouse X chromosome that phenocopies the human syndrome. Male mice with a deletion of a 1.1 Mb Nxf2-Nxf3 X-chromosomal segment exhibit respiratory failure, neonatal lethality and cleft palate. In female mice, heterozygosity for the deletion manifests cleft palate, early postnatal lethality, postnatal growth delay and spontaneous seizures in surviving animals, apparently due to X-chromosome inactivation. Furthermore, loss of a 0.35 Mb subregion containing Armcx5, Gprasp1, Gprasp2 and Bhlhb9 is sufficient to cause the Xq22.1 syndrome phenotype. Our results support that the 1.1 Mb deletion of human Xq22.1 is the genetic cause of the associated syndrome.

Neuronal activity and glutamate uptake decrease mitochondrial mobility in astrocytes and position mitochondria near glutamate transporters.

Within neurons, mitochondria are nonuniformly distributed and are retained at sites of high activity and metabolic demand. Glutamate transport and the concomitant activation of the Na(+)/K(+)-ATPase represent a substantial energetic demand on astrocytes. We hypothesized that mitochondrial mobility within astrocytic processes might be regulated by neuronal activity and glutamate transport. We imaged organotypic hippocampal slice cultures of rat, in which astrocytes maintain their highly branched morphologies and express glutamate transporters. Using time-lapse confocal microscopy, the mobility of mitochondria within individual astrocytic processes and neuronal dendrites was tracked. Within neurons, a greater percentage of mitochondria were mobile than in astrocytes. Furthermore, they moved faster and farther than in astrocytes. Inhibiting neuronal activity with tetrodotoxin (TTX) increased the percentage of mobile mitochondria in astrocytes. Mitochondrial movement in astrocytes was inhibited by vinblastine and cytochalasin D, demonstrating that this mobility depends on both the microtubule and actin cytoskeletons. Inhibition of glutamate transport tripled the percentage of mobile mitochondria in astrocytes. Conversely, application of the transporter substrate d-aspartate reversed the TTX-induced increase in the percentage of mobile mitochondria. Inhibition of reversed Na(+)/Ca(2+) exchange also increased the percentage of mitochondria that were mobile. Last, we demonstrated that neuronal activity increases the probability that mitochondria appose GLT-1 particles within astrocyte processes, without changing the proximity of GLT-1 particles to VGLUT1. These results imply that neuronal activity and the resulting clearance of glutamate by astrocytes regulate the movement of astrocytic mitochondria and suggest a mechanism by which glutamate transporters might retain mitochondria at sites of glutamate uptake.

Suppression of InsP3 receptor-mediated Ca2+ signaling alleviates mutant presenilin-linked familial Alzheimer's disease pathogenesis.

Exaggerated intracellular Ca(2+) signaling is a robust proximal phenotype observed in cells expressing familial Alzheimer's disease (FAD)-causing mutant presenilins (PSs). The mechanisms that underlie this phenotype are controversial and their in vivo relevance for AD pathogenesis is unknown. Here, we used a genetic approach to identify the mechanisms involved and to evaluate their role in the etiology of AD in two FAD mouse models. Genetic reduction of the type 1 inositol trisphosphate receptor (InsP3R1) by 50% normalized exaggerated Ca(2+) signaling observed in cortical and hippocampal neurons in both animal models. In PS1M146V knock-in mice, reduced InsP3R1 expression restored normal ryanodine receptor and cAMP response element-binding protein (CREB)-dependent gene expression and rescued aberrant hippocampal long-term potentiation (LTP). In 3xTg mice, reduced InsP3R1 expression profoundly attenuated amyloid ß accumulation and tau hyperphosphorylation and rescued hippocampal LTP and memory deficits. These results indicate that exaggerated Ca(2+) signaling, which is associated with FAD PS, is mediated by InsP3R and contributes to disease pathogenesis in vivo. Targeting the InsP3 signaling pathway could be considered a potential therapeutic strategy for patients harboring mutations in PS linked to AD.

Cortical parvalbumin GABAergic deficits with α7 nicotinic acetylcholine receptor deletion: implications for schizophrenia.

Dysfunction of cortical parvalbumin (PV)-containing GABAergic interneurons has been implicated in cognitive deficits of schizophrenia. In humans microdeletion of the CHRNA7 (α7 nicotinic acetylcholine receptor, nAChR) gene is associated with cortical dysfunction in a broad spectrum of neurodevelopmental and neuropsychiatric disorders including schizophrenia while in mice similar deletion causes analogous abnormalities including impaired attention, working-memory and learning. However, the pathophysiological roles of α7 nAChRs in cortical PV GABAergic development remain largely uncharacterized. In both in vivo and in vitro models, we identify here that deletion of the α7 nAChR gene in mice impairs cortical PV GABAergic development and recapitulates many of the characteristic neurochemical deficits in PV-positive GABAergic interneurons found in schizophrenia. α7 nAChR null mice had decreased cortical levels of GABAergic markers including PV, glutamic acid decarboxylase 65/67 (GAD65/67) and the α1 subunit of GABAA receptors, particularly reductions of PV and GAD67 levels in cortical PV-positive interneurons during late postnatal life and adulthood. Cortical GABAergic synaptic deficits were identified in the prefrontal cortex of α7 nAChR null mice and α7 nAChR null cortical cultures. Similar disruptions in development of PV-positive GABAergic interneurons and perisomatic synapses were found in cortical cultures lacking α7 nAChRs. Moreover, NMDA receptor expression was reduced in GABAergic interneurons, implicating NMDA receptor hypofunction in GABAergic deficits in α7 nAChR null mice. Our findings thus demonstrate impaired cortical PV GABAergic development and multiple characteristic neurochemical deficits reminiscent of schizophrenia in cortical PV-positive interneurons in α7 nAChR gene deletion models. This implicates crucial roles of α7 nAChRs in cortical PV GABAergic development and dysfunction in schizophrenia and other neuropsychiatric disorders.

Protracted postnatal development of sparse, specific dentate granule cell activation in the mouse hippocampus.

The dentate gyrus (DG) is a critical entry point regulating function of the hippocampus. Integral to this role are the sparse, selective activation characteristics of the principal cells of the DG, dentate granule cells (DGCs). This sparse activation is important both in cognitive processing and in regulation of pathological activity in disease states. Using a novel, combined dynamic imaging approach capable of resolving sequentially both synaptic potentials and action potential firing in large populations of DGCs, we characterized the postnatal development of firing properties of DG neurons in response to afferent activation in mouse hippocampal-entorhinal cortical slices. During postnatal development, there was a protracted, progressive sparsification of responses, accompanied by increased temporal precision of activation. Both of these phenomena were primarily mediated by changes in local circuit inhibition, and not by alterations in afferent innervation of DGCs because GABA(A) antagonists normalized developmental differences. There was significant θ and γ frequency-dependent synaptic recruitment of DGC activation in adult, but not developing, animals. Finally, we found that the decision to fire or not fire by individual DGCs was robust and repeatable at all stages of development. The protracted postnatal development of sparse, selective firing properties, increased temporal precision and frequency dependence of activation, and the fidelity with which the decision to fire is made are all fundamental circuit determinants of DGC excitation, critical in both normal and pathological function of the DG.

Cortical synaptic NMDA receptor deficits in α7 nicotinic acetylcholine receptor gene deletion models: implications for neuropsychiatric diseases.

Microdeletion of the human CHRNA7 gene (α7 nicotinic acetylcholine receptor, nAChR) as well as dysfunction in N-methyl-d-aspartate receptors (NMDARs) have been associated with cortical dysfunction in a broad spectrum of neurodevelopmental and neuropsychiatric disorders including schizophrenia. However, the pathophysiological roles of synaptic vs. extrasynaptic NMDARs and their interactions with α7 nAChRs in cortical dysfunction remain largely uncharacterized. Using a combination of in vivo and in vitro models, we demonstrate that α7 nAChR gene deletion leads to specific loss of synaptic NMDARs and their coagonist, d-serine, as well as glutamatergic synaptic deficits in mouse cortex. α7 nAChR null mice had decreased cortical NMDAR expression and glutamatergic synapse formation during postnatal development. Similar reductions in NMDAR expression and glutamatergic synapse formation were revealed in cortical cultures lacking α7 nAChRs. Interestingly, synaptic, but not extrasynaptic, NMDAR currents were specifically diminished in cultured cortical pyramidal neurons as well as in acute prefrontal cortical slices of α7 nAChR null mice. Moreover, d-serine responsive synaptic NMDAR-mediated currents and levels of the d-serine synthetic enzyme serine racemase were both reduced in α7 nAChR null cortical pyramidal neurons. Our findings thus identify specific loss of synaptic NMDARs and their coagonist, d-serine, as well as glutamatergic synaptic deficits in α7 nAChR gene deletion models of cortical dysfunction, thereby implicating α7 nAChR-mediated control of synaptic NMDARs and serine racemase/d-serine pathways in cortical dysfunction underlying many neuropsychiatric and neurodevelopmental disorders, particularly those associated with deletion of human CHRNA7.

Engraftment of nonintegrating neural stem cells differentially perturbs cortical activity in a dose-dependent manner.

Neural stem cell (NSC) therapy represents a potentially powerful approach for gene transfer in the diseased central nervous system. However, transplanted primary, embryonic stem cell- and induced pluripotent stem cell-derived NSCs generate largely undifferentiated progeny. Understanding how physiologically immature cells influence host activity is critical to evaluating the therapeutic utility of NSCs. Earlier inquiries were limited to single-cell recordings and did not address the emergent properties of neuronal ensembles. To interrogate cortical networks post-transplant, we used voltage sensitive dye imaging in mouse neocortical brain slices, which permits high temporal resolution analysis of neural activity. Although moderate NSC engraftment largely preserved host physiology, subtle defects in the activation properties of synaptic inputs were induced. High-density engraftment severely dampened cortical excitability, markedly reducing the amplitude, spatial extent, and velocity of propagating synaptic potentials in layers 2-6. These global effects may be mediated by specific disruptions in excitatory network structure in deep layers. We propose that depletion of endogenous cells in engrafted neocortex contributes to circuit alterations. Our data provide the first evidence that nonintegrating cells cause differential host impairment as a function of engrafted load. Moreover, they emphasize the necessity for efficient differentiation methods and proper controls for engraftment effects that interfere with the benefits of NSC therapy.

Deterministic and stochastic neuronal contributions to distinct synchronous CA3 network bursts.

Computational studies have suggested that stochastic, deterministic, and mixed processes all could be possible determinants of spontaneous, synchronous network bursts. In the present study, using multicellular calcium imaging coupled with fast confocal microscopy, we describe neuronal behavior underlying spontaneous network bursts in developing rat and mouse hippocampal area CA3 networks. Two primary burst types were studied: giant depolarizing potentials (GDPs) and spontaneous interictal bursts recorded in bicuculline, a GABA(A) receptor antagonist. Analysis of the simultaneous behavior of multiple CA3 neurons during synchronous GDPs revealed a repeatable activation order from burst to burst. This was validated using several statistical methods, including high Kendall's coefficient of concordance values for firing order during GDPs, high Pearson's correlations of cellular activation times between burst pairs, and latent class analysis, which revealed a population of 5-6% of CA3 neurons reliably firing very early during GDPs. In contrast, neuronal firing order during interictal bursts appeared homogeneous, with no particular cells repeatedly leading or lagging during these synchronous events. We conclude that GDPs activate via a deterministic mechanism, with distinct, repeatable roles for subsets of neurons during burst generation, while interictal bursts appear to be stochastic events with cells assuming interchangeable roles in the generation of these events.

Axonal α7 nicotinic ACh receptors modulate presynaptic NMDA receptor expression and structural plasticity of glutamatergic presynaptic boutons.

In association with NMDA receptors (NMDARs), neuronal α7 nicotinic ACh receptors (nAChRs) have been implicated in neuronal plasticity as well as neurodevelopmental, neurological, and psychiatric disorders. However, the role of presynaptic NMDARs and their interaction with α7 nAChRs in these physiological and pathophysiological events remains unknown. Here we report that axonal α7 nAChRs modulate presynaptic NMDAR expression and structural plasticity of glutamatergic presynaptic boutons during early synaptic development. Chronic inactivation of α7 nAChRs markedly increased cell surface NMDAR expression as well as the number and size of glutamatergic axonal varicosities in cortical cultures. These boutons contained presynaptic NMDARs and α7 nAChRs, and recordings from outside-out pulled patches of enlarged presynaptic boutons identified functional NMDAR-mediated currents. Multiphoton imaging of presynaptic NMDAR-mediated calcium transients demonstrated significantly larger responses in these enlarged boutons, suggesting enhanced presynaptic NMDAR function that could lead to increased glutamate release. Moreover, whole-cell patch clamp showed a significant increase in synaptic charge mediated by NMDAR miniature EPSCs but no alteration in the frequency of AMPAR miniature EPSCs, suggesting the selective enhancement of postsynaptically silent synapses upon inactivation of α7 nAChRs. Taken together, these findings indicate that axonal α7 nAChRs modulate presynaptic NMDAR expression and presynaptic and postsynaptic maturation of glutamatergic synapses, and implicate presynaptic α7 nAChR/NMDAR interactions in synaptic development and plasticity.

Astrocytic regulation of glutamate homeostasis in epilepsy.

Astrocytes play a critical role in regulation of extracellular neurotransmitter levels in the central nervous system. This function is particularly prominent for the excitatory amino acid glutamate, with estimates that 80-90% of extracellular glutamate uptake in brain is through astrocytic glutamate transporters. This uptake has significance both in regulation of the potential toxic accumulation of extracellular glutamate and in normal resupply of inhibitory and excitatory synapses with neurotransmitter. This resupply of neurotransmitter is accomplished by astroglial uptake of glutamate, transformation of glutamate to glutamine by the astrocytic enzyme glutamine synthetase (GS), and shuttling of glutamine back to excitatory and inhibitory neurons via specialized transporters. Once in neurons, glutamine is enzymatically converted back to glutamate, which is utilized for synaptic transmission, either directly, or following decarboxylation to γ-aminobutyric acid. Many neurologic and psychiatric conditions, particularly epilepsy, are accompanied by the development of reactive gliosis, a pathology characterized by anatomical and biochemical plasticity in astrocytes, accompanied by proliferation of these cells. Among the biochemical changes evident in reactive astrocytes is a downregulation of several of the important regulators of the glutamine-glutamate cycle, including GS, and possibly also glutamate transporters. This downregulation may have significance in contributing both to the aberrant excitability and to the altered neuropathology characterizing epilepsy. In the present review, we provide an overview of the normal function of astrocytes in regulating extracellular glutamate homeostasis, neurotransmitter supply, and excitotoxicity. We further discuss the potential role reactive gliosis may play in the pathophysiology of epilepsy.

Hippocampal microcircuit dynamics probed using optical imaging approaches.

Mammalian cortical structures are endowed with the capacity for plasticity, which emerges from a combination of the dynamics of circuit connectivity and function, and the intrinsic function of the neurons within the circuit. However, this capacity is accompanied by a significant risk: the capability to generate seizure discharges is also a property of all mammalian cortices. How do cortical circuits reconcile the requirement to maintain plasticity, but at the same time control seizure initiation? These issues come into particular focus in the hippocampus. The hippocampus is one of the main plasticity engines in the brain, and is also a structure frequently implicated in the generation of epileptic seizures, with temporal lobe epilepsy constituting the most prevalent form of epilepsy in the adult population. One aspect of hippocampal circuitry that is particularly prominent is its intimate interconnections with the entorhinal cortex. These interconnections create a number of excitatory synaptic loops within the limbic system, which, in addition to being important in cognitive function, can support reentrant activation and seizure generation. In the present review, using optical imaging approaches to elucidate circuit processing at high temporal and spatial resolution, we examine how two targets of entorhinal cortical input within the hippocampus, the dentate gyrus and area CA1, regulate these synaptic pathways in ways that can maintain functions important in generation of normal activity patterns, but that dampen the ability of these inputs to generate seizure discharges.

Functional NMDA receptors are expressed by human pulmonary artery smooth muscle cells.

N-methyl-D-aspartate (NMDA) receptors are widely expressed in the central nervous system. However, their presence and function at extraneuronal sites is less well characterized. In the present study, we examined the expression of NMDA receptor subunit mRNA and protein in human pulmonary artery (HPA) by quantitative polymerase chain reaction (PCR), immunohistochemistry and immunoblotting. We demonstrate that both GluN1 and GluN2 subunit mRNAs are expressed in HPA. In addition, GluN1 and GluN2 (A-D) subunit proteins are expressed by human pulmonary artery smooth muscle cells (HPASMCs) in vitro and in vivo. These subunits localize on the surface of HPASMCs and form functional ion channels as evidenced by whole-cell patch-clamp electrophysiology and reduced phenylephrine-induced contractile responsiveness of human pulmonary artery by the NMDA receptor antagonist MK801 under hypoxic condition. HPASMCs also express high levels of serine racemase and vesicular glutamate transporter 1, suggesting a potential source of endogenous agonists for NMDA receptor activation. Our findings show HPASMCs express functional NMDA receptors in line with their effect on pulmonary vasoconstriction, and thereby suggest a novel therapeutic target for pharmacological modulations in settings associated with pulmonary vascular dysfunction.

Modular, Circuit-Based Interventions Rescue Hippocampal-Dependent Social and Spatial Memory in a 22q11.2 Deletion Syndrome Mouse Model.

BACKGROUND: 22q11.2 deletion syndrome (22qDS) manifests with myriad symptoms, including multiple neuropsychiatric disorders. Complications associated with the polygenic haploinsufficiency make 22qDS symptoms particularly difficult to manage with traditional therapeutic approaches. However, the varying mechanistic consequences often culminate to generate inappropriate regulation of neuronal circuit activity. We explored whether managing this aberrant activity in adults could be a therapeutically beneficial strategy. METHODS: To assess and dissect hippocampal circuit function, we performed functional imaging in acute slices and targeted eloquent circuits (specific subcircuits tied to specific behavioral tasks) to provide relevant behavioral outputs. For example, the ventral and dorsal CA1 regions critically support social and spatial discrimination, respectively. We focally introduced chemogenetic constructs in 34 control and 24 22qDS model mice via adeno-associated viral vectors, driven by excitatory neuron-specific promoter elements, to manipulate circuit recruitment in an on-demand fashion. RESULTS: 22qDS model mice exhibited CA1 excitatory ensemble hyperexcitability and concomitant behavioral deficits in both social and spatial memory. Remarkably, acute chemogenetic inhibition of pyramidal cells successfully corrected memory deficits and did so in a regionally specific manner: ventrally targeted constructs rescued only social behavior, while those expressed dorsally selectively affected spatial memory. Additionally, manipulating activity in control mice could recapitulate the memory deficits in a regionally specific manner. CONCLUSIONS: These data suggest that retuning activity dysregulation can rescue function in disease-altered circuits, even in the face of a polygenetic haploinsufficiency with a strong developmental component. Targeting circuit excitability in a focal, modular manner may prove to be an effective therapeutic for treatment-resistant symptoms of mental illness.

Aged heterozygous Cdkl5 mutant mice exhibit spontaneous epileptic spasms.

CDKL5 deficiency disorder (CDD) is a devastating neurodevelopmental disorder characterized by early-onset epilepsy, severe intellectual disability, cortical visual impairment and motor disabilities. Epilepsy is a central feature of CDD, with most patients having intractable seizures, but seizure frequency and severity can vary. Clinical reports demonstrate a diversity in seizure semiology and electrographic features, with no pattern diagnostic of CDD. Although animal models of CDD have shown evidence of hyperexcitability, spontaneous seizures have not been previously reported. Here, we present the first systematic study of spontaneous seizures in mouse models of CDD. Epileptic spasms, the most frequent and persistent seizure type in CDD patients, were recapitulated in two mouse models of CDD carrying heterozygous mutations, Cdkl5R59X and Cdkl5KO. Spasm-like events were present in a significant proportion of aged heterozygous female mice carrying either of the two Cdkl5 mutations with significant variability in seizure burden. Electrographically, spasms were most frequently associated with generalized slow-wave activity and tended to occur in clusters during sleep. CDD mice also showed interictal and background abnormalities, characterized by high-amplitude spiking and altered power in multiple frequency bands. These data demonstrate that aged female heterozygous Cdkl5 mice recapitulate multiple features of epilepsy in CDD and can serve to complement existing models of epileptic spasms in future mechanistic and translational studies.

Deficits in phosphorylation of GABA(A) receptors by intimately associated protein kinase C activity underlie compromised synaptic inhibition during status epilepticus.

Status epilepticus (SE) is a progressive and often lethal human disorder characterized by continuous or rapidly repeating seizures. Of major significance in the pathology of SE are deficits in the functional expression of GABA(A) receptors (GABA(A)Rs), the major sites of fast synaptic inhibition in the brain. We demonstrate that SE selectively decreases the phosphorylation of GABA(A)Rs on serine residues 408/9 (S408/9) in the beta3 subunit by intimately associated protein kinase C isoforms. Dephosphorylation of S408/9 unmasks a basic patch-binding motif for the clathrin adaptor AP2, enhancing the endocytosis of selected GABA(A)R subtypes from the plasma membrane during SE. In agreement with this, enhancing S408/9 phosphorylation or selectively blocking the binding of the beta3 subunit to AP2 increased GABA(A)R cell surface expression levels and restored the efficacy of synaptic inhibition in SE. Thus, enhancing phosphorylation of GABA(A)Rs or selectively blocking their interaction with AP2 may provide novel therapeutic strategies to ameliorate SE.