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Additional file 1: Supplemental figures S1-S9. Figure S1. Nlgn3−/y rats display reduced classic freezing behaviour in a contextual fear conditioning paradigm. Figure S2. Freezing analysed as “paw immobility response” (all four paws unmoving but allowing for movement of head and neck). Figure S3. WT and Nlgn3−/y rats show similar activity in an open field, rotational platform & marble interaction tasks. Figure S4. Effect of repeated footshocks & thermal stimulus on WT and Nlgn3−/y rats. Figure S5. Intrinsic properties of PAG cells recorded from WT and Nlgn3−/y rats. Figure S6. Hyperexcitability of dorsal, but not ventral PAG neurons in 8-10 week old Nlgn3−/y rats. Figure S7. PAG LFPs during fear recall are significantly shorter duration in Nlgn3−/y rats. Figure S8. Defensive reactions were not elicited by electrical stimulation of primary somatosensory cortex in WT or Nlgn3−/y rats. Figure S9. Western blots showing lack expression of NLGN3 in Nlgn3−/y rats both in sensory cortex and periaqueductal grey.

Additional file 2: Supplemental table 1. Summary of statistics for main figures 2–7. Supplemental table 2. Summary of statistics for supplemental figures 1–7.

Key points NMDA receptors are neurotransmitter‐gated ion channels that are critically involved in brain cell communicationVariations in genes encoding NMDA receptor subunits have been found in a range of neurodevelopmental disorders.We investigated a de novo genetic variant found in patients with epileptic encephalopathy that changes a residue located in the ion channel pore of the GluN2A NMDA receptor subunit.We found that this variant (GluN2AN615K) impairs physiologically important receptor properties: it markedly reduces Mg2+ blockade and channel conductance, even for receptors in which one GluN2AN615K is co‐assembled with one wild‐type GluN2A subunit.Our findings are consistent with the GluN2AN615K mutation being the primary cause of the severe neurodevelopmental disorder in carriers. Abstract NMDA receptors are ionotropic calcium‐permeable glutamate receptors with a voltage‐dependence mediated by blockade by Mg2+. Their activation is important in signal transduction, as well as synapse formation and maintenance. Two unrelated individuals with epileptic encephalopathy carry a de novo variant in the gene encoding the GluN2A NMDA receptor subunit: a N615K missense variant in the M2 pore helix (GRIN2A C1845A). We hypothesized that this variant underlies the neurodevelopmental disorders in carriers and explored its functional consequences by electrophysiological analysis in heterologous systems. We focused on GluN2AN615K co‐expressed with wild‐type GluN2 subunits in physiologically relevant triheteromeric NMDA receptors containing two GluN1 and two distinct GluN2 subunits, whereas previous studies have investigated the impact of the variant in diheteromeric NMDA receptors with two GluN1 and two identical GluN2 subunits. We found that GluN2AN615K‐containing triheteromers showed markedly reduced Mg2+ blockade, with a value intermediate between GluN2AN615K diheteromers and wild‐type NMDA receptors. Single‐channel conductance was reduced by four‐fold in GluN2AN615K diheteromers, again with an intermediate value in GluN2AN615K‐containing triheteromers. Glutamate deactivation rates were unaffected. Furthermore, we expressed GluN2AN615K in cultured primary mouse cortical neurons, observing a decrease in Mg2+ blockade and reduction in current density, confirming that the variant continues to have significant functional impact in neuronal systems. Our results demonstrate that the GluN2AN615K variant has substantial effects on NMDA receptor properties fundamental to the roles of the receptor in synaptic plasticity, even when expressed alongside wild‐type subunits. This work strengthens the evidence indicating that the GluN2AN615K variant underlies the disabling neurodevelopmental phenotype in carriers., Key points NMDA receptors are neurotransmitter‐gated ion channels that are critically involved in brain cell communicationVariations in genes encoding NMDA receptor subunits have been found in a range of neurodevelopmental disorders.We investigated a de novo genetic variant found in patients with epileptic encephalopathy that changes a residue located in the ion channel pore of the GluN2A NMDA receptor subunit.We found that this variant (GluN2AN615K) impairs physiologically important receptor properties: it markedly reduces Mg2+ blockade and channel conductance, even for receptors in which one GluN2AN615K is co‐assembled with one wild‐type GluN2A subunit.Our findings are consistent with the GluN2AN615K mutation being the primary cause of the severe neurodevelopmental disorder in carriers.

Glycogen synthase kinase 3 (GSK3) is a critical enzyme in neuronal physiology; however, it is not yet known whether it has any specific role in presynaptic function. We found that GSK3 phosphorylates a residue on the large GTPase dynamin I (Ser-774) both in vitro and in primary rat neuronal cultures. This was dependent on prior phosphorylation of Ser-778 by cyclin-dependent kinase 5. Using both acute inhibition with pharmacological antagonists and silencing of expression with short hairpin RNA, we found that GSK3 was specifically required for activity-dependent bulk endocytosis (ADBE) but not clathrin-mediated endocytosis. Moreover we found that the specific phosphorylation of Ser-774 on dynamin I by GSK3 was both necessary and sufficient for ADBE. These results demonstrate a presynaptic role for GSK3 and they indicate that a protein kinase signaling cascade prepares synaptic vesicles for retrieval during elevated neuronal activity.

The influence that neurons exert on astrocytic function is poorly understood. To investigate this, we first developed a system combining cortical neurons and astrocytes from closely related species, followed by RNA-seq and in silico species separation. This approach uncovers a wide programme of neuron-induced astrocytic gene expression, involving Notch signalling, which drives and maintains astrocytic maturity and neurotransmitter uptake function, is conserved in human development, and is disrupted by neurodegeneration. Separately, hundreds of astrocytic genes are acutely regulated by synaptic activity via mechanisms involving cAMP/PKA-dependent CREB activation. This includes the coordinated activity-dependent upregulation of major astrocytic components of the astrocyte–neuron lactate shuttle, leading to a CREB-dependent increase in astrocytic glucose metabolism and elevated lactate export. Moreover, the groups of astrocytic genes induced by neurons or neuronal activity both show age-dependent decline in humans. Thus, neurons and neuronal activity regulate the astrocytic transcriptome with the potential to shape astrocyte–neuron metabolic cooperation.

Summary Objective Absence seizures in childhood absence epilepsy are initiated in the thalamocortical (TC) system. We investigated if these seizures result from altered development of the TC system before the appearance of seizures in mice containing a point mutation in γ‐aminobutyric acid A (GABAA) receptor γ2 subunits linked to childhood absence epilepsy (R43Q). Findings from conditional mutant mice indicate that expression of normal γ2 subunits during preseizure ages protect from later seizures. This indicates that altered development in the presence of the R43Q mutation is a key contributor to the R43Q phenotype. We sought to identify the cellular processes affected by the R43Q mutation during these preseizure ages. Methods We examined landmarks of synaptic development at the end of the critical period for somatosensory TC plasticity using electrophysiologic recordings in TC brain slices from wild‐type mice and R43Q mice. Results We found that the level of TC connectivity to layer 4 (L4) principal cells and the properties of TC synapses were unaltered in R43Q mice. Furthermore, we show that, although TC feedforward inhibition and the total level of GABAergic inhibition were normal, there was a reduction in the local connectivity to cortical interneurons. This reduction leads to altered inhibition during bursts of cortical activity. Significance This altered inhibition demonstrates that alterations in cortical circuitry precede the onset of seizures by more than a week.

The molecular basis of how chromosome 16p13.11 microduplication leads to major psychiatric disorders is unknown. Here we have undertaken brain imaging of patients carrying microduplications in chromosome 16p13.11 and unaffected family controls, in parallel with iPS cell-derived cerebral organoid studies of the same patients. Patient MRI revealed reduced cortical volume, and corresponding iPSC studies showed neural precursor cell (NPC) proliferation abnormalities and reduced organoid size, with the NPCs therein displaying altered planes of cell division. Transcriptomic analyses of NPCs uncovered a deficit in the NFκB p65 pathway, confirmed by proteomics. Moreover, both pharmacological and genetic correction of this deficit rescued the proliferation abnormality. Thus, chromosome 16p13.11 microduplication disturbs the normal programme of NPC proliferation to reduce cortical thickness due to a correctable deficit in the NFκB signalling pathway. This is the first study demonstrating a biologically relevant, potentially ameliorable, signalling pathway underlying chromosome 16p13.11 microduplication syndrome in patient-derived neuronal precursor cells.

Parvalbumin containing inhibitory interneurons (PV + INs) are a key element controlling co-ordinated activity of neuronal ensembles in the mammalian central nervous system (CNS). This article is protected by copyright. All rights reserved.

KEY POINTS: Neurodegenerative disorders can exhibit dysfunctional mitochondrial respiratory chain complex IV activity. Conditional deletion of cytochrome c oxidase, the terminal enzyme in the respiratory electron transport chain of mitochondria, from hippocampal dentate granule cells in mice does not affect low-frequency dentate to CA3 glutamatergic synaptic transmission. High-frequency dentate to CA3 glutamatergic synaptic transmission and feedforward inhibition are significantly attenuated in cytochrome c oxidase-deficient mice. Intact presynaptic mitochondrial function is critical for the short-term dynamics of mossy fibre to CA3 synaptic function.ABSTRACT: Neurodegenerative disorders are characterized by peripheral and central symptoms including cognitive impairments which have been associated with reduced mitochondrial function, in particular mitochondrial respiratory chain complex IV or cytochrome c oxidase activity. In the present study we conditionally removed a key component of complex IV, protohaem IX farnesyltransferase encoded by the COX10 gene, in granule cells of the adult dentate gyrus. Utilizing whole-cell patch-clamp recordings from morphologically identified CA3 pyramidal cells from control and complex IV-deficient mice, we found that reduced mitochondrial function did not result in overt deficits in basal glutamatergic synaptic transmission at the mossy-fibre synapse because the amplitude, input-output relationship and 50 ms paired-pulse facilitation were unchanged following COX10 removal from dentate granule cells. However, trains of stimuli given at high frequency (> 20 Hz) resulted in dramatic reductions in short-term facilitation and, at the highest frequencies (> 50 Hz), also reduced paired-pulse facilitation, suggesting a requirement for adequate mitochondrial function to maintain glutamate release during physiologically relevant activity patterns. Interestingly, local inhibition was reduced, suggesting the effect observed was not restricted to synapses with CA3 pyramidal cells via large mossy-fibre boutons, but rather to all synapses formed by dentate granule cells. Therefore, presynaptic mitochondrial function is critical for the short-term dynamics of synapse function, which may contribute to the cognitive deficits observed in pathological mitochondrial dysfunction.