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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.

Summary The GluN2 subtype (2A versus 2B) determines biophysical properties and signaling of forebrain NMDA receptors (NMDARs). During development, GluN2A becomes incorporated into previously GluN2B-dominated NMDARs. This “switch” is proposed to be driven by distinct features of GluN2 cytoplasmic C-terminal domains (CTDs), including a unique CaMKII interaction site in GluN2B that drives removal from the synapse. However, these models remain untested in the context of endogenous NMDARs. We show that, although mutating the endogenous GluN2B CaMKII site has secondary effects on GluN2B CTD phosphorylation, the developmental changes in NMDAR composition occur normally and measures of plasticity and synaptogenesis are unaffected. Moreover, the switch proceeds normally in mice that have the GluN2A CTD replaced by that of GluN2B and commences without an observable decline in GluN2B levels but is impaired by GluN2A haploinsufficiency. Thus, GluN2A expression levels, and not GluN2 subtype-specific CTD-driven events, are the overriding factor in the developmental switch in NMDAR composition., Graphical Abstract, Highlights • Mutating the GluN2B CaMKII site affects phosphorylation of its C-terminal domain • The developmental changes in NMDAR composition and synaptogenesis occur normally • Changes in NMDAR composition do not require distinct GluN2 C-terminal domains • Developmental changes in NMDAR composition are primarily sensitive to GluN2A levels, An important milestone in forebrain neuronal development is the switch in composition of the NMDA receptor: GluN2A becomes incorporated into previously GluN2B-dominated NMDARs. Using knockin mice, McKay et al. find that, contrary to earlier proposed models, the switch does not require GluN2A and GluN2B to possess distinct C-terminal domain sequences.

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.

Excessive mRNA translation downstream of group I metabotropic glutamate receptors (mGlu1/5) is a core pathophysiology of fragile X syndrome (FX); however, the differentially translating mRNAs that contribute to altered neural function are not known. We used translating ribosome affinity purification (TRAP) and RNA-seq to identify mistranslating mRNAs in CA1 pyramidal neurons of the FX mouse model (Fmr1−/y) hippocampus, which exhibit exaggerated mGlu1/5-induced long-term synaptic depression (LTD). In these neurons, we find that the Chrm4 transcript encoding muscarinic acetylcholine receptor 4 (M4) is excessively translated, and synthesis of M4 downstream of mGlu5 activation is mimicked and occluded. Surprisingly, enhancement rather than inhibition of M4 activity normalizes core phenotypes in the Fmr1−/y, including excessive protein synthesis, exaggerated mGluR-LTD, and audiogenic seizures. These results suggest that not all excessively translated mRNAs in the Fmr1−/y brain are detrimental, and some may be candidates for enhancement to correct pathological changes in the FX brain.

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.

Genetic mutations known to cause intellectual disabilities (ID) are concentrated in specific sets of genes including both those encoding synaptic proteins and those expressed during early development. We have characterised the effect of genetic deletion of Dlg3, an ID-related gene encoding the synaptic NMDA-receptor interacting protein SAP102, on development of the mouse somatosensory cortex. SAP102 is the main representative of the PSD-95 family of postsynaptic MAGUK proteins during early development and is proposed to play a role in stabilising receptors at immature synapses. Genetic deletion of SAP102 caused a reduction in the total number of thalamocortical (TC) axons innervating the somatosensory cortex, but did not affect the segregation of barrels. On a synaptic level SAP102 knockout mice display a transient speeding of NMDA receptor kinetics during the critical period for TC plasticity, despite no reduction in GluN2B-mediated component of synaptic transmission. These data indicated an interesting dissociation between receptor kinetics and NMDA subunit expression. Following the critical period NMDA receptor function was unaffected by loss of SAP102 but there was a reduction in the divergence of TC connectivity. These data suggest that changes in synaptic function early in development caused by mutations in SAP102 result in changes in network connectivity later in life.

Recent advances in techniques for manipulating genomes have allowed thegeneration of transgenic animals other than mice. These new models enable crossmammalian comparison of neurological disease from core cellular pathophysiology to circuit and behavioural endophenotypes. Moreover they will enable us to directly test whether common cellular dysfunction or behavioural outcomes of a genetic mutation are more conserved across species. Using a new rat model of Fragile X Syndrome (FXS), we report that Fmr1 knockout (KO) rats exhibit elevated basal protein synthesis and an increase in mGluR-dependent long-term depression (LTD) in CA1 of the hippocampus that is independent of new protein synthesis. These defects in plasticity are accompanied by an increase in dendritic spine density selectively in apical dendrites and subtle changes in dendritic spine morphology of CA1 pyramidal neurons. Behaviourally, Fmr1 KO rats show deficits in hippocampal-dependent, but not hippocampal-independent, forms of associative recognition memory indicating that the loss of FMRP causes defects in episodic-like memory. In contrast to previous reports from mice, Fmr1 KO rats show no deficits in spatial reference memory reversal learning. One-trial spatial learning in a delayed matching to place water maze task was also not affected by the loss of FMRP in rats. This is the first evidence for conservation across mammalian species of cellular and physiological hippocampal phenotypes associated with the loss of FMRP. Furthermore, while key cellular phenotypes are conserved they manifest in distinct behavioural dysfunction. Finally, our data reveal novel information about the selective role of FMRP in hippocampus-dependent associative memory.