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Abstract Background Mutations in the X-linked gene cyclin-dependent kinase-like 5 (CDKL5) cause a severe neurological disorder characterised by early-onset epileptic seizures, autism and intellectual disability (ID). Impaired hippocampal function has been implicated in other models of monogenic forms of autism spectrum disorders and ID and is often linked to epilepsy and behavioural abnormalities. Many individuals with CDKL5 deficiency disorder (CDD) have null mutations and complete loss of CDKL5 protein, therefore in the current study we used a Cdkl5 −/y rat model to elucidate the impact of CDKL5 loss on cellular excitability and synaptic function of CA1 pyramidal cells (PCs). We hypothesised abnormal pre and/or post synaptic function and plasticity would be observed in the hippocampus of Cdkl5 −/y rats. Methods To allow cross-species comparisons of phenotypes associated with the loss of CDKL5, we generated a loss of function mutation in exon 8 of the rat Cdkl5 gene and assessed the impact of the loss of CDLK5 using a combination of extracellular and whole-cell electrophysiological recordings, biochemistry, and histology. Results Our results indicate that CA1 hippocampal long-term potentiation (LTP) is enhanced in slices prepared from juvenile, but not adult, Cdkl5 −/y rats. Enhanced LTP does not result from changes in NMDA receptor function or subunit expression as these remain unaltered throughout development. Furthermore, Ca2+ permeable AMPA receptor mediated currents are unchanged in Cdkl5 −/y rats. We observe reduced mEPSC frequency accompanied by increased spine density in basal dendrites of CA1 PCs, however we find no evidence supporting an increase in silent synapses when assessed using a minimal stimulation protocol in slices. Additionally, we found no change in paired-pulse ratio, consistent with normal release probability at Schaffer collateral to CA1 PC synapses. Conclusions Our data indicate a role for CDKL5 in hippocampal synaptic function and raise the possibility that altered intracellular signalling rather than synaptic deficits contribute to the altered plasticity. Limitations This study has focussed on the electrophysiological and anatomical properties of hippocampal CA1 PCs across early postnatal development. Studies involving other brain regions, older animals and behavioural phenotypes associated with the loss of CDKL5 are needed to understand the pathophysiology of CDD.

Abstract Background Autism spectrum condition or ‘autism’ is associated with numerous genetic risk factors including the polygenic 16p11.2 microdeletion. The balance between excitatory and inhibitory neurons in the cerebral cortex is hypothesised to be critical for the aetiology of autism making improved understanding of how risk factors impact on the development of these cells an important area of research. In the current study we aim to combine bioinformatics analysis of human foetal cerebral cortex gene expression data with anatomical and electrophysiological analysis of a 16p11.2 +/- rat model to investigate how genetic risk factors impact on inhibitory neuron development. Methods We performed bioinformatics analysis of single cell transcriptomes from gestational week (GW) 8–26 human foetal prefrontal cortex and anatomical and electrophysiological analysis of 16p11.2 +/- rat cerebral cortex and hippocampus at post-natal day (P) 21. Results We identified a subset of human interneurons (INs) first appearing at GW23 with enriched expression of a large fraction of risk factor transcripts including those expressed from the 16p11.2 locus. This suggests the hypothesis that these foetal INs are vulnerable to mutations causing autism. We investigated this in a rat model of the 16p11.2 microdeletion. We found no change in the numbers or position of either excitatory or inhibitory neurons in the somatosensory cortex or CA1 of 16p11.2 +/- rats but found that CA1 Sst INs were hyperexcitable with an enlarged axon initial segment, which was not the case for CA1 pyramidal cells. Limitations The human foetal gene expression data was acquired from cerebral cortex between gestational week (GW) 8 to 26. We cannot draw inferences about potential vulnerabilities to genetic autism risk factors for cells not present in the developing cerebral cortex at these stages. The analysis 16p11.2 +/- rat phenotypes reported in the current study was restricted to 3-week old (P21) animals around the time of weaning and to a single interneuron cell-type while in human 16p11.2 microdeletion carriers symptoms likely involve multiple cell types and manifest in the first few years of life and on into adulthood. Conclusions We have identified developing interneurons in human foetal cerebral cortex as potentially vulnerable to monogenic autism risk factors and the 16p11.2 microdeletion and report interneuron phenotypes in post-natal 16p11.2 +/- rats.

Abstract Background Fragile X syndrome (FXS) is a common single gene cause of intellectual disability and autism spectrum disorder. Cognitive inflexibility is one of the hallmarks of FXS with affected individuals showing extreme difficulty adapting to novel or complex situations. To explore the neural correlates of this cognitive inflexibility, we used a rat model of FXS (Fmr1 −/y ). Methods We recorded from the CA1 in Fmr1 −/y and WT littermates over six 10-min exploration sessions in a novel environment—three sessions per day (ITI 10 min). Our recordings yielded 288 and 246 putative pyramidal cells from 7 WT and 7 Fmr1 −/y rats, respectively. Results On the first day of exploration of a novel environment, the firing rate and spatial tuning of CA1 pyramidal neurons was similar between wild-type (WT) and Fmr1 −/y rats. However, while CA1 pyramidal neurons from WT rats showed experience-dependent changes in firing and spatial tuning between the first and second day of exposure to the environment, these changes were decreased or absent in CA1 neurons of Fmr1 −/y rats. These findings were consistent with increased excitability of Fmr1 −/y CA1 neurons in ex vivo hippocampal slices, which correlated with reduced synaptic inputs from the medial entorhinal cortex. Lastly, activity patterns of CA1 pyramidal neurons were dis-coordinated with respect to hippocampal oscillatory activity in Fmr1 −/y rats. Limitations It is still unclear how the observed circuit function abnormalities give rise to behavioural deficits in Fmr1 −/y rats. Future experiments will focus on this connection as well as the contribution of other neuronal cell types in the hippocampal circuit pathophysiology associated with the loss of FMRP. It would also be interesting to see if hippocampal circuit deficits converge with those seen in other rodent models of intellectual disability. Conclusions In conclusion, we found that hippocampal place cells from Fmr1 −/y rats show similar spatial firing properties as those from WT rats but do not show the same experience-dependent increase in spatial specificity or the experience-dependent changes in network coordination. Our findings offer support to a network-level origin of cognitive deficits in FXS.

Fragile X syndrome and autism spectrum disorders are associated with circuit hyperexcitability, however, its cellular and synaptic bases are not well understood. Here, the authors report abnormal synaptogenesis with an increased prevalence of polysynaptic spines with normal morphology in a mouse model of fragile X.

Somatosensory hypersensitivity in Fmr-1 knockout mice is thought to arise from an increase in cortical circuit excitability. Here, the authors report that the loss of precision of sensory encoding in the Layer 4 of barrel cortex is the primary developmental circuit alteration that drives the other compensatory circuit dysfunction.

Summary: This protocol allows repeated whole-cell patch-clamp recordings from individual rodent CA1 hippocampal neurons, followed by immunohistological labeling of the axon initial segment. This overcomes the need to maintain whole-cell recordings over the timescales required for homeostatic modification to cellular excitability, allowing for correlative analysis of the structure and function of neurons. Moreover, this protocol allows for paired analysis of physiological properties assessed before and after pharmacological treatment, thus providing increased statistical power, despite the relatively low-throughput nature of the recordings.For complete details on the use and execution of this protocol, please refer to Booker et al. (2020a).

Summary: Cellular hyperexcitability is a salient feature of fragile X syndrome animal models. The cellular basis of hyperexcitability and how it responds to changing activity states is not fully understood. Here, we show increased axon initial segment length in CA1 of the Fmr1−/y mouse hippocampus, with increased cellular excitability. This change in length does not result from reduced AIS plasticity, as prolonged depolarization induces changes in AIS length independent of genotype. However, depolarization does reduce cellular excitability, the magnitude of which is greater in Fmr1−/y neurons. Finally, we observe reduced functional inputs from the entorhinal cortex, with no genotypic difference in the firing rates of CA1 pyramidal neurons. This suggests that AIS-dependent hyperexcitability in Fmr1−/y mice may result from adaptive or homeostatic regulation induced by reduced functional synaptic connectivity. Thus, while AIS length and intrinsic excitability contribute to cellular hyperexcitability, they may reflect a homeostatic mechanism for reduced synaptic input onto CA1 neurons.

Summary: Inhibition provided by local GABAergic interneurons (INs) activates ionotropic GABAA and metabotropic GABAB receptors (GABABRs). Despite GABABRs representing a major source of inhibition, little is known of their function in distinct IN subtypes. Here, we show that, while the archetypal dendritic-inhibitory somatostatin-expressing INs (SOM-INs) possess high levels of GABABR on their somato-dendritic surface, they fail to produce significant postsynaptic inhibitory currents. Instead, GABABRs selectively inhibit dendritic CaV1.2 (L-type) Ca2+ channels on SOM-IN dendrites, leading to reduced calcium influx and loss of long-term potentiation at excitatory input synapses onto these INs. These data provide a mechanism by which GABABRs can contribute to disinhibition and control the efficacy of extrinsic inputs to hippocampal networks. : Booker et al. show that GABAB receptors are highly expressed on somatostatin interneuron dendrites. Rather than activating Kir3 channels, they preferentially co-cluster with, and negatively couple to, L-type calcium channels inhibiting long-term potentiation at excitatory inputs. Keywords: GABAergic interneurons, feedback inhibition, GABAB receptors, dendrites, Cav1.2 channels, synaptic plasticity, hippocampus, electron microscopy, whole-cell recording, multi-photon imaging

Information processing in cortical neuronal networks relies on properly balanced excitatory and inhibitory neurotransmission. A ubiquitous motif for maintaining this balance is the somatostatin interneuron (SOM-IN) feedback microcircuit. Here, we investigated the modulation of this microcircuit by presynaptic GABAB receptors (GABABRs) in the rodent hippocampus. Whole-cell recordings from SOM-INs revealed that both excitatory and inhibitory synaptic inputs are strongly inhibited by GABABRs, while optogenetic activation of the interneurons shows that their inhibitory output is also strongly suppressed. Electron microscopic analysis of immunogold-labelled freeze-fracture replicas confirms that GABABRs are highly expressed presynaptically at both input and output synapses of SOM-INs. Activation of GABABRs selectively suppresses the recruitment of SOM-INs during gamma oscillations induced in vitro. Thus, axonal GABABRs are positioned to efficiently control the input and output synapses of SOM-INs and can functionally uncouple them from local network with implications for rhythmogenesis and the balance of entorhinal versus intrahippocampal afferents.

miR-128, a brain-enriched microRNA, has been implicated in the control of neurogenesis and synaptogenesis but its potential roles in intervening processes have not been addressed. We show that post-transcriptional mechanisms restrict miR-128 accumulation to post-mitotic neurons during mouse corticogenesis and in adult stem cell niches. Whereas premature miR-128 expression in progenitors for upper layer neurons leads to impaired neuronal migration and inappropriate branching, sponge-mediated inhibition results in overmigration. Within the upper layers, premature miR-128 expression reduces the complexity of dendritic arborization, associated with altered electrophysiological properties. We show that Phf6, a gene mutated in the cognitive disorder Börjeson-Forssman-Lehmann syndrome, is an important regulatory target for miR-128. Restoring PHF6 expression counteracts the deleterious effect of miR-128 on neuronal migration, outgrowth and intrinsic physiological properties. Our results place miR-128 upstream of PHF6 in a pathway vital for cortical lamination as well as for the development of neuronal morphology and intrinsic excitability.

Activity of cortical principal cells is controlled by the GABAergic system providing inhibition in a compartmentalized manner along their somatodendritic axis. While GABAAR-mediated inhibitory synaptic transmission has been extensively characterized in hippocampal principal cells, little is known about the distribution of postsynaptic effects of GABABRs. In the present study, we have investigated the functional localization of GABABRs and their effector inwardly rectifying potassium (Kir3) channels by combining electrophysiological recordings in acute rat hippocampal slices, high-resolution immunoelectron microscopic analysis and single cell simulations. Pharmacologically-isolated slow inhibitory postsynaptic currents were elicited in the three major hippocampal principal cell types by endogenous GABA released by electrical stimulation, photolysis of caged-GABA, as well as the canonical agonist baclofen, with the highest amplitudes observed in the CA3. Spatially restricted currents were assessed along the axis of principal cells by uncaging GABA in the different hippocampal layers. GABABR-mediated currents were present along the entire somatodendritic axis of principal cells, but non-uniformly distributed: largest currents and the highest conductance densities determined in the simulations were consistently found on the distal apical dendrites. Finally, immunocytochemical localization of GABABRs and Kir3 channels showed that distributions overlap but their densities diverge, particularly on the basal dendrites of pyramidal cells. GABABRs current amplitudes and the conductance densities correlated better with Kir3 density, suggesting a bottlenecking effect defined by the effector channel. These data demonstrate a compartmentalized distribution of the GABABR-Kir3 signaling cascade and suggest differential control of synaptic transmission, dendritic integration and synaptic plasticity at afferent pathways onto hippocampal principal cells.