IntroductionGABAergic interneurons (IN) are critical for the precise timing and flow of information in cortical circuits. Loss of GABAergic IN function has been suggested as a potential translationally relevant mechanism of neuropathology in Fragile X Syndrome (FXS). Indeed, in rodent models of FXS, some IN populations may display reduced number, while genes associated with other IN type upregulated. However, it remains unknown how these cell populations, and their cell-type specific gene expression patterns are regulated in early development across other mammalian models of FXS.MethodsHere we utilise an outbred rat model of FXS, in which we have performed single-nucleus RNA sequencing analysis in neonatal development of the somatosensory cortex. We then use immunohistochemistry to measure the number and distribution of neurochemically identified GABAergic IN subtypes from early development until adolescence in the somatosensory cortex.ResultsWe find that GABAergic INs in a rat model of FXS display clear evidence of transcriptomic alteration compared to wild-type littermates in early brain development. These effects are most profound in putative parvalbumin INs, but with modest changes in other cell types. From immunohistochemistry, we find that parvalbumin INs appear largely unaffected in density in distribution, but we observe a large upregulation in the number of somatostatin-expressing INs.ConclusionGABAergic INs may display cell-type specific transcriptomic regulation in response to the loss of FMRP. Our data suggests minimal alteration of parvalbumin IN density or distribution, but upregulated somatostatin cell numbers. These data partially agree with previous observations in mouse models of FXS.

Abstract Cortical circuits rely on a precise balance of inhibitory and excitatory neurotransmission to encode information reliably and prevent pathology. Metabotropic GABA B receptors (GABA B Rs) are key regulators of inhibitory signalling in mammalian neurons. In GABAergic interneurons (INs), GABA B R activation reduces inhibition overall, leading to disinhibitory mechanisms. In the hippocampus, somatostatin-expressing (SST) INs form a major subtype that provides feedback inhibition to the distal dendrites of principal cells (PCs) and other INs. Plasticity of SST INs is well established as a mechanism controlling hippocampal circuit function through both inhibitory and disinhibitory pathways and depends on metabotropic glutamate receptors (mGluRs) and GABA B Rs. However, whether activation of GABABRs induces metaplastic changes in SST INs, and how this influences circuit function and behaviour, remains unclear. Here, we combined quantitative SDS-digested freeze-fracture replica immunoelectron microscopy, ex vivo electrophysiology, in vivo behavioural testing, and pharmacological manipulation of GABA B Rs. We show that receptor activation directly regulates SST IN plasticity via protein phosphatase 2A (PP2A)-dependent internalisation. GABA B R activation not only controls its own surface expression but also regulates membrane levels of mGluR1α and high-voltage-activated Ca v 1.2 (L-type) Ca 2+ channels. This GABA B R-dependent metaplasticity shifts circuit plasticity toward greater enhancement of long-range inputs to the CA1 region and disrupts contextual memory formation. These findings demonstrate that receptor-mediated surface dynamics in SST INs are critical for maintaining physiological neurotransmission and proper hippocampal microcircuit function.

IntroductionFragile X Syndrome is a common, inherited single gene cause of intellectual disability, associated with autism, epilepsy, anxiety, and sensory disturbances. Many of these features have been attributed to cellular dysfunction leading to impaired synaptic plasticity, in particular through metabotropic glutamate and GABA receptor signalling. The function of these pathways in inhibitory interneurons has not been fully elucidated. In this study we test the hypothesis that somatostatin interneurons (SST-INs) display impaired synaptic plasticity, which leads to circuit-level plasticity deficits.MethodsWe use a combination of whole-cell and extracellular recordings in acute hippocampal brain slices prepared from adult, male wild-type and Fmr1-/y mice. ResultsWe find that long-term potentiation in SST-INs is enhanced in Fmr1-/y mice, and that this plasticity is susceptible to GABAB receptor activation. However, long-term potentiation at temporoammonic inputs to CA1 region is not impaired in Fmr1-/y mice following tetanic stimulation. We find that temporoammonic long-term potentiation is equivalently modified by metabotropic glutamate and GABA receptor pharmacology, despite changes in presynaptic function.DiscussionThese data show that while SST-IN function is impaired in Fmr1-/y mice, circuit level plasticity is maintained. This study provides new insights into the function of drugs proposed for the treatment of Fragile X Syndrome.

Maintaining neuronal output with respect to input in the physiological range relies on the ability of neurons to update their responsiveness to inputs dependent on changing activity levels. Termed homeostatic plasticity, the mechanisms that neurons employ to control their responsiveness are varied, and proposed to include structural changes to a key neuronal structure – the axon initial segment (AIS). As the site of action potential initiation, the AIS has been postulated to rapidly change its length in response to increased or decreased cellular and circuit activity. To date, AIS structural plasticity has only been tested in tissue cultures and rodent models. In our current study, we assess the ability of neurons to alter their AIS length over a variety of timescales in ex vivo rodent and human brain slices, human neurons derived from induced pluripotent stem cells, and in mice dark-reared during early life; using a combination of electrophysiology and immunohistochemistry. We find no evidence for changes to AIS length following depolarisation for up to 3 hours, despite positive controls confirming modulated activity. However, we do find that neuronal physiological properties are altered by changes in activity – but these are largely independent of action potential initiation associated with the AIS. In summary, we find no evidence supporting a role for AIS structural plasticity in mouse, rat, or human cortical neurons. ### Competing Interest Statement The authors have declared no competing interest. Medical Research Council, https://ror.org/03x94j517, MR/Y014529/1 Simons Initiative for the Developing Brain, https://ror.org/01gghaa40 Patrick Wild Centre Medical Research Scotland, https://ror.org/00saj4962 RS McDonald Trust Dyson Foundation Race Against Dementia, ARUK-RADF-2019a-00 Alzheimer's Society, AS-PG-21-006

Cortical circuit activity is controlled by GABA-mediated inhibition in a spatiotemporally restricted manner. GABAB receptor (GABABR) signalling exerts powerful slow inhibition that controls synaptic, dendritic and neuronal activity. But, how GABABRs contribute to circuit-level inhibition over the lifespan of rodents and humans is poorly understood. In this study, we quantitatively determined the functional contribution of GABABR signalling to pre- and postsynaptic domains in rat and human cortical principal cells. We find that postsynaptic GABABR differentially control pyramidal cell activity within the cortical column as a function of age in rodents, but minimally change over adult life in humans. Presynaptic GABABRs exert stronger inhibition in humans than rodents. Pre- and postsynaptic GABABRs contribute to co-ordination of local information processing in a layer- and species-dependent manner. Finally, we show that GABABR signalling is elevated in patients that have received the anti-seizure medication Levetiracetam. These data directly increase our knowledge of translationally relevant local circuit dynamics, with direct impact on understanding the role of GABABRs in the treatment of seizure disorders.

Quantitative methods for assessing neural anatomy have rapidly evolved in neuroscience and provide important insights into brain health and function. However, as new techniques develop, it is not always clear when and how each may be used to answer specific scientific questions posed. Dendritic spines, which are often indicative of synapse formation and neural plasticity, have been implicated across many brain regions in neurodevelopmental disorders as a marker for neural changes reflecting neural dysfunction or alterations. In this Perspective we highlight several techniques for staining, imaging, and quantifying dendritic spines as well as provide a framework for avoiding potential issues related to pseudoreplication. This framework illustrates how others may apply the most rigorous approaches. We consider the cost-benefit analysis of the varied techniques, recognizing that the most sophisticated equipment may not always be necessary for answering some research questions. Together, we hope this piece will help researchers determine the best strategy toward using the ever-growing number of techniques available to determine neural changes underlying dendritic spine morphology in health and neurodevelopmental disorders.

BackgroundAutism 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.MethodsWe 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.ResultsWe 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.LimitationsThe 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.ConclusionsWe 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.

Recently, Se-substituted selenocysteine conjugates were proposed as potential prodrugs to target biologically active selenol compounds to tissues containing high activities of cysteine conjugate beta-lyases, such as the kidneys. However, several selenium compounds are known to be relatively toxic compounds. In the present study, the cytotoxicity of 14 selenocysteine Se-conjugates was determined in freshly isolated rat renal proximal tubular cells (RPTC). The results of this study show that four selenocysteine Se-conjugates with alkyl substituents (methyl, ethyl, n-propyl, and n-butyl) did not cause significant cytotoxicity to RPTC up to concentrations of 500 microM after 90 min of incubation. Also, no effect was observed on mitochondrial functioning as indicated by the unaffected mitochondrial membrane potential (delta psi). Se-(i-Propyl)-selenocysteine, however, appeared to be a cytotoxic compound, causing time- and dose-dependent cytotoxicity, and caused a decrease of delta psi in remaining viable cells. Aminooxyacetic acid (AOAA) provided significant protection against cell death of Se-(i-propyl)-selenocysteine, pointing to involvement of cysteine conjugate beta-lyase. AOAA, however, did not prevent the decrease of delta psi. Differentially substituted Se-(phenyl)-L-selenocysteine and Se-(benzyl)-L-selenocysteine conjugates appeared to be cytotoxic to RPTC at a concentration of 200 microM, as indicated by increased cell death and a decreased delta psi in remaining viable cells. Within the Se-benzyl-series, Se-(4-methoxybenzyl)-L-selenocysteine was the most toxic conjugate, whereas Se-(4-chlorophenyl)-L-selenocysteine was the most toxic conjugate of the Se-phenyl compounds. The selenocysteine Se-conjugates with nonsubstituted phenyl and benzyl substituents were nontoxic at 200 microM, but caused significant cell death at a concentration of 500 microM. Preincubation with AOAA, an inhibitor of cysteine conjugate beta-lyase, provided only partial protection against the cytotoxicity of Se-(phenyl)-L-selenocysteine (500 microM) and Se-(4-methoxybenzyl)-L-selenocysteine (200 microM). AOAA did not protect against cytotoxicity of the other conjugates, suggesting direct effects of these compounds or involvement of alternative routes of bioactivation. This study demonstrates that cytotoxicity of selenocysteine Se-conjugates is strongly dependent on the nature of the Se-bound substituent. The nontoxic Se-(alkyl)-Se-conjugates may be promising candidates for further evaluation for chemopreventive activities.

ABSTRACTAutism spectrum condition or ‘autism’ is associated with numerous monogenic and polygenic genetic risk factors including the polygenic 16p11.2 microdeletion. A central question is what neural cells are affected. To systematically investigate we analysed single cell transcriptomes from gestational week (GW) 8-26 human foetal prefrontal cortex and identified a subset of interneurons (INs) first appearing at GW23 with enriched expression of a disproportionately large fraction of risk factor transcripts. This suggests the hypothesis that these INs are disproportionately 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. This study prompts deeper investigation of IN development as a convergent target for autism genetic risk factors.

The ability of neurons to produce behaviorally relevant activity in the absence of pathology relies on the fine balance of synaptic inhibition to excitation. In the hippocampal CA1 microcircuit, this balance is maintained by a diverse population of inhibitory interneurons that receive largely similar glutamatergic afferents as their target pyramidal cells, with EPSCs generated by both AMPA receptors (AMPARs) and NMDA receptors (NMDARs). In this study, we take advantage of a recently generated GluN2A-null rat model to assess the contribution of GluN2A subunits to glutamatergic synaptic currents in three subclasses of interneuron found in the CA1 region of the hippocampus. For both parvalbumin-positive and somatostatin-positive interneurons, the GluN2A subunit is expressed at glutamatergic synapses and contributes to the EPSC. In contrast, in cholecystokinin (CCK)-positive interneurons, the contribution of GluN2A to the EPSC is negligible. Furthermore, synaptic potentiation at glutamatergic synapses on CCK-positive interneurons does not require the activation of GluN2A-containing NMDARs but does rely on the activation of NMDARs containing GluN2B and GluN2D subunits.

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).