Current Best Practices for Analysis of Dendritic Spine Morphology and Number in Neurodevelopmental Disorder Research.

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.

Identifying foetal forebrain interneurons as a target for monogenic autism risk factors and the polygenic 16p11.2 microdeletion.

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.

Experience-dependent changes in hippocampal spatial activity and hippocampal circuit function are disrupted in a rat model of Fragile X Syndrome.

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.

Contribution of NMDA Receptors to Synaptic Function in Rat Hippocampal Interneurons.

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.

Repeated whole-cell patch-clamp recording from CA1 pyramidal cells in rodent hippocampal slices followed by axon initial segment labeling.

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).