A transcriptomic axis predicts state modulation of cortical interneurons.

Transcriptomics has revealed that cortical inhibitory neurons exhibit a great diversity of fine molecular subtypes1-6, but it is not known whether these subtypes have correspondingly diverse patterns of activity in the living brain. Here we show that inhibitory subtypes in primary visual cortex (V1) have diverse correlates with brain state, which are organized by a single factor: position along the main axis of transcriptomic variation. We combined in vivo two-photon calcium imaging of mouse V1 with a transcriptomic method to identify mRNA for 72 selected genes in ex vivo slices. We classified inhibitory neurons imaged in layers 1-3 into a three-level hierarchy of 5 subclasses, 11 types and 35 subtypes using previously defined transcriptomic clusters3. Responses to visual stimuli differed significantly only between subclasses, with cells in the Sncg subclass uniformly suppressed, and cells in the other subclasses predominantly excited. Modulation by brain state differed at all hierarchical levels but could be largely predicted from the first transcriptomic principal component, which also predicted correlations with simultaneously recorded cells. Inhibitory subtypes that fired more in resting, oscillatory brain states had a smaller fraction of their axonal projections in layer 1, narrower spikes, lower input resistance and weaker adaptation as determined in vitro7, and expressed more inhibitory cholinergic receptors. Subtypes that fired more during arousal had the opposite properties. Thus, a simple principle may largely explain how diverse inhibitory V1 subtypes shape state-dependent cortical processing.

Disruption of NEUROD2 causes a neurodevelopmental syndrome with autistic features via cell-autonomous defects in forebrain glutamatergic neurons.

While the transcription factor NEUROD2 has recently been associated with epilepsy, its precise role during nervous system development remains unclear. Using a multi-scale approach, we set out to understand how Neurod2 deletion affects the development of the cerebral cortex in mice. In Neurod2 KO embryos, cortical projection neurons over-migrated, thereby altering the final size and position of layers. In juvenile and adults, spine density and turnover were dysregulated in apical but not basal compartments in layer 5 neurons. Patch-clamp recordings in layer 5 neurons of juvenile mice revealed increased intrinsic excitability. Bulk RNA sequencing showed dysregulated expression of many genes associated with neuronal excitability and synaptic function, whose human orthologs were strongly associated with autism spectrum disorders (ASD). At the behavior level, Neurod2 KO mice displayed social interaction deficits, stereotypies, hyperactivity, and occasionally spontaneous seizures. Mice heterozygous for Neurod2 had similar defects, indicating that Neurod2 is haploinsufficient. Finally, specific deletion of Neurod2 in forebrain excitatory neurons recapitulated cellular and behavioral phenotypes found in constitutive KO mice, revealing the region-specific contribution of dysfunctional Neurod2 in symptoms. Informed by these neurobehavioral features in mouse mutants, we identified eleven patients from eight families with a neurodevelopmental disorder including intellectual disability and ASD associated with NEUROD2 pathogenic mutations. Our findings demonstrate crucial roles for Neurod2 in neocortical development, whose alterations can cause neurodevelopmental disorders including intellectual disability and ASD.

Intrinsic Neuronal Activity during Migration Controls the Recruitment of Specific Interneuron Subtypes in the Postnatal Mouse Olfactory Bulb.

Neuronal activity has been identified as a key regulator of neuronal network development, but the impact of activity on migration and terminal positioning of interneuron subtypes is poorly understood. The absence of early subpopulation markers and the presence of intermingled migratory and postmigratory neurons make the developing cerebral cortex a difficult model to answer these questions. Postnatal neurogenesis in the subventricular zone (SVZ) offers a more accessible and compartmentalized model. Neural stem cells regionalized along the border of the lateral ventricle produce two main subtypes of neural progenitors, granule cells and periglomerular neurons that migrate tangentially in the rostral migratory stream (RMS) before migrating radially in the olfactory bulb (OB) layers. Here, we used targeted postnatal electroporation to compare the migration of these two populations in male and female mice. We do not observe any obvious differences regarding the mode of tangential or radial migration between these two subtypes. However, we find a striking increase of intrinsic calcium activity in granule cell precursors (GC-Ps) when they switch from tangential to radial migration. By decreasing neuronal excitability in GC-Ps, we find that neuronal activity has little effect on migration but is required for normal positioning and survival of GC-Ps in the OB layers. Strikingly, decreasing activity of periglomerular neuron precursors (PGN-Ps) did not impact their positioning or survival. Altogether these findings suggest that neuronal excitability plays a subtype specific role during the late stage of migration of postnatally born OB interneurons.SIGNIFICANCE STATEMENT While neuronal activity is a critical factor regulating different aspects of neurogenesis, it has been challenging to study its role during the migration of different neuronal subpopulations. Here, we use postnatal targeted electroporation to label and manipulate the two main olfactory bulb (OB) interneuron subpopulations during their migration: granule cell and periglomerular neuron precursors (PGN-Ps). We find a very striking increase of calcium activity only in granule cell precursors (GC-Ps) when they switch from tangential to radial migration. Interestingly, blocking activity in GC-Ps affected mainly their positioning and survival while PGN-Ps were not affected. These results suggest that neuronal activity is required specifically for the recruitment of GC-Ps in the OB layers.

Neuronal integration in the adult mouse olfactory bulb is a non-selective addition process.

Adult neurogenesis in the olfactory bulb (OB) is considered as a competition in which neurons scramble during a critical selection period for integration and survival. Moreover, newborn neurons are thought to replace pre-existing ones that die. Despite indirect evidence supporting this model, systematic in vivo observations are still scarce. We used two-photon in vivo imaging to study neuronal integration and survival. We show that loss of new neurons in the OB after arrival at terminal positions occurs only at low levels. Moreover, long-term observations showed that no substantial cell death occurred at later stages. Neuronal death was induced by standard doses of thymidine analogs, but disappeared when low doses were used. Finally, we demonstrate that the OB grows throughout life. This shows that neuronal selection during OB-neurogenesis does not occur after neurons reached stable positions. Moreover, this suggests that OB neurogenesis does not represent neuronal turnover but lifelong neuronal addition.

Direct and efficient transfection of mouse neural stem cells and mature neurons by in vivo mRNA electroporation.

In vivo brain electroporation of DNA expression vectors is a widely used method for lineage and gene function studies in the developing and postnatal brain. However, transfection efficiency of DNA is limited and adult brain tissue is refractory to electroporation. Here, we present a systematic study of mRNA as a vector for acute genetic manipulation in the developing and adult brain. We demonstrate that mRNA electroporation is far more efficient than DNA electroporation, and leads to faster and more homogeneous protein expression in vivo Importantly, mRNA electroporation allows the manipulation of neural stem cells and postmitotic neurons in the adult brain using minimally invasive procedures. Finally, we show that this approach can be efficiently used for functional studies, as exemplified by transient overexpression of the neurogenic factor Myt1l and by stably inactivating Dicer nuclease in vivo in adult born olfactory bulb interneurons and in fully integrated cortical projection neurons.

Time-Specific Effects of Spindle Positioning on Embryonic Progenitor Pool Composition and Adult Neural Stem Cell Seeding.

The developmental mechanisms regulating the number of adult neural stem cells (aNSCs) are largely unknown. Here we show that the cleavage plane orientation in murine embryonic radial glia cells (RGCs) regulates the number of aNSCs in the lateral ganglionic eminence (LGE). Randomizing spindle orientation in RGCs by overexpression of Insc or a dominant-negative form of Lgn (dnLgn) reduces the frequency of self-renewing asymmetric divisions while favoring symmetric divisions generating two SNPs. Importantly, these changes during embryonic development result in reduced seeding of aNSCs. Interestingly, no effects on aNSC numbers were observed when Insc was overexpressed in postnatal RGCs or aNSCs. These data suggest a new mechanism for controlling aNSC numbers and show that the role of spindle orientation during brain development is highly time and region dependent.