Whole-brain annotation and multi-connectome cell typing quantifies circuit stereotypy in Drosophila.

The fruit fly Drosophila melanogaster combines surprisingly sophisticated behaviour with a highly tractable nervous system. A large part of the fly's success as a model organism in modern neuroscience stems from the concentration of collaboratively generated molecular genetic and digital resources. As presented in our FlyWire companion paper 1 , this now includes the first full brain connectome of an adult animal. Here we report the systematic and hierarchical annotation of this ~130,000-neuron connectome including neuronal classes, cell types and developmental units (hemilineages). This enables any researcher to navigate this huge dataset and find systems and neurons of interest, linked to the literature through the Virtual Fly Brain database 2 . Crucially, this resource includes 4,552 cell types. 3,094 are rigorous consensus validations of cell types previously proposed in the hemibrain connectome 3 . In addition, we propose 1,458 new cell types, arising mostly from the fact that the FlyWire connectome spans the whole brain, whereas the hemibrain derives from a subvolume. Comparison of FlyWire and the hemibrain showed that cell type counts and strong connections were largely stable, but connection weights were surprisingly variable within and across animals. Further analysis defined simple heuristics for connectome interpretation: connections stronger than 10 unitary synapses or providing >1% of the input to a target cell are highly conserved. Some cell types showed increased variability across connectomes: the most common cell type in the mushroom body, required for learning and memory, is almost twice as numerous in FlyWire as the hemibrain. We find evidence for functional homeostasis through adjustments of the absolute amount of excitatory input while maintaining the excitation-inhibition ratio. Finally, and surprisingly, about one third of the cell types proposed in the hemibrain connectome could not yet be reliably identified in the FlyWire connectome. We therefore suggest that cell types should be defined to be robust to inter-individual variation, namely as groups of cells that are quantitatively more similar to cells in a different brain than to any other cell in the same brain. Joint analysis of the FlyWire and hemibrain connectomes demonstrates the viability and utility of this new definition. Our work defines a consensus cell type atlas for the fly brain and provides both an intellectual framework and open source toolchain for brain-scale comparative connectomics.

A Role for Thalamic Projection GABAergic Neurons in Circadian Responses to Light.

The thalamus is an important hub for sensory information and participates in sensory perception, regulation of attention, arousal and sleep. These functions are executed primarily by glutamatergic thalamocortical neurons that extend axons to the cortex and initiate cortico-thalamocortical connectional loops. However, the thalamus also contains projection GABAergic neurons that do not extend axons toward the cortex. Here, we have harnessed recent insight into the development of the intergeniculate leaflet (IGL) and the ventral lateral geniculate nucleus (LGv) to specifically target and manipulate thalamic projection GABAergic neurons in female and male mice. Our results show that thalamic GABAergic neurons of the IGL and LGv receive retinal input from diverse classes of retinal ganglion cells (RGCs) but not from the M1 intrinsically photosensitive retinal ganglion cell (ipRGC) type. We describe the synergistic role of the photoreceptor melanopsin and the thalamic neurons of the IGL/LGv in circadian entrainment to dim light. We identify a requirement for the thalamic IGL/LGv neurons in the rapid changes in vigilance states associated with circadian light transitions.SIGNIFICANCE STATEMENT The intergeniculate leaflet (IGL) and ventral lateral geniculate nucleus (LGv) are part of the extended circadian system and mediate some nonimage-forming visual functions. Here, we show that each of these structures has a thalamic (dorsal) as well as prethalamic (ventral) developmental origin. We map the retinal input to thalamus-derived cells in the IGL/LGv complex and discover that while RGC input is dominant, this is not likely to originate from M1ipRGCs. We implicate thalamic cells in the IGL/LGv in vigilance state transitions at circadian light changes and in overt behavioral entrainment to dim light, the latter exacerbated by concomitant loss of melanopsin expression.

Dual midbrain and forebrain origins of thalamic inhibitory interneurons.

The ubiquitous presence of inhibitory interneurons in the thalamus of primates contrasts with the sparsity of interneurons reported in mice. Here, we identify a larger than expected complexity and distribution of interneurons across the mouse thalamus, where all thalamic interneurons can be traced back to two developmental programmes: one specified in the midbrain and the other in the forebrain. Interneurons migrate to functionally distinct thalamocortical nuclei depending on their origin: the abundant, midbrain-derived class populates the first and higher order sensory thalamus while the rarer, forebrain-generated class is restricted to some higher order associative regions. We also observe that markers for the midbrain-born class are abundantly expressed throughout the thalamus of the New World monkey marmoset. These data therefore reveal that, despite the broad variability in interneuron density across mammalian species, the blueprint of the ontogenetic organisation of thalamic interneurons of larger-brained mammals exists and can be studied in mice.

Brain-synthesized oestrogens regulate cortical migration in a sexually divergent manner.

Oestrogens play an important role in brain development where they have been implicated in controlling various cellular processes. Several lines of evidence have been presented showing that oestrogens can be synthesized locally within the brain. Studies have demonstrated that aromatase, the enzyme responsible for the conversion of androgens to oestrogens, is expressed during early development in both male and female cortices. Furthermore, 17ß-oestradiol has been measured in foetal brain tissue from multiple species. 17ß-oestradiol regulates neural progenitor proliferation as well as the development of early neuronal morphology. However, what role locally derived oestrogens play in regulating cortical migration and, moreover, whether these effects are the same in males and females are unknown. Here, we investigated the impact of knockdown expression of Cyp19a1, which encodes aromatase, between embryonic day (E) 14.5 and postnatal day 0 (P0) had on neural migration within the cortex. Aromatase was expressed in the developing cortex of both sexes, but at significantly higher levels in male than female mice. Under basal conditions, no obvious differences in cortical migration between male and female mice were observed. However, knockdown of Cyp19a1 resulted in an increase in cells within the cortical plate, and a concurrent decrease in the subventricular zone/ventricular zone in P0 male mice. Interestingly, the opposite effect was observed in females, who displayed a significant reduction in cells migrating to the cortical plate. Together, these findings indicate that brain-derived oestrogens regulate radial migration through distinct mechanisms in males and females.