The cellular architecture of the ventral tegmental area (VTA), the main hub of the brain reward system, remains only partially characterized. To extend the characterization to inhibitory neurons, we have identified three distinct subtypes of somatostatin (Sst)-expressing neurons in the mouse VTA. These neurons differ in their electrophysiological and morphological properties, anatomical localization, as well as mRNA expression profiles. Importantly, similar to cortical Sst-containing interneurons, most VTA Sst neurons express GABAergic inhibitory markers, but some of them also express glutamatergic excitatory markers and a subpopulation even express dopaminergic markers. Furthermore, only some of the proposed marker genes for cortical Sst neurons were expressed in the VTA Sst neurons. Physiologically, one of the VTA Sst neuron subtypes locally inhibited neighboring dopamine neurons. Overall, our results demonstrate the remarkable complexity and heterogeneity of VTA Sst neurons and suggest that these cells are multifunctional players in the midbrain reward circuitry.

No abstract

No abstract

Recent work suggests that cortical interneuron diversity arises from genetic mechanisms guided by the interplay of intrinsic developmental patterning and local extrinsic cues. Individual genetic programs underlying subtype identity are at least partly established in postmitotic neural precursors, prior to their tangential migration and integration in the cortical circuitry. Nevertheless, it is unclear how distinct interneuron identities are maintained during their migration and maturation. Sox6 is a transcription factor with an established role in MGEderived interneuron maturation and positional identity. To determine its role in maintaining somatostatin (Sst)-expressing interneurons' subtype identity, we conditionally removed Sox6 in migrating Sst interneurons and assessed the effects on their mature identity using singlecell RNA-sequencing (scRNAseq), in situ hybridization and electrophysiology. Sox6 removal prior to migration in Sst-expressing neurons reduced subtype diversity without affecting overall number of neurons. Seven out of nine Sst-expressing molecular subtypes were absent in the mature primary somatosensory cortex of Sox6-cKO mice, including the Chodl-Nos1expressing type which has been shown to be specified at, or shortly after, cell cycle exit. The remaining Sst-expressing subtypes in the Sox6-cKO cortex comprised three molecular subtypes, Crh-C1ql3 and Hpse-Cbln4, and a third subtype that seemed to be a molecular hybrid of these subtypes. Moreover, Sox6-cKO cells still expressed genes enriched within the entire class of Sst-expressing neurons, such as Sst, Lhx6, Satb1, Elfn1 and Mafb. Removal of Sox6 at P7, after cells have reached their final destination and begin integration into the network, did not disrupt Chodl-Nos1 marker expression. Our findings suggest that expression 2 of Sox6 during the migratory phase of cortical interneurons is necessary for maintenance of Sst + subtype identity, indicating that subtype maintenance during migration requires active transcriptional programs.

15The cellular architecture of the ventral tegmental area (VTA), the main hub of the brain reward 16 system, remains only partially characterized. To extend the characterization to inhibitory neurons, 17 we have identified three distinct subtypes of somatostatin (Sst)-expressing neurons in the mouse 18 VTA. These neurons differ in their electrophysiological and morphological properties, anatomical 19 localization, as well as mRNA expression profiles. Importantly, similar to cortical Sst-containing 20 interneurons, most VTA Sst neurons express GABAergic inhibitory markers, but some of them also 21 express glutamatergic excitatory markers and a subpopulation even express dopaminergic 22 markers. Furthermore, only some of the proposed marker genes for cortical Sst neurons were 23 expressed in the VTA Sst neurons. Physiologically, one of the VTA Sst neuron subtypes locally 24 inhibited neighboring dopamine neurons. Overall, our results demonstrate the remarkable 25 complexity and heterogeneity of VTA Sst neurons and suggest that these cells are multifunctional 26 players in the midbrain reward circuitry. 27 28 29 List of abbreviations 30