Genetically Defined Subtypes of Layer 5 Somatostatin-Containing Cortical Interneurons

Of the four main subclasses of inhibitory cortical interneurons, somatostatin-containing (SOM) interneurons are the most diverse. Earlier studies identified layer 1-projecting (Martinotti) cells in layer 5/6 of the X98 and the Chrna2-cre transgenic lines, and two groups of non-Martinotti cells - long-range projecting SOM cells in layers 2 and 6, and layer 4-projecting X94 cells in layers 4 and 5. Later in-vivo and ex-vivo studies described two morphological types of Martinotti cells which appear to have opposing roles in behaving animals. More recently, large-scale transcriptomic studies attempting to classify all cortical neurons by their gene expression profiles and by their morphological and electrophysiological phenotypes divided all SOM interneurons into 13 morpho-electro-transcriptomic (MET) types. It remains unclear, however, how the previously identified SOM subtypes relate to each other, and how they map onto the suggested MET classification scheme. Importantly, only a small number of Cre or Flp driver line are available to target SOM interneurons, and there are currently no genetic tools to target the majority of the proposed MET types for recording, imaging or optogenetic manipulations, severely hindering progress on understanding the roles SOM interneurons play in sensorimotor processing or in learning and memory. To begin to overcome these barriers, we undertook a systematic examination of SOM interneuron subtypes in layer 5 of mouse somatosensory cortex. We generated 4 intersectional triple-transgenic genotypes, by crossing the Sst-IRES-Flp line with 4 different Cre lines and with a dual-color reporter that labels all Cre expressing SOM cells with GFP and all other SOM cells in the same brain with tdTomato. Brains from adult mice of both sexes were retrogradely labeled by epipial dye deposits, processed histologically, and immunostained for 3 marker proteins known to be expressed in different SOM subsets. By correlating fluorescent protein expression, retrograde label and marker proteins in the same neurons, we found that Cre-expressing SOM cells in the Calb2-IRES-Cre and in the Chrna2-Cre lines, and GFP expressing neurons in the X94 line, comprise three non-overlapping SOM populations which together account for about half of all SOM cell in layer 5. Using whole-cell recordings ex-vivo, we show that they also exhibit electrophysiological properties which are distinctly different from each other. This multimodal convergence of axonal projection target, marker protein expression and electrophysiological properties strongly suggests that these three populations can be considered bona-fide SOM subtypes. Indeed, each of the three subtypes appears to map onto a unique MET type. Our findings call for a renewed effort to generate additional driver lines that can be used combinatorially to provide genetic access to the many remaining SOM subtypes and uncover their roles in cortical computations. ### Competing Interest Statement The authors have declared no competing interest.