Inhibitory interneurons play a crucial role in proper development and function of the mammalian cerebral cortex. Of the different inhibitory subclasses, dendritic-targeting, somatostatin-containing (SOM) interneurons may be the most diverse. Earlier studies used GFP-expressing and recombinase-expressing mouse lines to characterize genetically defined subtypes of SOM interneurons by morphological, electrophysiological and neurochemical properties. More recently, large-scale studies classified SOM interneurons into 13 morpho-electro-transcriptomic (MET) types. It remains unclear, however, how these various classification schemes relate to each other, and experimental access to MET types has been limited by the scarcity of specific mouse driver lines. To address these issues we crossed Flp and Cre driver lines with a dual-color intersectional reporter, allowing experimental access to several combinatorially defined SOM subsets. Brains from adult mice of both sexes were retrogradely dye-labeled from the pial surface to identify layer 1-projecting neurons and immunostained against several marker proteins, revealing correlations between genetic label, axonal target and marker protein expression in the same neurons. Lastly, using whole-cell recordings ex-vivo, we analyzed and compared electrophysiological properties between different intersectional subsets. We identified two layer 1-targeting subtypes with non-overlapping marker protein expression and electrophysiological properties which, together with a previously characterized layer 4-targeting subtype, account for >50% of all layer 5 SOM cells and >40% of all SOM cells, and appear to map onto 5 of the 13 MET types. Genetic access to these subtypes will allow researchers to determine their synaptic inputs and outputs and uncover their roles in cortical computations and animal behavior.SIGNIFICANCE STATEMENTInhibitory neurons are critically important for proper development and function of the cerebral cortex. Although a minority population, they are highly diverse, which poses a major challenge to investigating their contributions to cortical computations and animal and human behavior. As a step towards understanding this diversity we crossed genetically modified mouse lines to allow detailed examination of combinatorially defined groups of somatostatin-containing interneurons. We identified and characterized three somatostatin-containing subtypes in the deep cortical layers with distinct anatomical, neurochemical and electrophysiological properties. Future studies could now use these genetic tools to examine how these different subtypes are integrated into the cortical circuit and what roles they play during sensory, cognitive or motor behavior.

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.

Perceiving, recognizing and remembering 3-dimensional (3-D) objects encountered in the environment has a very high survival value; unsurprisingly, this ability is shared among many animal species, including humans. The psychological, psychophysical and neural basis for object perception, discrimination, recognition and memory has been extensively studied in humans, monkeys, pigeons and rodents, but is still far from understood. Nearly all 3-D object recognition studies in the rodent used the “novel object recognition” paradigm, which relies on innate rather than learned behavior; however, this procedure has several important limitations. Recently, investigators have begun to recognize the power of behavioral tasks learned through reinforcement training (operant conditioning) to reveal the sensorimotor and cognitive abilities of mice and to elucidate their underlying neural mechanisms. Here, we describe a novel method for training and testing mice in visual and tactile object discrimination, recognition and memory, and use it to begin to examine the underlying sensory basis for these cognitive capacities. A custom-designed Y maze was used to train mice to associate one of two 3-D objects with a food reward. Out of nine mice trained in two cohorts, seven reached performance criterion in about 20–35 daily sessions of 20 trials each. The learned association was retained, or rapidly re-acquired, after a 6 weeks hiatus in training. When tested under low light conditions, individual animals differed in the degree to which they used tactile or visual cues to identify the objects. Switching to total darkness resulted only in a transient dip in performance, as did subsequent trimming of all large whiskers (macrovibrissae). Additional removal of the small whiskers (microvibrissae) did not degrade performance, but transiently increased the time spent inspecting the object. This novel method can be combined in future studies with the large arsenal of genetic tools available in the mouse, to elucidate the neural basis of object perception, recognition and memory.

Oscillations of extracellular voltage, reflecting synchronous rhythmic activity in large populations of neurons, are a ubiquitous feature in the mammalian brain and are thought to subserve critical, if not fully understood cognitive functions. Oscillations at different frequency bands are hallmarks of specific brain or behavioral states. At the higher end of the scale, ultrafast (400-600 Hz) oscillations in the somatosensory cortex, in response to peripheral stimulation, were observed in human and a handful of animal studies; however, their synaptic basis and functional significance remain largely unexplored. Here we report that brief optogenetic activation of thalamocortical axons ex-vivo elicited precisely reproducible, ~410 Hz local field potential wavelets ("ripplets") in middle layers of mouse somatosensory (barrel) cortex. Fast-spiking (FS) inhibitory interneurons were exquisitely synchronized with each other and fired spike bursts in anti-phase with ripplets, while excitatory neurons fired only 1-2 spikes per stimulus. Both subtypes received shared excitatory inputs at ripplet frequency, and bursts in layer 5 FS cells required intact connection with layer 4, suggesting that layer 4 excitatory cells were driving FS bursts in both layers. Ripplets may be a ubiquitous cortical response to exceptionally salient sensory stimuli, and could provide increased bandwidth for encoding and transmitting sensory information. Lastly, optogenetically-induced ripplets are a uniquely accessible model system for studying synaptic mechanisms of fast and ultrafast oscillations.