SummaryAnatomical and physiological experiments have outlined a blueprint for the feed-forward flow of activity in cortical circuits: signals are thought to propagate primarily from the middle cortical layer, L4, up to L2/3, and down to the major cortical output layer, L5. Pharmacological manipulations, however, have contested this model and suggested that L4 may not be critical for sensory responses of neurons in either superficial or deep layers. To address these conflicting models we reversibly manipulated L4 activity in awake, behaving mice using cell-type specific optogenetics. In contrast to both prevailing models, we show that activity in L4 directly suppresses L5, in part by activating deep, fast spiking inhibitory neurons. Our data suggest that the net impact of L4 activity is to sharpen the spatial representations of L5 neurons. Thus we establish a novel translaminar inhibitory circuit in the sensory cortex that acts to enhance the feature selectivity of cortical output.

Inhibitory neurons play a fundamental role in cortical computation and behavior. Recent technological advances, such as two photon imaging, targeted in vivo recording, and molecular profiling, have improved our understanding of the function and diversity of cortical interneurons, but for technical reasons most work has been directed towards inhibitory neurons in the superficial cortical layers. Here we review current knowledge specifically on layer 5 (L5) inhibitory microcircuits, which play a critical role in controlling cortical output. We focus on recent work from the well-studied rodent barrel cortex, but also draw on evidence from studies in primary visual cortex and other cortical areas. The diversity of both deep inhibitory neurons and their pyramidal cell targets make this a challenging but essential area of study in cortical computation and sensory processing.

Summary: Understanding how cortical activity generates sensory perceptions requires a detailed dissection of the function of cortical layers. Despite our relatively extensive knowledge of their anatomy and wiring, we have a limited grasp for what each layer contributes to cortical computation. We need to develop a theory of cortical function that is rooted solidly in each layer’s component cell types and fine circuit architecture, and produces predictions that can be validated by specific perturbations. Here, we briefly review progress toward such a theory, and suggest an experimental roadmap toward this goal. We discuss new methods for the all-optical interrogation of cortical layers, for correlating in vivo function with precise identification of transcriptional cell type, and for mapping local and long range activity in vivo with synaptic resolution. The new technologies that can crack the function of cortical layers are finally on the immediate horizon.

The neocortex is functionally organized into layers. Layer four receives the densest bottom up sensory inputs, while layers 2/3 and 5 receive top down inputs that may convey predictive information. A subset of cortical somatostatin (SST) neurons, the Martinotti cells, gate top down input by inhibiting the apical dendrites of pyramidal cells in layers 2/3 and 5, but it is unknown whether an analogous inhibitory mechanism controls activity in layer 4. Using high precision circuit mapping, in vivo optogenetic perturbations, and single cell transcriptional profiling, we reveal complementary circuits in the mouse barrel cortex involving genetically distinct SST subtypes that specifically and reciprocally interconnect with excitatory cells in different layers: Martinotti cells connect with layers 2/3 and 5, whereas non-Martinotti cells connect with layer 4. By enforcing layer-specific inhibition, these parallel SST subnetworks could independently regulate the balance between bottom up and top down input.

The connectivity patterns of excitatory and inhibitory microcircuits are fundamental to computation in the neocortex. Highly specific excitatory projections form a stereotyped microcircuit linking the six cortical layers, but it is unclear whether inhibitory circuits are structured according to a similar layer-based logic or instead wire up non-selectively across the different layers. Understanding principles of inhibitory wiring is critical, since they constrain the computational operations that cortical inhibition can perform. If subnetworks of inhibitory neurons target specific functional components of cortical circuits (e.g. cortical input and output layers), these targets could be independently modulated, enabling a richer repertoire of inhibitory computations. Here we use one and two photon optogenetic circuit mapping techniques to demonstrate that two distinct subtypes of spatially intermingled Layer 5 (L5) somatostatin (SOM) interneurons form exquisitely selective and complementary intracortical circuits. One subtype connects predominantly with L4 and L6 -the primary cortical input layers, while a second subtype connects nearly exclusively with L2/3 and L5 -the primary cortical 2 output layers. This highly specific architecture suggests that separate SOM networks could differentially modulate processing at the input and output stages of the neocortical microcircuit.

The descending microcircuit from layer 2/3 (L2/3) to layer 5 (L5) is one of the strongest excitatory pathways in the cortex, presumably forming a core component of its feedforward hierarchy. To date, however, no experiments have selectively tested the impact of L2/3 activity on L5 during active sensation. We used optogenetic, cell-type-specific manipulation of L2/3 neurons in the barrel cortex of actively sensing mice (of either sex) to elucidate the significance of this pathway to sensory coding in L5. Contrary to standard models, activating L2/3 predominantly suppressed spontaneous activity in L5, whereas deactivating L2/3 mainly facilitated touch responses in L5. Somatostatin interneurons are likely important to this suppression because their optogenetic deactivation significantly altered the functional impact of L2/3 onto L5. The net effect of L2/3 was to enhance the stimulus selectivity and expand the range of L5 output. These data imply that the core cortical pathway increases the selectivity and expands the range of cortical output through feedforward inhibition.The primary sensory cortex contains six distinct layers that interact to form the basis of our perception. While rudimentary patterns of connectivity between the layers have been outlined quite extensively in vitro, functional relationships in vivo, particularly during active sensation, remain poorly understood. We used cell-type-specific optogenetics to test the functional relationship between layer 2/3 and layer 5. Surprisingly, we discovered that L2/3 primarily suppresses cortical output from L5. The recruitment of somatostatin-positive interneurons is likely fundamental to this relationship. The net effect of this translaminar suppression is to enhance the selectivity and expand the range of receptive fields, therefore potentially sharpening the perception of space.

Developing valid tools that assess key determinants of canine healthspan such as frailty and health-related quality of life (HRQL) is essential to characterizing and understanding aging in dogs. Additionally, because the companion dog is an excellent translational model for humans, such tools can be applied to evaluate gerotherapeutics and investigate mechanisms underlying longevity in both dogs and humans. In this multi-center, cross sectional study, we investigated the use of a clinical questionnaire (Canine Frailty Index; CFI; Banzato et al., 2019) to assess frailty and an owner assessment tool (VetMetrica HRQL) to evaluate HRQL in 451 adult companion dogs. Results demonstrated validity of the tools by confirming expectations that frailty and HRQL deteriorate with age. CFI scores were significantly higher (higher frailty) and HRQL scores significantly lower (worse HRQL) in old dogs (≥ 7 years of age) compared to young dogs (≥ 2 and < 6 years of age). Body size (small < 25lbs or large > 50lbs) was not associated with CFI or total HRQL score. However, older, larger dogs showed faster age-related decline in HRQL scores specific to owner-reported activity and comfort. Findings suggest that the clinician-assessed CFI and owner-reported VetMetrica HRQL are useful tools to evaluate two determinants of healthspan in dogs: the accumulation of frailty and the progressive decline in quality of life. Establishing validated tools that operationalize the assessment of canine healthspan is critical for the advancement of geroscience and the development of gerotherapeutics that benefit both human and veterinary medicine.

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