Development of genetic approaches to study the organization and function of neocortical layer 1 - Summary Neocortical layer 1 (L1), the outermost layer of the cortex and the “crowning mystery” of David Hubel, is a major target of cortico-cortical and thalamocortical projections that carry “top-down” information (behavioral saliency, expectations, predictions, and memories) to be integrated with the “bottom-up” sensory information received by pyramidal cells, the output neurons of the cortex, in order to produce percepts and context-dependent behavior. Understanding how top-down and bottom-up information streams are integrated is necessary to understand perception and behavior, and is of medical significance since disturbances in these processes are likely at the root of neurocognitive diseases such as autism and schizophrenia. L1 is unique among neocortical layers; it is the only layer that lacks excitatory neurons but instead contains the distal (tuft) dendrites of the pyramidal cells (PCs) located in L2-5. These apical dendrites receive the diverse long-range projections arriving in L1 and mediate the integration of these top-down inputs with the feedforward sensory input arriving at the basal dendrites of the PCs. L1 also contains a specialized population of GABAergic interneurons (INs) that sculpt how top-down information is delivered to the PC distal dendrites, but the mechanism by which these neurons regulate top-down processing is poorly understood. L1 INs are distinct from those in other layers that have been the subject of intense research in recent years. Our understanding of the cellular and circuit mechanisms of top- down signaling has lagged behind our knowledge of bottom-up sensory processing, largely due to the lack of molecular genetic tools that have helped elucidate IN and PC circuitry in other layers. We and others have recently discovered that both in mice and humans, the majority of L1 INs specifically express the marker neuron- derived neurotrophic factor (NDNF). Characterization of the properties of NDNF INs in the mouse revealed that there are two main subtypes: L1 neurogliaform cells (NGFCs) and an IN subtype that is unique to L1, the canopy cells. The evolutionary conservation of these two L1 NDNF IN subtypes has recently been confirmed in humans. The availability of genetic reagents to specifically label and manipulate these IN populations would provide unprecedented opportunities to advance our understanding of the circuit mechanisms of top-down processing. Thus, the goal of this exploratory R21 application is to develop and validate novel genetic approaches for the study of L1 NDNF INs. In Aim 1, we will utilize electrophysiology and neuronal reconstructions to assess the specificity and efficacy of a novel intersectional genetic targeting strategy (NDNF/Cxcl14) for the selective labeling and manipulation of canopy cells (Aim 1A). In Aim 1B, we will utilize our NDNF/Cxcl14 intersectional genetics to study the efferent connectivity of canopy cells using optogenetics. In Aim 2, we will leverage a recently developed intersectional inhibitory DREADD reporter mouse line with our intersectional NDNF/Cxcl14 genetic strategy to suppress the output of canopy cells. These experiments will allow us to generate hypotheses about the role of these neurons in regulating the integration of top-down information in the cerebral cortex.
