DESCRIPTION (provided by applicant): The mammalian visual cortex is necessary for vision and has been used over the decades as a model system for the rest of the cerebral cortex. In spite of this, little is known about its microcircuits, and one could argue that to really understad what the visual cortex does one needs to "open the black box" and decipher them. Moreover, the visual cortex is composed of many different cell types, and, it is likely that each cell type hs a particular circuit function. In the last cycle of the award, using a novel two-photon uncaging technique, we studied the GABAergic interneurons and discovered that two of their major subtypes, PARV and SOM positive cells, are connected in a very dense pattern to cells in their vicinity, often making synaptic connections with every single neighboring neuron. This was surprising since it implies that interneurons connect promiscuously, as if their function was not specific. We now propose to test if this is the case, by examining the input and output connectivity of the four major subtypes of interneurons in V1 (PARV, SOM, VIP and chandeliers). Using two-photon photoactivation with a novel caged compound and a novel channelrhodopsin, and transgenic mouse strains that label subtypes of interneurons specifically, we will map connections to and from interneurons in a systematic fashion, in order to arrive at the inhibitory "circuit blueprint" of mouse V1. In the last aim, using a novel SLM two-photon microscope, we will image the responses of these four subtypes of interneurons in anesthetized and awake animals, under spontaneous activity and visual stimulation. These data will be computationally analyzed to search for spatiotemporal patterns of activity indicative of specific subcircuits. Our work will provide a systematic description of the anatomical and functional connectivity of cortical interneurons, one that will enable to examine whether their function is specific or not and to understand how they regulate the activity of the visual cortex. Given the key role that interneurons appear to play in developmental plasticity, the generated knowledge could also help to understand the pathophysiology of developmental deficits in vision and to design novel therapeutic strategies to treat amblyopia.

PUBLIC HEALTH RELEVANCE: The role of inhibitory neurons in the visual cortex is still poorly understood, yet appears to be essential for processing of visual information and visual plasticity. In the last cycle of the grant we discovered that some inhibitory neurons are very densely connected in mouse cortex, and we now propose to obtain the complete circuit blueprint of all major types of inhibitory neurons in the visual cortex and understand whether they form specific circuits in vivo. This work could help to understand how neural circuits in the visual cortex operate and change and also help to design novel strategies to ameliorate amblyopia. __SpecificAimsTextDelimiter__ 10. SPECIFIC AIMS Imaging Functional Connectivity in Visual Cortex- EY11787 The primary visual cortex (V1) probably constitutes the best understood part of the neocortex, due to a sustained research effort, pioneered by Hubel and Wiesel and continued by generations of investigators. At the same time, in spite of enormous progress, it is fair to say that the exact mechanisms by which information is processed by V1, as well as the computational algorithms used, or how they are implemented, are still mostly unknown. Part of the problem lies in the apparently forbidding complexity of its synaptic microcircuits, akin to a "black box", built with many different neuronal cell types, connected in patterns that are still quite mysterious. Following in the tradition of anatomical studies, the goal of our research program is to open this "black box" and decipher the structure and functional connectivity of these microcircuits, something that appears necessary to properly understand how the visual cortex functions and changes in response to developmental plasticity or injury. But rather than traditional anatomical methods, we use imaging techniques in living sample. For the last three cycles of this award, we chose mouse primary visual cortex as our experimental preparation and applied a variety of two-photon techniques to answer basic questions relating to the structure and functional properties of intra- and inter-laminar circuits. We focused on GABAergic interneurons and recently investigated chandelier interneurons, a subtype of GABAergic cell that could serve as circuit switches to prevent (or perhaps trigger) cortical activity. We took advantage of mouse genetics to identify chandelier cells in living brain slices, characterized their morphological and electrophysiological properties, and, by activating or inactivating them, we tested their role in the spontaneous and thalamic-evoked activity of the circuit. We found that chandelier cells have a dual function: they excite resting pyramidal neurons, but provide strong inhibition of active ones. In addition, we developed a two-photon photoactivation technique to map the inhibitory inputs to pyramidal cells, discovering that two subtypes of interneurons, the parvalbumin- and somatostatin-positive (PARV and SOM) cells connect to local pyramidal neurons in a dense and unspecific fashion. We now propose to extend our approach to all major subtypes of neocortical interneurons (including PARV, SOM, chandeliers and VIP-positive cells), mapping what inputs they receive, how they connect to each other and how they respond to visual stimulation in vivo. Our central hypothesis is that all subtypes of interneurons have a reciprocal dense connectivity with the local excitatory circuit, but that they can still form part of specific subnetworks, due to differences in synaptic strength. In this view, interneurons form a structurally completely connected network, but one sculpted by developmental plasticity and activity into functional subdomains. Mouse transgenic lines with GFP-labeled PARV and SOM cells, and with Cre-PARV, SOM, Nkx2.1 and VIP promoters will enable us to selectively map the connections and synaptic weights of these four types of interneurons and image their activity under anesthetized, resting and visually-evoked activity in awake, head- restrained animals. Two-photon activation of interneurons and pyramidal cells will be performed with RuBiGlutamate, a novel caged compound and C1V1, a novel optogenetic construct. Two-photon calcium imaging will be carried out with calcium indicators using spatial light modulators (SLMs) to enable fast scan- less imaging. Finally, a novel computational framework will be used to infer the functional connectivity of interneurons in vivo and compare it with the functional properties of the neurons. Our specific aims are: Aim 1: Mapping outputs from V1 interneurons Hypothesis: Promiscuous innervation is common to all interneurons subtypes 1.1. Quantitative classification of V1 interneurons 1.1. Two-photon mapping of connections from VIP interneurons onto pyramidal cells 1.1. Mapping outputs from chandelier neurons onto pyramidal cells Aim 2: Mapping inputs onto V1 interneurons Hypothesis: Dense, unspecific local sampling of local inputs by interneurons 1.1. Two-photon mapping of excitatory inputs onto interneurons 1.2. Two-photon mapping of inhibitory inputs onto interneurons Aim 3: Imaging functional connectivity of V1 interneurons in vivo Hypothesis: Interneurons in vivo are activated in specific subnetworks 1.1. Two-photon SLM imaging of interneuron activity in anesthetized animals. 1.1. Two-photon SLM imaging of interneuron activity in awake animals 1.2. Computational analysis of functional connectivity. Outcomes and Impact Our work will provide a detailed blueprint of the structural and functional inhibitory connectivity in the visual cortex for all major subtypes of GABAergic interneurons. This blueprint could represent a "canonical microcircuit" for inhibition, i.e., a common and modular structural and functional circuit motif for neocortex, and could help understand the role that inhibition play in visual processing, developmental and adult visual plasticity, and central deficits of vision such as amblyopia.