David C. Lyon - US grants

DESCRIPTION (provided by applicant): The aim of this proposal is to identify the underlying intrinsic mechanisms of contextual modulation in primary visual cortex (V1). Though the basic drive of orientation selectivity of V1 neurons is known to derive from retinal relays of the lateral geniculate nucleus (LGN), modulation of this classical receptive field (CRF) is generated through lateral long-range connections within V1. This surround modulation can either suppress or enhance the response of a neuron to its CRF. Previous work from the Sur lab and others has shown that the direction of the modulation depends on the orientation of the surround and the contrast of the CRF. Furthermore, orientation preference maps are arranged so that transitions between preferred orientation domains can be smooth or fractured (where incongruent orientation domains are found adjacent to each other). There are indications that long-range inputs to these fracture sites come from an in-homogenous array of other orientation domains, whereas smooth, or iso-orientation domains are known to receive lateral connections primarily from like iso-orientation domains. The proposed experiments will use optical imaging to identify fracture sites for fluorescent tracer injections, and two-photon microscopy will be used to identify the resulting clusters of labeled cells in V1. By matching the sites of labeled cells with the orientation preference map we can define specifically which regions provide modulatory input. With this information we can discreetly stimulate these regions with optimal orientation displays and systematically measure the effects on the CRF.

DESCRIPTION (provided by applicant): The neocortex is comprised of a dense population of excitatory neurons that are balanced by a diverse and more sparsely distributed array of inhibitory neurons. As such, cortical inhibitory neurons provide a gain control that works under a myriad of conditions. In primary visual cortex (V1), for example, inhibition is essential for severl basic functional characteristics of individual neurons, including preferences for stimulus contrast, size, and orientation. While inhibitory neurons serve to control excitation it is not knon whether inhibitory and excitatory cell-types are mediated through the same set of cortical circuits, nor whether the functional selectivity of these circuits differ. Determining the origins f the neural circuits to these two major cell-types will provide tremendous insight into the basic mechanisms of cortical processing. However, in order to do so it is necessary to develop a technique for cell-type specific neuroanatomical tracing. The proposal here is to develop such a technique by taking advantage of the neurotropism of recombinant viral vectors (adeno associated virus and lentivirus) and selectivity of cell-type specific promoters (GAD-67 and ¿-CamKII) to deliver and allow for expression of two key genes (TVA and RabG). These genes will thereby be selectively expressed in either inhibitory or excitatory neocortical cells, but not both. The TVA and RabG proteins expressed in these specific cell types will allow for targeted viral infection and trans-complementation of a novel genetically modified and pseudo-typed rabies virus (EnvA-¿RabG) that acts as a monosynaptic retrograde tracer (Wickersham, Lyon et al., 2007, Neuron). This highly innovative combination of cell-type specific expression of these genes and the targeted selectivity of the EnvA-¿RabG rabies virus will enable independent tracing of the connections of local clusters of inhibitory or excitatory cells. Moreover, once developed it will be used to trace the long-range connectivity of inhibitory and excitatory neurons in cat V1 with respect to the orientation map; maps that will be derived through intrinsic signal optical imaging. In this way, the functional preference of inputs to inhibitory neurons in neocortex can be determined for the first time and compared to the preference of inputs to excitatory cell populations. Finally, while the goal of this proposal is to ultimately implement th new technique to understand intrinsic circuits in cat V1, this technique will be available to other for use in any mammalian species and any region of neocortex. PUBLIC HEALTH RELEVANCE: The method proposed to be developed here will allow for selective neuroanatomical tracing of inputs to the two major classes of neurons in the mammalian brain, inhibitory and excitatory, rather than indiscriminant tracing of both types together, as current techniques do. This method involves the development and combination of a number of highly innovative tools including recombinant viruses, cell- type specific promoters, a genetically modified and pseudo-typed rabies virus, and intrinsic signal optical imaging. Once developed the technique can be used in any region of neocortex to determine, for the first time, how neural circuits of inhibitory and excitatory neurons compare and then relate these circuits to differences in cell type function.

DESCRIPTION (provided by applicant): Specific cell types and their connectivity are a key determinant in neural function and selectivity. Primary visual cortex (V1) is one of the largest and most complex structures in the brain and several recent technological advances have enabled more detailed probing of cell type specific relationships to connectivity for a range of V1 cell functions, including orientation selectivity, aperture tuning, contrast response functions, an gain control. Nevertheless, technical limitations remain that have largely limited these studies to transgenic mice which lack more complex organization found in higher visual species like cat and monkey. We recently developed viral strategies for accessing specific cell types and specific circuits in non-transgenic species (Liu et al., 2013, Curr Biol) opening the door for unprecedented fine scale study of structure-function relationships in highly visual mammals. For this proposal we will apply these new strategies to enable optogenetic manipulation of specific cell types and circuits in cat V1, including superficial layer inhibitory neurons (Aim 1), long-ran lateral inputs to superficial inhibitory neurons (Aim 2), and layer 5 and 6 subcortical projection neurons that directly (or indirectly) interact with superficial layer neurons (Aim 3). Specific questions that we will address include whether and how orientation selectivity and surround suppression interact and are mediated by each of these circuits. These studies will represent the most direct in vivo assessment of inhibitory neurons and underlying intra- and inter-laminar circuitry of a large, highly visual mammal, advancing our understanding of how basic visual processes arise and depend on complex cortical structure.

Project Summary Here we propose to develop a novel rabies virus based strategy to express two channelrhodopsin proteins independent manipulation of excitatory and inhibitory transmembrane potentials in the same population of neurons. Combined with the use of cell-type specific promoters in helper viruses and rabies pseudotyping, the new suite of viruses will enable full control of excitation and inhibition over specific neural network subtypes of non-transgenic animals. We will apply the new technique here to determine cell type specific contributions of feedforward cortical integration on the emergence of complex visual feature selectivity using highly visual animal models. While our goal is primarily to develop this novel method to study structure-function organization of visual cortex, this technique will be readily applicable to study any brain region. Our proposed technique combines recently available channelrhodopsin cation and anion channel with our recently our developed viral strategies for accessing specific cell types and specific circuits in non- transgenic species (Liu et al., 2013, Curr Biol) opening the door for unprecedented fine scale study of structure-function relationships in highly visual mammals. For this proposal we will apply these new strategies to enable optogenetic manipulation of specific cell types involved in higher order visual cortex receptive field formation. These studies will represent the most direct in vivo assessment of inhibitory neurons and underlying intra- and inter-laminar circuitry of a large, highly visual mammal, advancing our understanding of how basic visual processes arise and depend on complex cortical structure.