ABSTRACT BRAIN Initiative-funded, large-scale approaches to classify neurons based on transcriptomic, morphological and electrical properties have unveiled dozens of unique cell classes in the mouse brain. However, whether they represent functionally diverse populations of relevance to animal perception and behavior remains an open question. Dissecting their individual roles requires the integration of targeted recording techniques and optogenetic manipulation approaches, which operate at the physiologically relevant spatiotemporal scales (i.e. cellular resolution and millisecond timescales). Here, we propose to use high-speed (0.4-1 kHz), genetically encoded fluorescence voltage imaging to understand the role of distinct interneuronal populations in attentional modulation of visual processing during visually guided behavior. First, we will establish the optical instrumentation for high-speed, dual channel voltage imaging with non-overlapping structured illumination. We will further validate the use of our second-generation, fluorescence resonance energy transfer (FRET)-opsin indicators Ace-mNeon2 and VARNAM2 and their reverse response polarity variants pAce and pAceR, for concurrent voltage recordings from pairs of interneuronal ensembles and pyramidal neurons in awake, running mice. Thereafter, using simultaneous triple-population voltage imaging, we will assess the effects of attention on the firing rates of the three cell classes during visually guided behavior and compute the spatiotemporal correlations in the activation patterns of neighboring neurons. Separately, we will measure the visual tuning properties of the same neurons during presentations of drifting grating stimuli. We will further draw a correlation between neuronal attention modulation index and feature selectivity to test the applicability of the feature similarity gain model. Lastly, to establish causal roles in the attentional modulation of visual responses, we will optogenetically manipulate the activity of select interneurons in a spatially precise manner, while recording the voltage responses in neighboring pyramidal cells when mice are engaged in the behavioral task. Our proposed work will (1) elucidate the role of distinct interneuron-types in visual attention and enable functional cross-comparisons within the same animals and at increased spatiotemporal resolution; (2) uncover synergistic and antagonistic relationships between neighboring pyramidal neuron-interneuron pairs for neurons that are positively versus negatively modulated by attention; (3) test the applicability of the feature similarity gain model in rodents and (4) establish causal roles for distinct interneuronal populations in attentional modulation of visual processing. Together, our work will establish simultaneous, multipopulation voltage imaging as the preferred modality to unravel the real-time functional differences between neuron-types in perception and behavior.

PROJECT NARRATIVE To dissect the functional roles of distinct neuron-types in animal perception and behavior, targeted recording techniques that operate at the physiologically relevant spatiotemporal scales should complement current approaches of functional manipulation. Here, we propose to use our new suite of genetically encoded fluorescent voltage indicators for concurrent, high-speed, multipopulation recordings to understand the role of distinct visual cortical interneuron-types in attentional modulation of sensory processing during visually guided behavior. Our proposed work will enable real-time comparisons of the voltage dynamics among and between distinct interneuronal ensembles that contribute to visual gain modulation and establish multipopulation voltage imaging as the preferred method to reveal these interactions at cellular-resolution and millisecond timescales.