Cellular and functional heterogeneity of interhemispheric connections in the anterior olfactory nucleus

Abstract

The olfactory system in most mammals begins with two unique air flows separated by a nasal septum. Olfactory signals are then relayed to the brain in two separate olfactory bulbs, from which the brain produces a unitary perception of the olfactory environment. The mechanisms by which the brain combines these separate inputs to obtain perceptual unity remain unknown. The anterior olfactory nucleus (AON) is the earliest olfactory cortical area to project contralaterally, making it an excellent candidate for the combination of bilateral olfactory information. The AON is also implicated in social behavior and olfactory memory. However, the specific neuron types involved in the interhemispheric AON connection are unknown, as are the functional consequences of contralateral input to the AON. Identifying the underlying circuitry within the AON is critical to understanding its role in the olfactory cortex and olfactory-dependent behaviors. Interhemispheric projections between the AON pars principalis in mice are heterogeneous, with projection density varying widely between subregions. I have demonstrated that interhemispheric projections appear to be from a subtype of excitatory neurons expressing vesicular glutamate transporter-1 (VGLUT1-positive) and form synapses with both excitatory and inhibitory cells in the contralateral AON. These contralaterally projecting neurons exhibit distinct patterns of projections to downstream olfactory and non-olfactory cortical areas when compared to all VGLUT1-positive neurons in the AON. Additionally, I propose genetic markers for the identification of contralaterally projecting neurons based on spatial transcriptomic data and single-nucleus RNA sequencing. These findings suggest that contralaterally-projecting neurons form a distinct network capable of inducing specific and complex patterns of activity throughout the brain. I investigated the functional properties of contralateral AON projections both in vitro, through slice electrophysiology, and in vivo, through behavioral manipulation. Stimulation of contralaterally-projecting fibers results in monosynaptic excitation followed by polysynaptic inhibition. The response probability for the connection was 0.45, with 21/47 recorded cells displaying EPSCs. The same responses were also seen in VGAT-positive neurons in the AON, corroborating the anatomical findings of synapses onto both excitatory and inhibitory neurons. In a pilot behavioral study, I also suggest that contralateral projections are necessary for the transfer of learned odorant associations by mice with unilateral naris occlusion in a go-nogo task. Mice whose anterior commissures were severed were both slower to learn the initial task and showed inefficient transfer of learning to a new odor set. Overall, this work characterizes the circuitry underlying interhemispheric communication in the AON, investigates the resulting activity from its activation, and provides insights into the role of interhemispheric projections in olfactory behavior. This work will contribute not just to our understanding of the AON but also to the processing and computation done by the olfactory cortex. Insights from the cellular and functional diversity within the AON will guide future research on the role of the AON in olfactory-related disorders.