Identifying molecular markers and local axonal projections of novel neuron types in the auditory midbrain

The National Institute on Deafness and Other Communication Disorders awarded the Regents of the University of Michigan $214,993 on September 30, 2025, under the Research Related to Deafness and Communication Disorders program (CFDA 93.173) to identify molecular markers and map local axonal projections of novel neuron types in the auditory midbrain's inferior colliculus.

The award funds research to characterize two newly identified candidate molecular markers—secreted phosphoprotein 1 (SPP1) and prodynorphin (PDYN)—as markers of previously unidentified neuron types in the inferior colliculus. Using molecular, anatomical, and electrophysiological approaches, the recipient will determine the distribution, morphology, physiology, and stereotyped patterns of local axonal projections of SPP1 and PDYN neurons. The research leverages next-generation sequencing data from the Allen Brain Cell Atlas and preliminary in situ hybridization results showing that SPP1 and PDYN label separate populations of excitatory neurons distinct from those previously identified in the midbrain auditory pathway.

Work is performed in Ann Arbor, Michigan, with a period of performance from September 30, 2025, through April 30, 2029. The assistance type is Project Grant.

Abstract

In the inferior colliculus (IC), the midbrain hub of the auditory pathway, most neurons have local axon collaterals, suggesting that the IC contains complex local circuits that support critical auditory computations. To understand how local circuits in the IC support auditory computations, IT is essential to determine the wiring diagram by which specific types of IC neurons interconnect. However, IT has proven difficult to reliably identify IC neuron types and map the local axonal connections using conventional approaches. Recently, molecular markers for four IC neuron types have been identified by screening transgenic mouse lines, but the identity of most IC neuron types remains unknown. By leveraging next generation sequencing data from the allen brain cell atlas, we have identified two new candidate molecular markers for IC neuron types: secreted phosphoprotein 1 (SPP1) and prodynorphin (pdyn). Our preliminary in situ hybridization results show that SPP1 and pdyn label two separate populations of excitatory neurons distinct from those previously identified in the IC. Based on our preliminary results we hypothesize that SPP1 and pdyn are markers of IC neuron types characterized by internally consistent morphological, physiological, and anatomical features, and exhibiting stereotyped patterns of local axonal projections. To test this hypothesis, in aim 1 we will use molecular, anatomical and electrophysiological approaches to identify the distribution, morphology and physiology of SPP1 and pdyn neurons. We expect aim 1 will demonstrate that SPP1 and pdyn mark two previously unidentified IC neuron types. Previous studies have shown that neurons in the central nucleus of the IC tend to form local axon projections that follow one of three patterns: remain in the same isofrequency lamina as the soma, project to a specific isofrequency lamina distal to the soma or spread across multiple isofrequency laminae. However, IT is not known whether individual IC neuron types exhibit homogenous or heterogenous patterns of local projections and whether those local projections preferentially synapse onto inhibitory or excitatory neurons. Based on precedents in other brain regions, we hypothesize that SPP1 and pdyn neurons each have a strong tendency to follow one of the aforementioned local axon projection patterns and to preferentially target a subset of IC neurons. We will test this hypothesis in aim 2 using two approaches. First, using sparse labeling, clearing and light sheet microscopy, we will determine the axonal and dendritic morphology of SPP1 and pdyn neurons across the entirety of the IC. Second, to determine postsynaptic target patterns, we will use expansion microscopy and immunolabeling to map the distribution of SPP1 and pdyn synapses on inhibitory and excitatory postsynaptic densities. The expected results from aim 2 will allow US to generate hypotheses about the functional roles of SPP1 and pdyn neurons in local IC circuits. Overall, our results will reveal the fundamental physiological and anatomical properties of two IC neuron types and provide insight into how these neuron types contribute to local circuits function in the IC.