Efferent connections and phenotype of somatostatinergic neurons of lateral parafacial region of mice.

• Grant number: 25/13565-3
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate (Direct)
• Start date: January 31, 2026
• End date: January 30, 2027
• Field of knowledge: Biological Sciences - Physiology
• Principal Investigator: Davi José de Almeida Moraes
• Grantee: Nathalia Salim
• Supervisor: Erica Levitt
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Institution abroad: University of Michigan, United States
• Associated to the scholarship: 23/02560-5 - Involvement of the somatostatinergic interneurons of the lateral parafacial region in the generation of active expiration in mice, BP.DD

Abstract

The lateral parafacial region (pFL) contains glutamatergic neurons that control active expiration. Under baseline conditions, these neurons are silent due to GABAergic and glycinergic inhibition but become active during hypercapnia. Studies have identified inhibitory neurons in the parafacial region with distinct phenotypes, such as somatostatin (Sst), whose inhibition leads to increases in ventilation. Sst is an inhibitory neuromodulator widely distributed throughout the central nervous system and acts via G-protein-coupled receptors to reduce neuronal excitability. Evidence demonstrated that the parafacial neurons project to the respiratory centers, such as the preBötzinger Complex (preBötC) and the Kölliker-Fuse nucleus, and that Sst exerts an inhibitory effect on their activity, affecting the respiratory frequency. However, the projections and functional effects of pFL Sst-positive neurons remain poorly understood. In this sense, we hypothesized that pFL Sst-positive neurons project to both preBötC and the KF nucleus and exert an inhibitory control over their activity. To test this, our initial approach will be to map the axonal projections of pFL Sst-positive neurons. We will inject a Cre-dependent adeno-associated viral (AAV) vector into the pFL of transgenic mice that express Cre recombinase specifically in Sst-positive neurons (Sst-Cre). This approach will enable selective expression of green fluorescent protein (GFP) in pFL Sst-positive neurons, allowing us to visualize their soma and axonal projections to the brainstem. Subsequently, we will use the RNAscope in situ hybridization technique to characterize the molecular identity of transfected pFL Sst-positive neurons. To investigate whether these neurons form functional inhibitory connections, we will perform microinjections of a Cre-dependent AAV encoding channelrhodopsin-2 (ChR2) into the pFL of Sst-Cre mice. Acute brainstem slices and whole-cell patch clamp recordings of preBötC and KF neurons will be performed, while the pFL Sst-positive axon will be optogenetically stimulated. Together, these results will improve understanding of the processes that control active expiration through pFL connectivity within the respiratory network.