Modulating Breathing: Identifying transcriptionally-defined projections to the preBotzinger complex and their effect on breathing

Breathing relies on rhythmic activity generated by the preBötzinger complex (preBötC), a small brainstem region. Because breathing is influenced by many different physiological, behavioral, and emotional contexts, the preBötC is not only highly connected with nearby respiratory-related regions in the hindbrain, but also has widespread connections with other mid- and forebrain regions not generally implicated in the regulation of breathing. The specific functions for many of these preBötC inputs remain unclear. Although afferent pathways indiscriminately target excitatory and inhibitory neurons in the preBötC, we hypothesized that inputs to the preBötC would map to distinct brain regions based on the inhibitory or excitatory phenotype of the projecting neurons. To test this, mice that express tdTomato only when both Cre and FlpO recombinases are present (Ai65) were bred with mice that express Cre in either inhibitory ( VgatCre ) or excitatory ( Vglut2Cre ) neurons. Adult offspring ( VgatCre or Vglut2Cre; Ai65) received a unilateral preBötC injection of a retrograde AAV that expresses FlpO recombinase (AAVrg-FlpO), thereby specifically activating tdTomato expression in either inhibitory or excitatory neurons that project directly to the preBötC. This combination of transgenic and viral techniques can identify subpopulations of projecting neurons with high efficiency because only a small amount of a single virus needs to reach the cell body to induce strong reporter labeling. Whole-brain imaging revealed distinct populations of labeled neurons with little overlap between inhibitory and excitatory projections. As expected, there were many projections within the ipsilateral preBötC and projecting neurons clustered in the contralateral preBötC. The tracing confirmed known connections through the ventral respiratory group (VRG) and identified projections from mid- and forebrain regions. Vgat+ projections were found primarily in the rostral ventromedial medulla, zona incerta, amygdala, and the bed nucleus of the stria terminalis. On the other hand, the major areas with Vglut2+ projections were the locus ceruleus, parabrachial nucleus, sensory cortex, hypothalamus, and motor cortex. In all areas there was a bias towards ipsilateral projections. This technique is highly versatile and can be used with many different Cre lines. Previous research has shown that preBötC neurons express the μ-opioid receptor, but it is unclear where the endogenous opioids originate from. We have started preliminary experiments repeating the double recombinase technique with a Cre line attached to Penk, a gene that encodes an endogenous opioid called enkephalin. These experiments have identified clear opioidergic projections to the preBötC from the nucleus tractus solitarius (NTS), rostral ventromedial medulla and parabrachial nucleus. Defining these cell-type-specific inputs to the preBötC has provided an essential foundation for current functional studies to determine how and under what circumstances these circuits influence breathing, using recombinase-dependent optogenetics and whole-body plethysmography. R01 HL1660317 (Baertsch) This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.