Characterizing the phenotype of pre-Bötzinger Complex neurons in rats

The pre-Bötzinger Complex (preBötC) is a key medullary region responsible for generating breathing. In rodents, preBötC neurons are divided almost evenly between excitatory neurons, which express vesicular glutamate transporter 2 (VGluT2), and inhibitory neurons expressing either glutamic acid decarboxylase (GAD) or glycine transporter 2 (GlyT2). The interaction between excitatory and inhibitory neurons plays a significant role in rhythmic breathing and its coordination with other physiological functions. However, comprehensive knowledge about the classification and the physiological roles of preBötC neuronal subpopulations in adults is limited. This arises due to the complex interconnections of preBötC with adjacent regions, undefined anatomical boundaries of the region, diverse neurochemical signatures without clear functional distinctions, and the predominant reliance on prenatal mouse data. In this study, we aimed to enhance the understanding of the neurochemical signatures of preBötC neurons and their proportions by rigorously defining the boundaries of the preBötC in adult male rats (n = 3). For this, we employed RNAscope in situ hybridization to identify, and anatomically and systematically characterize, the subgroups of preBötC neurons expressing VGluT2, somatostatin (SST), GAD1, vesicular GABA transporter (VGAT) and/or reelin. We observed that most SST-expressing neurons are glutamatergic and comprise over 50% of the excitatory population of preBötC. In addition, a considerable proportion of SST-expressing neurons express GAD1. Our results also show that approximately half of SST-expressing neurons co-express reelin, and that most reelin-expressing neurons are glutamatergic. A key finding is that the combination of immunohistochemistry for reelin with parvalbumin, is a reliable marker to define the anatomical location of preBötC.