RESUMO
In mammals, the ventral respiratory column (VRC) plays a pivotal role in integrating neurochemically diverse inputs from brainstem and forebrain regions to generate respiratory motor patterns. VRC microinjection of the neuropeptide galanin has been reported to dampen carbon dioxide (CO2)-mediated chemoreflex responses. Additionally, we previously demonstrated that galaninergic neurons in the retrotrapezoid nucleus (RTN) are implicated in the adaptive response to hypercapnic stimuli, suggesting a link between RTN neuroplasticity and increased neuronal drive to the VRC. VRC neurons express galanin receptor 1, suggesting potential regulatory action by galanin, however, the precise galaninergic chemoreceptor-VRC circuitry remains to be determined. This study aimed to identify sources of galaninergic input to the VRC that contribute to central respiratory chemoreception. We employed a combination of retrograde neuronal tracing, in situ hybridisation and immunohistochemistry to investigate VRC-projecting neurons that synthesise galanin mRNA. In an additional series of experiments, we used acute hypercapnia exposure (10% CO2, 1 h) and c-Fos immunohistochemistry to ascertain which galaninergic nuclei projecting to the VRC are activated. Our findings reveal that a total of 30 brain nuclei and 51 subnuclei project to the VRC, with 12 of these containing galaninergic neurons, including the RTN. Among these galaninergic populations, only a subset of the RTN neurons (approximately 55%) exhibited activation in response to acute hypercapnia. Our findings highlight that the RTN is the likely source of galaninergic transmission to the VRC in response to hypercapnic stimuli.
RESUMO
Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within cells. Neurochemical phenotyping of functionally identified neurons usually requires concurrent labelling with multiple antibodies (targeting protein) using immunohistochemistry (IHC) and optimization of in situ hybridization (targeting RNA), in tandem. A "neurochemical signature" to characterize particular neurons may be achieved however complicating factors include the need to verify FISH and IHC targets before combining the methods, and the limited number of RNAs and proteins that may be targeted simultaneously within the same tissue section. Here we describe a protocol, using both fresh frozen and fixed mouse brain preparations, which detects multiple mRNAs and proteins in the same brain section using RNAscope FISH followed by fluorescence immunostaining, respectively. We use the combined method to describe the expression pattern of low abundance mRNAs (e.g., galanin receptor 1) and high abundance mRNAs (e.g., glycine transporter 2), in immunohistochemically identified brainstem nuclei. Key considerations for protein labelling downstream of the FISH assay extend beyond tissue preparation and optimization of FISH probe labelling. For example, we found that antibody binding and labelling specificity can be detrimentally affected by the protease step within the FISH probe assay. Proteases catalyze hydrolytic cleavage of peptide bonds, facilitating FISH probe entry into cells, however they may also digest the protein targeted by the subsequent IHC assay, producing off target binding. The subcellular location of the targeted protein is another factor contributing to IHC success following FISH probe assay. We observed IHC specificity to be retained when the targeted protein is membrane bound, whereas IHC targeting cytoplasmic protein required extensive troubleshooting. Finally, we found handling of slide-mounted fixed frozen tissue more challenging than fresh frozen tissue, however IHC quality was overall better with fixed frozen tissue, when combined with RNAscope.
