PSer40 tyrosine hydroxylase immunohistochemistry identifies the anatomical location of C1 neurons in rat RVLM that are activated by hypotension

Abstract

Identification of neurons, and their phenotype, that are activated in response to specific stimuli is a critical step in understanding how neural networks integrate inputs to produce specific outputs. Here, we developed novel mouse monoclonal antibodies of different IgG isotypes that are specific to tyrosine hydroxylase (TH), and to tyrosine hydroxylase activated at its serine 40 position (pSer40TH), in order to assess changes in the activity of phenotypically identified cardiovascular neurons using fluorescence immunohistochemistry. We find that the proportion of C1 pSer40TH-positive neurons in the central and medial region of the rat rostral ventrolateral medulla (RVLM) increases dramatically following hydralazine treatment, whereas phenylephrine treatment does not significantly change the pSer40TH/TH ratio in these regions compared to control. This finding suggests that there is a mediolateral topology associated with the activation of C1 neurons following baroreceptor loading or unloading. Overall, we conclude first, that our newly characterized monoclonal antibodies are specific, and selective, against TH and pSer40TH. Secondly, that they can be used to label TH and pSer40TH immunoreactive neurons simultaneously, and thirdly that that they can be used to identify the activation state of catecholamine synthetizing neurons after physiological stimuli. Fourthly, we find that there is basal level of activation of TH neurons in the lateral, medial and central regions (~70%, 30% and 45%, respectively) of the C1 area, but that following unloading of the baroreceptors there is a marked increase in activation of central (~80%) and medial (~90%) C1 neurons in the RVLM. Support or Funding Information This work was supported by the Australian Research Council (Discovery Early Career Researcher Award DE120100992), the National Health and Medical Research Council of Australia (Grants 1065485 and 1082215 and Fellowship 1024489), Sydney University, Macquarie University and the Heart Research Institute. TH staining, pTH staining and merged staining in rat brainstem.