pSer40 tyrosine hydroxylase immunohistochemistry identifies the anatomical location of C1 neurons in rat RVLM that are activated by hypotension

• Two monoclonal antibodies (mAbs) were made: one to TH and one to phosphorylated TH.
• The mAbs were of different isotypes allowing dual fluorescence immunohistochemistry.
• At rest, C1 neurons have a modest level of pTH, located mostly in the lateral RVLM.
• Following hypotension, the majority of C1 neurons in the RVLM become pTH-positive.
• These mAbs will enable the study of catecholamine neurons through the body.

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. Finally, we find that there is basal level of activation of TH neurons in the lateral, central and medial 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.