OBJECTIVE: Previous studies have shown the significant contribution of sympathoinhibition in response to sodium loading to prevent increases in mean arterial blood pressure in salt resistant phenotypes. It has also been shown that brain Gαi2 protein gated signal transduction plays a major role in this pathway, however, the specific mechanisms through which this pathway is activated remain less well understood. The purpose of this study was to elucidate the relative contribution of increased sodium in either the plasma or the cerebrospinal fluid (CSF) to the regulation of mean arterial pressure and natriuresis. Additionally we explored the potential for using the novel CLARITY Imaging technique to identify the relative activity of neurons in areas of the brain thought to play a major role in body fluid homeostasis in response to salt. METHODS: Rats that were pre-treated with either scrambled or Gαi2 oligodeoxynucleotides (ODN), to selectively down regulate brain Gαi2 proteins, were challenged either peripherally or centrally with sodium. Upon sodium loading physiological parameters were measured for two hours after which the animal's brains were recovered for immunohistochemical (IHC) analysis of the paraventricular nucleus, a known regulatory center for body fluid homeostasis and blood pressure regulation. Additionally we adapted a version of the published CLARITY Imaging protocols for optically clearing tissue through application of electrophoretic tissue clearing (ETC) to a larger rat model. RESULTS: In scrambled ODN pre-treated rats we observed a temporary increase in MAP in response to both the peripheral and central sodium challenge. In the Gαi2 ODN pre-treated animals we saw some form of attenuation to this response in both studies, however, where in the peripheral challenge there was an increase in the amount of time that it took the rats to return to normotension with no alteration in natriuresis, in the central challenge there was a large attenuation in natriuresis with no differences in the time to return to baseline MAP. Our IHC analysis also showed a decrease in neuronal activation of paraventricular medial parvocellular neurons in Gαi2 pre-treated rats that were challenged peripherally vs their SCR pre-treated counterparts. No such difference was observed in either of the pre-treatment groups from the central sodium challenge study. In the CLARITY study we found that it is possible to adapt the method for optically clearing tissue to the larger model, however, we encountered several issues related to tissue swelling and peripheral tissue damage. CONCLUSION: Based on our current results it seems evident that there are at least two different mechanisms that activate the cardiovascular regulatory control centers in the brain that prevent long term increases in mean arterial pressure in response to increased salt. It also appears that these two different mechanisms are triggered either by increases in plasma or CSF salinity, though which of these two mechanisms may be directly responsible for the development of salt sensitive hypertension requires further investigation. While we had some success at optically clearing larger tissue volumes through ETC, problems we encountered with maintaining tissue integrity for investigations of intact neural networks prevented us from applying this technique, in its current form, to our investigation of salt sensitive hypertension.