Quantification of Nucleus Tractus Solitarius neurons projecting to Hypothalamic Paraventricular Nucleus

Background: Nucleus tractus solitarius (NTS) neurons integrate and relay visceral afferent inputs to various sites in the brain, notably the hypothalamic paraventricular nucleus (PVN). Understanding connectivity between the NTS and PVN will provide insight into possible interactions between the two areas in normal and pathophysiology. Purpose: To quantify the number of NTS neurons that project to the PVN using the retrograde transport agent cholera toxin B (CTB). Methods: Adult male Sprague-Dawley rats (n=3) were anesthetized intraperitoneally with ketamine (75mg/kg) and Dexdomitor (0.5mg/kg) then placed in a stereotaxic frame. Under aseptic conditions, the cranium overlying the PVN was exposed. Using a glass electrode, 100nL of 0.25% CTB in isotonic saline was slowly injected into the PVN bilaterally and the electrode withdrawn after 5 minutes. The skin was sutured and animals were allowed to recover. Three weeks later, the rats were transcardially perfused with 4% paraformaldehyde and their brains were harvested. PVN sections (40um thick) were used to verify the injection site. NTS sections (40um thick) were processed using immunohistochemistry. Polyclonal anti-CTB antibody (1:2000, Millipore) and secondary antibody Cy3 Donkey Anti-Goat (1:800, Jackson ImmunoResaerch Laboratories, Inc.) were used to visualize NTS neurons with axonal projections to PVN. Monoclonal anti-tyrosine hydroxylase (TH) antibody (1:1000, Millipore) and secondary antibody Alexa Fluor 488 Donkey Anti-Mouse (1:800, Jackson ImmunoResearch Laboratories, Inc.) were used to visualize catecholaminergic neurons. The number of CTB-immunoreactive, TH-immunoreactive, and co-labeled CTB and TH neurons were counted manually using Image J. Analysis was restricted to those NTS sections that had CTB-immunoreactivity and ranged from 15-21 sections each in the 3 rats. CTB injection sites were within the boundaries of the PVN. Results: We found 110±6 CTB immunoreactive and 346±33 TH immunoreactive neurons in NTS. 29±5 neurons were dual labeled with CTB and TH immunoreactivity indicating catecholaminergic NTS neurons projected to PVN. Conclusions: The majority of NTS neurons that project to PVN appear to be non-catecholaminergic. Approximately 10% of catecholaminergic NTS neurons project to PVN. Results will be useful in future studies using laser capture microdissection to examine gene expression in NTS neurons that project to PVN under a variety of conditions (e.g., hypoxia, hypertension).