Variability in the occurrence of nitric oxide synthase immunoreactivity in different populations of rat sympathetic preganglionic neurons

Abstract

The proportion of sympathetic preganglionic neurons (SPN) showing nitric oxide synthase (NOS) immunoreactivity appears to vary with innervation target and blood pressure level. For normotensive Sprague-Dawley rats (SD), we evaluated peroxidase immunolabelling for choline acetyltransferase (ChAT) plus NOS in spinal cord segments T1-L2 and assessed NOS immunofluorescence in SPN retrogradely labelled with cholera toxin B subunit from the adrenal medulla (AM) or superior cervical (SCG), coeliac (CG), or major pelvic (MPG) ganglia. We also compared the distributions and numbers of NOS-positive and NOS-negative/ChAT-positive lateral horn neurons in SD with those in normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). In SD, WKY, and SHR, rostrocaudal, dorsoventral, and mediolateral differences occurred in the distributions of NOS-positive and NOS-negative/ChAT-positive neurons in the intermediolateral cell column (IML), whereas the two groups were similarly distributed throughout the central autonomic area (CAA). Among the four retrogradely labelled populations of SPN, the percentages showing NOS immunoreactivity differed (CG-projecting, 54.8% +/- 0.7%; SCG-projecting, 75.3% +/- 1.2%; MPG-projecting, 89% +/- 1.1% and AM-projecting, 98.6% +/- 0.2%). Within each retrogradely labelled group of SPN, the NOS-positive proportion also varied with subnuclear location (e.g., 25.5% +/- 4.0% of CG-projecting SPN in the CAA vs. 82.7% +/- 7.6% of CG-projecting SPN in the dorsolateral funiculus). The numbers of NOS-positive and NOS-negative/ChAT-positive neurons in T9-T11 were the same in SD and SHR but differed in WKY. Our results show that the expression of NOS within SPN varies depending on the target that they innervate and also on their subnuclear location. Our data indicate that there are no anatomical differences between nitric oxide-synthesizing SPN in normotensive SD and hypertensive SHR.

Innervation of the rat uterus at estrus: A study in full‐thickness, immunoperoxidase‐stained whole‐mount preparations

“…The supplier has also shown that preadsorption of the anti-VAChT with the peptide used for antibody production abolished labeling. We have found that the Chemicon goat antiVAChT stains neurons in the spinal cord that have distributions and morphologies that are consistent with our previous immunohistochemical demonstrations of cholinergic neurons using an antibody against choline acetyltransferase (Llewellyn-Smith et al, 2007;Hinrichs and Llewellyn-Smith, 2009). A number of studies on central and peripheral cholinergic neurons have also used this antibody (e.g., Padley et al, 2007;Nangle et al, 2009).…”

“…In subsequent experiments, the dilution showing minimal nonspecific staining and the maximum number and best-stained immunoreactive axons was used. We have previously carried out adsorption control experiments on the anti-NPY (Llewellyn-Smith et al, 1990), anti-SP (Llewellyn-Smith andMinson, 1992), and anti-NOS (Hinrichs and Llewellyn-Smith, 2009) antisera used here. These experiments were done on cryostat sections of perfused medulla and showed that staining was abolished by preadsorption of the antisera with the antigens against which they were raised.…”

“…No. 1720-9007) on sections of medulla fixed with 4% formaldehyde and antibody-labeled using an immunoperoxidase method that is standard in our laboratory (e.g., Llewellyn-Smith et al, 2007;Hinrichs and Llewellyn-Smith, 2009). We found that a 1:10,000 dilution of the anti-CGRP antiserum preadsorbed with 10 lg/mL of CGRP 1-37 from rat (Auspep Cat.…”

Hydralazine administration activates sympathetic preganglionic neurons whose activity mobilizes glucose and increases cardiovascular function

“…Polyclonal sheep anti-digoxigenin (DIG) antibody recognizes the DIG-11-UTP incorporated into the riboprobe during in vitro transcription. Polyclonal anti-choline acetyltransferase (ChAT) antibody was used to identify SPN, which were determined by size and location in nests within the intermediolateral cell column (IML) of the spinal cord (Fenwick et al, 2006;Hinrichs and Llewellyn-Smith, 2009;Parker et al, 2013). ChAT is also found in ventral horn motoneurons which were not analyzed.…”

“…A range of neurochemicals have been described in SPN, particularly in those projecting to the adrenal gland and celiac ganglia, including pituitary adenylate cyclase activating polypeptide (PACAP) mRNA, pre-proenkephalin (PPE) mRNA, nitric oxide synthase (NOS), cocaine and amphetamine-regulated transcript (CART) and calretinin (Edwards et al, 1996;Fenwick et al, 2006;Hinrichs and Llewellyn-Smith, 2009;Kumar et al, 2010;Parker et al, 2013). We have shown previously that glucoprivation, induced by 2-deoxy-D-glucose, which does not change blood pressure, activates all SPN projecting to the adrenal glands and celiac ganglia that express PPE mRNA and only activates adrenergic chromaffin cells (Parker et al, 2013) suggesting PPE mRNA codes SPN regulating glycemic but not cardiovascular responses.…”