Immunohistochemical assessment of cyclic guanosine monophosphate (cGMP) and soluble guanylate cyclase (sGC) within the rostral ventrolateral medulla.

Functional evidence suggests that nitric oxide (NO) signalling in the rostral ventrolateral medulla (RVLM) is cGMP-dependent and that this pathway is impaired in hypertension. We examined cGMP expression as a marker of active NO signalling in the C1 region of the RVLM, comparing adult (>18 weeks) Wistar-Kyoto (WKY, n = 4) and spontaneously hypertensive rats (SHR, n = 4). Double label immunohistochemistry for cGMP-immunoreactivity (IR) and C1 neurons [as identified by phenylethanolamine N-methyltransferase (PNMT-IR) or tyrosine hydroxylase TH-IR)], or neuronal NO synthase (nNOS) neurones, failed to reveal cGMP-IR neurons in the RVLM of either strain, despite consistent detection of cGMP-IR in the nucleus ambiguus (NA). This was unchanged in the presence of isobutylmethylxanthine (IBMX; 0.5 mM, WKY, n = 4, SHR n = 2) and in young animals (WKY, 10-weeks, n = 3). Incubation of RVLM-slices (WKY, 10-weeks, n = 9) in DETA-NO (100 mum; 10 min) or NMDA (10 muM; 2 min) did not uncover cGMP-IR. In all studies, cGMP was prominent within the vasculature. Soluble guanylate cyclase (sGC)-IR was found throughout neurones of the RVLM, but did not co-localise with PNMT, TH or nNOS-IR neurons (WKY, 10-weeks, n = 6). Results indicate that within the RVLM, cGMP is not detectable using immunohistochemistry in the basal state and cannot be elicited by phosphodiesterase inhibition, NMDA receptor stimulation or NO donor application.

Distinct subpopulations of cyclic guanosine monophosphate (cGMP) and neuronal nitric oxide synthase (nNOS) containing sympathetic preganglionic neurons in spontaneously hypertensive and Wistar-Kyoto rats.

The sympathetic preganglionic neurons (SPN) of the intermediolateral cell column (IML) play a critical role in the maintenance of vascular tone. We undertook a comparative neuroanatomical analysis of neuronal nitric oxide synthase (nNOS) expression in the SPN of the mature normotensive Wistar Kyoto (WKY) and spontaneously hypertensive rat (SHR). The anatomical relationship between nNOS and the NO signaling molecule cyclic guanosine monophosphate (cGMP) was also determined. All animals were male, age > 6 months. Fluorogold (FG) retrograde labeling of SPN (detected with immunohistochemistry) was combined with NADPH-diaphorase histochemistry for NOS in the thoracic spinal cord (T1-11, n = 5 WKY, 5 SHR). There was no difference in the total number of FG-labeled SPN (WKY 6,542 +/- 828, SHR 6,091 +/- 820), but the proportion of FG-labeled cells expressing NOS was significantly less in the SHR (WKY 64.4 +/- 5.1 vs. SHR 55.6 +/- 2.1, P < 0.05). Fluorescence immunohistochemistry for nNOS/cGMP (n = 4 WKY, 4 SHR) was also performed. Confocal microscopy revealed that all nNOS-positive SPN contain cGMP and confirmed a strain-specific anatomical arrangement of SPN cell clusters. A novel subpopulation of cGMP-only cells were also identified. Double labeling for cGMP and choline acetyltransferase (n = 3 WKY, 3 SHR), confirmed these cells as SPN in both WKY and SHR. These results suggest that cGMP is a key signaling molecule in SPN, and that a reduced number of NOS neurons in the SHR may play a role in the increase in sympathetic tone associated with hypertension in these animals.

Unique levels of expression of N-methyl-D-aspartate receptor subunits and neuronal nitric oxide synthase in the rostral ventrolateral medulla of the spontaneously hypertensive rat.

The rostral ventrolateral medulla (RVLM) is the major brainstem region contributing to sympathetic control of blood pressure. We have compared the expression of N-methyl-d-aspartate (NMDA) receptor subunits (NR1, NR2A-D), NR1 splice variants (NR1-1a/1b, -2a/2b, -3a/3b, -4a/4b), and the neuronal and inducible isoforms of NO synthase (nNOS and iNOS) in the RVLM of Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR), based on the hypothesis that altered NMDA receptor make-up or altered expression of endogenous NO may be associated with the increase in sympathetic output described from this site in hypertension. Total RNA was extracted and reverse transcribed from the RVLM of mature male WKY and SHR (16-23 weeks). Conventional polymerase chain reaction (PCR) indicated that only the NR1 splice variants NR1-2a, NR1-2b, NR1-4a and NR1-4b were expressed in the RVLM of either species. Quantitative real-time PCR indicated that for both strains of rat, mRNA for the NR1 subunit (all splice variants) was the most abundant (16.5-fold greater, P< or =0.05, relative to the NR2A subunit). Amongst the NR2A-D subunits, NR2C was the most abundant (7- and 1.7-fold greater relative to the NR2A subunit, P< or =0.05, WKY and SHR, respectively). Relative to WKY, mRNA levels for the NR2C and NR2D subunits in the SHR RVLM were significantly lower (0.3- and 0.25-fold less, P< or =0.05), while nNOS was significantly higher (1.76-fold greater, P< or =0.05). This was confirmed immunohistochemically for nNOS expression. These results demonstrate differential expression levels of NMDA receptor subunits and NOS isoforms in the RVLM region of SHR when compared to WKY rats.

Heterogeneous distribution of basal cyclic guanosine monophosphate within distinct neuronal populations in the hypothalamic paraventricular nucleus.

The supraoptic (SON) and the paraventricular (PVN) hypothalamic nuclei constitute major neuronal substrates underlying nitric oxide (NO) effects on autonomic and neuroendocrine control. Within these nuclei, constitutively produced NO restrains the firing activity of magnocellular neurosecretory and preautonomic neurons, actions thought to be mediated by a cGMP-dependent enhancement of GABAergic inhibitory transmission. In the present study, we expanded on this knowledge by performing a detailed anatomical characterization of constitutive NO-receptive, cGMP-producing neurons within the PVN. To this end, we combined tract-tracing techniques and immunohistochemistry to visualize cGMP immunoreactivity within functionally, neurochemically, and topographically discrete PVN neuronal populations in Wistar rats. Basal cGMP immunoreactivity was readily observed in the PVN, both in neuronal and vascular profiles. The incidence of cGMP immunoreactivity was significantly higher in magnocellular (69%) compared with preautonomic ( approximately 10%) neuronal populations (P < 0.01). No differences were observed between oxytocin (OT) and vasopressin (VP) magnocellular neurons. In preautonomic neurons, the incidence of cGMP was independent of their subnuclei distribution, innervated target (i.e., intermediolateral cell column, nucleus tractus solitarii, or rostral ventrolateral medulla) or their neurochemical phenotype (i.e., OT or VP). Finally, high levels of cGMP immunoreactivity were observed in GABAergic somata and terminals within the PVN of eGFP-GAD67 transgenic mice. Altogether, these data support a highly heterogeneous distribution of basal cGMP levels within the PVN and further support the notion that constitutive NO actions in the PVN involve intricate cell-cell interactions, as well as heterogeneous signaling modalities.