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NO-responsive, cGMP-producing structures are abundantly present in the cervical spinal cord. NO-mediated cGMP synthesis has been implicated in nociceptive signaling and it has been demonstrated that cGMP has a role establishing synaptic connections in the spinal cord during development. As cGMP levels are controlled by the activity of soluble guanylyl cyclase (synthesis) and the phosphodiesterase (PDE) activity (breakdown), we studied the influence of PDE activity on NO-stimulated cGMP levels in the rat cervical spinal cord. cGMP-immunoreactivity (cGMP-IR) was localized in sections prepared from slices incubated in vitro. A number of reported PDE isoform-selective PDE inhibitors was studied in combination with diethylamineNONOate (DEANO) as a NO-donor including isobutyl-methylxanthine (IBMX) as a non-selective PDE inhibitor. We studied 8-methoxy-IBMX as a selective PDE1 inhibitor, erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and BAY 60-7550 as selective PDE2 inhibitors, sildenafil as a selective PDE5 inhibitor, dipyridamole as a mixed type PDE5 and PDE10 inhibitor, rolipram as a PDE4 inhibitor, and SCH 81566 as a selective PDE9 inhibitor. cGMP-IR structures (nerve fibers, axons, and terminals) were characterized using the following neurochemical markers: vesicular transporter molecules for acetylcholine, GABA, and glutamate (type 1 and type 2), parvalbumin, glutamate transporter molecule EAAT3, synaptophysin, substance P, calcitonin gene-related peptide, and isolectin B4. Most intense cGMP-IR was observed in the dorsal lamina. Ventral motor neurons were devoid of cGMP-IR. cGMP-IR was observed in GABAergic, and glutamatergic terminals in all gray matter laminae. cGMP-IR was abundantly colocalized with anti-vesicular glutamate transporter 2 (vGLUT2), however not with the anti-vesicular glutamate transporter 1 (vGLUT1), suggesting a functional difference between structures expressing vGLUT1 or vGLUT2. cGMP-IR did not colocalize with substance P- or calcitonin-gene related peptide-IR structures, however did partially colocalize with isolectin B4 in the dorsal horn. cGMP-IR in cholinergic structures was observed in dorsal root fibers entering the spinal cord, occasionally in laminae 1-3, in laminae 8 and 9 in isolated boutons and in the C-type terminals, and in small cells and varicosities in lamina 10. This latter observation suggests that the proprioceptive interneurons arising in lamina 10 are also NO-responsive. No region-specific nor a constant co-expression of cGMP-IR with various neuronal markers was observed after incubation of the slices with one of the selected PDE inhibitors. Expression of the mRNA of PDE2, 5, and 9 was observed in all lamina. The ventral motor neurons and the ependymal cells lining the central canal expressed all three PDE isoforms. Incubation of the slices in the presence of IBMX, DEANO in combination with BAY 41-2272, a NO-independent activator of soluble guanylyl cyclase, provided evidence for endogenous NO synthesis in the slice preparations and enhanced cGMP-IR in all lamina. Under these conditions cGMP-IR colocalized with substance P in a subpopulation of substance P-IR fibers. It is concluded that NO functions as a retrograde neurotransmitter in the spinal cord but that also postsynaptic structures are NO-responsive by producing cGMP. cGMP-IR in a subpopulation of isolectin B4 positive fibers and boutons is indicative for a role of NO-cGMP signaling in nociceptive processing. cGMP levels in the spinal cord are controlled by the concerted action of a number of PDE isoforms, which can be present in the same cell.

cGMP synthesis in cholinergic neurons of the basal forebrain, the caudate putamen, and the tegmento-pedunculopontine nucleus of the rat was studied during development after birth at P1, P4, P10, and P21, in the adult, and during aging. NO-mediated cGMP synthesis in these neurons was studied using the approach of in vitro incubation of brain slices in combination with cGMP-immunocytochemistry. The percentage of NO-responsive, cGMP-synthesizing cholinergic cells in the septum and diagonal band of Broca decreased from 75% to 6% in adult animals and to 2% in aged ones. In the caudate putamen, this decrease was from 81% to 21% in adult and 11% in aged animals. Cholinergic cells of the tegmento-pedunculopontine nucleus were unresponsive to NO and never showed cGMP-immunoreactivity. In addition, it was observed that the amount of NO-responsive, cGMP-synthesizing cholinergic fibers in the hippocampus declined in parallel with the maturation of the septal-hippocampal cholinergic pathway, whereas in the caudate putamen, this colocalization became complete 2 weeks after birth. It is concluded that the property of NO-mediated cGMP synthesis in the cholinergic nuclei of the forebrain is developmentally regulated after birth and that NO-cGMP signal transduction has a role in establishing cholinergic neuronal connections in the hippocampus and caudate putamen.

Possible differences in the localization of the cGMP response were investigated in rat brain coronal slices after in vitro incubation and NO-dependent or NO-independent stimulation of soluble guanylyl cyclase (sGC). Dose-dependent stimulation of cGMP synthesis by the NO donors, sodium nitroprusside, S-nitrosoglutathione, 3-morpholinosydnonimine and diethylamino NONOate was studied in the somatoparietal cortex, the hippocampus and the thalamus. cGMP accumulation was evaluated using a radioimmunoassay and by measuring cGMP-immunofluorescence using image analysis. All four NO donors induced similar cGMP staining patterns in the somatoparietal cortex, the hippocampus and the thalamus. NO-mediated cGMP synthesis in the cortical areas colocalized predominantly with the acetylcholine transporter and occasionally with parvalbumin (GABAergic cells) or the neuronal glutamate transporter. Incubation of the slices in the combined presence of a NO donor and the NO-independent activators YC-1 or BAY 41-2272 strongly potentiated cGMP synthesis and induced abundant cGMP-immunoreactivity in cortical GABAergic and glutamatergic cells. These findings indicate that the mechanism of NO release from the NO donors used does not determine the location of the cGMP response. The results suggest that YC-1 and BAY 41-2272 trigger a NO-sensing mechanism in cells in which the sGC is otherwise not sensitive to NO.

In this study, we report the cloning of the rat cGMP-specific phosphodiesterase type 9 (PDE9A) and its localization in rat and mouse brain by non-radioactive in situ hybridization. Rat PDE9A was 97.6% identical to mouse PDE9A1 and showed 92.1% similarity on the amino acid level to the human homologue. PDE9A mRNA was widely distributed throughout the rat and mouse brain, with the highest expression observed in cerebellar Purkinje cells. Furthermore, strong staining was detected in areas such as cortical layer V, olfactory tubercle, caudate putamen and hippocampal pyramidal and granule cells. Comparison of PDE9A mRNA expression by double staining with the cellular markers NeuN and glial fibrillary acidic protein demonstrated that PDE9A expression was mainly detected in neurons and in a subpopulation of astrocytes. Using cGMP-immunocytochemistry, the localization of cGMP was investigated in the cerebellum in which the highest PDE9 expression was demonstrated. Strong cGMP immunoreactivity was detected in the molecular layer in the presence of the non-selective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX). After treatment with soluble guanylyl cyclase activators the granular layer also showed cGMP staining, whereas no clear immunostaining was detected in Purkinje cells under all conditions investigated, which might be due to the presence of the IBMX-insensitive PDE9A in these cells. The present findings indicate that PDE9A is highly conserved between species and is widely distributed throughout the rodent brain. PDE9A is probably involved in maintenance of low cGMP levels in cells and might play an important role in a variety of brain functions involving cGMP-mediated signal transduction.

Whole brain spheroids provide a suitable model to study neurodevelopment. In the literature a role for the nitric oxide (NO)-cyclic guanosine 3',5'-monophosphate (cGMP) signalling pathway during development has frequently been suggested. In this study we investigated whether functional cGMP pathways were present in differentiated spheroids. In 3-week-old spheroids soluble guanylate cyclase was stimulated with N-methyl D-aspartic acid or sodium nitroprusside (NO donor). The results showed that the NO synthase-cGMP pathway is present in the culture system. Soluble guanylate cyclase-dependent cGMP formation was found in NO synthase containing neurons, in neurons of the GABAergic, glutamatergic and cholinergic system, and in astroglia and oligodendroglia. Activation of particulate guanylate cyclase by atrial natriuretic peptide also triggered an increase in cGMP production. Particulate guanylate cyclase was found in astroglia and in microglia as well as in glutamic acid decarboxylase and calbindin containing structures and neuronal NO synthase containing neurons. Chronic inhibition of NO synthase during culture development had no effect on soluble or particulate guanylate cyclase functioning. Similarly, inhibition of soluble guanylate cyclase during culture development did not have any effect on NO synthase and particulate guanylate cyclase functioning. It is concluded that NO synthase and both soluble and particulate guanylate cyclase are present in whole brain spheroid cultures and that their activity can be influenced by several stimuli. The spheroid culture system constitutes a suitable model to study the NO-cGMP pathway during brain development in mammals.

Recent studies have provided evidence that nitric oxide (NO) has a role in certain forms of memory formation. Spatial learning is one of the cognitive abilities that has been found to be impaired after systemic administration of an NO-synthase inhibitor. As the hippocampus has a pivotal role in spatial orientation, the present study examined the role of hippocampal NO in spatial learning and reversal learning in a Morris task in adult rats. It was found that N omega-nitro-L-arginine infusions into the dorsal hippocampus affected the manner in which the rats were searching the submerged platform during training, but did not affect the efficiency to find the spatial location of the escape platform. Hippocampal NO-synthase inhibition did not affect the learning of a new platform position in the same water tank (i.e. reversal learning). Moreover, no treatment effects were observed in the probe trials (i.e. after acquisition and after reversal learning), indicating that the rats treated with N omega-nitro-L-arginine had learned the spatial location of the platform. These findings were obtained under conditions where the NO synthesis in the dorsal hippocampus was completely inhibited. On the basis of the present data it was concluded that hippocampal NO is not critically involved in place learning in rats.

The urothelium plays a sensory role responding to deformation of the bladder wall; this involves the release of adenosine trisphosphate (ATP) and nitric oxide (NO), which affect afferent nerve discharge and bladder sensation. The urothelial cells responsible for producing ATP and NO and the cellular targets, other than afferent nerves, for ATP and NO remain largely unexplored. Sub-urothelial interstitial cells (SU-ICs) lie immediately below the urothelium and respond to NO with a rise in cGMP. To determine which cells might target SU-ICs by producing NO, areas of dome, lateral wall and base wall were treated with isobutyl-methyl-xanthine, exposed to the NO donor diethylamino NONOate and then fixed for immunohistochemistry. Surface urothelial cells (SUCs) in the base and dome expressed neuronal nitric oxide synthase (nNOS), whereas those in the lateral wall did not. Distinct populations of SUCs were present in the bladder base. SUCs with significant amounts of nNOS lay adjacent to cells with low levels of nNOS. In specific base regions, the few SUCs present contained nNOS within discrete intracellular particles. In the basal urothelial cell (BUC) layer of the lateral wall, nNOS-positive (NOS+) BUCs neither showed an elevation in cGMP in response to NO, nor expressed the β1 sub-unit of soluble guanylate cyclase, protein kinase I or protein kinase II. Thus, they produced but did not respond to NO. The BUC layer also stained for the stem cell factor c-Kit suggesting its involvement in urothelial cell development. No NOS+ BUCs were present in the SUC-sparse region in the bladder base. Exogenous NO produced an elevation in cGMP in SUCs and SU-ICs. The distribution and proportion of these target cells varied between the dome, lateral wall and base. cGMP+ SU-ICs were present as a dense layer in the bladder base but were rarely seen in the lateral wall, which contained nNOS+ BUCs. No nNOS+ BUCs and cGMP+ SU-ICs were apparent in the dome. The degree of complexity in nNOS distribution and NO target cells is therefore greater than has previously been described and may reflect distinct physiological functions that have yet to be identified. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO)-mediated cGMP synthesis is localized throughout the rat brain in close proximity to the NO-synthase-containing structures. However, characterization of the cGMP synthesizing structures in terms of co-localization with the classical neurotransmitter systems has not yet been reported. Here we present evidence, using double immunostaining for cGMP and the vesicular acetylcholine transporter, that virtually all of the cholinergic fibers in the cerebral cortex and the majority of the cholinergic fibers in the basal ganglia accumulate cGMP in response to a NO donor. In these areas, only few cGMP-containing fibers were observed not to be part of the cholinergic system. Co-localization between cGMP and the vesicular acetylcholine transporter was only observed to a minor degree in the ventral forebrain, the hippocampus, the reticular thalamic nucleus, and the nucleus ambiguus. No association of cGMP synthesis with the cholinergic system was observed to a similar extent in other brain areas. These results, in combination with literature data on the distribution of cholinergic receptors in the rat brain, suggest that NO has an anterograde and/or retrograde signaling function on subsets of cholinergic neurons. [ABSTRACT FROM AUTHOR]

Abstract Nitric oxide synthase (NOS) activity and NO-mediated cGMP synthesis were studied in the rat forebrain of control animals and animals which had received a unilateral lesioning of dopaminergic or serotonergic pathways. Lesioning of the dopaminergic innervation using 6-hydroxydopamine resulted in a 50% decrease in NOS activity in the lesioned frontal cortex and caudate putamen. Lesioning of the serotonergic innervation using 5,7-dihydroxytryptamine had no effect on NOS activity. NO-mediated cGMP accumulation in rat forebrain slices was not affected by 6-hydroxydopamine or 5,7,-dihydroxytryptamine lesioning. Using cGMP immunocytochemistry, it was demonstrated that NO-mediated cGMP synthesis was absent from dopaminergic, serotonergic, GABA-ergic and neuronal NOS-containing nerve fibres. A minor colocalization of cGMP immunoreactivity was found in parvalbumin-containing fibres in the cortex. Extensive colocalization between cGMP immunoreactivity and the acetylcholine transporter was found in all cortical areas and in the caudate putamen. There was no effect of the lesions on this colocalization. These results demonstrate NO-mediated cGMP accumulation in cholinergic fibres in the forebrain of the rat and suggest an anterograde signalling function of NO in cholinergic neuronal systems in the cortex and caudate putamen of the rat. [ABSTRACT FROM AUTHOR]