Immunocytochemistry of cGMP in the Cerebellum of the Immature, Adult, and Aged Rat: the Involvement of Nitric Oxide. A Micropharmacological Study

Abstract

In this study we describe the localization of formaldehyde-fixed cGMP-immunoreactivity (cGMP-IR) in rat cerebellar tissue slices incubated in vitro. In the absence of phosphodiesterase inhibition, cGMP-immunofluorescence was of low intensity in tissue slices prepared from immature cerebella. Addition of isobutylmethylxanthine (IBMX) to the incubation medium resulted in the appearance of cGMP-IR in clusters of astrocytes in the internal granular layer. Addition of N-methyl-d-aspartate (NMDA), kainic acid, atrial natriuretic factor (ANF), or sodium nitroprusside (SNP) gave an intense cGMP-IR in Bergmann fibres, Bergmann cell bodies, and astrocytes in the internal granular layer. Astrocytes in the white matter showed cGMP-IR after incubation of the slice in the presence of ANF or nitroprusside, but not after NMDA or kainic acid. In addition, after SNP stimulation of cGMP production, cGMP-IR was found in fibres which were not positive for glial fibrillary acidic protein (GFAP). In the adult cerebellar slice, intense basal cGMP-immunostaining was observed in Bergmann fibres, Bergmann cell bodies, and astrocytes in the granular layer. No cGMP-IR was observed in Purkinje cells. Stimulation of the cGMP-content in the glial structures by NMDA, ANF, or SNP, was suggested by the immunocytochemical results. However, when measured biochemically, only the effect of SNP was statistically significant, and immunocytochemistry showed that SNP clearly stimulated cGMP synthesis in neuronal cell structures. In the cerebellum of the aged rat a reduced cGMP-IR was found compared to the adult, in the same structures which showed cGMP-IR in the adult. Basal cGMP-immunostaining was reduced in the presence of haemoglobin, methylene blue, by inhibiting nitric oxide synthesis with NG-monomethyl-l-arginine (NGMAr), or by depletion of external Ca2+. Also the stimulatory effect of NMDA and of ANF (partly) on the cGMP-IR was inhibited by these compounds. cGMP-IR after stimulation of guanylate cyclase by SNP was reduced by the concomitant presence of haemoglobin or methylene blue, but not by NGMAr, or by omission of Ca2+. Our results point to an important role for cGMP in the functioning of glial tissue in the cerebellum and also suggest a role for nitric oxide as an intercellular mediator in the functioning of glutamate and ANF in the cerebellum.

Hydrolysis of N-Methyl-d-aspartate Receptor-Stimulated cAMP and cGMP by PDE4 and PDE2 Phosphodiesterases in Primary Neuronal Cultures of Rat Cerebral Cortex and Hippocampus

“…Immunocytochemical studies done in cerebellar slice preparations also have shown potentiation by IBMX of NOdependent cGMP formation in granule neurons (de Vente et al, 1990;Southam et al, 1992). These studies suggest that PDEs are regulators of cGMP in neurons; however, it is not clear which PDE families are involved in the hydrolysis of cGMP formed by NMDA receptor stimulation.…”

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

“…The lack of detectable cGMP expression in neurons of the RVLM is in accord with a detailed study by De Vente et al [22], who similarly observed cGMP in the NA and nucleus tractus solitarii (NTS) in the brain stem region, but do not comment on its expression in the RVLM, despite the use of invitro stimulation paradigms as applied in this study. In the work by De Vente et al, the importance of IBMX to reveal otherwise non-detectable cGMP populations was stressed [22,47] and this has also been noted by other authors [48]. For this reason, IBMX was used in a subset of studies in perfusion fixed rats and for the brainstem slice in-vitro incubation experiments.…”

“…Given the time period available for anaesthetic washout and the demonstration of an increase of detectable cGMP in surrounding tissues, it is likely the effects of anaesthetic were minimal in this study. The use of NMDA and NO donors to increase detectable cGMP in brain slice in-vitro preparations has been demonstrated previously in various tissues including the paraventricular nucleus [52] cerebellum [22,47] and hippocampus and thalamus [46]. The choice of DETA-NO as a donor was based on its ability to spontaneously liberate NO in aqueous solutions in a stable, time-controlled fashion [53].…”

Neuronal-glial Interactions Define the Role of Nitric Oxide in Neural Functional Processes

“…This is mainly due to the fact that astrocytes are highly responsive to NO-mediated cGMP production [61][62][63][64]. Some results suggest that the cGMP rise, readily occurring in astrocytes in response to NO stimulation, results in an increase of calcium concentration, in GFAP expression, the main intermediate filament of astrocytes, and in cytoskeleton remodeling [65][66][67][68].…”