Marjanne Markerink-van Ittersum

mRNA expression patterns of the cGMP-hydrolyzing phosphodiesterases types 2, 5, and 9 during development of the rat brain

Recent evidence indicates that cGMP plays an important role in neural development and neurotransmission. Since cGMP levels depend critically on the activities of phosphodiesterase (PDE) enzymes, mRNA expression patterns were examined for several key cGMP‐hydrolyzing PDEs (type 2 [PDE2], 5 [PDE5], and 9 [PDE9]) in rat brain at defined developmental stages. Riboprobes were used for nonradioactive in situ hybridization on sections derived from embryonic animals at 15 days gestation (E15) and several postnatal stages (P0, P5, P10, P21) until adulthood (3 months). At all stages PDE9 mRNA was present throughout the whole central nervous system, with highest levels observed in cerebellar Purkinje cells, whereas PDE2 and PDE5 mRNA expression was more restricted. Like PDE9, PDE5 mRNA was abundant in cerebellar Purkinje cells, although it was observed only on and after postnatal day 10 in these cells. In other brain regions, PDE5 mRNA expression was minimal, detected in olfactory bulb, cortical layers, and in hippocampus. PDE2 mRNA was distributed more widely, with highest levels in medial habenula, and abundant expression in olfactory bulb, olfactory tubercle, cortex, amygdala, striatum, and hippocampus. Double immunostaining of PDE2, PDE5, or PDE9 mRNAs with the neuronal marker NeuN and the glial cell marker glial fibrillary acidic protein revealed that these mRNAs were predominantly expressed in neuronal cell bodies. Our data indicate that three cGMP‐hydrolyzing PDE families have distinct expression patterns, although specific cell types coexpress mRNAs for all three enzymes. Thus, it appears that differential expression of PDE isoforms may provide a mechanism to match cGMP hydrolysis to the functional demands of individual brain regions. J. Comp. Neurol. 467:566–580, 2003.

Nitric oxide-mediated cGMP synthesis in oligodendrocytes in the developing rat brain.

We investigated the nature of cGMP‐synthesizing cells in the developing rat forebrain using cGMP‐immunocytochemistry in combination with in vitro incubation of brain slices. When brain slices of immature rats, aged between 1 and 4 weeks, were incubated with sodium nitroprusside (SNP), a nitric oxide (NO) donor compound, in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX), small round cells with a few processes in and around the corpus callosum were visualized with the cGMP‐antibody. The morphology and the distribution of the cGMP‐positive cells were consistent with the criteria for oligodendrocytes. Furthermore, the cGMP‐positive cells expressed 2′3′‐cyclic nucleotide 3′‐phosphodiesterase (CNPase) and gelsolin, which are marker proteins for oligodendrocytes. Therefore, we concluded that the cGMP‐positive cells were oligodendrocytes. A subpopulation of the oligodendrocyte was found to be cGMP‐immunoreactive also when slices were incubated in the absence of SNP. Furthermore, incubation of the slice in the presence of L‐NAME, an inhibitor of NO synthase, but in the absence of SNP abolished cGMP immunostaining. In addition, some populations of neurons and astrocytes in restricted brain areas produced cGMP in response to the incubation with SNP as previously reported, whereas both ameboid and ramified microglial cells did not respond to the treatment. Atrial natriuretic peptide, a stimulator of particulate guanylyl cyclase, enhanced cGMP synthesis in astrocytes in some brain regions but not in oligodendrocytes. These findings indicate that oligodendrocytes in the immature rat brain express soluble guanylyl cyclase. No cGMP‐positive oligodendrocytes were found in the mature rat brain, suggesting that cGMP may mediate signals related to myelinogenesis in the rat brain. GLIA 19:286–297, 1997.

Expression of the cGMP-specific phosphodiesterases 2 and 9 in normal and Alzheimer's disease human brains

We studied the mRNA expression of cGMP‐hydrolysing phosphodiesterases (PDEs) in selected brain areas of normal elderly people and patients with Alzheimers disease. Using radioactive in‐situ hybridization histochemistry we found a widespread distribution of the mRNA for PDE2 and PDE9, whereas no specific hybridization signal was observed for PDE5. We observed PDE2 and PDE9 mRNA in all cortical areas studied (insular cortex, entorhinal cortex and visual cortex), although to a different extent. PDE2 mRNA was high in the claustrum, whereas PDE9 mRNA was moderate. PDE2 and PDE9 mRNAs was present in the putamen. No cGMP‐hydrolysing PDE expression was observed in the globus pallidus. PDE2 and PDE9 mRNA was observed in all subareas of the hippocampus; however, there were significant differences in the amount of expression. In the Purkinje and cerebellar granule cells only PDE9 expression was observed. PDE2 and PDE9 mRNA expression was not significantly different in Alzheimers disease brains.

Species differences in the localization of cGMP-producing and NO-responsive elements in the mouse and rat hippocampus using cGMP immunocytochemistry

The aim of the study was to compare the localization of the nitric oxide (NO)–cGMP pathway in hippocampus of mice and rats using cGMP‐ and soluble guanylyl cyclase (GC) immunocytochemistry and in situ hybridization of the cGMP‐hydrolysing phosphodiesterase types 2, 5 and 9. In vitro incubation of hippocampus slices in the absence of a guanylyl cyclase stimulator or a phosphodiesterase inhibitor resulted in cGMP‐positive astrocytes mainly in the CA1 area in mouse slices. In contrast, no cGMP immunoreactivity was observed under these conditions in the rat hippocampus. Treatment with an NO synthase inhibitor or inhibitors of soluble or particulate GC did not abolish cGMP immunoreactivity in astrocytes. Incubation with the NO donors sodium nitroprusside or diethylamino NONOate, or with the NO‐independent activators of soluble GC, YC‐1 and BAY 41‐2272, in combination with phosphodiesterase inhibitors, resulted in an increase in cGMP immunoreactivity in numerous astrocytes throughout the mouse hippocampus. In contrast, under these conditions cGMP immunoreactivity was primarily observed in varicose fibers in rat hippocampus. Comparison of the cellular localization of the β1 subunit of soluble GC and the mRNAs of PDE2, PDE5 and PDE9 revealed that in both species the β1 subunit was observed in pyramidal and granule cells, which also expressed the mRNAs of the three phosphodiesterase families. Although the β1 subunit was observed in astrocytes, none of the phosphodiesterases were detected in these cells. We conclude that, although the expression profiles of the soluble GC β1 subunit and cGMP‐hydrolysing phosphodiesterase mRNAs were identical, the cellular patterns of cGMP immunoreactivity differ between rat and mouse hippocampus.

Interstitial cells and phasic activity in the isolated mouse bladder

To describe the distribution of interstitial cells (ICs, defined as cells which show an increase in cGMP in response to nitric oxide, NO) in the isolated mouse bladder, and changes in phasic contractile activity after exposure to a NO donor.

Nitric oxide synthase, cGMP, and NO-mediated cGMP production in the olfactory bulb of the rat.

High levels of nitric oxide synthase and cyclic 3′,5′‐guanosine monophosphate (cGMP) in the olfactory bulb suggest that nitric oxide, acting as a diffusible intercellular messenger molecule inducing increased synthesis of cGMP, plays an important role in olfaction. The localization of cGMP after sodium nitroprusside stimulation of in vitro slices of rat olfactory bulb was compared with the distribution of nicotinamide adenine dinucleotide phosphate‐diaphorase, nitric oxide synthase, and glial fibrillary acidic protein. cGMP was detected immunohistochemically in cryostat sections. In the presence of the phosphodiesterase blocker isobutyl methylxanthine, cGMP was present in neurons in the glomerular layer, axons in the external and internal plexiform layers, and in a few somata and axons of the granule cell layer. This staining was blocked by NG‐nitro‐L‐arginine methylester hydrochloride or hemoglobin. After sodium nitroprusside stimulation, the olfactory nerve layer was intensely stained, as were the glomeruli and periglomerular cells. In the external plexiform layer, axonal staining was increased substantially, and there were occasional multipolar cGMP‐positive neurons. In the internal plexiform and granule cell layers, axonal staining was greatly increased. Many granule cells were also cGMP positive after sodium nitroprusside stimulation. cGMP and nitric oxide synthase‐positive neuronal elements overlapped in the glomerular and granule cell layers, but staining was not colocalized. cGMP was not found in astrocytes. The glutamatergic antagonists D‐2‐amino‐5‐phosphonovalerate and 6‐cyano‐7‐nitroquinoxaline caused differential inhibition of cGMP accumulation in layers of the olfactory bulb. These findings support the hypothesis that nitric oxide is an intercellular messenger in the olfactory bulb (Breer and Shepherd [1993] Trends Neurosci. 16:5–9).

The effects of exogenous prostaglandins and the identification of constitutive cyclooxygenase I and II immunoreactivity in the normal guinea pig bladder

To establish the functional consequences of exposing the isolated whole bladder preparation to exogenous prostaglandins (PGE1, PGE2, PGF2α) and to determine which cells express cyclooxygenase (COX) types I and II, to generate PG to effect these changes in vivo.

Local inhibition of hippocampal nitric oxide synthase does not impair place learning in the Morris water escape task in rats

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 Nω‐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 Nω‐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.

Interstitial cells and cholinergic signalling in the outer muscle layers of the guinea‐pig bladder

To explore the relationship between cholinergic mechanisms and interstitial cells (ICs) in the outer muscle layer of the bladder.

Presence of soluble and particulate guanylyl cyclase in the same hippocampal astrocytes

The localisation of particulate and soluble guanylyl cyclase was studied in hippocampal astrocytes. Counting the colocalisation of cGMP immunoreactivity with the astrocytic marker glial fibrillary acidic protein after stimulation of brain slices with sodium nitroprusside (0.1 mM) or atrial natriuretic peptide (100 nM), we were able to show that at least 67% of the hippocampal astrocytes contained both guanylyl cyclase isoforms. In addition, it was shown that a large number of atrial natriuretic peptide, brain-derived natriuretic peptide or sodium nitroprusside responsive cells contain the beta1-subunit of the soluble guanylyl cyclase. The results show that, in at least a subset of hippocampal astrocytes, soluble and particulate guanylyl cyclases are simultaneously present in the same cells.