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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 beta1 subunit of soluble GC and the mRNAs of PDE2, PDE5 and PDE9 revealed that in both species the beta1 subunit was observed in pyramidal and granule cells, which also expressed the mRNAs of the three phosphodiesterase families. Although the beta1 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 beta1 subunit and cGMP-hydrolysing phosphodiesterase mRNAs were identical, the cellular patterns of cGMP immunoreactivity differ between rat and mouse hippocampus.

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., (Copyright 2003 Wiley-Liss, Inc.)

The present study investigated whether 3-nitrotyrosine is an early marker for neurodegenerative processes involving oxidative stress. We characterized the 3-nitrotyrosine formation after 3-nitropropionic acid (3-NP) exposure in the whole brain spheroid culture model and in a rat model, using Lewis and Wistar rats. Increased 3-nitrotyrosine concentration in spheroid cultures from Lewis rats was observed at lower dose of and shorter exposure time to 3-NP as compared to alterations in glial fibrillary acidic protein concentration, decrease in glutamine synthetase activity or cell loss. Five days of exposure to 3-NP (5 mM) resulted in decreased staining of GABAergic processes, while neuronal nitric oxide synthase staining was preserved. In addition, staining of EAAC1, anti-2',3'-cyclic nucleotide 3'-phosphohydrolase and ED1 was diminished after treatment of spheroid cultures with 3-nitropropionic acid (5 mM), while isolectin B4 staining was increased. Dithiothreitol and vitamin E inhibited the increased formation of 3-nitrotyrosine. Interestingly, N(G)-nitro-L-arginine methyl ester increased the 3-nitrotyrosine formation. No increased 3-nitrotyrosine concentration was shown after exposure to 3-nitropropionic acid during 5 days in spheroid cultures obtained from Wistar rats. In the striatum of 3-NP-exposed Lewis and Wistar rats, no change in 3-nitrotyrosine concentration was observed, whereas only in Wistar rats the glial fibrillary acidic protein concentration was increased in addition to activation of microglial cells. It is concluded that 3-nitrotyrosine was a more sensitive marker for oxidative stress-induced neurodegeneration than glial fibrillary acidic protein and glutamine synthase in spheroid cell cultures of Lewis rats. Finally, the similarities between the 3-NP spheroid model and the vivo model indicate that the spheroid cultures provide a good alternative for chronic exposure of animals to neurotoxins.

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.

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.

In the present study we investigated the effects of acute treatment with acetyl-L-carnitine (50 mg/kg, i.v. 90 min before the sodium cyanide injection) on a sodium cyanide-induced behavioral deficit in the Morris water escape task. In a first experiment the spatial discrimination performance of the rats was found to be dose-dependently impaired after an i.c.v. injection of sodium cyanide (2.5 and 5.0 microg). Acute treatment with acetyl-L-carnitine was found to increase the behavioral deficit after sodium cyanide. These findings were replicated in a second experiment. Based on these results it can be argued that an acute administration of acetyl-L-carnitine appears to potentiate a sodium cyanide-induced behavioral deficit. An additional in vitro experiment with rat brain synaptosomes showed clear effects of administered sodium cyanide on the energy-dependent incorporation of inositol into phosphoinositides and on the ATP concentration. In vitro acetyl-L-carnitine administration had no effect on the sodium cyanide-induced energy depletion. The negative behavioral findings are in contrast with our previously found protective effect of chronic treatment with acetyl-L-carnitine (via drinking water) on the sodium cyanide-induced behavioral deficit. Since chronic acetyl-L-carnitine treatment has no effect on the phosphoinositide metabolism it was suggested that acetyl-L-carnitine may act via the formation of an ATP-independent reservoir of activated acyl groups. Thus, fatty acids as acylated derivatives can be used for reacylation processes during an acute period of energy depletion. However, we have no clear explanation for the discrepancy in behavioral results between the chronic vs acute treatment of acetyl-L-carnitine at present. Further research is needed to characterize the mechanism of action of acetyl-L-carnitine in relation to sodium cyanide.

Previous immunohistochemical staining procedures of the brain and pituitary in Xenopus laevis, using an antiserum against neuronal nitric oxide (NO) synthase (nNOS) and nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry, have revealed NOS activity in neurons and fibers in a number of brain areas, as well as in fibers in the pituitary. In the present study we have localized the target structures of the NOergic system in the Xenopus brain by visualizing the sites of NO-sensitive cyclic 3',5'-guanosine monophosphate (cGMP) accumulation, according to a method for cGMP visualization in rat brain slices. Brain slices of unfixed Xenopus are incubated in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine and the NO donor sodium nitroprusside, followed by fixation and cryosectioning. Sections were then processed for immunohistochemistry using rabbit and sheep antisera against cGMP and a sheep antiserum against nNOS. Visualization of single and double labeling of cGMP immunoreactive and/or nNOS immunoreactive structures was performed with combined CY3/fluorescein isothiocyanate fluorescence microscopy. Following this procedure, we provide immunohistochemical evidence for the distribution of cGMP-accumulating neurons in the brain of adult Xenopus. In most brain areas, the distribution of nNOS and cGMP immunoreactive structures (neuron somata and fibers) is distinct and separate, for instance in the dorsal pallium, the lateral thalamic nuclei, the optic tectum, the locus coeruleus and the reticular formation. However, nNOS and cGMP immunoreactive structures are often found in the vicinity of each other, and in the optic tectum even in adjacent neuron fibers and somata. The present observations are in line with the presence of an NO-dependent soluble guanylate cyclase in distinct brain areas of Xenopus laevis, corroborating similar data in the mammalian brain. Further, our observations may add to the understanding of the anatomical connectivity pattern and functional relevance of the NOergic system in the amphibian brain.

The striatum is rich in nitric oxide synthase (NOS). It is present in a dense fiber network and in a few medium-sized non-spiny interneurons. Previous work showed chronic overexpression of NOS in the rat striatum after a severe perinatal asphyctic (SPA) insult. This was prevented by hypothermia. We investigated whether the overexpression of NOS was accompanied by increased NOS activity. As nitric oxide (NO) is a potent activator of the soluble isoform of guanylyl cyclase, we measured striatal 3',5'-cyclic monophosphate (cyclic GMP) synthesis in 10-day-old (P10) rat pups that were subjected to SPA during normothermia or hypothermia during or after the insult. Cyclic GMP levels in striatal tissue from control pups were approximately 25.8 pmol/mg protein and in the SPA group approximately 38.1 pmol/mg protein (p<0.01). Hypothermia, during as well as after insult, prevented this increase of cyclic GMP. Nomega-nitro-L-arginine (L-NAME) (0.1 mM) decreased cyclic GMP levels in control, SPA and hypothermia treated pups to similar low levels (approximately 8% of level without L-NAME). Sodium nitroprusside (SNP) stimulated cyclic GMP showed no differences between the four groups. This indicates that high cyclic GMP levels in the striatum of rats subjected to SPA are caused by increased NOS activity. Hypothermia after an asphyctic insult could be a promising treatment., (Copyright 1998 Elsevier Science B.V.)

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.

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 phosphatediaphorase, 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).