In vivo expression of inducible nitric oxide synthase in cerebellar neurons.

In the CNS, nitric oxide (NO) functions as both neuromodulator and neurotoxic agent. In vivo neuronal expression of NO synthase (NOS) has been attributed to constitutive NOS--both the neuronal and the endothelial types. The other class of NOS--the inducible NOS (iNOS)--is known to mediate toxic effects of NO in various tissues. In this study, we show for the first time that direct intracerebellar injection of endotoxin and cytokine (lipopolysaccharide and interferon-gamma) induced in vivo neuronal expression of the iNOS gene, as demonstrated by fluorescent in situ hybridization and immunohistochemical staining analyzed by confocal laser-scanning microscopy. This raises the possibility that neuronal iNOS might contribute significantly to the vulnerability of the brain to various insults.

Expression of inducible nitric oxide synthase by neurones following exposure to endotoxin and cytokine.

In the CNS, nitric oxide (NO) has been implicated as both a mediator of neurotoxicity and a neuromodulator. The inducible NO synthase (iNOS), thought to mediate toxic effects of NO, has been attributed to glial cells in the CNS. We now report that cerebellar granule cell neurones can be stimulated by lipopolysaccharide and interferon-gamma to express iNOS in vitro, as demonstrated by reverse transcription-polymerase chain reaction and fluorescent in situ hybridisation. The expression of both constitutive NO synthase (cNOS) and iNOS by neurones suggests that NO has diverse functions in the brain, and supports the possibility that iNOS plays a role in neuronal damage and inflammation following activation of brain microglia and production of cytokines.

Expression of constitutive nitric oxide synthase in a primary neuronal culture.

Nitric oxide (NO), a short-lived, highly diffusible free radical, is a messenger molecule produced through the conversion of arginine to citrulline by NO synthase (NOS). In the CNS, NO functions as an important neuromodulator. We now report that cerebellar granule cells in culture express the constitutive form of NOS (cNOS). The expression is demonstrated both at the level of RNA, by RNA-specific reverse transcription-polymerase chain reaction (RT-PCR), and of protein, by immunostaining and by enzymatic activity. Cerebellar granule cells can thus serve as a tool to study the regulation of expression and activity of cNOS in a defined environment.

Cell damage by excess CuZnSOD and Down's syndrome.

Down's Syndrome (DS), the phenotypic expression of human trisomy 21, is presumed to result from overexpression of certain genes residing on chromosome 21 at the segment 21q22-the Down locus. The "housekeeping" enzyme CuZn-superoxide dismutase (CuZnSOD) is encoded by a gene from that region and its activity is elevated in DS patients. Moreover, the recent discovery that familial ALS is associated with mutations in the gene encoding CuZnSOD, focused attention on the entanglement of oxygen-free radicals in cell death and neuronal disorders. To investigate the involvement of CuZnSOD gene dosage in the etiology of the syndrome we have developed both cellular and animal models which enabled us to investigate the physiological consequences resulting from overexpression of the CuZnSOD gene. Rat PC12 cells expressing elevated levels of transfected human CuZnSOD gene were generated. These transformants (designated PC12-hSOD) closely resembled the parental cells in their morphology, growth rate, and response to nerve growth factor, but showed impaired neurotransmitter uptake. The lesion was localized to the chromaffin granule transport mechanism. These results show that elevation of CuZnSOD activity interferes with the transport of biogenic amines into chromaffin granules. Since neurotransmitter uptake plays an important role in many processes of the central nervous system, CuZnSOD gene-dosage may contribute to the neurobiological abnormalities of Down's Syndrome. As an approach to the development of an animal model for Down's Syndrome, several strains of transgenic mice which carry the human CuZnSOD gene have been prepared. These animals express the transgene as an active enzyme with increased activity from 1.6 to 6.0-fold in the brains of four transgenic strains and to an equal or lesser extent in several other tissues. To investigate the contribution of CuZnSOD gene dosage in the neuropathological symptoms of Down's Syndrome, we analyzed the tongue muscle of the transgenic-CuZnSOD mice. The tongue neuromuscular junctions (NMJ) in the transgenic animals exhibited significant pathological changes; withdrawal and destruction of some terminal axons and the development of multiple small terminals. The ratio of terminal axon area to postsynaptic membranes decreased, and secondary folds were often complex and hyperplastic. The morphological changes in the transgenic NMJ were similar to those previously seen in the transgenic NMJ and were similar to those previously seen in muscles of aging mice and rats as well as in tongue muscles of patients with Down's Syndrome. The findings suggest that CuZnSOD gene dosage is involved in the pathological abnormalities of tongue NMJ observed in Down's Syndrome patients.(ABSTRACT TRUNCATED AT 400 WORDS)

Synthesis of nitric oxide in CNS glial cells.

Attention has focused on particular neurons as the source of nitric oxide (NO) within the parenchyma of the CNS. In contrast, glial cells have been viewed mainly as potential reservoirs of L-arginine, the substrate for nitric oxide synthase (NOS), and as likely targets for neuronally derived NO because of their proximity and their expression of soluble guanylyl cyclase (sGC). However, it is becoming evident that astrocytes display both constitutive and inducible NOS activity under various conditions, and that activated microglia express an inducible NOS. The NO-producing capacity of oligodendrocytes is not yet known. Glial-derived NO has significant implications for CNS pathophysiology, given the anatomical location and abundance of these cells, and the wide variety of potential interactions that NO can have with cellular biochemistry. Our intention here is to evaluate the evidence for NO production from non-neuronal CNS sources and thus prompt discussion about potential 'nitrinergic' roles for glial cells.

Gene dosage of CuZnSOD and Down's syndrome: diminished prostaglandin synthesis in human trisomy 21, transfected cells and transgenic mice.

Patients with Down's syndrome (DS) exhibit elevated activity of copper zinc superoxide dismutase (CuZnSOD) caused by the trisomy 21 state. To investigate the possible involvement of CuZnSOD gene dosage in perturbation of prostaglandin biosynthesis we analyzed transfected cells and transgenic mice that express elevated levels of human CuZnSOD. It was found that the synthesis of prostaglandin E2 (PGE2) was diminished in transfected PC12-CuZnSOD cells as well as in fibroblasts from DS patients. Primary cells derived from transgenic CuZnSOD mice showed similar reduction. Impaired biosynthesis of prostaglandins was not confined to cells grown in culture since secretion of PGE2 and PGD2 by kidney and cerebellum of transgenic CuZnSOD was significantly lower than in non-transgenic littermate mice. These findings strongly suggest that overexpression of the CuZnSOD gene induces a demotion in PGE2 and PGD2 formation and establish a connection between alteration of prostaglandin biosynthesis in trisomy 21 cells and gene dosage of CuZnSOD.

Cysteine sulfinic acid-induced release of D-[3H]aspartate and [14C]GABA in hippocampus slices: the role of sodium channels and cAMP.

Cysteine sulfinic acid, a putative transmitter in the brain induces release of D-[3H]aspartate and [14C]GABA without the help of any general depolarizing agent. Tetrodotoxin partially blocks the release of D-[3H]aspartate and completely blocks the induced release of [14C]GABA. Withdrawal of Ca2+ from the medium does not affect the D-[3H]aspartate release, but increases the extent of inhibition by tetrodotoxin. In contrast, removal of Ca2+ increases the cysteine sulfinic acid-induced [14C]GABA release, which remains totally blocked by the toxin. Anemonia sulcata toxin type II, which slows down Na+ channel inactivation, acts in synergism with cysteine sulfinic acid to increase the rate of release of both of the labeled amino acids. Comparison of glutamate with cysteine sulfinic acid in the same experiments indicates a different action pattern of the two acidic amino acids. Forskolin plus isobutyl methyl xanthine, which are known to raise intracellular cyclic adenosine monophosphate (cyclic AMP) levels, caused little release of the labeled amino acids on their own, but strongly enhanced the cysteine sulfinic acid-induced release. The experiments conducted by double labeling with D-[3H]aspartate and [14C]GABA, revealed several characteristic differences between the glutamatergic and the GABAergic neurons. It is tentatively concluded that cysteine sulfinic acid brings about excitation of the glutamatergic as well as the GABAergic neurons, leading to opening of Na+ channels which play a role in the release of both systems. Cyclic AMP, presumably by initiating phosphorylation of a specific component, has a remarkable potentiating effect on the release.

Release of D-[3H]aspartate and [14C]GABA in rat hippocampus slices: effects of fatty acid-free bovine serum albumin and Ca2+ withdrawal.

Extended incubation of hippocampus slices in the presence of fatty acid-free bovine serum albumin (FAF-BSA) strongly enhanced the release of D-[3H]aspartate and [14C]GABA induced by veratridine. Saturation of the FAF-BSA with oleic acid abolished the enhancing effect. Spontaneous release and K+-induced release were not significantly changed by the addition of FAF-BSA. Amino-oxyacetic acid in the medium enhanced the veratridine-induced release of D-[3H] aspartate. The spontaneous release of [14C]GABA was greatly increased by Ca2+ withdrawal. With the further addition of EGTA the spontaneous release in the absence of Ca2+ increased more than 8-fold over the measured in the presence of 1.5 mM Ca2+. The enhanced release caused by Ca2+ withdrawal was totally blocked by tetrodotoxin. The toxin was effective even when added after the spontaneous release in the absence of Ca2+ was already proceeding at a high rate. The veratridine-induced release of [14C]GABA was also considerably augmented by Ca2+ withdrawal. D-[3H]aspartate release, studied simultaneously with [14C]GABA by double labeling, did not show enhanced spontaneous release upon Ca2+ withdrawal. The findings provide evidence that the enhanced [14C]GABA release caused by Ca2+ withdrawal is mediated by voltage-dependent Na+ channels.

D-[3H]aspartate release from hippocampus slices studied in a multiwell system: controlling factors and postnatal development of release.

Medium components and various factors were tested to define optimal conditions for D-[3H]aspartate release. Isolation of the hippocampus and preparation of the slices in a medium without Ca2+ increased the release of D-[3H]aspartate in response to veratridine when subsequently tested in a regular Ca2+ containing medium. Apparently, the absence of Ca2+ during preparation of the slices reduced irreversible damage due to hypoxic conditions which prevail throughout the interval between killing the animal and immersion of the slices in a well oxygenated medium. Substitution of 10 mM Mg2+ for Ca2+ was an efficient procedure to test for Ca2+ dependence of D-[3H]aspartate release induced by veratridine. The inhibition was readily reversible when Ca2+ was readded. Veratridine (50 microM) was superior to high K+ (45 mM) in inducing D-[3H]aspartate release under all conditions tested in slices of mature animals. Furthermore, veratridine-induced release could be completely blocked by tetrodotoxin while K+-induced release was essentially unaffected by this toxin. Postnatal development of the D-[3H]aspartate release induced by veratridine was found to require 40-45 days, whereas release induced by K+ reached about 80% of maximum at postnatal day 22. K+-induced release appears to reach maturation when most hippocampal cells have been formed while veratridine-induced release probably requires completion of the neural circuit, involving also extensive sodium channel formation. These investigations were conveniently performed using a modified plastic culture box in which 24 slice systems can be studied simultaneously.