Nitric oxide targets oligodendrocytes and promotes their morphological differentiation.

In the central nervous system, nitric oxide (NO) transmits signals from one neurone to another, or from neurones to astrocytes or blood vessels, but the possibility of oligodendrocytes being physiological NO targets has been largely ignored. By exploiting immunocytochemistry for cGMP, the second messenger generated on activation of NO receptors, oligodendrocytes were found to respond to both exogenous and endogenous NO in cerebellar slices from rats aged 8 days to adulthood. Atrial natriuretic peptide, which acts on membrane-associated guanylyl cyclase-coupled receptors, also raised oligodendrocyte cGMP in cerebellar slices. The main endogenous source of NO accessing oligodendrocytes appeared to be the neuronal NO synthase isoform, which was active even under basal conditions and in a manner that was independent of glutamate receptors. Oligodendrocytes in brainstem slices were also shown to be potential NO targets. In contrast, in the optic nerve, oligodendrocyte cGMP was raised by natriuretic peptides but not NO. When cultures of cerebral cortex were continuously exposed to low NO concentrations (estimated as 40-90 pM), oligodendrocytes responded with a striking increase in arborization. This stimulation of oligodendrocyte growth could be replicated by low concentrations of 8-bromo-cGMP (maximum effect at 1 µM). It is concluded that oligodendrocytes are probably widespread targets for physiological NO (or natriuretic peptide) signals, with the resulting rise in cGMP serving to enhance their growth and maturation. NO might help coordinate the myelination of axons to the ongoing level of neuronal activity during development and could potentially contribute to adaptive changes in myelination in the adult.

Picomolar nitric oxide signals from central neurons recorded using ultrasensitive detector cells.

Nitric oxide (NO) is a widespread signaling molecule with potentially multifarious actions of relevance to health and disease. A fundamental determinant of how it acts is its concentration, but there remains a lack of coherent information on the patterns of NO release from its sources, such as neurons or endothelial cells, in either normal or pathological conditions. We have used detector cells having the highest recorded NO sensitivity to monitor NO release from brain tissue quantitatively and in real time. Stimulation of NMDA receptors, which are coupled to activation of neuronal NO synthase, routinely generated NO signals from neurons in cerebellar slices. The average computed peak NO concentrations varied across the anatomical layers of the cerebellum, from 12 to 130 pm. The mean value found in the hippocampus was 200 pm. Much variation in the amplitudes recorded by individual detector cells was observed, this being attributable to their location at variable distances from the NO sources. From fits to the data, the NO concentrations at the source surfaces were 120 pm to 1.4 nm, and the underlying rates of NO generation were 36-350 nm/s, depending on area. Our measurements are 4-5 orders of magnitude lower than reported by some electrode recordings in cerebellum or hippocampus. In return, they establish coherence between the NO concentrations able to elicit physiological responses in target cells through guanylyl cyclase-linked NO receptors, the concentrations that neuronal NO synthase is predicted to generate locally, and the concentrations that neurons actually produce.

Signaling from blood vessels to CNS axons through nitric oxide.

Brain function is usually perceived as being performed by neurons with the support of glial cells, the network of blood vessels situated nearby serving simply to provide nutrient and to dispose of metabolic waste. Revising this view, we find from experiments on a rodent central white matter tract (the optic nerve) in vitro that microvascular endothelial cells signal persistently to axons using nitric oxide (NO) derived from the endothelial NO synthase (eNOS). The endogenous NO acts to stimulate guanylyl cyclase-coupled NO receptors in the axons, leading to a raised cGMP level which then causes membrane depolarization, apparently by directly engaging hyperpolarization-activated cyclic nucleotide-gated ion channels. The tonic depolarization and associated endogenous NO-dependent cGMP generation was absent in optic nerves from mice lacking eNOS, although such nerves responded to exogenous NO, with raised cGMP generation in the axons and associated depolarization. In addition to the tonic activity, exposure of optic nerves to bradykinin, a classical stimulator of eNOS in endothelial cells, elicited reversible NO- and cGMP-dependent depolarization through activation of bradykinin B2 receptors, to which eNOS is physically complexed. No contribution of other NO synthase isoforms to either the action of bradykinin or the continuous ambient NO level could be detected. The results suggest that microvascular endothelial cells participate in signal processing in the brain and can do so by generating both tonic and phasic NO signals.

Dynamics of nitric oxide during simulated ischaemia-reperfusion in rat striatal slices measured using an intrinsic biosensor, soluble guanylyl cyclase.

Nitric oxide (NO) may act as a toxin in several neuropathologies, including the brain damage resulting from cerebral ischaemia. Rat striatal slices were used to determine the mechanism of enhanced NO release following simulated ischaemia and, for estimating the NO concentrations, the activity of guanylyl cyclase served as a biosensor. Exposure of the slices for 10 min to an oxygen- and glucose-free medium caused a 70% fall in cGMP levels. On recovery, cGMP increased 2-fold above basal, where it remained for 40 min before declining. The pattern of changes matched those of cGMP or NO oxidation products measured during and after brain ischaemia in vivo. The increase observed during the recovery period was blocked by inhibition of NO synthase or NMDA receptors and was curtailed by tetrodotoxin, implying that it was caused by glutamate release leading to activation of the NMDA receptor-NO synthase pathway. Calibration of the cGMP levels against NO-stimulated guanylyl cyclase yielded a basal NO concentration of 0.6 nm. The peak NO concentration achieved on recovery from simulated ischaemia was estimated as 0.8 nm. These values are compatible with the low micromolar concentrations of NO oxidation products (chiefly nitrate) found by microdialysis in vivo, providing the NO inactivation rate (forming nitrate) is accounted for. NO at a concentration around 1 nm is unlikely to be toxic to cells. However, if the NO inactivation mechanism were to fail (as it can) the NO production rate normally providing only subnanomolar NO could readily generate toxic (microM) NO concentrations.

Glutamate Toxicity: An Experimental and Theoretical Analysis.

In slices of 8-day-old rat cerebellum, the lowest concentration of glutamate that induced toxicity (30 min exposure; 90 min recovery) was 100 microM, but the damage only occurred in the outermost regions. As the concentration was raised, the band of necrosis became progressively deeper until, at 3 mM, it was uniform across the slice thickness. At a test concentration of 300 microM, the width of the necrotic band did not change when either the exposure time or the recovery period was varied between 30 min and 3 h. These results are predicted by a theoretical model in which the diffusion of glutamate into brain tissue is countered by cellular uptake of the amino acid, and they argue against the idea that glutamate toxicity is inherently self-propagating. When slices were examined immediately after exposure (300 microM), a prominent swelling of glial cells was present at the slice surface. Swelling per se did not appear to compromise their uptake function, and the model predicts that cellular swelling, by reducing the rate of diffusion of glutamate, protects against glutamate toxicity. The damage produced by 3 mM glutamate, which was primarily exerted against granule cells, was prevented by N-methyl-d-aspartate (NMDA) receptor blockade, whereas antagonists acting at alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors were ineffective. Under conditions of energy deprivation, the neurotoxic potency of glutamate was markedly enhanced and a normally non-toxic concentration (30 microM) became maximally toxic towards granule cells. Dark vacuolar degeneration of Purkinje cells was also present, and this could be inhibited by blocking AMPA receptors. The results and theoretical analysis suggest that intact brain tissue is remarkably resistant to glutamate toxicity, chiefly because of the formidable properties of the uptake system. However, under special circumstances, glutamate can become a potent neurotoxin and its toxicity can then involve both NMDA and AMPA receptors.

AMPA Neurotoxicity in Rat Cerebellar and Hippocampal Slices: Histological Evidence for Three Mechanisms.

Excitatory amino acid-induced death of central neurons may be mediated by at least two receptor types, the so-called NMDA (N-methyl-d-aspartate) and AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate) receptors. We have studied the neurodegenerative mechanisms set in motion by AMPA receptor activation using incubated slices of 8-day-old rat cerebellum and hippocampus. In both preparations, AMPA induced a pattern of degeneration that differed markedly from the one previously shown to be elicited by NMDA. In cerebellar slices, AMPA induced the degeneration of most Purkinje cells together with a population of Golgi cells; in hippocampal slices the neurons were affected in the order CA3 > CA1 > dentate granule cells. Three mechanisms could be discerned: an acute one in which neurons (e.g. cerebellar Golgi cells) underwent a rapid degeneration; a delayed one in which the neurons (Purkinje cells and hippocampal neurons) appeared to be only mildly affected immediately after a 30 min exposure but then underwent a protracted degeneration during the postincubation period (1.5 - 3 h); and finally a slow toxicity, which took place during long (2 h) exposures to AMPA (3 - 30 microM). Although Purkinje cells were vulnerable in both cases, the efficacy of AMPA was higher for the delayed mechanism than for the slow one. The pathology displayed by the acutely destroyed Golgi neurons was a classical oedematous necrosis, whereas most neurons vulnerable to the delayed and slow mechanisms displayed a 'dark cell degeneration', whose cytological features bore a close resemblance to those of neurons irreversibly damaged by ischaemia, hypoglycaemia or status epilepticus in vivo.

Mechanisms of AMPA Neurotoxicity in Rat Brain Slices.

The mechanisms underlying the neurodegenerative effects of the glutamate receptor agonist, AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate), were studied using brain slice preparations of young rat (8 - 9 days old) cerebellum and hippocampus. Rapid AMPA toxicity (exerted on some cerebellar interneurons) was inhibited by including the appropriate receptor blocker, CNQX (6-cyano-7-nitroquinoxaline-2,3-dione, 10 microM), in the exposing solution. The degeneration of other neurons, including Purkinje cells and hippocampal pyramidal neurons, persisted. It could, however, be largely prevented if CNQX was included for 1.5 h during the post-incubation period, suggesting that an enduring 'rebound' AMPA receptor activation was responsible for this delayed type of degeneration, not the exposure itself. In cerebellar slices, independent evidence for the occurrence, postexposure, of persisting AMPA receptor stimulation was obtained electrophysiologically. Omission of Ca2+ during the exposure period (and for 10 min beforehand) markedly reduced rapid AMPA toxicity but was ineffective in protecting most of the Purkinje cells. However, if the slices were previously starved of Ca2+ for 1 h, then most of these neurons survived, even if the ion was reinstated during the recovery period. Slow AMPA toxicity, which takes place during long (2 h) exposures, could be inhibited either by CNQX or by omission of Ca2+ (30 min preincubation). The results indicate that the rapid oedematous necrosis induced by AMPA, like that caused by N-methyl-d-aspartate and kainate, is likely to involve excessive influx of Ca2+. In contrast, the induction of the delayed mechanisms, as well as its 'expression' during the postincubation period, probably depends on intracellular Ca2+, rather than Ca2+ influx.