Biomedical subjects
Publications and source records attributed to J Garthwaite.
A new mGluR antagonist, MAP4 (the alpha-methyl derivative of L-AP4), was found to antagonize the synaptic depressant actions of L-AP4 at the lateral perforant path synapse, in rat hippocampal slices.
Nitric oxide synthase (NOS) enzyme activity was determined in a comprehensive selection of regions of the rat brain. The effects of lateral ventricular administration of N omega-nitro-L-arginine (L-NA, 30 micrograms) and its methyl ester (L-NAME, 3-100 micrograms) on NOS activity were examined in the ipsilateral and contralateral areas of 4 of these brain regions and in the cerebellum. NOS activity was determined using a new and rapid ex vivo assay method which ensures minimal dissociation of the enzyme-inhibitor complex. Following infusion of L-NAME, NOS activity was rapidly and dose-dependently inhibited in all brain regions studied (cerebral cortex, striatum, hippocampus, cerebellum and thalamus). However, NOS activity of brain regions within the contralateral hemisphere was inhibited significantly less than in ipsilateral regions, with the exception of the thalamus. The degree of NOS inhibition varied markedly between brain regions within each hemisphere and correlated with their ventricular proximity to the site of NOS inhibitor administration. Therefore, NOS in the thalamus was inhibited most effectively and NOS in the cerebral cortex the least. Within the cerebral cortex further regional differences could be observed, with NOS in the frontal/parietal areas inhibited more effectively than NOS in the temporal/occipital areas. Maximal inhibition of NOS was sustained for approx 6 hr after administration of 30 and 100 micrograms L-NAME. No inhibition of NOS was observed 24 hr after administration. Lateral ventricular administration of the metabolite and active moiety of L-NAME, L-NA, resulted in a similar degree of inhibition and time of inhibitory onset. In contrast, when L-NAME was administered i.p., a significant delay in the onset of NOS inhibition was observed in the above brain regions compared to L-NA. However, no regional or hemispheric differences in NOS inhibition were detected following peripheral administration of these inhibitors. These results indicate that central administration of NOS inhibitors yields a complex pattern of NOS inhibition and that data obtained on brain physiology following the i.c.v. administration of NOS inhibitors, or for that matter any other CNS effector, should therefore be interpreted with extreme caution.
The diffusible second messenger, nitric oxide, is synthesised in central neurons in response to activation of glutamate receptors or other stimuli that increase cytosolic Ca2+ concentrations. Among the many roles suggested for nitric oxide in the central nervous system is that of mediating synaptic plasticity. For example, long-term potentiation in the CA1 region of the rat hippocampus was reported to be blocked by inhibitors of nitric oxide synthase and exogenous nitric oxide has been claimed to induce an enduring enhancement of synaptic strength under certain conditions. These findings, however, are controversial and even when a participation of nitric oxide is evident, the transduction mechanism is unclear. A well-known action of nitric oxide is to stimulate the soluble form of guanylyl cyclase, thereby evoking an accumulation of cyclic GMP in target cells but several other mechanisms have been proposed, including stimulation of ADP ribosyltransferase or cyclooxygenase, and nitrosylation of protein thiol residues. The identification of a selective inhibitor of soluble guanylyl cyclase, the oxadiazoloquinoxaline derivative, ODQ, provides, for the first time, the means to investigate the importance of the cyclic GMP pathway in nitric oxide signal transduction. We find that ODQ and the nitric oxide synthase inhibitor, nitroarginine, reduce hippocampal long-term potentiation in an equal and mutually exclusive manner, suggesting that the actions of nitric oxide in this phenomenon are entirely mediated through cyclic GMP. The experiments also show that there is a component of long-term potentiation that involves neither nitric oxide nor cyclic GMP.
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In brain and other tissues, nitric oxide (NO) operates as a diffusible second messenger that stimulates the soluble form of the guanylyl cylase enzyme and so elicits an accumulation of cGMP in target cells. Inhibitors of NO synthesis have been used to implicate NO in a wide spectrum of physiological and pathophysiological mechanisms in the nervous system and elsewhere. The function of cGMP in most tissues, however, has remained obscure. We have now identified a compound, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), that potently and selectively inhibits NO-stimulated guanylyl cyclase activity. In incubated slices of cerebellum, ODQ reversibly inhibited the NO-dependent cGMP response to glutamate receptor agonists (IC50 approximately nM) but did not affect NO synthase activity. The compound did not affect synaptic glutamate receptor function, as assessed in hippocampal slices, nor did it chemically inactivate NO. ODQ did, however, potentially inhibit cGMP generation in response to NO-donating compounds. An action on NO-stimulated soluble guanylyl cyclase was confirmed in studies with the purified enzyme. ODQ failed to inhibit NO-mediated macrophage toxicity, a phenomenon that is unrelated to cGMP, nor did it affect the activity of particulate guanylyl cyclase or adenylyl cyclase. ODQ is the first inhibitor that acts selectively at the level of a physiological NO "receptor" and, as such, it is likely to prove useful for investigating the function of the cGMP pathway in NO signal transduction.
The ability of exogenous nitric oxide (NO) to modify synaptic transmission was investigated in area CA1 of the rat hippocampal slice. The NO donors S-nitroso-N-acetylpenicillamine (SNAP) and S-nitrosoglutathione (SNOG) depressed field excitatory postsynaptic potentials evoked by low frequency stimulation of the Schaffer collateral-commissural pathway. Upon washout of the NO donors, synaptic transmission rapidly returned to control levels. A similar reversible synaptic depression was produced by SNAP when tetanic stimulation (100 Hz; 1 s) was delivered in its presence. The effect of SNAP was not mimicked by its precursor or breakdown product and was blocked by haemoglobin, indicating that the effect involved NO. Roussin's black salt, a photolabile NO donor, also depressed transiently field excitatory postsynaptic potentials following photolysis. The depression was induced rapidly following a flash of UV light (20 s duration) focused onto the slice using a confocal microscope. The depressant effect of the NO donors on synaptic transmission was mimicked by zaprinast, a specific cGMP-phosphodiesterase inhibitor. Zaprinast depressed to a similar extent both the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate and N-methyl-D-aspartate receptor-mediated components of excitatory postsynaptic currents without affecting passive membrane properties, indicating a presynaptic locus of action. SNAP, SNOG and zaprinast all elevated cGMP levels in rat hippocampal slices. Immunocytochemical staining revealed that the cGMP accumulation was mainly in a network of varicose fibres running throughout the CA1 region, consistent with a presynaptic site of action of NO. We conclude that NO, possibly through activation of guanylate cyclase, may be involved in transient presynaptic depression in the CA1 region of the hippocampus.
Incubation of slices of neonatal rat spinal cord with nitric oxide donor compounds produced marked elevations in cyclic guanosine 3',5' monophosphate (cGMP) levels. The excitatory amino acid receptor agonists N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) produced smaller increases, which were blocked by the nitric oxide synthase (NOS) inhibitor L-NG-nitroarginine (NOArg), indicating that these cGMP responses were mediated by nitric oxide. Immunocytochemistry revealed that, in response to NMDA, cGMP accumulated in a population of small cells and neuropil in laminae II and III of the dorsal horn. This area was also shown, by reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry, to contain NOS. These observations suggest that, in the rat spinal cord, NMDA receptor activation is linked to the formation of NO and, hence, of cGMP. This pathway is located selectively in the superficial dorsal horn, consistent with a role in the processing of nociceptive signals.
Nitric oxide (NO) functions as a diffusible messenger molecule in many different tissues, including the brain. To create a conceptual framework for understanding the behaviour of NO in a biological (particularly neurobiological) scenario, we have developed theoretical models describing the kinetic and concentration profiles for NO generated from single or multiple sources. It is predicted that the physiological sphere of influence of a single point source of NO that emits for 1-10 sec has a diameter of about 200 microns corresponding to a volume of brain enclosing 2 million synapses. Inactivation of NO (imposed as a half-life of 0.5-5 sec) has only relatively minor effects because diffusion is so fast. When there are multiple simultaneously-active NO sources within a tissue volume, and in the absence of decay of NO or of a time-dependent reduction in source strength, the concentration of NO simply rises linearly with time, indicating the likely importance of negative feedback by NO on NO synthesis. Distant sources (200-500 microns away) make significant contributions to the steady-state NO concentrations in this situation even when the half-life of NO is short (0.5-5 sec). The models predict the results of several pharmacological experiments that were interpreted to suggest that a NO-containing molecule, rather than NO itself, is the endogenous messenger. Accordingly, invoking the presence of a hypothetical "NO carrier" on the basis of these experimental results is unnecessary.
Nitric oxide (NO), generated upon glutamate receptor activation, elicits cyclic GMP accumulation through stimulation of guanylyl cyclase. NO is also a potential cytotoxin that has been suggested, on the basis of tissue culture experiments, to mediate neuronal damage associated with excessive activity of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor. We have investigated the involvement of NO in the toxicity of glutamate receptor agonists in brain slice preparations. Slices of cerebellum and hippocampus from the developing rat exhibited neuronal necrosis following exposure (5-30 min) to NMDA (100 microM or 1 mM). When the exposures were carried out in the presence of NO synthase inhibitors, at concentrations suppressing NMDA-induced NO formation (as judged by measurements of cyclic GMP accumulation), the extent of injury was unaffected. To determine if exogenous NO is able to replicate NMDA toxicity, the slices were exposed to high concentrations of NO donating compounds for up to 2 hr. No damage was detectable. NO donors, moreover, neither reduced NMDA toxicity, nor potentiated the degeneration caused by just suprathreshold NMDA concentrations. The toxicities of non-NMDA agonists, or of glutamate itself, were also unaltered by NO synthase inhibitors or NO donors. Similar results were obtained using hippocampal slices from more mature animals. We conclude that the acute neurodegeneration mediated by NMDA or non-NMDA receptors in the slice preparations is not mediated by NO, nor is NO neuroprotective under these conditions.
Glutamate, the major excitatory neurotransmitter in the central nervous system, acts through two broad classes of receptors: ion channel-linked (ionotropic) receptors, which include N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, and metabotropic receptors which couple via G-proteins to intracellular messenger cascades. Seven subtypes of mGluR are known to exist but their roles in synaptic physiology are poorly understood. In cerebellar Purkinje cells, application of the mGluR agonist, trans-1-aminocyclopentane-1,3-dicarboxylic acid, or the active enantiomer, 1S,3R-ACPD, results in a depolarization associated with an inward current and an elevation of intracellular Ca2+ (for review see Ref. 29). Moreover, using an extracellular (grease-gap) technique that monitors population responses, we have previously discovered that, in Purkinje cells of adult rat cerebellum, brief tetanic stimulation of the glutamatergic parallel fibre input gives rise to a slow depolarising synaptic potential that is resistant to ionotropic glutamate receptor blockers and to antagonists acting at GABA receptors. It was suggested that this novel potential is mediated by metabotropic receptors. The advent of antagonists for metabotropic receptors has allowed us to test this hypothesis. We find that the S-enantiomer of alpha-methyl-4-carboxyphenylglycine stereoselectively antagonizes the slow synaptic potential recorded using the grease-gap method. The results were confirmed by intracellular recording from Purkinje cells. To our knowledge this is the first direct evidence of an mGluR-mediated EPSP in intact brain tissue.
Nitric oxide is a novel signalling molecule in the brain and a potent activator of the cyclic GMP-synthesising enzyme, soluble guanylate cyclase. To determine if stimulation of cyclic GMP formation is a widespread mechanism of nitric oxide signal transduction, we have compared the distribution of the nitric oxide-generating enzyme (nitric oxide synthase) with that of nitric oxide-stimulated cyclic GMP accumulation, throughout the rat brain. The former was done using NADPH diaphorase histochemistry and the latter by cyclic GMP immunohistochemistry following perfusion of the nitric oxide donor, nitroprusside, in vivo. At a gross level, there was generally a good match when the two were compared in adjacent sections. Although the relative staining intensity varied from area to area, in no grey matter region did we observe cyclic GMP accumulation in the absence of nitric oxide synthase staining. In detail, the locations were complementary rather than identical. In some areas, nitric oxide synthase was found in postsynaptic structures and cyclic GMP accumulation in presynaptic elements and fibres; in others, the locations were reversed. Glial cells and their processes also accumulated cyclic GMP in the cerebellum. The results suggest that soluble guanylate cyclase is a major nitric oxide "receptor" throughout the brain. They also support the hypothesis that nitric oxide generated therein primarily functions as a mediator of cell-cell signaling rather than as a conventional second messenger acting within the cells in which it is produced. The types of communication subserved by nitric oxide appear to be extraordinarily diverse.
Glutamate receptors of both the ionotropic (ion channel-linked) and metabotropic (enzyme-linked) categories are abundantly expressed by Purkinje cells in the cerebellum but the functional significance of the latter receptors is unknown. We have tested the possibility that they are activated by the parallel fibre input by recording from Purkinje cells within a biplanar cerebellar slice preparation using the grease-gap technique. Under conditions where ionotropic (NMDA and non-NMDA) glutamate and GABA receptors were blocked pharmacologically, electrical stimulation of parallel fibres gave rise to two very slow potentials. The first peaked about 400 msec from the start of stimulation and was depolarising. It was not evident with single stimuli but reached maximum amplitude after 6 shocks delivered at 50 Hz. The wave was abolished when the slices were perfused with Ca(2+)-free solution or with drugs that inhibit synaptic transmission, but it was resistant to blockade of GABAB receptors, acetylcholine receptors and adrenergic receptors. Next came a slow hyperpolarising potential that peaked about 30 sec after stimulation and which was also Ca(2+)-dependent. The sequence of potentials was replicated by perfusion of an exogenous agonist acting selectively on metabotropic glutamate receptors. We conclude that parallel fibre-to-Purkinje cell synaptic transmission involves not only fast signals generated through ionotropic non-NMDA receptors but also much slower potentials that are likely to be mediated by metabotropic glutamate receptors. These potentials are likely to be significant both for shorter-term (seconds to minutes) Purkinje cell excitability as well as for the induction of longer-term synaptic plasticity.
Using NADPH-diaphorase staining as a marker for the enzyme nitric oxide synthase (NOS) we have investigated the possible sites of nitric oxide (NO) synthesis in a number of cranial ganglia in the rat. Intense staining was found in the majority of neurones in the sphenopalatine ganglion, suggesting a major role for NO in postganglionic parasympathetic systems in the head. In contrast the neurones of the superior cervical ganglion were not stained by this histochemical procedure but were enveloped by a mesh of intensely staining fibres. As preganglionic sympathetic neurones in the intermediolateral horn of the spinal cord stain for NADPH-diaphorase, our results would suggest that NO acts as a neurotransmitter between pre- and post-ganglionic sympathetic neurones.
The electrophysiological actions of the metabotropic glutamate receptor agonist 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) on Purkinje and granule cells were studied in immature and adult cerebellar slices. ACPD elicited a depolarising response when applied to Purkinje cells (EC50 approximately 20 microM). Granule cells hyperpolarised when exposed to low (3-10 microM) concentrations of ACPD; higher concentrations produced a depolarisation (EC50 approximately 40 microM) that was rapidly curtailed by a hyperpolarisation. The hyperpolarisation was abolished when Ca2+ was removed. In Purkinje cells, the amplitude of the depolarisation was greater in adult slices compared to those in immature slices. The responses were not blocked by ionotropic glutamate receptor antagonists or (L)-2-amino-3-phosphonopropionate (AP3).
Amino acid levels were measured in perfusates from biplanar slices of rat cerebellum installed in a Krebs-filled three-compartment chamber. The two lateral compartments housed the white matter and a section containing parallel fibres respectively. The central compartment housed cortical structures, including the Purkinje cell and granule cell bodies. This arrangement allows selective electrical stimulation of the parallel fibre or mossy fibre pathways, recording of the evoked responses to such stimulation and collection of the perfusion medium passing through the central chamber for amino acid analysis using high-pressure liquid chromatography (HPLC). Both, 2-Hz and 5-Hz stimulation of white matter caused a delayed increase in arginine levels in the perfusate. Since L-arginine is the physiological precursor of nitric oxide, a neuronal messenger in the brain, the data suggest that physiological stimuli can result in the release of this precursor, possibly to supply the nitric oxide synthase.
Nitric oxide (NO) mediates cell-cell signalling in the brain and stimulates cyclic GMP (cGMP) production in target cells. We have used NADPH-diaphorase (reduced nicotinamide adenine dinucleotide phosphate-diaphorase) histochemistry to identify NO-producing neurones and cGMP immunohistochemistry to locate the targets of NO in rat cerebellum. NADPH-diaphorase staining was prominent in granule cells and in the molecular layer. cGMP immunostaining in cerebellar slices stimulated with the NO donors, nitroprusside and SIN-1, was found in granule cells, glomeruli, fibres, Bergmann glia and in other astrocytes. The results provide visible evidence that NO mediates neuron-neuron and neuron-glia communication.
The distribution of NADPH-diaphorase and cyclic GMP in neonatal dorsal root ganglia in vitro has been investigated under control conditions and in response to incubation with either sodium nitroprusside or N-methyl-D-aspartate. NADPH-diaphorase activity which reveals the distribution of nitric oxide (NO) synthase in neurons was found to be intense in some dorsal root ganglion neurones and present at a lower level in the majority. Basal levels of cGMP were found to be low but when stimulated by sodium nitroprusside were found to be selectively increased in satellite cells. The results suggest that NO may function as a signalling system between neurones and satellite cells in sensory ganglia.