Eric Southam

Sources and targets of nitric oxide in rat cerebellum

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 Nitric Oxide - Cyclic GMP Pathway and Synaptic Depression in Rat Hippocampal Slices

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. Roussins 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 α‐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.

Nitric oxide may act as a messenger between dorsal root ganglion neurones and their satellite cells.

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.

Intercellular action of nitric oxide in adult rat cerebellar slices.

Nitric oxide (NO) is a novel messenger molecule that is produced following glutamate receptor activation and which stimulates cyclic GMP (cGMP) formation. To determine if the mode of action of NO is predominantly intra- or inter-cellular in intact brain tissue, we tested the ability of haemoglobin (Hb), a NO-binding protein that remains extracellular, to inhibit cGMP accumulation induced by glutamate receptor agonists in adult rat cerebellar slices. Responses to agonists acting selectively on N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors were inhibited by Hb (IC50 approximately 20 microM) suggesting that NO functions predominantly as a mediator of communication between cells. The effects of NO-donating drugs (e.g. nitroprusside) were also inhibited by Hb, implying that they yield NO extracellularly.

Climbing Fibres as a Source of Nitric Oxide in the Cerebellum

Exposure of adult rat cerebellar slices to a moderately raised K+ concentration (15 mM) caused a large (30‐fold) rise in the levels of cyclic GMP. Excitatory amino acid antagonists failed to inhibit this response, nor could it be mimicked by agonists active at a number of other transmitter receptors. It was, however, inhibited by the nitric oxide (NO) synthase antagonist, l‐methylarginine (lC50= 10 μM), and also by tetrodotoxin (1 μM) implying that underlying the cyclic GMP response was an action potential‐dependent formation of NO. Prelesioning of climbing fibres resulted in a loss of ∼50% of the response to K+ but failed to influence the effects of glutamate receptor agonists or the NO‐donor, nitroprusside. These findings point to a new mechanism for the formation of NO in the central nervous system and suggest that, in the cerebellum, climbing fibres are a source of NO.

NADPH-diaphorase staining in autonomic and somatic cranial ganglia of the rat.

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 nitric oxide‐cyclic GMP pathway and synaptic plasticity in the rat superior cervical ganglion

1 We have investigated the possibility that nitric oxide (NO) and soluble guanylyl cyclase, an enzyme that synthesizes guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) in response to NO, contributes to plasticity of synaptic transmission in the rat isolated superior cervical ganglion (SCG). 2 Exposure of ganglia to the NO donor, nitroprusside, caused a concentration‐dependent accumulation of cyclic GMP which was augmented in the presence of the phosphodiesterase inhibitor, 3‐isobutyl‐1‐methylxanthine. The compound, 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ), a selective inhibitor of soluble guanylyl cyclase, completely blocked this cyclic GMP response. 3 As assessed by extracellular recording, nitroprusside (100 μm) and another NO donor, S‐nitrosoglutathione (30 μm) increased the efficacy of ganglionic synaptic transmission in response to electrical stimulation of the preganglionic nerve, an effect that was reversible and which could be replicated by the cyclic GMP analogue, 8‐bromo‐cyclic GMP. Ganglionic depolarizations resulting from stimulation of nicotinic receptors with carbachol were not increased by nitroprusside. The potentiating actions of the NO donors on synaptic transmission, but not that of 8‐bromo‐cyclic GMP, were inhibited by ODQ. 4 Brief tetanic stimulation of the preganglionic nerve resulted in a long‐term potentiation (LTP) of synaptic transmission that was unaffected by ODQ, either in the absence or presence of the NO synthase inhibitor, NG‐nitro‐L‐arginine (L‐NOARG, 100 μm). A lack of influence of L‐NOARG was confirmed in intracellular recordings of LTP of the excitatory postsynaptic potential. Furthermore, under conditions where tetanically‐induced LTP was saturated, nitroprusside was still able to potentiate synaptic transmission, as judged from extracellular recording. 5 We conclude that NO is capable of potentiating ganglionic neurotransmission and this effect is mediated through the stimulation of soluble guanylyl cyclase and the accumulation of cyclic GMP. However, this potentiation is distinct from LTP of nicotinic synaptic transmission, in which neither NO nor soluble guanylyl cyclase appear to participate.

The NO-cGMP Pathway in Neonatal Rat Dorsal Horn

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 α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionate (AMPA) produced smaller increases, which were blocked by the nitric oxide synthase (NOS) inhibitor Ml‐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.

[12] Nitric oxide-cyclic GMP pathway in brain slices

Publisher Summary This chapter describes the preparation of rat cerebellar and hippocampal slices, the in vitro maintenance, methods for the measurement of cyclic guanosine monophosphate (cGMP) levels and nitric oxide synthase (NOS) activity, and a simple histochemical technique for locating NOS within brain slices. Brain slice techniques are applied to almost every area of the central nervous system (CNS). However, it is the cerebellum and hippocampus that are particularly well suited to slicing because, when sectioned in an appropriate plane, disruption to the constituent neurons and their synaptic connections is limited because of the highly organized anatomy. Consequently, parasagittal slices of rodent cerebellar vermis and transverse slices of hippocampus are commonly used in in vitro investigations into the role of NO in the CNS. The cGMP response in rat cerebellar slices is caused by activation of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor is mediated by the messenger molecule NO, a variety of biochemical, histological, and electrophysiological techniques are used to investigate the synthesis and action of NO in brain slices. The method for determining citrulline formation in slices of adult rat cerebellum is L-citrulline assay of NO synthase activity.

Inter-Cellular Signalling by Nitric Oxide

It has been known for many years that excitation in the central nervous system is associated with marked elevations in the levels of the second messenger, cyclic GMP (cGMP) (Drummond, 1983). These are now known to be mediated through the release of the novel messenger molecule, nitric oxide (NO), which functions as a powerful activator of the soluble form of the cGMP synthesising enzyme, guanylate cyclase (Garthwaite, 1991). NO has a number of properties which set it apart from conventional signalling molecules, not least of which is its ability to diffuse readily across membranes and so act on cellular elements located some distance from the site of its formation. As discussed below, the targets for neuronally-derived NO include other neurons and neighbouring glial cells.