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The distribution of the different glutamate receptor subunits in human spinal cord has yet to be fully elucidated. The aim of this study was to examine the distribution of the N-methyl-D-aspartate (NMDA) glutamate receptor modulatory subunit NR2A, in control human spinal cord and to examine in parallel the expression of the mRNA in amyotrophic lateral sclerosis (ALS). The aetiology of ALS is poorly understood, although abnormalities in glutamate and glycine transport have been reported as well as alterations in NMDA receptors including the NR1 subunit; suggesting a role for glutamate in the disease process. We have used the technique of in situ hybridisation to localise this receptor subunit to the laminae of human spinal cord and have found that it shows a widespread distribution similar to that previously reported for the universal NMDA receptor subunit NR1. Quantitation of mRNA expression in control and ALS cases showed a significant widespread loss of NR2A from both dorsal and ventral horns with losses of 55% and 78%, respectively, in ALS as compared to control. These results were substantiated by analysis of spinal cord homogenates, which showed a significant total decrease of 50% in ALS spinal cord as compared to control.

We have examined the expression of the NR-1 subunit of the glutamate NMDA receptor and the immediate early gene c-jun in lumbar spinal cord following neonatal common peroneal nerve crush. The expression of these two genes was studied up to 12 days post-injury (crush occurring at neonatal day P2). The levels of both NR-1 and c-jun mRNA were increased in spinal cord ipsilateral to the site of crush, the induction of mRNA was shown to occur in a time-dependent manner, peaking at 5 days post-injury. The level of NR-1 mRNA showed the most substantial change following nerve crush, increasing 5 times from 4 h to 5 days post-crush. An increase in expression of NR-1 was also observed in spinal cord contralateral to the injury, although quantitatively this was a smaller effect. These results indicate that early postnatal injury causes a significant increase in the expression of NR-1 mRNA which is most marked at 5 days after injury. This period coincides with that of maximum cell death and indicates that the selective induction of NR-1 could underlie the mechanism of this cell death.

In this study we have characterized the induction of c-fos mRNA in cerebral cortex in response to unilateral kainate injection into the nucleus basalis. This treatment is associated with an intense stimulation of the ascending pathway and the subsequent induction of ornithine decar☐ylase (ODC) enzyme activity and ODC mRNA in ipsilateral cerebral cortex which is sensitive to treatment with MK-801 and dihydropyridine antagonists. Unilateral injection of kainate into nucleus basalis caused a marked induction of c-fos mRNA in ipsilateral cortex which was detectable at 1 h, reached a maximal value at 8 h where c-fos mRNA levels were 16 times those in unoperated animals and then returned to control values by 24 h. However, the early induction of c-fos mRNA at 1 h was not related to a specific effect of kainate since, at this time point, sham-operated animals also showed a significant increase in the level of c-fos mRNA in ipsilateral cerebral cortex. No significant induction of c-fos mRNA was detected in ipsilateral cortex in sham-operated animals at 4 and 8 h after injection of vehicle. Treatment with the N-methyl- d -aspartate (NMDA) receptor antagonist, MK-801 (3 mg/kg) significantly attenuated the response obtained at 4 h and 8 h after kainate injection by 73% and 55% respectively, but did not influence the level of c-fos mRNA induced at 1 h. Delaying administration of MK-801 by 30 min reduced the effectiveness of this treatment on the response obtained at 4 h. The dihydropyridine antagonist, nimodipine had no effect on the induction of c-fos mRNA in response to kainate injection at 1, 4 or 8 h, indicating that ODC and c-fos mRNA are not induced by a common mechanism. These results demonstrate the induction of c-fos mRNA by two distinct mechanisms, one which is a rapid and transient response to tissue damage, and a second delayed and more substantial response which is initiated by excitotoxin stimulation and is mediated by NMDA receptors. Thus, NMDA receptors can regulate the expression of c-fos in cerebral cortex and hence may mediate effects produced in response to persistent neuronal excitation.

Discrete sections applied to one hemisphere of the rat brain cut the pathway from cerebral cortex to corpus striatum unilaterally. Comparison of crude synaptosomal fractions from the corpus striatum of each side showed a decrease in protein yield (47%) and a significant decrease in glutamate content (27%) when expressed in terms of protein content. Aspartate and GABA content did not significantly change. Decrease in the potassium-evoked release of glutamate (21%) and aspartate (33%) were also detected, whilst hemispheres over the two week post-operation period studied.

Excitotoxin lesion of the nucleus basalis of the rat causes a substantial reduction in the K + -evoked release of [ 3 H]acetylcholine (ACh) from tissue slices of ipsilateral frontal and parietal cortex. Decreases in the K + -evoked release of [ 3 H]ACh (nCi/mg protein) of 71% and 47% are seen 21 days after lesion in the frontal and parietal cortex respectively. However, in later periods a considerable reversal of this deficit occurred which was complete in parietal cortex at 102 days, when the K + -evoked release of [ 3 H]ACh was not significantly different from the controls. In the frontal cortex where a greater decrease in acetylcholinesterase (AChE) is obtained, a similar but attenuated reversal occurred, the K + -evoked release of [ 3 H]ACh reaching 75% of the control value at 128 days after lesion. The compensatory changes in [ 3 H]ACh release were paralleled by changes in AChE in both regions. Despite the reduced level of release following lesion, the K + -evoked release of [ 3 H]ACh was responsive to the inhibitory effect of the cholinergic agonist, oxotremorine. Lesions outside the nucleus basalis produced no changes in the release of [ 3 H]ACh from frontal or parietal cortex. All lesions were verified histologically. The compensatory changes reported are discussed in terms of the growth of ‘sprouts’ from the spared cholinergic axons. This study clearly demonstrates that compensatory changes in complex systems such as transmitter release which markedly affect cholinergic function do occur in response to lesion in common with other presynaptic parameters.

Levels of muscarinic receptors were measured in the nucleus accumbens of rat following 0.8 microgram 6-hydroxydopamine or vehicle injections (0.2 microliter into the ventral tegmental area to investigate whether the dopaminergic terminals destroyed by this procedure bear muscarinic receptors. Dopamine levels in the nucleus accumbens ipsilateral to the injection of 6-hydroxydopamine were substantially reduced by 83% as compared to the unlesioned side after 7 days. Significant decreases in the specific binding of [3H]N-methylscopolamine of 9 and 15% were also seen in the nucleus accumbens ipsilateral to the lesion after 7 and 14 days respectively. The class of muscarinic receptor depleted by the lesion was further investigated using [3H]oxotremorine-M to label the 'super high' affinity binding sites. The percentage occupancy of total muscarinic receptors by [3H]oxotremorine-M was significantly decreased by lesion e.g. 23% after 7 days indicting a selective loss of 'super high' affinity binding sites. The lesion caused no change in the affinity constant for the muscarinic antagonist, propylbenzilylcholine. Studies of the binding of the agonist carbachol and oxotremorine-M by competition with [3H] propylbenzilylcholine showed little change in the concentrations or affinity constants of the 'high' and 'low' affinity binding sites with the 6-hydroxydopamine lesion.

Two compartments of striatal synaptosome dopamine were identified by differential labelling with the isotopic presursors, L-tyrosine and Dopa, and from specific radioactivity measurement. Either, endogenous or exogenous L-tyrosine could provide a source for the dopamine pool synthesised and released in response to K+ depolarization, whereas external DOPA did not enter this pool. Acetylcholine (0.1 mM) in the presence of neostigmine (0.1 mg/ml) increased dopamine turnover as shown by increased formation of [14C] dopamine and [14C] DOPAC from [14C] DOPA. Haloperidol (0.65 mM) did not affect the size of dopamine pools but increased the conversion of [14C] DOPA to [14C] dopamine and the formation of [14C] DOPAC. Acetylcholine stimulated the release of dopamine from synaptosomes, which effect could be modified by both muscarinic and nicotinic antagonists. In the presence of the muscarinic antagonist, atropine, acetylcholine stimulated dopamine release, whereas in the presence of the nicotinic antagonists, hexamethonium (0.2 mM) or alpha-bungaro-toxin (0.188 muM), acetylcholine inhibited dopamine release. This showed that presynaptic cholinergic receptors were operational, excitatory nicotinic receptors in the former case and inhibitory muscarinic in the latter. The nicotinic receptor was shown to be saturable and to bind specifically 11.2 fmoles of [3H] alpha-bungarotoxin per mg. protein which could be prevented by hexamethonium or D-tubocurarine.

Cholinergic neurones located in the nucleus basalis of Meynert (NBM) in the substantia innominata (SI) of primates are known to project to cerebral cortex and cell loss in NBM is thought to be associated with the cholinergic deficit seen in Alzheimer's disease. We have examined in rats the effect of lesion of SI with kainate (1 microgram/0.5 microliter) on acetylcholine esterase (AChE) activity, muscarinic receptor number and subtypes in cerebral cortex at 1, 2 and 4 weeks. The area of lesion was assessed histologically. AChE activity was significantly reduced in frontal and parietal cortex ipsilateral to the lesion compared to the contralateral side by 37 and 30%, respectively, at 1 week. The reduction in parietal cortex at 4 weeks (16%) was significantly attenuated. Muscarinic receptor number was reduced in cerebral cortex ipsilateral to the lesion at the 3 time periods measured, being reduced by 14 and 17% in the frontal and parietal cortex, respectively, at 1 week. Changes in receptor number and AChE activity correlated with the size of lesion. Low affinity agonist binding sites and high affinity pirenzepine binding sites were also analyzed and found to be significantly reduced by lesion of SI. The proportions of high and low affinity agonist binding sites and subtypes of pirenzepine binding sites were, however, not significantly affected by lesion.

The distributions of 8 peptides were studied in the 4 major segmental levels (cervical, thoracic, lumbar, sacral) of the spinal cord in 52 neurologically normal cases. Similar regions from 36 cases of motor neurone disease (MND) were compared using the same procedures to determine possible changes in the distribution of peptides in areas associated with sensory, motor and autonomic function. In normal spinal cords, calcitonin gene-related peptide (CGRP)-, the C-flanking peptide of neuropeptide Y (CPON)-, enkephalin-, galanin-, neurokinin-like-, somatostatin- and vasoactive intestinal polypeptide (VIP)-immunoreactive fibres were abundant in the dorsal horn. Numerous somatostatin-immunoreactive cell bodies were also present. In the ventral horn, immunoreactive fibres were less abundant. Most motoneurones were closely apposed by fibres immunoreactive for enkephalin, neurokinin, somatostatin and thyrotrophin-releasing hormone (TRH). A subpopulation of motoneurones, most notable in lumbar segments, displayed CGRP immunoreactivity. In common with autonomic nuclei, Onuf's nucleus, which is thought to innervate perineal striated muscle and external urethral and anal sphincters, was densely innervated with CPON-, enkephalin-, and in particular somatostatin-immunoreactive fibres, thus suggesting Onuf's nucleus may have an autonomic component. In the diseased cords, there was a reduction in the area of the ventral horn and numbers of motoneurones as revealed by conventional histological staining and immunostaining of neurofilament triplet proteins. No changes in the distribution of peptides was noted in the dorsal horn or autonomic nuclei. By contrast, in the ventral horn, neurokinin-, enkephalin-, somatostatin- and TRH-immunoreactive fibres, which are normally found associated with motoneurones, were absent. Therefore, not only are motoneurones lost in MND, but also the fibres which innervate them. CGRP-immunoreactive motoneurones were not observed, a finding consistent with the proposed role of this peptide as a muscle-trophic factor. In contrast to the large motoneurone groups in the ventral horn, the neuronal integrity of Onuf's nucleus and the peptides associated with it were spared. These data further imply that Onuf's nucleus is not a typical motor nucleus and it is not purely somatic. The coincident loss of peptide immunoreactivity and motoneurones from the large motor nuclei and sparing of Onuf's nucleus and its peptide-containing constituents in the diseased state suggests that peptides contribute to maintenance of neural integrity.