Postsynaptic nicotinic receptors on dopaminergic neurons in the substantia nigra pars compacta of the rat

Previous studies have shown that application of nicotinic agonists in the substantia nigra pars compacta increases the firing rate of dopaminergic neurons. We have used intracellular recordings to show that the response of these neurons to nicotine is postsynaptic, since it persists in the presence of low-calcium buffer containing tetrodotoxin. Burst firing in the presence of nicotine was not observed. The presence of postsynaptic nicotinic receptors was confirmed by immunohistochemical localization of the alpha4 nicotinic receptor subunit on dendrites in the substantia nigra pars compacta. The majority of tyrosine hydroxylase-immunopositive neurons in the substantia nigra pars compacta were also immunopositive for the alpha4 subunit. Immunohistochemical localization of the alpha4 and beta2 subunits in adjacent brain sections produced similar patterns of staining. Electron micrographs clearly indicated the presence of alpha4 subunit at postsynaptic densities. The predominant role of nicotinic receptors in the central nervous system has been suggested to be the presynaptic modulation of neurotransmitter release [McGehee D. S. and Role L. W. (1995) A. Rev. Physiol. 57, 521-546]. Although several postsynaptic nicotinic responses have also been reported in the literature, it is unclear as to whether the postsynaptic nicotinic receptors mediating responses to exogenously applied agonists are involved in synaptic transmission. From our electrophysiological and immunohistochemical results, we conclude that alpha4-containing nicotinic receptors are found at synapses on dopaminergic neurons. These synapses are similar to the cholinergic synapses described at these neurons, suggesting that nicotinic receptors are important in modulating the excitability of dopaminergic neurons by direct synaptic transmission.     

Compensatory effects of glutamatergic inputs to the substantia nigra pars compacta in experimental parkinsonism

The effect of transitory blockage of substantia nigra pars compacta glutamatergic inputs by intracranial injections of kynurenic acid were evaluated in two monkey treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The MPTP protocol was designed to mimic the gradual evolution of human Parkinson's disease. No effects were observed before MPTP treatment or in the first stage of treatment. Once clinical signs appeared, however, motor abnormalities were significantly aggravated by blockage of these inputs (P < 0.001). Conversely, after full Parkinsonism was established, blockage no longer had any behavioural effect. These results confirm the postulated compensatory role of the glutamatergic pathways feeding the substantia nigra pars compacta. This added insight into the physiopathology of the basal ganglia, when compared with previous data on the presymptomatic revelation of experimental Parkinsonism, should help elucidation of the time pattern of evolution of Parkinson's disease.     

Dopamine-degrading activity of monoamine oxidase is not detected by histochemistry in neurons of the substantia nigra pars compacta of the rat

Monoamine oxidase (MAO) activity was examined in neurons of the substantia nigra pars compacta (SNC) of the rat using a histochemical method, and compared to MAO activity in neurons of the locus coeruleus (LC) and dorsal raphe nucleus (DR). Using dopamine as a substrate, dopamine-degrading MAO activity was not detected in any SNC neurons, although LC and DR neurons were intensely stained for this activity. We further examined MAO activity in these neurons using other substrates, including serotonin (an MAO type A preferential substrate), beta-phenylethylamine (an MAO type B preferential substrate), and tyramine (a substrate common to both MAO types A and B). As for dopamine, no SNC neurons were stained for MAO activity using any of these other substrates. In contrast, LC neurons were intensely stained when either serotonin or tyramine was used, and DR neurons were darkly stained when either beta-phenylethylamine or tyramine was used. The lack of evidence of MAO activity in the SNC is surprising given that there are densely packed tyrosine hydroxylase (TH)-immunoreactive neurons in the SNC (i.e., dopaminergic neurons). By comparison, in the LC and DR the distribution patterns of the MAO-stained neurons were similar to those of TH-immunolabeled neurons (i.e., noradrenergic neurons) and serotonin-immunoreactive neurons, respectively. Our results suggest that dopamine-degrading MAO activity and MAO types A and B activities in SNC dopamine neurons are very low compared to MAO activity in LC noradrenaline neurons and in DR serotonin neurons.     

Expression of metabotropic glutamate receptor 1 isoforms in the substantia nigra pars compacta of the rat

Metabotropic glutamate receptors, which are linked via G-proteins to second messenger systems, have been implicated in the physiological regulation of dopaminergic neurons of the substantia nigra pars compacta as well as in neurodegeneration. Of the eight known metabotropic glutamate receptors, metabotropic glutamate receptor 1 is the most abundant subtype in the substantia nigra pars compacta. Metabotropic glutamate receptor 1 is alternatively spliced at the carboxy terminal region to yield five variants: 1a, 1b, 1c, 1d and a form recently identified in human brain, 1g. We used an antibody recognizing metabotropic glutamate receptor 1, and another recognizing the splice form la only, to study the localization of these receptors in dopaminergic neurons identified by the presence of tyrosine hydroxylase. Metabotropic glutamate receptor immunoreactivity was present within the somata, axons, and dendrites of substantia nigra pars compacta neurons. The 1a splice form specific antibody, however, did not label these cells, suggesting that they express a metabotropic glutamate receptor 1 splice form different from 1a. In situ hybridization with splice form-specific oligonucleotide probes was used to determine which of the other known metabotropic glutamate receptor 1 splice forms might be present in the substantia nigra pars compacta. Each probe produced a very distinct labelling pattern in the rat brain with the exception of the 1g specific probe which produced only background signal. Substantia nigra pars compacta neurons were most intensely labelled by the metabotropic glutamate receptor 1d splice form specific probe. Metabotropic glutamate receptor 1a was expressed weakly whereas there was no detectable 1b, c, or g signal in the substantia nigra pars compacta. These data demonstrate that metabotropic glutamate receptor 1 protein is present within the perikarya and processes of dopaminergic neurons in the substantia nigra pars compacta. The majority of this protein is not the 1a splice form, which is abundant in other brain regions, and may be the 1d isoform. Since splicing alters the carboxy terminus of the receptor, it is likely to affect the interaction of the receptor with intracellular signalling systems.     

Hyperpolarization induces a rise in intracellular sodium concentration in dopamine cells of the substantia nigra pars compacta

We investigated the effect of changes in membrane-voltage on intracellular sodium concentration ([Na+]i) of dopamine-sensitive neurons of the substantia nigra pars compacta in a slice preparation of rat mesencephalon. Whole-cell patch-clamp techniques were combined with microfluorometric measurements of [Na+]i using the Na+-sensitive probe, sodium-binding benzofuran isophthalate (SBFI). Hyperpolarization of spontaneously active dopamine neurons (recorded in current-clamp mode) caused the cessation of action potential firing accompanied by an elevation in [Na+]i. In dopamine neurons voltage-clamped at a holding potential of -60 mV elevations of [Na+]i were induced by long-lasting (45-60 s) voltage jumps to more negative membrane potentials (-90 to -120 mV) but not by corresponding voltage jumps to -30 mV. These hyperpolarization-induced elevations of [Na+]i were depressed during inhibition of I(h), a hyperpolarization-activated inward current, by Cs+. Hyperpolarization-induced elevations in [Na+]i might occur also in other cell types which express a powerful I(h) and might signal lack of postsynaptic activity.     

The effects of L-deprenyl treatment, alone and combined with GM1 ganglioside, on striatal dopamine content and substantia nigra pars compacta neurons

A novel amperometric HPLC detection method for the cyanobacterial (blue-green algal) peptide toxins microcystin-LR, -YR and -RR was developed. Purified microcystins and cyanobacterial extracts were chromatographed using an internal surface reversed-phase column with acetate- and phosphate-based mobile phase systems. Electrochemical oxidation reactions at 1.20 V vs. Ag/AgCl (glassy carbon working electrode) were show to originate in arginine and tyrosine residues of microcystins.     

Baclofen inhibition of the hyperpolarization-activated cation current, Ih, in rat substantia nigra zona compacta neurons may be secondary to potassium current activation

1. The properties of the hyperpolarization-activated cation current (Ih), and its modulation by gamma-aminobuturic acid-B (GABAB) receptor activation and protein kinase A, were investigated using whole cell voltage clamp of substantia nigra zona compacta principal neurons in rat midbrain slices in vitro. 2. At 30 degrees C, Ih activated between -75 and -155 mV, with a V1/2 of -115 mV. At 35 degrees C, the activation curve shifted positive by 10 mV. Ih had an estimated reversal potential of -27 mV. Ion substitution experiments showed that the current was carried by Na+ and K+. 3. Application of the GABAB receptor agonist baclofen (30 microM) induced an outward potassium current (GIRK), increased neuronal membrane conductance and inhibited Ih. The inhibition of Ih was voltage independent. Baclofen induced an 11-mV positive shift in the reversal potential of Ih. 4. Extracellular barium (300 microM) markedly reduced the baclofen-evoked outward current and associated increase in membrane conductance due to GIRK activation. There was also very little inhibition of Ih by baclofen in the presence of barium. When cesium was the major intracellular cation, both the increase in membrane conductance due to GIRK activation and the inhibition of Ih evoked by baclofen were reduced by a similar extent. 5. Neither forskolin (10 microM) nor the protein kinase A inhibitor, H89 (10 microM), had any effect on Ih or its inhibition by baclofen. 6. These data suggest that the inhibition of Ih by baclofen is secondary to the activation of GIRK, i.e., due directly to alteration of membrane conductance, rather than a distinct effect, and is not mediated by inhibition of adenylyl cyclase.     

Site-specific effects of local pH changes in the substantia nigra pars reticulata on flurothyl-induced seizures

Local cerebral changes of acid-base balance may interfere with neuronal communication. Acidosis enhances and alkalosis suppresses GABAA receptor neurotransmission while there are opposite effects on NMDA receptor transmission. In this study, we determined site-specific effects of acidified solutions of Na-HEPES-artificial cerebrospinal fluid infused into the anterior or posterior area of the substantia nigra pars reticulata (SNR) in rats. Two levels of pH were compared: 6.7 and 7.4. Rats were challenged with flurothyl and the threshold for clonic and tonic-clonic seizures was determined. In the anterior SNR, there were no differences between the effects of the solution with pH 6.7 and 7.4 on flurothyl seizures. In contrast in the posterior SNR, microinfusions with pH 6.7 had proconvulsant effects. The results suggest that local pH changes may have site-specific effects on seizure susceptibility in vivo.     

The distribution of neurons in the substantia nigra pars reticulata with input from the motor, premotor and prefrontal areas of the cerebral cortex in monkeys

We investigated the distribution of neurons in the substantia nigra pars reticulata (SNr) which received cortical input. The activities of single SNr neurons were studied extracellulary in awake monkeys. SNr neurons showed excitatory and/or inhibitory responses to cortical stimulation. These responses were considered to be mediated by the subthalamic nucleus and striatum, respectively. The neurons receiving inhibitory input from the motor, premotor and supplementary motor areas (Motor-related cortical areas) were located in the lateral part of the SNr, whereas those with input from the medial, dorsal and orbital areas of the prefrontal cortex (PFmdo) were frequently found in the rostro-medial part of this nucleus. SNr neurons with inhibitory input from the ventral periprincipal area (PSv) were mainly distributed in the intermedio-lateral portion, with some degree of overlap with input from other cortical areas. The distribution of the excitatory input was almost similar to that of inhibitory one, but the excitatory input from the PSv was much stronger than that from the PFmdo. Some SNr neurons receiving cortical input were proved to project to the thalamus. Our results support the existence of several parallel organization of the cortico-basal ganglia loop circuits [G.E. Alexander, M.R. DeLong, P.L. Strick, Parallel organization of functionally segregated circuits linking basal ganglia and cortex, Ann. Rev. Neurosci., 9, 1986, pp. 357-381.], but interaction between the loops can not be ignored.     

Metabotropic glutamate receptor-mediated inhibition and excitation of substantia nigra dopamine neurons

Microiontophoretic drug application and extracellular recording techniques were used to evaluate the effects of the selective metabotropic glutamate receptor (mGluR) agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate(1S,3R-ACPD) on dopamine (DA) neurons in the substantia nigra zona compacta (SNZC) of chloral hydrate-anesthetized rats. 1S,3R-ACPD had a biphasic effect on the firing rate of DA cells, initially decreasing, then increasing the firing rate. 1S,3R-ACPD also increased the burst-firing activity of DA neurons. Application of the ionotropic receptor (iGluR) agonists (R,S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) or N-methyl-D-aspartate (NMDA) increased the firing rates of neurons which had responded to 1S,3R-ACPD, indicating that mGluRs and iGluRs reside on the same neurons. The initial inhibitory period was not antagonized by systemic haloperidol or iontophoretic bicuculline, indicating a lack of DA or gamma-amino-n-butyric acid (GABA) involvement in this effect. Combined application of the AMPA antagonist, 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX), and the NMDA antagonist, (I)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphoric acid (CPP), at currents which antagonized AMPA and NMDA, did not antagonize either the inhibitory or excitatory effects of 1S,3R-ACPD. Application of the metabotropic antagonist (S)-4-carboxy-phenylglycine antagonized both the inhibitory and excitatory effects of 1S,3R-ACPD. These results indicate that mGluRs may play a role in the modulation of dopaminergic activity in the SNZC.     

Occurrence of heat shock response in deafferented neurons in the substantia nigra of rats

The substantia nigra is innervated by massive inhibitory GABAergic projections from the striatum and globus pallidus, deafferentation of which is supposed to lead to anterograde trans-synaptic degeneration of the nigral neurons. An immunohistochemical method was used to examine the induction of 72,000 mol. wt heat shock protein in the substantia nigra following cerebral hemitransection or transient middle cerebral artery occlusion. At three and four days post-transection, strong immunoreactivity for 72,000 mol. wt heat shock protein was found in the ipsilateral substantia nigra pars reticulata. Light microscopic observation revealed a number of pars reticulata neurons showing strong immunoreactivity for 72,000 mol. wt heat shock protein in their perikarya and proximal processes. In addition, Golgi-like stained neurons with dystrophic features were occasionally observed in the ipsilateral substantia nigra pars reticulata. The immunoreactivity for 72,000 mol. wt heat shock protein in the ipsilateral pars reticulata gradually declined and almost disappeared by 15 days after transection. No apparent induction of 72,000 mol. wt heat shock protein was found in the substantia nigra pars compacta throughout the time period examined. Massive striatal ischemic injury produced by transient middle cerebral artery occlusion also induced expression of 72,000 mol. wt heat shock protein in the pars reticulata neurons three and four days postoperatively. These findings suggest that deafferentation of the striatal or striatopallidal inputs per se is a harmful stress for the substantia nigra pars reticulata neurons, inducing 72,000 mol. wt heat shock protein synthesis. The present data may contribute to our understanding of the molecular basis of the pathomechanism of the transneuronal regression of substantia nigra pars reticulata neurons, which may occur after removal of inhibitory GABAergic inputs.     

(-)-Stepholidine acts as a D1 partial agonist on firing activity of substantia nigra pars reticulata neurons in 6-hydroxydopamine-lesioned rats

This cross-sectional survey was conducted in 20 randomly selected streets in Moradbad city in North India to determine the association of magnesium and antioxidant vitamins with risk of ageing. There were 595 subjects (314 males, 281 females) between 50-84 years of age inclusive. The overall prevalence of hypo-magnesemia was 11.8 per cent (n = 60) with a prevalence of 13.2 per cent (n = 33) in males and 10.6 per cent (n = 27) in females. The prevalence of hypomagnesemia showed significant declining trend in the concentration of serum magnesium, vitamin C, vitamin E and beta-carotene and a rising trend in lipid peroxides and diene conjugates with increase in age from 50-59 years to 70-84 years in both men and women. Multivariate logistic regression analysis showed that serum magnesium, vitamin C, vitamin E and beta-carotene were significant risk factors of ageing in both men and women. The findings suggest that some urban populations of India can benefit by consuming higher dietary magnesium, potassium and antioxidant vitamins for prevention of ageing.     

Single subthalamic nucleus neurons project to both the globus pallidus and substantia nigra in rat

The organization of the major efferents of the rat subthalamic nucleus (STN) was investigated using a fluorescent retrograde double-labeling technique. Red fluorescent Evans Blue was injected into the globus pallidus and blue fluorescent DAPI-Primuline was injected into the substantia nigra. After retrograde axonal transport many double-labeled neurons were seen throughout the STN. Occasionaly double-labeled cells were seen in the lateral hypothalamus just medial to the STN and in a thin lateral strip of neurons extending laterally from the STN. Evidence for a mediolateral topography in both the STN-pallidal and STN-nigral pathways was obtained. The STN contains few, if any, local interneurons. Cell counts revealed that at least 94% of, and possibly all, STN neurons send axon collaterals to both the globus pallidus and substantia nigra.     

Localization of ionotropic and metabotropic glutamate receptors in distinct neuronal elements of the rat substantia nigra

PURPOSE: The contingent valuation method (CVM) is a survey-based approach for eliciting consumer's monetary valuations for programme benefits for use in cost-benefit analysis (CBA). We used the conceptual framework of O'Brien and Gafni (1996) to classify and critically appraise health care CVM studies. METHODS: Search of computerized health care and economic citation databases (e.g. MEDLINE, ECONLIT) and manual search for papers published between 1984 1996 reporting primary data valuing health programme benefits in monetary units by CVM using willingness-to-pay (WTP) or accept (WTA). We classified studies using both empirical (i.e. who was surveyed and how) and conceptual criteria (i.e. which measure of consumer utility was measured and why). RESULTS: 48 CVM studies were retrieved; the majority (42) undertook money valuation in the context of cost benefit analysis (CBA), with the remainder being pricing/demand studies. Among the 42 CBA studies, the consumer utility being measured (i.e. compensating (CV) vs. equivalent variation (EV) was explicitly stated in only three (7%) studies). WTP was measured in 95% of studies and WTA in 5%. By cross-tabulation, 42 (91%) studies were designed as WTP/CV, two (4%) were WTP/EV, two (4%) were WTA/CV and no studies used WTA/EV. Most studies were administered by mail (52%) with 38% being in-person interviews. Value elicitation techniques included open-ended questions (38%), payment cards (19%) discrete choice questions (26%) or bidding games (29%). Some form of construct validation tests, particularly associations between WTP and income, were done in 21 studies (50%). CONCLUSIONS: (i) The number of health care CVM studies is growing rapidly and the majority are done in the context of CBA; (ii) there is wide variation among health care CVM studies in terms of the types of questions being posed and the elicitation formats being used; (iii) classification and appraisal of the literature is difficult because reporting of methods and their relationship with the conceptual framework of CBA is poor; (iii) the applicability to health care of the CVM guidelines issued by the National Oceanic and Atmospheric Administration (NOAA) panel for environmental economics is unclear.     

A quantitative correlation between single unit activity and fluorescence intensity of dopamine neurones in zona compacta of substantia nigra, as demonstrated under the influence of nicotine and physostigmine

In order to investigate the possible relationship between neuronal activity and cellular fluorescence intensity, extracellular recordings of single unit activity and determinations of fluorescence intensity of dopamine (DA) neurones by histochemical microfluorimetry were performed in the same (rostral) part of zona compacta of substantia nigra in male rats. In urethane anaesthesia, zona compacta neurones characteristically showed a slow and fairly regular type of firing. Nicotine (1 mg/kg s.c.) induced a transient decrease in unit activity for 1 min followed by a sustained increase in firing rate. During that stage, 4-5 neurones/rat were recorded at different anteroposterior levels, each during 200 sec. Microfluorimetric examination of the fluorescence intensity developed at the end of the 30-min observation period by the DA neurones of the same area revealed a marked rise in cellular fluorescence intensity. Similar results were obtained with a lower dose of nicotine and/or a shorter observation period. Additional microiontophoretic experiments supported the view that extracellular recordings of the correlative electrophysiological-microfluorimetric investigation belonged to DA neurones. Release of DA from terminals was indicated by an increase in HVA concentration of caudate-putamen in rats subjected to the same nicotine treatment. When tested on one cell during a prolonged period of time, physostigmine (0.25 mg/kg i.p.) caused an initial increase in firing rate of zona compacta neurones (5-10 min) followed by a decrease of unit activity (15-23 min). In agreement with previous observations in mice, fluorescence intensity of nigral DA neurones likewise showed a biphasic change with an initial rise and subsequent decrease (examined at 9.5 and 22-23.5 min, respectively). When mean unit activity and mean fluorescence intensity of individual rats out of various experimental groups were related to each other, a highly significant positive correlation between neuronal fluorescence intensity and firing rate was found. The results obtained with physostigmine demonstrate that mean intensity closely paralleled mean unit activity in time, so that this correlation was maintained. These findings indicate that cellular fluorescence intensity of DA neurone groups can be used as an index of the level of neuronal activity, except for cases where a drug treatment interferes directly with catecholamine synthesis or storage mechanisms.     

Functional coupling between substantia nigra and basal ganglia homologues in amphibians.

Neuroanatomical and pharmacological experiments support the existence of a homologue of the mammalian substantia nigra-basal ganglia circuit in the amphibian brain. Demarcation of borders between the striatum and pallidum in frogs, however, has been contentious, and direct evidence of functional coupling between the putative nigral and striatal homologues is lacking. To clarify basal ganglia function in anurans, the authors used expression of immediate-early gene egr-1 as a marker of neural activation in the basal ganglia of túngara frogs (Physalaemus pustulosus). Regional variation in egr-1 mRNA levels distinguished striatal and pallidal portions of the basal ganglia and supported the grouping of the striatopallidal transition zone with the dorsal pallidum. As further evidence for a functional coupling between the dopaminergic cells in the posterior tuberculum (the putative substantia nigra homologue) and the basal ganglia, a positive relationship was demonstrated between the size of the dopaminergic cell population and the neural activation levels within the dorsal pallidum. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Effects of full D1 dopamine receptor agonists on firing rates in the globus pallidus and substantia nigra pars compacta in vivo: tests for D1 receptor selectivity and comparisons to the partial agonist SKF 38393

Many studies have used the D1 agonist SKF 38393 to characterize D1 receptor influences on firing rates in basal ganglia nuclei in vivo. However, SKF 38393 is a partial agonist and so may not be ideal for delineating D1 receptor effects. This study characterizes the effects of four full D1 agonists, SKF 82958 (chloro-APB), SKF 81297 (6-chloro-PB), dihydrexidine and A-77636, on the firing rates of midbrain dopamine and globus pallidus neurons. Recordings were done in fully anesthetized or paralyzed, locally anesthetized rats, and drugs were given systemically intravenously. Dihydrexidine, SKF 81297 and A-77636 were free of rate effects on midbrain dopamine neurons (up to 10.2 mg/kg) and also did not antagonize the inhibitory effects of quinpirole. In contrast, SKF 82958 strongly inhibited dopamine cells through activation of D2 autoreceptors (ED50 = 0.70 mg/kg). Of these drugs, SKF 82958 also was the only one to increase pallidal unit firing rates when given alone (at 5.0 but not 1.0 mg/kg); the other compounds appeared to be selective for postsynaptic D1 receptors. The results suggest that SKF 82958 may be more properly classified as a mixed D1/D2 agonist. In addition, all four agonists strongly potentiated the pallidal response to quinpirole, demonstrating a D1 receptor potentiation of D2 receptor effects. The results support the role of D1 receptors in the midbrain and globus pallidus as previously characterized with SKF 38393. The similar actions of partial and full D1 agonists in these systems support evidence for a D1 receptor reserve and possibly an effector system other than adenylate cyclase.     

Excitotoxic activation of the NMDA receptor results in inhibition of calcium/calmodulin kinase II activity in cultured hippocampal neurons

Neurotoxic effects of excitatory amino acids have been implicated in various neurological disorders, and have been utilized for excitotoxic models of delayed neuronal cell death. The excitotoxic glutamate-induced, delayed neuronal cell death also results in inhibition of calcium/calmodulin-dependent kinase II (CaM kinase II). In this report, we characterized the glutamate-induced inhibition of CaM kinase II in relation to loss of intracellular calcium regulation and delayed neuronal cell death. Glutamate (500 microM for 10 min), but not KCl (50 mM), exposure resulted in a significant inhibition of CaM kinase II activity. The inhibition of CaM kinase II activity was observed immediately following excitotoxic glutamate exposure and present at every time point measured. Glutamate-induced inhibition of kinase activity and delayed neuronal cell death was dependent upon both the activation of the NMDA glutamate receptor subtype and the presence of extracellular calcium. The relationship between inhibition of CaM kinase II activity and loss of intracellular calcium regulation was also examined. Experimental conditions which resulted in significant neuronal cell death and inhibition of CaM kinase II activity also resulted in a long-term loss of intracellular calcium regulation. Thus, inhibition of CaM kinase II activity occurred under experimental conditions which resulted in loss of neuronal viability and loss of neuronal calcium regulation. Since the glutamate-induced inhibition of CaM kinase II activity preceded neuronal cell death, the data support the hypothesis that inhibition of CaM kinase II activity may play a significant role in excitotoxicity-dependent, delayed neuronal cell death.