Persistent cognitive and morphological alterations induced by repeated exposure of adolescent rats to the abused inhalant toluene.

While thepsychoactive inhalant toluene causes behavioral effects similarto those produced by other drugs of abuse, the persistent behavioral and anatomical abnormalities induced by toluene exposure are not well known. To mimic human "binge-like" inhalant intoxication, adolescent, male Sprague-Dawley rats were exposed to toluene vapor (5700ppm) twice daily for five consecutive days. These rats remained in their home cages until adulthood (P60), when they were trained in operant boxes to respond to a palatable food reward and then challenged with several different cognitive tasks. Rats that experienced chronic exposure to toluene plus abstinence ("CTA") showed enhanced performance in a strategy set-shifting task using a between-session, but not a within-session test design. CTA also blunted operant and classical conditioning without affecting responding during a progressive ratio task. While CTA rats displayed normal latent inhibition, previous exposure to a non-reinforced cue enhanced extinction of classically conditioned approach behavior of these animals compared to air controls. To determine whether CTA alters the structural plasticity of brain areas involved in set-shifting and appetitive behaviors, we quantified basal dendritic spine morphology in DiI-labeled pyramidal neurons in layer 5 of the medial prefrontal cortex (mPFC) and medium spiny neurons in the nucleus accumbens (NAc). There were no changes in dendritic spine density or subtype in the mPFC of CTA rats while NAc spine density was significantly increased due to an enhanced prevalence of long-thin spines. Together, these findings suggest that the persistent effects of CTA on cognition are related to learning and memory consolidation/recall, but not mPFC-dependent behavioral flexibility.

Altered Activity of Lateral Orbitofrontal Cortex Neurons in Mice following Chronic Intermittent Ethanol Exposure.

The lateral orbitofrontal cortex (LOFC) is thought to encode information associated with consumption of rewarding substances and is essential for flexible decision-making. Indeed, firing patterns of LOFC neurons are modulated following changes in reward value associated with an action outcome relationship. Damage to the LOFC impairs behavioral flexibility in humans and is associated with suboptimal performance in reward devaluation protocols in rodents. As chronic intermittent ethanol (CIE) exposure also impairs OFC-dependent behaviors, we hypothesized that CIE exposure would alter LOFC neuronal activity during alcohol drinking, especially under conditions when the reward value of ethanol was modulated by aversive or appetitive tastants. To test this hypothesis, we monitored LOFC activity using GCaMP6f fiber photometry in mice receiving acute injections of ethanol and in those trained in operant ethanol self-administration. In naive mice, an acute injection of ethanol caused a dose-dependent decrease in the frequency but not amplitude of GCaMP6f transients. In operant studies, mice were trained on a fixed ratio one schedule of reinforcement and were then separated into CIE or Air groups. Following four cycles of CIE exposure, GCaMP6f activity was recorded during self-administration of alcohol, alcohol+quinine (aversive), or alcohol+sucrose (appetitive) solutions. LOFC neurons showed discrete patterns of activity surrounding lever presses and surrounding drinking bouts. Responding for and consumption of ethanol was greatly enhanced by CIE exposure, was aversion resistant, and was associated with signs of LOFC hyperexcitability. CIE-exposed mice also showed altered patterns of LOFC activity that varied with the ethanol solution consumed.

Brain-derived neurotrophic factor activation of extracellular signal-regulated kinase is autonomous from the dominant extrasynaptic NMDA receptor extracellular signal-regulated kinase shutoff pathway.

NMDA receptors bidirectionally modulate extracellular signal-regulated kinase (ERK) through the coupling of synaptic NMDA receptors to an ERK activation pathway that is opposed by a dominant ERK shutoff pathway thought to be coupled to extrasynaptic NMDA receptors. In the present study, synaptic NMDA receptor activation of ERK in rat cortical cultures was partially inhibited by the highly selective NR2B antagonist Ro25-6981 (Ro) and the less selective NR2A antagonist NVP-AAM077 (NVP). When Ro and NVP were added together, inhibition appeared additive and equal to that observed with the NMDA open-channel blocker MK-801. Consistent with a selective coupling of extrasynaptic NMDA receptors to the dominant ERK shutoff pathway, pre-block of synaptic NMDA receptors with MK-801 did not alter the inhibitory effect of bath-applied NMDA on ERK activity. Lastly, in contrast to a complete block of synaptic NMDA receptor activation of ERK by extrasynaptic NMDA receptors, activation of extrasynaptic NMDA receptors had no effect upon ERK activation by brain-derived neurotrophic factor. These results suggest that the synaptic NMDA receptor ERK activation pathway is coupled to both NR2A and NR2B containing receptors, and that the extrasynaptic NMDA receptor ERK inhibitory pathway is not a non-selective global ERK shutoff.

Alterations in glutamatergic and gabaergic ion channel activity in hippocampal neurons following exposure to the abused inhalant toluene.

Toluene, a representative member of the large class of abused inhalants, decreases neuronal activity and depresses behavior in both animals and humans. The sites of action of toluene are not completely known but recent studies suggest that ion channels that regulate neuronal excitability may be particularly sensitive. Previous studies with recombinant receptors showed that toluene decreases currents carried by N-methyl-D-aspartate (NMDA)-glutamate receptors without affecting those gated by non-NMDA receptors. In addition, toluene increases currents generated by GABA and glycine receptors. In the present study, primary cultures of rat hippocampal neurons were used to investigate the effects of acute and chronic toluene exposure on native excitatory and inhibitory ligand-gated ion channels. Toluene dose-dependently inhibited NMDA-mediated currents (IC50 1.5 mM) but had no effect on responses evoked by the non-NMDA agonist kainic acid. Prolonged treatment of neurons with toluene (1 mM; 4 days) increased whole-cell responses to exogenously applied NMDA, reduced those evoked by GABA but did not alter responses generated by kainic acid. Immunoblot analysis revealed that prolonged toluene exposure increased levels of NR2A and NR2B NMDA receptor subunits with no change in NR1. Immunohistochemical analysis with confocal imaging showed that toluene-treated neurons had significant increases in the density of NR1 subunits as compared with control neurons. Toluene exposure increased the amplitude of synaptic NMDA currents and decreased those activated by GABA. The results from this study suggest that toluene induces compensatory responses in the functional expression of ion channels that regulate neuronal excitability.

The effects of thiol reduction and oxidation on the inhibition of NMDA-stimulated neurotransmitter release by ethanol.

The N-methyl-D-aspartate (NMDA) receptor is sensitive to inhibition by pharmacologically relevant concentrations of ethanol and may be an important target for the actions of ethanol in the brain. The mechanisms responsible for ethanol's action are unknown but may involve perturbation of one of the multiple regulatory sites that have been identified on the receptor. Previous studies have shown that thiol reducing and oxidizing agents selectively after the activity of the NMDA receptor via a redox modulatory site. In this study, these agents were tested for their ability to modify the inhibitory actions of ethanol on NMDA-stimulated neurotransmitter release from rat brain slices and the potentiation of NMDA-stimulated release by the glycine agonist D-serine. Treatment of hippocampal slices with the thiol reducing agent dithiothreitol (DTT) significantly potentiated (> 2-fold) the NMDA-stimulated release of tritiated norepinephrine ([3H]NE) from rat hippocampal slices. Slices treated with the thiol oxidizing agent dithionitrobenzoic acid (DTNB) had slightly lower (10-15%) levels of NMDA-stimulated neurotransmitter release. Ethanol (25-200 mM) dose-dependently inhibited the NMDA-stimulated release of [3H]NE from control slices with a calculated IC50 value of 73.1 mM. The inhibitory potency for ethanol was increased following DTNB treatment (IC50 of 40.9 mM) while DTT treatment slightly reduced ethanol's potency (IC50 of 85.6 mM). The ability of D-serine to augment NMDA-stimulated neurotransmitter release was tested in the presence and absence of ethanol following treatment of slices with either DTT or DTNB to examine any potential interaction between these modulatory sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential effects of nitric oxide gas and nitric oxide donors on depolarization-induced release of [3H]norepinephrine from rat hippocampal slices.

The NO-generating compounds sodium nitroprusside (NP), nitroglycerin (NTG), and isosorbide dinitrate (ISDN) all significantly inhibited N-methyl-D-aspartate (NMDA)-stimulated release of tritiated norepinephrine ([3H]NA) from preloaded hippocampal slices of adult male Sprague-Dawley rats with IC50's of 114 microM, 1.2 mM, and 1.7 mM respectively. NTG and ISDN also inhibited KCl-stimulated release, while NP had no significant effect on KCl-stimulated release. Although these results suggest that the inhibitory effects of these compounds were mediated by release of NO, NTG and ISDN did not generate detectable levels of NO, and iron-cyanide complexes similar in structure to NP but lacking NO also inhibited release. In contrast, both S-nitroso-N-acetyl-D,L-penicillamine (SNAP) and authentic NO gas significantly enhanced NMDA-stimulated release of [3H]NA (EC50's: 331 and 3.4 microM respectively). This enhancement was not selective for NMDA-stimulated release, since both SNAP and NO potentiated KCl-stimulated release as well. In addition, NO gas significantly enhanced NMDA-stimulated release of tritiated dopamine ([3H]DA) from striatal slices and [3H]NA from cortical and cerebellar slices. Analogs of cyclic guanosine monophosphate (cGMP) had no significant effect on NMDA-stimulated transmitter release, suggesting that the observed increase in release is via a cGMP-independent mechanism. While exogenous NO enhanced both NMDA- and KCl-stimulated neurotransmitter release, it appears that endogenous NO does not play a role in this depolarization-induced release since NO synthase inhibitors did not significantly reduce NMDA-stimulated [3H]NA release. The possibility remains that endogenous NO could modulate neurotransmitter release in other circumstances.

Mechanism for nitric oxide's enhancement of NMDA-stimulated [3H]norepinephrine release from rat hippocampal slices.

Previous studies in our laboratory have shown that nitric oxide (NO) gas enhances NMDA-stimulated release of preloaded tritiated norepinephrine ([3H]NA) from rat brain slices in a dose-dependent, oxygen-sensitive, and cyclic GMP-independent manner. In this study we have attempted to determine the mechanism for the enhancement of neurotransmitter release seen with NO. No-enhanced transmitter release was not due to buffer acidification or generation of NO degradation products, since reducing buffer pH below 7.3 inhibited NMDA-stimulated [3H]NA release and nitrite or nitrate ions (3-100 microM) had no significant effect on release. Carbon monoxide (CO, 10-300 microM), another diatomic gas with properties similar to NO including heme binding and guanylate cyclase activation, had no significant effect on depolarization-induced [3H]NA release. The NO effect was probably not due to mono-ADP-ribosylation of cellular proteins, since the ADP-ribosyltransferase (ADPRT) inhibitors nicotinamide (10 microM-10 microM) and luminol (1 microM-1mM) did not diminish the enhancement of transmitter release seen with NO. The NA reuptake inhibitor desmethylimipramine (DMI, 10 nM-10 microM) neither mimicked nor blocked the effect of NO, suggesting that NO was not acting via inhibition or reversal of the NA transporter. Similar to NO, the metabolic inhibitors sodium azide (NaN3, 0.1-3 mM), potassium cyanide (KCN, 0.1-3 mM), and 2,4-dinitrophenol (2,4-DNP, 10-300 microM) also dose-dependently enhanced NMDA-stimulated [3H]NA release. These results suggest that NO may enhance neurotransmitter release by inhibiting cellular respiration and perhaps ultimately via altering calcium homeostasis.

Ethanol sensitivity of heteromeric NMDA receptors: effects of subunit assembly, glycine and NMDAR1 Mg(2+)-insensitive mutants.

In the current study, dimeric and trimeric combinations of N-methyl-D-aspartate (NMDA) receptor subunits were expressed in Xenopus oocytes and their sensitivity to ethanol was examined using conventional two electrode voltage clamp methods. In oocytes expressing the NR1/2A subunits, ethanol (25, 50 and 100 mM) inhibited NMDA (100 microM)/glycine (10 microM) induced currents by 21, 31 and 47%; respectively. NMDA-stimulated currents in oocytes expressing NR1/2B currents were inhibited by 13, 25 and 45% while NR1/NR2C currents were inhibited by 6, 11 and 24%. Ethanol inhibition of NMDA-stimulated currents in oocytes injected with NR1/2A/2B or NR1/2A/2C was not significantly different from that observed in NR1/2B or NR1/2C injected oocytes, respectively. With all receptor combinations, ethanol inhibition was rapid, reversible and not altered by pre-incubation. In the absence of ethanol, glycine enhanced NMDA-induced currents with an EC50 of 1.42 microM for the NR1/NR2A combination and 0.51 microM for the NR1/NR2C combination. Ethanol inhibited NMDA-induced currents at all glycine concentrations tested (1-100-microM) and did not significantly alter the EC50 value for glycine suggesting that ethanol does not compete for the glycine site on the NMDA receptor. Finally, three NR1 mutants which have been previously shown by others to possess either decreased Mg2+ sensitivity and Ca2+ permeability (N616Q and N616R) or reduced current amplitude (F609L) were tested for their ethanol sensitivity when expressed in combination with the NR2A subunit. Substitution of the wild-type NR1 with F609L did not alter the sensitivity of the receptor to ethanol.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of volatile solvents on recombinant N-methyl-D-aspartate receptors expressed in Xenopus oocytes.

1. We have previously shown that toluene dose-dependently inhibits recombinant N-methyl-D-aspartate (NMDA) receptors at micromolar concentrations. This inhibition was rapid, almost complete and reversible. The NR1/2B combination was the most sensitive receptor subtype tested with an IC(50) value for toluene of 0.17 mM. 2. We now report on the effects of other commonly abused solvents (benzene, m-xylene, ethylbenzene, propylbenzene, 1,1,1-trichlorethane (TCE) and those of a convulsive solvent, 2,2,2-trifluoroethyl ether (flurothyl), on NMDA-induced currents measured in XENOPUS oocytes expressing NR1/2A or NR1/2B receptor subtypes. 3. All of the alkylbenzenes and TCE produced a reversible inhibition of NMDA-induced currents that was dose- and subunit-dependent. The NR1/2B receptor subtype was several times more sensitive to these compounds than the NR1/2A subtype. 4. The convulsant solvent flurothyl had no effect on NMDA responses in oocytes but potently inhibited ion flux through recombinant GABA receptors expressed in oocytes. 5. Overall, these results suggest that abused solvents display pharmacological selectivity and that NR1/2B NMDA receptors may be an important target for the actions of these compounds on the brain.

Ethanol and NMDA receptor signaling.

NMDA receptors are a multi-subunit family of ionotropic receptors activated by the neurotransmitter glutamate. Localized primarily postsynaptically in neurons, they play an important role in mediating excitatory synaptic neurotransmission and are implicated in a wide variety of important calcium-dependent neuronal processes. Experimental animals expressing mutant forms of NMDA subunits display abnormal behaviors and locomotor and cognitive impairments. Over the last 10 years, a wealth of studies has indicated that NMDA receptors are an important site of action for ethanol in the brain. The effects of acute ethanol on NMDA receptor function is discussed herein, with particular focus on efforts to define a molecular site of action of ethanol on the receptor. While it is clear that the ethanol sensitivity of NMDA receptors is influenced by subunit composition, it is also apparent that posttranslational factors such as phosphorylation and protein-protein interactions are important in modulating this sensitivity.

Increased free radical production due to subdural hematoma in the rat: effect of increased inspired oxygen fraction.

Acute subdural hematoma (ASDH) complicates about 15%-20% of severe head injury patients and is one of the major causes for bad outcome, yet the pathomechanisms involved are not well understood. This study has employed a recently developed technique to determine whether ASDH induces free radicals in the underlying brain. We also studied the effect of increased inspired oxygen fraction (FiO2) on free radical production, both in the normal rat brain and after ASDH induction. Twelve male Sprague Dawley rats were studied over 5 h (2 h of FiO2 = 30%, 3 h of FiO2 = 100%). Hydroxyl radical production was measured with microdialysis using the salicylate trapping technique by quantitating the 2,3 dihydroxy benzoic acid (2,3 DHBA) and 2,5 dihydroxy benzoic acid (2,5 DHBA), degradation products, in either noninjured brain (n = 6) or after ASDH (n = 6). Both 2,3 DHBA and 2,5 DHBA increased significantly by 39% and 108%, respectively, after the induction of the SDH (p < 0.05). By increasing the FiO2 to 100%, 2 h after ASDH induction, the 2,3 DHBA and 2,5 DHBA further increased only slightly (ns). After increasing the FiO2 to 100% in the noninjured group, the mean level of 2,3 DHBA increased by 56% (p = 0.06, ns). The level of 2,5 DHBA in the dialysate increased significantly by 56% (p < 0.05), when the FiO2 was increased to 100% ASDH results in a significant increase in free radical production. At the same time, prolonged increase in FiO2 does not lead to further increase in free radical production in the injured brain.

Effects of ethanol on three endogenous membrane conductances present in Xenopus laevis oocytes.

The Xenopus oocyte expression and recording system has allowed a detailed analysis of the physiology and pharmacology of neuronal ion channels including their sensitivity to ethanol. It is important however, to ascertain the effects of a particular drug on the channels inherently expressed by oocytes to ensure that drug effects ascribed to the expressed recombinant receptors are manifested solely through those receptors. In this study, the effects of ethanol were determined on three endogenous currents that can be elicited in oocytes and other cells by various manipulations. The inward cation current, IC, was activated by perfusing naive oocytes with a divalent-free recording solution. Ethanol (25-100 mM) modestly inhibited IC with 100 mM ethanol producing a 7-8% inhibition of steady state currents. The store-operated or capacitative calcium current (I(SOC)) was activated in thapsigargin-treated oocytes by switching from a calcium-free solution to one containing 10 mM calcium. In thapsigargin-treated oocytes also injected with EGTA to block calcium-activated chloride currents, ethanol (100 mM) had no effect on the store-operated calcium current. In contrast, ethanol (10-100 mM) dose-dependently inhibited the calcium-dependent chloride current (I(Cl(Ca)) in thapsigargin-treated oocytes. A voltage-jump protocol was used to separate the two components of I(Cl(Ca)), I(Cl-1) and I(Cl-2). Under these conditions, ethanol (100 mM) inhibited I(Cl-1) currents to a greater extent (38%) than it did I(Cl-2) currents (14%). These results show that Xenopus oocytes express endogenous ion channels that are differentially sensitive to ethanol.

Ethanol sensitivity of recombinant human N-methyl-D-aspartate receptors.

In this study, the ethanol sensitivity of human N-methyl-D-aspartate (NMDA) receptors stably expressed in L(tk-) cells, or transiently expressed in HEK 293 cells and Xenopus oocytes was determined. NMDA receptor function was measured using fura-2 calcium imaging for L(tk-) cells, whole cell voltage-clamp for HEK 293 cells, and two-electrode voltage clamp for oocytes. Ethanol inhibited NMDA receptor function in all three expression system, but was less potent for receptors expressed in L(tk-) cells. NMDA receptors composed of NR1a/2B subunits were inhibited to a greater extent by ethanol than NR1a/2A receptors when expressed in L(tk-) cells and HEK 293 cells, but not in oocytes. These results suggest that the method of receptor expression and assay system used may influence the degree of ethanol inhibition of recombinant NMDA receptors.

The significance of nitric oxide production in the brain after injury.

Glutamate toxicity has been implicated in many aspects of brain injury including traumatic, ischemic, and hemorrhagic damage. We have used in vitro as well as in vivo methods to measure NO production and to examine the role of NO in glutamate toxicity. In building our recombinant system, we used human kidney embryonic cells, HEK 293, as host for transfection of nNOS and NMDA receptor proteins. Cells cotransfected with NMDA and nNOS were more resistant to glutamate toxicity. This resistance correlated with NO production as measured by citrulline assay. Meanwhile, the production of NO did not significantly change the response of the NMDA receptor as seen by calcium studies. Moreover, in vivo, NO production was directly correlated with brain tissue oxygen tension in subarachnoid hemorrhage patients. These data and others point toward the importance of NO production in the response of brain to injury.

A quantitative estimate of the role of striatal D-2 receptor proliferation in dopaminergic behavioral supersensitivity: the contribution of mesolimbic dopamine to the magnitude of 6-OHDA lesion-induced agonist sensitivity in the rat.

Rats with unilateral depletions of neostriatal dopamine display increased sensitivity to dopamine agonists estimated to be 30 to 100 x in the 6-hydroxydopamine (6-OHDA) rotational model. Given that mild striatal dopamine D-2 receptor proliferation occurs (20-40%), it is difficult to explain the extent of behavioral supersensitivity by a simple increase in receptor density. This study was designed to investigate the quantitative aspects of the rotational behavior model utilizing constrained non-linear curve fitting routines. A dose-response curve for the rotational response arising from apomorphine stimulation of the normosensitive striatum was obtained in animals bearing unilateral lesions of striatal efferents (predominantly the striato-nigral pathway as previously described). After the control dose-response experiment, rats received a dopamine- (DA) depleting lesion in the contralateral hemisphere. In one group, 6-OHDA was infused into the medial forebrain bundle (MFB), a placement which is common in the literature and is known to deplete DA in both the striatum and nucleus accumbens. In a second group of rats, 6-OHDA was infused into the globus pallidus at a site which depletes caudate DA, but leaves n. accumbens DA relatively intact. The two experimental groups were tested in identical apomorphine-induced rotation dose-response experiments. The ED50's of the MFB- and caudate-lesioned rats were reduced by 36 and 5.8 fold, respectively, as compared to the control dose-response curve. The MFB and caudate lesions depleted striatal DA and produced a 30 and 36% increase in striatal D-2 binding sites, respectively. Modeling the behavioral and biochemical data with the null model for receptor occlusion indicated that increased striatal D-2 receptor density could account for the magnitude of behavioral supersensitivity in neither the MFB-lesioned group, nor even in the caudate-lesioned group. Thus simple up-regulation or D-2 receptors is unlikely to account for supersensitization as measured in the rotational model. Further, we suggest that quantitative modeling of such hypotheses is a valuable experimental technique for assessing relationships between biochemical and behavioral variables.

Bay K 8644 stimulation of calcium entry and endogenous dopamine release in rat striatal synaptosomes antagonized by nimodipine.

The calcium channel agonist Bay K 8644 (0.1-100 nM) significantly increased the fast-phase entry of calcium and release of endogenous dopamine from rat striatal synaptosomes partially depolarized with 15 mM KCl. This increase was completely blocked by 10 nM nimodipine which had no inhibitory effect on calcium influx and dopamine release in the absence of Bay K 8644. Bay K 8644's agonist effect was attenuated with higher KCl concentrations. These findings suggest that Bay K 8644, in combination with partial KCl depolarization, may expose brain synaptosomal calcium channels which are sensitive to nanomolar concentrations of dihydropyridine calcium channel blockers.

Differential sensitivity of synaptosomal calcium entry and endogenous dopamine release to omega-conotoxin.

The presynaptic neurotoxin omega-conotoxin (omega-CgTx) was tested for its ability to inhibit voltage-dependent calcium flux and transmitter release in rat brain synaptosomes. Conotoxin (0.001-10 microM) had no effect on calcium uptake or endogenous dopamine release from rat striatal synaptosomes in the absence of potassium depolarization. Fast-phase potassium stimulated calcium influx was only partially (20-30%) inhibited by conotoxin at concentrations between 1 nM and 10 microM. The fast-phase release of endogenous dopamine from the same synaptosomal preparation was inhibited by approximately 25% at 0.01 microM and by 60% at 10 microM. These results suggest that a subgroup of high affinity omega-CgTx-sensitive calcium channels may be involved in regulating the release of endogenous dopamine from brain synaptosomes.

Calcium-dependent and -independent release of endogenous dopamine from rat striatal synaptosomes.

We examined the role of calcium in the stimulus-secretion coupling process of brain neurons by measuring the potassium-stimulated release of endogenous dopamine from striatal synaptosomes in the presence and absence of extracellular calcium. Intracellular free calcium levels were also monitored under these conditions using the fluorescent calcium chelator, fura-2. The fast-phase (less than 3 s) of potassium-stimulated dopamine release was completely blocked by removing calcium from the external medium. Elimination of calcium from the medium with EGTA only partially blocked the slow phase (60 s) of K+-stimulated dopamine release. Depolarization of synaptosomes in the presence of extracellular calcium significantly increased intracellular calcium levels as measured by fura-2. No changes in intracellular calcium were observed during depolarization in calcium free-medium. Reductions in the sodium concentration of the extracellular medium produced a significant increase in the basal release of dopamine under calcium-free conditions. Depolarization of synaptosomes under these conditions markedly enhanced the release of dopamine. These results suggest that the slow-phase of dopamine release from synaptosomes does not require calcium but may be mediated via the reversal of the sodium-linked dopamine transport system.

Correlation of rates of calcium entry and endogenous dopamine release in mouse striatal synaptosomes.

The time course of simultaneous Ca2+ entry and endogenous dopamine release was examined in mouse striatal synaptosomes depolarized by 30 mM KCl. Ca2+ entry and endogenous dopamine release exhibited fast and slow phase processes. The fastest rates occurred between 0 and 1 s. Ca2+ uptake and dopamine release dropped off quickly with 5-15 s rates at 13 and 10%, respectively, of the 0-1 s rate. Both processes were maintained at relatively high rates at the 1-3 and 3-5 s intervals suggesting mixed fast and slow phase processes. Uptake of Ca2+ and release of dopamine occurred in parallel over the entire 30 s measurement period; however, approximately 70% of the Ca2+ uptake and dopamine release occurred within the first 5 s following depolarization. A calculated ratio of Ca2+ entry versus dopamine release showed that approximately 1-2 Ca2+ ions were required to cause the release of one dopamine molecule. This ratio remained constant from 1 to 15 s following depolarization. Our results suggest that Ca2+ entry is coupled to endogenous dopamine release for both the fast and slow phase process.

Chronic ethanol treatment uncouples striatal calcium entry and endogenous dopamine release.

Chronic ethanol treatment resulted in a marked reduction in the release of dopamine from striatal synaptosomes in response to depolarization. Calcium entry into the same synaptosomal preparations was not altered. Calculation of the ratio of calcium entry vs dopamine release showed that, under control conditions, approximately 15 calcium ions were required to cause the release of 1 dopamine molecule. Chronic ethanol treatment increased this ratio to more than 80:1, suggesting that chronic ethanol administration altered the coupling between calcium entry and dopamine release. Addition of ethanol in vitro to synaptosomes isolated from chronic ethanol-treated rats returned this ratio to approximately 20:1. These results suggest that chronic ethanol treatment results in dependence which is reflected biochemically in striatum through changes in the coupling between voltage-dependent calcium entry into nerve endings and subsequent neurotransmitter release.