Effect of cocaine on dopamine transporter receptors depends on routes of chronic cocaine administration.

Investigation of residual behavioral states produced by withdrawal from different routes of cocaine administration indicates that depending on the mode of intake, chronic cocaine produces either tolerance or sensitization to subsequent challenge doses of cocaine. We studied the effect of routes of cocaine administration on the dopamine transporter receptors (DATR), the presumed neuronal mediator of cocaine reward, using the diphenyl substituted piperazine derivative, [3H]GBR 12935 and the cocaine analogue [3H]WIN 35,428. Alzet osmotic mini-pumps filled with either cocaine (100 mg/ml) or saline were surgically implanted on rats into a subcutaneous pocket at the dorsal midline, continuously infusing cocaine at the rate of 40 mg/kg/day. The pumps were removed 14 days later, and the rats were killed 7 days after the removal of pumps. For the intermittent administration, two groups of rats were injected daily either with 40 mg/kg of cocaine or saline for 14 days. Rats were killed 7 days following the last injection. Continuous infusion of cocaine did not alter the [3H]GBR 12935 dopamine transporter (DAT) binding either in the caudate nucleus or in the prefrontal cortex, but it enhanced the density of [3H]WIN 35,428-labeled DAT receptor sites in the caudate putamen. In contrast, intermittent administration of cocaine resulted in a selective alteration of [3H]GBR 12935 binding in the prefrontal cortex but not in the caudate; the cocaine-injected rats had a 48% decrease in [3H]GBR 12935, Bmax (p < .05), without changing the KD. The contrast between the lack of effect of cocaine on [3H]GBR 12935-DATR and the increased binding of [3H]WIN 35,428-DATR highlights the differential sensitivities of the two binding sites to the continuous presence of cocaine.

Association of nerve growth factor mRNA levels with MK-801-induced explosive behaviors in mice.

MK-801, a noncompetitive antagonist of the N-methyl-D-aspartate (NMDA) receptor, stimulated an outbred strain of NIH Swiss mice to display discrete episodes of explosive jumping behavior, designated as "popping." The rapid onset of the MK-801-induced "popping" seems to follow the rapid distribution of the drug to the frontal cortex, the area that contains high levels of NMDA receptors. We examined the effect of this drug on the levels of mRNA coding for nerve growth factor (NGF) in the frontal cortex in relation to the exhibited "popping" episodes. Mice treated with 1 mg/kg MK-801 could be split into two groups based on the total number of "popping" episodes in a 30 min post-injection period. These groups also differed in the steady-state levels of frontal cortex NGF mRNA. Animals that exhibited low numbers of "popping" had levels of NGF mRNA significantly higher than saline treated controls or mice that exhibited high numbers of "popping." Mice treated with 10 mg/kg MK-801 had a high frequency of "popping" that was impossible to separate into episodes. In addition, these mice had levels of frontal cortex NGF mRNA that were significantly lower than either group of mice treated with 1 mg/kg MK-801. These data indicated that there was an increased level of NGF mRNA under conditions where MK-801 induced a low level of "popping" behavior. However, when "popping" intensified, NGF mRNA levels were decreased, suggesting a possible behavioral antagonism of the NGF response.

Alterations in brain monoamines and GABAA receptors in transgenic mice overexpressing TGF alpha.

This study investigated the possibility that overexpression of transforming growth factor alpha (TGF alpha) changes those neurotransmitter systems that have been associated with behaviors found to be altered in the transgenic TGF alpha CD-1 mice. The female TGF alpha mice showed elevated levels of norepinephrine (NE) in the hypothalamus and serotonin (5-HT) in the cortex and brain stem when compared with nontransgenic CD-1 females. The concentrations of monoamines were not altered in the male transgenic brain. The 5-hydroxyindoleacitic acid (5-HIAA)/5-HT ratio was significantly reduced in the brain stem of the male TGF alpha mice and frontal cortex in the female transgenics. The binding of the [3H]GBR 12935-labeled DA transporter was lower in the frontal cortex in the transgenic male TGF alpha mice than in the female TGF alpha mice. No gender difference in dopamine (DA) transporter binding was noted between the nontransgenic male and female mice. Serotonin and GABAA receptors were measured only in males. No differences in the number of 5-HT1A and 5-HT2 receptors were found in the cortex or hippocampus. Maximal GABA stimulation of [3H]flunitrazepam binding in the forebrain hemispheres and cerebellar binding of an imidazobenzodiazepine, [3H]Ro 15-4513, were not different between transgenic and nontransgenic male mice. However, forebrain [35S]TBPS binding in male TGF alpha mice was less affected by the blockade of the GABA agonist sites by the specific GABAA antagonists SR 95531 and bicuculline than the binding of the controls, suggesting either altered endogenous GABA concentrations or a change in receptor populations.(ABSTRACT TRUNCATED AT 250 WORDS)

Age-related changes in [3H]GBR 12935 binding site density in the prefrontal cortex of controls and schizophrenics.

We investigated dopamine transporter receptor ligand binding in the prefrontal cortex as a function of age in schizophrenic and control postmortem brains. [3H]GBR 12935 binding constants were calculated by Scatchard analysis from the autopsied brains from 29 individuals with schizophrenia, and 28 control subjects. There were wide interindividual variations in Bmax and KD that were not related to gender, age, or postmortem interval (PMI) in controls. While there were no significant associations between gender, PMI, and Bmax, or KD in individuals with schizophrenia, there was a significant negative correlation between age and Bmax (r = -.44, p = .02). The slope of the regression lines between age and Bmax for the two groups was significantly different. The results suggest a differential effect of age, or something associated with age, on [3H]GBR 12935 binding sites in the prefrontal cortex of controls and individuals with schizophrenia.

Regional effects of amphetamine, cocaine, nomifensine and GBR 12909 on the dynamics of dopamine release and metabolism in the rat brain.

1. The effects of single-dose regimens of amphetamine, cocaine, nomifensine and GBR 12909 on the dynamics of dopamine (DA) release and metabolism were evaluated in the frontal cortex, hypothalamus, nucleus accumbens and striatum. The regimens selected are known to produce substantial behavioural effects. 2. 3-Methoxytyramine (3MT) and 3,4-dihydroxyphenylacetic acid (DOPAC) rates of formation were used to assess DA metabolism by catechol-O-methyltransferase and monoamine oxidase respectively. The rate of formation of 3MT was used as an index of synaptic DA. The ratio and sum, respectively, of 3MT and DOPAC rates of formation were used to assess DA reuptake inhibition and turnover. 3. The effects of amphetamine on 3MT production and DOPAC steady-state levels were similar in all regions, suggesting similar pharmacodynamic actions. Amphetamine increased 3MT formation and steady-state levels, and reduced DOPAC steady-state levels. DOPAC formation was significantly reduced only in the nucleus accumbens and striatum. Total DA turnover remained unchanged except in the nucleus accumbens. Apparently, the amphetamine-induced increase in DA release occurred at the expense of intraneuronal DA metabolism and did not require stimulation of synthesis. 4. Nomifensine elevated 3MT formation in all regions. A similar effect was produced by cocaine except in the nucleus accumbens. GBR 12909 elevated 3MT production only in the hypothalamus, the striatum and the nucleus accumbens. 5. Cocaine selectively reduced DOPAC formation in the frontal cortex. Nomifensine increased and reduced, respectively, DOPAC formation in striatum and hypothalamus. GBR 12909 elevated DOPAC formation in all regions except the cortex, where pargyline did not reduce DOPAC levels in GBR 12909-treated rats. 6. Ratios and sum of 3MT and DOPAC rates of formation also exhibited wide regional variations for each drug. In contrast to the other drugs, the ratio was not increased after GBR 12909. Apparently, the DA uptake properties of this drug are poorly related to its in vivo effects on the ratio of 3MTproduction to that of DOPAC, which should increase when DA reuptake is inhibited.7. Total DA turnover was increased by GBR 12909 in the hypothalamus, nucleus accumbens and striatum, while cocaine and nomifensine increased it only in the nucleus accumbens and striatum respectively.8. It is concluded that:(a) 3MT and DOPAC rates of formation provide better indices of DA release and metabolism than do their steady-state concentrations.(b) Some effects of DA uptake blockers on DA transmission, especially those of nomifensine and cocaine, may be attributed to increased DA release.(c) Patterns of regional effects of psychostimulants on the dynamics of DA release and metabolism may be better biochemical correlates of stimulant-induced behaviours than would changes in any single region.

Fewer dopamine transporter receptors in the prefrontal cortex of cocaine users.

The authors investigated dopamine transporter receptor binding in the post-mortem prefrontal cortex of 13 subjects with histories of cocaine use who had positive blood screens for cocaine at autopsy and 13 comparison subjects with no history of cocaine use and negative blood screens for cocaine at autopsy. Synaptosomes from pulverized prefrontal cortex were assayed with [3H]GBR 12935 for dopamine transporter receptor. There was a 38% decrease in number of binding sites but no change in affinity constants in the cocaine users.

Allosteric effects of a GABA receptor-active steroid are altered by stress.

Recently, ring A reduced metabolites of naturally occurring steroids have been shown to act as allosteric modulators of GABA-gated chloride ion conductance. Specifically, 5 alpha-pregnane-3 alpha,21-diol-20-one (allotetrahydrodeoxycorticosterone; 5 alpha-THDOC) was shown to be a positive allosteric effector. For example, 5 alpha-THDOC enhances the specific binding of [3H]flunitrazepam, a benzodiazepine receptor agonist, among other pharmacological actions. Swim stress has been shown to reduce the ability of flurazepam, a prototypic benzodiazepine agonist, to antagonize the electrical precipitation of tonic hindlimb extension in mice. This stress-induced reduction in flurazepam's antiseizure efficacy persists for up to 72 h and is associated with alterations in the specific binding of ligands to the GABAA receptor complex. In the current study, the potentiation of [3H]flunitrazepam binding by 5 alpha-THDOC was greater in cerebral cortical membranes prepared from stressed mice compared with unstressed controls. Moreover, nanomolar concentrations of 5 alpha-THDOC that were ineffective in potentiating the specific binding of [35S]TBPS in cerebral cortical membranes prepared from unstressed control mice were capable of potentiating this binding in membranes prepared from stressed animals. Specifically, 50 nM 5 alpha-THDOC caused a 23% increase in the specific binding of [35S]TBPS in membranes from stressed mice, whereas it was without any significant effect in unstressed controls. This apparent ability of 5 alpha-THDOC to distinguish between the binding of [35S]TBPS to crude membranes prepared from stressed and unstressed control mice was eliminated in the presence of a 5 microM concentration of GABA.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular, functional and biochemical characteristics of the dopamine transporter: regional differences and clinical relevance.

The carrier molecule that transports dopamine (DA) across the synaptic membrane is known as the dopamine transporter (DAT). Depending on the ionic conditions, DAT may function as a mediator of both the inward directed DA transport known as the "reuptake" and the outward directed DA transport known as the "release." The functional significance of DAT is in the regulation of DA neurotransmission by terminating the action of DA in the synapse via reuptake. With use of DAT binding as a presynaptic marker to measure altered DA innervation, abnormalities of the DAT binding have been demonstrated in idiopathic Parkinson's disease, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity, and progressive supranuclear palsy. Moreover, the identification of DAT as the neuronal element that mediates the addictive properties of cocaine highlights its significance in cocaine addiction. Cocaine binding in the brain is heterogeneous, and there is an uneven distribution of the high- and low-affinity binding sites across the anatomical regions. Regional differences in ligand binding are observed by using both [3H]cocaine and the diphenyl-substituted piperazine derivatives known as the "GBR series" of ligands. The identification of compounds that inhibit the binding of medications for cocaine abuse. Furthermore, clarification of the various binding domains that may be relevant to transporter function in human neuropsychiatric disorders may lead to the development of new medications for schizophrenia, Tourette's disease, and drug addiction.

Regional differences in rat brain dopamine transporter binding: function of time after chronic cocaine.

Chronic administration of cocaine to rats has been shown to produce a persistent decrease in dopamine (DA) and its metabolites in the brain and periphery. To further explore the alterations in the DA system following repeated administration of cocaine, we studied the regional differences in DA transporter binding as a function of time after the last injection of cocaine. Two groups of rats were treated with cocaine (10 mg/kg twice a day) or saline for 7 days. Rats were sacrificed 1, 2, 3, 6, and 12 weeks after the last injection. The corpus striatum and the frontal cortex were dissected and assayed with [3H]GBR 12935 for DA transporter binding. Time-related differences were observed in the frontal cortex but not in the striatum of the saline-treated control rats. Cocaine treatment prevented the time-dependent increase in Bmax over the course of 6 weeks, but not over the course of 12 weeks following withdrawal. Although there was no difference between the cocaine- and saline-treated group in the striatum at any of the time points, cocaine in the frontal cortex produced a 33% reduction in Bmax during weeks 2 and 3 and a 57% reduction in Bmax at week 6 of withdrawal; the reduction persisted for > or = 12 weeks. The KD was not affected by cocaine or time in either brain region. These findings may be functionally related to cocaine craving because the DA transporter has been identified as the neuronal structure and the medial prefrontal cortex as the anatomical site mediating cocaine reinforcement.

Swim stress selectively alters the specific binding of a benzodiazepine antagonist in mice.

The ability of flurazepam to antagonize the electrical precipitation of tonic hindlimb extension is reduced 24 h after mice are forced to swim for 10 min in cold water (6 degrees C). Presumably, this reduction in flurazepam's antiseizure efficacy reflects an environmental stress-induced modification of the GABAA receptor complex. The current study employed a variety of complementary in vitro approaches to characterize the delayed effects of cold-water swim stress on binding parameters of the GABAA receptor complex that may be associated with flurazepam's reduced antiseizure efficacy. The specific binding of [3H]flunitrazepam and the potentiation of this binding by chloride ions did not change after stress in the cerebral cortex, hippocampus, and cerebellum. Moreover, swim stress did not alter the ability of GABA to inhibit the binding of [35S]t-butylbicyclophosphorothionate (TBPS), a ligand that is a specific biochemical marker of the GABA-associated chloride ionophore, to crude membranes prepared from the cerebral cortex and cerebellum. Swim stress was associated with alterations of the specific binding of [3H]Ro 15-1788, a benzodiazepine receptor antagonist, to crude hippocampal and cerebellar membranes. The results are considered in the context of new insights derived from molecular cloning studies of the GABAA receptor complex.

Differentiation between MK-801- and apomorphine-induced stereotyped behaviors in mice.

The ability of phencyclidine (PCP) to model schizophreniform psychosis is believed to be related to its ability to produce both hypoglutamatergia and hyperdopaminergia. As such, identification of PCP-stimulated behaviors may be important for the development of animal models of schizophrenia. In this study, MK-801 [(+)-5-methyl-10,11-dihydro-5H- dibenzo[a,d]cycloheptane-5,10-imine maleate], a high-affinity PCP analogue, was administered to mice in order to stimulate "PCP behaviors." These PCP behaviors were compared with behaviors stimulated by apomorphine, a dopamine agonist. Stereotyped behavior was assessed by both visual observations and automated measurements. Visual observations showed highly intense gnawing and sniffing in apomorphine-treated mice and the absence of gnawing in MK-801-treated mice. Automated stereotypic measures showed that, compared with vehicle-treated controls, there were frequent dissociations between MK-801 and apomorphine. Conceivably, a compound that attenuates PCP-stimulated behaviors while sparing apomorphine-stimulated behaviors would possess both antipsychotic efficacy and be devoid of undesirable side effects associated with dopamine blockade.

Measurement of an explosive behavior in the mouse, induced by MK-801, a PCP analogue.

MK-801, a noncompetitive antagonist of the N-methyl-D-aspartate (NMDA) receptor complex that binds with high-affinity to the phencyclidine (PCP) binding site, stimulated an outbred strain of NIH Swiss mice to display discrete episodes of explosive jumping behavior, designated as "popping." The episodes of this behavior were characterized with respect to their dose dependency, latency, and duration. The number of mice displaying this behavior increased with increasing doses of MK-801. The intensity of the popping behavior was sensitive to dose-dependent inhibition by haloperidol, a conventional antipsychotic medication, and clozapine, an atypical antipsychotic medication. In view of PCP's ability to precipitate a schizophreniform psychosis in humans, the behavior may serve as a useful preclinical paradigm for the screening of potentially novel antipsychotic medications.

GABA-active steroids: endogenous modulators of GABA-gated chloride ion conductance.

Naturally occurring 3-alpha-hydroxy ring A-reduced metabolites of progesterone and deoxycorticosterone and their synthetic analogues bind to specific sites within the hydrophobic channel domain of the GABAA receptor complex. Acting at these sites, these ligands function as positive allosteric effectors of the complex; they potentiate GABA-stimulated membrane chloride ion conductance, enhance the binding of [3H]muscimol and [3H]flunitrazepam, and displace the binding of [35S]t-butylbicyclophosphorothionate ([35S]TBPS), a channel ligand that is a specific marker of the GABA-associated chloride ionophore. Moreover, steroid metabolites (namely pregnenolone sulfate and dehydroepiandrosterone sulfate) have been identified that display properties of GABA-negative allosteric effectors. The identification of this membrane-associated steroid binding should stimulate development of new classes of anxiolytic, sedative-hypnotic, anticonvulsant, anesthetic, and muscle-relaxant medications that may be devoid of many of the side effects associated with benzodiazepines and barbiturates. Also, elucidation of the physiologic role of this binding site should contribute both to our understanding of endogenous mechanisms for modulating inhibitory neurotransmission, and the pathophysiologic role of the GABAA receptor complex in a variety of neuropsychiatric disorders.

Plasma beta-endorphin levels in post-traumatic stress disorder: a preliminary report on response to exercise-induced stress.

Post-traumatic stress disorder (PTSD) may be associated with changes in endogenous opioid peptide function. To test this hypothesis, 10 male Vietnam combat veterans with PTSD and 8 age-matched male controls underwent a standard grade-incremented exercise treadmill stress test. Plasma beta-endorphin measurements were obtained at rest and following maximal exercise. Resting plasma beta-endorphin levels were comparable between groups. Post-exercise plasma beta-endorphin levels were significantly higher than resting levels in the PTSD patients only (P less than 0.05). These pilot data suggest a differential alteration in plasma beta-endorphin response to exercise in PTSD.

Characteristics of [3H]GBR 12935 binding in the human and rat frontal cortex.

Binding characteristics of the selective dopamine uptake inhibitor [3H]GBR 12935 have been described for the striatum but not for the frontal cortex. We have developed assay conditions for quantifying [3H]GBR 12935 binding in the frontal cortex. In both the rat and human frontal cortex, the assay required four times more tissue (8 mg/ml) than in the striatum (2 mg/ml). [3H]GBR 12935 binding in the frontal is complex, as it involves multiple binding sites. The high-affinity binding site is sodium dependent and is inhibited by sodium. In human but not in rat frontal cortex, addition of K+ reversed the sodium inhibition. The pharmacological profile of the high-affinity [3H]GBR 12935 binding site is consistent with that of the dopamine transporter, because drugs with the most selective dopamine reuptake blocking activities are the most potent displacers of [3H]GBR 12935 binding. There is a positive correlation between the rat and human inhibitory constants, a finding indicating that there are similar pharmacological profiles across at least these two species. Rats with a 6-hydroxydopamine lesion had a 47% decrease in number of [3H]GBR 12935 binding sites, a result indicating that at least a portion of these sites had been on presynaptic dopamine terminals.

Phenytoin potentiation of neuroleptic-induced dyskinesias.

Clinically phenytoin-induced movement disorders may resemble neuroleptic-induced tardive dyskinesia (TD). Neuroleptic-induced TD is thought to be related to an increase in the number and affinity of dopamine D2 receptors. We investigated the interaction of phenytoin with dopaminergic systems by measuring dopamine behavior and receptor changes in animals treated with phenytoin alone or in combination with chlorpromazine. Phenytoin alone or in combination produced significant behavioral supersensitivity without alteration of dopamine D2 receptor density or affinity. This animal study suggests that phenytoin may aggravate neuroleptic-induced tardive dyskinesia through mechanisms other than the dopamine D2 receptors.

Concurrent treatment with benztropine and haloperidol attenuates development of behavioral hypersensitivity but not dopamine receptor proliferation.

Groups of male rats (n = 16 each) were treated with normal saline, haloperidol (0.75 mg/kg), benztropine (1.8 mg/kg) or haloperidol and benztropine once a day for 24 days. Following a 96 hour drug free interval, subsets of these animals were assessed for apomorphine-induced (0.75 mg/kg) stereotypic behavior, sacrificed and analyzed for striatal dopamine biochemistry or sacrificed and analyzed for spiroperidol binding sites. Benztropine cotreatment attenuated the development of behavioral hypersensitivity to haloperidol but did not alter either the dopamine receptor proliferation or the striatal dopamine biochemical changes induced by haloperidol. These results suggest that behavioral hypersensitivity is not an automatic manifestation of dopamine receptor proliferation but must depend, in part, on other factors.

Drug levels and antiparkinsonian drugs in neuroleptic-treated schizophrenic patients.

The limitations of antiparkinsonian treatment strategy when using anticholinergic drugs are determined by their side effects induced through excessive inhibition of parasympathetic functions. In the present study we have investigated the peripheral effects of antiparkinsonian agents on blood levels of concomitantly administered neuroleptic drugs. We have compared the anticholinergic and a dopamine mimetic antiparkinsonian agent in their effects on serum neuroleptic activity (SNA) and serum anticholinergic activity (SAA). Sixteen schizophrenic patients on chronic neuroleptic therapy with steady state neuroleptic levels were receiving either amantadine, 200 mg/day, or anticholinergic drugs (trihexyphenidyl, 10 mg/day, or benztropine, 6 mg/day) for the first 2 weeks, after which the amantadine group was crossed over to anticholinergic and the anticholinergic group to amantadine for the following 2 weeks. Blood samples were obtained once a week along with clinical testing. The results indicate that SAA was fivefold higher with benztropine than with trihexyphenidyl and that amantadine had no effect on SAA. Moreover, SNA was not altered either by anticholinergics or amantadine coadministration, indicating that the therapeutic blood neuroleptic levels are not compromised by antiparkinsonian administration.