The effects of acute and chronic morphine administration on GABA receptor binding

The effect of acute and chronic morphine administration and naloxone-precipitated withdrawal on the binding of [3H]GABA to its receptor sites in rat brain membranes was investigated. Acute morphine (25 mg/kg) administration produced a decrease in the GABA binding in cerebellum, cortex and striatum. This decrease appears to be due to a selective decrease in the number of high-affinity GABA receptor binding sites. In contrast, rats chronically treated with morphine by pellet implantation did not exhibit any changes in GABA receptor binding, except for an increase in pons medulla. However, in rats which were physically dependent, as indicated by naloxone-precipitated withdrawal, GABA binding was decreased significantly in cerebellum and striatum, relative to chronic morphine treatment or placebo pellet controls. This decrease was due to a decrease in the number of low affinity GABA receptor binding sites. Both chronic morphine and naloxone-precipitated withdrawal treatments produced an increase in GABA binding in the pons medulla. These results suggest that morphine may produce some of its effects by modulating GABAergic systems and that high and low affinity GABA receptor sites may play a differential role during various morphine treatments.     

Effect of chronic treatment of ethanol on benzodiazepine and picrotoxin sites on the GABA receptor complex in regions of the brain of the rat

Ethanol has been shown to enhance gamma-aminobutyric acid (GABA)ergic transmission. In this study an examination was made of the effect of chronic treatment with ethanol and its withdrawal at 24 h on the binding of [3H]flunitrazepam and [35S]t-butylbicyclophosphorothionate (TBPS) to brain regions in rat. Rats were rendered tolerant to, and dependent on, ethanol by an intragastric intubation method. The affinity (KD) or the binding capacity (Bmax) of [3H]flunitrazepam or [35S]TBPS was not altered by chronic treatment with ethanol or during withdrawal from ethanol. Neither the enhancing effect of GABA on the binding of [3H]flunitrazepam nor its inhibitory effect on the binding of [35S]TBPS were affected by chronic treatment with ethanol or its withdrawal at 24 h. These results suggest that the sensitivity of benzodiazepine and picrotoxin sites on the oligomeric GABA receptor complex is not affected during tolerance to, or withdrawal from ethanol. It is suggested that the effects of ethanol on GABAergic transmission may be produced at the level of coupled chloride ion channels.     

Stress prevents the chronic ethanol-induced delta opiod receptor supersensitivity in the rat brain

The effects of ethanol administration on binding characteristics of the highly selective mu and delta opioid receptor agonists 8H-[D-Ala2-MePhe4-Gly5-ol]enkephalin (3H-DAGO) and 3H-[D-Pen2, D-Pen5] enkephalin (3H-DPDPE), respectively, were investigated in the rat brain. Chronic but not acute ethanol administration profoundly increased affinity of 3H-DPDPE without changing the number of delta receptors. Stress, applied before each ethanol administration, prevents the above changes. On the other hand, chronic treatment with ethanol did not affect the binding characteristics of 3H-DAGO. These results suggest particular sensitivity of the delta opioid receptor to chronic ethanol administration. Furthermore, a possible involvement of endogenous opioid peptide systems in the enhancement of delta opioid receptor sensitivity is postulated.     

The effect of acute ethanol administration on GABA receptor binding in cerebellum and hypothalamus

The effect of the intraperitoneal administration of ethanol on [3H]GABA binding and glutamic acid decarboxylase (GAD) in cerebellum and hypothalamus was investigated. Acute ethanol administration produced an increase in the binding capacity of the high affinity GABA binding sites and a decrease in the binding capacity of low affinity sites. A decrease in the binding capacity of the high affinity GABA binding sites and an increase in the binding capacity of the low affinity sites were observed in the hypothalamus. No apparent changes were detected in the binding affinities for the two types of GABA receptor sites in both brain areas following ethanol treatment. Ethanol enhanced GAD activity in the cerebellum and reduced GAD activity in the hypothalamus. Changes in GABA binding may be involved in some of the neuropharmacological effects of ethanol.     

Acute and chronic ethanol administration on specific 3H-GABA binding in different rat brain areas

Acute ethanol treatment produces a significant decrease of specific ³H-GABA binding in cerebellum while no changes were detectable in other brain areas. Scatchard analysis shows a decrease in receptor affinity but not in the number of GABA binding sites. On the other hand, chronic administration of ethanol selectively increases specific ³H-GABA binding in the striatum. Kinetic analysis of these data shows that ethanol chronic administration produces a significant increase in the number of GABA binding sites. These data may be useful for the understanding of clinical pictures following acute and chronic ethanol intoxication.     

Gender impacts behavioral and neurochemical adaptations in ethanol-dependent rats

Previous investigations have found gender differences in the effects of chronic ethanol exposure on ethanol withdrawal behaviors as well as GABA(A) receptor gene expression. The present investigation extended these studies with additional behavioral and neurochemical measures of ethanol dependence and withdrawal. No significant gender differences in the elevated plus-maze assessment of ethanol withdrawal anxiety behaviors were found. However, the neuroactive steroid, 3alpha,5alpha-THP, increased exploratory behavior in ethanol withdrawn female, but not male, rats. GABA(A) receptor binding assays showed potent competition of [35S]TBPS binding by 3alpha,5alpha-THP. Control females displayed a decreased affinity for 3alpha,5alpha-THP compared to control males, as evidenced by a nearly 30% increase in the IC50 value. There was no significant effect of ethanol withdrawal on 3alpha,5alpha-THP modulation of [35S]TBPS binding. However, gender differences were observed in the effects of chronic ethanol exposure on GABA(A) receptor subunit peptide levels in the hypothalamus. Female rats had a significant increase in peptide levels for the alpha2 and alpha3 but not alpha4 subunit, whereas male rats displayed a significant increase in alpha4 and alpha3 but not alpha2 subunits compared to pair-fed control levels. Chronic ethanol-induced alterations in gene expression in the hypothalamus did not coincide with previous findings in the cerebral cortex. In particular, male rats showed an increase in alpha1 subunit peptide levels in the hypothalamus, whereas significant decreases in this subunit have been observed in the cerebral cortex. Both female and male rats showed significant increases in the alpha3 subunit in the hypothalamus but not the cerebral cortex. Taken together, these studies provide additional support for gender-selective effects of chronic ethanol-elicited adaptations at the molecular level.     

Physiological levels of glutamine prevent morphine-induced preconditioning in the isolated rat heart

We examined neuroadaptive changes in gamma-aminobutyric acid (GABA)(B) receptor binding following cocaine self-administration and its withdrawal in several rat brain structures using a "yoked" procedure and a quantitative autoradiographic analysis. In order to estimate the distribution of GABA(B) receptors in several brain areas, we used ([S-(R,R)]-[3-[[1-(3,4-diclorophenyl)ethyl]amino]-2-hydroxypropyl]([3,4-(3)H]-cyclohexylmethyl) phosphinic acid) ([(3)H]CGP 54626), a GABA(B) receptor antagonist. The binding of [(3)H]CGP 54626 in the nucleus accumbens subareas and the amygdala was decreased by ca. 20% in rats that actively self-administered cocaine. Similar decreases in the nucleus accumbens were seen in the animals passively receiving cocaine. These animals also showed a reduction in GABA(B) receptor binding in the prefrontal and frontal cortices, septum and dorsal striatum. The [(3)H]CGP 54626 binding in several rat brain areas was decreased after 10-day withdrawal from self-administered cocaine. In summary, the decreases in the GABA(B) receptor binding seem to reflect the effects of chronic administration of cocaine per se and not the motivated process of reinforced responding. Furthermore, withdrawal from cocaine self-administration evoked changes in the GABA(B) receptor binding in several rat brain areas that may indicate neurobiological adaptations.     

Inhibition by miltirone of up-regulation of GABAA receptor alpha4 subunit mRNA by ethanol withdrawal in hippocampal neurons

Miltirone, a tanshinone isolated from the root of Salvia miltiorrhiza, has been characterized as a low-affinity ligand for central benzodiazepine receptors. We have now shown that this compound bound with low affinity (micromolar range) to central benzodiazepine recognition sites but did not interact with peripheral benzodiazepine receptors. It failed to potentiate Cl(-) currents induced by gamma-aminobutyric acid (GABA) both in Xenopus oocytes expressing recombinant human GABA(A) receptors and in cultured rat hippocampal pyramidal cells, but it inhibited the ability of diazepam to potentiate the effect of GABA in these systems. Miltirone (1-10 microM) also partially inhibited the increase in the abundance of the mRNA for the alpha(4) subunit of the GABA(A) receptor induced by ethanol withdrawal in cultured hippocampal neurons. These results suggest that miltirone might ameliorate the symptoms associated with discontinuation of long-term administration of ethanol or of other positive modulators of the GABA(A) receptor.     

Evidence for GABA tolerance in barbiturate-dependent and withdrawn mice

Mice were rendered tolerant and dependent to barbital by a chronic feeding schedule of barbital over 5 weeks. The behavioural effects of muscimol, imidazole-acetic acid (ImAA), and gamma-hydroxybutyric acid (GHB) were measured in control, barbital-dependent, and mice dependent on barbital 48 hr after withdrawal of the drug. The sedative effects of the GABA-mimetics imidazole-acetic acid and muscimol were increased in dependent mice, but reduced in withdrawn mice. Subanaesthetic doses of barbital, given acutely, also increased the sedative effects of imidazole-acetic acid and muscimol but not of gamma-hydroxybutyric acid. Assay of plasma barbital levels by GLC indicated that a negligible amount of barbital was present 48 hr after withdrawal compared to levels of between 60 and 120 micrograms/ml during chronic treatment with barbital. The binding of [3H]GABA to membrane preparations from brain indicated that the values of Kd and Bmax for low affinity binding were not significantly altered in mice withdrawn from chronic treatment with barbital, but that the Kd for high affinity binding was significantly increased from 4.38 to 6.06 nM in barbital-withdrawn mice. There was no difference in the enhancement of GABA binding by pentobarbital between the two groups. It is concluded that barbital-tolerant and dependent mice are cross-tolerant to GABA and that this is possibly mediated by a change in the affinity of the GABA receptor for its ligand.     

Chronic ethanol administration increases the binding of the benzodiazepine inverse agonist and alcohol antagonist [3H]RO15-4513 in rat brain

Chronic ethanol treatment which produced intoxication and physical dependence in rats, produced an increase in the specific binding of ethanol antagonist [3H]RO15-4513 in rat brain cerebral cortex and cerebellum, but not in hippocampus and striatum. The increase in both the regions was due to an increase in the number (Bmax) of receptor sites. These results suggest that the RO15-4513 binding sites on the oligomeric GABA receptor complex are altered following chronic ethanol administration, and support the notion of a unique role of RO15-4513 as an ethanol antagonist.     

Activation of adenosine A1 receptors reduces anxiety-like behavior during acute ethanol withdrawal (hangover) in mice.

Elevated signs of anxiety are observed in both humans and rodents during withdrawal from chronic as well as acute ethanol exposure, and it represents an important motivational factor for ethanol relapse. Several reports have suggested the involvement of brain adenosine receptors in different actions produced by ethanol such as motor incoordination and hypnotic effects. In addition, we have recently demonstrated that adenosine A1 receptors modulate the anxiolytic-like effect induced by ethanol in mice. In the present study, we evaluated the potential of adenosine A1 and A2A receptor agonists in reducing the anxiety-like behavior during acute ethanol withdrawal (hangover) in mice. Animals received a single intraperitoneal administration of saline or ethanol (4 g/kg) and were tested in the elevated plus maze after an interval of 0.5-24 h. The results indicated that hangover-induced anxiety was most pronounced between 12 and 18 h after ethanol administration, as indicated by a significant reduction in the exploration of the open arms of the maze. At this time interval, ethanol was completely cleared. The acute administration of 'nonanxiolytic' doses of adenosine and the selective adenosine A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA), but not the adenosine A2A receptor agonist N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl]adenosine (DPMA), at the onset of peak withdrawal (18 h), reduced this anxiogenic-like response. In addition, the effect of CCPA on the anxiety-like behavior of ethanol hangover was reversed by pretreatment with the selective adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). These results reinforce the notion of the involvement of adenosine receptors in the anxiety-like responses and indicate the potential of adenosine A1 receptor agonists to reduce the anxiogenic effects during ethanol withdrawal.     

Effect of ethanol on mouse cerebral cortical beta-adrenergic receptors

Low concentrations of ethanol (10-100 mM), added to assays in vitro, altered agonist (isoproterenol) binding to mouse cerebral cortical beta-adrenergic receptors in a reversible manner. Ethanol decreased the affinity of the high affinity form of the receptor for isoproterenol but had no effect on the affinity of the low affinity form of the receptor, the proportion of high and low affinity forms of the receptor, the total number of agonist-binding sites, or antagonist binding. The selective effect of ethanol on the properties of the high affinity agonist-binding site suggested that ethanol alters the characteristics of the complex of the receptor and Gs, the guanine nucleotide-binding protein. In cerebral cortical membranes of mice that had ingested ethanol chronically, isoproterenol binding data were best fit by a one-site model, even in the absence of guanine nucleotides. This change, when considered together with previously reported changes in adenylate cyclase activity, is reminiscent of heterologous desensitization of the beta-adrenergic receptor. Thus, both acute and chronic ethanol administration may produce changes in adrenergic function in brain.     

Effects of acute and chronic ethanol intake on synaptosomal glutamate binding activity

The effects of acute and chronic ethanol administration in vivo, on brain synaptosomal glutamate binding activity were explored. In the l-glutamic acid concentration range in which high affinity stereospecific binding to synaptic membranes takes place, both acute and chronic ethanol administration led to progressively greater glutamate binding depending on the chronicity of exposure to ethanol (2 hr to 16 days). These changes appeared to be primarily due to an increase in the maximum binding capacity of these membrane binding sites rather than to changes in their affinity towards the ligand. The withdrawal from ethanol following 16 days of continuous exposure brought about a slow reversal of the increased glutamate-binding activity over a period of 6 days. A brief (2 hr) exposure to ethanol in vitro, produced a small decrease in glutamate binding, whereas prolonged exposure to the alcohol during equilibrium dialysis had a biphasic effect on ligand binding to synaptosomal membranes. These results are suggestive of a possible role for l-glutamic acid in the nervous system during ethanolism and the post-withdrawal reaction.     

The effect of ethanol on 35S-TBPS binding to mouse brain membranes in the presence of chloride

The effect of in vitro and in vivo administration of ethanol on the binding of 35S-t-butyl-bicyclophosphorothionate (35S-TBPS) to cortical brain membranes of C57Bl mice was investigated using KCl (100 mM) containing assay media. The in vitro addition of ethanol produced a dose-dependent inhibition of basal 35S-TBPS binding. In the presence of chloride ions, GABA and pentobarbital had a biphasic action (stimulation followed by inhibition) on 35S-TBPS binding, whereas diazepam only stimulated the binding. Ethanol reduced the stimulatory effects of GABA and pentobarbital in a dose-dependent manner, but had no effect on the enhancement of 35S-TBPS binding produced by diazepam. 35S-TBPS binding to cortical brain membranes was inhibited by the putative Cl- channel blocking agent DIDS. This inhibitory action of DIDS was significantly, and dose-dependently reduced by ethanol (greater than or equal to 100 mM ethanol). Chronic ethanol ingestion in vivo, which produced tolerance to and physical dependence on ethanol in the animals, did not alter the stimulatory and inhibitory effects of GABA and pentobarbital on 35S-TBPS binding. The enhancement of 35S-TBPS binding produced by diazepam was slightly, but significantly, enhanced in brain membranes from animals which had undergone 24 hours of ethanol withdrawal. Chronic ethanol treatment did not change the potency of picrotoxin and of the peripheral BDZ-receptor ligand RO 5-4864 to competitively inhibit 35S-TBPS binding. Our results suggest that in vitro addition of ethanol alters the activity of the GABA/benzodiazepine (BDZ) receptor complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of chronic haloperidol administration on GABA receptor binding

The effect of chronic administration of haloperidol for 30 days and its subsequent withdrawal for 0,4 and 8 days on the binding of [³H]-GABA and [³H]-muscimol to GABA binding sites in rat brain membranes obtained from six discrete regions of the brain and spinal cord was investigated. The chronic administration of haloperidol resulted in a marked increase in the number of GABA binding sites within the substantia nigra, but did not affect GABA binding sites in other regions of rat brain or spinal cord. The increase in GABA binding sites in the substantia nigra was evident for at least 4 days following termination of haloperidol treatment; however, 8 days following withdrawal from haloperidol, when dopaminergic supersensitivity had become maximally expressed, the number of GABA binding sites in the substantia nigra had declined and was the same as that observed in the control rats. These observations suggest that blockade of dopamine receptors in the striatum by haloperidol results in a compensatory decrease in the activity of the GABAergic strionigral system that leads to an increase in the number of GABA binding sites within the substantia nigra. This increase in the number of GABA binding sites is gradually reversed when dopamine is again allowed to interact with its receptors.     

Changes in benzodiazepine-GABA receptor coupling in an accumbens-habenula circuit after chronic diazepam treatment.

1. The effects of subacute and of chronic diazepam treatment upon binding to the GABAA receptor have been examined by use of receptor autoradiography for determining flunitrazepam (FNZP) binding, GABA enhancement of FNZP binding. SR 95531 2-(3'-carboxy-2',propyl)-3-amino-6-p-methoxyphenylpyridazinium bromide) binding and GABA binding in parallel sections from rat brain. Prior to the autoradiographic procedures, a behavioural assessment of the rats was made in the elevated plus-maze test of anxiety. 2. Rats receiving diazepam either subacutely (3 days) or chronically (28 days) by both continuous release, from previously implanted subcutaneous silastic capsules, or by daily injection (5 mg kg-1) did not display changes in FNZP or GABA binding in any of the 47 brain structures analysed. Similarly, there were no significant effects of treatment upon mean total entries or on the open:total ratio for entries in the elevated plus-maze. 3. There were reductions in the GABA enhancement of FNZP binding in the nucleus accumbens and central grey after subacute diazepam treatment. This effect persisted in the nucleus accumbens after chronic treatment. Less marked effects occurred in the lateral habenula, dorsal raphe and substantia nigra pars compacta. In the dorsal tegmental nucleus, GABA enhancement of FNZP binding was enhanced after chronic treatment and this was accompanied by reductions in SR 95531 binding. Treatment did not otherwise affect SR 95531 binding, with the exception of the dorsal raphe where binding was decreased after subacute treatment. 4. In general, the patterns of binding produced by the two different treatment routes were very similar. However, SR 95531 binding was lower in certain hippocampal fields in the i.p. treated animals compared to the rats implanted with silastic capsules. 5. It is concluded that repeated administration of diazepam evokes changes in benzodiazepine and GABA receptor coupling, and to a lesser extent changes in low affinity GABA binding, in certain interrelated brain structures of which an accumbens-habenula circuit is a central feature. These changes occur soon after the initiation of diazepam treatment, suggesting that they are unlikely to account for tolerance to the anxiolytic effects of diazepam but may trigger and/or accompany other critical neurochemical events.     

Differences in GABA activity between ethanol withdrawal seizure prone and resistant mice

Withdrawal seizure prone (WSP) and withdrawal seizure resistant (WSR) lines of mice have been genetically selected based on the severity of handling-induced convulsions after identical chronic ethanol exposure. The present experiments showed that naive WSP mice were more sensitive than WSR mice to a subconvulsant dose of picrotoxin, bicuculline or pentylenetetrazole as measured by the ability of these drugs to exacerbate handling-induced convulsions. This may reflect a difference between lines in the GABA-chloride channel. The density and affinity of [35S]t-butylbicyclophosphorothionate (TBPS) binding sites, a cage convulsant which binds to the picrotoxin site on the GABA-chloride channel, was measured in the frontal cortex, remainder of the cortex, cerebellum and hippocampus. The binding properties of [3H]flunitrazepam and the potency of gamma-aminobutyric acid (GABA) to enhance flunitrazepam binding was characterized in whole brain samples. There were no differences between lines. The behavioral results suggest a role for the GABA-chloride channel in the differential ethanol withdrawal seizure behavior of WSR and WSP mice, but this is not due to changes in receptor densities or affinities.     

Hyposensitivity to Gamma-Aminobutyric Acid in the Ventral Tegmental Area During Alcohol Withdrawal: Reversal by Histone Deacetylase Inhibitors

Putative dopaminergic (pDAergic) ventral tegmental area (VTA) neurons have an important role in alcohol addiction. Acute ethanol increases the activity of pDAergic neurons, and withdrawal from repeated ethanol administration produces a decreased sensitivity of pDAergic VTA neurons to GABA. Recent studies show that behavioral changes induced by chronic alcohol are reversed by inhibitors of histone deacetylases (HDACs). Whether HDAC-induced histone modifications regulate changes in GABA sensitivity of VTA pDAergic neurons during withdrawal is unknown. Here, we investigated modulation of withdrawal-induced changes in GABA sensitivity of pDAergic VTA neurons by HDAC inhibitors (HDACi), and also measured the levels of HDAC2, histone (H3-K9) acetylation, and GABA-Aα1 receptor (GABA (A-α1) R) subunit in VTA during ethanol withdrawal. Mice were injected intraperitoneally (ip) with either ethanol (3.5 g/kg) or saline twice daily for 3 weeks. In recordings from pDAergic VTA neurons in brain slices from ethanol-withdrawn mice, sensitivity to GABA (50–500 μM) was reduced. In brain slices from ethanol-withdrawn mice incubated with the HDACi SAHA (vorinostat) or trichostatin A (TSA) for 2 h, the hyposensitivity of pDAergic VTA neurons to GABA was significantly attenuated. There was no effect of TSA or SAHA on GABA sensitivity of pDAergic VTA neurons from saline-treated mice. In addition, ethanol withdrawal was associated with an increase in levels of HDAC2 and a decrease in histone (H3-K9) acetylation and levels of GABA (A-α1) R subunits in the VTA. Therefore, blockade of upregulation of HDAC2 by HDACi normalizes GABA hyposensitivity of pDAergic neurons developed during withdrawal after chronic ethanol treatment, which suggests the possibility that inhibition of HDACs can reverse ethanol-induced neuroadaptational changes in reward circuitry.     

Unraveling the identity of benzodiazepine binding sites in rat hipppocampus and olfactory bulb

The goals of the work reported here were (i) to identify distinct GABA(A)/benzodiazepine receptors in the rat hippocampus and olfactory bulb using receptor binding assays, and (ii) to determine the affinities and selectivities of benzodiazepine receptor ligands from structurally diverse chemical families at each site identified. These studies were aided by the use of software AFFINITY ANALYSIS SYSTEM, developed in our laboratory for analysis of receptor binding data that allows the determination of receptor heterogeneity using non-selective radioligands. Saturation binding assays using [3H]RO15-4513 (ethyl 8-azido-6-dihydro-5-methyl-6-oxo-4H-imidazo[1, 5-a]-[1,4]benzodiazepine-3-carboxylate) revealed two binding sites in each of these two tissues. The higher affinity site corresponds to alpha(5) subunit-containing GABA(A) receptor and the lower affinity site to a combination of alpha(1), alpha(2), and alpha(3) subunit-containing receptors. These results should be useful in the challenging task of identifying the various functional GABA(A) receptors in the central nervous system, and in providing a link between receptor affinities and in vivo activities of the GABA(A)/benzodiazepine receptor ligands studied.     

Time and brain region specific up-regulation of low affinity neuronal nicotinic receptors during chronic nicotine administration in mice

We studied the effects of chronic oral nicotine on brain low affinity nicotine binding sites. Mice received nicotine in the drinking water for 4 or 7 weeks. Receptor binding was measured at 24 or 48 h after cessation of nicotine administration with [3H]methyllycaconitine, an antagonist in alpha7 and alpha3/alpha6beta2beta3* nicotinic receptors in striatum, midbrain, hippocampus and cortex. Chronic nicotine for 4 weeks resulted in a significant increase in the [3H]methyllycaconitine binding in the striatum and cortex, whereas after 7 weeks the increase in binding could be found in the hippocampus but not in the other brain areas studied. For comparison, high affinity nicotine binding sites (mostly alpha4beta2) were measured with [3H]epibatidine after 7-week chronic nicotine treatment. [3H]Epibatidine binding sites were increased in the hippocampus, midbrain and cortex, but not in the striatum. The up-regulation of [3H]methyllycaconitine binding was significant at 24 h but that of [3H]epibatidine binding sites was not observed until at 48 h after cessation of chronic nicotine. These results suggest that up-regulation of low affinity nicotine binding sites does occur during chronic nicotine administration. Furthermore, the low affinity and high affinity binding differ clearly as regards regions and duration suggesting that different nicotinic receptors respond differently to nicotine administration.