Alcohol selectivity of ß3-containing GABAA receptors: evidence for a unique extracellular alcohol/imidazobenzodiazepine Ro15-4513 binding site at the α+ß- subunit interface in αß3δ GABAA receptors.

GABAA receptors (GABARs) have long been the focus for acute alcohol actions with evidence for behaviorally relevant low millimolar alcohol actions on tonic GABA currents and extrasynaptic α4/6, δ, and ß3 subunit-containing GABARs. Using recombinant expression in oocytes combined with two electrode voltage clamp, we show with chimeric ß2/ß3 subunits that differences in alcohol sensitivity among ß subunits are determined by the extracellular N-terminal part of the protein. Furthermore, by using point mutations, we show that the ß3 alcohol selectivity is determined by a single amino acid residue in the N-terminus that differs between GABAR ß subunits (ß3Y66, ß2A66, ß1S66). The ß3Y66 residue is located in a region called "loop D" which in γ subunits contributes to the imidazobenzodiazepine (iBZ) binding site at the classical α+γ2- subunit interface. In structural homology models ß3Y66 is the equivalent of γ2T81 which is one of three critical residues lining the benzodiazepine binding site in the γ2 subunit loop D, opposite to the "100H/R-site" benzodiazepine binding residue in GABAR α subunits. We have shown that the α6R100Q mutation at this site leads to increased alcohol-induced motor in-coordination in alcohol non-tolerant rats carrying the α6R100Q mutated allele. Based on the identification of these two amino acid residues α6R100 and ß66 we propose a model in which ß3 and δ containing GABA receptors contain a unique ethanol site at the α4/6+ß3- subunit interface. This site is homologous to the classical benzodiazepine binding site and we propose that it not only binds ethanol at relevant concentrations (EC50-17 mM), but also has high affinity for a few selected benzodiazepine site ligands including alcohol antagonistic iBZs (Ro15-4513, RY023, RY024, RY80) which have in common a large moiety at the C7 position of the benzodiazepine ring. We suggest that large moieties at the C7-BZ ring compete with alcohol for its binding pocket at a α4/6+ß3- EtOH/Ro15-4513 site. This model reconciles many years of alcohol research on GABARs and provides a plausible explanation for the competitive relationship between ethanol and iBZ alcohol antagonists in which bulky moieties at the C7 position compete with ethanol for its binding site. We conclude with a critical discussion to suggest that much of the controversy surrounding this issue might be due to fundamental species differences in alcohol and alcohol antagonist responses in rats and mice.

Trace and contextual fear conditioning is enhanced in mice lacking the alpha4 subunit of the GABA(A) receptor.

The GABA(A)R alpha4 subunit is highly expressed in the dentate gyrus region of the hippocampus at predominantly extra synaptic locations where, along with the GABA(A)R delta subunit, it forms GABA(A) receptors that mediate a tonic inhibitory current. The present study was designed to test hippocampus-dependent and hippocampus-independent learning and memory in GABA(A)R alpha4 subunit-deficient mice using trace and delay fear conditioning, respectively. Mice were of a mixed C57Bl/6J X 129S1/X1 genetic background from alpha4 heterozygous breeding pairs. The alpha4-knockout mice showed enhanced trace and contextual fear conditioning consistent with an enhancement of hippocampus-dependent learning and memory. These enhancements were sex-dependent, similar to previous studies in GABA(A)R delta knockout mice, but differences were present in both males and females. The convergent findings between alpha4 and delta knockout mice suggests that tonic inhibition mediated by alpha4betadelta GABA(A) receptors negatively modulates learning and memory processes and provides further evidence that tonic inhibition makes important functional contributions to learning and behavior.

A novel alpha subunit in rat brain GABAA receptors.

Two cDNAs (alpha 1 and alpha 4) from rat brain cDNA libraries encode isoforms of the alpha subunit of the GABA/benzodiazepine receptor, which differ at 30% of their amino acid residues. Northern blot analysis and in situ hybridization histochemistry show that alpha 1 and alpha 4 mRNAs have distinct sizes and distinct regional and cellular distributions in rat brain: both mRNAs are found in the cortex and hippocampus; however, only the alpha 1 mRNA is detected in the cerebellum. We injected RNA transcribed from alpha 1 and alpha 4 cDNAs into Xenopus oocytes, together with an RNA for a rat beta subunit. We obtained GABA-dependent inward currents that were reversibly blocked by picrotoxin. Picrotoxin alone, applied to oocytes producing the alpha and beta polypeptides, elicited an outward current. We suggest that these polypeptides together produce GABA-gated ion channels that can also open spontaneously.

Physiology and pharmacology of alcohol: the imidazobenzodiazepine alcohol antagonist site on subtypes of GABAA receptors as an opportunity for drug development?

Alcohol (ethanol, EtOH) has pleiotropic actions and induces a number of acute and long-term effects due to direct actions on alcohol targets, and effects of alcohol metabolites and metabolism. Many detrimental health consequences are due to EtOH metabolism and metabolites, in particular acetaldehyde, whose high reactivity leads to nonspecific chemical modifications of proteins and nucleic acids. Like acetaldehyde, alcohol has been widely considered a nonspecific drug, despite rather persuasive evidence implicating inhibitory GABA(A) receptors (GABA(A)Rs) in acute alcohol actions, for example, a GABA(A)R ligand, the imidazobenzodiazepine Ro15-4513 antagonizes many low-to-moderate dose alcohol actions in mammals. It was therefore rather surprising that abundant types of synaptic GABA(A)Rs are generally not responsive to relevant low concentrations of EtOH. In contrast, delta-subunit-containing GABA(A)Rs and extrasynaptic tonic GABA currents mediated by these receptors are sensitive to alcohol concentrations that are reached in blood and tissues during low-to-moderate alcohol consumption. We recently showed that low-dose alcohol enhancement on highly alcohol-sensitive GABA(A)R subtypes is antagonized by Ro15-4513 in an apparently competitive manner, providing a molecular explanation for behavioural Ro15-4513 alcohol antagonism. The identification of a Ro15-4513/EtOH binding site on unique GABA(A)R subtypes opens the possibility to characterize this alcohol site(s) and screen for compounds that modulate the function of EtOH/Ro15-4513-sensitive GABA(A)Rs. The utility of such drugs might range from novel alcohol antagonists that might be useful in the emergency room, to drugs for the treatment of alcoholism, as well as alcohol-mimetic drugs to harness acute positive effects of alcohol.

gamma-Aminobutyric acid-stimulated chloride permeability in crayfish muscle.

gamma-Aminobutyric acid selectively increased Cl- permeability in isolated strips of crayfish abdominal muscle. Muscle fibers incubated in Van Harrevald's solution at room temperature took up 36Cl- to the extent of 700 ml/kg wet weight with a halftime of 2.5 min. During 15-S incubations, the control 36Cl- uptake space was 131 +/- 4 ml/kg (n = 60) and this was significantly increased by gamma-aminobutyric acid at 200 muM or higher concentrations to 177 +/- 4 ml/kg (n = 48, P less than 0.05). This effect was specific for chloride since gamma-aminobutyric acid did not increase the uptake by crayfish muscle of radioactive sucrose, inositol, or propionate. gamma-Aminobutyric acid stimulation of 36Cl- uptake is mediated by receptor-ionophore function since the process shows pharmacological properties virtually identical to those observed by electrophysiological techniques. The gamma-aminobutyric acid stimulation of Cl- permeability is dose dependent with 50% of the maximal effect at 40 muM gamma-aminobutyric acid and the dose vs. response curve is somewhat sigmoid. The gamma-aminobutyric acid agonist muscimol causes the same maximal effect on Cl- uptake as gamma-aminobutyric acid, but acts at 5-fold lower concentrations, i.e. is more potent. However, the partial agonist gamma-amino, beta-hydroxybutyric acid produced little or no stimulation of 36Cl- flux. The response to gamma-aminobutyric acid was blocked by 2 mM beta-guanidinopropionate or gamma-guanidinobutyrate, 0.5 mM bicuculline, and 10 muM picrotoxinin. Picrotoxinin inhibition was dose dependent with 50% inhibition occurring at 4 muM. Antagonists did not affect control 36Cl- uptake. These results confirm electrophysiological observations that the postsynaptic response to the inhibitory neurotransmitter gamma-aminobutyric acid involves a rapid increase in membrane permeability to Cl-.

Functional domains of GABAA receptors.

The transmitter-gated ion channels mediate rapid synaptic transmission, for example, at the neuromuscular junction using acetylcholine and in the CNS using primarily the amino acids glutamate and GABA. GABAA-receptor Cl- channels share sequence homology with a superfamily of these channels including nicotinic acetylcholine receptor and inhibitory glycine receptor. In this article, Geoffrey Smith and Richard Olsen discuss recent affinity labelling and site-directed mutagenesis studies on GABAA receptors that have identified amino acid residues essential for binding of agonists and allosteric modulators as well as the ion channel wall formation. The structural domains identified are consistent with results obtained with other members of the transmitter-gated ion channel superfamily and suggest that structural models for one member of the family may apply to the others as well.

gamma-Aminobutyric acid receptors in normal human brain and Huntington disease.

GABA receptor binding curves were measured in well-washed membrane homogenates from eleven regions of normal human brain and four regions from brains of Huntington disease patients. Computer analysis suggested two populations of receptor sites of different affinity (KD = 10 nM and 240 nM) present in all brain regions but in variable quantity (cerebellar cortex and cerebral cortex greater than basal ganglia greater than deeper brain structures). The number of GABA receptor sites in the caudate-putamen region of Huntington brain was less than normal, but the number of sites was increased in Huntington disease substantia nigra. No differences from normal were found in cerebellar cortex or frontal cortex from Huntington disease, and no significant changes in binding affinity were observed for any of the four regions tested under the conditions used.

Deduction of amino acid residues in the GABA(A) receptor alpha subunits photoaffinity labeled with the benzodiazepine flunitrazepam.

Peptide mapping and microsequencing were used to infer the site of photoaffinity labeling by the gamma-aminobutyric acidA receptor modulator [3H]flunitrazepam. Peptide mapping with and without N-deglycosylation was used to restrict the domain for photoaffinity labeling to residues 74-123 of the bovine alpha1 subunit, in agreement with a previously predicted labeling domain between residues 59-148 based on cyanogen bromide fragmentation. Edman degradation of partially purified photolabeled peptides gave release of 3H counts in the ninth cycle of a tryptic peptide sequence. A second V8/chymotryptic peptide produced an impure sequence with release of 3H counts in the seventh through ninth cycle of sequence. The combined data support those previously reported, i.e., that the primary site for photoaffinity labeling by [3H]flunitrazepam is His102 of the bovine alpha1 subunit. In addition we also detected possible secondary labeling of Pro97.

Synthesis and properties of 3-(2-hydroxyethyl)-3-n-pentyldiazirine, a photoactivable general anesthetic.

To overcome the difficulties of locating the molecular sites of general anesthetic action, we synthesized a novel photoactivable general anesthetic, 3-(2-hydroxyethyl)-3-n-pentyldiazirine (3-diazirinyloctanol), which anesthetized tadpoles with an ED(50) of 160 microM. Subanesthetic concentrations of 3-diazirinyloctanol enhanced GABA-induced currents in GABA(A) receptors, an effect that has been implicated in general anesthetic action. It also enhanced [(3)H]muscimol binding to this receptor. In muscle nicotinic acetylcholine receptors (nAcChoR), it inhibited the response to acetylcholine with an IC(50) of 33 microM. 3-Diazirinyloctanol's pharmacological actions were comparable to those of octanol. 3-(2-Hydroxyethyl)-3-[4,5-(3)H(2)]-n-pentyldiazirine photoincorporated into Torpedo nAcChoR-rich membranes mainly in the alpha subunit with 70% being in a proteolytic fragment containing the M4 transmembrane segment. Agonist enhanced the photolabeling 10-fold in a fragment containing the M1, M2, and M3 transmembrane segments. Thus, 3-diazirinyloctanol is a novel general anesthetic that acts on, and can be photoincorporated into, postsynaptic receptors.

Tyrosine kinase phosphorylation of GABAA receptors.

Phosphorylation of purified bovine brain GABAA receptors by the tyrosine kinase, pp60v-src was examined. pp60v-src phosphorylated two bands of 54-62 kDa and 48-51 kDa that migrated to approximately the same position as bands recognized by antisera against the beta 2 and gamma 2 GABAA receptor subunits, respectively. Bacterially expressed proteins containing the putative large cytoplasmic loops of the beta 1 and gamma 2L subunits were phosphorylated by pp60v-src, indicating that the phosphorylation sites are located in these subunit domains. The tyrosine kinase inhibitors, genistein and the tyrphostins B-42 and B-44, inhibited muscimol-stimulated 36Cl- uptake in mouse brain membrane vesicles (microsacs). magnitude of the tyrphostin B-44-induced inhibition of muscimol-stimulated 36Cl- uptake was significantly reduced in microsacs that were lysed and resealed under conditions that inhibit phosphorylation. GABA-gated Cl- currents were also inhibited by genistein and tyrphostin B-44 in Xenopus oocytes expressing alpha 1 beta 1 and alpha 1 beta 1 gamma 2L subunits. Consequently, protein tyrosine kinase-dependent phosphorylation appears to be another mechanism of regulating the function of GABAA receptors.

GABAA receptor subtypes: autoradiographic comparison of GABA, benzodiazepine, and convulsant binding sites in the rat central nervous system.

The regional distribution of radioactive ligand binding in rat brain for the different receptors of the gamma-aminobutyric acidA (GABAA)-benzodiazepine receptor/chloride channel complex was measured on tissue sections by autoradiography. Seven ligands were employed including [3H]muscimol for high-affinity GABA agonist sites; [3H]bicuculline methochloride and [3H]SR-95531 for the low-affinity GABA sites; [3H]flunitrazepam for benzodiazepine sites, and [3H]2-oxo-quazepam for the 'BZ1'-type subpopulation; and [35S]t-butyl bicyclophosphorothionate (TBPS) and [3H]t-butyl bicyclo-orthobenzoate (TBOB) for convulsant sites associated with the chloride channel. Allosteric interactions of benzodiazepine receptor ligands with [35S]TBPS binding also were examined in membrane homogenates. Comparison of 19 brain regions indicated areas of overlap between these ligands, but also significant lack of correspondence in some regions between any two ligands compared. In particular, the cerebellum, thalamus, hippocampus, substantia nigra and superior colliculus showed enrichment in the binding of some ligands compared to others, and other brain regions showed smaller discrepancies. In addition to the previously observed discrepancies between high-affinity GABA agonists binding and benzodiazepine receptor distribution, especially in the cerebellum, and the well-documented differences in 'BZ1'-selective versus non-selective ligands, significant differences were observed in comparing GABA agonists with antagonists, one antagonist with another, GABA ligands with benzodiazepine or convulsant sites, and even between the two convulsants TBPS and TBOB. The major factor in regional variations within one ligand and between ligands involves differences in binding site densities, although other factors such as endogenous ligands and conformational flexibility may contribute to these findings. The lack of correspondence between components of the GABAA-receptor complex is most consistent with the existence of subtypes that vary in their binding affinities or even binding capabilities. At least four such subtypes are required to explain the regional dissimilarities between ligands. It is likely that these subtypes based on binding alone correspond to different gene products demonstrated recently by molecular cloning and protein chemistry, indicating a pharmacological heterogeneity that might be exploited with subtype-specific drugs showing desirable clinical profiles.

Molecular determinants of desensitization and assembly of the chimeric GABA(A) receptor subunits (alpha1/gamma2) and (gamma2/alpha1) in combinations with beta2 and gamma2.

Two gamma-aminobutyric acid(A) (GABA(A)) receptor chimeras were designed in order to elucidate the structural requirements for GABA(A) receptor desensitization and assembly. The (alpha1/gamma2) and (gamma2/alpha1) chimeric subunits representing the extracellular N-terminal domain of alpha1 or gamma2 and the remainder of the gamma2 or alpha1 subunits, respectively, were expressed with beta2 and beta2gamma2 in Spodoptera frugiperda (Sf-9) cells using the baculovirus expression system. The (alpha1/gamma2)beta2 and (alpha1/gamma2)beta2gamma2 but not the (gamma2/alpha1)beta2 and (gamma2/alpha1)beta2gamma2 subunit combinations formed functional receptor complexes as shown by whole-cell patch-clamp recordings and [3H]muscimol and [3H]flunitrazepam binding. Moreover, the surface immunofluorescence staining of Sf-9 cells expressing the (alpha1/gamma2)-containing receptors was pronounced, as opposed to the staining of the (gamma2/alpha1)-containing receptors, which was only slightly higher than background. To explain this, the (alpha1/gamma2) and (gamma2/alpha1) chimeras may act like alpha1 and gamma2 subunits, respectively, indicating that the extracellular N-terminal segment is important for assembly. However, the (alpha1/gamma2) chimeric subunit had characteristics different from the alpha1 subunit, since the (alpha1/gamma2) chimera gave rise to no desensitization after GABA stimulation in whole-cell patch-clamp recordings, which was independent of whether the chimera was expressed in combination with beta2 or beta2gamma2. Surprisingly, the (alpha1/gamma2)(gamma2/alpha1)beta2 subunit combination did desensitize, indicating that the C-terminal segment of the alpha1 subunit may be important for desensitization. Moreover, desensitization was observed for the (alpha1/gamma2)beta2gamma2 receptor with respect to the direct activation by pentobarbital. This suggests differences in the mechanism of channel activation for pentobarbital and GABA.

The identification of GABA receptor binding sites in insect ganglia.

The binding of [(3)H]muscimol to membrane fractions from central nervous tissue from fly (Musca domestica) and locust (Schistocerca gregaria) was measured. Specific saturable binding was observed with K(d)s of 10 nM (locust) and 40 nM (fly) and B(max) values of 20 fmol/mg protein (fly) and 60 fmol/mg protein (locust). Binding was Na(+)-independent and in locust tissue an additional Na(+)-dependent binding component was observed. Pharmacological properties of the binding sites in both species are consistent with a GABA receptor site rather than an uptake/transport site. We conclude that insect central nervous tissue contains receptors that show some similarities to the GABA receptor complex of mammalian brain.

Opiate receptor binding in the brain of the seizure sensitive Mongolian gerbil (Meriones unguiculatus).

Opiate receptor binding was studied in seizure sensitive (SS) and seizure resistant (SR) strains of the Mongolian gerbil. Cryostat sections of the brain were labeled with [3H]-dihydromorphine, subjected to autoradiography and analysed by microdensitometry. SS gerbils, prior to seizure induction, demonstrated overall greater brain opiate binding when compared to SR animals. Immediately following a seizure, binding in the interpeduncular nucleus fell to levels found in SR animals. The increased opiate binding in the SS (pre-seizure) compared to SR gerbils could reflect a deficit of endogenous ligand which could underlie the seizure diathesis in the gerbil.

[3H]AMPA binding to glutamate receptor subpopulations in rat brain.

The glutamate analog (RS)-alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA), displaced 11% of the binding of L-[3H]glutamate to rat brain membranes, amounting to 22% of the specific binding displaceable by excess non-radioactive glutamate. AMPA-sensitive L-[3H]glutamate binding was additive with that displaced by kainic acid (1 microM) plus N-methyl-D-aspartate (10 microM) when low concentrations of non-radioactive AMPA (1 microM) were employed to determine non-specific background, but partially overlapped when higher concentration of AMPA (100 microM) were used. [3H]AMPA binding was 21% specific (displaceable by non-radioactive 0.1 mM AMPA) in sodium-, calcium- and chloride-free buffer, but increased to over 30% in the presence of 0.1 M chloride. AMPA-sensitive glutamate binding and AMPA binding were both stimulated dramatically by thiocyanate and by several other anions. [3H]AMPA binding activity was resistant to freezing and thawing, optimal at 0-4 degrees C, and detectable at slightly reduced levels by filtration assays and in tissue section autoradiography. AMPA showed a heterogeneous affinity in displacement of L-[3H]glutamate, and [3H]AMPA binding showed heterogeneity with respect to AMPA, quisqualate, and glutamic acid diethyl ester. Scatchard plots gave a best fit for two sites with Kd values of 28 and 500 nM and Bmax values of 200 and 1800 fmol/mg protein, respectively. [3H]AMPA was inhibited by quisqualate (IC50 = 60 nM), L-glutamate (2 microM), (RS)-3-hydroxy-4,5,6,7-tetrahydroisoxazolo-[5,4-c]-pyridine-7-carboxylic acid (7-HPCA, 5 microM), kainic acid (20 microM) and glutamic acid diethyl ester (21 microM) but insensitive to L-aspartate, ibotenic acid, N-methyl-D-aspartate, (RS)-2-amino-phosphonobutyric acid and (RS)-2-amino-phosphonovaleric acid. This is consistent with labeling of a quisqualate-specific subpopulation of glutamate receptors. The high affinity (28 nM) and intermediate affinity (0.5 microM) AMPA sites had similar pharmacological specificity and brain regional distribution as determined by autoradiography. The latter revealed high densities of [3H]AMPA binding in the superficial layers of the cerebral cortex; stratum pyramidale, stratum radiatum, and stratum oriens of the hippocampus; and stratum moleculare of the dentate gyrus. Within the cerebellum, higher densities of binding were observed in the molecular layer than in the granule cell layer. In many regions, [3H]AMPA binding had a similar distribution to that of L-[3H]glutamate binding displaced by AMPA (1 microM).(ABSTRACT TRUNCATED AT 400 WORDS)

Dihydropicrotoxinin binding sites in mammalian brain: interaction with convulsant and depressant benzodiazepines.

The specific binding of [3H] alpha-dihydropicrotoxinin to rat brain membranes was inhibited competitively and potently (IC50 congruent to 100 nM) by a convulsant benzodiazepine drug, RO5-3663. This compound did not inhibit high affinity flunitrazepam binding to the same tissue under similar conditions, and its reported pharmacological activity as an antagonist of GABAergic synaptic transmission, which resembles that of picrotoxinin, appears to involve the picrotoxinin binding sites. Other benzodiazepines such as diazepam, in micromolar concentrations, inhibited picrotoxinin binding in a stereospecific and chemically specific manner. However, the order of potency of a series of depressant benzodiazepines did not correlate well with pharmacological activities nor with reported activities for displacement of high affinity benzodiazepine 'receptor' binding sites (although heterogeneity of both picrotoxinin and benzodiazepine binding site populations may make difficult such comparisons). A comparison of benzodiazepine-displaceable benzodiazepine binding and benzodiazepine-displaceable picrotoxinin binding for different brain regions and subcellular fractions revealed a very similar though not identical distribution of these two classes of drug receptor, again suggesting that the two are not identical. Both classes of drug binding site also showed a very similar distribution to sodium-independent GABA receptor binding sites, which is consistent with other evidence that at least part of these 3 receptor types may be found at least sometimes coupled together in the postsynaptic membrane GABA receptor-ionophore complex.

Studies on the neuropharmacological activity of bicuculline and related compounds.

Bicuculline and 3 chemical derivatives were assayed on a variety of biological systems. Consistent with reports of studies on other animals, some of these compounds caused convulsions in insects and blocked inhibitory postsynaptic potentials in insect muscle. They all potently inhibited mouse brain acetylcholinesterase. Bicuculline and its analogs inhibited the binding of GABA in vitro to sites in crayfish muscle membranes which have properties of receptor sites; this site of action could explain the activity of bicuculline at arthropod neuromuscular junctions. These compounds, at high concentrations (over 100 muM), also inhibited GABA uptake by mouse brain homogenates at 0 degrees C apparently non-competitively. Bicucine methyl ester inhibited GABA transport by brain at 37 degrees C, consistent with non-specific membrane effects at high concentrations of drug. These and other observations cast doubt upon the specificity of bicuculline-like compounds for action on GABA synapses, especially for in vitro studies at high drug concentrations (over 10 muM). The neuroactivity of low doses of bicuculline is apparently not explained by these in vitro effects, and could very well be due to inhibition of GABA synapses at either receptor or ionophore sites. At physiological conditions of pH and temperature, bicuculline is hydrolyzed at its lactone moiety to the less active compound bicucine; this could lead to underestimates of the biological activity of bicuculline. More stable analogs studied so far are not more potent, however.

Length of continuous cocaine exposure determines the persistence of muscarinic and benzodiazepine receptor alterations.

The effects of varied durations of cocaine (1, 3 or 5 days) on muscarinic (MSC) and benzodiazepine (BZD) binding sites in striatum and hippocampus were investigated using homogenate receptor binding. The progressive alterations in these receptor sites from a 5 day cocaine administration were also examined 12 h, 2 days or 21 days after drug exposure. Neither a one nor a three day exposure to cocaine produced any long-term alteration in BZD binding in either structure whereas a 5 day administration produced significant increases in binding. Decreases in MSC receptor binding were apparent in striatum from either a 3 or 5 day cocaine exposure and in hippocampus from a 5 day exposure. The 5 day cocaine exposure produced immediate increases in striatal and hippocampal BZD binding which persisted for 21 days. Conversely, 5 days of cocaine produced a short-term increase in MSC receptor binding in both structures which then became significantly decreased 21 days later. Based on the divergent pattern of changes in BZD and MSC receptor types over time in these structures, it appears that cocaine may induce such changes via separate mechanisms. In addition, it is apparent that changes in the numbers of these receptor sites after cocaine exposure can be quite dynamic, changing rapidly over time.

MPTP is proconvulsant acutely but has no long-term effect in rodent models of seizure and epilepsy.

1-Methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) was used to lesion the substantia nigra of rodents to look for changes in various animal models of epilepsy and seizures. MPTP, acutely administered to C57BL/6J mice, could cause seizures at high doses and enhanced maximal electroshock seizures at lower doses. Older mice were more sensitive to MPTP toxicity. MPTP given over 1 week to produce a 75% drop of striatal dopamine had no effect on seizure thresholds to pentylenetetrazol or picrotoxin and did not change the maximal electroshock seizure. Epileptic gerbils given maximally tolerated doses of MPTP had only a slight striatal dopamine reduction (32%) while seizures remained unaltered. The data are consistent with the hypothesis that chronic dysfunction of dopamine containing substantia nigra neurons have no significant influence on seizures in these animal models.