In vivo autoradiography of [3H]SCH 39166 in rat brain: selective displacement by D1/D5 antagonists.

The purpose of this study was to examine the receptor occupancy of D1/D5 antagonists for D1-like dopamine receptors in rat brain using [3H]SCH 39166, a highly selective D1/D5 antagonist with low affinity for 5HT2 receptors. A single concentration of triated SCH 39166 was administered to rats, with or without competing doses of the Dl/D5 antagonist SCH 23390 and unlabeled SCH 39166. the D2-like antagonists haloperidol or the 5-HT, antagonist ketanserin. The bound radioactivity in the cortex, striatum, nucleus accumbens and olfactory tubercle was then quantified using an in vivo autoradiographic procedure. The results indicated that [3H]SCH 39166 was dose dependently displaced by the Dl/D5 antagonists in regions associated with both the nigro-striatal pathway and the mesolimbic dopamine pathway, particularly the nucleus accumbens. Neither haloperidol nor ketanserin displaced [3H]SCH 39166 in any of the regions examined. The data were compared with previously published data examining the in vivo binding of [3H]SCH 39166 in rat brain homogenates. The relative values obtained were comparable to values detected in rat brain homogenates after in vivo binding of [3H]SCH 39166.

Felbamate increases [3H]glycine binding in rat brain and sections of human postmortem brain.

The anticonvulsant compound felbamate (2-phenyl-1,3-propanediol dicarbamate; FBM) appears to inhibit the function of the N-methyl-D-aspartate (NMDA) receptor complex through an interaction with the strychnine-insensitive glycine recognition site. Since we have demonstrated previously that FBM inhibits the binding of [3H]5, 7-dichlorokynurenic acid (DCKA), a competitive antagonist at the glycine site, we assessed the ability of FBM to modulate the binding of an agonist, [3H]glycine, to rat forebrain membranes and human brain sections. In contrast to its ability to inhibit [3H]5,7-DCKA binding, FBM increased [3H]glycine binding (20 nM; EC50 = 485 microM; Emax = 211% of control; nH = 1.8). FBM, but not carbamazepine, phenytoin, valproic acid or phenobarbital, also increased [3H]glycine binding (50 nM; EC50 = 142 microM; Emax = 157% of control; nH = 1.6) in human cortex sections. Autoradiographic analysis of human brain slices demonstrated that FBM produced the largest increases in [3H]glycine binding in the cortex, hippocampus and the parahippocampal gyrus. Because various ions can influence the binding of glycine-site ligands, we assessed their effects on FBM-modulation of [3H]glycine binding. FBM-enhanced [3H]glycine binding was attenuated by Zn++ and not inhibited by Mg++ in human brain. These results suggest that FBM increases [3H]glycine binding in a manner sensitive to ions which modulate the NMDA receptor. These data support the hypothesis that FBM produces anticonvulsant and neuroprotective effects by inhibiting NMDA receptor function, likely through an allosteric modulation of the glycine site.

Lack of long-term changes in cocaine and monoamine concentrations in rat CNS following chronic administration of cocaine.

In previous studies, we reported time-dependent and dose-dependent changes in the rat dopaminergic receptor system following chronic administration of cocaine. The aim of the present investigation was to monitor the concentration of monoamines (using HPLC-ECD) and cocaine (using GC-PCI/MS) in rat CNS following a dose schedule of 5, 10, 15, 20 and 25 mg/kg, i.p., b.i.d. for 21 days. 12 h after the last cocaine injection, cortical and striatal concentrations of monoamines and their metabolites were not significantly different in saline vs cocaine treated animals. In addition, the cocaine concentration in the brain regions examined did not change with the different doses used. Accumulation of a metabolite of cocaine (ecgonine methyl ester) was the only alteration found. These results indicate that alterations in the dopaminergic receptor system following chronic cocaine administration are not due to changes in neurotransmitter concentration or accumulation of cocaine in the brain.

Time dependent changes in DA uptake sites, D1 and D2 receptor binding and mRNA after 6-OHDA lesions of the medial forebrain bundle in the rat brain.

Quantitative receptor autoradiography and in situ hybridization techniques were used to examine the temporal pattern of changes in dopamine uptake sites, D1 and D2 receptors and their transcripts in the striata of animals lesioned with 6-hydroxydopamine. Animals were unilaterally lesioned in the medial forebrain bundle and the brains were analyzed at 1, 2, 4, 6, 8, and 16 weeks postlesion. Degeneration of the nigrostriatal pathway induced a significant loss of dopamine uptake sites in the ipsilateral caudate putamen of all lesioned animals. D1 receptor binding was significantly increased in the caudate putamen on the lesioned side from 1 week to 16 weeks postlesion, whereas the expression of D1 receptor mRNA did not show any change during this period. There was a significant upregulation of D2 receptor binding as well as D2 mRNA from 2 weeks to 8 weeks postlesion. However, at 16 weeks postlesion, D2 receptor binding continued to increase, whereas the mRNA appeared to compensate. These studies show that a different regulatory mechanism may exist between these two DA receptor subtypes. D1 receptor changes occur at the post-transcriptional or translational level, whereas D2 alterations occur by both transcriptional and translational processes. These studies also indicate that the postsynaptic supersensitivity observed in D1 receptors may not be accompanied by a corresponding increase in D1 receptor mRNA.

Interaction of felbamate with [3H]DCKA-labeled strychnine-insensitive glycine receptors in human postmortem brain.

The dicarbamate felbamate has been shown to be capable of competing for the binding of 5,7-[3H]dichlorokynurenic acid ([3H]DCKA) to strychnine-insensitive glycine receptors in sections of human postmortem brain. The IC50 for this interaction was 305.8 microM and the inhibition was complete at 1 mM. Autoradiographic localization of [3H]DCKA binding revealed many regions of human brain in which strychnine-insensitive glycine receptors are manifest. The specific binding in most of these areas was markedly reduced in the presence of 625 microM felbamate. In many regions, [3H]DCKA binding was reduced to background in the presence of felbamate, but some areas retained binding by as much as 41% (i.e., the CA2 region of the hippocampus). This is in contrast to the binding of [3H]DCKA in the presence of carbamazepine, phenytoin, or valproic acid. The binding of the glycine receptor antagonist was not affected by any of these latter agents to the same degree as felbamate. Strychnine-insensitive glycine receptors represent a site of action of felbamate in the human brain.

Receptor alterations in subcortical structures after bilateral middle cerebral artery infarction of the cerebral cortex.

Ischemic damage to the prefrontal, motor, and somatosensory cortex induces alterations in the receptor systems of the caudate putamen and some subcortical brain regions. These alterations may represent an attempt of various neuronal systems to compensate for the reduction in innervation caused by the cortical infarction. Assessment of the receptor changes induced by cortical infarction define neuropharmacologic correlates of cortical damage, indicate possible neurotransmitters associated with neuroanatomically defined subcortical pathways, and suggest possible pharmacologic interventions to counteract the consequences of stroke. Bilateral cortical infarction, induced by ligation of both middle cerebral arteries and temporary occlusion of both common carotid arteries, was investigated in the rat. The infarction resulted in dramatic alterations in subcortical receptor populations as determined by autoradiography. Sodium-dependent, high-affinity, choline-uptake (SDHACU) sites and D1-dopamine receptors in the caudate putamen were unaffected by the infarction, whereas muscarinic and glutamate receptors were increased and D2 receptors were decreased in this structure. A reduction in SDHACU sites caused by the lesion was found in regions including the medial septum, vertical nucleus of the diagonal band, thalamus, nucleus basalis magnocellularis, and basolateral amygdala. M1 receptors were increased in the basolateral and central amygdaloid nuclei whereas non-M1 receptors were increased in the basolateral and central amygdaloid nuclei, but were diminished in the medial septum, vertical nucleus of the diagonal band, thalamus, and nucleus basalis magnocellularis. No significant alterations in muscarinic binding were observed in the various laminae of the hippocampus and dentate gyrus.

Dopamine deficiency in a genetic mouse model of Lesch-Nyhan disease.

We have examined several aspects of neurotransmitter function in the brains of mice carrying a deletion mutation in the gene encoding the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT). During the first 6 weeks of postnatal development, dopamine levels in whole-brain extracts from the mutant mice (HPRT-) failed to increase at rates comparable to normal animals, resulting in 40% lower dopamine levels throughout adulthood. Regional analysis in adult animals showed the caudoputamen to be the most severely affected region, with dopamine deficits of 48-64%. Dopamine levels in other regions were normal or less severely affected. The decrease in dopamine was accompanied by a decrease in tyrosine hydroxylase (TH) activity, the rate-limiting step in dopamine synthesis. Kinetic analysis of TH extracted from the caudoputamen of normal and HPRT- mice demonstrated a 45% decrease in Vmax with an increased affinity for the tetrahydropterin cofactor in the mutants. Labeling of midbrain dopamine neurons using TH immunohistochemistry revealed no obvious deficits in the number of midbrain dopamine neurons, but quantitative autoradiographic studies revealed significant reductions in the binding of 3H-N-[1-(2-benzo(beta)thiophenyl)cyclohexyl]piperidine (3H-BTCP) to dopamine uptake sites in the forebrain of the mutants. In contrast to these abnormalities of the dopamine systems in the mutant mice, other neurotransmitter systems appeared relatively unaffected. Norepinephrine, 5-HT, tryptophan hydroxylase, and glutamic acid decarboxylase were present at normal levels in the brains of the mutants. ChAT activity was slightly lower than normal in the caudoputamen of the mutant animals, but was normal in all other brain regions examined. These results indicate that HPRT deficiency is associated with a relatively specific deficit in basal ganglia dopamine systems that emerges during the first 2 months of postnatal development.

Use of [3H]5,7 dichlorokynurenic acid to identify strychnine-insensitive glycine receptors in human postmortem brain.

[3H]5,7 Dichlorokynurenic acid ([3H]DCKA) was used to define conditions for obtaining selective binding to strychnine-insensitive glycine receptors. The parameters were established in sections of human brain prior to localizing the receptors sites by autoradiography. The binding of [3H]DCKA was of high affinity (Kd = 14.5 nM), readily reversible (K-1 = 0.216 min-1), and specific (60% specific binding determined by inhibition with 100 microM glycine or D-serine). High levels of strychnine-insensitive glycine receptors were identified in several brain areas including portions of the cerebral cortex (Bmax in middle temporal gyrus: 174.0 fmol/mg tissue), basal ganglia, hippocampal formation, and midbrain. These results identify regions where glycine receptors may be involved in modulating NMDA-mediated channel activity.

A sensitive and rapid HPLC-ECD method for the simultaneous analysis of norepinephrine, dopamine, serotonin and their primary metabolites in brain tissue.

This report details a very sensitive, rapid and accurate ion-pair HPLC-ECD method for the analysis of biogenic amines and their metabolites in brain tissue. The method described enables detection of picogram amounts of 3-methoxy-4-hydroxy phenethyleneglycol (MHPG) (37 pg), 3,4-dihydroxy phenylacetic acid (DOPAC) (18 pg), norepinephrine (NE) (12 pg), epinephrine (E) (6 pg), 5-hydroxyindoleacetic acid (5-HIAA) (18 pg), 3,4-dihydroxyphenylethylamine (DA) (6 pg), 3-methoxy-4-hydroxyphenylacetic acid (HVA) (12 pg) and 5-hydroxytryptamine (5-HT) (12 pg). The linearity of the method is from 18.75 ng/mL to 300 ng/mL for MHPG; 9.37 to 150 ng/mL for DOPAC and 5-HIAA; 6.25 to 100 ng/mL for NE, HVA and 5-HT; and from 3.12 to 100 ng/mL for E and DA. The reproducibility, expressed as coefficient of variance (CV%) within-run and between-run groups, was 2.25% and 19.49% for MHPG; 3.84% and 29.84% for DOPAC; 0.89% and 8.97% for NE; 1.26% and 5.61% for E; 1.07% and 28.77% for 5-HIAA; 2.65% and 10.65% for DA; 5.97% and 24.38% for HVA; and 4.44% and 9.45% for 5-HT.

Reduction in striatal D2 dopamine receptor mRNA and binding following AF64A lesions.

Unilateral lesions by a cholinotoxin, receptor autoradiography, and in situ hybridization techniques were employed to determine if dopaminergic receptors are located on cholinergic interneurons in the caudate-putamen (CPu). Lesion of the CPu with small amounts of the cholinotoxin AF64A resulted in a significant decrease in D2 receptor mRNA and D2 receptor binding. The loss was more pronounced in lateral and central portions of the CPu. Results obtained using [3H] SCH23390 binding to D1 receptors indicated that there was no change in this dopamine receptor subtype in the AF64A-lesioned CPu. A decrease in D2 receptor mRNA and receptor binding in AF64A-lesioned animals indicates that a population of postsynaptic D2 receptors is associated with the cholinergic interneurons. Lack of any change in [3H]SCH23390 binding in the AF64A-lesioned animals suggests that D1 receptors are not located on cholinergic neurons. These results provide evidence to support the selectivity of the lesion when used as indicated.

Alterations in the dopaminergic receptor system after chronic administration of cocaine.

Several studies suggest that one of the most important factors contributing to cocaine dependence is an alteration in the actions of the neurotransmitter dopamine in the central nervous system. In order to understand some of the neuroreceptor consequences of cocaine administration, groups of rats were injected with cocaine (2 daily doses of 15 mg/kg) for 1 to 21 days. Binding of [3H]cocaine, [3H]SCH23390, [3H]raclopride, and [3H]BTCP in striatal and cortical tissue from the treated animals was compared to controls. [3H]Cocaine binding was increased by the drug in the striatum and cortex at days 14 and 21, respectively. The binding of [3H]SCH23390 to D1 dopamine receptors was significantly increased at day 3 of cocaine exposure. In striatal membranes, [3H]BTCP binding to dopamine uptake sites was significantly increased after day 7, whereas binding in cortical membranes was increased from day 1. [3H]Raclopride binding to D2 dopamine receptors remained unchanged throughout the study in both cortical and striatal tissues. These results indicate that repeated exposure to cocaine produces an upregulation (possible supersensitivity) in cortical D1, cocaine, and DA-uptake sites which occurs in a time-dependent manner. These increases are coupled with an upregulation in striatal D1, cocaine, and DA-uptake sites, without simultaneous changes in D2 receptors. Thus, cocaine's effects are not uniformly distributed across all brain regions, but rather are focused within areas of the dopamine system.

Characterization of the binding and comparison of the distribution of benzodiazepine receptors labeled with [3H]diazepam and [3H]alprazolam.

The binding characteristics of [3H]diazepam and [3H]alprazolam were obtained by in vitro analysis of sections of rat brain. Dissociation, association, and saturation analyses were performed to optimize the conditions for obtaining selective labeling of benzodiazepine receptors with the two tritiated compounds. Both drugs approached equilibrium rapidly in vitro. Rosenthal analysis (Scatchard plot) of the saturation data indicated a similar finite number of receptors was being occupied by both ligands. Competition studies, using various ligands to inhibit both [3H]diazepam and [3H]alprazolam indicated that these two compounds bind to the tissue sections as typical benzodiazepine drugs and apparently do not overlap onto other subtypes of receptors. These experiments were performed by both binding assay in tissue sections and by light microscopic autoradiography. The major difference between the labeling of the two compounds is represented by the peripheral benzodiazepine sites, which are recognized by [3H]diazepam, but not occupied by [3H]alprazolam (at nanomolar concentrations). This difference was readily apparent in the autoradiograms. Other pharmacokinetic or pharmacodynamic properties must distinguish these two benzodiazepines.

Unilateral ibotenic acid lesion of the caudate putamen results in D2 receptor alterations on the contralateral side.

Dopaminergic projections to the caudate putamen (CPu) involve fibers in the nigrostriatal pathway from the ipsilateral substantia nigra-pars compacta. Post-synaptic receptor populations on cells receiving this information are composed of both D1 and D2 dopamine receptor subtypes. In the present study, unilateral lesions of the CPu, with ibotenic acid, caused a significant reduction in D2 receptor mRNA on the ipsilateral side, as evidenced by in situ hybridization. Similarly, a reduction of D2 receptor binding (as demonstrated with [3H]raclopride) was observed on the lesioned side. As expected, there was no significant change in the D2 receptor binding on the contralateral side. However, a significant increase of D2 receptor mRNA (> 100%) was found in the CPu on the contralateral side when compared to sham-lesioned animals. These results indicate that compensatory changes may be occurring on the unlesioned side of the brain. These changes may reflect elevated transcription from DNA to mRNA or decreased translation of the D2 mRNA to protein following unilateral damage in the CPu. The observation of bilateral influence in the striatal dopamine receptor system may be of paramount importance in understanding movement disorders. These findings could influence the interpretation of results obtained in animal models of human disease in which the dopamine receptor system of the basal ganglia is compromised.

Alpha 2-agonist binding sites in brain: [125I]para-iodoclonidine versus [3H]para-aminoclonidine.

The localization of alpha 2-receptors was determined by quantitative autoradiography using [125I]para-iodoclonidine ([125I]PIC) and [3H]para-aminoclonidine ([3H]PAC). In cortical tissue, [125I]PIC and [3H]PAC were equipotent in their capacity to bind sites recognized by oxymetazoline (preferentially binds to the alpha 2A receptor subtype). The iodinated ligand was about 10 times more potent than [3H]PAC for binding to the heterogenous receptor population labeled by ARC-239 (alpha 2B and alpha 2C). The density of [125I]PIC binding was found to be two-fold higher than that of [3H]PAC in many brain areas and the disparity was even greater in regions such as the dentate gyrus, stria terminalis, and granular layer of the cerebellum. By contrast, other regions of the brain such as the laterodorsal thalamic nucleus, the locus coeruleus, and several amygdaloid areas had equivalent levels of binding. These observations indicate that [3H]PAC has selectivity for the alpha 2A receptor subtype and thus offer a restricted view of alpha 2-adrenergic receptor distribution. The iodinated ligand provides a more complete picture of the overall alpha 2 receptor population.

Effects on monoamine levels in rat CNS after chronic administration of cocaine.

We have previously reported time-dependent and dose-dependent changes in the rat dopaminergic receptor system following chronic administration of cocaine (upregulation of cocaine, D1, and DA-uptake sites). We have now evaluated the effects of chronic cocaine exposure on the central catecholamine/indolamine neurotransmitter systems. Groups of rats were injected with cocaine (15 mg/kg, i.p., b.i.d.) or saline for 1, 3, 7, 14 or 21 days. Cortical and striatal tissues were analyzed for norepinephrine, dopamine, serotonin and their primary metabolites using a HPLC-ECD method. Chronic administration of cocaine did not change the cortical and striatal concentrations of the neurotransmitters under study; except, for a transient increase in the cortical MHPG concentration on day 3. These results suggest that changes in the dopaminergic receptor system following chronic cocaine exposure are not due to changes in the neurotransmitter concentrations.

D1-receptor antagonists: comparison of [3H]SCH39166 to [3H]SCH23390.

A radiolabeled form of the benzonaphthazephine, SCH39166 was used to characterize the binding of this D1 antagonist in cortex, and an autoradiographic comparison of the localization of [3H]SCH39166 to [3H]SCH23390 (D1 antagonist and forerunner of SCH39166) binding was performed. The Kd for [3H]SCH39166, calculated from dissociation and association rate constants (1.09 nM), was comparable to the Kd value derived from Scatchard analyses of saturation data (1.74 nM). [3H]SCH39166 binds to brain tissue in a saturable manner with high affinity and low non-specific binding. Inhibition of [3H]SCH39166 binding by dopaminergic and serotonergic agents supports the hypothesis that this is indeed a D1-specific compound with little overlap onto serotonin (5-HT) receptors. The affinity of [3H]SCH39166 for 5-HT2 and 5-HT1c receptors is at least an order of magnitude lower than the affinity of [3H]SCH23390 for these same receptor sites. Quantitative autoradiographic analysis of [3H]SCH39166 and [3H]SCH23390 binding indicates high D1-receptor density in the caudate-putamen, nucleus accumbens, olfactory tubercle, substantia nigra and entopeduncular nucleus. Low levels of binding (not significantly above background) were detected with [3H]SCH39166 in lamina IV of the cortex and in choroid plexus; areas which had significant [3H]SCH23390 binding and are known to have a high density of 5-HT (5-HT2 and 5-HT1c respectively) receptors.

CNS distribution of D1 receptors: use of a new specific D1 receptor antagonist, [3H]SCH39166.

D1 dopamine receptors have been localized using a radioactive form of a new specific antagonist, [3H]SCH39166. This compound has been shown, in in vitro binding studies, to be highly selective for the D1 receptor subtype; more so than its predecessor, [3H]SCH23390. These ligand binds saturably, reversibly and with high affinity. Use of appropriate conditions produces a high signal to noise binding ratio to D1 receptors in slide-mounted tissue sections. Autoradiographic localization of radiolabeled receptors shows high densities of the D1 receptor subtype in such brain structures as the caudate-putamen, nucleus accumbens, entopeduncular nucleus, and the substantia nigra pars reticulata. A lower density of receptors is found in a few other areas including lamina VI of the cerebral cortex. A distinct paucity of binding was apparent in lamina IV of the cerebral cortex and in the choroid plexus, two areas thought to have D1 receptors. SCH39166 thus represents a superior ligand for obtaining selective labeling of D1 receptors in autoradiographic and binding studies.