Dopamine receptor binding predicts clinical and pharmacological potencies of antischizophrenic drugs.

Tritiated haloperidol and tritiated dopamine label postsynaptic dopamine receptors in mammalian brain. Clinical potencies of butyrophenones, phenothiazines, and related drugs correlate closely with their ability to inhibit tritiated haloperidol binding. These binding methods provide a simple in vitro means for evaluating new drugs as potential antischizophrenic agents.

Dopamine receptor binding enhancement accompanies lesion-induced behavioral supersensitivity.

The binding of [3H]haloperidol to rat striatal dopamine receptors increases after lesion (made by injection of 6-hydroxydopamine) of the nigrostriatal dopamine pathway in those rats which are behaviorally supersensitive, as reflected by apomorphine-induced contralateral rotations. The enhanced binding is associated with an increased number of receptor sites with no change in their affinity.

Antischizophrenic drugs: chronic treatment elevates dopamine receptor binding in brain.

Chronic treatment of rats with the neuroleptic drugs haloperidol, fluphenazine, and reserpine elicits a 20 to 25% increase in striatal dopamine receptor binding assayed with [3H]haloperidol. This increase in receptor sites may account for behavioral supersensitivity to dopamine receptor stimulants in such animals and for tardive dyskinesia in patients treated with these drugs.

Increased brain dopamine and dopamine receptors in schizophrenia.

In postmortem samples of caudate nucleus and nucleus accumbens from 48 schizophrenic patients, there were significant increases in both the maximum number of binding sites (Bmax) and the apparent dissociation constant (KD) for tritiated spiperone. The increase in apparent KD probably reflects the presence of residual neuroleptic drugs, but changes in Bmax for tritiated spiperone reflect genuine changes in receptor numbers. The increases in receptors were seen only in patients in whom neuroleptic medication had been maintained until the time of death, indicating that they may be entirely iatrogenic. Dopamine measurements for a larger series of schizophrenic and control cases (n greater than 60) show significantly increased concentrations in both the nucleus accumbens and caudate nucleus. The changes in dopamine were not obviously related to neuroleptic medication and, unlike the receptor changes, were most severe in younger patients.

Dopamine receptor binding in bovine intermediate lobe pituitary membranes.

[3H]Spiroperidol ([3H]SPIRO) and [3H]N-n-propylnorapomorphine ([3H]NPA), a dopamine antagonist and agonist, respectively, were found to bind stereospecifically to bovine intermediate lobe pituitary membranes. The specific binding was saturable ([3H]SPIRO maximum number of binding sites, 11.2 pmol/g tissue; [3H]NPA maximum number of binding sites, 5.0 pmol/g tissue) and of high affinity affinity ([3H]SPIRO dissociation constant, 0.17 nM; [3H]NPA dissociation constant, 0.35 nM). The rank order of catecholamines, phenothiazines, and related drugs in competing for [3H]SPIRO and [3H]NPA binding is consistent with interactions at a dopamine receptor. Guanine nucleotides were found to selectively decrease agonist but not antagonist affinities for these binding sites. With the addition of 0.1 mM GTP, the ability of agonists to displace [3H]SPIRO binding is decreased 5- to 7-fold, while the dissociation constant for [3H]NPA is increased to 0.8 nM.

Low neuroleptic serum levels in patients receiving fluphenazine decanoate.

The authors monitored serum levels of fluphenazine in nine patients following injections of fluphenazine decanoate ranging from 10 to 75 mg. Levels were detected by a radioreceptor assay based on the ability of the drug to complete with 3H-spiroperidol for binding to dopamine receptors in rat caudate membranes. Serum levels of fluphenazine were quite stable over a 2- to 3-week period following single intramuscular injections of the decanoate and correlated with injected dose. Following decanoate treatment serum levels of fluphenazine are substantially lower than levels observed for most other neuroleptics administered orally. This raises questions as to how fluphenazine decanoate can exert therapeutic actions.

Clinical state and serum neuroleptic levels measured by radioreceptor assay in schizophrenia.

The authors measured serum neuroleptic levels by radioreceptor assay in 30 schizophrenic patients receiving haloperidol, fluphenazine, chlorpromazine, molindone, thiothixene, or trifluoperazine. Neuroleptic levels were significantly correlated with clinical state, monitored by an abbreviated version of the Present State Examination (the mini-PSE). Poor therapeutic responses were associated with serum levels under 50 ng/ml chlorpromazine equivalents. There was no correlation between neuroleptic dosage and mini-PSE score or between neuroleptic dose and serum neuroleptic levels.

Sulpiride isomers exhibit reversed stereospecificity for D-1 and D-2 dopamine receptors in the CNS.

We have investigated the stereospecificity of the interaction of (-) and (+)sulpiride with [3H]cis-flupentixol and [3H]spiperone binding to D-1 and D-2 dopamine receptors respectively in rat striatum. Both isomers of sulpiride compete more potently at D-2 vs. D-1 dopamine receptors. (-)Sulpiride is 50-fold more potent than (+)sulpiride in blocking D-2 receptors, while (+)sulpiride is 3-fold more potent than (-)sulpiride at D-1 receptors. This reversed stereospecificity of sulpiride interactions with CNS D-1 and D-2 dopamine receptors is similar to the stereospecificity of sulpiride interactions at DA1 and DA2 dopamine receptors in peripheral vascular beds.

Differential regulation of dopamine receptors after chronic typical and atypical antipsychotic drug treatment.

Changes in dopamine receptor subtype binding in different brain regions were examined after 28 days treatment of rats with haloperidol, raclopride, clozapine or SCH23390 using in vitro receptor autoradiography. [3H]7-hydroxy-N,N-di-n-propyl-2-aminotetralin binding to dopamine D3 receptors was not changed in any brain region by any of the drug treatments. [3H]SCH23390 was only increased by chronic SCH23390 treatment. Haloperidol significantly increased [3H]nemonapride and [3H]spiperone binding to dopamine D2-like receptors in the caudate putamen. In contrast, haloperidol caused a small, significant increase in [3H]raclopride binding in the lateral caudate putamen only. Raclopride also elevated, but to a lesser extent [3H]nemonapride and [3H]spiperone binding in caudate putamen, whereas it did not affect [3H]raclopride binding. Clozapine did not significantly change D2-like striatal binding of [3H]nemonaipride, [3H]spiperone or [3H]raclopride. The differences in radioligand binding suggest that [3H]nemonapride and [3H]spiperone may be binding to additional subsets of dopamine D2-like receptors (including D4-like receptors) that are not recognized by [3H]raclopride, which has high affinity for D2 and D3 receptors only. Quantification of [3H]nemonapride or [3H]spiperone binding in the presence of 300 nM raclopride (to block D2 and D3 receptors) revealed that haloperidol, raclopride and clozapine up-regulated D4-like receptors in the caudate putamen using either radioligand. These results suggest that D4-like receptors may be a common site of action of both typical and atypical antipsychotics.

Differential regulation of expression of rat hippocampal muscarinic receptor subtypes following fimbria-fornix lesion.

Quantitative RNase protection assays were performed to determine the levels of muscarinic receptor subtype (m1-m5) mRNAs in rat hippocampi. Results showed that the m1, m3, and m4 subtype mRNAs were expressed at relatively high levels, but the levels of the m2 and m5 subtype were very low. Three weeks following aspiration lesions of the fimbria-fornix to produce cholinergic denervation of the hippocampus, non-M1 receptors (non-pirenzepine displaceable [3H]quinuclindinyl benzilate binding sites) in the hippocampus were increased significantly, which correlated with increases in the levels of hippocampal m3 and m4 receptor mRNAs (m3: +24% and m4: +41%). These findings indicate that multiple muscarinic receptor subtypes are expressed in the hippocampus with the m3 and m4 subtypes predominantly postsynaptic to the septohippocampal cholinergic terminals.

Reduction in muscarinic receptors by antisense oligodeoxynucleotide.

Treatment of rat superior cervical ganglion cells in culture for 4 days with an antisense oligodeoxynucleotide corresponding to a partial sequence of the mRNA coding the m1 muscarinic receptor decreased m1 receptor mRNA by 64% without affecting m2 receptor mRNA. [3H]Pirenzepine binding to M1 receptors was decreased by 43%. Intraventricular infusion of this antisense oligodeoxynucleotide reduced muscarinic receptor density in M1 but not M2 receptor-rich brain regions.

Interactions of agonists with D-2 dopamine receptors: evidence for a single receptor population existing in multiple agonist affinity-states in rat striatal membranes.

Several lines of evidence previously indicated that [3H]spiroperidol (SPIRO) or [3H]domperidone (DOMP) might label heterogeneous populations of striatal dopamine receptors in radioligand binding studies. We have examined this possibility in rat striatum using computerized non-linear curve fitting and a method to block the unwanted [3H]SPIRO binding to S-2 serotonergic receptors. In the absence of a serotonergic antagonist, [3H]SPIRO saturation data produce curved Scatchard plots which are best computer fit by assuming the presence of two sites of different [3H]SPIRO affinities. In the presence of appropriate concentrations of ketanserin to block S-2 serotonergic binding, Scatchard plots are linear, with data modeling best to a single population of homogeneous binding sites. The D-2 dopamine receptor Bmax and [3H]SPIRO KD determined in this fashion are indistinguishable from that obtained for the higher affinity binding site by computer analysis of data obtained in the absence of ketanserin. [3H]DOMP produced indistinguishable values for D-2 receptor Bmax as well. Competitions by (-)sulpiride, metoclopramide, and DOMP for [3H]SPIRO binding sites in the presence of ketanserin are steep (Hill slope approximately 1), demonstrating that the previously observed heterogeneity of these sites is due entirely to serotonergic [3H]SPIRO binding. In contrast, agonist/3H-antagonist competition curves in the presence of ketanserin are best computer fit by assuming two independent receptor sites of high (RH) and low (RL) agonist affinity. With the addition of 5'-guanylylimidodiphosphate [Gpp(NH)p] computer analyses of agonist/3H-antagonist competition curves show an increased ratio RL/RH concomitant with apparent decreases in agonist affinities for both sites. Under some conditions, some agonist/3H-antagonist competition curves are best fit by a single site model in which agonist affinity is indistinguishable from the agonist's affinity at RL determined in the absence of Gpp(NH)p. These data are consistent with the presence of a single dopaminergic 3H-butyrophenone binding site, representing the D-2 receptor, which exists in two interconverting states differing in agonist affinity.

GTP regulation of (-)-stepholidine binding to R(H) of D1 dopamine receptors in calf striatum.

(-)-Stepholidine (SPD) exhibits antagonist effects on normosensitive dopamine (DA) receptors, but it has an agonist action on rotation in unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rats. The present work endeavors to further elucidate the mechanism of its agonist action on D1 receptors. [3H]R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-be nzazepine ([3H]SCH-23390) and [3H]spiperone were used, respectively, as radioligands in D1 and D2 DA receptor binding assays in calf striatal membranes. Experimental data were analyzed by a non-linear regression computer program, GraphPAD InPlot 3.15. The competition curves were fitted first by a single-site equation and then by a two-site equation. The results showed that both apomorphine (APO) and SPD competitively inhibited [3H]SCH-23390 binding. Their competition curves fitted best to the two-site equation (P < 0.05) with a high-affinity site (R(H)) and a low-affinity site (R(L)) to DA receptors. The K(H) and K(L) values (nM) were 2.7 +/- 0.45 and 378 +/- 62 for APO, and 3.9 +/- 2.2 and 126 +/- 25 for SPD, respectively. In contrast, the competition curve of SCH-23390, a selective D1 DA receptor antagonist, fitted best to a single-site model with a Ki value of 1.7 +/- 0.5 nM. The R(H) of APO or SPD could be decreased by the addition of 450 microM GTP. In the [3H]spiperone binding test, the APO curve was modeled best by the two-site equation, while the SPD curve fitted best to a single-site model. In the rotational behavior test, APO induced 441 +/- 20 turns/30 min in the 6-OHDA-lesioned rats, and SPD induced 310 +/- 42 turns/30 min, while SCH-23390 antagonized the SPD-induced rotation but did not induce rotational behavior. These results suggest that SPD possesses agonist actions on D1 but antagonist effects on D2 DA receptors.

Regulation of ionotropic glutamate receptors following subchronic and chronic treatment with typical and atypical antipsychotics.

Quantitative in vitro receptor autoradiography was used to examine changes in three ionotropic glutamate receptor subtypes using 3H-MK801 (NMDA-R antagonist), 3H-CNQX (AMPA-R antagonist) and 3H-kainic acid (kainate-R agonist) following subchronic (28 days) and chronic (8 months) treatment of rats with a typical antipsychotic, haloperidol (1.5 mg/kg per day), atypical antipsychotic, clozapine (25 mg/kg per day), the dopamine D2/D3 receptor antagonist, raclopride (10 mg/kg per day), and the dopamine D1 (D1/D5) receptor antagonist SCH23390 (0.5 mg/kg per day). Subchronic and chronic drug treatments did not significantly alter 3H-CNQX or 3H-kainate binding in any of brain regions examined. Subchronic SCH23390 treatment elevated 3H-MK801 binding in the hippocampal formation with significant increases in the CA1 and dentate gyrus, suggesting a specific role for dopamine D1 receptors in the regulation of hippocampal NMDA receptor function. Subchronic, but not chronic, haloperidol and clozapine treatment significantly reduced 3H-MK801 binding in the medial prefrontal cortex. This suggests that typical and atypical antipsychotics may exert some of their clinical effects by affecting NMDA receptors in the medial prefrontal cortex. Both subchronic and chronic clozapine treatment decreased 3H-MK801 binding in the caudate putamen. The minimal extrapyramidal side effects produced by clozapine may result, in part, from the reduction in NMDA receptor binding in the caudate putamen.

Expression of rat striatal D1 and D2 dopamine receptor mRNAs: ontogenetic and pharmacological studies.

The developmental characteristics of D1 and D2 dopamine receptor mRNA levels were determined by Northern blot analyses. Striatal D1 and D2 receptor mRNAs of male Fischer 344 rats were about 60% of adults levels at postnatal day 1 and reached their highest measured levels at day 30 (126-139% adults levels) and then decreased by day 120 (100%). D1 and D2 receptor densities at day 30 were about 8-fold higher than at day 1, while mRNA levels showed only a 2-fold increase. The highest level of D2 receptor mRNA in midbrain as reached at day 14 (195% adult level) while levels at day 1 were 31% higher than dose at day 120. Treatment with selective D2 receptor antagonist, haloperidol (0.5 mg/kg/day, s.c., for 2 h, 7, 14, 21 days or 21 days + 3 days withdrawal) had no effect on Sprague-Dawley rat striatal D2 receptor mRNA levels in spite of significant increases in receptor density at the later time points. However, the selective D1 receptor antagonist, SCH 23390 (0.5 mg/kg/day, s.c.) produced increases in striatal D1 receptors and mRNA levels after treatments of 7, 14 and 21 days + 3 days withdrawal.

Modulation of alpha-1 adrenergic receptors in rat brain following chronic reserpine.

Functional denervation of the central adrenergic receptors by 30 daily injections of reserpine (0.25 mg/kg/day s.c.) produced an increase in the B(max) of alpha-l adrenergic receptor binding sites labeled by [(3)H]prazosin. A similar increase was also observed for the alpha-1 adrenergic receptor component of [(3)H]WB4101 binding in the hippocampus but not in the cortex. No change in the lower affinity [(3)H]WB4101 binding site, which identifies S-l serotonin receptors was detected after this treatment. These data support the hypothesis that alpha-1 receptors are regulated by their neurotransmitter and may explain why previous studies have not detected alpha-1 receptor increases following 6-hydroxydopamine lesions of the dorsal bundle and locus coeruleus.

Regulation of putative muscarinic cholinergic receptor subtypes in rat brain.

Transection of the fimbria/fornix, producing a 75% reduction in the activity of the cholinergic marker choline-o-acetyltransferase (CAT EC. 2.3.1.6) in rat hippocampus, did not change the binding characteristics of the non-subtype selective, muscarinic cholinergic receptor antagonist ligand [(3)H](?)quinuclidinyl benzilate {[(3)H](?)QNB}. Pirenzepine competition for [(3)H](?)QNB binding in the hippocampus was best described by a computer derived model assuming two binding sites of high affinity (putative M(1) receptors) and low affinity (putative M(2) receptors). There was no change in the proportion of high and low affinity pirenzepine binding sites in the hippocampus following cholinergic deafferentation. Thus, these data provide no evidence for a discrete localization of either putative subtype of muscarinic receptor discriminated by pirenzepine restricted to the terminals of CAT containing neurons innervating the rat hippocampus. Chronic scopolamine treatment produced a 48% increase in the B(max) of [(3)H](?)QNB binding in the hippocampus, but again there was no change in the proportions of the sites discriminated by pirenzepine demonstrating that both putative subtypes were regulated identically. Similarly, carbachol competition for [(3)H](?)QNB was unaltered following cholinergic deafferentation or chronic scopolamine treatment. Furthermore, similar guanylyl-5?-imidodiphosphate [Gpp(NH)p] modulation of the proportions of high and low affinity carbachol binding sites was found in the hippocampus following transection of the fimbria/fornix or chronic scopolamine treatment. Thus there is no adaptation of receptor-effector coupling following these treatments that is reflected by changes in receptor recognition site characteristics. Carbachol competition for [(3)H]pirenzepine binding to putative M(1) receptors in the hippocampus was biphasic and was also modulated by Gpp(NH)p. In the brainstem, there was a homogeneous population of putative M(2) [(3)H](?)QNB binding sites having low affinity for pirenzepine. Carbachol competition for these binding sites was also biphasic and modulated by guanine nucleotides. Thus, both putative M(1) and M(2) muscarinic receptors, as defined by high or low affinity for pirenzepine respectively, may mediate their effects in rat brain via a guanine nucleotide regulatory subunit.

The pharmacological and anatomical substrates of the amphetamine response in the rat.

Bilateral 6-hydroxydopamine microinjections into the substantia nigra abolished both the locomotor and stereotyped responses to d-amphetamine in adult rats. The lesions resulted in a depletion of over 99 per cent of striatal tyrosine hydroxylase activity (indicating a near total lesion of the nigro-striatal dopamine pathway) as well as severe noradrenaline depletions. However, lesion of the dorsal or ventral noradrenergic pathways resulted in similar noradrenaline depletions but with no effect on striatal tyrosine hydroxylase levels and without the concomitant blockage of the amphetamine response. The substantia nigra lesioned rats were behaviourally supersensitive to apomorphine and L-DOPA and did not show a locomotor response to cocaine. The substantia nigra lesioned rats were not aphagic or adipsic. It was concluded that both the locomotor and stereotyped responses induced by amphetamine are dependent on the functional integrity of the nigro-striatal dopamine pathway.

Alteration of nicotinic cholinergic agonist binding sites in hippocampus after fimbria transection.

Nicotinic cholinergic agonist binding sites were studied in rat hippocampus by the binding of [3H]acetylcholine in the presence of 1.5 microM atropine sulfate. Following transection of the fimbria/fornix there was a 49% increase in the binding of [3H]acetylcholine reflecting an increase in the affinity of the receptor binding site from Kd = 18.82 +/- 3.6 nM in control animals to Kd = 9.06 +/- 1.2 nM in experimental tissue. Chronic administration of the agonist nicotine (4 mg/kg/day) by osmotic minipumps produced an increase in the binding of 10 nM [3H]acetylcholine after 14 days (49% increase over control) and after 28 days (141% increase over controls). These data are consistent with the suggestion that [3H]acetylcholine labels a nicotinic cholinergic receptor in rat brain. Further they support the notion that some of the termination sites of the septal-cholinergic projection to the hippocampus are nicotinic.