Comparison of Neuroleptic Binding Characteristics in Rat Striatum and Renal Cortex

Abstract

The binding characteristics of the dopaminergic ligand, ³H- spiperone, were compared in renal cortical and striatal membrane homogenates of the rat. This ligand labelled a single class of high affinity binding sites in striatum with an apparent dissociation constant (Kd) of 0.13 nM and a maximal number of binding sites (Bmax) of 890 fmol/mg protein representing D-2 receptors. In the renal cortex, ³H-spiperone identified a population of binding sites with a Bmax and a Kd of 310 fmol/mg protein and 5.1 nM, respectively. The antagonist displacing profile suggests the dopaminergic nature of the renal binding site. The affinities of dopamine antagonists for the peripheral ³H-spiperone binding site were in general in the micromolar range while the affinities of D-2 or D-2/D-1 dopamine antagonists in striatum were in the nanomolar range. Moreover, these sites showed differential stereoselectivity for (+)- and (-)-isomers of sulpiride. In conclusion, the presence of a D-2/DA-2 dopamine receptor population in renal cortex could not be confirmed. The pharmacological properties of the peripheral ³H-spiperone binding site are also different from the DA-1 receptor but seem to resemble those previously reported for dopamine receptors in sympathetic ganglia and adrenal medulla.     

Brain neurotransmitter receptors after long-term haloperidol: dopamine, acetylcholine, serotonin, alpha-noradrenergic and naloxone receptors.

Since long-term neuroleptic therapy is known to alter brain dopaminergic sensitivity, we tested the effects of chronic haloperidol administration (10 mg/kg/day for over 3 weeks) on the amount of the dopamine receptors (using ³H-apomorphine and ³H-haloperidol) in various regions of the rat brain. To test whether the changes in dopamine receptors were selectively produced, we also assayed acetylcholine receptors (with ³H-quinuclidinyl benzilate or ³H-QNB), alpha-noradrenergic receptors (with ³H-WB-4101), ³H-serotonin receptors and ³H-naloxone receptors.The specific binding of ³H-haloperidol increased significantly by 34% in the striatum and by 45% in the mesolimbic region after long-term haloperidol. The specific binding of ³H-apomorphine also increased significantly by 77% in the striatum and 55% in the mesolimbic area. Although there was a small significant increase of 20% in specific ³H-serotonin binding in the striatum, no such increment occurred in the hippocampus or the cerebral cortex. No significantly different binding occurred for the other ³H-ligands in these brain regions except for a 13% increase in alpha-noradrenergic binding in the cerebral cortex. These results indicate that long-term haloperidol treatment produces rather selective increases in dopamine/neuroleptic receptors, without much change in 4 other types of receptors. Such relatively selective increments in these receptors may be the basis of dopaminergic supersensitivity (e.g. tardive dyskinesia) after long-term haloperidol.     

Effect of age on adrenergic and dopaminergic receptor binding in rat brain

The effects of age on receptor binding of adrenergic and dopaminergic ligands were studied in rat cerebral cortex and striatum respectively. Compared to rats 5 months of age, 25-month old rats had a significant decrease in specific binding of the β-adrenergic antagonist ligand ³H-DHA, the α-adrenergic ligand ³H-WB-4101 in cortex, and the dopaminergic antagonist ³H-spiperone in striatum. Scatchard analysis of ligand binding indicated that the decrease in specific binding was due to a decrease in the number of receptors and not to a change in the affinity of the ligand for the receptor.     

The effect of chronic neuroleptic administration on cerebral dopamine receptor function

Acute administration of neuroleptic drugs causes blockade of cerebral dopamine receptors. It has been discovered that chronic administration of neuroleptic drugs may have different effects on cerebral dopamine systems. Initial antagonism of dopamine mediated behaviour, such as stereotypy, disappears and may be replaced by supersensitivity to dopamine agonists. Changes also occur in biochemical indices of dopamine receptors, such as in the number and affinity of specific binding sites identified by ³H-ligands labelling D-2 receptors, and in dopamine-stimulated adenylate cyclase activity. All these changes occur obviously in the striatum in response to chronic administration of a range of neuroleptic drugs. Lesser changes take place in the mesolimbic dopamine system. What happens in the mesocortical dopamine pathways is unknown. The consequence of such adaptive responses to chronic neuroleptic therapy may be of importance to understanding of tardive dyskinesia and schizophrenia.     

Characterization of specific in vivo binding of neuroleptic drugs in rat brain

$\text{In vivo}$ binding of ³H-spiperone is saturable in the striatum, the limbic system and the frontal cortex but not in the cerebellum. A specific binding is different in all the brain regions thus the amount of labelling in the cerebellum may not be considered as a blank value.³H-spiperone binding revealed a specific subcellular distribution only when a very low dose was injected into rats.$\text{Ex vivo}$ experiments allow the assessment of biochemical profiles of neuroleptic drugs according to their relative affinity for dopamine or serotonin receptors.     

Alterations in central neurotransmitter receptor binding sites following estradiol implantation in female rats

The subcutaneous implantation of an estradiol pellet (10 mg) into female rats induced a hypophyseal hyperplasia with hyperprolactinaemia. Examination of neurotransmitter receptors in the hippocampus, striatum and cerebral cortex one month after the implantation revealed that estrogenization was associated with: an increased density of ³H-domperidone binding sites (D₂ receptors) in the striatum and reduced numbers of ³H-serotonin high affinity sites (5-HT₁ receptors) in the hippocampus and of ³H-muscimol binding sites (GABA receptors) in the hippocampus, striatum and cerebral cortex. In contrast, the characteristics of ³H-spiperone binding to 5-HT₂ receptors (in the cerebral cortex) and those of ³H-flunitrazepam binding to benzodiazepine sites (in the three brain regions examined) were not significantly different in estrogenized and in control female rats. However, the enhancing effect of GABA on ³H-flunitrazepam binding was markedly reduced in brain membranes from estrogenized animals. The respective roles of estradiol and prolactin in mediating these changes in neurotransmitter receptors are discussed notably with regard to the regional heterogeneity of estradiol binding capacity in the rat brain.     

The effects of standard neuroleptic compounds on the binding of 3H-spiroperidol in the striatum and mesolimbic system of the rat in vitro.

Neuroleptics are reported to produce their antipsychotic activity and extrapyramidal side effects by blocking dopamine receptors in the mesolimbic system and striatum respectively. We have thus looked at the characteristics of the binding of ³H-spiroperidol to specific binding sites in these two areas of rat brain and the ability of a number of neuroleptics to displace it from these sites.The ³H-spiroperidol binding sites in the striatum and mesolimbic area are different and evidence has been obtained for an involvement of 5-HT receptors, particularly in the latter area.In the striatum the order of activity of neuroleptics in displacing ³H-spiroperidol binding parallels their clinical potency. This is not the case in the mesolimbic system. Also the ratio of activity of a neuroleptic in the two brain areas does not correlate with its ability to produce extrapyramidal disturbance in man. This may be due to the interaction of neuroleptics, particularly in the mesolimbic system, with receptors not involved in the expression of antipsychotic activity.     

Different kinetic properties of neuroleptic receptor binding in the rat striatum and frontal cortex.

³H-spiperone binding in the striatum is 80% stereospecific and 6 % non-stereospecific. In the frontal cortex stereospecific sites account for 60 % of the total binding and non-stereospecific sites for 25 %. Stereospecific sites are of different nature in both brain regions : dopaminergic in the striatum and serotonergic in the frontal cortex. The non-stereospecific sites are saturable and can only be detected by the use of appropriate blanks.Release experiments demonstrate the occurrence of positive coöperative effects in the dissociation of spiperone from striatal receptors while this is not detectable in the frontal cortex. In both brain regions a rapidly and a slowly dissociating component have been observed. In the frontal cortex the rapid component is ascribed to the dissociation of spiperone from the stereospecific sites and the slow component to the dissociation from non-stereospecific sites. The nature of these sites is yet to be identified.     

Effect of long-term L-dopa administration on the dopaminergic and cholinergic (muscarinic) receptors of striatum in 6-hydroxydopamine lesioned rats

L-Dihydroxyphenylalanine (L-Dopa) (200 mg/kg/day) was administered for 30 days to the rats whose nigrostriatal dopamine pathway was lesioned unilaterally with 6-hydroxydopamine and the receptor binding of ³H-spiperone and ³H-quinuclidinyl benzilate (³HQNB) was measured in the dopaminergic and muscarinic cholinergic receptors of the striatum. ³H-spiperone binding increased by 73% and ³HQNB binding decreased by 14% in the lesioned side when compared to the control side of L-Dopa-non-treated rats. ³H-spiperone binding was measured in the lesioned sides of L-Dopa-treated and L-Dopa-non-treated rats and was found to have decreased by 21% in the former. In the control side of the L-Dopa-treated lesioned rats, however, ³H-spiperone binding increased by 27% when compared to the opposite striatum of the same rats. ³HQNB binding in the lesioned side of L-Dopa-treated rats was not significantly different from that of the control side statistically. These results suggest that changes in functional equilibrium between the dopaminergic and cholinergic mechanisms influence the muscarinic cholinergic receptors and that supersensitivity of dopamine receptors after lesion of the nigrostriatal pathway also remains after long-term L-Dopa treatment.     

The effect of chronic L-DOPA administration on supersensitive pre- and postsynaptic dopaminergic receptors in rat brain

Chronic administration of haloperidol induced supersensitivity of the pre- and postsynaptic dopaminergic receptors in rat brain. The response of the presynaptic receptors was determined by an enhanced inhibitory effect of apomorphine on dopamine synthesis after gamma-butyrolactone injection. This change in the receptor function was detected both in the nigrostriatal and mesolimbic pathways. Haloperidol also increased the ³H-spiperone binding sites in striatal membranes, indicating supersensitivity of the postsynaptic receptors. Subsequent prolonged treatment with high doses of L-DOPA/carbidopa resulted in a decrease in ³H-spiperone binding sites, but had no effect on the supersensitive presynaptic receptors. It is suggested that tardive dyskinesia may be a state of both pre- and postsynaptic dopamine receptor supersensitivity and that chronic L-DOPA treatment may have a differential effect on these sites.     

In vivo studies of dopamine receptor ontogeny

Studies of the ontogeny of dopamine and neuroleptic receptors in the central nervous system of the rat were carried out $\text{in vivo}$ using ³H-spiperone as ligand. It was demonstrated that intraperitoneal injections can be successfully used to label these receptors in rat pups up to at least 30 days of age. The time course and characteristics of ³H-spiperone binding in the brain of 5, 15 and 30 day old rat pups were determined and found to include appropriate regional distribution, saturability and appropriate pharmacology. The developmental pattern of ³H-spiperone binding paralleled what has been seen using $\text{in vitro}$ techniques. In addition preliminary autoradiographic studies describe the neuroanatomical pattern of dopamine receptor ontogeny in the striatum.     

Neurotensin interacts with dopaminergic neurons in rat brain

Neurotensin (NT) injected intracerebroventricularly in rat increases dopamine (DA) turnover in the corpus striatum and nucleus accumbens. Significant increases in 3,4-dihydroxyphenylacetic acid (DOPAC) levels occurred within 15 minutes after injection with peak levels at 60 minutes. The effect on NT on DOPAC and homovanillic acid (HVA) accumulation was dose-dependent at 3–100 μg. NT, like haloperidol, stimulated 3,4-dihydroxyphenylalanine (DOPA) accumulation in striatal neurons, in the presence of DOPA decarboxylase inhibitor, after injection of gamma-butyrolactone (GBL). NT had a similar stimulatory effect on DOPA levels in the accumbens while haloperidol (0.25 mg·kg^?1) had no significant effect in this brain region. NT did not block the inhibitory effect of apomorphine on DOPA accumulation in both the striatum and accumbens, while haloperidol inhibited apomorphine effect in both regions. NT also failed to displace ³H-spiperone from DA receptors and the presence of NT in the binding assay did not alter the ability of DA to displace ³H-spiperone in either brain region. These experiments demonstrate that NT increases DA turnover in both the nigrostriatal and mesolimbic pathways.     

Direct binding of 3H-lisuride to adrenergic and serotonergic receptors

The characteristics of the specific binding of ³H-lisuride hydrogen maleate (³H-LHM) to homogenates of rat striatum and bovine frontal cortex tissue were investigated. In rat striatum 50% of ³H-LHM binding was inhibited potently by spiperone and haloperidol indicating a component of ³H-LHM binding to D₂ dopamine receptors. Specific ³H-LHM binding was detected in rat striatum after selective blockade of the D₂ dopamine component indicating specific ³H-LHM binding to other striatal sites. In bovine frontal cortex clonidine and serotonin competition curves for specific ³H-LHM binding included high affinity phases indicating alpha₂ adrenergic and high affinity serotonergic components of binding. Blockade of the adrenergic component by 10^?7M clonidine resulted in the specific ³H-LHM binding exhibiting distinctly serotonergic characteristics. Conversely, blockade of the serotonergic component by 2 × 10^?7M serotonin resulted in the specific ³H-LHM binding exhibiting distinct alpha₂ receptor characteristics. These data demonstrate the specific binding of ³H-LHM to alpha₂ adrenergic receptors, to a high affinity serotonin site, and to D₂ dopamine receptors.     

Enhancement of [m-methoxy3H]MDL100907 binding to 5HT2A receptors in cerebral cortex and brain stem of streptozotocin induced diabetic rats

5-Hydroxytryptamine_2A (5-HT_2A) receptor kinetics was studied in cerebral cortex and brain stem of streptozotocin (STZ) induced diabetic rats. Scatchard analysis with [³H] (±) 2,3dimethoxyphenyl-1-[2-(4-piperidine)-methanol] ([³H]MDL100907) in cerebral cortex showed no significant change in maximal binding (B_max) in diabetic rats compared to controls. Dissociation constant (K_d) of diabetic rats showed a significant decrease (p < 0.05) in cerebral cortex, which was reversed to normal by insulin treatment. Competition studies of [³H]MDL100907 binding in cerebral cortex with ketanserin showed the appearance of an additional low affinity site for 5-HT_2A receptors in diabetic state, which was reversed to control pattern by insulin treatment. In brain stem, scatchard analysis showed a significant increase (p < 0.05) in B_max accompanied by a significant increase (p < 0.05) in K_d. Competition analysis in brain stem also showed a shift in affinity towards a low affinity State for 5-HT_2A receptors. All these parameters were reversed to control level by insulin treatment. These results show that in cerebral cortex there is an increase in affinity of 5-HT_2A receptors without any change in its number and in the case of brain stem there is an increase in number of 5HT_2A receptors accompanied by a decrease in its affinity during diabetes. Thus, from the results we suggest that the increase in affinity of 5-HT_2A receptors in cerebral cortex and upregulation of 5-HT_2A receptors in brain stem may lead to altered neuronal function in diabetes.     

A behavioural and biochemical comparison of dopamine receptor blockade produced by haloperidol with that produced by substituted benzamide drugs

Haloperidol inhibited dopamine (DA) mediated behaviours and induced pronounced catalepsy in rodents. Metoclopramide, sulpiride, sultopride, tiapride and clebopride, in general, also inhibited these behaviours but only clebopride induced marked catalepsy. Haloperidol displaced ³H-haloperidol and ³H-spiperone from striatal binding sites and inhibited DA stimulated cyclase from striatal and mesolimbic regions. In general, substituted benzamide drugs displaced labelled ligands, but did not inhibit adenylate cyclase. Elevations of striatal HVA produced by haloperidol and sulpiride, but not other benzamide drugs, were partially reversed by atropine. Hypophysectomy did not prevent the elevation of forebrain HVA produced by sulpiride and metoclopramide. Substituted benzamide drugs appear to act on cerebral DA receptors that are independent of DA-sensitive adenylate cyclase and are not balance by a cholinergic input.     

Asynchrony in the Diurnal Rhythms of Dopaminergic D2 Receptors in Different Brain Regions of the Rat

Diurnal variations of dopaminergic D₂ receptors have been described in the striatum of rats, while other dopaminergic regions remain unstudied. Diurnal variations of dopamine D₂ receptors in the striatum, frontal cortex, and amygdala of the rat, were characterized by the stereospecific binding of [³H]-spiperone. Clear rhythms were found in all these areas, but asynchronous to each other. Striatal receptors had diurnal variations with a single peak at 00:00 hours. Frontal cortex receptors showed two peaks at 00:00 and 12:00 hours. Amygdaline complex receptors had two peaks at 18:00 and 06:00 hours. Saturation binding curves and their Scatchard analysis indicated that the diurnal variations in [³H]-spiperone binding are related to changes in receptor density rather than its affinity. The diurnal variations asynchrony in [³H]-spiperone binding to dopaminergic D₂ receptors from different neural regions, suggest different regulation in each area. Other functional implications of these rhythms remains to be established.     

Age-related changes in dopamine receptor densities in the brain of the honey bee, Apis mellifera

Changes in the levels of binding of ³H-SCH-23390, a vertebrate D1 dopamine receptor ligand, and ³H-spiperone, a vertebrate D2 dopamine receptor ligand were investigated in the brain of the worker honey bee during metamorphic adult development and during the lifetime of the adult bee. Age-related fluctuations in binding levels were markedly different for these two ligands. ³H-SCH-23390 and ³H-spiperone binding sites were present at low levels during metamorphic adult development. After adult emergence, however, ³H-SCH-23390 binding levels, in contrast to those of ³H-spiperone, increased significantly. Within the first 48 h of adult life ³H-SCH-23390 binding reached a level not significantly different from that detected in forager bees. No significant fluctuations in the levels of ³H-spiperone binding were observed during the adult lifetime of the bee. Measurements of dopamine levels in the brains of pupal and adult bees revealed no direct correlation between fluctuations in endogenous amine levels and the amount of binding of either ³H-SCH-23390 or ³H-spiperone. These results provide evidence for subtype-specific patterns of expression of dopamine receptors in the insect brain and show that D1- and D2-like receptors are expressed not only in the adult CNS, but also in the developing brain of the bee. Accepted: 4 June 1997     

Dopamine receptor binding: Specificity,localization and regulation by ions and guanyl nucleotides

³H-Spiroperidol labels dopamine receptors in rats striatum but in frontal cortex and hippocampus ³H-spiroperidol labels serotonin receptors. The agonists ³H-ADTN and ³H-apomorphine label rat striatal dopamine receptors. Comparison with calf striatal binding indicates a species difference in ³H-apomorphine binding. Drug displacement and lesion studies suggest that in the rat ³H-apomorphine labels two distinct dopamine receptors, one associated with the dopamine-sensitive adenylate cyclase and the other with presynaptic dopamine receptors also labeled by ³H-spiroperidol. Whereas divalent cations increase specific dopamine receptor binding of ³H-agonists and ³H-antagonists, ³H-agonist binding is selectively decreased by some guanyl nucleotides.     

Melatonin reverses pinealectomy-induced decrease of benzodiazepine binding in rat cerebral cortex

Pinealectomy of rats resulted in significant depression of benzodiazepine receptors (assessed by [³H]flunitrazepam binding) in cerebral cortex 3–14 days after surgery without affecting their affinity significantly. A single s.c. injection of melatonin (800 μg/kg body wt) restored the depressed brain benzodiazepine receptor sites. Single melatonin injections (up to 1600 μg/kg) to intact rats did not affect brain benzodiazepine binding when injected at either morning or evening hours. Daily melatonin treatment to intact rats for 5 days augmented benzodiazepine receptor density in brain (morning injections) or its dissociation constant (evening injections). Melatonin added in vitro to rat cerebral cortex membranes only slightly depressed [³H]flunitrazepam binding at 100 μM concentrations. These results point out a link between pineal activity and benzodiazepine receptor function in rats. They also indicate that pharmacological doses of melatonin affect benzodiazepine binding sites in rat cerebral cortex.     

Age and Brain Structural Related Effects of Glutaric and 3-Hydroxyglutaric Acids on Glutamate Binding to Plasma Membranes During Rat Brain Development

(1) In the present study we determined the effects of glutaric (GA, 0.01–1 mM) and 3-hydroxyglutaric (3-OHGA, 1.0–100 μM) acids, the major metabolites accumulating in glutaric acidemia type I (GA I), on Na⁺-independent and Na⁺-dependent [³H]glutamate binding to synaptic plasma membranes from cerebral cortex and striatum of rats aged 7, 15 and 60 days. (2) GA selectively inhibited Na⁺-independent [³H]glutamate binding (binding to receptors) in cerebral cortex and striatum of rats aged 7 and 15 days, but not aged 60 days. In contrast, GA did not alter Na⁺-dependent glutamate binding (binding to transporters) to synaptic membranes from brain structures of rats at all studied ages. Furthermore, experiments using the glutamatergic antagonist CNQX indicated that GA probably binds to non-NMDA receptors. In addition, GA markedly inhibited [³H]kainate binding to synaptic plasma membranes in cerebral cortex of 15-day-old rats, indicating that this effect was probably directed towards kainate receptors. On the other hand, experiments performed with 3-OHGA revealed that this organic acid did not change Na⁺-independent [³H]glutamate binding to synaptic membranes from cerebral cortex and striatum of rats from all ages, but inhibited Na⁺-dependent [³H]glutamate binding to membranes in striatum of 7-day-old rats, but not in striatum of 15- and 60-day-old rats and in cerebral cortex of rats from all studied ages. We also provided some evidence that 3-OHGA competes with the glutamate transporter inhibitor L-trans-pyrrolidine-2,4-dicarboxylate, suggesting a possible interaction of 3-OHGA with glutamate transporters on synaptic membranes. (3) These results indicate that glutamate binding to receptors and transporters can be inhibited by GA and 3-OHGA in cerebral cortex and striatum in a developmentally regulated manner. It is postulated that a disturbance of glutamatergic neurotransmission caused by the major metabolites accumulating in GA I at early development may possibly explain, at least in part, the window of vulnerability of striatum and cerebral cortex to injury in patients affected by this disorder.