Is clozapine selective for the dopamine D4 receptor?

Binding of ³H-spiperone and ³H-raclopride to membranes of cells stably-transfected with a human dopamine D₂ receptor clone was investigated, as was that of ³H-spiperone to those stably-transfected with a human D₄ receptor clone. ³H-spiperone and ³H-raclopride labeled the same number of sites in the D₂ receptor preparation. The inhibition of binding by clozapine, spiperone, (−) eticlopride, haloperidol and the novel substituted benzamide 1192U90 was also investigated. Clozapine and 1192U90 showed greater inhibition of ³H-raclopride binding than ³H-spiperone binding to the D₂ receptor. Comparison with inhibition of ³H-spiperone binding to the D₄ receptor revealed that clozapine and 1192U90 displayed apparent selectivity (as assessed by K_i ratios) for the D₄ receptor when compared with binding of ³H-spiperone, but not ³H-raclopride, to the D₂ receptor.     

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.     

Regional distribution of monoamines in the nucleus accumbens of the rat

Monoamine concentrations were low in the rostral area of the nucleus accumbens. Their distributions were not identical. Differences were observed in the medial area. DA concentrations were high in both medial and caudal areas. Noradrenaline (NA) and serotonin (5-HT) concentrations were considerably lower than the dopamine (DA) concentration. The NA concentration was highest in the caudal area of the nucleus accumbens and the (5-HT) concentration was highest in the ventrocaudal area. There was a rostrocaudal decrease in the 3,4-dihydroxyphenylacetic acid (DOPAC)/DA and 5-hydroxyindole-3-acetic acid (5-HIAA)/5-HT ratios. Uptake of [³H]DA and [¹⁴C]choline was lowest in the rostral area. The K⁺-stimulated release of [¹⁴C]acetylcholine (ACh) was also lowest rostrally, but there was no rostrocaudal difference in the K⁺-stimulated release of [³H]DA. These results provide further evidence of the heterogeneity of the nucleus accumbens.     

Brain tissue transplanted to the anterior chamber of the eye

Summary Small pieces of fetal rat brain selected to contain a high number of noradrenaline (NA), dopamine (DA), or 5-hydroxytryptamine (5-HT) neuroblasts were transplanted to the anterior chamber of the eye of adult rats. The sympathetic ground plexus of the host iris was removed by superior cervical ganglionectomy so that transmitter mechanisms of the different central monoamine fibers innervating the iris could be selectively studied after intraocular maturation. Such irides, containing NA, DA, or 5-HT nerve terminals were incubated with radiolabelled transmitters and then stimulated by an electrical field while superfused, to investigate the spontaneous and stimulation-induced release of amine, both in drug-free buffer and buffer containing drugs acting on monoamine receptors.The central monoamine neurons of all three types were able to take up exogenous amines and release them upon stimulation by an electrical field, in much the same way as corresponding nerves in situ in slices of cerebral cortex (NA, 5-HT) or olfactory tubercle (DA).The -adrenergic receptor blocking agent phentolamine increased the stimulation-induced release of ³H-NA from central NA fibers on the iris significantly. The dopamine receptor stimulating agent apomorphine decreased the stimulation-induced release of ³H-DA from central DA fibers on the iris. Pimozide, a DA receptor blocking drug tended to increase the ³H-DA release. The 5-HT receptor stimulating agent ergocornine tended to reduce the stimulation-induced release of ³H-5-HT from central 5-HT fibers on the iris. It was concluded that all three types of central monoamine nerve fibers develop essentially normal transmitter storage and release mechanisms also in an environment completely devoid of normal postsynaptic receptors. The drug experiments add strong support to the view that there are presynaptic monoamine receptors (autoreceptors) able to modulate transmitter release present on the monoamine nerve terminals.Supported by the Swedish Medical Research Council (04X-3185 and 04X-2330) and by grants from Magnus Bergvalls Stiftelse and Karolinska Institutets Fonder, we thank Miss Ingrid Strömberg and Miss Ulla Enberg for skilful technical assistance.     

Stimulation by neurotensin of dopamine and 5-hydroxytryptamine (5-HT) release from rat prefrontal cortex: Possible role of NTR1 receptors in neuropsychiatric disorders

The modulation of cortical dopaminergic and serotonergic neurotransmissions by neurotensin (NT) was studied by measuring the release of dopamine (DA) and 5-hydroxytryptamine (5-HT) from the prefrontal cortex (PFC) of freely moving rats. The samples were collected via transversal microdialysis. Dopamine and 5-HT levels in the dialysate were measured using high-performance liquid chromatography (HPLC) with an electrochemical detector. Local administration of neurotensin (1 μM or 0.1 μM) in the PFC via the dialysis probe produced significant, long-lasting, and concentration-dependent increase in the extracellular release of DA and 5-HT. The increase produced by 1 μM neurotensin reached a maximum of about 210% for DA and 340% for 5-HT. A high-affinity selective neurotensin receptor (NTR1) antagonist {2-[(1-(7-chloro-4-quinolinyl)-5-(2,6-dimethoxyphenyl)pyrazol-3yl)carbonylamino tricyclo (3.3.1.1.^3.7) decan-2-carboxylic acid} (SR 48692), perfused locally at a concentration of 0.1 μM and 0.5 μM in the PFC antagonized the effects of 1 μM neurotensin. Our in vivo neurochemical results indicate, for the first time, that neurotensin is able to regulate cortical dopaminergic and serotonergic neuronal activity in freely moving rats. These effects are possibly mediated by interactions of neurotensin with neurons releasing DA or 5-HT, projecting to the PFC from the ventrotegmental area (VTA) and from the dorsal raphe nuclei (DRN), respectively. The potentiating effects of neurotensin on DA and 5-HT release in the PFC are regulated by NTR1 receptors, probably located on dopaminergic and serotonergic nerve terminals or axons.     

Serotonin,dopamine, noradrenaline and their metabolites: Levels in the brain of the house cricket (Acheta domesticus L.) during a 24-hour period and after administration of quipazine—a 5-HT2 receptor agonist

1. The levels of 5-HT, DA, NA and DA metabolites (NADA, DOPAC) measured by HPLC (with electrochemical detection) in the brain of the house cricket did not change over a 24-hr period. The level of 5-HIAA, a 5-HT metabolite, was below the limit of detection.2. The 5-HT and DOPAC levels decreased and NADA increased after quipazine injection but DA and NA levels did not change after it.3. [³H]Ketanserin was used to identify serotonin receptors bound to sites in the house cricket brain with a K_d of 5 nM and a concentration of B_max 180 fmol/mg protein.     

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.     

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.     

Electrochemical measurement of release of dopamine and 5-hydroxytryptamine from synaptosomes.

Liquid chromatography with electrochemical detection has been used to determine endogenous dopamine (DA) and serotonin (5-HT) in a striatal crude synaptosomal fraction isolated by centrifugal methods. The electrochemical determinations are accomplished with a new type of electrode material, pressure annealed pyrolytic graphite. The analysis scheme permits direct quantitation of DA and 5-HT with minimal sample pretreatment. The distribution of endogenous 5-HT in each subfraction throughout the centrifugation procedure is found to be approximately parallel to that of endogenous DA. The release of DA and 5-HT from the crude synaptosome preparation exhibits the properties expected for stimulus-coupled, depolarization-induced release. The release of both neurotransmitters is Ca²⁺ dependent and is induced by high K⁺ or veratridine in the external medium. The latter is blocked by tetrodotoxin. Release is independent of ascorbic acid, but is dependent on the temperature of incubation.     

Release of endogenous dopamine, 3,4-dihydroxyphenylacetic acid,and amino acid transmitters from rat striatal slices

The release of endogenous dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) was measured in superfused striatal slices of the rat and the results compared with data obtained for the release of endogenous (a) DA and DOPAC in the cerebral cortex, nucleus accumbens and thalamus; (b) 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), GABA, and glutamate in the striatum; and (c) GABA, glutamate and 5-HT in the cerebral cortex. In superfused slices of all four CNS regions, there appeared to be a Ca²⁺-dependent, K⁺-stimulated release of endogenous DA. In addition, in slices of the striatum and nucleus accumbens there also appeared to be a Ca²⁺-dependent, 60 mM K⁺ stimulated release of endogenous DOPAC. In the striatum, 16 mM Mg²⁺ was as effective as 2.5 mM Ca²⁺ in promoting the 60 mM K⁺-stimulated release of DOPAC. In addition, 16 mM Mg²⁺ appeared to function as a weak Ca²⁺ agonist since it also promoted the release of DA to approximately 40% of the level attained with Ca²⁺ in the presence of 60 mM K⁺. On the other hand, in the striatum, 16 mM Mg²⁺ inhibited the Ca²⁺-dependent, 60 mM K⁺-stimulated release of GABA and glutamate. Similar Mg²⁺-inhibition was observed in the cerebral cortex not only for GABA and glutamate but also for DA and 5-HT. With the use of -methyl -tyrosine (tyrosine hydroxylase inhibitor), cocaine (uptake inhibitor) and pargyline (monoamine oxidase inhibitor), it was determined that (a) most of the released DA and DOPAC was synthesized in the slices during the superfusion; (b) DOPAC was not formed from DA which had been released and taken up; and (c) DA and DOPAC were released from DA nerve terminals. In addition, the data indicate a difference in the release process between the amino acids and the monoamines from striatal slices since Mg²⁺ inhibited the Ca²⁺-dependent, K⁺-stimulated release of GABA and glutamate and appeared to promote the release of DA and 5-HT.     

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     

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.     

Regulation of cyclic AMP in heart and gill of Aplysia by the putative neurotransmitters dopamine and serotonin.

Serotonin (5-HT) and dopamine (DA), but not several other putative neurotransmitters, stimulate cyclic adenosine-3',5'-monophosphate (cAMP) production in slices of $\text{Aplysia}$ gill. Furthermore, 5-HT but not DA increases cAMP in slices of the heart of $\text{Aplysia}$. Several lines of evidence indicate that the receptors are distinct entities; however, no drugs were found to block one receptor without affecting the other.     

Alpha-ethyltryptamines as dual dopamine–serotonin releasers

The dopamine (DA), serotonin (5-HT), and norepinephrine (NE) transporter releasing activity and serotonin-2A (5-HT_2A) receptor agonist activity of a series of substituted tryptamines are reported. Three compounds, 7b, (+)-7d and 7f, were found to be potent dual DA/5-HT releasers and were >10-fold less potent as NE releasers. Additionally, these compounds had different activity profiles at the 5-HT_2A receptor. The unique combination of dual DA/5-HT releasing activity and 5-HT_2A receptor activity suggests that these compounds could represent a new class of neurotransmitter releasers with therapeutic potential.     

α2-Adrenergic Receptor Blockade Markedly Potentiates Duloxetine- and Fluoxetine-Induced Increases in Noradrenaline, Dopamine, and Serotonin Levels in the Frontal Cortex of Freely Moving Rats

Abstract: Evidence exists that a reinforcement in monoaminergic transmission in the frontal cortex (FCX) is associated with antidepressant (AD) properties. Herein, we examined whether blockade of α₂-adrenergic receptors modified the influence of monoamine reuptake inhibitors on FCX levels of serotonin (5-HT), noradrenaline (NAD), and dopamine (DA). The selective α₂-adrenergic receptor agonist S 18616 (0.16 mg/kg, s.c.) suppressed extracellular levels of NAD, DA, and 5-HT (by 100, 51, and 63%, respectively) in single dialysates of FCX of freely moving rats. In contrast, the selective α₂-adrenergic receptor antagonists atipamezole (0.16 mg/kg, s.c.) and 1-(2-pyrimidinyl)piperazine (1-PP; 2.5 mg/kg, s.c.) increased levels of NAD (by 180 and 185%, respectively) and DA (by 130 and 90%, respectively), without affecting 5-HT levels. Duloxetine (5.0 mg/kg, s.c.), a mixed inhibitor of 5-HT and NAD reuptake, and fluoxetine (10.0 mg/kg, s.c.), a selective 5-HT reuptake inhibitor, both increased levels of 5-HT (by 150 and 120%, respectively), NAD (by 400 and 100%, respectively), and DA (by 115 and 55%, respectively). Atipamezole (0.16 mg/kg, s.c.) markedly potentiated the influence of duloxetine and fluoxetine on levels of 5-HT (by 250 and 330%, respectively), NAD (by 1,030 and 215%, respectively), and DA (by 370 and 170%, respectively). 1-PP similarly potentiated the influence of duloxetine on 5-HT, NAD, and DA levels (by 290, 1,320, and 600%, respectively). These data demonstrate that α₂-adrenergic receptors tonically inhibit NAD and DA and phasically inhibit 5-HT release in the FCX and that blockade of α₂-adrenergic receptors strikingly potentiates the increase in FCX levels of 5-HT, NAD, and DA elicited by reuptake inhibitors. Concomitant α₂-adrenergic receptor antagonism and inhibition of monoamine uptake may thus provide a mechanism allowing for a marked increase in the efficacy of AD agents.     

Butanol extracts from myelin fragments. VI. Lack of specificity of 5-hydroxytryptamine binding to recombinate fraction with sulphatides and phosphatidylinositol

To clarify the participation of sulphatides and PI for the binding of 5-HT to myelin butanol extracts, binding experiments of ¹⁴C·5-HT to the recombinate fraction of these two acidic lipids were performed. The recombinate fraction was incubated with 5 × 10^?7 M of ¹⁴C·5-HT, and elution profile of ¹⁴C·5-HT, sulphatides and PI was examined by Sephadex LH₂₀ column chromatography. Three components were eluted with CM 4:1 and elution areas of those relatively corresponded to each other. On the other hand, when 3 fold excess volumes of solvents were used ¹⁴C·5-HT was also eluted with CM 4:1 but the clear difference was observed in the elution pattern of sulphatides and PI. In the myelin butanol extracts, we have reported that the 5-HT binding macromolecules present in the myelin extracts would be composed of saturable and non-saturable components, and ACh, DA and tryptamine specifically inhibited the saturable 5-HT binding. Therefore, on the recombinate system of two acidic lipids the specificity of ¹⁴C·5-HT binding was investigated by displacement experiments. The results indicated that only ACh slightly inhibited the ¹⁴C·5-HT binding but 5-HT, DA and tryptamine had no effect. All these observations suggest that other component(s) besides sulphatides and PI may be implicated in the binding of 5-HT to myelin butanol extracts.     

Chronic neuroleptic treatment specifically alters the number of dopamine receptors in rat brain

Trifluoperazine dihydrochloride (2.8–4.0 mg/kg/day) was administered continuously to rats in drinking water for six months. Animals killed at this time exhibited an increase in the number of dopamine receptors in the striatum and mesolimbic area, with a corresponding decrease in affinity (increase in the dissociation constant) for ³H-spiperone binding. In frontal cortex, ³H-spiperone binding to 5-HT receptors indicated no apparent change in numbers of receptors, but a slight increase in the dissociation constant. There was no obvious alteration in ³H-apomorphine binding in the striatum and mesolimbic area, but the individual results were very variable. The number and binding affinity of muscarinic receptors in striatum, mesolimbic area and cerebral cortex as identified by ³H-dexetemide were unchanged. Nor was there any alternation in the number or binding affinity of H-1 receptors identified by ³H-mepyramine, or of α-noradrenergic receptors identified by ³H-WB 4101, in cerebral cortex. The number and binding affinity of GABA receptors in the cerebellum identified by ³H-muscimol also was not altered.Chronic neuroleptic administration to rats appears to alter specifically the number of cerebral dopamine receptors.     

Differential effects of chronic clorgyline and amfonelic acid on desensitization of striatal dopamine receptors

Chronic treatment of rats with the MAOI clorgyline significantly reduced the density (Bmax) of cortical beta-adrenergic receptors but did not alter either the Bmax or dissociation constant (Kd) of 3H-spiperone binding to striatal DA receptors. Clorgyline co-treatment also did not significantly affect either behavioral supersensitivity to apomorphine or the increase in 3H-spiperone binding induced by chronic haloperidol. In contrast, repeated treatment with the DA uptake inhibitor amfonelic acid elicited behavioral subsensitivity and reduced striatal 3H-spiperone binding. Furthermore, amfonelic acid co-treatment prevented haloperidol-induced behavioral and receptor binding changes. The possible relevance of these findings in relation to drug choice in clinical trials of receptor sensitivity modification are discussed.     

Cation specificity of 3H-sulpiride binding involves alteration in the number of striatal binding sites

Specific ³H-sulpiride binding to rat striatal membranes shows an absolute requirement for the presence of sodium ions in the incubation buffer. Potassium, rubidium and caesium ions were unable to initiate specific ³H-sulpiride binding in a sodium free buffer, and lithium ionscould only partially replace sodium ions. Specific ³H-spiperone binding was unaffected by variation of the cation content of the incubation buffer. The alteration in ³H-sulpiride binding caused by sodium and lithium ions was due predominantly to an increase in the number of available binding sites, rather than to altered receptor affinity. Sodium ions may be essential for the accessability of ³H-sulpiride to a single site labelled also by ³H-spiperone. However, the Ki value for sulpiride displacement of ³H-spiperone in the presence of sodium ions was 20 times greater than the K_D value for ³H-sulpiride binding. So, ³H-sulpiride may interact with a highly sodium dependent binding site distinct from that labelled by ³H-spiperone.