Apomorphine induced biting and fighting behaviour in reserpinized rats and an approach to the mechanism of action

Various patterns of aggressive behaviour have been induced by drugs. In the present study, the biting and the fighting responses were induced in rats by apomorphine alone, and reserpine plus apomorphine combination respectively, and these could be blocked completely by a dopamine receptor blocking agent. Dopamine, norepinephrine and clonidine given intraperitoneally or intraventricularly failed to induce these responses. Chemical agents known to increase the concentration of dopamine in the brain, induced the biting, but not the fighting response, whereas these behavioural patterns were more intense due to apomorphine in the rats pretreated with reserpine and dopamine or α-methyltyrosine and reserpine combinations. In amphetamine pretreated rats, apomorphine induced intense biting after 10 min and a few bouts of fighting after 30 min. It is suggested that (i) the receptors on which apomorphine acts may be called ‘Apomorphine Receptor’ rather than ‘Dopamine Receptor’, (ii) dopamine incompletely activates these receptors which are sensitised in the absence of catecholamines and induce a higher degree of stereotyped behaviour i.e. fighting, due to apomorphine.     

Use of siRNA in knocking down of dopamine receptors, a possible therapeutic option in neuropsychiatric disorders

Heightened dopaminergic activity has been shown to be implicated in some major neuropsychiatric disorders such as schizophrenia. Use of dopaminergic antagonists was limited by some serious side effects related to unspecific blocking of dopamine receptors. Thus a target specific dopamine receptor gene silencing method such as using small interfering RNA (siRNA) might be useful. In this study recombinant plasmids expressing siRNA against dopamine receptors (D1-D5DRs) were produced, and their efficiency in knocking down of receptors in were assessed in rat neuroblastoma cell line (B65), using Real-time PCR method. Furthermore, D2DR siRNA expressing plasmid was injected into the rat nucleus accumbens bilaterally to investigate whether it can prevent the hyperactivity induced by apomorphine. Locomotion was measured in 10 min intervals, 50 min before and 60 min after apomorphine injection (0.5 mg/kg, S.C). Our results indicated that the mRNA level of dopamine receptors were reduced between 25 and 75% in B65 cells treated with the plasmids in vitro. In behavioral tests, locomotion was lower at least in the second 10 min after apomorphine injection in rats treated with plasmid expressing D2DR siRNA compare to control group [F (4,24) = 2.77, (P < 0.05)]. The spontaneous activity of treated rats was normal. In conclusion, dopamine receptors can be downregulated by use of siRNA expressing plasmids in nucleus accumbens. Although our work may have some possible clinical applications; the potentially therapeutic application of siRNA in knocking down of dopamine receptors needs further studies.     

Dopamine receptors in the central nervous system.

Dopamine receptors in the central nervous system can be studied by measuring the specific binding of [3H]dopamine, [3H]haloperidol, d-[3H]LSD, [3H]dihydroergocryptine or [3H]apomorphine. The receptors are stereoselectively blocked by +)-butaclamol, a neuroleptic. All neuroleptics inhibit the specific binding of [3H]haloperidol in relation to their clinical potencies. The radioligand that desorbs most slowly from the receptor is [3H]apomorphine, thus making it a reliable ligand for dopamine receptors. Dopamine agonists that compete for [3H]apomorphine binding do so at concentrations that correlate with their potency in stimulating striatal adenylate cyclase. Structure-activity analysis, using [3H]apomorphine, confirms that the active dopamine-mimetic conformation is the beta rotamer of dopamine. Prolonged exposure in vitro of caudate homogenate to high concentrations of dopamine leads to increased binding of [3H]apomorphine or [3H]haloperidol, suggesting receptor "sensitization." Chronic haloperidol treatment of rats leads to an increased number of dopamine/neuroleptic receptors in the striatum, but a decrease in the pituitary.     

Effect of cyclo(Leu-Gly) on the supersensitivity of dopamine receptors in spontaneously hypertensive rats

Spontaneously hypertensive rats exhibited dopamine receptor supersensitivity as evidenced by a greater hypothermic response to apomorphine in comparision with normotensive Wistar-Kyoto rats. A single injection of cyclo(Leu-Gly) given prior to apomorphine administration did no alter apomorphine induced hypothermia in either the normotensive or the hypertensive rats. Chronic administration of cyclo(Leu-Gly) for 7 days did not affect apomorphine response in normotensive rats, but blocked the exaggerated response to apomorphine in the hypertensive rats. These studies suggest that cyclo(Leu-Gly) interacts with the dopamine receptors and that the central dopamine receptors may play a role in the pathophysiology of hypertension.     

INHIBITION BY APOMORPHINE OF DOPAMINE DEAMINATION IN THE RAT BRAIN

Abstract— Apomorphine (A) inhibited dopamine deamination by rat brain mitochondria, but did not influence catechol- O -methyltransferase (COMT) activity by brain homogenates. The administration of apomorphine (10mg/kg i.p.) to normal rats increased brain dopamine (DA) by 34 per cent and decreased homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC) by 60 per cent. In rats treated with reserpine 15 min prior to A, the latter prevented the rise of cerebral HVA and DOPAC and the depletion of DA produced by the former. Finally, A decreased the L-DOPA-induced accumulation of HVA and DOPAC in the rat basal ganglia. These results indicate that A inhibits DA deamination by monoamine oxidase.
This inhibition seems to be specific since apomorphine did not influence 5-HIAA levels in normal rats and prevented neither central 5-HT depletion nor 5-HIAA rise induced by reserpine.     

7-[3-(4-[2,3-Dimethylphenyl]piperazinyl)propoxy]-2(1H)-quinolinone (OPC-4392), a presynaptic dopamine autoreceptor agonist and postsynaptic D2 receptor antagonist

7-[3-(4-[2,3-dimethylphenyl]piperazinyl)propoxy]-2(1H)-quinolinone (OPC-4392), was synthesized in our laboratories and compared with apomorphine, 3-(3-hydroxyphenyl)-N-n-propylpiperidine (3-PPP) and dopamine antagonists in a series of tests designed to characterize dopamine receptor activation and inhibition. The assertion that OPC-4392 acts as an agonist at presynaptic dopamine autoreceptors is supported by the following behavioral and biochemical observations: OPC-4392, 3-PPP and apomorphine inhibited the reserpine-induced increase in DOPA accumulation in the forebrain of mice and in the frontal cortex, limbic forebrain and striatum of rats. In addition, the gamma-butyrolactone (GBL)-induced increase in DOPA accumulation in the mouse forebrain was also inhibited by OPC-4392, 3-PPP and apomorphine. Haloperidol antagonized the inhibitory effect of OPC-4392 in both instances. The inhibitory effect of OPC-4392 on GBL-induced DOPA accumulation lasted for at least 8 hours after oral administration to mice, while that of 3-PPP and apomorphine disappeared in 4 hours after subcutaneous injection. OPC-4392 failed to increase spontaneous motor activity in reserpinized mice, enhance spontaneous ipsilateral rotation in rats with unilateral striatal kainic acid (KA) lesions, induce contralateral rotation in rats with unilateral striatal 6-hydroxydopamine (6-OHDA) lesions and inhibit 14C-acetylcholine (Ach) release stimulated by 20 mM KCl in rat striatal slices. In addition, OPC-4392 appears to block postsynaptic D2 receptors since OPC-4392, as well as dopamine antagonists, was able to inhibit stereotyped behavior and climbing behavior induced by apomorphine in mice, displace the 3H-spiroperidol binding to rat synaptosomal membranes in vitro and reverse the inhibitory effect of apomorphine on Ach release in rat striatal slices. These results suggest that OPC-4392 acts as a dopamine agonist at presynaptic autoreceptors related to dopamine synthesis and acts as dopamine antagonist at postsynaptic D2 receptors.     

Dopamine receptor sensitivity after repeated morphine administrations to rats.

It was studied in rats, if chronic morphine treatment induces a supersensitivity of dopamine receptors in brain. The rats were treated twice daily for 8–11 days with single doses of morphine, increasing from 10 to 20 mg/kg i.p. The experiments were carried out 16–20 hours after the last injection of morphine. After chronic morphine treatment, the potency of apomorphine in lowering the striatal dopamine turnover was increased. On the other hand, apomorphine was not more potent in inducing stereotypies (sniffing, licking, gnawing) after chronic morphine administration than in saline controls. Finally, dopamine activated the adenylate cyclase in striatal homogenates of rats after chronic morphine treatment to a similar extent as in homogenates of control rats. The results suggest that a supersensitivity of dopamine receptors in brain is not necessarily involved in symptoms of an increased dopaminergic activity after chronic morphine application.     

Significance of the dopamine-blocking and m-choline-blocking components in the antiapomorphine action of neuroleptics

In experiments on rats and mice the correlation between the ability of neuroleptics to antagonize apomorphine induced stereotypy and to block central dopamine and muscarinic acetylcholine receptors was studied. The analysis showed significant correlation (v = 0.76; P less than 0.05) between antistereotypic effects of drugs and their ability to inhibit 3H-spiperone binding to rat striated tissue. However, no correlation was found between antistereotypic effect of neuroleptics and their ability to block 3H-quinuclidinyl benzylate binding or arecoline-induced tremor.     

Antagonism between L-DOPA and apomorphine in their effects on rat behavior

The effect of simultaneous injections of apomorphine and l-DOPA on stereotypy, aggressiveness, on the exploratory motor activity, the threshold of emotional reactivity and aggressiveness elicited by painful electrical stimulation was studied in experiments on male albino rats. When injected separately, in control experiments, both compounds had similar effects on the exploratory-motor activity and the emotional behaviour, but when injected simultaneously in various doses a distinct antagonism between l-DOPA and apomorphine, according to all the behaviour tests, was noted (a decrease of sterotypy, aggressiveness and emotional reactivity). Against the background of l-DOPA apomorphine (5 mg/kg) increased the dopamine content in the forebrain and in the diencephalon even more. It is suggested that the increased level of the functionally active mediator suppressed the activity of postsynaptic receptors sensitive to it.     

Chronic treatment with antidepressants prevents the inhibitory effect of small doses of apomorphine on dopamine synthesis and motor activity.

In control rats small doses of apomorphine (25 to 100 μg/kg) decreased motor activity and reduced DOPAC content in the caudate nucleus. A larger dose (500 μg/kg) increased motor activity and elicited stereotypy. Chronic treatment with imipramine, amitryptiline and mianserine (10, 10 and 2.5 mg/kg twice daily for 10 days respectively) counteracted or reversed the effect of small doses of apomorphine on motor activity, left DOPAC content unchanged and potentiated the central stimulant response to the larger dose of apomorphine. Changes in apomorphine responses were observed after ten but not after two days of imipramine treatment and persisted unaltered up to 4 days after imipramine withdrawal. It is suggested that chronic treatment with antidepressants induces persistent subsensitivity in presynaptic dopamine receptors. The relevance of the findings in the therapeutic effect of these drugs is discussed.     

Two types of "spike-wave" discharges in the electrocorticogram of WAG/Rij rats, the genetic model of absence epilepsy

WAG/Rij rats were injected with apomorphine (0.5 mg/kg, i.p.), an agonist of D2 receptors. Two types of spike-wave discharges (generalized and local) were found in the baseline ECoG of the intact and injected rats. Injections of apomorphine led to a suppression of the generalized (type 1) for about 30 minutes and a 8-10-fold increase in the local spike-wave discharges (type 2) within 4-6 minutes. Since it has been shown earlier that haloperidol, which acts on dopamine receptors oppositely to apomorphine, enhance the generalized spike-wave activity and suppress the local discharges. Thus, the different pharmacological characteristics of the two types of spike-wave activity suggest the controlling role of the dopaminergic system in the processes of spike-wave generation.     

Enhanced response to apomorphine in rats treated with multiple injections of human beta endorphin and its blockade by Pro-Leu-Gly-NH2 and cyclo (Leu-Gly)

Repeated intraventricular injections of human β-endorphin to rats every eight hours for three days resulted in the development of super-sensitivity of brain dopamine receptors as evidenced by an enhanced locomotor activity response to apomorphine, a dopamine receptor agonist. Daily subcutaneous injections of Pro-Leu-Gly-NH₂ or its cyclic analog, cyclo (Leu-Gly) blocked the enhanced apomorphine-induced stimulation of locomotor activity in β-endorphin treated rats. Thus, the development of brain dopamine receptor supersensitivity induced by chronic β-endorphin administration may be regulated by the hypothalamic peptides.     

Antioxidant activity of propionyl-L-carnitine in liver and heart of spontaneously hypertensive rats

Oxidative stress plays an important role in arterial hypertension and propionyl-L-carnitine (PLC) has been found to protect cells from toxic reactive oxygen species. In this work, we have evaluated the antioxidant capacity of chronic PLC treatment in spontaneously hypertensive rats (SHR) by measuring the activity of antioxidant enzymes and the lipid peroxidation in liver and cardiac tissues. The activity of glutathione peroxidase was decreased in liver and cardiac tissues of SHR when compared with their normotensive controls, Wistar- Kyoto (WKY) rats, this alteration being prevented by PLC treatment. Glutathione reductase activity was increased in hypertensive rats and no effect was observed after the treatment. No significant changes in superoxide dismutase activity were observed among all experimental groups. Liver of hypertensive rats showed higher catalase activity than that of normotensive rats, and PLC enhanced this activity in both rat strains. Thiobarbituric acid reactive substances, determined as a measure of lipid peroxidation, were increased in SHR compared with WKY rats, and PLC treatment decreased these values not only in hypertensive rats but also in normotensive ones. The content of carnitine in serum, liver and heart was higher in PLC-treated rats, but PLC did not prevent the hypertension development in young SHR. In addition, triglyceride levels, which were lower in SHR than WKY rats, were reduced by chronic PLC treatment in both rat strains. These results demonstrate: i) the hypotriglyceridemic effect of PLC and ii) the antioxidant capacity of PLC in SHR and its beneficial use protecting tissues from hypertension-accompanying oxidative damage.     

Potent dopamine agonist activity of a novel ergoline, 6-Ethyl-9-oxaergoline (EOE)

6-Ethyl-9-oxaergoline (EOE) and its enantiomers were compared with apomorphine in a number of tests designed to measure dopamine (DA) agonist activity within the central nervous system. In rats, the tests were: interaction with DA receptors labeled with ³H-apomorphine or ³H-spiroperidol; the effects on DA synthesis as assessed by the

-butyrolactone procedure; turning in 6-OHDA lesioned animals; stereotypy; and, slowing of DA cell firing rates. In the mouse, locomotor activity, hypothermia and postural asymmetry in Caudectomized animals were studied. Emesis in the beagle was also examined. The (-)-enantiomer of EOE was more potent than either the (+)-enantiomer or the racemate in all tests. With the exception of inducing stereotypy and the displacement of ³H-apomorphine from rat striatal membranes, (-)-EOE was equi- or more potent than apomorphine in all test procedures. (-)-EOE was effective following oral administration and exhibited a longer duration of action than apomorphine. The results indicate EOE is a potent DA agonist.     

Dopaminergic modulation of footshock induced aggression in paired rats.

Footshock induced aggression (FIA) was induced in weight matched paired rats and three paradigms of aggressive behaviour was recorded, namely, the latency to fight (LF), total period of physical contact (TPP) and cumulative aggression scores (CAS). Dopamine (DA), administered centrally, and peripherally administered L-dopa (with benserazide, a peripheral decarboxylase inhibitor), a DA precursor, and the postsynaptic D2 receptor agonists, apomorphine, N-n-propyl-norapomorphine (PNA), bromocriptine, lisuride and pergolide, induced a dose-related facilitation of FIA characterized by decrease in LF and increase in TPP and CAS. However, the DA presynaptic receptor agonist, BHT-920, induced a biphasic effect with inhibition of FIA being induced by a lower dose and facilitation of the aggressive behaviour produced by a higher dose. The postsynaptic D2 receptor antagonists, haloperidol, spiperone and pimozide, induced a dose-related attenuation of FIA, an effect not seen with domperidone, a peripheral DA receptor antagonist. The results indicate that central dopaminergic postsynaptic D2 receptors have a modulatory facilitative effect on FIA, while the presynaptic DA autoreceptors mitigate aggressive behaviour. However, the presynaptic DA receptor agonist, BHT-920, appears to lose its receptor specificity on dose increment. Long term administration of haloperidol, followed by withdrawal, or desipramine, induced per se augmentation of FIA and potentiated the aggression-facilitative effects of L-dopa, apomorphine and PNA. Since both these treatments are known to induce supersensitivity of central postsynaptic dopamine D2 receptors, the effects are likely to be related to augmented function of dopamine neurones. The findings, in conjunction with a recent report from this laboratory indicating an increase in rat brain DA levels in FIA, support the contention that the central DA system has a facilitative effect on FIA.     

Further prove on oxidative stress in alloxan diabetic rat tissues

After intravenous administration of alloxan monohydrate (AL) diabetes developed in rats. Forty-eight hours after the injection the animals were sacrificed, their blood was collected in heparin containing tubes and the tissues were dissected and frozen (-70 degrees C) until their homogenization for pro- and antioxidant testing. Our results can be summarised as follows: (i) In the blood hemolysate the lipid peroxidation slightly elevated and the activity of antioxidant enzymes and reduced glutathione decreased. (ii) Similar phenomena could be observed in the different examined organ homogenates. The organs tested for pro- and antioxidant system were as follows: the liver, heart, skeletal muscle, kidney and pancreas. In our present work we attempt to confirm the data in support of the oxidative predominance over antioxidants in oxidative stress of AL diabetic rats.     

Behavioral and radioligand binding evidence for irreversible dopamine receptor blockade by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline

N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), an irreversible alpha adrenergic antagonist, also acts as a potent and longlasting in vivo antagonist of D-2 dopamine receptors. Rats given EEDQ 3-10 mg/kg i.p. exhibit catalepsy and greatly reduced apomorphine-induced stereotypy, behavioral effects associated with D-2 dopamine receptor blockade. These effects are apparent up to 4 days after drug administration, with scores returning to control level by day 7. In vitro receptor binding assays of striatal membrane preparations from these animals using the radioligand 3H-spiroperidol directly demonstrate that EEDQ is a potent D-2 dopamine receptor antagonist, revealing the apparent basis of the behavioral effects of EEDQ. This antagonism proceeds via a reduction in D-2 receptor Bmax, with no change in the observed KD for 3H-spiroperidol, and is resistant to extensive washing of the membrane preparation after in vivo EEDQ exposure. These observations suggest that EEDQ inhibition of D-2 receptors is irreversible. Administration of behaviorally active doses of EEDQ effect a reduction of 50-85% in D-2 receptor number. Recovery of this loss roughly parallels recovery of normal catalepsy and apomorphine stereotypy scores. These doses of EEDQ also reduce binding of 3H-flupentixol to D-1 and 3H-dopamine to D-3 type dopaminergic binding sites, putative dopamine receptors with no known behavioral correlates. Recovery of D-1 and D-3 binding also occurs with a similar timecourse. Because of the apparent covalent nature of its interaction with dopamine receptors and because of its activity after peripheral administration, EEDQ may prove useful in the study of the function and turnover of dopamine receptors.     

125I](+/-)FISCH: a new CNS D-1 dopamine receptor imaging ligand

Radiolabeling and in vitro and in vivo evaluation of an iodinated benzazepine: [125I] FISCH 7-Chloro-8-hydroxy-1-(4'-iodophenyl)-3-methyl-2,3,4,5- tetrahydro-1H-3-benzazepine, as a potential imaging agent for CNS D-1 dopamine receptors in animals, were investigated. After an iv injection, this benzazepine derivative showed good brain uptake in rats (2.70, 1.28, 0.48 %dose/whole brain at 2, 15 and 60 min, respectively). The striatum/cerebellum ratio was 2.50 at 60 min after the injection. The regional distribution in rat brain, as measured by ex vivo autoradiography, displayed highest uptake in the regions of the striatal complex and the substantia nigra, regions known to have a high concentration of D-1 dopamine receptors. Furthermore, this localized regional cerebral distribution was blocked by pretreatment with SCH-23390, a selective D-1 dopamine receptor antagonist. The in vitro binding affinity of this agent in rat striatum tissue preparation displayed a Kd of 1.43 +/- 0.15 nM. Competition data (in vitro) showed the following rank order of potency: SCH-23390 greater than (+/-)IBZP much greater than apomorphine greater than WB 4101 greater than ketanserin approximately spiperone. The preliminary data suggest that this analog of SCH-23390 shows similar selectivity for the CNS D-1 receptor.     

The effects of apomorphine and haldol on PCP induced behavioral and motor abnormalities in the rat

Behavioral abnormalities and motor dysfunctions induced by the administration of PCP to rats is antagonized by both the dopamine agonist apomorphite and the dopamine antagonist haloperidol. In the test situation used, however, the antagonistic effects of apomorphine were much more rapid than those of haloperidol. These results suggest that PCP may be acting on specific presynaptic dopamine receptors and that this reaction is effectively antagonized by low doses of apomorphine in a manner similar to its effects in reducing schizophrenic symptoms and choreiform movement disorders.     

Pharmacological evaluation of selected arylpiperazines with atypical antipsychotic potential

Six active compounds, among previously synthesized and screened arylpiperazines, were selected and evaluated for the binding affinity to rat dopamine, serotonin and alpha(1) receptors. Two compounds with benztriazole group had a 5-HT(2A)/D(2) binding ratio characteristic for atypical neuroleptics (>1, pK(i) values). Compound 2, 5-[2-[4-(2,3-dimethyl-phenyl)-piperazin-1-yl]ethyl]1H-benzotriazole, expressed clozapine-like in vitro binding profile at D(2), 5-HT(2A) and alpha 1 receptors and a higher affinity for 5-HT(1A) receptors than clozapine. Also, it exhibited the noncataleptic behavioural pattern of atypical antipsychotics and antagonized d-amphetamine-induced hyperlocomotion in rats.