The effect of cyclo (Leu-Gly) on chemical denervation supersensitivity of dopamine receptors induced by intracerebroventricular injection of 6-hydroxydopamine in mice

Chemical denervation supersensitivity of postsynaptic dopamine receptors was induced in mice by intracerebroventricular injection of 6-hydroxydopamine. Fourteen days after the 6-hydroxydopamine injection, mice exhibited greater spontaneous locomotor activity and hypothermic response when challenged intraperitoneally with apomorphine. Whole brain levels of dopamine were reduced by 80%. Daily subcutaneous administration of cyclo (Leu-Gly) (50 μg/mouse/day) for 14 days inhibited the development of dopamine receptor supersensitivity induced by 6-hydroxydopamine as evidenced by the blockade of an apomorphine induced locomotor and hypothermic effect. Cyclo (Leu-Gly) did not alter the depletion of brain dopamine induced by 6-hydroxydopamine. These data suggest that cyclo (Leu-Gly) can block the development of dopamine receptor supersensitivity induced by 6-hydroxydopamine without protecting the neurons from dopamine depletion.     

Age related alterations of motor behavior and release of dopaminergic supersensitivity in rats

Rats aged greater than or equal to 18 months show, aside from clearly diminished motor parameters (exploratory behavior, resting activity, nocturnal activity profile, rotation behavior), after intracerebral dopamine injection a considerably lower apomorphine hypermotility than young animals. The characteristic alteration of activity occurring in young rats during and following chronic administration of haloperidol (1 mg/kg . day, s.c. for 21 days) as an expression of developing dopaminergic supersensitivity does not appear in older animals. Repeated application of amphetamine (2 X 2 mg/kg daily) caused a significant increase in hypermotility. The results are interpreted as the consequence of a age-related reduction of the activity of central-dopaminergic transmission systems and are discussed with regard to possible differences in the realization of agonist- or antagonist-induced supersensitivity.     

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.     

Behavioral evidence for dopaminergic supersensitivity after chronic haloperidol

Chronic administration for 16 days of haloperidol (in increasing doses up to 20 mg/kg/day) results in a supersensitivity of dopamine receptors. This supersensitivity is manifested by an enhanced stereotypy and aggression in response to small, otherwise ineffective, doses of apomorphine. Maximum aggression is observed 7 days after the last dose of haloperidol when 2.5 mg/Kg of apomorphine is administered. In addition, “wet shakes”, reminiscent of withdrawal from morphine, are observed in these animals after the cessation of the haloperidol administration. These shakes are blocked by morphine. These results may be interpreted to mean that “wet shakes” and drug induced aggression are the results of hyperactivity of the dopaminergic system.     

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.     

Ethanol induces rotational behavior in 6-hydroxydopamine lesioned mice

Mice with unilateral striatal lesions created by 6-hydroxydopamine (6HDA) injection were screened for rotational (circling) behavior in response to injection of amphetamine and apomorphine. Those that rotated ipsilaterally in response to amphetamine and contralaterally in response to apomorphine were subsequently challenged with 1 to 3 g/kg (i.p.) ethanol. Surprisingly, ethanol induced dose related contralateral (apomorphine-like) rotation which, despite gross intoxication, was quite marked in most animals. No significant correlation was found between the number of turns made following ethanol and made after apomorphine or amphetamine.     

Reverse tolerance to amphetamine of mice bearing unilateral striatal lesions: effect upon the circling response to apomorphine

When mice bearing unilateral lesions of the striatum receive weekly doses of amphetamine, their circling responses increase with successive doses, i.e., they become reverse tolerant (1). The present work was undertaken to ascertain whether presynaptic dopaminergic changes might underlie this phenomenon. This was approached by observing whether the response to a direct dopaminergic agent would be affected by the development of reverse tolerance to amphetamine. The circling responses of unilaterally electrothermally lesioned mice to apomorphine, 0.4 mg/kg s.c., increased significantly after four weekly 4 mg/kg s.c. doses of d-amphetamine. The responses to apomorphine of mice not exposed to amphetamine declined over the same interval. The conclusion drawn is that presynaptic effects are not likely to underlie the phenomenon of reverse tolerance.     

Choline chloride in animal models of tardive dyskinesia

Rats treated chronically with haloperidol show evidence of supersensitive dopamine receptors by increased stereotypy when challenged with apomorphine. When such animals are treated acutely with choline chloride at the time of challenge, no changes in stereotypy were observed. Chronic treatment, either during or after induction of supersensitivity, mitigated stereotypy after challenge. This model of dopaminergic supersensitivity is pertinent to the development of tardive dyskinesia in man after treatment with neuroleptic drugs. Our results with choline chloride in the animal model are consistent with their therapeutic action in tardive dyskinesia.     

Synaptogenesis of hippocampal neurons in primary cell culture

Hippocampal neurons in dissociated cell culture are one of the most extensively used model systems in the field of molecular and cellular neurobiology. Only limited data are however available on the normal time frame of synaptogenesis, synapse number and ultrastructure of excitatory synapses during early development in culture. Therefore, we analyzed the synaptic ultrastructure and morphology and the localization of presynaptic (Bassoon) and postsynaptic (ProSAP1/Shank2) marker proteins in cultures established from rat embryos at embryonic day 19, after 3, 7, 10, 14, and 21 days in culture. First excitatory synapses were identified at day 7 with a clearly defined postsynaptic density and presynaptically localized synaptic vesicles. Mature synapses on dendritic spines were seen from day 10 onward, and the number of synapses steeply increased in the third week. Fenestrated or multiple synapses were found after 14 or 21 days, respectively. So-called dense-core vesicles, responsible for the transport of proteins to the active zone of the presynaptic specialization, were seen on cultivation day 3 and 7 and could be detected in axons and especially in the presynaptic subcompartments. The expression and localization of the presynaptic protein Bassoon and of the postsynaptic molecule ProSAP1/Shank2 was found to correlate nicely with the ultrastructural results. This regular pattern of development and maturation of excitatory synapses in hippocampal culture starting from day 7 in culture should ease the comparison of synapse number and morphology of synaptic contacts in this widely used model system.     

An increase in putative voltage dependent calcium channel number following reserpine treatment

Rats were treated with reserpine (0.2 mg/kg) on days 1, 3, and 5. On day 6, binding parameters for alpha-1 adrenergic receptors (3H-prazosin) and putative voltage dependent calcium channels, VDCC (3H-nitrendipine), were determined. There was an increase in both the number (2.1 fold) and affinity (1.8 fold) of alpha-1 adrenergic receptors following reserpine treatment. In addition, there was a 2.7 fold increase in the number of VDCCs, but no change in VDCC binding affinity, following reserpine treatment. These data are consistant with the development of smooth muscle supersensitivity following reserpine treatment in a variety of tissues, and suggest that VDCC number may be modulated by the cell in response to tonic levels of catecholamines. Changes in the number of VDCCs may be an important regulatory mechanism for cell function in physiologic and pathologic states.     

Reserpine-induced hypothermia and its reversal by dopamine agonists

Prior treatments with reserpine altered the thermic response of mice to subsequently administered apomorphine and amphetamine. Thus, normal mice exhibited hypo- and hyper-thermic responses to apomorphine and (+)-amphetamine, respectively but did not respond to (-)-amphetamine. These responses were each readily attenuated by haloperidol. Reserpinized mice, on the other hand, exhibited hyperthermic responses to all three agonists and these responses were not attenuated by haloperidol. In addition to its hypothermic action, reserpine also produced hypoactivity which was reversed by (+)-amphetamine. This reversal of hypoactivity was attenuated by haloperidol. These data suggest that reversal of reserpine-induced hypothermia by dopamine agonists results through activation of mechanisms which are separate from those normally associated with agonist-induced thermic responses. Reversal of hypoactivity, on the other hand, appears to be due to reactivation of those systems which normally regulate locomotor activity.     

Effects of the potential neuroleptic peptide des-tyrosine1-γ-endorphin and haloperidol on apomorphine-induced behavioural syndromes in rats and mice

The effects of haloperidol and Des-Tyr¹-γ-endorphin (DTγE) were studied on climbing induced in mice by high doses of apomorphine and on the yawning syndrome induced in rats by low doses of apomorphine. Haloperidol in a dose of 0.0046 mg/kg s.c. potentiated climbing whereas at higher doses climbing was inhibited (ED₅₀=0.03 mg/kg). DTγE had no effect on climbing under normal conditions in doses up to 2 mg/kg s.c.. After three days of handling and saline pre-injections DTγE potentiated climbing in doses from 0.1 to 1 mg/kg.Haloperidol inhibited yawning induced by low doses of apomorphine (ED₅₀=0.01 mg/kg). DTγE, on the other hand, potentiated yawning induced by low apomorphine at doses of 0.02 and 0.04 mg/kg s.c.. From the point of view that low doses of apomorphine predominantly activate presynaptic dopamine autoreceptors while higher doses predominantly activate postsynaptic dopamine receptors the following tentative conclusions are drawn. 1) Haloperidol blocks presynaptic dopamine autoreceptors at low doses and postsynaptic dopamine receptors at higher doses. 2) DTγE sensitizes presynaptic dopamine autoreceptors at low doses, thereby strengthening the local feedback mechanism at the dopaminergic nerve ending, and sensitizes postsynaptic dopamine receptors at higher doses.     

Tolerance to hypokinesia elicited by dopamine agonists in mice: Hyposensitization of autoreceptors?

In control mice, dopamine agonists like apomorphine and amphetamine have biphasic actions on motor activity : low doses elicit hypokinesia which disappears or is replaced by hyperkinesia at a higher dosage. In mice pretreated with a single dose of apomorphine, the hypokinesia is durably suppressed whereas the hyperkinesia elicited by amphetamine is not only present but facilitated. These observations could be explained on the assumption that, following their stimulation, hyposensitivity develops on autoreceptors regulating the activity of dopaminergic neurons whereas the sensitivity of postsynaptic receptors is not modified.     

Amphetamine effects on acetylcholine release and mammalian neuromuscular transmission

The mechanisms by which (+)-amphetamine biphasically modifies neuromuscular transmission were studied in the rat phrenic nervediaphragm preparation. Low to moderate amphetamine concentrations (30–300 μM) enhanced twitch height and potentiated the nerve stimulated release of acetylcholine (ACh) by up to 4.8-fold from the phrenic nerve. Higher amphetamine concentrations depressed muscle twitch and ACh release. Using a cannulated diaphragm preparation, amphetamine enhanced the twitch response to nerve stimulation but markedly depressed the contractions elicited by a pulsed injection of ACh. Amphetamine-induced enhancement of ACh release was prevented by pretreatment of animals with α-methyl-p-tyrosine, suggesting that amphetamine may be acting indirectly by releasing catecholamines. These results support the hypothesis that amphetamine enhancement results from a presynaptic increase in ACh release and the blocking actions are mediated by a postsynaptic inhibitory effect.     

Chronic treatment with metoclopramide induces behavioral supersensitivity to apomorphine and enhances specific binding of 3H-spiroperidol to rat striata

Metoclopramide is a dopamine (DA) antagonist with a potency and pharmacologic profile similar to chlorpromazine, but is paradoxical insofar as it is virtually inactive as an antagonist of DA-stimulated adenylate cyclase and has an extremely low affinity for DA/neuroleptic binding sites in membranes prepared from DA-rich brain regions. Chronic administration of metoclopramide to rats induced behavioral supersensitivity to apomorphine which was associated with enhanced specific binding of ³H-spiroperidol to striatal membranes. These changes were seen when metoclopramide was administered to rats in their drinking water for 39 days, but not when it was given i.p. once daily for 14 days. These findings are discussed in terms of their implications regarding the validity of currently used receptor models as predictors of DA antagonism as well as the implicit assumption that DA/neuroleptic binding sites truly represent functionally relevant DA receptors.     

Enhanced striatal 3H-spiroperidol binding induced by chronic haloperidol treatment inhibited by peptides administered during the withdrawal phase

Chronic intragastric administration of haloperidol (1.5 mg/kg/day) for 21 days followed by a 3-day withdrawal period resulted in the development of enhanced locomotor activity response to apomorphine, and an increase in the number of binding sites for 3H-spiroperidol in the striatal membranes of the rat brain. Subcutaneous administration of Pro-Leu-Gly-NH2 or cyclo(Leu-Gly) in doses of 2 mg/kg/day given for 3-days after termination of haloperidol treatment inhibited the enhanced response to apomorphine, as well as the increases in the number of 3H-spiroperidol binding sites in the striatum. If indeed, the supersensitivity of striatal dopamine receptors is one of the mechanisms in the development of tardive dyskinesia symptoms, the present results suggest that the above peptides may be helpful in ameliorating some of the symptoms of tardive dyskinesia induced by neuroleptic drugs.     

Morphological and biochemical changes in supersensitive smooth muscle.

The effect of postganglionic denervation on the incidence of nexal contacts in the smooth muscle of the rat vas deferens was investigated. The chronically denervated tissue exhibited twice as many nexuses as control. This increase in the incidence of cell contacts may contribute to the supersensitivity and/or the increase in maximum response of the denervated vas deferens. The effects of denervation, decentralization, and pretreatment with reserpine on the concentration of adenosine triphosphate (ATP) in vasa deferentia of rats and guinea pigs were also investigated. One day after denervation there was a substantial decrease in the endogenous norepinephrine and ATP concentrations. The norepinephrine concentration remained low (less than 10% of control) throughout subsequent days (up to 14 days) whereas the ATP concentration, after the first postoperative day, rose significantly. The rise in ATP concentration was temporally correlated with the development of postjunctional supersensitivity. Decentralization and pretreatment with reserpine both resulted in a significant increase in ATP concentration which preceded by 2 to 3 days a significant increase in sensitivity of the vas deferens. It appears that a change in the tissue concentration of ATP may be one of the initial events that occurs following interruption of the neural contact to the smooth muscle of the vas deferens.     

Effect of reserpine administered during infancy on brain catecholamines and adult behaviour in the rat

Reserpine (0.1 mg/kg/day) was administered to rats from 11 through 30 days of age. During and after administration of reserpine, concentrations of catecholamines, epinephrine and norepinephrine, in the brain were estimated. Levels of catecholamines were about 30 per cent of normal during the period of reserpine administration. Approximately 3 weeks were required for these levels to return to normal. When animals were 95-100 days of age, they were deprived of food and were trained to press a bar for food. When the rate of responding became stable, the animals were subjected to three successive extinctions at daily intervals and the increase in response rate after the onset of each extinction was determined. This extinction-induced increase in response rate was greater for previously reserpinized animals than controls during the second and third extinctions, but not the first. These findings are interpreted as a decreased ability of the animals, reserpinized during infancy, to learn to respond discriminatively during non-reinforcement (extinction). Thus, an effect of reserpine administration during infancy on a type of behaviour in the adult has been demonstrated. This occurs after the catecholamine-depleting effect of the reserpine has been fully dissipated.     

Dopaminergic supersensitivity in G protein-coupled receptor kinase 6-deficient mice

Brain dopaminergic transmission is a critical component in numerous vital functions, and its dysfunction is involved in several disorders, including addiction and Parkinson's disease. Responses to dopamine are mediated via G protein-coupled dopamine receptors (D1-D5). Desensitization of G protein-coupled receptors is mediated via phosphorylation by members of the family of G protein-coupled receptor kinases (GRK1-GRK7). Here we show that GRK6-deficient mice are supersensitive to the locomotor-stimulating effect of psychostimulants, including cocaine and amphetamine. In addition, these mice demonstrate an enhanced coupling of striatal D2-like dopamine receptors to G proteins and augmented locomotor response to direct dopamine agonists both in intact and in dopamine-depleted animals. The present study indicates that postsynaptic D2-like dopamine receptors are physiological targets for GRK6 and suggests that this regulatory mechanism contributes to central dopaminergic supersensitivity.     

RGS mRNA expression in rat striatum: modulation by dopamine receptors and effects of repeated amphetamine administration

Single injections of cocaine, amphetamine, or methamphetamine increased RGS2 mRNA levels in rat striatum by two- to fourfold. The D1 dopamine receptor-selective antagonist SCH-23390 had no effect by itself but strongly attenuated RGS2 mRNA induction by amphetamine. In contrast, the D2 receptor-selective antagonist raclopride induced RGS2 mRNA when administered alone and greatly enhanced stimulation by amphetamine. To examine the effects of repeated amphetamine on RGS2 expression, rats were treated with escalating doses of amphetamine (1.0-7.5 mg/kg) for 4 days, followed by 8 days of multiple daily injections (7.5 mg/kg/2 h x four injections). Twenty hours after the last injection the animals were challenged with amphetamine (7.5 mg/kg) or vehicle and killed 1 h later. In drug-naive animals, acute amphetamine induced the expression of RGS2, 3, and 5 and the immediate early genes c-fos and zif/268. RGS4 mRNA levels were not affected. Prior repeated treatment with amphetamine strongly suppressed induction of immediate early genes and RGS5 to a challenge dose of amphetamine. In sharp contrast, prior exposure to amphetamine did not reduce the induction of RGS2 and RGS3 mRNAs to a challenge dose of amphetamine, indicating that control of these genes is resistant to amphetamine-induced tolerance. These data establish a role for dopamine receptors in the regulation of RGS2 expression and suggest that RGS2 and 3 might mediate some aspects of amphetamine-induced tolerance.