Non-equivalent stringency of ethical review in the Baltic States: a sign of a systematic problem in Europe?

We analyse the system of ethical review of human research in the Baltic States by introducing the principle of equivalent stringency of ethical review, that is, research projects imposing equal risks and inconveniences on research participants should be subjected to equally stringent review procedures. We examine several examples of non-equivalence or asymmetry in the system of ethical review of human research: (1) the asymmetry between rather strict regulations of clinical drug trials and relatively weaker regulations of other types of clinical biomedical research and (2) gaps in ethical review in the area of non-biomedical human research where some sensitive research projects are not reviewed by research ethics committees at all. We conclude that non-equivalent stringency of ethical review is at least partly linked to the differences in scope and binding character of various international legal instruments that have been shaping the system of ethical review in the Baltic States. Therefore, the Baltic example could also serve as an object lesson to other European countries which might be experiencing similar problems.

Apomorphine-induced behavioural sensitization in rats: individual differences, role of dopamine and NMDA receptors.

Apomorphine-induced behavioural sensitization was studied in male Wistar rats. The acute administration of apomorphine (0.5 mg/kg s.c.), a dopamine agonist, did not affect the locomotor activity of rats, but it caused stereotyped behaviour characterized by repeated gnawing, licking and sniffing. A significant increase in the locomotor activity became evident after repeated treatments with apomorphine (0.5 mg/kg twice daily for 14 days). However, there were marked individual differences in the sensitization of rats to apomorphine. One third of animals did not react with increased locomotor activity even after the 2-week administration of apomorphine, whereas the other one third needed only a few injections to display increased behavioural response to apomorphine. The behavioural response of the remaining one third of rats was between weak and strong responders. Simultaneously, the stereotyped behaviour occurred earlier and its intensity tended to be lower after repeated treatment with apomorphine. Nevertheless, the established changes of stereotyped behaviour did not correlate with the increase of locomotor activity. The administration of amphetamine (2.5 mg/kg, s.c.), an indirect dopamine agonist, but not a non-competitive NMDA antagonist dizocilpine (0.25 mg/kg i.p.), tended to cause a similar response profile with apomorphine in sensitized rats. The ED50 values of the dopamine antagonists blocking apomorphine-induced increase in the locomotor activity were the following: 0.09 mg/kg for raclopride (dopamine D2 antagonist), 0.023 mg/kg for SCH 23390 (dopamine D1 antagonist), 6.42 mg/kg for clozapine (dopamine D4 antagonist). This supports the involvement of D1 and D1 receptors in the expression of apomorphine-induced behavioural sensitization. The concomitant administration of dizocilpine (0.5 mg/kg), SCH 23390 (0.05 mg/kg), raclopride (0.1 mg/kg) and clozapine (20 mg/kg) with apomorphine (0.5 mg/kg twice daily for 2 weeks) antagonized the development of behavioural sensitization to apomorphine. Accordingly, at least three different molecular targets, namely dopamine D1 and D2, and NMDA receptors, are involved in the development of apomorphine-induced behavioural sensitization.

BOC-CCK-4, CCK(B)receptor agonist, antagonizes anxiolytic-like action of morphine in elevated plus-maze.

This study investigated a role of cholecystokinin (CCK) in the anxiolytic-like action of morphine, an agonist of mu-opioid receptors, in the rat plus-maze model of anxiety. The acute administration of morphine (1 mg/kg) induced a significant increase of exploratory activity in the plus-maze, but did not affect the locomotor activity in the motility test. The higher dose of morphine (2.5 mg/kg) tended to decrease the locomotor activity and, therefore, did not cause the anxiolytic-like action in the plus-maze. The other drugs (naloxone, BOC-CCK-4, L-365,260) and their combinations with morphine (0.5-1 mg/kg) did not affect the locomotor activity of rats. The opioid antagonist naloxone itself (0.5 mg/kg) did not change the exploratory activity in the plus-maze, but potently antagonized the anxiolytic-like action of morphine (1 mg/kg). An agonist of CCK(B)receptors BOC-CCK-4 (1-50 microgram/kg) induced a dose-dependent anxiogenic-like action in the plus-maze. Nevertheless, only one dose of BOC-CCK-4 (10 microgram/kg) completely reversed the action of morphine. Also, one dose of CCK(B)receptor antagonist L-365,260 (10 microgram/kg) was effective to modify the behaviour of rats in the elevated plus-maze. Namely, this dose of L-365,260 increased the ratio between open and total arm entries, a behavioural measure believed to reflect the anxiolytic-like action in the elevated plus-maze. The combination of L-365,260 (100 microgram/kg) with the sub-effective dose of morphine (0.5 mg/kg) caused the anxiolytic-like action in the plus-maze not seen if the drugs were given alone. In conclusion, morphine induces a potent anxiolytic-like action in the elevated plus-maze and CCK is acting as an endogenous antagonist of this effect of morphine.

L-Arginine abolishes the anxiolytic-like effect of diazepam in the elevated plus-maze test in rats.

The involvement of nitrergic mechanisms in the behavioural effects of diazepam in rats was studied in the elevated plus-maze, open-field and rotarod tests. Administration of the nitric oxide (NO) precursor L-arginine (100 mg/kg, i.p.), assumed to increase the synthesis of NO, abolished the anxiolytic-like effect of diazepam (2 mg/kg, i.p.) in the elevated plus-maze, whereas the inactive enantiomer D-arginine (100 mg/kg) did not. Neither diazepam alone nor in combination with L- or D-arginine affected the exploratory activity of animals in the open field. Pretreatment with L-arginine (100 and 200 mg/kg) did not modify the motor impairment of rats after diazepam (3 mg/kg) in the rotarod test. Diazepam (2 mg/kg i.p.) did not inhibit the cortical or hippocampal cytosolic NO synthase activity measured ex vivo by [3H]L-arginine assay. Diazepam was similarly ineffective in in vitro studies at concentrations up to 10 microM. We conclude that a suppression of NO synthase activity may be important in the anxiolytic-like effect of benzodiazepines. However, diazepam does not inhibit NO synthase directly, but may affect NO synthase activity indirectly via some unknown mechanism.

Opioid antagonist naloxone potentiates anxiogenic-like action of cholecystokinin agonists in elevated plus-maze.

This study investigated the interplay of cholecystokinin (CCK) and endogenous opioid peptides in the regulation of anxiety. The acute administration of non-selective CCK agonist caerulein (1 and 5 microg/kg) and a selective CCK(B) receptor agonist BOC-CCK-4 (1, 10 and 50 microg/kg) induced a dose-dependent anxiogenic-like action in the plus-maze model of anxiety. BOC-CCK-4 displayed a similar efficacy with caerulein, indicating that the described effect was mediated via CCK(B) receptor subtype. The opioid antagonist naloxone itself (0.5 mg/kg) did not change the exploratory activity of rats in the plus-maze. However, the combination of naloxone with the sub-effective doses of caerulein (1 microg/kg) and BOC-CCK-4 (1 microg/kg) induced a significant inhibition of exploratory behaviour in rats. Accordingly, CCK and endogenous opioid peptides have an antagonistic role in the exploratory model of anxiety in rats.

7-Nitroindazole, a nitric oxide synthase inhibitor, has anxiolytic-like properties in exploratory models of anxiety.

The action of the novel nitric oxide synthase (NOS) inhibitor 7-nitroindazole (7-NI) was studied in different exploratory models of anxiety. In the rat plus-maze test, 7-NI potently increased time spent on open arms and percentage of open arm visits in a dose dependent manner with the minimal effective dose of 40 mg/kg. 7-NI caused an anxiolytic-like effect in the rat social interaction test. The minimal dose increasing social interaction time was 20 mg/kg. However, the drug also produced a clear sedative effect occurring even at smaller doses (10 mg/kg) in the open field test. 7-NI also showed an anxiolytic-like profile in the mouse light-dark compartment test and in the elevated plus-maze test, but the doses required were higher (80-120 mg/kg) than in rat models. Also, the sedative effect occurred at these doses in open field. We failed to demonstrate any effect of L-arginine either in the rat elevated plus-maze test or in the open field test at doses up to 600 mg/kg IP. These results indicate that there are no major interspecies differences between rats and mice in respect of action of 7-NI. The clear anxiolytic-like action of the nitric oxide synthase inhibitor in four different models shows that nitric oxide is involved in the process of anxiety and that NOS could be a new target in developing anxiolytic drugs.

Relation of exploratory behavior of rats in elevated plus-maze to brain receptor binding properties and serum growth hormone levels.

Forty-five male Wistar rats were selected according to their behavior in the elevated plus-maze. They were separated as follows: animals with low exploratory activity ('anxious'), an 'intermediate' group and animals having high exploratory activity ('non-anxious'). Various receptor binding studies and hormonal assays were also performed in these selected rats. The affinity of 5-hydroxytryptamine 5-HT2A receptors in the frontal cortex was lower in the 'anxious' rats compared to home-cage controls and 'non-anxious' animals. Moreover, the number of cholecystokinin (CCK) receptors in the hippocampus was significantly elevated in the 'anxious' group compared to home-cage control animals. The blood levels of growth hormone (GH) were significantly lower in the 'non-anxious' rats compared to 'anxious' counterparts. In conclusion, it seems likely that the decreased exploratory activity of rats is related to the increased 5-hydroxytryptamine (5-HT) and CCK mediated neurotransmission in the brain. The different serum levels of GH in the selected rats probably reflect alterations in the activity of 5-HT and CCK.

Nitric oxide mediates caerulein-induced suppression of locomotor activity.

Caerulein, a non-selective agonist of cholecystokinin (CCK) receptors, is shown to suppress locomotor activity in rodents via stimulation of CCK(A) receptors. In the present study we examined the possible involvement of nitric oxide (NO) in caerulein-induced hypolocomotion in rats. Caerulein (10 microg/kg) markedly decreased the horizontal and vertical components of locomotor activity in rats measured in dark motility boxes. Pretreatment with a nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME), at 5 mg/kg i.p., abolished the inhibiting action of caerulein on the horizontal activity, but did not affect the reduced frequency of rearing. The other doses of L-NAME (1, 10 and 20 mg/kg) were ineffective against caerulein. As L-NAME at this dose range does not stimulate locomotor activity, it is likely that NO is involved in the motor suppressant effect of systemically administered caerulein.

Cell type specific regulation of COUP-TF II promoter activity.

COUP-TF family orphan receptors regulate activity of ligand-activated nuclear hormone receptors or function independently in the regulation of gene expression. COUP-TF II has a complex expression pattern suggesting that different mechanisms are involved in the regulation of its expression. We isolated the 5' regulatory region of the mouse COUP-TF II gene and demonstrated that the basal promoter is localized in a -200 bp region 5' from the transcription start site. All-trans retinoic acid and dibutyryl cyclic AMP have cell type specific effects on COUP-TF II promoter activity. The effect of cyclic AMP is mediated by the cyclic AMP response element that is localized 74 nucleotides upstream from the major transcriptional start. In vitro promoter analyses also demonstrated that the effect of all-trans RA is not directly mediated by the binding of RARs or RXRs to the promoter sequence.

Orphan receptor COUP-TF I antagonizes retinoic acid-induced neuronal differentiation.

Chicken ovalbumin upstream promoter-transcription factors (COUP-TF) are expressed in the developing nervous system and interact with nuclear hormone receptors to regulate expression of different genes. The role of COUP-TF orphan receptors in neurogenesis is virtually unknown. To study the possible function of COUP-TF I during neuronal differentiation, we generated COUP-TF I overexpressing teratocarcinoma PCC7 cell lines and analyzed retinoic acid (RA)-induced neuronal differentiation of these cells. COUP-TF I overexpression results in the blockade of morphological differentiation after induction to differentiate. COUP-TF I represses expression of microtubule-associated protein 2 (MAP2) gene and delays induction of growth-associated protein 43 (GAP43) gene expression. In contrast, expression of the neurofilament light subunit (NF-L) gene is not affected by COUP-TF I overexpression during neuronal differentiation. Also, cells overexpressing COUP-TF I do not stop proliferating after RA and dBcAMP treatment and possess suppressed transcriptional activation from different RA response elements. These results suggest that COUP-TF I plays an important role in regulating RA-induced neuronal differentiation.

Isolation of genes which block neuronal differentiation of teratocarcinoma PCC7 cells.

Since several viral oncoproteins block differentiation and induce proliferation of differentiated cells, we developed an expression screening method to isolate cDNAs which block neuronal differentiation and induce proliferation of teratocarcinoma cells. Mouse E2F1, RNP-1, and RNP-2 (Regulator of Neuronal Proliferation) were isolated using the developed screening method. Overexpression of E2F1, RNP-1, and RNP-2 cDNAs in neuronally differentiated teratocarcinoma PCC7 cells results in blocking differentiation and initiation of proliferation. Also, expression of RNP-1 and RNP-2 blocks the expression of neurofilament-L and GAP-43 genes in PCC7 cells.

Role of N-methyl-D-aspartic acid and cholecystokinin receptors in apomorphine-induced aggressive behaviour in rats.

We studied the aggressive behaviour induced by repeated treatment with apomorphine, a dopamine agonist (0.5 mg/kg s.c. twice daily, 10 days), in rats. The first signs of defensive aggressiveness appeared on the third day of apomorphine treatment and were generally seen on the 7th day. Aggressiveness induced by a challenge dose of apomorphine (0.5 mg/kg s.c.) on the 11th day was antagonized by haloperidol (0.05 and 0.1 mg/kg i.p.) and clozapine (10 mg/kg i.p.). An antagonist of N-methyl-D-aspartate (NMDA)-gated channels, dizocilpine (MK-801), also blocked the aggressive behaviour at 0.25 and 0.5 mg/kg i.p. but caused ataxia. When dizocilpine (0.25 mg/kg i.p.) and apomorphine were coadministered for 10 days, aggressive behaviour did not develop. At 0.025 mg/kg i.p., dizocilpine even accelerated the appearance of apomorphine-induced aggressive behaviour, which manifested on the 3rd day in all rats. In a separate study, a 7-day treatment with dizocilpine (0.25-1 mg/kg i.p.) of rats, sensitized by a prior 10-day apomorphine treatment, did not reverse the established aggressive behaviour. The coadministration of apomorphine and cholecystokinin (CCK) -A or -B antagonists, devazepide or L-365,260 (0.01-2.5 mg/kg i.p.) respectively, neither affected development of apomorphine-induced aggressive behaviour nor intensity of aggressiveness in the sensitized rats. In binding studies neither density nor affinity of striatal dopamine D2 receptors was changed by acute or chronic apomorphine treatment. The number of [3H]pCCK-8 binding sites in the frontal cortex increased already after a single injection of apomorphine.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential expression and distinct DNA-binding specificity of ME1a and ME2 suggest a unique role during differentiation and neuronal plasticity.

Class A basic-helix-loop-helix (bHLH) proteins have been referred to as ubiquitous and are believed to have redundant functions. They are involved in the control of several developmental pathways, such as neurogenesis and myogenesis. To rationalize the existence of multiple class A bHLH proteins, we evaluated the differences and similarities between ME1a and ME2, two class A bHLH proteins, highly expressed in differentiating neuronal cells. In situ hybridization analyses reveal that ME1a and ME2 are characterized by distinguishable patterns of expression in areas of the adult mouse brain where neuronal plasticity occurs. Also, DNA-binding assays show that both proteins bind to E-boxes as homodimers and heterodimers, and show differences in their DNA-binding specificities, which suggest selective interactions with different binding sites of target genes. In addition, in vitro DNA-binding assays demonstrate that Id2 forms heterodimers with ME1a and ME2. As a result of these interactions, their DNA-binding activity is abolished. Furthermore, overexpression of Id2 in neuronal cells suppresses ME1a and ME2 transcriptional activity. Based on our data, we hypothesize that ME1a and ME2 may activate gene expression of different target genes and therefore are likely to be differently involved during neurogenesis.

Pharmacological comparison of antipsychotic drugs and sigma-antagonists in rodents.

We compared antipsychotic drugs (haloperidol, chlorpromazine and clozapine) and sigma antagonists (remoxipride, cinuperone, alpha-(4-fluorophenyl)-4-(-fluoro-2-pyrimidinyl)-1-piperazine butanol (BMY 14802) and rimcazole) in the radio-ligand binding and behavioural experiments in rodents. A good correlation was established between the affinity of compounds at dopamine2-receptors in the striatum and their ability to block apomorphine-, amphetamine- and quipazine-induced behavioural effects in rodents. By contrast, no correlation was found between the behavioural effects of these drugs and their affinity at dopamine1-5-HT2- and sigma receptors. The rank order of potency among the studied antipsychotic drugs in the behavioural tests and at dopamine2-receptors was following: haloperidol >> chlorpromazine > or = clozapine. The effectiveness of chlorpromazine and clozapine was nearly similar against apomorphine-induced aggressiveness and yawning, whereas at 5-HT2-receptors clozapine was more active than chlorpromazine. The weak activity of sigma antagonists at dopamine2 receptors could be a possible reason why these compounds were less effective in the behavioural studies compared to antipsychotic drugs. However, the antagonism of remoxipride against apomorphine-induced stereotypy and aggressiveness is not related to its activity at sigma receptors, because the other sigma antagonists did not block these effects of apomorphine. It is probable that remoxipride exerts its action through blocking of dopamine2 receptors. In conclusion, the present study revealed only weak activity of sigma antagonists in the behavioural models widely used to study the antipsychotic drugs. Therefore, the antipsychotic activity of sigma antagonists is doubtful.

Expression of basic-helix-loop-helix transcription factor ME2 during brain development and in the regions of neuronal plasticity in the adult brain.

We report the isolation of a cDNA encoding the mouse class A bHLH transcription factor ME2 and the analysis of its expression. ME2 is expressed in the cerebral cortex, Purkinje and granule cell layers of the cerebellum, olfactory neuroepithelium, pyramidal cells of hippocampal layers CA1-CA4, and in the granular cells of the dentate gyrus. The specific expression of ME2 during development and in the regions of neuronal plasticity in the adult brain suggest that ME2 may have a regulatory function in developmental processes as well as during neuronal plasticity.

Anti-exploratory effect of N-methyl-D-aspartate in elevated plus-maze. Involvement of NMDA and CCK receptors.

N-Methyl-D-aspartate (NMDA, 20 mg/kg) produced a clear decrease in mouse exploration in open parts of an elevated plus-maze. Paradoxically, 40 mg/kg NMDA did not modify the behavior of the mice in the plus-maze. NMDA at a dose of 80 mg/kg again depressed the exploratory activity of mice, but this effect was accompanied with tremor and compulsive tail biting. The 'anti-exploratory' dose of NMDA (20 mg/kg) increased, whereas the 'tremorigenic' dose (80 mg/kg) significantly decreased the number of cholecystokinin (CCK) binding sites in the mouse cerebral cortex. The competitive NMDA antagonist (+/-)-CPP (2.5-5 mg/kg) and the non-competitive antagonist MK-801 (0.25 mg/kg) antagonized the anti-exploratory effect of NMDA (20 mg/kg). The tricyclic antidepressant imipramine (5 mg/kg, but not 1 or 10 mg/kg) also attenuated the inhibition of exploratory activity induced by NMDA. Of three CCK receptor antagonists tested, the unselective CCK antagonist proglumide (1 mg/kg, but not 0.1 and 10 mg/kg) significantly opposed the anti-exploratory action of NMDA. The selective CCK antagonists L-365,260 (1 microgram/kg) and devazepide (1 microgram/kg) were evidently weaker antagonists of NMDA. Furthermore, 10 micrograms/kg of L-365,260, a CCK-B receptor antagonist, and 1 mg/kg of devazepide, a CCK-A receptor antagonist, even tended to augment the effect of NMDA in the plus-maze. The results of the present study seem to give some support to the notion that not only NMDA receptors, but also CCK-ergic mechanisms are involved in the modulation of anti-exploratory action of NMDA in the elevated plus-maze.

Changes at cholecystokinin receptors induced by long-term treatment with diazepam and haloperidol.

Fourteen days administration of haloperidol (1 mg/kg daily) prevented the motor depressant effect of caerulein (an agonist at cholecystokinin receptors, 15 micrograms/kg) and the antagonistic effect of caerulein (100 micrograms/kg) against (+)-amphetamine (5 mg/kg) induced hyperlocomotion in mice. The antiaggressive effect of caerulein (40 micrograms/kg) in saline-treated mice was replaced by increased aggressiveness after long-term haloperidol and diazepam (5 mg/kg daily) treatment. The anticonvulsant effect of caerulein (125 micrograms/kg) against picrotoxin (10 mg/kg) induced seizures was abolished after 14 days diazepam, but not after haloperidol, treatment. The above described changes in the mouse behaviour are probably related to the development of subsensitivity at CCKA receptors, whereas the CCKB receptor subtype becomes more sensitized to the action of caerulein after long-term haloperidol and diazepam treatment.

The involvement of sigma and phencyclidine receptors in the action of antipsychotic drugs.

An atypical antipsychotic drug clozapine and a selective sigma antagonist BMY 14802 were significantly less effective in the behavioural experiments (against apomorphine, d-amphetamine and MK-801), as well in the radioligand binding studies against 3H-spiperone (dopamine2-receptors) and 3H-haloperidol (sigma receptors) in the rat brain, as compared to a typical antipsychotic compound haloperidol. Contrary to haloperidol and BMY 14802, clozapine was a relatively selective antagonist of MK-801-induced motor excitation in the mouse. A nearly 3-fold lower dose of clozapine was needed to block the effect of MK-801 (6.4 mumol/kg) as compared to the action of amphetamine (17 mumol/kg). Haloperidol and clozapine, but not BMY 14802, antagonized apomorphine-induced aggressiveness in the rat. After long-term treatment (for 15 days) with BMY 14802 (10 mg/kg daily), haloperidol (0.5 mg/kg daily) and clozapine (10 mg/kg daily) the motor depressant effect of apomorphine (0.15 mg/kg) was reversed. Chronic haloperidol treatment, but not administration of BMY 14802 and clozapine, increased the number of dopamine2-receptors in the rat brain. BMY 14802 caused upregulation of sigma receptors in frontal cortex, whereas haloperidol induced the opposite change in cerebellum. Repeated treatment with clozapine significantly augmented the motor stimulating effect of MK-801 in rats. Simultaneously with a behavioural change the density of 3H-TCP binding sites in the rat forebrain was elevated after long-term treatment with clozapine, probably indicating the involvement of PCP binding sites at NMDA channel in the action of clozapine.

Differential involvement of CCK-A and CCK-B receptors in the regulation of locomotor activity in the mouse.

The influence of the CCK-A antagonist devazepide and the CCK-B/gastrin antagonist L-365,260 on the locomotor activity of mice was studied. Devazepide and L-365,260 had opposite effects on spontaneous locomotor activity, and on caerulein- and apomorphine-induced hypomotility in the mouse. Devazepide in high doses (0.1-1 mg/kg IP) reduced spontaneous motor activity, whereas L-365,260 at a high dose (1 mg/kg IP) increased the activity of mice. Devazepide (0.1-10 micrograms/kg) moderately antagonized the sedative effect of apomorphine (0.1 mg/kg SC) and caerulein (25 micrograms/kg SC), whereas L-365,260 (1-10 micrograms/kg) significantly potentiated the actions of dopamine and CCK agonists. Concomitant administration of caerulein (15 micrograms/kg SC) and apomorphine (0.1 mg/kg SC) caused an almost complete loss of locomotor activity in the mouse. Devazepide and L-365,260 (0.1-10 micrograms/kg) were completely ineffective against caerulein-induced potentiation of apomorphine hypomotility. Devazepide in high doses (0.1-1 mg/kg), reducing the spontaneous motor activity of mice, counteracted the motor excitation induced by d-amphetamine (5 mg/kg IP). The CCK agonist caerulein (100 micrograms/kg SC) had a similar antiamphetamine effect. Devazepide (1-100 micrograms/kg) and L-365,260 (1 micrograms/kg) reversed completely the antiamphetamine effect of caerulein. The results of present study reflect apparently distinct role of CCK-A and CCK-B receptors in the regulation of motor activity. The opposite effect of devazepide and L-365,260 on caerulein- and apomorphine-induced hypolocomotion is probably related to the antagonistic role of CCK-A and CCK-B receptor subtypes in the regulation of mesencephalic dopaminergic neurons. The antiamphetamine effect of caerulein is possibly linked to the stimulation of CCK-A receptors in the mouse brain, whereas the blockade of both subtypes of the CCK-8 receptor is involved in the antiamphetamine effect of devazepide.

Similar behavioral and biochemical effects of long-term haloperidol and caerulein treatment in albino mice.

Behavioral and biochemical experiments on male albino mice have revealed similar effects after the cessation of repeated (15 days) haloperidol (0.5 mg/kg daily IP) and caerulein (0.1 mg/kg daily SC) treatment. Tolerance developed to the action of muscimol (a GABA-A agonist, 1 mg/kg IP), caerulein (a CCK-8 agonist, 15 micrograms/kg SC) and flumazenil (a benzodiazepine antagonist, 10 mg/kg IP). Muscimol and caerulein were not able to suppress the motor activity of mice after 15 days treatment with haloperidol and caerulein. Flumazenil, which increased motor activity in saline-treated animals, also failed to affect activity after extended haloperidol or caerulein treatment. In contrast, the motor excitation induced by amphetamine (an indirect dopamine agonist, 3 mg/kg IP) was increased after haloperidol or caerulein administration. In radioligand binding studies the density of dopamine-2-receptors in striatum, opioid receptors in mesolimbic structures, and benzodiazepine and GABA-A receptors in brainstem was significantly elevated after long-term haloperidol or caerulein treatment. Simultaneously, the number of CCK-8, benzodiazepine and GABA-A receptors in cerebral cortex was decreased. It is probable that CCK-8-ergic mechanisms are involved closely in the action of repeated haloperidol treatment. CCK-8 seems to modulate the action of haloperidol through altering the sensitivity of dopamine, opioid, GABA-A and benzodiazepine receptors.