Altered pain sensitivity and morphine-induced anti-nociception in mice lacking CCK<Subscript>2</Subscript> receptors

Rationale. Cholecystokinin (CCK) interacts with the endopioid system in the regulation of various physiological functions, including the control of pain sensitivity, motor activity and emotional behaviour. Objective. The aim of the present work was to study the pain sensitivity, morphine-induced antinociception and density of opioid receptors in mice lacking CCK₂ receptors. Methods. Plantar analgesia and hotplate tests were used to evaluate pain sensitivity and morphine-induced antinociception. The parameters of opioid receptors were analysed by using [³H]-diprenorphine binding. Results. In the plantar analgesia test the latency of hind paw withdrawal was significantly increased in CCK₂ receptor deficient mice compared to wild-type (+/+) littermates. The treatment with saline reversed the reduced pain sensitivity in heterozygous (+/−) and homozygous (−/−) mice. The administration of morphine (1 mg/kg) induced a significantly stronger antinociceptive effect in homozygous (−/−) mice compared with wild-type (+/+) animals. In the hotplate test, only homozygous (−/−) mutant mice displayed the delayed latency of hind paw licking/shaking in comparison with wild-type (+/+) mice. The injection of saline and isolation of mice for 30 min reversed the delayed response in homozygous (−/−) mice. However, in this test, the anti-nociceptive action of morphine (5–10 mg/kg) in mutant mice did not differ from that in wild-type (+/+) littermates. By contrast, the jump latency was decreased in both homozygous (−/−) and heterozygous (+/−) mice in the hotplate test. The increased density of opioid receptors was established in the striatum of homozygous (−/−) mice. Conclusion. It is apparent that the targeted mutagenesis of the CCK₂ receptor gene has different effects on the sensitivity of opioid receptors in various brain structures. This is a probable reason for the altered pain sensitivity and morphine-induced antinociception in mutant mice compared to wild-type (+/+) littermates. Electronic Publication     

Pharmacological analysis of the interaction of antimuscarinic drugs at M<Subscript>2</Subscript> and M<Subscript>3</Subscript> muscarinic receptors in vivo using the pithed rat assay

Muscarinic receptor antagonists form the mainstay of the therapeutic options for airway, bladder, and gastrointestinal smooth muscle disorders. Both M₂ and M₃ muscarinic receptors are involved in mediating smooth muscle contractility, although the relative functional contribution of each subtype, especially in the disease state, is unclear. Because the potency and selectivity of compounds for a given receptor in an in vivo setting can be dissimilar to that observed in an in vitro system, we developed an in vivo assay to simultaneously determine the absolute potency and selectivity of muscarinic receptor antagonists at M₂ and M₃ receptors using the pithed rat. Methacholine (MCh)-induced bradycardia and depressor responses were used as surrogate functional endpoints for M₂ and M₃ receptor activation, respectively. The influence of the muscarinic antagonists, tolterodine, oxybutynin, darifenacin, Ro 320-6206, solifenacin, or tiotropium on the MCh-induced responses were studied. The estimated DR₁₀ values (dose producing a tenfold shift in the MCh curve) of tolterodine, oxybutynin, darifenacin, Ro 320-6206, solifenacin, and tiotropium for the M₂ muscarinic receptor-mediated bradycardia were 0.22, 1.18, ∼2.6, 0.025, 0.40, and 0.0026 mg/kg, respectively, and 0.14, 0.18, 0.11, 3.0, 0.18, and 0.0017 mg/kg, respectively, for the M₃ muscarinic receptor-mediated depressor response. In a separate set of experiments, a single intravenous dose of tiotropium was administered before a MCh curve at 1, 3, 6, or 9 h to determine if tiotropium exhibited time-dependent selectivity for the M₃ receptor as has been reported from in vitro studies. The results indicate a slight preference of tiotropium for the M₃ receptor at later time points. The pithed rat assay may serve useful for elucidating the functional contribution of M₂ and M₃ receptors to the in vivo pharmacological effects of antagonists in disease animal models.     

The role of M<Subscript>1</Subscript> muscarinic cholinergic receptors in the discriminative stimulus properties of <Emphasis Type="Italic">N</Emphasis>-desmethylclozapine and the atypical antipsychotic drug clozapine in rats

Rationale  The discriminative stimulus properties of clozapine (CLZ) have been studied for decades because it remains the prototype for atypical antipsychotic drug effects and yet is unique in many ways, including increased efficacy in treatment-resistant schizophrenia and in reducing suicidality. Recent studies have indicated that the active CLZ metabolite N-desmethylclozapine (NDMC) may play a role in mediating the cognitive efficacy of CLZ and may also have atypical antipsychotic properties. Objectives  The present study sought to determine if NDMC has discriminative stimulus properties similar to that of its parent drug CLZ. Materials and methods  Rats were trained to discriminate 1.25 mg/kg CLZ from vehicle in a two-choice drug discrimination task. Results  Although NDMC (2.5–20.0 mg/kg) failed to substitute for CLZ, the combination of NDMC (5.0 and 10.0 mg/kg) with a low dose (0.3125 mg/kg) of CLZ produced full substitution (>80% CLZ-appropriate responding) for the 1.25 mg/kg CLZ training dose. Co-administration of the M1-preferring receptor antagonist trihexyphenidyl (6.0 mg/kg) with a 5.0 mg/kg dose of NDMC produced partial substitution (>60% to <80% CLZ-appropriate responding) for CLZ, while administration of trihexyphenidyl alone (0.3–12.0 mg/kg) failed to substitute for CLZ. Conclusions  These findings suggest that NDMC produces discriminative stimulus effects that are different from those elicited by its parent drug CLZ. This difference may be due to the agonist properties of NDMC at M₁ muscarinic cholinergic receptors.     

Targeted invalidation of CCK<Subscript>2</Subscript> receptor gene induces anxiolytic-like action in light–dark exploration,but not in fear conditioning test

Rationale Evidence suggests that γ-aminobutyric acid (GABA) and cholecystokinin (CCK) have opposite roles in the regulation of anxiety. Objectives The aim of our work was to study the behaviour of CCK₂ receptor deficient mice in light–dark exploration and fear conditioning tests. Moreover, the action of diazepam and methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM), having the opposite effect on GABA_A receptors, was evaluated on the exploratory behaviour in these mice. Expression levels of GABA_A receptor subunit genes were also measured. Methods Light–dark exploration and fear conditioning tests were used to determine changes in anxiety of mice. The action of diazepam (0.5–2 mg/kg i.p.) and DMCM (0.25–1 mg/kg i.p.) was studied in the light–dark box. The effect of DMCM was also evaluated in the motor activity test to demonstrate that its anti-exploratory action was not related to motor suppression. Expression levels of GABA_A receptor subunit genes were determined by means of real-time polymerase chain reaction (qRT-PCR). Results Female mice lacking CCK₂ receptors displayed increased exploratory activity in the light–dark box compared to their wild-type (+/+) littermates. Locomotor activity in the motility boxes and the intensity of freezing did not differ in wild-type (+/+) and homozygous (−/−) mice. Treatment with diazepam (0.5 mg/kg) increased the number of transitions in wild-type (+/+) animals, whereas in homozygous (−/−) mice diazepam (0.5–2 mg/kg) reduced exploratory activity. Administration of DMCM (0.25–1 mg/kg) induced an anxiogenic-like effect in homozygous (−/−) mice, but did not change their locomotor activity. Gene expression analysis established a 1.6-fold increase in the expression of the α2 subunit of GABA_A receptors in the frontal cortex of homozygous (−/−) mice. Conclusion Genetic invalidation of CCK₂ receptors induced an anxiolytic-like action in exploratory, but not in conditioned models of anxiety. The observed reduction in anxiety in homozygous (−/−) mice is probably related to an increased function of GABAergic system in the brain.     

Locomotor and discriminative-stimulus effects of cocaine in dopamine D<Subscript>5</Subscript> receptor knockout mice

Rationale Dopamine D₁-like antagonists block several effects of cocaine, including its locomotor-stimulant and discriminative-stimulus effects. Because these compounds generally lack selectivity among the dopamine D₁ and D₅ receptors, the specific roles of the subtypes have not been determined. Objectives Dopamine D₅ receptor knockout (DA D₅R KO), heterozygous (HET) and wild-type (WT) mice were used to study the role of D₅ dopamine receptors in the effects of cocaine. In addition, effects of the D₁-like antagonist, SCH 39166 were also studied to further clarify the roles of D₁ and D₅ dopamine receptors in the discriminative-stimulus effects of cocaine. Methods DA D₅R KO, HET and WT mice were treated with cocaine (3–30 mg/kg) or vehicle and their horizontal locomotor activity was assessed. The mice were also trained to discriminate IP injections of saline from cocaine (10 mg/kg) using a two-lever food-reinforcement (FR10) procedure. Doses of cocaine (1.0–10 mg/kg) were administered 5 min before 15-min test-sessions. Results Cocaine dose-dependently stimulated activity in each genotype, with the highest level of activity induced in the DA D₅R WT mice. Both DA D₅R KO and HET mice showed reduced levels of horizontal activity compared to WT mice. All three genotypes acquired the discrimination of 10 mg/kg cocaine; doses of 1.0–10.0 mg/kg produced dose-related increases in the number of cocaine-appropriate responses. SCH 39166, at inactive to fully active doses (0.01–0.1 mg/kg) produced predominately saline-appropriate responding. SCH 39166 produced a dose-dependent rightward shift in the cocaine dose-effect curve in all genotypes, with similar apparent affinities. Conclusions The present data suggest an involvement of DA D₅R in the locomotor stimulant effects of cocaine. In addition, the data indicate that there is little involvement of the DA D₅R in the discriminative-stimulus effects of cocaine. In addition, the antagonism data suggest a role of the D₁ receptor in the behavioral effects of cocaine.     

Impaired M3 and enhanced M2 muscarinic receptor contractile function in a streptozotocin model of mouse diabetic urinary bladder

We investigated the contractile roles of M₂ and M₃ muscarinic receptors in urinary bladder from streptozotocin-treated mice. Wild-type and M₂ muscarinic receptor knockout (M₂ KO) mice were given a single injection of vehicle or streptozotocin (125 mg kg⁻¹) 2–24 weeks prior to bladder assays. The effect of forskolin on contractions elicited to the muscarinic agonist, oxotremorine-M, was measured in isolated urinary bladder (intact or denuded of urothelium). Denuded urinary bladder from vehicle-treated wild-type and M₂ KO mice exhibited similar contractile responses to oxotremorine-M, when contraction was normalized relative to that elicited by KCl (50 mM). Eight to 9 weeks after streptozotocin treatment, the EC₅₀ value of oxotremorine-M increased 3.1-fold in urinary bladder from the M₂ KO mouse (N = 5) compared to wild type (N = 6; P < 0.001). Analogous changes were observed in intact bladder. In denuded urinary bladder from vehicle-treated mice, forskolin (5 μM) caused a much greater inhibition of contraction in M₂ KO bladder compared to wild type. Following streptozotocin treatment, this forskolin effect increased 1.6-fold (P = 0.032). At the 20- to 24-week time point, the forskolin effect increased 1.7-fold for denuded as well as intact bladders (P = 0.036, 0.01, respectively). Although streptozotocin treatment inhibits M₃ receptor-mediated contraction in denuded urinary bladder, muscarinic contractile function is maintained in wild-type bladder by enhanced M₂ contractile function. M₂ receptor activation opposes forskolin-induced relaxation of the urinary bladder, and this M₂ function is enhanced following streptozotocin treatment.     

The 5-HT<Subscript>1A</Subscript> receptor agonist MKC-242 reverses isolation rearing-induced deficits of prepulse inhibition in mice

Rationale Prepulse inhibition (PPI) of startle provides an operational measure of sensorimotor gating in which a weak stimulus presented prior to a startling stimulus reduces the startle response. PPI deficits observed in schizophrenia patients can be modeled in rats by individual housing from weaning until adulthood. The deficits in PPI produced by isolation rearing can be reversed by antipsychotics. We previously found that (S)-5-[3-[(1,4-benzodioxan-2-ylmethyl)amino]propoxy]-1,3-benzodioxole HCl (MKC-242), a highly potent 5-HT_1A receptor agonist, reduced aggressive behavior selectively in isolation-reared mice. Objective This study examines whether isolation rearing of mice produces PPI deficits and whether PPI deficits are attenuated by 5-HT_1A receptor activation. Methods Male ddY mice, 4 weeks old, were housed for more than 6 weeks singly or in groups of five or six. The PPI of the acoustic startle response was measured using SR-LAB systems. Results The PPI was less in isolation-reared mice than in group-reared mice. Oral administration of MKC-242 at 0.1–0.3 mg/kg reversed PPI deficits in isolation-reared mice, although it did not affect PPI in group-reared mice. MKC-242 did not affect MK-801-induced and apomorphine-induced PPI deficits in group-reared mice. The reversal by MKC-242 of isolation-induced PPI deficits was antagonized by the 5-HT_1A receptor antagonist WAY100635 at low doses. Conclusion These results suggest that isolation rearing produces deficits in sensorimotor gating in mice that are reversible by activation of 5-HT_1A receptors, probably somatodendritic 5-HT_1A autoreceptors.     

Clozapine binds preferentially to cortical D1-like dopamine receptors in the primate brain: a PET study

Rationale The D₁-like dopamine receptors have been suggested to play a role in the pathophysiology and treatment of schizophrenia. Previous positron emission tomography studies have demonstrated that the atypical antipsychotic clozapine occupies D₁-like dopamine receptors in the striatum in clozapine-treated patients. Objectives The aim of the present study was to compare striatal and cortical D₁-like dopamine receptor occupancy by clozapine in the primate brain. Methods Three monkeys were each examined three times at the same day with the radioligand (+)−[¹¹C]NNC 112. The first measurement was at baseline conditions, the second after 1.5 mg/kg and the third after 6 mg/kg clozapine IV. To compare regional levels of nonspecific binding in brain regions, an additional monkey was examined using the inactive enantiomer (−)−[¹¹C]NNC 112. Receptor occupancy was calculated using both the equilibrium–ratio analysis and the simplified reference tissue model. Results After 1.5 mg/kg the D₁-like dopamine receptor occupancy ranged from 30 to 38% in the striatum, whereas the range was 51 to 57% in the frontal cortex. After 6.0 mg/kg the occupancy was 53 to 64% in the striatum and 63 to 83% in the frontal cortex. The differences between striatal and cortical D₁-like receptors occupancy were between 12 and 25%. The study with (−)−[¹¹C]NNC 112 did not show regional differences in nonspecific binding that might explain the regional differences in occupancy. Conclusions The higher D₁-like dopamine receptor occupancy in the frontal cortex may reflect a different distribution of the D₁ and D₅ dopamine receptor subtypes among brain regions and different affinity of clozapine for the two subtypes. The finding supports the suggestion that binding to D₁-like dopamine receptors may explain clozapine’s atypical drug actions.     

Activation of GABAB receptors reverses spontaneous gating deficits in juvenile DBA/2J mice

Rationale Gamma-amino-butyric acid (GABA)_B receptors play a key role in the pathophysiology of psychotic disorders. We previously reported that baclofen, the prototypical GABA_B agonist, elicits antipsychotic-like effects in the rat paradigm of prepulse inhibition (PPI) of the startle, a highly validated animal model of schizophrenia. Objectives We studied the role of GABA_B receptors in the spontaneous PPI deficits displayed by DBA/2J mice. Materials and methods We tested the effects of baclofen (1.25–5 mg/kg, intraperitoneal [i.p.]) in DBA/2J and C57BL/6J mice, in comparison to the antipsychotic drugs haloperidol (1 mg/kg, i.p.) and clozapine (5 mg/kg, i.p.). Furthermore, we investigated the expression of GABA_B receptors in the brain of DBA/2J and C57BL/6J mice by quantitative autoradiography. Results Baclofen dose-dependently restored PPI deficit in DBA/2J mice, in a fashion similar to the antipsychotic clozapine (5 mg/kg, i.p.). This effect was reversed by pretreatment with the GABA_B antagonist SCH50211 (50 mg/kg, i.p.). In contrast, baclofen did not affect PPI in C57BL/6J mice. Finally, quantitative autoradiographic analyses assessed a lower GABA_B receptor expression in DBA/2J mice in comparison to C57BL/6J controls in the prefrontal cortex and hippocampus but not in other brain regions. Conclusions Our data highlight GABA_B receptors as an important substrate for sensorimotor gating control in DBA/2J mice and encourage further investigations on the role of GABA_B receptors in sensorimotor gating, as well as in the pathophysiology of psychotic disturbances. M. Paola Castelli, Giampaolo Mereu, and Francesco Marrosu have contributed equally to the study.     

Targeted mutation of CCK2 receptor gene modifies the behavioural effects of diazepam in female mice

Rationale Evidence suggests that GABA and CCK have opposite roles in the regulation of anxiety. Objective The aim of the present work was to study diazepam-induced anxiolytic-like action and impairment of motor co-ordination, and the parameters of benzodiazepine receptors in mice lacking CCK₂ receptors. Methods The action of diazepam (0.5–3 mg/kg IP) was studied in the elevated plus-maze model of anxiety and rotarod test using mice lacking CCK₂ receptors. The parameters of benzodiazepine receptors were analysed using [³H]-flunitrazepam binding. Results In the plus-maze test, the exploratory activity of the homozygous (−/−) mice was significantly higher compared to their wild-type (+/+) littermates. However, the wild-type (+/+) mice displayed higher sensitivity to the anxiolytic-like action of diazepam. Even the lowest dose of diazepam (0.5 mg/kg) induced a significant increase of open arm entries in the wild-type (+/+) mice. A similar effect in the homozygous (−/−) mice was established after the administration of diazepam 1 mg/kg. The highest dose of diazepam (3 mg/kg) caused a prominent anxiolytic-like effect in the wild-type (+/+) mice, whereas in the homozygous (−/−) animals suppression of locomotor activity was evident. The performance of the homozygous (−/−) mice in the rotarod test did not differ from that of the wild-type (+/+) littermates. However, a difference between the wild-type (+/+) and homozygous (−/−) animals became evident after treatment with diazepam. Diazepam (0.5 and 3 mg/kg) induced significantly stronger impairment of motor co-ordination in the homozygous (−/−) mice compared to their wild-type (+/+) littermates. The density of benzodiazepine binding sites was increased in the cerebellum, but not in the cerebral cortex and hippocampus, of the homozygous (−/−) mice. Conclusions Female mice lacking CCK₂ receptors are less anxious than their wild-type (+/+) littermates. The reduced anxiety in homozygous (−/−) mice probably explains why the administration of a higher dose of diazepam is necessary to induce an anxiolytic-like action in these animals. The highest dose of diazepam (3 mg/kg) induced significantly stronger suppression of locomotor activity and impairment of motor co-ordination in the homozygous (−/−) mice compared to the wild-type (+/+) littermates. The increase in the action of diazepam is probably related to the elevated density of benzodiazepine receptors in the cerebellum of homozygous (−/−) mice. The present study seems to be in favour of increased tone of the GABAergic system in mice without CCK₂ receptors.     

Comparison of the effects of clozapine and 8-hydroxy-2-(di-<Emphasis Type="Italic">n</Emphasis>-propylamino)tetralin (8-OH-DPAT) on progressive ratio schedule performance: evidence against the involvement of 5-HT<Subscript>1A</Subscript> receptors in the behavioural effects of clozapine

Rationale Performance on progressive ratio schedules has been proposed as a means of assessing the effects of drugs on the efficacy of reinforcers. A mathematical model (Killeen PR (1994) Mathematical principles of reinforcement. Behav Brain Sci 17:105–172) affords a basis for quantifying the effects of drugs on progressive ratio schedule performance. The model postulates a bitonic function relating response rate and ratio size. One parameter of the function, a, expresses the motivational effect of the reinforcer, whereas another parameter, δ, expresses the minimum time needed to execute a response, and is regarded as an index of ‘motor capacity’. Previously we found that the atypical antipsychotic clozapine increased a, indicating an increase in reinforcer efficacy; a similar effect was observed with the 5-hydroxytryptamine (5-HT)_1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). It has been suggested that some of clozapine’s behavioural effects are mediated by agonistic action at 5-HT_1A receptors. Objective This study was conducted to compare the effects of clozapine and 8-OH-DPAT on progressive ratio schedule performance. Methods Rats were trained under a time-constrained progressive ratio schedule (50-min sessions). In experiment 1, they received acute doses of clozapine (4 mg kg⁻¹) and 8-OH-DPAT (100 μg kg⁻¹), alone and in combination with the 5-HT_1A receptor antagonist N-[2-(4-[2-methoxyphenyl]-1-piperazinyl)ethyl]-N-2-yridinylcyclohexanecarboxamide (WAY-100635; 30 μg kg⁻¹). In experiment 2, the effects of clozapine (2, 4 and 8 mg kg⁻¹) and 8-OH-DPAT (25, 50 and 100 μg kg⁻¹) were compared between intact rats and rats whose 5-HTergic pathways had been ablated by 5,7-dihydroxytryptamine (5,7-DHT). Results In both experiments, clozapine and 8-OH-DPAT increased a and δ. In experiment 1, WAY-100635 abolished the effect of 8-OH-DPAT on a and δ, but did not alter clozapine’s effects on these parameters. In experiment 2, the effects of clozapine and 8-OH-DPAT did not differ between sham-lesioned and 5,7-DHT-lesioned rats. Conclusions The results confirm previous findings on the effects of clozapine and 8-OH-DPAT on progressive ratio schedule performance. 8-OH-DPAT’s effects are probably mediated by post-synaptic 5-HT_1A receptors; clozapine’s effects are mediated by a different mechanism, which does not appear to involve 5-HT_1A receptors and which does not depend upon an intact 5-HTergic pathway. Jonathan Francis Rickard (1977–2003), a gifted and dedicated Ph.D. student, made a major contribution to this work     

Differential regulation of rat peripheral 5-HT<Subscript>2A</Subscript> and 5-HT<Subscript>2B</Subscript> receptor systems: influence of drug treatment

Most studies of 5-HT₂ receptor regulation have been carried out on the central nervous system (CNS) (which expresses 5-HT_2A and 5-HT_2C receptors); very few in vitro studies have addressed the peripheral receptors 5-HT_2A and 5-HT_2B. The aim of this investigation was to compare the possible short- and long-term processes regulating these peripheral receptors in the rat.The in vitro contractile response elicited by serotonin (5-HT, 10 µM) in the rat gastric fundus (5-HT_2B receptor system) was rapid and followed by a partial fade to a steady state, in contrast with the rat thoracic aorta response (5-HT_2A receptor system), which was more stable, slower and sustained. To characterize drug-receptor interactions, cumulative concentration/response curves (CCRCs) for 5-HT were constructed ex vivo for rat tissues treated with drugs acting at these receptors. Rats were examined 4 or 24 h after a single, i.p. administration of (±)1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane [(±)DOI, 1 or 2.5 mg/kg], clozapine, cyproheptadine or rauwolscine (10 mg/kg), 48 h after a single i.p. administration of (±)DOI (2.5 mg/kg), clozapine or cyproheptadine (10 mg/kg) or 24 h after the last of with 15 daily i.p. administrations of (±)DOI (1 or 2.5 mg/kg), clozapine, cyproheptadine or rauwolscine (10 mg/kg). In the aorta, E_max (the maximum response elicited by 5-HT) was unchanged 4 h after a single dose of any of the drugs tested. However, 24 h after a single dose, E_max was lower in animals treated with (±)DOI (2.5 mg/kg), clozapine or cyproheptadine than in controls, whilst 48 h after a single dose of (±)DOI (2.5 mg/kg), clozapine or cyproheptadine there was no difference in E_max between experimental and control animals. After chronic treatment with (±)DOI (2.5 mg/kg), clozapine and cyproheptadine, E_max was lower than in controls. In the gastric fundus, E_max 4 h after a single dose of each drug was lower than in controls, and the response recovered by 24 or 48 h. Following chronic treatment, E_max was significantly lower than in controls for each drug used.These findings suggest first, that regulation of peripheral 5-HT₂ receptors (5-HT_2A and 5-HT_2B) is a functionally significant phenomenon in vivo, and occurs after administration of both agonists and antagonists. Second, the kinetics of peripheral 5-HT₂ receptor regulation were similar in both in vivo and ex vivo experiments. The 5-HT_2B receptors in rat gastric fundus are more sensitive to drug-induced regulation than the 5-HT_2A rat aortic receptors. Finally, long-term regulation of both receptors stabilizes short-term desensitization for longer.     

Potentiation of prepulse inhibition of the startle reflex in rats: pharmacological evaluation of the procedure as a model for detecting antipsychotic activity

Prepulse inhibition (PPI) of the startle reflex – whereby presentation of a weak prepulse preceding a startling pulse diminishes the amplitude of the startle reflex – is disrupted by dopamine (DA) agonists; this disruption can be reversed by antipsychotics. There are also some indications in the literature that a few antipsychotics (most notably clozapine and haloperidol) may, on their own, have effects opposite to those of DA agonists, i.e. may enhance PPI. In order to explore these antipsychotic-induced potentiations of PPI more thoroughly, we assessed, in Sprague-Dawley rats, the effects of IP administration of various clinically effective antipsychotics in a PPI procedure with levels of PPI (ranging from 5 to about 40%) low enough to facilitate detection of PPI-potentiating effects of drugs. Both clozapine (5–20 mg/kg) and haloperidol (0.25–1 mg/kg) robustly and dose-dependently potentiated PPI. A similar effect was not seen with risperidone (0.1–1 mg/kg) or with the three substituted benzamides amisulpride (10–60 mg/kg), raclopride (0.1–3 mg/kg) and remoxipride (1–10 mg/kg). As risperidone is known to have prominent 5-HT₂ antagonistic activity, these results do not indicate a role for 5-HT₂ receptors in the clozapine and haloperidol PPI-enhancing effects. The absence of effects with the benzamides and with risperidone, at doses with known anti-dopaminergic activity, suggests that DA antagonist activity is not involved. The demonstration that prazosin (3–20 mg/kg), a non-antipsychotic with α₁ adrenoceptor antagonistic properties, dose-dependently potentiated PPI indicates that α₁ receptors might mediate the clozapine and haloperidol PPI-enhancing activity. Additionally, the finding that diazepam (1–10 mg/kg) did not enhance, but on the contrary reduced PPI, argues against a sedation- or general depressant-mediated effect of clozapine, haloperidol and prazosin. The negative results with four clinically active antipsychotics (risperidone and the benzamides), and the positive result with the non-antipsychotic prazosin, indicate that this PPI-enhancing procedure has poor predictive validity as a screening tool for potential antipsychotics. Received: 14 November 1996/Final version: 6 February 1997     

Pro-cognitive effects of 5-HT<Subscript>6</Subscript> receptor antagonists in the social recognition procedure in rats: implication of the frontal cortex

Rationale 5-HT₆ receptor antagonists improve cognitive processes in rodents. However, their site(s) of action remains unexplored and their influence upon social memory has been little investigated. Objectives We examined the influence of 5-HT₆ receptor ligands upon social memory in rats by use of systemic or local administration into the frontal cortex (FCX), striatum, or nucleus basalis magnocellularis (NBM). Materials and methods The social recognition test is based upon the ability of an adult rat to recognize a younger conspecific during the second of two 5-min sessions. In a procedure without an inter-session interval, the actions of drugs alone and the ability to reverse “amnesia” induced by the muscarinic antagonist, scopolamine (1.25 mg/kg, s.c.), were examined. The potential promnesic effect of drugs was also investigated in another procedure where a spontaneous deficit of recognition was induced by a 120-min inter-session interval. Results The 5-HT₆ receptor agonist, WAY-181187 (10.0 mg/kg, i.p.), significantly impaired social recognition. This effect was abolished by the 5-HT₆ receptor antagonists, SB-271046 (20.0 mg/kg, i.p.) and SB-258585 (10.0 mg/kg, i.p.). These agents also abolished scopolamine-induced amnesia (10.0 and 2.5 mg/kg, i.p., respectively) and reversed the delay-induced deficit (10.0–20.0 and 2.5–10.0 mg/kg, i.p., respectively). WAY-181187 into the FCX significantly impaired social recognition (0.16–0.63 μg/side). Conversely, SB-271046 into the FCX (2.5–5.0 μg/side), but neither into the striatum nor the NBM, significantly reversed spontaneous deficit. Conclusion These results indicate that 5-HT₆ receptors modulate social recognition by actions in the FCX and underpin their pertinence as targets for the treatment of psychiatric disorders in which cognitive function is compromised.     

Enhanced effect of dopaminergic stimulation on prepulse inhibition in mice deficient in the alpha subunit of G<Subscript>z</Subscript>

Rationale G_z is a member of the G_i G protein family associated with dopamine D2-like receptors; however, its functions remain relatively unknown. The aim of the present study was to investigate prepulse inhibition (PPI) of acoustic startle, locomotor hyperactivity and dopamine D2 receptor binding in mice deficient in the α subunit of G_z.Methods We used automated startle boxes to assess startle and PPI after treatment with saline, amphetamine, apomorphine or MK-801. We used photocell cages to quantitate locomotor activity after amphetamine treatment. Dopamine D2 receptor density was determined by autoradiography.Results Startle responses and baseline PPI were not different between the Gα_z knockout mice and wild-type controls (average PPI 46±4 vs 49±3%, respectively). Amphetamine treatment caused a marked disruption of PPI in Gα_z knockouts (average PPI 22±2%), but less so in controls (average PPI 42±3%). Similar genotype-dependent responses were seen after apomorphine treatment (average PPI 23±3% vs 40±3%), but not after MK-801 treatment (average PPI 29±5 vs 33±2%). Amphetamine-induced locomotor hyperactivity was greater in Gα_z knockouts than in controls. There was no difference in the density of dopamine D2 receptors in nucleus accumbens.Conclusions Mice deficient in the α subunit of G_z show enhanced sensitivity to the disruption of PPI and locomotor hyperactivity caused by dopaminergic stimulation. These results suggest a possible role for G_z in neuropsychiatric illnesses with presumed dopaminergic hyperactivity, such as schizophrenia.     

Selective dopamine D4 receptor antagonists reverse apomorphine-induced blockade of prepulse inhibition

 Recent evidence suggests that the dopamine D₄ receptor may play a role in schizophrenia, and that the atypical properties of the antipsychotic clozapine may be attributable in part to its antagonistic actions at this receptor. In the present study, clozapine and three other compounds having D₄ dopamine receptor antagonist properties were examined for their effectiveness in reducing losses in prepulse inhibition (PPI) induced in rats by the dopamine receptor agonist apomorphine. Previously, activity in the PPI model has been shown to correlate highly with the antipsychotic potency of a number of neuroleptics. As previously reported, clozapine (1–5.6 mg/kg) significantly reduced apomorphine-induced PPI deficits. The three D₄-selective compounds, CP-293,019 (5.6–17.8 mg/kg), U-101,387 (3–30 mg/kg) and L-745,870 (1–10 mg/kg), also significantly blocked the losses in PPI produced by apomorphine. Taken together, these results suggest that dopamine receptor antagonists with selectivity for the D₄ dopamine receptor subtype may be effective in the treatment of schizophrenia, while being less likely to produce dyskinesias associated with D₂ receptor antagonists. Received: 13 May 1997/Final version: 15 July 1997     

Involvement of decreased muscarinic receptor function in prepulse inhibition deficits in mice reared in social isolation

BACKGROUND AND PURPOSE

We have previously reported that galantamine, a weak acetylcholinesterase inhibitor, improves prepulse inhibition (PPI) deficits in mice reared in social isolation. ACh receptors are involved in the underlying mechanism of PPI, but whether rearing in social isolation causes dysfunction of the cholinergic system is unknown. In this study, we examined the involvement of muscarinic receptors in the improvement of PPI deficits induced by galantamine, and whether the cholinergic system is altered in mice reared in isolation.

EXPERIMENTAL APPROACH

Three-week-old male ddY mice were housed in isolated cages for 6 weeks before the initiation of experiments to create PPI deficits. Cholinergic functions were determined by measuring the behavioural and neurochemical responses to nicotinic and muscarinic receptor agonists.

KEY RESULTS

The improvement by galantamine of social isolation-induced PPI deficits was blocked by scopolamine, a non-selective muscarinic antagonist, and telenzepine, a preferential M₁ receptor antagonist. Activation of M₁ receptors improved social isolation-induced PPI deficits. Social isolation did not affect choline acetyltransferase and acetylcholinesterase activities in the prefrontal cortex and hippocampus, but it reduced the locomotor-suppressive response to muscarinic agonist oxotremorine, but not to nicotine. The isolation also attenuated the M₁ receptor agonist N-desmethylclozapine-induced increase in prefrontal dopamine release.

CONCLUSIONS AND IMPLICATIONS

Galantamine improves PPI deficits of mice reared in social isolation via activation of M₁ receptors. Social isolation reduces the muscarinic, especially M₁, receptor function and this is involved in PPI deficits.     

The selective dopamine D<Subscript>3</Subscript> receptor antagonists SB-277011A and NGB 2904 and the putative partial D<Subscript>3</Subscript> receptor agonist BP-897 attenuate methamphetamine-enhanced brain stimulation reward in rats

Rationale We have previously reported that selective antagonism of brain D₃ receptors by SB-277011A or NGB 2904 significantly attenuates cocaine- or nicotine-enhanced brain stimulation reward (BSR). Objective In the present study, we investigated whether the selective D₃ receptor antagonists SB-277011A and NGB 2904 and the putative partial D₃ agonist BP-897 similarly reduce methamphetamine (METH)-enhanced BSR. Materials and methods Rats were trained to respond for rewarding electrical self-stimulation of the medial forebrain bundle. To assess the degree of drug-induced changes in BSR, a rate–frequency curve shift paradigm was used to measure brain-reward threshold (θ ₀). Results METH (0.1–0.65 mg/kg, i.p.) dose-dependently lowered (∼10–50%) BSR thresholds, producing an enhancement of BSR. Pretreatment with SB-277011A (12 mg/kg, but not 24 mg/kg, i.p.) significantly attenuated METH-enhanced BSR. NGB 2904 (0.1–1.0 mg/kg, but not 10 mg/kg) also attenuated METH-enhanced BSR. SB-277011A or NGB 2904 alone, at the doses tested, had no effect on BSR. Pretreatment with BP-897 (0.1–5 mg/kg) dose-dependently attenuated METH-enhanced BSR. However, when the dose was increased to 10 mg/kg, BP-897 shifted the stimulation–response curve to the right (inhibited BSR itself) in the presence or absence of METH. Conclusions Selective antagonism of D₃ receptors by SB-277011A or NGB 2904 attenuates METH-enhanced BSR in rats, while the METH-enhanced BSR attenuation produced by BP-897 may involve both D₃ and non-D₃ receptors. These findings support a potential use of selective D₃ receptor antagonists for the treatment of METH addiction.     

Antipsychotic effects of N-desmethylclozapine on sensorimotor gating function in rats--possible involvement of activation of M(1) muscarinic receptors

N-desmethylclozapine (NDMC), one of the major metabolites of clozapine, has been demonstrated to exhibit partial agonistic activity at M(1) muscarinic receptors in vitro. Behavioral effects of NDMC were examined to determine whether NDMC contributed to the antipsychotic effects of clozapine via activation of muscarinic receptors. Both NDMC (10-30 mg/kg) and its parent compound clozapine (3-10 mg/kg) antagonized the disruption of prepulse inhibition (PPI) caused by the indirect dopamine agonist methamphetamine (3 mg/kg) in rats. However, NDMC (30 mg/kg) did not increase plasma levels of prolactin in rats. The same dose ranges of NDMC antagonized the disruption of PPI caused by the N-methyl-D-aspartate receptor antagonist ketamine (5 mg/kg) in rats. Furthermore, NDMC in the same dose ranges antagonized the disruption of PPI caused by the muscarinic receptor antagonist scopolamine (0.3 mg/kg) in rats. These findings suggest that NDMC has potent antipsychotic effects in animal models to examine sensorimotor gating function, and that NDMC may act through the activation of a muscarinic receptor for the treatment of schizophrenia.     

Determination of muscarinic agonist potencies at M1 and M2 muscarinic receptors in a modified pithed rat preparation

Summary The agonistic potencies of (±)muscarine, (±)cis - 2 - methyl - 5 - [(dimethylamino)methyl] - 1,3 -oxathiolane methiodide (cis-oxathiolane) and its two enantiomers were determined at muscarinic M₁ and M₂ receptors in the pithed rat. In non-pretreated animals, i.v. administration of these agents produced bradycardic effects mediated by cardiac M₂ receptors followed by increases in heart rate mediated by M1 receptors in sympathetic ganglia. As these responses have been shown to partly overlap, “true” M₁ and M₂ potencies were determined after selective blockade of M₁ and M₂ receptors by pirenzepine and methoctramine, respectively. A similar rank order of agonist potencies was obtained at M₁ and M₂ receptors: (+)cis-oxathiolane > (±)cis-oxathiolane > (±)muscarine > (-)cis-oxathiolane. At both receptor subtypes, (+)cis-oxathiolane was considerably more potent (ca. 30-fold) than its corresponding (−) enantiomer indicating that the agonist binding sites of the two receptor subtypes may have similar stereochemical properties. While (±)muscarine showed similar potencies at M₁ and M₂ receptors, racemic cis-oxathiolane and its two enantiomers showed a slight selectivity (3–7 fold) for M₁ receptors indicating the potential usefulness of these compounds in the development of selective M₁ receptor agonists. Send offprint requests to F. Cantalamessa at the above address