Chronic Administration Of Clozapine Research Articles

Regional brain volume changes following chronic antipsychotic administration are mediated by the dopamine D2 receptor

BackgroundNeuroanatomical alterations are well established in patients suffering from schizophrenia, however the extent to which these changes are attributable to illness, antipsychotic drugs (APDs), or their interaction is unclear. APDs have been extremely effective for treatment of positive symptoms in major psychotic disorders. Their therapeutic effects are mediated, in part, through blockade of D2-like dopamine (DA) receptors, i.e. the D2, D3 and D4 dopamine receptors. Furthermore, the dependency of neuroanatomical change on DA system function and D2-like receptors has yet to be explored. MethodsWe undertook a preclinical longitudinal study to examine the effects of typical (haloperidol (HAL)) and atypical (clozapine (CLZ)) APDs in wild type (WT) and dopamine D2 knockout (D2KO) mice over 9-weeks using structural magnetic resonance imaging (MRI). ResultsChronic typical APD administration in WT mice was associated with reductions in total brain (p = 0.009) and prelimbic area (PL) (p = 0.02) volumes following 9-weeks, and an increase in striatal volume (p = 0.04) after six weeks. These APD-induced changes were not present in D2KOs, where, at baseline, we observed significantly smaller overall brain volume (p < 0.01), thinner cortices (q < 0.05), and enlarged striata (q < 0.05). Stereological assessment revealed increased glial density in PL area of HAL treated wild types. Interestingly, in WT and D2KO mice, chronic CLZ administration caused more limited changes in brain structure. ConclusionsOur results present evidence for the role of D2 DA receptors in structural alterations induced by the administration of the typical APD HAL and that chronic administration of CLZ has a limited influence on brain structure.

Chronic administration of haloperidol and clozapine induces differential effects on the expression of Arc and c-Fos in rat brain.

The modulation of genes implicated in synaptic plasticity following administration of antipsychotic drugs has been instrumental in understanding their possible mode of action. Arc (Arg 3.1) is one such gene closely associated with changes in synaptic plasticity. In this study we have investigated the changes in expression of Arc protein following acute and chronic administration of a typical antipsychotic (haloperidol) and an atypical antipsychotic (clozapine) by means of immunohistochemistry compared to the prototypic gene marker c-Fos. In dorsal striatum haloperidol (1 mg/kg) significantly increased Arc expression following both acute and chronic (21 day) administration with evidence of modulation in induction after repeated dosing. No significant changes were observed following either acute or chronic administration of clozapine (20 mg/kg). In the nucleus accumbens shell both clozapine and haloperidol induced Arc expression following acute administration, again with evidence of modulation after chronic dosing. The pattern of induction of Arc expression following haloperidol and clozapine in both dorsal and ventral striatum was similar to that for c-Fos. In medial prefrontal and cingulate cortex, Arc expression was significantly decreased by clozapine but not haloperidol without any indication of modulation following chronic dosing, whereas no significant changes in c-Fos expression were observed with either drug. Since synaptic modulation mediated by Arc is associated with down-regulation of the AMPA glutamate receptor, this study suggests a mechanism whereby enhanced glutamate receptor efficacy in medial cortical areas may be a component of antipsychotic drug action.

Effects of chronic clozapine administration on markers of arachidonic acid cascade and synaptic integrity in rat brain

The mode of action of clozapine, an atypical antipsychotic approved for treating schizophrenia (SZ) and used for bipolar disorder (BD) mania, remains unclear. We tested for overlap with the actions of the mood stabilizers, lithium, carbamazepine and valproate, which downregulate arachidonic acid (AA) cascade markers in rat brain and upregulate BDNF. AA cascade markers are upregulated in BD and SZ postmortem BD brain in association with neuroinflammation and synaptic loss, while BDNF is decreased. Rats were injected intraperitoneally with a therapeutically relevant dose of clozapine (10 mg/kg/day) or with saline for 30 days, and AA cascade and synaptic markers and BDNF were measured in the brain. Compared with saline-injected rats, chronic clozapine increased brain activity, mRNA and protein levels of docosahexaenoic acid (DHA)-selective calcium-independent phospholipase A₂ type VIA (iPLA₂), mRNA and protein levels of BDNF and of the postsynaptic marker, drebrin, while decreasing cyclooxygenase (COX) activity and concentration of prostaglandin E₂ (PGE₂), a proinflammatory AA metabolite. Activity and expression of AA-selective calcium-dependent cytosolic cPLA₂ type IVA and of secretory sPLA₂ Type II were unchanged. These results show overlap with effects of mood stabilizers with regard to downregulation of COX activity and PGE₂ and to increased BDNF and suggest a common action against the reported neuropathology of BD and SZ. The increased iPLA₂ expression following clozapine suggests increased production of anti-inflammatory DHA metabolites, and, with increased BDNF and drebrin, clear neuroprotective action.

Effect of chronic administration of clozapine on arachidonic acid (AA) turnover

Effect of chronic administration of clozapine on arachidonic acid (AA) turnover

Serum brain-derived neurotrophic factor and clozapine daily dose in patients with schizophrenia: A positive correlation

Brain-derived neurotrophic factor (BDNF) plays a critical role in neurodevelopment and neuroplasticity. Altered BDNF signaling is thought to contribute to the pathogenesis of schizophrenia (SZ) especially in relation to cognitive deficits. Clozapine (CLZ) has been shown a beneficial effect on cognition in SZ in some studies and a detrimental effect in others. To examine serum BDNF, two groups of chronically medicated DSM-IV SZ patients (n=44), on treatment with clozapine (n=31) and typical antipsychotics (n=13) had 5ml blood samples collected by venipuncture. Serum BDNF levels were significantly correlated with CLZ daily dose (r=0.394, p=0.028), but not with typical antipsychotic daily dose (r=0.208, p=0.496). This study suggests that serum BDNF levels are correlated with CLZ daily dose, and this may lead to the cognitive enhancement as seen in patients with SZ under CLZ. Despite the strong evidence that chronic administration of CLZ is effective for patients with SZ, it is still unknown whether atypical antipsychotic drugs regulate BDNF expression. Serum BDNF levels concentration in SZ merits further investigations with regard to the role of neurotrophins in the cognitive response to treatment with CLZ and other atypical antipsychotics.

Effects of chronic Clozapine administration on apolipoprotein D levels and on functional recovery following experimental stroke

Elevated brain levels of apolipoprotein D (ApoD) correlate with improved neurological recovery after experimental stroke. Hence, a pharmacological induction of ApoD in the postischemic brain could be beneficial for recovery after stroke. Here we investigated the effect of Clozapine, a compound that increases the expression of ApoD, in two rat models of experimental stroke. Rats were subjected to permanent occlusion of the middle cerebral artery (pMCAO) and treated with Clozapine (i.p. 10 mg/kg body weight) or saline for 8 or 28 days starting on the second day after MCAO. ApoD levels increased by 35% in the peri-infarct area after 10 and 30 days after pMCAO, mainly in neuron-specific nuclear protein (NeuN) positive neurons and glial fibrillary acidic protein (GFAP) positive astrocytes . Clozapine did not affect the neurological deficit assessed by the rotating pole test and a grip strength test at 7 days, 14 days, 21 days, and 28 days after pMCAO. Functional outcome and the infarct size were similar in rats subjected to transient MCAO and injected with Clozapine (i.p. 10 mg/kg body weight) or saline for 26 days starting on the second day after tMCAO. We conclude that Clozapine affects cellular processes involved in peri-infarct tissue reorganization, but does not affect functional recovery after MCAO.

Further neurochemical and behavioural investigation of Brattleboro rats as a putative model of schizophrenia

Brattleboro (BRAT) rats are a mutant variant of the Long-Evans (LE) strain deficient in the neurohormone vasopressin. BRAT rats show behavioural alterations relevant to schizophrenia. In particular, BRAT rats show deficits in prepulse inhibition (PPI) and alterations in various measures of cognition. The aim of this study was to replicate the reported PPI deficits in BRAT rats and its reversal by antipsychotic drugs and to investigate other behavioural and neurochemical characteristics. Acoustic startle reactivity, PPI, spontaneous and amphetamine-induced locomotor activity (LMA) and ex-vivo steady state neurochemistry were measured in male homozygous BRAT rats and LE rats. The effects of antipsychotics on PPI deficits were also determined. Relative to LE, BRAT rats showed enhanced startle reactivity, hyperactivity to a novel environment, PPI deficits and decreased levels of dopamine and DOPAC (dihydroxyphenylacetic acid) in the frontal cortex. BRAT and LE rats showed similar levels of hyperactivity following amphetamine (0.26 mg/kg s.c.). PPI deficits were attenuated by acute clozapine (5-10 mg/kg s.c.), risperidone (0.1-1 mg/kg i.p.), haloperidol (0.1-0.5 mg/kg p.o.) and less robustly by olanzapine (0.3-3 mg/kg s.c.). Chronic administration of clozapine (5 mg/kg s.c., once daily) attenuated baseline hyperactivity and elevated PPI of both strains. Clozapine concentrations were higher in BRAT brains compared with LE rats. These data confirm the reported PPI deficit in BRAT rats and its reversal by antipsychotic drugs, suggesting BRAT rats may represent a potential model for identifying novel antipsychotic drugs.

Effect of chronic treatment with clozapine and haloperidol on 5-HT 2A and 2C receptor mRNA expression in the rat brain

This study examined regional changes of 5-HT 2A and 2C receptor mRNA expression in the rat brain after chronic administration of clozapine (1.5 mg/kg/day) and haloperidol (2.0 mg/kg/day) for 36 days. 5-HT 2A and 2C receptor mRNA expression and distributions were detected by in situ hybridization after rats were sacrificed either 2 or 48 h after the last drug administration to examine both immediate and delayed effects following drug withdrawal. Following 2 h of drug withdrawal, it showed that clozapine administration significantly decreased 5-HT 2A receptor mRNA, predominantly in the nucleus accumbens (65%), hippocampus (80%), lasteral septal nucleus (61%) and striatum (68%) compared to controls, whilst rebound increases were observed in most of these regions 48 h later. In contrast, no change in 5-HT 2A receptor mRNA expression was found in the haloperidol treated groups either 2 h or 48 h after drug withdrawal. Clozapine also decreased 5-HT 2C receptor mRNA expression in the posteromedial cortical amygdala (32%) and substantia nigra (35%) 2 h after the last drug administration, while rebound effects were also observed 48 h later. 5-HT 2C receptor mRNA was only decreased in the substantia nigra at both 2 h (42%) and 48 h (54%) after the last haloperidol administration. Alterations in serotonin receptor expression in limbic system region such as the nucleus accumbens, hippocampus and lateral septal nucleus as well as the striatum may represent the specific regional targets that mediate the clinical effects of antipsychotics via the serotonin system.

Clozapine-Induced Alteration of Glucose Homeostasis in the Rat: The Contribution of Hypothalamic-Pituitary-Adrenal Axis Activation

Background/Aims: To our knowledge, a suitable animal model to investigate how atypical antipsychotics may induce diabetes in patients has not received much attention. Methods: We investigated the effects of acute as well as subchronic administration of clozapine on food intake, body weight gain, glucose tolerance and insulin secretion in response to glucose in Sprague-Dawley rats. We then evaluated the effects of clozapine on corticosterone secretion and 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD-1) and phosphoenolpyruvate carboxykinase (PEPCK) expression in the liver. We investigated the in vitro effects of clozapine on glucose uptake and development of differentiated myotubes in skeletal muscle cell (C2C12) cultures. Results: Clozapine administration caused hyperglycemia (p < 0.05) in female rats. In male rats, the increase of plasma glucose levels after clozapine injection was not statistically significant. The increase of plasma insulin concentrations and the intraperitoneal glucose tolerance test results proved that clozapine reduced insulin sensitivity in female rats. These endocrine and metabolic effects of clozapine were not related to changes in feeding behavior of fat accumulation. We observed a stimulatory effect of clozapine on corticosterone (p < 0.01) secretion in both female and male rats. Chronic clozapine administration upregulated PEPCK and 11β-HSD-1 expression in rat liver. Clozapine did not inhibit basal and insulin-induced glucose transport in murine myotubes but it was able to antagonize the stimulatory effect of α-methyl-5-hydroxytryptamine on glucose uptake. Conclusion: Clozapine induces sex-related alterations of glucose homeostasis and insulin sensitivity in rodents. We discussed the possible contribution of clozapine-induced activation of HPA and clozapine antagonistic activity at peripheral 5-HT_2A receptors to the observed metabolic alterations.

Cyproheptadine resembles clozapine in vivo following both acute and chronic administration in rats

Cyproheptadine is a cheap, widely available anti-allergy drug with a broad receptor binding profile which resembles that of clozapine. In rats discriminating clozapine from vehicle cyproheptadine mimicked clozapine very closely. Acutely it induced full generalization in the absence of response suppression, as observed with clozapine. Chronic administration of clozapine and cyproheptadine induced tolerance and cross-tolerance respectively to the clozapine stimulus. This was characterized by circa 3.5-fold parallel shifts to the right in the clozapine generalization curves. Such tolerance and cross-tolerance was spontaneously reversible, suggesting that it was pharmacodynamic, and that clozapine and cyproheptadine induce similar neuroadaptations when administered chronically. Administration of chlordiazepoxide at a very high dose induced no cross-tolerance to the clozapine stimulus showing the pharmacological specificity of tolerance. The clozapine stimulus is a compound cue involving actions at various receptors, and various clozapine-like antipsychotic (APD) drugs generalize fully to it. These data demonstrate that in vivo cyproheptadine resembles clozapine both acutely and chronically. Our findings, in conjunction with other actions of cyproheptadine -- induction of weight gain, alleviation of clozapine withdrawal, anxiolytic actions, alleviation of 'typical' APD-induced motoric side effects, and some preliminary clinical findings -- suggest that further study of cyproheptadine in conjunction with a 'typical' APD for the possible treatment of schizophrenia is merited at both pre-clinical and clinical levels.

Chronic administration of clozapine alleviates reversal-learning impairment in isolation-reared rats

Isolation rearing has been used for inducing schizophrenia-like symptoms in rats. Human schizophrenics have deficits in prefrontal-dysfunction-related cognitive/behavioral flexibility. Rats with lesions of the medial prefrontal cortex perform poorly in reversal learning. It is uncertain whether isolation rearing, however, causes reversal-learning impairment in adult rats. Using the rotating T maze, this study examined the effect of chronic administration of clozapine on visual discrimination learning and reversal learning in isolation-reared and socially reared adult rats. The results show that isolation-reared rats without clozapine injection performed significantly worse than socially reared rats in reversal learning but not in acquisition learning. Chronic injection of clozapine (5 or 10 mg/kg) in isolation-reared rats significantly improved reversal learning but had no effects on acquisition learning. Further data analyses show that in both the inhibition phase and the new-strategy-acquisition phase of reversal learning, isolation-reared rats needed significantly more correct-response trials to reach the criterion than socially reared rats, and clozapine significantly reduced the isolation-induced impairment of reversal learning only in the new-strategy-acquisition phase. In socially reared rats, clozapine had a dose-related interfering effect on reversal learning but not acquisition learning. This study supports the use of isolation rearing as a model for investigating the neurodevelopmental hypothesis of schizophrenia.

In rats chronically treated with clozapine, tyrosine depletion attenuates the clozapine-induced in vivo increase in prefrontal cortex dopamine and norepinephrine levels

We previously reported that depletion of brain tyrosine attenuated the acute clozapine (CLZ)-induced increase in medial prefrontal cortex (MPFC) dopamine (DA) levels. This effect was now examined after chronic CLZ treatment. Male rats received CLZ (10 mg kg(-1) day(-1)) in drinking water for 21 days. On day 18, a cannula was stereotaxically implanted over the MPFC. A microdialysis probe was inserted on day 20. On day 21 after a stable baseline was reached, rats received an acute injection of vehicle (VEH) or a tyrosine- and phenylalanine-free mixture of neutral amino acid [NAA(-)] (total 1 g kg(-1), i.p., two injections, 1 h apart) followed by CLZ (10 mg kg(-1), i.p.) or VEH. Basal tyrosine or norepinephrine (NE) levels were not different between the groups, but basal DA was higher in the group treated chronically with CLZ (p<0.05). Acute CLZ (10 mg kg(-1), i.p.) increased MPFC DA and NE levels to 370% and 510% of baseline, respectively, and similarly in rats chronically pretreated with CLZ or VEH. NAA(-) did not affect basal MPFC DA or NE levels but significantly attenuated acute CLZ-induced DA (220% of baseline) and NE (330% of baseline) levels (p<0.01) in rats pretreated chronically with CLZ or with VEH. These data demonstrate that even after chronic CLZ administration, the acute CLZ-induced increases in MPFC DA and NE levels depend on the availability of brain tyrosine. Judicious manipulation of brain tyrosine levels may provide a useful probe as well as a mechanism for enhancing psychotropic drug actions.

Psychotropic drugs affect Ser9-phosphorylated GSK-3β protein levels in rodent frontal cortex

Glycogen synthase kinase (GSK)-3beta, a serine/threonine kinase highly abundant in brain is a negative regulator of signal transduction cascades including the phosphatidylinositol-3-kinase (PI3-K)/Akt and the Wnt. GSK-3beta has recently been suggested to be an intracellular target of the mood stabilizers lithium and valproate and of the typical and atypical antipsychotic agents haloperidol and clozapine. We have previously shown that these agents do not alter frontal cortex GSK-3beta protein levels or activity. The current study was conducted to assess the effect of psychotropic drugs on phospho-Ser9-GSK-3beta levels in rodents. Chronic administration of haloperidol to rats resulted in a significant reduction in frontal cortex phospho-Ser9-GSK-3beta protein levels and no change in those of GSK-3alpha, while chronic administration of clozapine or subchronic administration of valproate caused significant elevation of GSK-3beta protein levels. Mice treated chronically with lithium exhibited the most prominent elevation in phospho-Ser9-GSK-3beta. The results support the notion that GSK-3beta may be a common target for mood stabilizers and neuroleptics.

Effects of clozapine and 2,5-dimethoxy-4-methylamphetamine [DOM] on 5-HT 2A receptor expression in discrete brain areas

Activation of 5-HT 2A receptors has been shown to be an essential component of the discriminative stimulus effects of indoleamine and phenethylamine hallucinogens. The objective of the present study was to determine the neuroanatomical location of the 5HT 2A receptors which may be responsible for the stimulus effects of the phenethylamine hallucinogen [−]2,5-dimethoxy-4-methylamphetamine (DOM). It was hypothesized that brain areas containing altered 5-HT 2A receptor expression in the context of a similar alteration in DOM-induced stimulus control might be important in mediating the stimulus effects of DOM. Fisher 344 rats were treated with either clozapine (25 mg/kg/day) or DOM (2 mg/kg/day) for 7 days, and the consequences of these drug treatment regimens on DOM-induced stimulus control and on 5-HT 2A receptor expression in several brain areas were determined. Chronic administration of clozapine was associated with a wide-spread decrease in levels of 5-HT 2A/2C receptors. Conversely, treatment with DOM had varied effects including a neuroanatomically selective decrease in 5-HT 2A/2C receptor levels that was restricted to the olfactory nucleus. Both chronic treatment with DOM and clozapine decreased the stimulus effects of DOM. The present findings suggest a role for the olfactory nucleus in producing the stimulus effects of DOM.

The effect of chronic treatment with typical and atypical antipsychotics on working memory and jaw movements in three- and eighteen-month-old rats

The effects of chronic treatment with typical and atypical antipsychotics on acquisition, working memory, motor activity, and rat tardive dyskinesia (TD) were studied in 3- and 18-month-old Sprague–Dawley rats. Acquisition and working memory were studied in eight-arm radial mazes. TD liability of antipsychotic drugs (APD) was evaluated in rat model of TD in which spontaneous repetitive jaw movements (RJM) occur during withdrawal from neuroleptic treatment. Motor behavior was assessed using the traverse beam test. D1 and D2 receptor occupancy was determined in the rat brain during treatment with typical and atypical antipsychotics. Chronic administration of clozapine, haloperidol, and risperidone impaired acquisition of the eight-arm radial maze in both young and aging rats while olanzapine had no effect . Retention tests showed that aging rats made more errors than the adults and that the antipsychotics haloperidol and risperidone significantly impaired retention in both age groups. Evaluation of motor behavior revealed that typical and atypical antipsychotics used in comparable doses in young rats had no effect on motor behavior, whereas in aging rats performance was impaired by clozapine, haloperidol, and risperidone but not by olanzapine. RJM responses were increased during washout from haloperidol treatment in young and aging rats whereas olanzapine, clozapine, and risperidone had no effect. D2 receptor occupancy in haloperidol- and risperidone-treated rats was above 70% while olanzapine and clozapine receptor occupancy was below 70%, which is the threshold for the appearance of extrapyramidal syndrome (EPS) and TD.

Enhancement of Laminar FosB Expression in Frontal Cortex of Rats Receiving Long Chronic Clozapine Administration

The frontal cortex (FrC) and cingulate cortex (CgC) are critical sites for normal cognitive function, as well as cognitive dysfunction in schizophrenia. Thus, modulation of synaptic transmission within these cortical areas may, in part, account for the therapeutic actions of antipsychotic drugs such as haloperidol and clozapine. FosB and ΔFosB are immediate–early gene (IEG) products sensitive to changes in response to chronic neuroleptic drug administration. We quantitatively examine whether there are light microscopic regional and/or laminar variations in FosB or ΔFosB in the FrC or CgC of normal adult rats, or animals receiving 6 months administration of either drinking water clozapine, or depot haloperidol. Only animals receiving chronic haloperidol developed vacuous chewing movements, the equivalent of tardive dyskinesia in humans. In control animals, the deep and superficial layers of the FrC showed a higher area density of FosB, but not ΔFosB immunoreactive cells than the medial layers of FrC or any of the CgC layers. In animals receiving clozapine, but not haloperidol there was increase in the area density of FosB immunoreactive neurons in all FrC layers, but the major increase occurs in medial layers. These findings suggest that FosB expression identifies those FrC neurons that are most active during normal waking behaviors and are further activated following chronic administration of atypical neuroleptics without motor side effects. The results also indicate that the actions of clozapine are attributed in large part to modulation of the output of frontal cortical pyramidal neurons residing in the medial layers.

Additivities of compounds that increase the numbers of high affinity [3H]muscimol binding sites by different amounts define more than 9 GABA(A) receptor complexes in rat forebrain: implications for schizophrenia and clozapine research.

The numbers of [3H]MUS binding sites were reported to be elevated in layers II and III, but not V or VI, in cingulate cortex of schizophrenic brains post mortem. These increases in [3H]MUS binding sites are probably due to compensatory up-regulation of GABA(A) receptors on pyramidal cells as a consequence of a selective loss of GABAergic interneurons in layer II of cingulate cortex. The number of [3H]flunitrazepam binding sites was reported to be reduced in schizophrenic cingulate cortex, and this may directly reflect the loss of GABAergic interneurons. Chronic administration of clozapine to rats was reported to significantly reduce the numbers of [3H]MUS binding sites in temporal cortex and hippocampus which may be due to selective blockade of GABA(A) receptors on GABAergic interneurons that make synaptic contact with pyramidal cells. Basket cells are GABAergic interneurons that make synaptic contact with pyramidal cells as well as other interneurons. Basket cells can also generate both theta and gamma oscillations. Clozapine increases the power of theta and gamma EEG. Schizophrenic patients show reduced EEG power at 40 Hz (gamma frequency) but not at lower frequencies during auditory stimulation. The GABA(A) receptor blocker bicuculline at 10 nM, but not 10 microM, was reported to increase the amplitude of slow oscillations (< or =1 Hz) in rat hippocampal slices. It therefore seems possible that clozapine, by selectively blocking another GABA(A) receptor, could increase the amplitude of gamma oscillations. Twenty-six compounds that inhibit [35S]TBPS binding in ways that are reversible by 10 nM R-5135 were found to increase [3H]MUS binding to membranes prepared from rat whole forebrain. In almost all cases the increases in binding were due to increases in the number of binding sites with little effect on affinity (Kd) for [3H]MUS. Concentration-response curves for the compounds revealed maximum increases in [3H]MUS (Esat) binding ranging from 140% (for meclizine) to 313% of control for honokiol. Additivity experiments showed that propofol (44% above control) and diflunisal (50% above control) were almost entirely additive, but there was also a small, but significant overlap, suggesting the existence of three groups of [3H]MUS binding sites defined by propofol and diflunisal. Meclizine was entirely additive with both propofol and diflunisal, indicating the existence of a fourth [3H]MUS binding site. Alphaxalone is also completely additive with meclizine, and has an Esat value significantly larger than that for propofol + diflunisal suggesting a fifth [3H]MUS binding site. The Esat for mefenamate is significantly greater than the Esat for alphaxalone, and mefenamate is also completely additive with meclizine, suggesting the existence of a sixth [3H]MUS binding site. The Esat for magnolol is significantly greater then the Esat, for mefenamate, and the Esat for honokiol is greater than that for magnolol, suggesting, but not proving, the existence of a seventh and an eighth group of [3H]MUS binding sites. The binding of [3H]MUS alone, without enhancers may represent a ninth group of binding sites which is probably heterogeneous as indicated by the very low pseudo Hill coefficients for bicuculline and strychnine in displacing [3H]MUS without enhancer. Altogether, our results suggest the existence of more than 9 different [3H]MUS binding sites. Clozapine was a very weak overall displacer of [3H]MUS (IC50 = 280 microM). However, 5 microM clozapine reduced [3H]MUS binding 6% (P < 0.0001, n = 10) and significantly reduced [3H]MUS binding enhanced by propofol (approximately 14%) or clotrimazole (approximately 17%) but not 17 other compounds tested. In the absence of enhancers [3H]MUS may bind preferentially to GABA(A) receptors on pyramidal cells and less to interneurons in cerebral cortex. Conversely, [3H]flunitrazepam may bind preferentially to GABA(A) receptors (allosterically) on interneurons and less to pyramidal cells. Clozapine appears to selectively block a small fraction (10-20%) of [3H]MUS binding sites with an IC50 value in the low micromolar range. This fraction may be preferentially located on certain GABAergic interneurons (basket cells?) that make synaptic contact with pyramidal cells. The blockade of these GABA(A) receptors by clozapine would be expected to increase the firing rate of the interneurons and the release of GABA onto pyramidal cells. Such blockade would also increase the generation of gamma oscillations by the basket cells. Some of these interneurons appear to be destroyed selectively, probably during the second trimester of gestation by a non-paralytic polio virus, in individuals who wil

Effects of acute and chronic administration of olanzapine in comparison to clozapine and haloperidol on extracellular recordings of substantia nigra reticulata neurons in the rat brain.

Previously, we have shown that the atypical antipsychotics clozapine and risperidone, unlike haloperidol, decreased the firing rate of substantia nigra reticulata (SNR) neurons. As the SNR receives substantial input from the striatum, an area where motoric side-effects of antipsychotics are thought to be mediated, the SNR might be an interesting brain structure with regard to motor side-effects. The newly developed atypical antipsychotic olanzapine was studied for its effects on the firing rate of SNR cells. In addition, to gain insight in the implications of our experimental setup for clinical use, responses upon clozapine, olanzapine and haloperidol were studied after chronic treatment. In chloralhydrate-anaesthetized male Wistar rats, extracellular recordings were made from SNR neurons upon intravenously (i.v.) administered cumulative doses of the antipsychotics. Naive rats and rats that were subcutaneously (SC) injected for 21 days with an antipsychotic were used. Olanzapine (50-1600 mg/kg; i.v.), significantly inhibited the firing rate of the SNR neurons. Upon 21 days of treatment with a daily SC injection of 20 mg/kg clozapine, the challenge on day 22 with cumulative injections of clozapine (200 6400 mg/kg; i.v.) significantly inhibited the firing rate of the SNR neurons. Olanzapine (50-1600 mg/kg; i.v.) also significantly inhibited the SNR activity when pretreated with olanzapine in an SC administered dose of 1 mg/kg, but not 5 mg/kg. Haloperidol (12.5-800 microg/kg; i.v.) did not significantly affect the SNR activity in rats pretreated with SC administered 0.5 mg/kg haloperidol. Upon acute and chronic administration of clozapine and olanzapine versus haloperidol, differential effects on SNR neuronal firing could be obtained. The experimental setup seem to be valid for further studies into the mechanism of action of typical versus (relatively low doses of) atypical antipsychotics. The implications of the inhibitory effect of atypical antipsychotics on the SNR firing rate are presently unknown, but could be associated with the lower propensity to induced motoric side-effects. On the other hand, the SNR activity might also reflect non-motoric activity possibly related to negative symptoms.

Serotonergic antagonist effects on trafficking of serotonin 5-HT2A receptors in vitro and in vivo.

The mechanism by which antagonists down-regulate 5-HT2A receptors in unknown. We here report that a variety of 5-HT2A antagonists induce a change in the subcellular distribution of 5-HT2A receptors both in vitro and in vivo. In a stably transfected NIH 3T3 cell-line, brief exposure to 1 muM clozapine caused a 2.5-fold increase in intracellular 5-HT2A-like immunoreactivity, as measured by confocal microscopy. Confirmatory studies utilizing a biotin-trap technique, demonstrated that the increase in intracellular immunoreactivity results from internalization of receptor from the cell surface. Exposure of transfected cells to other 5-HT2A receptor antagonists produced similar increases in intracellular 5-HT2A-like immunoreactivity. In vivo administration of clozapine (20 mg/kg, sc, X 7 days) caused a greater than twofold increase in intracellular immunoreactivity in cell bodies of cortical pyramidal neurons. Additionally, chronic clozapine administration was associated with decrease in labeling of apical dendrites on pyramidal cells. These results show that clozapine causes a change in subcellular distribution of 5-HT2A receptors in vitro and in vivo.

Acute and chronic administration of clozapine produces greater proconvulsant actions than haloperidol on focal hippocampal seizures in freely moving rats

Acute and chronic administration of clozapine produces greater proconvulsant actions than haloperidol on focal hippocampal seizures in freely moving rats