An evaluation of the role of 5-HT2 receptor antagonism during subchronic antipsychotic drug administration in rats

A widely postulated mechanism of action for the atypical profile of many novel antipsychotic drugs (APDs) is their relatively high affinity for 5-HT₂ receptors. The present study investigated motor function and striatal dopamine (DA) efflux and metabolism in rats given 21 daily injections of drugs that differed in 5-HT₂ affinity. These drugs included: risperidone (high 5-HT_2A/2C/high D₂), clozapine (high 5-HT_2A/2C/low D₂), haloperidol (low 5-HT_2A/2C/high D₂), haloperidol+ritanserin (selective 5-HT_2A/2C), or vehicle. Rats injected with haloperidol (0.5 mg/kg) or haloperidol+ritanserin (0.5 mg/kg and 1.0 mg/kg, respectively) showed extreme catalepsy on day 1, but significantly decreased catalepsy when tested again on days 7 and 21. Acute or subchronic risperidone (0.05 or 0.5 mg/kg), clozapine (20 mg/kg), or vehicle did not induce significant catalepsy. Microdialysis performed 24 h after the last injection demonstrated that rats treated with risperidone, clozapine, or vehicle showed similar increases in DA efflux and metabolism following an acute injection of a selective DA D_2/3 antagonist (raclopride, 0.5 mg/kg). DA efflux showed an attenuated response to raclopride in the haloperidol alone group; this effect was less apparent in the haloperidol+ritanserin group. However, both of these groups showed a similar tolerance effect to the raclopride-induced increase in DA metabolites. These results suggest that the profile seen after subchronic risperidone more closely resembles clozapine than haloperidol. While ritanserin reduced the tolerance-like effects of haloperidol on striatal DA efflux, the overall results demonstrate that potent 5-HT₂ blockade alone may not entirely account for the distinctive profile of novel APDs.     

The role of 5-HT2C receptor polymorphisms in the pharmacogenetics of antipsychotic drug treatment

The development of novel, atypical antipsychotics has been welcomed due to their lower incidence of extrapyramidal side effects. However, as with all pharmacotherapy, patient response is often varied and these novel compounds are not without their own side effect profile, most notably weight gain. Inter-individual variations in response to drug treatment are, in part, due to polymorphisms of the genes encoding drug targets. The importance of the serotonin system in psychiatric symptomatology and several side effects of antipsychotic drugs is well established. Thus genetic polymorphisms of two central 5-HT receptors (5-HT2A and 2C), at which many of the newer antipsychotic drugs act, are prime candidates for pharmacogenetic analysis of the effects of these drugs in the treatment of schizophrenia. To date, much work has focussed on the 5-HT2A receptor subtype. However, pharmacological evidence and recent molecular genetic studies would suggest a role for the 5-HT2C receptor in the consequences of antipsychotic treatment, particularly in relation to the development of both drug-induced dyskinesias and weight gain. This review briefly examines the pharmacology and physiology of the 5HT2C receptor in the context of its genetics, with discussion of known polymorphisms of this receptor gene and its promoter region. Associations between these polymorphisms and the development of schizophrenia, the symptom responses to antipsychotic treatment and side effects of such treatment, notably tardive dyskinesia and weight gain, are assessed and related to the presumed functionality of these polymorphisms. Such studies clearly demonstrate the potential of pharmacogenetics in optimising treatment for the individual patient.     

Synthesis and evaluation of dimeric derivatives of 5-HT(2A) receptor (5-HT(2A)R) antagonist M-100907

It is now well accepted that at least some serotonin receptors exist in dimeric and oligmeric forms. The linking of receptor ligands has been shown to have potential in the development of selective agonists and antagonists for traditionally refractive receptors. Here we report the development of a dimeric version of the known 5-HT(2A)R antagonist, M-100907. Derivatives of M-100907 were synthesized to determine an appropriate site for the linker connection. Then, homodimers with polyether linkers of different lengths were functionally tested in a bioassay to determine the optimal linker length. Attachment at the catechol of M-100907 with linkers between 12 and 18 atoms in length proved to be optimal.     

5-HT2 receptor antagonism and slow-wave sleep in major depression.

Specific sleep disturbances such as reduced slow-wave sleep (SWS) and decreased serotonergic (5-HT) activity have been observed in depressive disorders. Ritanserin, a specific 5-HT2 receptor antagonist, has been shown to increase SWS in healthy subjects. This study explored the effects of a single dose or ritanserin (5 mg) on sleep electroencephalography in 18 major depressed patients and in 10 control subjects. Ritanserin affected SWS differently in the two groups. Although stage 3 increased significantly in the groups, in contrast to controls, there was no significant effect of ritanserin on stage 4 in depressed patients. In the depressed group, irritability and DSM-III-R melancholic type predicted 40% or the variance of stage 4 increment after ritanserin, as assessed by stepwise multiple regression. These results are in agreement with a potential 5-HT disturbance, particularly at the 5-HT2 receptor level, in some clinical forms of depression.     

Partial 5-HT(1A) receptor agonist activity by the 5-HT(2C) receptor antagonist SB 206,553 is revealed in rats spinalized as neonates

Modification of spinal serotonergic receptors caudal to spinal injury occurs in rats that received spinal cord transections as neonates. Evaluation of the serotonin syndrome, a group of motor stereotypies elicited by serotonergic (5-HT) agents in 5-HT-depleted animals, and open field locomotor behavior were used to assess behavioral consequences of injury and treatment. We extend these findings to show that a partial 5-HT(1A) agonist activity is revealed by the 5-HT(2C) receptor antagonist (SB 206,553) in this animal model, as measured by evaluation of serotonin syndrome behavior. Treadmill stimulation enhances this motor response, caudal to the injury, in the hindlimbs and tail. These results imply a broader modification of serotonergic receptors than previously thought and suggest a potential strategy by which serotonergic agents may enhance functional recovery following neonatal injury.     

Blockade of 5-HT(2) receptors in the periaqueductal grey matter (PAG) abolishes the anxiolytic-like effect of 5-HT(1A) receptor antagonism in the median raphe nucleus in mice

Several lines of evidence support the involvement of serotonergic (5-HT) neurons of the median raphe nucleus (MRN) in anxiety-like behaviour. In this context, it is known that blockade of 5-HT_1A somatodendritic autoreceptors in the midbrain raphe nuclei increases the firing rate of these neurons, disinhibiting 5-HT release in postsynaptic target areas such as amygdala, hippocampus and periaqueductal grey matter (PAG). However, while activation of 5-HT_1A or 5-HT₂ receptors in forebrain targets such as the amygdala or hippocampus enhances anxiety-like behaviours in rodents, stimulation of both receptor subtypes in the midbrain PAG markedly reduces anxiety-like behaviour. In view of these findings, the present study investigated whether the anti-anxiety effects induced by pharmacological disinhibition of 5-HT neurons in the MRN are attenuated by the blockade of 5-HT₂ receptors within the PAG. Mice received combined intra-PAG injection with ketanserin (10 nmol/0.1 μl), a 5-HT₂ receptor antagonist, followed by intra-MRN injection of WAY-100635 (5.6 nmol/0.1 μl), a highly selective 5-HT_1A receptor antagonist. They were then individually exposed to the elevated plus-maze (EPM), with the videotaped behavioural sessions subsequently scored for both conventional and ethological measures. The results confirmed that intra-MRN infusion of WAY100635 reduces behavioural indices of anxiety without significantly altering general activity measures, and further showed that this effect was completely blocked by intra-PAG pretreatment with an intrinsically-inactive dose of ketanserin. Together, these results suggest that 5HT₂ receptor populations located within the midbrain PAG play a significant role in the reduction of anxiety observed following disinhibition of 5-HT neurons in the MRN.     

Differential effects of 5-HT(2A) and 5-HT(2C) receptor blockade on strategy-switching

Recent experiments indicate that blockade of serotonin (5-HT) 2A and 2C receptors have differential effects on reversal learning. The present experiments investigated the effects of the 5-HT(2A) receptor antagonist, ketanserin and 5-HT(2C) receptor antagonist, SB242084 on acquisition and strategy-switching in a visual cue-response paradigm. Long-Evans rats were trained in a cross-maze to enter an arm based on color (visual cue version) or a specific turn response (response version). Systemic treatment with ketanserin did not affect initial learning of a visual cue or response discrimination, but ketanserin at 0.5 mg/kg significantly enhanced a switch between visual cue and response strategies. Ketanserin facilitated strategy-switching by inhibiting responses to a previously relevant strategy without affecting choices to never-reinforced strategies. Treatment with SB242084 (0.5, 1.0 or 2.0 mg/kg) did not affect acquisition of a visual cue or response discrimination. SB242084 treatment also did not affect strategy-switching. The present findings suggest that blockade of 5-HT(2A), but not 5-HT(2C), receptors enhance strategy switching.     

5-HT6 receptor binding sites in schizophrenia and following antipsychotic drug administration: autoradiographic studies with [125I]SB-258585

The 5-hydroxytryptamine (5-HT; serotonin)-6 receptor (5-HT6R) is a putative target of atypical antipsychotic drugs and its mRNA expression is altered in schizophrenia. [125I]SB-258585 is a selective 5-HT6R antagonist which has been well characterized for use in the rat brain. The present study evaluated its suitability for receptor autoradiography in the human brain and its application to quantitative studies. The affinity (K(d) approximately 1.2 nM) and relative distribution of binding sites (striatum > cortex approximately hippocampus) were similar to the rat. The distribution of [125I]SB-258585 binding in these regions was also consistent with that of 5-HT6R mRNA, determined in parallel using in situ hybridization. [125I]SB-258585 binding site densities were measured in dorsolateral prefrontal cortex of 20 patients with chronic schizophrenia and compared with 17 normal subjects. No differences were seen between groups. Neither were [125I]SB-258585 binding site densities affected in the frontal cortex or striatum of rats following 2 weeks' administration of the antipsychotic drugs haloperidol, chlorpromazine, olanzapine, risperidone, or clozapine. In summary, [125I]SB-258585 is a suitable radioligand for studies of human brain 5-HT6R binding sites and shows that their distribution is broadly similar to that of the rodent. The lack of effect of schizophrenia or antipsychotic drug administration on [125I]SB-258585 binding suggests that an altered receptor density does not contribute to any involvement which the 5-HT6R may have in the disease or its treatment.     

Effects of 5-HT(2A) and 5-HT(2C) receptor antagonists on acute and chronic dyskinetic effects induced by haloperidol in rats

An important limitation of classical antipsychotic drugs such as haloperidol (HAL) is their liability to induce extrapyramidal motor symptoms acutely and tardive dyskinetic syndromes when given chronically. These effects are less likely to occur with newer antipsychotic drugs, an attribute that is often thought to result from their serotonin-2 (5-HT(2)) receptor antagonistic properties. In the present study, we used selected doses of the 5-HT(2A) antagonist M100,907, the 5-HT(2C) antagonist SB242,084 and the mixed 5-HT(2A/C) antagonist ketanserin to re-examine the respective roles of 2A vs. 2C 5-HT(2) receptor subtypes in both acute and chronic motor effects induced by HAL. Acutely, SB242,084 (0.5 mg/kg) reduced HAL-induced catalepsy, while M100,907 (0.5 mg/kg) and ketanserin (1 mg/kg) were without effect. None of the drugs reduced HAL-induced Fos expression in the striatum or frontal cortex, and M100,907 actually potentiated HAL-induced Fos expression in the n. accumbens. In rats chronically treated with HAL, both ketanserin and SB242,084 attenuated vacuous chewing movements, while M100,907 had no effect. In addition, 5-HT(2C) but not 5-HT(2A) mRNA levels were altered in several brain regions after chronic HAL. These results highlight the importance of 5-HT2(2C) receptors in both acute and chronic motoric side effects of HAL, and suggest that 5-HT(2C) antagonism could be targeted as a key property in the development of new antipsychotic medications.     

Rodent data and general hypothesis: antipsychotic action exerted through 5-HT2A receptor antagonism is dependent on increased serotonergic tone

Summary. The locomotor stimulation induced by the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 (dizocilpine) in mice was regarded as a model of at least some aspects of schizophrenia. The serotonin synthesis inhibitor dl-p-chlorophenylalanine (PCPA) was used to evaluate the involvement of endogenous serotonin in (a) the induction of MK-801-induced hyperlocomotion in NMRI mice, and (b) the inhibition of MK-801-induced hyperlocomotion by each of five monoaminergic antagonists (M100907, clozapine, olanzapine, raclopride, SCH23390). Further, brain monoaminergic biochemistry was characterised in rats and mice after various drug treatments. PCPA pretreatment did not significantly reduce MK-801-induced hyperlocomotion in any of the experiments performed; however in a meta-analysis of six experiments, the locomotion displayed by MK-801-treated animals was diminished 17% by PCPA pretreatment. The selective 5-HT2A receptor antagonist M100907 exerted a dose-dependent inhibition of MK-801-induced hyperlocomotion. This effect was abolished in mice pretreated with PCPA, but could be restored in a dose-dependent manner by restitution of endogenous 5-HT by means of 5-hydroxytryptophan (5-HTP). On the other hand, the inhibition of MK-801-induced hyperlocomotion exerted by the selective dopamine D-2 receptor antagonist raclopride or the dopamine D-1 receptor antagonist SCH23390 was unaffected by PCPA pretreatment. The antipsychotics clozapine and olanzapine displayed a split profile. Hence, the inhibitory effect on MK-801-induced hyperlocomotion exerted by low doses of these compounds was diminished after PCPA pretreatment, while inhibition exerted by higher doses was unaffected by PCPA. These results suggest that (1) MK-801-induced hyperlocomotion is accompanied by an activation of, but is not fully dependent upon, brain serotonergic systems. (2) In the hypoglutamatergic state induced by MK-801, endogenous serotonin exerts a stimulatory effect on locomotion through an action at 5-HT2A receptors, an effect that is almost completely counterbalanced by a concomitant inhibitory impact on locomotion, mediated through stimulation of serotonin receptors other than 5-HT2A receptors. M100907, by blocking 5-HT2A receptors, unveils the inhibitory effect exerted on locomotion by these other serotonin receptors. (3) Dopamine D-2 receptor antagonistic properties of antipsychotic compounds, when they come into play, override 5-HT2A receptor antagonism. Possible implications for the treatment of schizophrenia with 5-HT2A receptor antagonists are discussed. It is hypothesized that treatment response to such agents is dependent on increased serotonergic tone. Accepted February 9, 1998; received December 16, 1997     

The role of serotonin (5-HT) in the control of TSH and prolactin release in euthyroid subjects as assessed by the administration of ketanserin (5-HT2 antagonist) and zimelidine (5-HT re-uptake inhibitor)

We have studied the effects of ketanserin, a specific 5-HT2 antagonist, and zimelidine, a selective inhibitor of 5-HT re-uptake into central nervous system presynaptic terminals, upon basal and TRH stimulated serum TSH and prolactin levels in euthyroid individuals. Ten normal volunteers were studied (five male and five female), each receiving oral ketanserin and oral zimelidine during respective seven day periods, separated by an interval of at least two weeks. A standard TRH test (200 micrograms i.v.) was carried out before and after each period of drug administration, serum samples being obtained for TSH and prolactin estimation. We were unable to demonstrate any modification of basal or TRH-stimulated TSH and prolactin levels by the oral administration of either ketanserin or zimelidine, suggesting that 5-HT has no significant role in the physiological release of either TSH or prolactin.     

Differential effects of subchronic treatments with atypical antipsychotic drugs on dopamine D2 and serotonin 5-HT2A receptors in the rat brain

Summary. The effects of 3-week treatment with a typical antipsychotic drug chlorpromazine and three atypical antipsychotic drugs (risperidone, olanzapine and perospirone) on the binding to dopamine D₂ and serotonin 5-HT_2A receptors were examined in the rat stratum and frontal cortex, respectively. Subchronic treatment with chlorpromazine (10 mg/kg) and perospirone (1 mg/kg) significantly increased D₂ receptors, while no increase was observed with lower dose of chlorpromazine (5 mg/kg), perospirone (0.1 mg/kg), risperidone (0.25, 0.5 mg/kg) or olanzapine (1, 2 mg/kg). On the other hand, 3-week administration of chlorpromazine (5, 10 mg/kg) and olanzapine (1, 2 mg/kg) significantly decreased 5-HT_2A receptors, but risperidone (0.25, 0.5 mg/kg) or perospirone (0.1, 1 mg/kg) had no effect. The measurement of in vivo drug occupation for D₂ and 5-HT_2A receptors using N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) suggested that high occupation of 5-HT_2A receptors with lower D₂ receptor occupancy might be involved in the absence of up-regulation of D₂ receptors after subchronic treatment with some atypical antipsychotic drugs. Received September 24, 1999; accepted December 1, 1999     

5-HT2 receptor antagonism in dysthymic disorder: a double-blind placebo-controlled study with ritanserin

Thirty patients suffering from dysthymic disorder participated in a 6-week double-blind trial comparing ritanserin 10 mg and placebo. After a single-blind placebo wash-out period of one week, the test medication was administered during 5 weeks on a double-blind basis. Twenty-three patients completed the study. At the end of the trial, ritanserin was significantly superior to placebo in its effect as manifested on the 19-item Hamilton Rating Scale for Depression, the Hamilton Rating Scale for Anxiety and the State Trait Anxiety Inventory X-1 and X-2. At the end of the study, the therapeutic effect was rated marked or moderate in 75% of the ritanserin-treated patients, but only in 18% of the controls. These data are consistent with the hypothesis of serotonin abnormalities in dysthymic disorder and suggest a therapeutic role of 5-HT2 antagonists. Ritanserin treatment was very well tolerated; no serious adverse experiences were reported.     

Central 5-HT(1) and 5-HT(2) binding sites in transgenic mice with reduced glucocorticoid receptor number

Transgenic mice bearing a transgene coding for a glucocorticoid receptor antisense mRNA, which partially blocks glucocorticoid receptor expression, were used in order to clarify the role of glucocorticoid receptors in the regulation of 5-HT(1A), 5-HT(1nonA) and 5-HT(2) binding sites labelled by quantitative autoradiography in the frontal and prefrontal cortex, striatum, hypothalamus, amygdala and raphe nuclei. We found that 1 nM [3H]8-hydroxy-2-[di-N-propylamino]tetralin ([3H]8-OH-DPAT) binding to 5-HT(1A) sites was decreased in strata oriens (-15.1+/-3.5%) and radiatum-lacunosum-moleculare (-13.3+/-4.3%) of the hippocampal CA(3) area, and 2 nM [3H]5-hydroxytryptamine binding to 5-HT(1nonA) sites in the presence of 100 nM 8-OH-DPAT and mesulergine was decreased in the dorsal subiculum (-17.8+/-6.9%). By contrast, 5-HT(2) sites labelled by 0.5 nM of (+/-)-1-(2, 5-dimethoxy-4-[125I]iodophenyl)-2-aminopropane was increased in the dorsal subiculum (+35.2+/-11.5%) and CA(2) area (+29.2+/-11.3%). The observed differences in binding to 5-HT(1) and 5-HT(2) sites were all located in areas of the hippocampus that contain both gluco- and mineralo-corticoid receptors, and no difference was observed in anatomical structures which contain only glucocorticoid receptors. Therefore, it seems that the important factor for the regulation of these 5-HT receptors is the interaction between gluco- and mineralo-corticoid receptors rather than the absolute density of glucocorticoid receptors. These results suggest that some of the alterations of the serotonergic neurotransmission observed in depressed patients might be secondary to an altered glucocorticoid receptor function.     

Increased binding at 5-HT(1A), 5-HT(1B), and 5-HT(2A) receptors and 5-HT transporters in diet-induced obese rats

5-Hydroxytryptamine (5-HT, serotonin), synthesized in midbrain raphe nuclei and released in various hypothalamic sites, decreases food intake but the specific 5-HT receptor subtypes involved are controversial. Here, we have studied changes in the regional density of binding to 5-HT receptors and transporters and the levels of tryptophan hydroxylase, in rats with obesity induced by feeding a palatable high-energy diet for 7 weeks. We mapped binding at 5-HT receptor subtypes and transporters using quantitative autoradiography and determined tryptophan hydroxylase protein levels by Western blotting. In diet-induced obese (DiO) rats, specific binding to 5-HT(1A) receptors ([3H]8-OH-DPAT) was significantly increased in the dorsal and median raphe by 90% (P<0.01) and 132% (P<0.05), respectively, compared with chow-fed controls. 5-HT(1B) receptor binding sites ([125I]cyanopindolol) were significantly increased in the hypothalamic arcuate nucleus (ARC) of DiO rats (58%; P<0.05), as were 5-HT(2A) receptor binding sites ([3H]ketanserin) in both the ARC (44%; P<0.05) and lateral hypothalamic area (LHA) (121%; P<0.05). However, binding to 5-HT(2C) receptors ([3H]mesulgergine) in DiO rats was not significantly different from that in controls in any hypothalamic region. Binding to 5-HT transporters ([3H]paroxetine) was significantly increased (P<0.05) in both dorsal and median raphe, paraventricular nuclei (PVN), ventromedial nuclei (VMH), anterior hypothalamic area (AHA) and LHA of DiO rats, by 47%-165%. Tryptophan hydroxylase protein levels in the raphe nuclei were not significantly different between controls and DiO rats. In conclusion, we have demonstrated regionally specific changes in binding to certain 5-HT receptor subtypes in obesity induced by voluntary overeating of a palatable diet. Overall, these changes are consistent with reduced 5-HT release and decreased activity of the 5-HT neurons. Reduction in the hypophagic action of 5-HT, possibly acting at 5-HT(1A), 5-HT(1B) and 5-HT(2A) receptors, may contribute to increased appetite in rats presented with highly palatable diet.     

Serotonin2 (5-HT2) receptor binding in the frontal cortex of schizophrenic patients

Summary Serotonin₂ (5-HT₂) receptor binding was studied, using³H-spiperone as the ligand, in post-mortem brain specimens obtained from schizophrenic patients (N=11) and non-psychiatric controls (N=11). The maximum number of binding sites (B_max) was significantly decreased in schizophrenic patients as compared to normal controls. This difference did not appear to be due to neuroleptic treatment. No difference in K_d (an inverse measure of the affinity of³H-spiperone to its binding sites) was observed between the two groups. However, studies with unmedicated schizophrenic patients are needed to draw any definite conclusion. The role of serotonergic processes in the psychobiology of schizophrenia is discussed.     

Role of 5-HT(2) receptor subtypes in depletion of 5-HT induced by p-chloroamphetamine in the mouse frontal cortex

A serotonin (5-hydroxytryptamine, 5-HT)-releasing drug, p-chloroamphetamine elicited decreases in 5-HT levels in the mouse frontal cortex. 5-HT reduction elicited by p-chloroamphetamine was inhibited by the 5-HT(2A/2B/2C) receptor antagonist, LY 53857 and the 5-HT(2A) receptor antagonist, ketanserin. However, the 5-HT(2B/2C) receptor antagonist, SB 206553, enhanced it. LY 53857 and ketanserin can inhibit hyperthermia elicited by p-chloroamphetamine, although SB 206553 enhances it. The effects of the 5-HT(2) receptor antagonists on neurotoxicity are very similar to those on hyperthermia. Since hyperthermia facilitates neurotoxicity induced by amphetamine analogue, these 5-HT(2) receptor antagonists may modify 5-HT depletion induced by p-chloroamphetamine through responses to body temperature.