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.

Differential expression of IEG mRNA in rat brain following acute treatment with clozapine or haloperidol: a semi-quantitative RT-PCR study.

Antipsychotic drugs have been shown to modulate immediate early gene (IEG) expression in rat brain regions that are associated with schizophrenia, which may be directly linked to their immediate therapeutic benefit. In this study, we analysed the expression profile of a series of IEGs (c-fos, c-jun, fra-1, Krox-20, Krox-24, arc, sgk-1, BDNF and NARP) in six rat brain regions (prefrontal cortex, hippocampus, striatum, nucleus accumbens, thalamus and cerebellum). Rats (n=5) were administered either clozapine (20 mg/kg i.p.), haloperidol (1 mg/kg i.p.) or the appropriate vehicle with pre-treatment times of 1, 6 and 24 h. IEG expression was analysed in these regions by Taqman RT-PCR. The spatial and temporal profile of IEG induction following antipsychotic drug treatment correlates with regions associated with the efficacy and side effect profile of each drug. In particular, sgk-1 expression levels after antipsychotic drug treatment may have predictive value when investigating the profile of a novel antipsychotic drug.

Medial prefrontal cortex volume loss in rats with isolation rearing-induced deficits in prepulse inhibition of acoustic startle.

Rearing rats in isolation produces perturbations in behavior and brain neurochemistry suggested to resemble those of schizophrenia. In particular, isolation-reared rats display deficits in prepulse inhibition of acoustic startle that in humans are associated with disorders including schizophrenia and are interpreted as abnormalities in sensorimotor gating. The prefrontal cortex is considered important in the regulation of prepulse inhibition of acoustic startle and postmortem studies suggest that neuropil and total volume, but not total number of neurons, are decreased in this region of the brains of schizophrenic patients. In this study we used design-based stereological techniques to examine the brains of Lister Hooded rats, reared in isolation and which displayed prepulse inhibition of acoustic startle deficits, for changes in morphology compared with the brains of their socially-reared littermates. Pooled data from three batches of animals revealed a significant 7% volume loss of the medial prefrontal cortex of isolation-reared rats whereas neuron number in this region was unchanged. In contrast, volume and total neuron number were unaffected in the rostral caudate putamen. The robust reduction in prefrontal cortical volume observed in isolation-reared rats, in the absence of reductions in total neuron number, suggest that there is a loss of volume of the neuropil. These changes parallel those reported in schizophrenia patients and therefore support the construct validity of this model.

The effects of ziprasidone on regional c-Fos expression in the rat forebrain.

RATIONALE: Typical and atypical antipsychotic drugs produce characteristic patterns of immediate early gene expression in rat forebrain that are considered to reflect their effects in schizophrenia subjects. OBJECTIVE: To use c-Fos immunohistochemistry to investigate the functional neuroanatomical profile of the newly introduced atypical agent ziprasidone. MATERIALS AND METHODS: c-Fos immunohistochemistry was performed on paraformaldehyde-fixed cryosections of rat brains obtained, initially, from animals 2, 4, or 6 h after oral administration of 10 mg/kg ziprasidone or vehicle and, subsequently, from animals 2 h after oral administration of 1, 3, or 10 mg/kg ziprasidone or vehicle. The density of immunoreactive nuclei was assessed in pre-determined forebrain regions. RESULTS: Ziprasidone induced a time-dependent increase in the density of c-Fos-positive nuclei that was maximal at 2 h. At the 2 h time-point, c-Fos expression was significantly (p<0.05) elevated in the shell and core of the nucleus accumbens, lateral and medial caudate putamen, and lateral septum. At 4 h post-dose, c-Fos expression was also significantly increased in the cingulate gyrus. Ziprasidone-induced c-Fos expression was dose-dependent with significant (p<0.05) c-Fos expression observed in the nucleus accumbens (shell and core) and caudate putamen (lateral and medial) at 3 and 10 mg/kg and in the lateral septum at 10 mg/kg. CONCLUSIONS: Increased c-Fos expression in the nucleus accumbens and lateral septum is considered to be predictive of activity against positive symptoms, in the caudate putamen of motor side effect liability, and in the cingulate gyrus of efficacy against negative symptoms. Thus, the observed pattern of c-Fos expression induced in rat brain by ziprasidone is consistent with its reported clinical effects, namely, efficacy against positive symptoms with a therapeutic window over motor side effects and with some activity against negative symptoms.

Long-term evaluation of isolation-rearing induced prepulse inhibition deficits in rats: an update.

RATIONALE: Rats reared in social isolation from weaning show prepulse inhibition (PPI) deficits which are thought to model the sensorimotor gating deficits seen in schizophrenia and other psychiatric disorders. We have previously reported that ten cohorts of Lister Hooded rats reared in isolation showed robust and reliable PPI deficits. OBJECTIVE: Our methodology differed from those used by others (Weiss and Feldon in Psychopharmacology 156(2-3):305-326, 2001), most notably in the weaning of pups at postnatal day (PND) 28 compared with PND20-22. Since our initial report, we have studied 18 more cohorts weaned at PND28 and one cohort weaned at PND21. METHOD: At weaning, male Lister Hooded pups were singly (isolates) or group (n=5) housed (grouped). Eight weeks later, startle and PPI responses of isolates and grouped rats were investigated using conditions of fixed inter-stimulus interval (ISI) (pulse=110 dB/50 ms; prepulse (PP)=75-80 dB/30 ms; ISI=100 ms). RESULTS: Isolates from 14 of the subsequent 18 cohorts demonstrated PPI deficits, giving an overall success rate of 86% for all 28 cohorts. %PPI ranged from 12 to 26% in the isolates and from 26 to 47% in the grouped for the successful cohorts, compared to 16-30% (isolates) and 19-35% (grouped) for those that failed. Only five out of the 19 subsequent cohorts demonstrated startle hyperreactivity, which was unrelated to PPI response. The isolates from the cohort weaned at PND21 did not show a significant deficit in PPI, suggesting, in our hands at least, a requirement for weaning at PND28. CONCLUSION: The data presented here reinforce our original findings that isolation-rearing of Lister Hooded rats provides a viable, non-pharmacological model of impaired PPI.

Long-term evaluation of isolation-rearing induced prepulse inhibition deficits in rats.

RATIONALE: Rats reared in social isolation from weaning show prepulse inhibition (PPI) deficits which are thought to model the sensorimotor gating deficits seen in schizophrenia and other psychiatric disorders. However, recent studies have questioned the robustness of this paradigm. OBJECTIVE: The existence of a substantial dataset generated over 4 years in our laboratory has allowed the investigation of the robustness and reliability of the procedure under a variety of environmental conditions. The effects of atypical antipsychotics (clozapine, olanzapine and risperidone) under different experimental conditions are also reported. METHOD: At weaning, Hooded Lister pups were singly (isolates) or group (n=5) housed (grouped). Eight weeks later, the startle and PPI response of isolates and grouped rats was investigated using conditions of fixed inter-stimulus interval (ISI) (pulse=110 dB/50 ms; prepulse=80 dB/30 ms; ISI=100 ms) or variable

Increased responsiveness of dopamine to atypical, but not typical antipsychotics in the medial prefrontal cortex of rats reared in isolation.

Dopaminergic hypofunction in the medial prefrontal cortex (mPFC) has been associated with the aetiology of negative symptoms and cognitive dysfunction of schizophrenia, which are both alleviated by clozapine and other atypical antipsychotics such as olanzapine. In rodents, early life exposure to stressful experiences such as social isolation produces a spectrum of symptoms emerging in adult life, which can be restored by antipsychotic drugs. The present series of experiments sought to investigate the effect of clozapine (5-10 mg/kg s.c.), olanzapine (5 mg/kg s.c.), and haloperidol (0.5 mg/kg s.c.) on dopamine (DA) and amino acids in the prelimbic/infralimbic subregion of the mPFC in group- and isolation-reared rats. Rats reared in isolation showed significant and robust deficits in prepulse inhibition of the acoustic startle. In group-reared animals, both clozapine and olanzapine produced a significant increase in DA outflow in the mPFC. Isolation-reared rats showed a significant increase in responsiveness to both atypical antipsychotics compared with group-reared animals. In contrast, the administration of haloperidol failed to modify dialysate DA levels in mPFC in either group- or isolation-reared animals. The results also show a positive relationship between the potency of the tested antipsychotics to increase the release of DA in the mPFC and their respective affinities for 5-HT1A relative to DA D2 or D3 receptors. Finally, isolation-reared rats showed enhanced neurochemical responses to the highest dose of clozapine as indexed by alanine, aspartate, GABA, glutamine, glutamate, histidine, and tyrosine. The increased DA responsiveness to the atypical antipsychotic drugs clozapine and olanzapine may explain, at least in part, clozapine- and olanzapine-induced reversal of some of the major behavioral components of the social isolation syndrome, namely hyperactivity and attention deficit.

Clozapine enhances breakpoint in common marmosets responding on a progressive ratio schedule.

RATIONALE: Motivational effects of psychotropic drugs may contribute to their therapeutic profile and progressive ratio (PR) schedules provide a method of measuring these effects in animals. OBJECTIVE: Determine effects of selected antipsychotic, psychotomimetic, anxiolytic and antidepressant drugs on PR performance in common marmosets. METHOD: Marmosets were trained to lever press for banana milkshake reinforcement using a PR schedule, in which the number of lever presses to achieve successive reinforcements increased by one until responding ceased (breakpoint). RESULTS: Clozapine administered intramuscularly (0.01-2 mg/kg IM; 30 min pretreatment time (ptt) or by oral gavage (0.1-4 mg/kg PO; 60 min ptt) significantly increased the breakpoint. Independent tests of fluid consumption failed to show enhanced fluid intake after clozapine pretreatment, suggesting this effect was not due to drug induced polydipsia. Neither haloperidol (0.005-0.1 mg/kg PO; 60 min ptt) nor risperidone (0.0025-0.05 mg/kg PO; 60 min ptt) altered breakpoint. Olanzapine (0.01-1 mg/kg PO; 60 min ptt) significantly enhanced the breakpoint at 0.05 mg/kg PO, but the effect was not robust. Amphetamine (0.2-2 mg/kg SC; 30 min ptt) significantly reduced the breakpoint at 2 mg/kg and fluoxetine (0.1-1 mg/kg PO; 60 min ptt) was without effect. Diazepam significantly increased the breakpoint at 0.5 mg/kg PO. Drug-induced polydipsia might play a role in this response as independent tests showed increased fluid consumption following diazepam. CONCLUSIONS: These results demonstrate that, unlike other antipsychotics, clozapine over a wide dose range increased the motivational state of marmosets to respond for banana milkshake. This motivational aspect of clozapine's actions may contribute to its unique clinical profile and the PR procedure may provide a method for detecting novel antipsychotics with a clozapine-like profile.

Pharmacological actions of a novel, high-affinity, and selective human dopamine D(3) receptor antagonist, SB-277011-A.

SB-277011-A (trans-N-[4-[2-(6-cyano-1,2,3, 4-tetrahydroisoquinolin-2-yl)ethyl]cyclohexyl]-4-quinolininecarboxamide), is a brain-penetrant, high-affinity, and selective dopamine D(3) receptor antagonist. Radioligand-binding experiments in Chinese hamster ovary (CHO) cells transfected with human dopamine D(3) or D(2 long) (hD(3), hD(2)) receptors showed SB-277011-A to have high affinity for the hD(3) receptor (pK(i) = 7.95) with 100-fold selectivity over the hD(2) receptor and over 66 other receptors, enzymes, and ion channels. Similar radioligand-binding data for SB-277011-A were obtained from CHO cells transfected with rat dopamine D(3) or D(2). In the microphysiometer functional assay, SB-277011-A antagonized quinpirole-induced increases in acidification in CHO cells overexpressing the hD(3) receptor (pK(b) = 8.3) and was 80-fold selective over hD(2) receptors. Central nervous system penetration studies showed that SB-277011-A readily entered the brain. In in vivo microdialysis studies, SB-277011-A (2. 8 mg/kg p.o.) reversed the quinelorane-induced reduction of dopamine efflux in the nucleus accumbens but not striatum, a regional selectivity consistent with the distribution of the dopamine D(3) receptor in rat brain. SB-277011-A (2-42.3 mg/kg p.o.) did not affect spontaneous locomotion, or stimulant-induced hyperlocomotion. SB-277011-A (4.1-42.2 mg/kg p.o.) did not reverse prepulse inhibition deficits in apomorphine- or quinpirole-treated rats, but did significantly reverse the prepulse inhibition deficit in isolation-reared rats at a dose of 3 mg/kg p.o. SB-277011-A (2.5-78. 8 mg/kg p.o.) was noncataleptogenic and did not raise plasma prolactin levels. Thus, dopamine D(3) receptor blockade produces few of the behavioral effects characteristic of nonselective dopamine receptor antagonists. The effect of SB-277011-A on isolation-induced prepulse inhibition deficit suggests that blockade of dopamine D(3) receptors may benefit the treatment of schizophrenia.

The profile of sabcomeline (SB-202026), a functionally selective M1 receptor partial agonist, in the marmoset.

1. Sabcomeline (SB-202026, 0.03 mg kg(-1), p.o.), a potent and functionally selective M1 receptor partial agonist, caused a statistically significant improvement in the performance of a visual object discrimination task by marmosets. No such improvement was seen after RS86 (0.1 mg kg(-1), p.o.). 2. Initial learning, which only required an association of object with reward and an appropriate response to be made, was not significantly affected. Reversal learning, which required both the extinction of the previously learned response and the acquisition of a new response strategy, was significantly improved after administration of sabcomeline (0.03 mg kg(-1), p.o.). 3. Sabcomeline (0.03 and 0.1 mg kg(-1), p.o.) had no significant effect on mean blood pressure measured for 2 h after administration in the conscious marmoset. 4. Sabcomeline (0.03 mg kg(-1), p.o.) caused none of the overt effects such as emesis or behaviours often seen after the administration of muscarinic agonists, e.g. face rubbing and licking. 5. This is the first study to demonstrate cognitive enhancement by a functionally selective M1 receptor partial agonist in a normal (i.e. non-cognitively impaired) non-human primate and this effect was seen at a dose which did not cause side effects. 6. Perseverative behaviour and deficient acquisition of new information are seen in patients with Alzheimer's disease (AD). Therefore the data suggest that sabcomeline might be of therapeutic benefit in the treatment of AD.

A comparison of rectal and subcutaneous body temperature measurement in the common marmoset.

Two methods of measuring body temperature were compared in common marmosets. Subcutaneous temperatures were measured remotely via previously implanted subcutaneous microchips (Plexx BV, IPTT-100) prior to measurement of rectal temperature using a conventional rectal probe. Marmosets were treated with saline or the brain penetrant, 5-HT1A/B/D receptor agonist SKF-99101H (3-(2-dimethylaminoethyl)-4-chloro-5-propoxyindole hemifumarate) (0.3-3 mg/kg SC), which has previously been shown to induce hypothermia in guinea pigs. Body temperature was sampled immediately before drug administration and at 30-min intervals thereafter for a period of 2.5 h. SKF-99101H dose-dependently induced hypothermia in the common marmoset and there was close agreement between rectal and subcutaneous body temperatures, with an average difference in absolute body temperature of 0.26+/-0.02 degrees C. The data show that subcutaneously implanted microchips provide a simple, reliable measure of body temperature in common marmosets which is sensitive to pharmacological intervention, minimizes handling induced stress, and is minimally invasive.

Marmoset conspecific confrontation: an ethologically-based model of anxiety.

A method of measuring confrontation-induced behavioural changes in common marmosets (Callithrix jacchus) together with automated monitoring of locomotor activity has been developed as a possible model of anxiety. Recording both affiliative and agonistic behaviours between male/female pairs of marmosets and using diazepam as a reference drug, it has been possible to define a profile of behavioural changes which could be regarded as representing an anxiolytic response. Unfamiliar male/female pairs of marmosets were brought into close (non-contact) proximity in a controlled environment, in which their locomotor activity was recorded automatically. Simultaneously, their interactive behaviour was assessed by an independent observer via closed-circuit television. The following behaviours were analysed: aggressive postures, allogrooming, scratching, anxiety-related behaviours, social contact and self-grooming. Administration of diazepam at 1 and 3.5 mg/kg PO induced a significant (compared to control) reduction in scratching, aggressive behaviours, anxiety-related behaviours and an increase in allogrooming without affecting locomotor activity during confrontation. Differing responses dependent on gender were not found, nor did gender influence the effect of treatment on behaviour. Habituation to repeated confrontation did not occur. The results from this study demonstrate that this method can be used to measure anxiolytic activity in an objective manner.

In vitro and in vivo profile of SB 206553, a potent 5-HT2C/5-HT2B receptor antagonist with anxiolytic-like properties.

1. SB 206553 (5-methyl-1-(3-pyridylcarbamoyl)-1,2,3,5-tetrahydropyrrolo[2 ,3-f]indole) displays a high affinity (pK1 7.9) for the cloned human 5-HT2C receptor expressed in HEK 293 cells and the 5-HT2B receptor (pA2 8.9) as measured in the rat stomach fundus preparation. SB 206553 has low affinity for cloned human 5-HT2A receptors expressed in HEK 293 cells (pK1 5.8) and (pK1 < 6) for a wide variety of other neurotransmitter receptors. 2. SB 206553 appears to be a surmountable antagonist of 5-HT-stimulated phosphoinositide hydrolysis in HEK 293 cells expressing the human 5-HT2C receptor (pKB 9.0). 3. The compound potently (ID50 5.5 mg kg-1, p.o., 0.27 mg kg-1, i.v.) inhibited the hypolocomotor response to m-chlorophenylpiperazine (mCPP), a putative model of 5-HT2C/5-HT2B receptor function in vivo. 4. At similar doses (2-20 mg kg-1, p.o.) SB 206553 increased total interaction scores in a rat social interaction test and increased punished responding in a rat Geller-Seifter conflict test. These effects are consistent with the possession of anxiolytic properties. 5. SB 206553 also increased suppressed responding in a marmoset conflict model of anxiety at somewhat higher doses (15 and 20 mg kg-1, p.o.) but also reduced unsuppressed responding. 6. These results suggest that SB 206553 is a potent mixed 5-HT2C/5-HT2B receptor antagonist with selectivity over the 5-HT2A and all other sites studied and possesses anxiolytic-like properties.