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The pituitary neuropeptide oxytocin promotes social behavior, and is a potential adjunct therapy for social deficits in schizophrenia and autism. Oxytocin may mediate pro-social effects by modulating monoamine release in limbic and cortical areas, which was investigated herein using in vivo microdialysis, after establishing a dose that did not produce accompanying sedative or thermoregulatory effects that could concomitantly influence behavior. The effects of oxytocin (0.03-0.3 mg/kg subcutaneous) on locomotor activity, core body temperature, and social behavior (social interaction and ultrasonic vocalizations) were examined in adult male Lister-hooded rats, using selective antagonists to determine the role of oxytocin and vasopressin V 1a receptors. Dopamine and serotonin efflux in the prefrontal cortex and nucleus accumbens of conscious rats were assessed using microdialysis. 0.3 mg/kg oxytocin modestly reduced activity and caused hypothermia but only the latter was attenuated by the V 1a receptor antagonist, SR49059 (1 mg/kg intraperitoneal). Oxytocin at 0.1 mg/kg, which did not alter activity and had little effect on temperature, significantly attenuated phencyclidine-induced hyperactivity and increased social interaction between unfamiliar rats without altering the number or pattern of ultrasonic vocalizations. In the same rats, oxytocin (0.1 mg/kg) selectively elevated dopamine overflow in the nucleus accumbens, but not prefrontal cortex, without influencing serotonin efflux. Systemic oxytocin administration attenuated phencyclidine-induced hyperactivity and increased pro-social behavior without decreasing core body temperature and selectively enhanced nucleus accumbens dopamine release, consistent with activation of mesocorticolimbic circuits regulating associative/reward behavior being involved. This highlights the therapeutic potential of oxytocin to treat social behavioral deficits seen in psychiatric disorders such as schizophrenia.

Rationale: Rearing rats in isolation from weaning is an established preclinical neurodevelopmental model which induces behavioural deficits with apparent translational relevance to some core symptoms of schizophrenia., Objective: This study evaluated the ability of the atypical antipsychotic risperidone to reverse behavioural deficits induced by post-weaning social isolation of rat pups and to further characterise the predictive validity of this model., Method: Forty-five male Lister hooded rats were housed in groups of 3-4 (n = 16) or singly (n = 29) for 4 weeks immediately after weaning on postnatal day (PND) 22-24. On PND 51, novel cage-induced locomotor activity (LMA) was assessed to subdivide rats into groups balanced for behavioural response. On PNDs 58, 59, 65 and 72, rats received either vehicle (1 ml/kg; i.p.) or risperidone (0.2 or 0.5 mg/kg; i.p.) 30 min prior to testing in LMA, novel object discrimination (NOD), prepulse inhibition (PPI) of acoustic startle and conditioned emotional response (CER) learning paradigms, respectively., Results: Isolation rearing had no effect on PPI, but produced LMA hyperactivity and impaired NOD and CER compared to group-housed controls. Risperidone caused a dose-dependent reduction in LMA, irrespective of rearing condition, but selectively reversed the NOD deficit in isolation-reared rats. Risperidone did not reverse the isolation rearing-induced CER deficit., Conclusions: Similar to its clinical profile, risperidone only partially reverses the schizophrenic symptomology; since it reversed some, but not all, of the learning and memory deficits induced by post-weaning isolation, the isolation rearing model may be useful to predict antipsychotic activity of novel therapeutic agents.

Many antipsychotics (APDs) have a high affinity for muscarinic receptors, which is thought to contribute to their therapeutic efficacy, or side effect profile. In order to define how muscarinic receptor gene expression is affected by atypical or typical APDs, rats were treated with chronic (2.58 mg/kg) PCP (a psychotomimetic) or vehicle, plus clozapine (20 mg/kg/day) or haloperidol (1 mg/kg/day), and M1, M2 and M3 receptor mRNA levels were determined in brain sections. Negligible changes in M2 or M3 muscarinic mRNA were detected in any region after clozapine or haloperidol. Chronic PCP administration increased M1 mRNA expression in the prefrontal cortex, which was not reversed by either chronic clozapine or haloperidol treatment. Chronic clozapine treatment in combination with PCP treatment decreased M1 receptor mRNA levels in the nucleus accumbens core, whereas chronic haloperidol in combination with PCP treatment increased M1 receptor mRNA levels in the ventromedial hypothalamus and medial amygdala. Thus M1 receptor gene expression is targeted by APDs, although the regions affected differ according to the APD treatment and whether PCP has been administered. The different brain circuitry modulated, may reflect the differing modes of action of typical and atypical APDs. These data provide support for the dysregulation of M1 receptors in schizophrenia, and furthermore, modulation by antipsychotic agents in the treatment of schizophrenia., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The ability of antipsychotic drugs to affect 5-HT(2A) receptor function has been widely suggested to contribute to their therapeutic properties. We have compared the ability of the antipsychotic drugs clozapine and haloperidol, alone and in combination with chronic phencyclidine (PCP), to modulate 5-HT(2A) receptor binding and mRNA. Acute (i.p. 45 min) and chronic (21-day) clozapine (osmotic minipump (OMP); 20 mg/kg/day) produced widespread decreases in 5-HT(2A) receptor binding (-60%-80%), measured using [(3)H]ketanserin autoradiography. Conversely, 5-HT(2A) mRNA levels, determined using in-situ hybridisation, were modestly increased by chronic clozapine treatment (+10%-30%). Chronic PCP treatment, at a dose (2.58 mg/kg i.p. intermittently for 28 days) that reproduces many of the neurochemical deficits of schizophrenia, decreased 5-HT(2A) receptor binding in the prefrontal cortex (PFC; -16%), consistent with the changes in post-mortem brain tissue from schizophrenic patients. Combined chronic PCP (i.p.) and clozapine (OMP) treatment down-regulated 5-HT(2A) receptor binding in many areas, similar to the effects of clozapine treatment alone and clozapine further enhanced the effects of PCP in the prefrontal cortex. In contrast 5-HT(2A) mRNA was not altered. Haloperidol treatment alone (1 mg/kg/day; OMP) and in combination with PCP (i.p.), generally produced no changes in 5-HT(2A) receptor protein or mRNA. Hence chronic PCP treatment, as employed here, mimics the decreased 5-HT(2A) receptor binding observed in the PFC of schizophrenic patients. Clozapine's enhancement of the natural response of PCP to down-regulate PFC 5-HT(2A) receptors may contribute to it's improved therapeutic profile against negative symptoms and cognitive deficits.

Numerous human imaging studies have revealed an absolute or relative metabolic hypofunction within the prefrontal cortex, thalamus and temporal lobes of schizophrenic patients. The former deficit correlates with cognitive deficits and negative symptoms, whereas the latter correlates with positive symptomologies. There is also general consensus that schizophrenia is associated with decreased parvalbumin expression in the prefrontal cortex. Since the drug phencyclidine can induce a psychosis resembling schizophrenia in humans, we have examined whether repeated phencyclidine (PCP) treatment to rats could produce similar metabolic and neurochemical deficits to those occurring in schizophrenia and whether these deficits could be modulated by antipsychotic drugs. We demonstrate here that chronic intermittent exposure to PCP (2.58 mg kg(-1) i.p.) elicits a metabolic hypofunction, as demonstrated by reductions in the rates of glucose utilization, within the prefrontal cortex, reticular nucleus of thalamus and auditory system, key structures displaying similar changes in schizophrenia. Moreover, chronic PCP treatment according to this regime also decreases parvalbumin mRNA expression in the rat prefrontal cortex and reticular nucleus of the thalamus. Chronic coadministration of haloperidol (1 mg kg(-1) day(-1)) or clozapine (20 mg kg(-1) day(-1)) with PCP did not modulate PCP-induced reductions in metabolic activity in the rat prefrontal cortex, but reversed deficits in the structures of the auditory system. Clozapine, but not haloperidol, reversed PCP-induced decreases in parvalbumin expression in prefrontal cortex GABAergic interneurons, whereas both drugs reversed the deficits in the reticular nucleus of the thalamus. These data provide important new information, which strengthen the validity of chronic PCP as a useful animal model of schizophrenia, when administered according to this protocol. Furthermore, we propose that reversal of PCP-induced reductions in parvalbumin expression in the prefrontal cortex may be a potential marker of atypical antipsychotic activity in relation to amelioration of cognitive deficits and negative symptoms of schizophrenia.

The 5-hydroxytryptamine (5-HT)(3) receptor is a member of the ligand-gated ion channel receptor family with significant homology to the nicotinic acetylcholine, gamma-aminobutyric acid(A), and glycine receptors. In this receptor class, the agonist binding site is formed by parts of the extracellular amino-terminal region. This study examines the effects of altering phenylalanine 107 (F107) of the 5-HT(3AL) subunit, obtained from NG108-15 cells, using site-directed mutagenesis. The wild-type (WT) and mutant receptors were expressed in HEK 293 cells and characterized using both whole-cell patch-clamp and radioligand binding. The tyrosine mutant F107Y exhibits a significantly lower affinity for the agonist 5-HT (K(i) = 203 versus 15.6 nM) and an increase of similar magnitude in the EC(50) value (10.6 versus 1.2 microM) compared with WT. The activation kinetics of the maximal currents generated by 5-HT with this mutant were markedly slower than those of the WT receptor, but application of supramaximal concentrations of the agonist markedly decreased the time to half-peak. The asparagine mutant F107N displayed a significantly higher affinity for 5-HT than the WT receptor (1.62 versus 15.6 nM), which was mirrored in direction and magnitude by changes in the EC(50) value for this agonist (0.2 versus 1.2 microM). In contrast to the WT receptor, the mutant F107N was activated by acetylcholine (EC(50) = 260 microM). The response to acetylcholine was blocked by the 5-HT(3) receptor antagonist renzapride with a similar IC(50) value as that determined against currents generated by 5-HT in the WT receptor. These data suggest that F107 is an important determinant of agonist recognition at the 5-HT(3) receptor.

The 5-HT3 receptor is a ligand-gated ion channel with significant structural similarity to the nicotinic acetylcholine receptor. Several regions that form the ligand binding site in the nicotinic acetylcholine receptor are partially conserved in the 5-HT3 receptor, presumably reflecting the conserved signal transduction mechanism. Specific amino acid differences in these regions may account for their distinct ligand recognition properties. Using site-directed mutagenesis, we have replaced one of these residues, glutamate 106 (E106), with aspartate (D), asparagine (N), alanine (A) or glutamine (Q) and characterized the ligand-binding and electrophysiological properties of the mutant receptors after transient expression in HEK-293 cells. The affinity for the selective 5-HT3 receptor antagonist [3H]GR65630 was decreased 14-fold in the mutant E106D (Kd = 3.69 +/- 0.32 nM) when compared to wildtype (WT, E106) 5-HT3 receptor (0.27 +/- 0.03 nM), while the affinity for E106N was unchanged (0.42 +/- 0.07 nM, means +/- SEM, n = 3-10). Decreased affinities for both E106D and E106N were observed for the antagonists granisetron, ondansetron and renzapride and for the agonists 5-HT (130- and 30-fold) and 2-methyl-5-HT (250- and 20-fold), respectively. Both mutants still formed 5-HT-activatable ion channels, but the high Hill coefficient of the concentration effect curves in wildtype (2.0) was decreased to unity in both cases. The EC50 of 5-HT was increased seven-fold in E106N (8.7 microM) when compared to wildtype (1.2 microM), but unchanged in E106D, and the potency of the antagonist ondansetron for both mutants was decreased. E106A and E106Q expressed poorly preventing a detailed characterization. These data suggest that E106 contributes to the ligand-binding site of the 5-HT3 receptor and may form an ionic or hydrogen bond interaction with the primary ammonium group of 5-HT.

1. The ability of angiotensin II to modulate dopamine release from rat striatal slices in vitro and in the intact rat striatum in vivo was assessed by the microdialysis technique. 2. In slices of rat striatum, angiotensin II (0.1-1.0 microM) induced a concentration-related increase in endogenous dopamine release which was maximal (approximately 250% above basal levels) within the first 2-4 min of agonist application and subsequently declined to near basal values. The angiotensin II-induced increase in dopamine release was Ca(2+)-dependent and was completely antagonized by the selective AT1 receptor antagonist, losartan (1.0 microM). In contrast, the AT2 receptor antagonist, PD123177 (1.0 microM) failed to modify the angiotensin II-induced response. Neither antagonist alone modified basal dopamine release from striatal slices. 3. In freely moving rats, angiotensin II (1.0-10 microM; administered via the microdialysis probe) induced a concentration-related increase in extracellular levels of dopamine which was maximal (approximately 150% above basal levels) within 20-40 min of agonist application and subsequently declined. The angiotensin II (10 microM)-induced increase in extracellular levels of dopamine was completely antagonized by the AT1 receptor antagonist, losartan (0.1-1.0 microM; administered via the microdialysis probe) but not by the AT2 receptor antagonist, PD123177 (1.0 microM; administered via the microdialysis probe). Neither antagonist alone modified basal extracellular levels of dopamine. 4. Homogenate radioligand binding studies with [125I]-angiotensin II (0.1 nm) identified relatively low levels of specific binding sites in rat striatal homogenates compared to homogenates of pyriform cortex (51.3 +/- 9.2 and 651.3 +/- 55.1 fmol g-1 wet weight, respectively, mean +/- s.e.mean, n = 3; non-specific binding defined by unlabelled angiotensin II). The majority of the specific [125I]-angiotensin II (0.1 nM) binding in the striatal and pyriform cortex homogenates was sensitive to the selective AT1 receptor antagonist, losartan (1.0 microM). 5. In conclusions the present study provides direct evidence that angiotensin II acting via the AT1 receptor subtype facilitates the release of dopamine in the rat striatum in vitro and in vivo. This receptor-mediated response may account for the modulation of dopamine-mediated behavioural responses by antagonists of the AT1 receptor and inhibitors of angiotensin converting enzyme.

1. The ability of 5-HT4 (5-hydroxytryptamine4) receptor ligands to modify dopamine release from rat striatal slices in vitro and in the striatum of freely moving rats was assessed by the microdialysis technique. 2. The release of dopamine from slices of rat striatum continually perfused with Krebs buffer was enhanced by 5-HT4 receptor agonists; 5-HT (10 microM), 5-methoxytryptamine (5-MeOT; 10 microM), renzapride (10 microM) and (S)-zacopride (10 microM) maximally increased dopamine release by 133 +/- 5, 214 +/- 25, 232 +/- 29 and 264 +/- 69%, respectively (mean +/- s.e.mean, n = 3-8). The drug-induced responses were maximal within the first 2 min of drug application, and subsequently declined. The non-selective 5-HT3/5-HT4 receptor antagonist, SDZ205-557 (10 microM), failed to modify basal dopamine release from striatal slices but completely antagonized the (S)-zacopride (10 microM)-induced increase in dopamine release. 3. To allow faster drug application, the modulation of dopamine release from rat striatal slices in a static release preparation was also investigated. The 5-HT4 receptor agonist, renzapride (10 microM) also enhanced dopamine release in this preparation (maximal increase = 214 +/- 35%, mean +/- s.e.mean, n = 14), whilst a lower concentration of renzapride (3 microM) was less effective. The renzapride-induced response was maximal within the first 2 min of drug application, before declining. In this preparation, the stimulation of dopamine release by renzapride (10 microM), was completely antagonized by the selective 5-HT4 receptor antagonist, GR113808 (100 nM). In addition, both the Na+ channel blocker, tetrodotoxin (100 nM) and the non-selective protein kinase A inhibitor, H7 (100 nM) completely prevented the stimulation of dopamine release induced by renzapride (10 microM). 4. In vivo microdialysis studies demonstrated that the 5-HT4 receptor agonists, 5-MeOT (10 microM), renzapride (100 microM) and (S)-zacopride (100 microM) maximally elevated extracellular levels of dopamine in the striatum by 220 +/- 20, 161 +/- 10 and 189 +/- 53%, respectively (mean +/- s.e.mean, n = 5-9). A lower concentration of renzapride (10 microM) was less effective. The elevation of extracellular striatal dopamine levels induced by either renzapride (100 microM) or (S)-zacopride (100 microM) were completely antagonized by the non-selective 5-HT4 receptor antagonist, SDZ205-557 (100 microM). In addition, the elevation of extracellular levels of dopamine induced by either 5-MeOT (10 microM) or renzapride (100 microM) was completely prevented by the selective 5-HT4 receptor antagonist, GR113808 (1 microM) and the renzapride (100 microM)-induced response was also completely prevented by the non-selective protein kinase A inhibitor, H7 (1 microM). In this in vivo preparation, both GR113808 (1 microM) and H7 (1 microM), when perfused alone, reduced extracellular levels of dopamine. 5. In conclusion, the present study provides evidence that the 5-HT4 receptor facilitates rat striatal dopamine release in vitro and in vivo.

1. The radioligand binding characteristics of the 3H-derivative of the novel 5-HT3 receptor antagonist BRL46470 were investigated and directly compared to the well characterized 5-HT3 receptor radioligand [3H]-granisetron, in tissue homogenates prepared from rat cerebral cortex/hippocampus, rat ileum, NG108-15 cells, HEK-5-HT3As cells and human putamen. 2. In rat cerebral cortex/hippocampus, rat ileum, NG108-15 cell and HEK-5-HT3As cell homogenates, [3H]-BRL46470 bound with high affinity (Kd (nM): 1.57 +/- 0.18, 2.49 +/- 0.30, 1.84 +/- 0.27, 3.46 +/- 0.36, respectively; mean +/- s.e. mean, n = 3-4) to an apparently homogeneous saturable population of sites (Bmax (fmol mg-1 protein): 102 +/- 16, 44 +/- 4, 968 +/- 32 and 2055 +/- 105, respectively; mean +/- s.e. mean, n = 3-4) but failed to display specific binding in human putamen homogenates. 3. In the same homogenates of rat cerebral cortex/hippocampus, rat ileum, NG108-15 cells, HEK-5-HT3As cells and human putamen as used for the [3H]-BRL46470 studies, [3H]-granisetron also bound with high affinity (Kd (nM): 1.55 +/- 0.61, 2.31 +/- 0.44, 1.89 +/- 0.36, 2.03 +/- 0.42 and 6.46 +/- 2.58 respectively; mean +/- s.e. mean, n = 3-4) to an apparently homogeneous saturable population of sites (Bmax (fmol mg-1 protein): 39 +/- 4, 20 +/- 2, 521 +/- 47, 870 +/- 69 and 18 +/- 2, respectively; mean +/- s.e. mean, n = 3-4). 4. Competition studies with a range of structurally different 5-HT3 receptor ligands indicated that in both rat cerebral cortex/hippocampus and rat ileum homogenates, [3H]-BRL46470 binding exhibited a pharmacological profile consistent with the labelling the 5-HT3 receptor with compounds competing with Hill coefficients close to unity.5 In HEK-5-HT3As cell homogenates, [3H]-BRL46470 and [3H]-granisetron associated rapidly((3.84+/-0.4)106 M-1S-1 and (5.85+/-0.2)106 M-1S-1, respectively, mean+/-s.e.mean, n=3-4) in an apparently monophasic manner. Following the establishment of equilibrium, both [3H]-BRL46470 and [3H]-granisetron at a saturating concentration ([3H]-BRL46470 approximately 16 nM; [3H]-granisetron approximately 18 nM) and at a sub-Kd concentration (approximately 1 nm for both radioligands)dissociated biphasically in HEK-5-HT3As cell homogenates (saturating concentration; [3H]-BRL464704.05 x 10-3+/-2.53 x I0-3 s-1 and 5.83 x 10-5+0.91 x I0-5 s-1; [3H]-granisetron 3.20 x 10-3+ 1.70 x IO-3 s-1 and18.58 x 10-5 +/- 4.19 x I0-5 s-1: sub-Kd concentration; [3H]-BRL46470 2.47 x 10-3+/- 1.18 x 10-3 s-1 and 9.30x 10-5+/-2.59x 10-5 S-1; [3H]-granisetron 65.91 x 10-3+/-22.14x I0-3 s-1 and 49.96x 10-5+/-12.26x 10-5s- 1 mean+/- s.e.mean, n = 4-8) when induced by a 300 fold dilution in ice-cold Tris/Krebs.6 In conclusion, the present study provides evidence that [3H]-BRL46470 specifically labels the 5-HT3receptor in rat cerebral cortex/hippocampus, rat ileum, NG108-15 cell and HEK-5-HT3As cell homogenates, but fails to label the 5-HT3 receptor expressed in human putamen. Whilst the pharmacological profile of the site labelled by [3H]-BRL46470 is directly comparable to that labelled by [3H]-granisetron, [3H]-BRL46470 consistently labelled approximately twice the density of sites compared to [3H]-granisetron in the same tissue homogenates prepared from rat cortex/hippocampus, ratileum, NG108-15 cells and HEK-5-HT3As cells.

1. The effects of ethanol, chloral hydrate and trichloroethanol upon the 5-HT3 receptor have been investigated by use of electrophysiological techniques applied to recombinant 5-HT3 receptor subunits (5-HT3R-A or 5-HT3R-As) expressed in Xenopus laevis oocytes. Additionally, the influence of trichloroethanol upon the specific binding of [3H]-granisetron to membrane preparations of HEK 293 cells stably transfected with the murine 5-HT3R-As subunit and 5-HT3 receptors endogenous to NG 108-15 cell membranes was assessed. 2. Ethanol (30-300 mM), chloral hydrate (1-30 mM) and trichloroethanol (0.3-10 mM), produced a reversible, concentration-dependent, enhancement of 5-HT-mediated currents recorded from oocytes expressing either the 5-HT3R-A, or the 5-HT3R-As subunit. 3. Trichloroethanol (5 mM) produced a parallel leftward shift of the 5-HT concentration-response curve, reducing the EC50 for 5-HT from 1 +/- 0.04 microM (n = 4) to 0.5 +/- 0.01 microM (n = 4) for oocytes expressing the 5-HT3R-A. A similar shift, from 2.1 +/- 0.05 microM (n = 11) to 1.3 +/- 0.1 microM (n = 4), was observed in oocytes expressing the 5-HT3R-As subunit. Trichloroethanol (5 mM) had little or no effect upon the maximum current produced by 5-HT for either recombinant receptor. 4. Trichloroethanol (5 mM) similarly reduced the EC50 for 2-methyl-5-HT from 13 +/- 0.4 microM (n = 4) to 4.6 +/- 0.2 microM (n = 4) and from 15 +/- 2 microM (n = 4) to 5 +/- 0.4 microM (n = 4) for oocytes expressing the 5-HT3R-A and 5-HT3R-As subunit respectively. Additionally, trichloroethanol (5 mM) produced a clear enhancement of the maximal current to 2-methyl-5-HT (expressed as a percentage of the maximal current to 5-HT) from 63 +/- 0.7% (n = 4) to 101 +/- 1.6% (n = 4) and from 9 +/- 0.2% (n = 4) to 74 +/- 2% (n = 4) for oocytes expressing the 5-HT3R-A and 5-HT3R-As subunit respectively. 5. Trichloroethanol (2.5 mM) had no effect upon the Kd, or Bmax, of specific [3H]-granisetron binding to membrane homogenates of NG 108-15 cells or HEK 293 cells. Similarly, competition for [3H]-granisetron binding by the 5-HT3 receptor antagonists ondansetron and tropisetron was unaffected. However, competition for [3H]-granisetron binding by the 5-HT3 receptor agonists, 5-HT, 2-methyl-5-HT and phenylbiguanide was enhanced by trichloroethanol (2.5 mM). 6 Unexpectedly, the competition for [3H]-granisetron binding by the 5-HT3 receptor antagonist,quipazine, was enhanced by 2.5 mM trichloroethanol. Quipazine (1 nM-0.3 microM) antagonized 5-HT evoked currents recorded from oocytes expressing the 5-HT3R-A subunit with an IC50 of 18 +/- 3 nM(n = 4). Additionally, quipazine (30 nM-0.3 microM) produced a small inward current which was greatly enhanced by 5 mM trichloroethanol and antagonized by 100 nM ondansetron. Collectively, these observations suggest that quipazine may act as a partial agonist.7. The demonstration that a recombinant homo-oligomeric receptor, expressed in a foreign membrane,retains a sensitivity to alcohols, together with the sequencing of alcohol-insensitive 5-HT3 receptor subunits, may lead to a better definition of the alcohol binding site(s).

The present study has demonstrated the distribution of [3H]granisetron-labelled 5-HT3 receptors in the human forebrain with relatively high levels of this receptor in homogenates of hippocampus, caudate nucleus, putamen, nucleus accumbens and amygdala. Lower levels of 5-HT3 receptors were found in other brain regions and the cervical vagus nerve. Pharmacological characterization of the labelled 5-HT3 receptor in human putamen homogenates identified a relatively low affinity for d-tubocurarine compared to the 5-HT3 receptor in NG108-15 neuroblastoma-glioma cell homogenates. In contrast, the affinities of 19 other 5-HT3 receptor ligands were not significantly different for the [3H]granisetron-labelled receptor in these two preparations. Such findings indicate that the human putamen 5-HT3 receptor displays a unique pharmacology which may have significance given the reported clinical potential of compounds active at this receptor when assessed in animal models of disease.

The binding of the tritiated derivative of the 5-HT3 receptor agonist meta-chlorophenylbiguanide ([3H]mCPBG) to rat cortical homogenates and whole rat brain sections was assessed in an attempt to further investigate the binding of agonists to the 5-HT3 receptor. In crude homogenates of rat cortex, no reproducible specific [3H]mCPBG (1.0 nM) binding (defined by either 10 microM granisetron, 100 microM 5-HT or 100 nM 'cold' mCPBG) was detected. Using autoradiographic techniques, in rat hindbrain sections, [3H]mCPBG (1.0 nM) labelled a differential distribution of specific binding sites (defined by the inclusion of granisetron, 1.0 microM). Specific binding was only detected within the dorsal vagal complex (nucleus tractus solitarius, area postrema and dorsal motor nucleus of the vagus nerve). An identical distribution of specific binding was detected in adjacent sections incubated with the selective 5-HT3 receptor radioligand, [3H](S)-zacopride (0.5 nM; non-specific binding defined by the inclusion of granisetron, 1.0 microM). No reproducible specific [3H]mCPBG (1.0 nM) binding (defined by the inclusion of granisetron, 1.0 microM) was detected within the rat forebrain. In contrast, [3H](S)-zacopride (0.5 nM) labelled specific sites (defined by the inclusion of granisetron, 1.0 microM) in some limbic brain structures (e.g. cerebral cortex, hippocampus, amygdala). These studies indicate that [3H]mCPBG labels the 5-HT3 receptor in rat brain tissue. However, the relatively high level of non-specific binding associated with this radioligand appears to mask the specific binding in regions which do not express relatively high densities of the 5-HT3 receptor.

The present study assessed 5-HT3 receptor recognition site levels in homogenates of putamen derived from patients with clinically and neurochemically diagnosed Huntington's disease or Parkinson's disease and those from age-, sex- and post-mortem delay-matched neurologically and psychiatrically normal patients to investigate the cellular location of 5-HT3 receptors in the human putamen. Specific [3H]granisetron (0.91 nM) binding (defined by ondansetron, 10 microM) was significantly reduced in putamen homogenates from eight out of ten patients with Huntington's disease compared to similar homogenates from 'control' patients (72 +/- 6 and 39 +/- 8 fmol/g wet weight, mean +/- S.E.M., n = 10 and 8, tissue from 'control' and Huntington's disease patients, respectively, P = 0.004). In contrast, specific [3H]granisetron (1.04 nM) binding levels were similar in putamen homogenates from patients with Parkinson's disease when compared to homogenates from 'control' patients. The present results indicate that at least a proportion of the 5-HT3 receptor population in the human putamen is located on neurones that have their cell bodies within this brain region and that these receptors are not primarily located on dopamine neurone terminals in the human putamen.

1. The content of amino acids (taurine, glycine, glutamic acid, gamma-aminobutyric acid (GABA) and aspartic acid) and monoamines (5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA) and noradrenaline) in homogenates of rat cortical and hippocampal tissue were measured by high performance liquid chromatography (h.p.l.c.) with fluorescent and electrochemical detection respectively, after two anxiogenic treatments: exposure to a phobic stimulus (cat odour) and withdrawal from chronic diazepam treatment. 2. In neither of the two anxiogenic situations was there a significant change in any amino acid content, in either brain area. 3. In the group withdrawn from chronic diazepam, cortical 5-HT and 5-HIAA levels and hippocampal 5-HT levels were significantly increased. Noradrenaline content was significantly decreased in the hippocampus. 4. The changes in 5-HT and 5-HIAA levels following cat odour exposure were area-specific in that they decreased in the hippocampus, but increased in the cortex. 5. Following cat odour exposure, noradrenaline levels appeared not to change in either area studied. However during exposure to cat odour, it was found that half the animals avoided the odour source and half were indifferent. The animals showing marked avoidance had significantly higher cortical noradrenaline content and this was significantly different from control, whereas hippocampal noradrenaline levels were not dependent upon the differences in avoidance of the odour source. 6. The results show clearly different neurochemical changes in the rat following exposure to a phobic stimulus and withdrawal from diazepam. It is hoped comparative studies such as this will enable better understanding of anxiety states in the rat which could parallel the different classes of anxiety recognised in the clinic

Autoradiographic and homogenate binding studies using the radioligand, [125I]angiotensin II, identified a heterogeneous distribution of specific binding sites (defined by angiotensin II, 1.0 microM) throughout the human forebrain. Highest AT receptor densities were detected in the paraventricular nucleus, median eminence, substantia nigra, putamen and caudate nucleus (2.4, 1.2, 1.0, 0.30 and 0.24 fmol/mg tissue equivalent, respectively). The AT1 receptor antagonist, losartan (1.0 microM) competed for the majority of the specific binding. [125I]Angiotensin II-specific binding (although not consistently above non-specific binding levels) was also detected in various other brain regions (e.g. amygdala, entorhinal cortex, frontal cortex, hippocampus, inferior colliculus, nucleus accumbens, parietal cortex, periaquaductal grey, superior colliculus, striate cortex, temporal cortex, thalamus). In the presence of losartan (1.0 microM), angiotensin II, saralasin, losartan and PD123177 competed for [125I]angiotensin II binding to membranes prepared from the cerebellum or substantia nigra with a rank order of affinity; angiotensin II = saralasin > PD123177 > losartan. In the presence of PD123177 (1.0 microM), the rank order of affinity of losartan and PD123177 was reversed. These studies indicate the presence of both AT1 and AT2 receptor subtypes within various regions of the human forebrain.