Social threat and novel cage stress-induced sustained extracellular-regulated kinase1/2 (ERK1/2) phosphorylation but differential modulation of brain-derived neurotrophic factor (BDNF) expression in the hippocampus of NMRI mice.

The extracellular signal-regulated kinase1/2 (ERK1/2) pathway has a key role in cell survival and brain plasticity, processes that are impaired following exposure to stressful situations. We have recently validated two repeated intermittent stress procedures in male NMRI mice, social threat and repeated exposure to a novel cage, which result in clear behavioral effects following 4 weeks of application. The present results demonstrate that both repeated intermittent stress procedures alter the activity of the ERK1/2 pathway in the brain, as shown by changes in phosphorylated ERK1/2 (phospho-ERK1/2) protein expression and in the expression of downstream proteins: phosphorylated cAMP response element binding protein (CREB) and brain-derived neurotrophic factor (BDNF), in the hippocampus, the frontal cortex and the hypothalamus. The hippocampus showed greater responsiveness to stress as the two stressors increased phospho-ERK1/2 and BDNF expression under acute condition. Following repeated stress, hyperphosphorylation of ERK1/2 was associated with up-regulation of hippocampal BDNF expression in the social threat group but not in mice exposed to novel cage. This lack of a pro-survival effect of ERK1/2 with repeated novel cage exposure may constitute an early event in stress-mediated brain pathology. The sustained BDNF up-regulation in the hippocampi of mice subjected to repeated social threat could be related to rewarding aspects of aggressive interactions, suggested by our previous studies.

Effects of fluoxetine on social behaviour and plasma corticosteroid levels in female Mongolian gerbils.

Gerbils are a highly social species and extremely sensitive to social manipulations. In this laboratory, separating male/female pairs has been found to produce significant effects on these animal's subsequent social behaviour. The present studies were conducted in order to examine the effects of a short period of individual housing in females of this species, as this may also be predicted to produce alterations in social responding. It was found that 21 days' individual housing induced a marked reduction in social behaviour directed towards an untreated male placed in the same arena. This was indicated by a highly significant increase in immobile-in-contact, a behaviour that involves females freezing while, and only while, they are being socially investigated. This represents the declining of an invitation to socially interact and so high levels of immobile-in-contact indicate low levels of social motivation. There was also an increase in evading, upon another animal's approach, and a decrease in social investigation of other animals. The effects of 15 days of fluoxetine were found to be highly dependent on housing condition. In individually housed females, 10 mg/kg increased their social investigation of other animals and markedly reduced the duration of immobile-in-contact. Twenty mg/kg also reduced levels of immobile-in-contact and increased the frequency of active approaches towards other animals. Fluoxetine therefore acts to increase social motivation in individually housed animals. By direct contrast, in group-housed female gerbils, fluoxetine had no effects on social behaviour and produced clear indications of sedation. While housing condition had no influence on levels of corticosterone, fluoxetine produced dose-related increases in corticosteroid levels in both group- and individually housed animals. These findings show that: (1) a short period of individual housing induces a significant reduction in these animals' motivation towards social behaviour; (2) the effects of fluoxetine on behaviour are greatly influenced by housing condition--prosocial effects are seen in individually housed animals but only sedative effects are seen in animals maintained in groups; and (3) while housing condition has no effects on levels of corticosterone, fluoxetine dose-dependently stimulates corticosteroid release. It can be concluded that the effects of fluoxetine on gerbil behaviour are independent of its stimulatory influence on HPA axis functioning, and that the prosocial effects of this selective serotonin reuptake inhibitor (SSRI) can only be seen in animals with a pre-existing social deficit.

Evidence implicating a role for orexin-1 receptor modulation of paradoxical sleep in the rat.

The neuropeptide orexin-A modulates the sleep-wake cycle such that central administration to rats increases arousal, reduces slow-wave-sleep (SWS) and paradoxical sleep (PS) and delays PS onset. The contribution of orexin-1 and -2 receptor (OXR) activation to this orexin-A response is still unknown. Using the OX(1)R antagonist SB-334867-A we investigated the role of this receptor in orexin-A-induced PS alteration. Male rats prepared for frontal-occipital electroencephalograph, nuchal muscle electromyograph recording and lateral ventricle cannulae received vehicle or orexin-A (10 microg icv) at lights on in combination with vehicle or SB-334867-A (10 or 30 mg/kg ip) 30 min pre-icv injection. The amount of arousal, SWS 1, SWS 2 and PS was determined during the 1st h post icv administration along with the latency to onset of the first> or =10 s epoch of PS. Orexin-A administration reduced the amount and increased the latency to onset of PS. SB-334867-A reversed this effect of orexin-A. The present study demonstrates that the OX(1)R also has a role in orexinergic sleep modulation.

Further evidence for the predictive validity of the unstable elevated exposed plus-maze, a behavioural model of extreme anxiety in rats: differential effects of fluoxetine and chlordiazepoxide.

The unstable elevated exposed plus-maze (UEEPM) is a novel model of extreme anxiety which elicits unconditioned flight/escape behaviour in rats. The current studies aimed to investigate further the predictive validity of the paradigm, by examining the effects on UEEPM behaviour of both clinically effective (chronic fluoxetine) and ineffective (acute fluoxetine and chlordiazepoxide) treatments for panic disorder. In the first experiment, male Brown Norway rats received a single injection of fluoxetine (1.0-10.0 mg/kg p.o.) or chlordiazepoxide (CDP, 1.0-10.0 mg/kg i.p.) 30 min prior to UEEPM exposure. In the second experiment, in order to assess the effects of chronic dosing or handling on baseline UEEPM behaviour, subjects received either 21 days vehicle injection (p.o.) or handling, before being exposed to the test. Finally, rats received 21 days fluoxetine (1.0-10.0 mg/kg) in their food, before being tested in the UEEPM. Acute CDP and fluoxetine had no effect on UEEPM behaviour. Chronic handling and vehicle administration (p.o.) significantly reduced escape in the UEEPM, hence preventing the effects of chronic fluoxetine administration from being investigated by p.o. dosing. Chronic fluoxetine in subjects' food (10.0 mg/kg) significantly attenuated animals' propensity to escape from the UEEPM. The results further support the pharmacological similarity between symptoms of panic in humans and escape in the UEEPM and suggest that the UEEPM may represent a paradigm to facilitate investigation into the neurochemical basis of extreme anxiety disorders.

Effect of SB-243213, a selective 5-HT(2C) receptor antagonist, on the rat sleep profile: a comparison to paroxetine.

5-HT(2) receptor antagonists promote slow wave sleep (SWS) in humans and rats, conversely 5-HT(2) agonists inhibit SWS in rats. These alterations are thought to be predominantly mediated via the 5-HT(2C) receptor subtype. It is evident that 5-HT(2) receptor function also plays an important role in depression. Here, we examine the acute effect of the selective 5-HT(2C) receptor antagonist 5-methyl-1-[[-2-[(2-methyl-3-pyridyl)oxy]-5-pyridyl]carbamoyl]-6-triflouromethylindoline hydrochloride (SB-243213-A) on rat sleep in comparison to the selective serotonin reuptake inhibitor (SSRI) paroxetine. Both SB-243213-A (10 mg/kg po) and paroxetine (3 mg/kg po) significantly increased deep SWS (SWS2) quantity (27% and 24%, respectively) and reduced paradoxical sleep (PS) quantity (35%) during the sleep period. Following SB-243213-A, SWS2 occurrence frequency was reduced (24.1%); however, elevated quantity of SWS2 can be attributed to an increase in occurrence duration (81%). Reduced PS quantity results from a decrease in occurrence frequency (46%). In comparison, paroxetine increased SWS2 occurrence frequency (50%), with decreased frequency (27%) and duration (21%) of PS. The data for SB-243213-A in the present study is consistent with that following ritanserin supporting 5-HT(2C) receptor subtype mediation of this response. The similar effect of SB-243213-A to paroxetine with regard to PS quantity provides further evidence that 5-HT(2C) receptor antagonists maybe beneficial in the treatment of depression/anxiety.

Evidence that orexin-A-evoked grooming in the rat is mediated by orexin-1 (OX1) receptors, with downstream 5-HT2C receptor involvement.

RATIONALE: Orexins A and B have recently been discovered and shown to be derived from preproorexin, primarily expressed in the rat hypothalamus. Orexin-A has been ascribed a number of in vivo functions in the rat after intracerebroventricular (ICV) administration, including hyperphagia, neuroendocrine modulation and, most recently, evidence for a behavioural response characterised by an increase in grooming. OBJECTIVES: Here, we have investigated the orexin-receptor subtypes involved in the grooming response to orexin-A (3 microg, ICV) in the rat. METHODS: Male rats, habituated to clear Perspex behavioural observation boxes, were pretreated with antagonists with mixed selectivity for OX1, OX2, 5-HT2B and 5-HT2C receptor subtypes prior to the administration of orexin-A and the intense grooming response elicited by this peptide assessed. RESULTS: Pretreatment of rats with a mixed OX1/5-HT2B/2C receptor antagonist 1-(4-methylsulfanylphenyl)-3-quinolin-4-ylurea (SB-284422), revealed a significant, but incomplete, blockade of orexin-A-induced grooming. Despite the low potency of orexin-A at 5-HT2B and 5-HT2C receptors in vitro (pKi<5), studies were undertaken to determine whether downstream 5-HT2B or 5-HT2C receptors mediate in the grooming-elicited by orexin-A. Whilst the selective 5-HT2B receptor antagonist, SB-215505 (3 mg/kg, PO, 5-HT2B, pKi=8.58; OX1, pKB < 5.15) failed to effect orexin-A-induced grooming, the selective 5-HT2C receptor antagonist, SB-242084 (1 mg/kg, IP, 5-HT2C, pKi = 8.95; OX1, pKB < 5.1) potently antagonised the grooming response to this peptide. This suggested that the partial blockade of orexin-A-induced grooming obtained with SB-284422 might be attributable to its 5-HT2C and/or OX1 receptor blocking activity. However, complete blockade of orexin-A-induced grooming by the subsequently identified selective OX1 receptor antagonist 1-(2-methylbenzoxazol-6-yl)-3-[1,5]naphthyridin-4-yl urea hydrochloride, SB-334867-A (OX1, pKB = 7.4; OX2, pKB = 5.7), devoid of appreciable affinity for either 5-HT2B (pKi < 5.3) or 5-HT2C (pKi < 5.4) receptors, provides the first definitive evidence that a central behavioural effect of orexin-A (grooming) is mediated by OX1 receptors. CONCLUSIONS: This data suggests that orexin-A indirectly activates 5-HT2C receptors downstream from OX1 receptors to elicit grooming in the rat. The use of SB-334867-A in vivo will enable the role of OX,1 receptors within the rat central nervous system to be further characterised.

Latency to paroxetine-induced anxiolysis in the rat is reduced by co-administration of the 5-HT(1A) receptor antagonist WAY100635.

We report here the use of rat high-light social interaction to model the temporal anxiolytic/antidepressant effects of SSRIs seen in the clinic. Compared to vehicle controls, 21, but not 14, days of paroxetine treatment (3 mg kg(-1), p.o., daily) produced a marked increase in rat social interaction (Vehicle=71.3+/-7.3 s; Paroxetine=116.7+/-14.7 s; P<0.01) with no concurrent effect on locomotor activity, consistent with anxiolysis. To assess whether concurrent 5-HT(1A) receptor blockade reduces the time to onset of anxiolysis seen with paroxetine alone (21 days), rats were implanted with osmotic minipumps to continuously infuse the 5-HT(1A) receptor antagonist WAY100635 (1 mg kg(-1) day(-1), s.c., 7 days) alone or in combination with paroxetine (3 mg kg(-1), p.o., daily, 7 days), prior to anxiety testing. Paroxetine (Veh/Par=61.9+/-7.9 s) or WAY100635 (WAY/Veh=71.6+/-4.7 s) alone, had no effect on social interaction time compared to vehicle treated controls (Veh/Veh=76.4+/-4.9 s), whilst coadministration of WAY100635 with paroxetine, produced a marked elevation in social interaction (WAY/Par=149.3+/-16.8 s; P<0.01) relative to all other groups with no concurrent change in locomotor activity. In contrast, acute administration of WAY100635 (0.03 mg kg(-1), s.c.) with paroxetine (3 mg kg(-1), p.o.) did not alter any behavioural measure, suggesting that the anxiolysis induced by the combination after 7 days is attributable to a CNS adaptive response. This data demonstrates that coadministration of a 5-HT(1A) receptor antagonist with paroxetine markedly reduces the latency to anxiolysis, in the rat. This study supports the use of the rat social interaction test to further delineate adaptive changes in the CNS responsible for the anxiolytic/antidepressant action of SSRIs seen in humans.

Evidence for accelerated desensitisation of 5-HT(2C) receptors following combined treatment with fluoxetine and the 5-HT(1A) receptor antagonist, WAY 100,635, in the rat.

Both pre-clinical and clinical studies suggest that additional treatment with 5-HT(1A) receptor antagonists may accelerate the antidepressant efficacy/onset of selective serotonin re-uptake inhibitors (SSRIs). Given that chronic SSRI treatment has been shown to desensitise 5-HT(2C) receptor mediated responses, we have used the rat social interaction test to determine if combined treatment with WAY 100,635, a selective 5-HT(1A) receptor antagonist, will accelerate this effect. In pairs of unfamiliar rats, acute administration of the 5-HT(2C) receptor agonist m-chlorophenylpiperazine (mCPP) or fluoxetine decreased the time spent in social interaction, responses which were reversed by the 5-HT(2C/2B) receptor antagonists SB 200646A and SB 221284. Similar reductions in social interaction were observed in rats treated with fluoxetine (10 mg/kg, i.p. daily) for 4, 7 and 14 days but was no longer apparent after 28 days of treatment. In contrast, only 7 days of combined treatment with WAY 100,635 (1 mg/kg/s.c./day) and fluoxetine were needed to reverse this response. The decrease in social interaction induced by an acute challenge of mCPP (1 mg/kg, i. p.) was also reduced after 6 days co-treatment with WAY 100,635 and fluoxetine. Thus, WAY 100,635 accelerates SSRI-induced desensitisation of 5-HT(2C) receptors, suggesting that this response might contribute towards the therapeutic effects of SSRIs in man.

Immunohistochemical localisation of the 5-HT2C receptor protein in the rat CNS.

5-HT2C receptor mRNA has a widespread distribution in the human and rat CNS but the absence of a specific high affinity ligand has made autoradiographic localisation of the receptor difficult. By raising polyclonal antibodies against the rat 5-HT2C receptor protein this study reports the immunohistochemical distribution of this receptor in the rat CNS. A sephadex purified 5-HT2C antiserum visualised a single immunopositive band (54 kDa) in Western blots of membranes prepared from several rat brain regions and caused intense membrane immunofluorescence in HEK 293 cells transfected with h5-HT2C cDNA, which were both attenuated by incubation with the antigenic peptide sequence (200-300 microM). 5-HT2C-like immunoreactivity was located on neurones throughout the CNS. The most abundant 5-HT2C-like immunoreactive cell bodies were in the anterior olfactory nucleus, medial and intercalated amygdaloid nuclei, hippocampus layers CA1 to CA3, laterodorsal and lateral geniculate thalamic nuclei, caudate-putamen and several areas of the cortex (including piriform and frontal), consistent with this receptor being located postsynaptic to serotonergic neurones. Immunopositive neurones were also found in the dorsal raphé, suggesting that 5-HT2C receptors may be on some serotonergic neurones. The overall distribution of 5-HT2C-like immunoreactivity complements previous findings with conventional radioligands and agrees well with reported levels of 5-HT2C receptor mRNA.

Activation of 5-HT2B receptors in the medial amygdala causes anxiolysis in the social interaction test in the rat.

In a recent study, we reported the presence of neurones expressing 5-HT2B receptor protein in the medial amygdaloid nucleus of the adult rat brain. In the present study, bilateral micro-injection of the 5-HT2B receptor agonist 1-[5-(2-thienylmethoxy)-1H-3-indolyl]propan-2-amine hydrochloride (BW 723C86, 0.09 and 0.93 nmol, 5 min pretest) into the medial amygdaloid nuclei increased the total interaction time of a pair of male rats in the social interaction test, to a comparable extent to chlordiazepoxide (5 mg/kg p.o., 1 hr pretest) without altering locomotor activity; indicative of anxiolytic activity. The increase in social interaction was prevented by pretreatment with the 5-HT2C/2B receptor antagonist N-(1-methyl-5-indoyl)-N'-(3-pyridyl) urea hydrochloride (SB 200646A, at 2 but not 1 mg/kg p.o., 1 hr pretest), which did not alter behaviour when given alone. Intra-amygdala BW 723C86 (0.09, 0.31 and 0.93 nmol, 5 min pretest) did not significantly alter the number of punished responses made when the same rats were examined seven days later in a Vogel punished drinking test, although chlordiazepoxide (5 mg/kg p.o., 1 hr pretest) produced the expected anxiolytic profile. The results are consistent with the proposal that activation of 5-HT2B receptors in the medial amygdala induces anxiolysis in the social interaction model but has little effect on behaviour in a punished conflict model of anxiety. These data suggest that serotonergic neurotransmission in this nucleus may selectively affect specific kinds of anxiety generated by different animal models.

Evidence for expression of the 5-hydroxytryptamine-2B receptor protein in the rat central nervous system.

The 5-hydroxytryptamine-2B receptor is the most recent addition to the 5-hydroxytryptamine-2 family of G-protein-coupled receptors. In the rat stomach fundus, 5-hydroxytryptamine-2B receptor activation causes contraction; however, its distribution and function in the rat CNS are unclear. By performing immunohistochemistry with an antiserum raised against the N-terminus of the 5-hydroxytryptamine-2B receptor protein, this study identifies receptor expression in longitudinal and circular smooth muscle in the rat stomach fundus and in neurons in discrete nuclei in the cerebellum, lateral septum, dorsal hypothalamus and medial amygdala. The potential function of this receptor in the CNS is discussed.

Central c-fos expression following 20kHz/ultrasound induced defence behaviour in the rat.

Exposure of rats to aversive stimuli produces specific defence behaviour including the emission of 20-27kHz ultrasonic calls. Recent studies in this laboratory have shown that rats exposed to a 20kHz ultrasound tone display flight behaviour similar to that seen naturally, or following stimulation of brain regions associated with anxiety and defence. The present study examines the effect of ultrasound exposure on the central expression of the immediate early gene c-fos in the rat, in order to examine the brain structures activated by such behaviour. Ultrasound presentation produced rapid locomotor activity characteristic of defence behaviour, including brisk running and jumping behaviour. Animals showed dense c-fos like immunoreactivity in the dorsal periaqueductal grey matter, basolateral, medial, central amygdala, paraventricular thalamic nuclei and the dorsomedial nuclei of the hypothalamus, which was significantly greater than in either home-cage or arena control rats. These results suggest that exposure to artificially generated ultrasound can induce defence behaviour which is associated with activity in brain regions important in mediating aversion. This technique offers the potential of generating unconditioned aversive behaviour in rats in a non invasive way.