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Though transduction mechanisms recruited by heterologously expressed 5-HT(2A) receptors have been extensively studied, their interaction with specific subtypes of G-protein remains to be directly evaluated in cerebral tissue. Herein, as shown by an immunocapture/scintillation proximity analysis, 5-HT, the prototypical 5-HT(2A) agonist, DOI, and Ro60,0175 all enhanced [(35)S]GTPgammaS binding to G alpha q/11 in rat cortex with pEC(50) values of 6.22, 7.24 and 6.35, respectively. No activation of G o or G s/olf was seen at equivalent concentrations of DOI. Stimulation of G alpha q/11 by 5-HT (30 microM) and DOI (30 microM) was abolished by the selective 5-HT(2A) vs. 5-HT(2C)/5-HT(2B) antagonists, ketanserin (pK(B) values of 9.11 and 8.88, respectively) and MDL100,907 (9.82 and 9.68). By contrast, 5-HT-induced [(35)S]GTPgammaS binding to G alpha q/11 was only weakly inhibited by the preferential 5-HT(2C) receptor antagonists, RS102,221 (6.94) and SB242,084 (7.39), and the preferential 5-HT(2B) receptor antagonist, LY266,097 (6.66). The antipsychotic, clozapine, which had marked affinity for 5-HT(2A) receptors, blocked the recruitment of G alpha q/11 by 5-HT and DOI with pK(B) values of 8.54 and 8.14, respectively. Its actions were mimicked by the "atypical" antidepressant and 5-HT(2A) receptor antagonist, mirtazapine, which likewise blocked 5-HT and DOI-induced G alpha q/11 protein activation with pK(B) values of 7.90 and 7.76, respectively. In conclusion, by use of an immunocapture/scintillation proximity strategy, this study shows that native 5-HT(2A) receptors in rat frontal cortex specifically recruit G alpha q/11 and that this action is blocked by clozapine and mirtazapine. Quantification of 5-HT(2A) receptor-mediated G alpha q/11 activation in frontal cortex should prove instructive in characterizing the actions of diverse classes of psychotropic agent.

Mirtazapine displayed marked affinity for cloned, human alpha2A-adrenergic (AR) receptors at which it blocked noradrenaline (NA)-induced stimulation of guanosine-5'-O-(3-[35S]thio)-triphosphate ([35S]-GTPgammaS) binding. Similarly, mirtazapine showed high affinity for cloned, human serotonin (5-HT)2C receptors at which it abolished 5-HT-induced phosphoinositide generation. Alpha2-AR antagonist properties were revealed in vivo by blockade of UK-14,304-induced antinociception, while antagonist actions at 5-HT2C receptors were demonstrated by blockade of Ro 60 0175-induced penile erections and discriminative stimulus properties. Mirtazapine showed negligible affinity for 5-HT reuptake sites, in contrast to the selective 5-HT reuptake inhibitor, citalopram. In freely moving rats, in the dorsal hippocampus, frontal cortex (FCX), nucleus accumbens and striatum, citalopram increased dialysate levels of 5-HT, but not dopamine (DA) and NA. On the contrary, mirtazapine markedly elevated dialysate levels of NA and, in FCX, DA, whereas 5-HT was not affected. Citalopram inhibited the firing rate of serotonergic neurons in dorsal raphe nucleus, but not of dopaminergic neurons in the ventral tegmental area, nor adrenergic neurons in the locus coeruleus. Mirtazapine, in contrast, enhanced the firing rate of dopaminergic and adrenergic, but not serotonergic, neurons. Following 2 weeks administration, the facilitatory influence of mirtazapine upon dialysate levels of DA and NA versus 5-HT in FCX was maintained, and the influence of citalopram upon FCX levels of 5-HT versus DA and NA was also unchanged. Moreover, citalopram still inhibited, and mirtazapine still failed to influence, dorsal raphe serotonergic neurons. In conclusion, in contrast to citalopram, mirtazapine reinforces frontocortical dopaminergic and corticolimbic adrenergic, but not serotonergic, transmission. These actions reflect antagonist properties at alpha2A-AR and 5-HT2C receptors.

In the present study, the comparative mechanisms of action of phencyclidine (PCP) and amphetamine were addressed employing the parameter of locomotion in rats. PCP-induced locomotion (PLOC) was potently blocked by the selective serotonin (5-HT)2A vs. D2 antagonists, SR46349, MDL100,907, ritanserin and fananserin, which barely affected amphetamine-induced locomotion (ALOC). In contrast, the selective D2 vs. 5-HT2A antagonists, eticlopride, raclopride and amisulpride, preferentially inhibited ALOC vs. PLOC. The potency of these drugs and 12 multireceptorial antipsychotics in inhibiting PLOC vs. ALOC correlated significantly with affinities at 5-HT2A vs. D2 receptors, respectively. Amphetamine and PCP both dose dependently increased dialysate levels of dopamine (DA) and 5-HT in the nucleus accumbens, striatum and frontal cortex (FCX) of freely moving rats, but PCP was proportionally more effective than amphetamine in elevating levels of 5-HT vs. DA in the accumbens. Further, whereas microinjection of PCP into the accumbens elicited locomotion, its introduction into the striatum or FCX was ineffective. The action of intra-accumbens PCP, but not intra-accumbens amphetamine, was abolished by SR46349 and clozapine. Parachloroamphetamine, which depleted accumbens pools of 5-HT but not DA, likewise abolished PLOC without affecting ALOC. In contrast, intra-accumbens 6-hydroxydopamine (6-OHDA), which depleted DA but not 5-HT, abolished ALOC but only partially attenuated PLOC. In conclusion, PLOC involves (indirect) activation of accumbens-localized 5-HT2A receptors by 5-HT. PLOC is, correspondingly, more potently blocked than ALOC by antipsychotics displaying marked affinity at 5-HT2A receptors.

(-)-Pindolol (2.5-20.0 mg/kg, s.c.) markedly increased extracellular levels of dopamine (DA) and noradrenaline (NA) in single dialysate samples of the frontal cortex (FCX) of freely-moving rats. This action was specific inasmuch as serotonin (5-HT) levels were not significantly modified. In contrast, (-)-propranolol (2.5) did not modify FCX dialysate levels of DA, NA (or 5-HT) alone and abolished the facilitatory influence of (-)-pindolol (10.0) upon levels of DA, though not NA. In contrast to (-)-propranolol, (-)-pindolol exerts, a facilitatory influence upon frontocortical dopaminergic and adrenergic, but not serotonergic, transmission.

The glycine B receptor partial agonists L 687,414, D-cycloserine and (+)-HA 966, and the glycine B receptor antagonists MDL 29,951 and 5,7-dichloro-2,4 dihydroxy-3-phenyl-quinoline dione (DCPQ) dose-dependently inhibited the late phase (LP) of formalin-induced licking (FIL) elicited by intraplantar formalin in mice at doses exerting little motor disruption in the rotarod test. In distinction, the early phase (EP) of FIL and the writhing response to intra-abdominal acetic acid were little influenced and, irrespective of stimulus intensity, they failed to modify the tail-flick response to phasic, thermal or mechanical stimulation of the tail. In contrast to glycine B ligands, competitive antagonists at the NMDA receptor recognition site (CPP, CGS 19755, CGP 34879 and 39551) and blockers of the associated ion channel ((+)-MK 801, (-)-MK 801, memantine and ketamine) all blocked both the LP and EP of FIL and induced ataxia at comparable doses. In conclusion, normalization of transmission at NMDA receptors by inhibition of the coupled glycine B site preferentially elicits antinociception against prolonged (chemical) noxious stimulation in the absence of a marked influence upon motor coordination.

The 5-HT1A partial agonists, buspirone, ipsapirone and gepirone did not affect the latency to respond in the tail flick test to heat. However, they strongly attenuated the antinociceptive action of the mu-opioids, morphine and sufentanil. The buspirone metabolite, 1-(2-pyrimidyl)pyridine (1-PP) was ineffective. BMY 7378, spiperone and alprenolol, putative antagonists at 5-HT1A sites, did not modify basal latencies or the action of morphine. TFMPP and mCPP, agonists at 5-HT1B and 5-HT1C sites, also did not affect basal latencies or morphine induced antinociception. These data show that 5-HT1A partial agonists attenuate morphine-evoked antinociception without affecting basal thresholds. They represent an interesting aspect of the interaction between opioids and serotonin in the control of nociception. In addition to opioids (Millan, 1986), serotonin (5-HT) is considered to play a major role in the control of pain and in the expression of opioid analgesia (Roberts, 1984). The identification of a multiplicity of binding sites for 5-HT in the CNS (Fozard, 1987) raises the question of their individual roles in nociceptive processes. The 5-HT1A site is of particular interest since it is present in high concentrations in the dorsal horn of the spinal cord (Daval, Verge, Basbaum, Bourgoin, and Hamon, 1987) and there are conflicting reports that it may mediate analgesia or hyperalgesia (Berge, Fasmer, Ogren, and Hole, 1985, Zemlan, Kow, and Pfaff, 1983). Indeed, the 5-HT1A agonist, 8-OH-DPAT, was reported to attenuate morphine-evoked antinociception in mice (Berge et al., 1985).(ABSTRACT TRUNCATED AT 250 WORDS)

Since its inception some two decades ago, microdialysis has rapidly assumed a crucial role as an interface between cellular models of drug actions in vitro and studies of their behavioural effects in vivo. Microdialysis provides invaluable information regarding the mechanisms of action of psychotropic agents and their influence upon endogenous modulators implicated in the aetiology and treatment of CNS disorders. In addition, measures of extracellular levels of specific neurotransmitters are complementary to behavioural parameters in the characterisation of experimental models of psychiatric and neurological diseases. Further, microdialysis can be used for quantification of levels of psychotropic agents themselves in specific brain regions. Though microdialysis techniques are increasingly being applied to a broad variety of cellular mediators, most studies have to date focussed on monoamines, acetylcholine and amino acids like glycine, glutamate and GABA; that is, neurotransmitters strongly implicated in the pathogenesis and control of depression, anxiety, schizophrenia and other psychiatric states. Recent years have seen substantial improvements in the sensitivity of systems for their detection, which now permit, for example, the simultaneous quantification of dopamine (DA), serotonin (5-HT) and noradrenaline (NA) levels in single dialysis samples; quantification of acetylcholine (ACh) levels in the absence of acetylcholinesterase inhibitors; and determination of glutamate and GABA levels concurrently with glycine and a miscellany of related amino acids. The present chapter provides an overview of how microdialysis can be applied to the discovery and evaluation of centrally active drugs. Furthermore, it specifically focuses on the application of novel microdialysis techniques to the characterisation of antipsychotic agents for the improved treatment of schizophrenia.

the differentiation of NG108−15 neuroblastoma cells and rat striatal neurons in primary culture, assessed by neurite outgrowth. Expression of voltage-gated Ca2+ channels was also analysed in NG108−15 cells using Fura2 imaging. The unpaired Student’s t-test and one-way ANOVA followed by Dunnett’s test were performed (using the Prism software) for two-sample and multiple comparisons, respectively. Expressing 5-HT6 receptors in NG108−15 neuroblastoma triggered neurite outgrowth and induced expression of functional voltage-gated Ca2+ channels. These effects were not further enhanced by an agonist (WAY181,187, 1mM) but were prevented by SB258,585 (10mM), a selective 5-HT6 antagonist. Thus, upon SB258,585 treatment, cells showed a decrease in neurite length of 48.4% compared to untreated cells (22.5±1.5 mm vs. 43.5±1mm, n = 4, p< 0.0001). Expression of a dominantnegative (DN) Cdk5 or treating cells with roscovitine (pharmacological inhibitor of Cdk5) likewise inhibited NG108−15 cells differentiation induced by 5-HT6 receptor expression (control: 69.8±3.6mm; DN Cdk5: 42.8±3mm, p< 0.0001; roscovitine: 28.1±1.5mm, n = 3, p< 0.0001). SB258,585 also impaired the association of 5-HT6 receptor with Cdk5 in NG108−15 cells, as determined by co-immunoprecipitation, suggesting that this interaction was necessary for induction of differentiation. Treating striatal neurons with either SB258,585 or roscovitine immediately after seeding decreased neurite length, as determined 24 hrs later (untreated: 29.9±0.7mm; SB258,585: 23.7±0.6mm, p< 0.0001; roscovitine: 20.2±1.0mm, n = 4, p< 0.0001). Conversely, exposure of neurons to WAY181,187 did not significantly affect neurite outgrowth (p = 0.43). Further supporting a role of endogenously expressed receptors in differentiation of striatal neurons, silencing 5-HT6 receptor expression in cultured neurons significantly reduced neurite length (control siRNA: 29.6±0.8mm, 5-HT6 receptor siRNA: 17.2±0.9mm, n = 3, p< 0.0001). Conclusions: Complementing work indicating that 5-HT6 receptors modulate neuronal migration [3], the present data show that 5-HT6 receptors developmentally promote neuronal differentiation and reveal a critical role for Cdk5 in this process. These novel insights into molecular substrates underlying neurodevelopmental effects of 5-HT6 receptors are of potential importance to the pathophysiology and treatment of early-onset CNS conditions like autism-spectrum disorder and schizophrenia.

The 5-HT2 receptor agonist, DOI, dose-dependently (0.16-10.0 mg/kg, s.c.) increased dialysate levels of dopamine (DA) and noradrenaline (NA), but not 5-HT, in the frontal cortex (FCX) of freely-moving rats. This action was abolished by the selective 5-HT2A antagonist, MDL100,907 (0.04), which did not, itself, modify levels of DA and NA. In contrast, the selective 5-HT2B/2C antagonist, SB206,553 (0.63), increased levels of DA and NA additively with DOI. Thus, in contrast to a tonic, inhibitory influence of 5-HT2C receptors (see Millan, M.J., Dekeyne, A., Gobert, A., 1998. Serotonin (5-HT)2C receptors tonically inhibit dopamine (DA) and noradrenaline (NAD), but not 5-HT, release in the FCX in vivo. Neuropharmacology 37, 953-955), 5-HT2A receptors exert a phasic, facilitatory influence upon FCX levels of DA and NA.

1. It has recently been suggested that opioid antagonists may be divided into those possessing negative intrinsic activity (e.g. naloxone) and those with neutral intrinsic activity (e.g. MR2266). 2. MR2266 was chronically administered to rats by subcutaneous infusion at a dose of 0.3 mg kg-1 h-1 for 1 week. 3. This dose reduced ingestive behaviour and blocked the antinociceptive effects of a kappa-agonist, indicating occupation of opioid receptors in vivo. 4. No supersensitivity could be detected to the antinociceptive actions of mu or kappa agonists, either one or two days after cessation of treatment. 5. No up-regulation of mu, delta or kappa binding sites was observed. 6. Since naloxone induces both supersensitivity and receptor up-regulation under equivalent conditions, the results suggest that negative intrinsic activity may be required for these phenomena to occur.