BACKGROUND

Reports have indicated that administration of nicotine inhibits, while withdrawal of chronically administered nicotine augments effects of serotonergic <em>5HT</em>2A/2C agonists.

OBJECTIVE

It was our objective to determine whether <em>5HT</em>2A/2C agonists can modulate the discriminative stimulus effects of nicotine in rats or its locomotor activity effects in mice.

METHODS

Adult male Sprague-Dawley rats were trained to discriminate 0.3 mg/kg nicotine base from saline in a two-lever, fixed-ratio (FR10), food-reinforced, operant-conditioning task during daily (Monday-Friday) 15-min experimental sessions. After characterizing a dose-response curve for nicotine, we tested the ability of the <em>5HT</em>(2A/2C) agonists (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCL (DOI; 0.18-1.0 mg/kg) and 1-(4-bromo-2, 5-dimethoxyphenyl)-2-aminopropane (DOB; 0.1-1.0 mg/kg), the <em>5HT</em>2C agonist 6-chloro-2-(1-piperazinyl)pyrazine hydrochloride (MK 212; 0.1 mg/kg-1.0 mg/kg), and the <em>5HT</em>1A agonist (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT; 0.01 mg/kg-1.0 mg/kg) to modulate nicotine's discriminative stimulus effects. After finding that DOI was able to attenuate the percentage nicotine lever responding (%NLR), we tested for it to also reverse nicotine's effects on locomotor activity in mice.

RESULTS

The <em>5HT</em>2A/2C agonists-in particular DOI-dose dependently attenuated %NLR. The effects of DOI were reversed by the <em>5HT</em>2A/2C antagonist ketanserin. MK 212 and 8-OH-DPAT had irregular effects among rats and only reduced %NLR to below 50% levels at doses markedly suppressing responding. DOI also dose dependently blocked nicotine's acute rate-lowering locomotor activity effects.

CONCLUSIONS

These results indicate that activation of serotonin <em>5HT</em>2A/2C receptors can blunt the discriminative stimulus and locomotor activity effects of nicotine and presents the possibility that activation of these receptors might also be able to attenuate other effects of nicotine.

Parkinson's disease has been traditionally thought of as a dopaminergic disease in which cells of the substantia nigra pars compacta (SNc) die. However, accumulating evidence implies an important role for the serotonergic system in Parkinson's disease in general and in physiological responses to levodopa therapy, the first line of treatment. We use a mathematical model to investigate the consequences of levodopa therapy on the serotonergic system and on the pulsatile release of dopamine (DA) from dopaminergic and serotonergic terminals in the striatum. Levodopa competes with tyrosine and tryptophan at the blood-brain barrier and is taken up by serotonin neurons in which it competes for aromatic amino acid decarboxylase. The DA produced competes with serotonin (<em>5HT</em>) for packaging into vesicles. We predict the time courses of LD, cytosolic DA, and vesicular DA in <em>5HT</em> neurons during an LD dose. We predict the time courses of DA and <em>5HT</em> release from <em>5HT</em> cell bodies and <em>5HT</em> terminals as well as the changes in <em>5HT</em> firing rate due to lower <em>5HT</em> release. We compute the time course of DA release in the striatum from both <em>5HT</em> and DA neurons and show how the time course changes as more and more SNc cells die. This enables us to explain the shortening of the therapeutic time window for the efficacy of levodopa as Parkinson's disease progresses. Finally, we study the effects <em>5HT</em>1a and <em>5HT</em>1b autoreceptor agonists and explain why they have a synergistic effect and why they lengthen the therapeutic time window for LD therapy. Our results are consistent with and help explain results in the experimental literature and provide new predictions that can be tested experimentally.

Global Burden of Disease 2010 study considers migraine as one of the most important noncommunicable diseases in the world, classifying it third in terms of global prevalence (14.70%): it sums up the 54.19% of all the years of life lived with disabilities caused by the rest of all neurological disorders. This Editorial provides an historical excursus of old and new-entry molecules in migraine therapeutic area. Drugs for acute treatment such as triptans date back to the early 1990s with the appearance of sumatriptan and the following six triptans in the years immediately after (zolmitriptan, rizatriptan, naratriptan, eletriptan, almotriptan, frovatriptan). Prophylaxis drugs, dedicated to patients with medium/high frequency of crises, show as last entries topiramate and botulinum toxin type A. The use of this preventative group, with its intrinsic limits, is mandatory to reduce the risk of migraine chronification, a highly harmful clinical phenomenon that produces as its natural consequence the medication overuse headache. The development of new acute and preventative compounds, such as <em>5HT</em> (serotonin) 1F receptor (5-HT1F) agonist lasmiditan, calcitonin gene related peptide (CGRP) peptide receptor antagonists, anti-CGRP monoclonal antibodies (LY2951742, ALD403, LBR101) and anti-CGRP-r monoclonal antibody (AMG334), is warranted and might be soon completed in order to offer new opportunities to migraine patients.

The 5-hydroxytryptamine 2C (5-HT(2C)) receptor subtype has received considerable attention as a target for drug discovery, having been implicated in a wide variety of disorders. Here, we describe the in vitro pharmacological profile of the novel 5-HT(2C) receptor-selective agonist vabicaserin [(-)-4,5,6,7,9,9a,10,11,12,12a-decahydrocyclopenta[c] [1,4]diazepino[6,7,1-ij]quinoline hydrochloride] (SCA-136), including a comprehensive strategy to assess 5-HT(2B) receptor selectivity using diverse preparations and assays of receptor activation. Vabicaserin displaced (125)I-(2,5-dimethoxy)phenylisopropylamine binding from human 5-HT(2C) receptor sites in Chinese hamster ovary cell membranes with a K(i) value of 3 nM and was >50-fold selective over a number of serotonergic, noradrenergic, and dopaminergic receptors. Binding affinity determined for the human 5-HT(2B) receptor subtype using [(3)H]<em>5HT</em> was 14 nM. Vabicaserin was a potent and full agonist (EC(50), 8 nM; E(max), 100%) in stimulating 5-HT(2C) receptor-coupled calcium mobilization and exhibited 5-HT(2A) receptor antagonism and 5-HT(2B) antagonist or partial agonist activity in transfected cells, depending on the level of receptor expression. In rat stomach fundus and human colonic longitudinal muscle endogenously expressing 5-HT(2B) receptors, vabicaserin failed to induce a 5-HT(2B) receptor-dependent contraction and produced a rightward shift of the 5-HT and α-methyl-5-HT concentration-response curves in these preparations, respectively, consistent with 5-HT(2B) competitive antagonism. Likewise, vabicaserin failed to induce a 5-HT(2B) receptor-mediated contraction in arteries from deoxycorticosterone acetate-salt-treated rats, a model of hypersensitized 5-HT(2B) receptor function, and produced a rightward shift in the 5-HT-induced response that was consistent with 5-HT(2B) receptor antagonism. In summary, vabicaserin is a novel, potent, and selective 5-HT(2C) receptor agonist.

This study evaluated the effects of intravitreal injections of 300 micrograms of 6-hydroxydopamine (6-OHDA) on electroretinogram (ERG) amplitudes and implicit response times of adult pigmented rabbits. One eye was injected intravitreally with 300 micrograms 6-OHDA and 600 micrograms ascorbic acid in a 0.3 ml 0.9% NaCl solution; the fellow eye received a similar solution containing only 600 micrograms ascorbic acid. Following this treatment ERG recordings were performed at 1, 4, and 7 days. After the last recordings, animals were sacrificed and retinas were isolated for biochemical analyses. Significant and progressive reductions in retinal concentrations of dopamine (DA) and its main metabolites homovanillic acid (HVA), and dihydroxyphenylacetic acid (DOPAC) were found in treated retinas. Concentrations of norepinephrine (NE), serotonin (<em>5HT</em>), and 5 hydroxyindoleacetic acid (5-HIAA) were not affected, thus demonstrating the specific neurotoxic action of 6-OHDA on retinal dopaminergic neurons. Concurrently, significant increases in ERG a- and b-wave amplitudes as well as in implicit response times were observed. These electrophysiological changes were progressive reaching a maximum 7 days after intravitreal injections. Changes in b-wave amplitudes and response times were more pronounced at low intensities of stimulation. These results clearly show that, in rabbits, selective decreases in retinal DA concentrations result in pronounced ERG changes, which offer additional evidence supporting a role for this transmitter in lateral inhibition in the retina.

Both acetylcholine (ACh) and 5-hydroxytryptamine (<em>5HT</em>) are used to examine nitric oxide (NO)-mediated vasodilatation in humans. Animal data suggest that both substances can also induce the release of prostacyclin (PGI ). This study was designed to investigate the role of the prostaglandin pathway in Ach- and <em>5HT</em>-induced vasodilation in humans. The experiments were done in three groups of healthy male volunteers. In group 1 (n = 6), ACh (100-1,000 ng/kg/min) and sodium-nitroprusside (10-100 ng/kg/min) were infused into the brachial artery alone, together with a continuous infusion of indomethacin (1.3 micro g/kg/min) and during a combined infusion of indomethacin and the competitive NO synthase inhibitor N -monomethyl-l-arginine (l-NMMA; 30 micro g/kg/min). In group 2 (n = 5), <em>5HT</em> (0.3-1.0 ng/kg/min) was infused alone and together with a continuous infusion of indomethacin and l-NMMA. In group 3 (n = 6), the synthetic prostaglandin analog iloprost (0.5-4.5 ng/kg/min) was infused together with a continuous infusion of saline, l-NMMA, and l-NMMA with indomethacin, respectively. The infusions of indomethacin and l-NMMA started 10 min before the infusion of ACh, <em>5HT</em>, iloprost, and sodium nitroprusside. Forearm blood flow was measured using computerized venous occlusion plethysmography. Both the Ach- and <em>5HT</em>-induced vasodilator responses were significantly attenuated by indomethacin (p < 0.05 for both), but not further influenced by a concomitant infusion of l-NMMA. The vasodilatation induced by iloprost was significantly inhibited by l-NMMA (p < 0.05) and not affected by indomethacin. The sodium nitroprusside-induced vasodilation was influenced by neither l-NMMA nor indomethacin. It is concluded that in the human forearm, the prostaglandin pathway is involved in both the Ach- and <em>5HT</em>-induced NO-mediated vasodilatation.

BACKGROUND

The human pupil may be a suitable physiological test system for the assessment of excessive daytime sleepiness (EDS), but pupillometric assessment could be confounded by medication for comorbid hypertension and mood disorders.

OBJECTIVE

We examined the profile of the <em>5HT</em>-2/alpha1/H1 antagonist ketanserin, the <em>5HT</em>1a agonist buspirone and the beta adrenoceptor antagonist propranolol on pupillary and other measures of arousal.

METHODS

Ketanserin (20 mg), buspirone (10 mg) and propranolol (40 mg) were administered in three independent experiments according to a crossover, placebo-controlled, double-blind design. Resting pupil diameter (RPD) was sampled over 5-min in darkness with infrared pupillometry. Tests also included critical flicker fusion frequency (CFFF), visual analogue scales (VAS), the pupillary light reflex and heart rate/blood pressure.

RESULTS

Ketanserin reduced RPD, CFFF, VAS-rated arousal and blood pressure and increased the light reflex amplitude. Buspirone reduced RPD and blood pressure. Propranolol reduced heart rate but had no effects on pupillary functions or any arousal measure.

CONCLUSIONS

Ketanserin but not propranolol had a fully sedative profile and may confound pupillometric assessment of EDS. Beta adrenergic receptors do not appear to participate in arousal and pupillary functions, while <em>5HT</em>1a receptors reduce pupil size without affecting arousal. Pupil size may not be used unequivocally as an index of the level of alertness in the case of drug-induced changes, when drugs interfere with the central pupil control mechanism in ways that are unrelated to their effects on arousal.

Aspects of the functional anatomy of the chromaffin cell since 1950 are reviewed--beginning with the identification of noradrenaline in the adrenal gland and the first descriptions of distinct adrenaline- and noradrenaline-storing cells. Reference is made to the identification of specific proteins and neuropeptides in chromaffin cells, of the storage of endogenous and exogenous amines including <em>5HT</em>. The influence of corticosteroids and nerve growth factor on cell phenotypes is discussed and a plea made for the specific use of descriptive terms with due regard to historical and functional context: this relating particularly to the appropriate labelling of elements as SIF cells. The review includes reference to recent quantitative studies on the rat adrenal medulla and adrenal chromaffin cells and to the innervation of the adrenal medulla by pre- and postganglionic sympathetic neurons, by spinal afferent neurons and by sensory and motor vagal fibers.

Vasoconstrictor responses mediated by the alpha 2-adrenoceptor agonist TL99, were particularly sensitive to blockade by the calcium antagonist drug diltiazem in isolated perfused tail arteries of spontaneously hypertensive rats (SHR). In contrast, the vasoconstrictor responses induced by the alpha 1-adrenoceptor agonist methoxamine were significantly more resistant to antagonism by diltiazem. At higher concentrations (greater than 300 nmol/l) diltiazem became an effective antagonist of all alpha-adrenoceptor mediated responses. In normotensive Wistar Kyoto (WKY) or Sprague-Dawley (SD) rats diltiazem was significantly less potent against vasoconstrictor responses to TL99 than in SHR. The blockade of alpha 1-adrenoceptor mediated vasoconstriction by diltiazem was not significantly different when normotensive rats and SHR were compared. The vasoconstrictor responses evoked by <em>5HT</em> in the perfused tail arteries were particularly resistant to blockade by diltiazem in SHR arteries. The responses to endogenously released noradrenaline, evoked by electrical field stimulation, were significantly antagonised by diltiazem (30 nmol/1-3 mumol/l) in SHR-tail arteries, while they were not modified in WKY-tail arteries. At the concentrations of diltiazem which blocked end organ responses to field stimulation, there was no modification of total tritium overflow from SHR-tail arteries after labelling the tissue with 3H-noradrenaline, indicating that diltiazem does not inhibit transmitter release at these concentrations. The tail artery preparation of SHR contains a population of postsynaptic alpha 2-adrenoceptors which mediate contraction in this blood vessel and the calcium entry blocker diltiazem is a potent antagonist of vasoconstrictor responses mediated by vascular alpha 2-adrenoceptors in hypertensive rats. These findings may be relevant to the antihypertensive action of diltiazem.

Bacopa monnieri (BM) has been used in Ayurvedic medicine as a nootropic, anxiolytic, antiepileptic and antidepressant. An n-butanol extract of the plant (nBt-ext BM) was analysed and found to contain Bacoside A (Bacoside A3, Bacopaside II and Bacopasaponin C). The effects of the BM extract were then studied on morphine-induced hyperactivity as well as dopamine and serotonin turnover in the striatum since these parameters have a role in opioid sensitivity and dependence. Mice were pretreated with saline or nBt-ext BM (5, 10 and 15 mg/kg, orally), 60 min before morphine administration and locomotor activity was subsequently recorded. Immediately after testing, striatal tissues were analysed for dopamine (DA), serotonin (<em>5HT</em>) and their metabolites using HPLC coupled with electrochemical detection. The results indicated that nBt-ext BM significantly (p < 0.001) decreased locomotor activity in both the saline and morphine treated groups. Additionally, nBt-ext BM significantly lowered morphine-induced dopamine (DA), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindole acetic acid (5-H1AA) upsurges in the striatum but failed to affect DA, 5-HT and their metabolites in the saline treated group. These findings suggest that nBt-ext BM has an antidopaminergic/serotonergic effect and may have potential beneficial effects in the treatment of morphine dependence.

Surgical interventional strategies for the treatment of obesity are being implemented at an increasing rate. The safety and feasibility of these procedures are questionable for most overweight or obese individuals. The use of long-term pharmacotherapy options, on the other hand, can target a greater portion of the obese population and provide early intervention to help individuals maintain a healthy lifestyle to promote weight loss. Medications that act on the central serotonergic pathways have been a relative mainstay for the treatment of obesity for the last 35 years. The clinical efficacy of these drugs, however, has been encumbered by the potential for drug-associated complications. Two drugs that act, albeit by different mechanisms, on the central serotonergic system to reduce food intake and decrease body weight are sibutramine and lorcaserin. Sibutramine is a serotonin and norepinephrine reuptake inhibitor, whereas lorcaserin is a selective <em>5HT</em>(2C) receptor agonist. The recent worldwide withdrawal of sibutramine and FDA rejection of lorcaserin has changed the landscape not only for serotonin-based therapeutics specifically, but for obesity pharmacotherapy in general. The purpose of this review is to focus on the importance of the serotonergic system in the control of feeding and its potential as a target for obesity pharmacotherapy. Advances in refining and screening more selective receptor agonists and a better understanding of the potential off-target effects of serotonergic drugs are needed to produce beneficial pharmacotherapy.

Currently, there is no effective strategy for the treatment of spinal cord injury (SCI). A combination of biomaterials and stem cell therapy seems to be a promising approach to increase regenerative potential after SCI. We evaluated the use of a cellpolymer construct based on a combination of the conditionally immortalized spinal progenitor cell line SPC-01_GFP3, derived from human fetal spinal cord tissue, with a serotonin-modified poly(2-hydroxyethyl methacrylate) hydrogel (pHEMA-<em>5HT</em>). We compared the effect of treatment with a pHEMA-<em>5HT</em> hydrogel seeded with SPC-01_GFP3 cells, treatment with a pHEMA-<em>5HT</em> only and no treatment on functional outcome and tissue reconstruction in hemisected rats. Prior to transplantation the cell-polymer construct displayed a high potential to support the growth, proliferation and differentiation of SPC-01 cells in vitro. One month after surgery, combined hydrogel-cell treatment reduced astrogliosis and tissue atrophy and increased axonal and blood vessel ingrowth into the implant; however, two months later only the ingrowth of blood vessels remained increased. SPC-01_GFP3 cells survived well in vivo and expressed advanced markers of neuronal differentiation. However, a majority of the transplanted cells migrated out of the lesion and only rarely remained in the hydrogel. No differences among the groups in motor or sensory recovery were observed. Despite the support of the hydrogel as a cell carrier in vitro, and good results in vivo one month postsurgery, there was only a small effect on long term recovery, mainly due to the limited ability of the hydrogels to support the in vivo growth and differentiation of cells within the implant. Further modifications will be necessary to achieve stable long term improvement in functional outcome.

(R)-5-[(1-Methyl-3-indolyl)carbonyl]-4,5,6,7-tetrahydro-1H-benzimidazole hydrochloride (YM060) is a structurally novel, extremely potent, and highly selective serotonin (<em>5HT</em>)3 receptor antagonist. Its <em>5HT</em>3 receptor blocking properties were compared with those of its enantiomer (S-form), granisetron and ondansetron, in the isolated distal colon of the guinea pig. YM060 competitively antagonized <em>5HT</em>- and 2-methyl-<em>5HT</em>-induced contraction of the colon, with pA2 values of 8.71 +/- 0.09 (n = 12) and 8.69 +/- 0.06 (n = 9), respectively. Its antagonistic activity was approximately 200, 5 and 50 times more potent than those of the S-form (pA2 = 6.33 +/- 0.06, n = 9 against <em>5HT</em>; 6.47 +/- 0.1, n = 9 against 2-methyl-<em>5HT</em>), granisetron (pA2 = 8.03 +/- 0.07, n = 9; 8.02 +/- 0.04, n = 9), and ondansetron (pA2 = 7.02 +/- 0.08, n = 9; 6.98 +/- 0.02, n = 9), respectively. Each pA2 value was constant and the isomeric activity ratio (R-form/S-form) was constant irrespective of the agonist used, suggesting that each drug acted on the same <em>5HT</em>3 receptor. YM060 failed to antagonize contractions induced by <em>5HT</em> in the saphenous vein of the dog (<em>5HT</em>1-like receptor) or in the aorta of the rabbit (<em>5HT</em>2 receptor. YM060 has a low affinity for alpha-1 (rabbit aorta; pA2 = 5.08 +/- 0.07, n = 8) and alpha-2 (guinea pig ileum; pA2 = 5.61 +/- 0.09, n = 11) adrenergic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

The factors that influence the timing of puberty and the onset of adult fertility are poorly understood. While focus on the juvenile period has provided insights into how growth-related cues affect pubertal timing, growth velocity during infancy that is sustained into the juvenile period may be important. On the other hand, social factors, specifically exposure to psychosocial stressors, can delay sexual maturation, possibly by altering growth velocities during development. Using female rhesus monkeys, the present study used a prospective analysis to determine how neonatal growth hormone (GH) inhibition with a sandostatin analog or suppression of the pituitary-gonadal axis with a GnRH analog affected growth and sexual maturation. A separate retrospective analysis was done assessing the effects of social dominance status during development on pubertal timing. Because a specific polymorphism in the gene encoding the serotonin (<em>5HT</em>) reuptake transporter increases vulnerability to psychosocial stressors, females were also genotyped and were then classified as socially dominant, having both alleles for the long promoter variant or having at least one allele for the short promoter variant, or as socially subordinate, having the long variant or having the short variant. Neonatal treatments were not balanced for social status or genotype, so analyses were performed separately. Although the neonatal treatments reduced GH secretion postnatally and through the juvenile period, neither growth nor sexual maturation was affected. In contrast, the retrospective analysis showed sexual maturation was delayed significantly in subordinate females carrying at least one allele of the short promoter variant in the gene encoding the <em>5HT</em> reuptake transporter, and this delay was associated with reduced GH and leptin secretion during the juvenile phase but not with differences in growth velocities from birth. These data suggest that decreased neonatal GH secretion does not adversely affect sexual maturation, but that polymorphisms in the gene encoding the <em>5HT</em> transporter modulate the adverse consequences of social subordination on the timing of puberty in female rhesus monkeys.

Dietary glutamate (Glu) stimulates to evoke the umami taste, one of the five basic tastes, enhancing food palatability. But it is also the main gut energy source for the absorption and metabolism for each nutrient, thus, only a trace amount of Glu reaches the general circulation. Recently, we demonstrated a unique gut sensing system for free Glu (glutamate signaling). Glu is the only nutrient among amino acids, sugars and electrolytes that activates rat gastric vagal afferents from the luminal side specifically via metabotropic Glu receptors type 1 on mucosal cells releasing mucin and nitrite mono-oxide (NO), then NO stimulates serotonin (<em>5HT</em>) release at the enterochromaffin cell. Finally released <em>5HT</em> stimulates <em>5HT</em>3 receptor at the nerve end of the vagal afferent fiber. Functional magnetic resonance imaging (f-MRI, 4.7 T) analysis revealed that luminal sensing with 1 % (w/v) monosodium L-glutamate (MSG) in rat stomach activates both the medial preoptic area (body temperature controller) and the dorsomedial hypothalamus (basic metabolic regulator), resulting in diet-induced thermogenesis during mealing without changes of appetite for food. Interestingly, rats were forced to eat a high fat and high sugar diet with free access to 1 % (w/w) MSG and water in a choice paradigm and showed the strong preference for the MSG solution and subsequently, they displayed lower fat deposition, weight gain and blood leptin. On the other hand, these brain functional changes by the f-MRI signal after 60 mM MSG intubation into the stomach was abolished in the case of total vagotomized rats, suggesting that luminal glutamate signaling contributes to control digestion and thermogenesis without obesity.

Numerous pathological processes have been linked to disorders of the central 5-hydroxytryptamine (<em>5HT</em>) system but the possibility of gene therapy through modulation of <em>5HT</em> system remained unexplored due to the lack of the specific targeting vectors. We explored the sequences upstream of the tryptophan hydroxylase-2 (TPH-2) gene, the key enzyme in neuronal <em>5HT</em> synthesis and generated lentiviral vectors, which were then tested in vivo using microinjections into the rat raphe. All fragments longer than 1 kb (called 2TPH, 3.6TPH and 6.7TPH) drove highly specific expression in <em>5HT</em> neurons which was, however too weak to be detectable without immunostaining. To enhance the level of expression, a two-step transcriptional amplification strategy was employed whereby the activity of a TPH-2 promoter fragment was potentiated by a chimeric enhancer GAL4/p65. Surprisingly, previously published implementations of this strategy compromised the specificity of expression. We therefore used a new approach where both, the transgene and GAL4/p65 are co-expressed using an internal ribosomal entry site and GAL4/p65 operates in a positive feedback manner to amplify the expression. Thus, we have generated new vectors, which are both, highly specific for <em>5HT</em> neurons and sufficiently powerful. They open a range of new opportunities for enquiries into genetic correction of <em>5HT</em>-related disorders.

Ruthenium Purple (RP), an analogue of Prussian Blue, has potentially advantageous electrochemical characteristics. We now demonstrate its use in microelectrode biosensors for the first time. An RP layer was grown on, and remained stably anchored to, the surface of gold microelectrodes at physiological pH ranges. Crucially, it retained its electrochemical activity in sodium-based phosphate buffers. The RP microelectrodes displayed electrocatalytic reduction of hydrogen peroxide at 0 to -50 mV (vs Ag/AgCl). To fabricate biosensors on the RP microelectrodes, we used a sol-gel film electrodeposition technique to create ATP and hypoxanthine biosensors as examples of the methodology. These RP-mediated biosensors displayed excellent performance including the following: high selectivity against interferences such as <em>5HT</em>, ascorbic acid, urate, and acetaminophen; high sensitivity with wide linear calibration range; and good stability. These attractive characteristics demonstrate that RP can be universally employed as an electron mediator in fabrication of highly selective oxidase-based microelectrode biosensors. Furthermore, given their ability to operate in the presence of physiological levels of Na+, the RP-mediated biosensors can be potentially applied to the in vitro and in vivo measurement of physiological signaling substances.

Initiation and termination of signaling of the type I angiotensin receptor (AT(1)-R) can lead to dynamic changes in its localization in plasma membrane microdomains. Several markers were recently developed to investigate membrane microdomains. Here, we used several YFP-labeled fusion constructs (i.e. raft or non-raft plasma membrane markers) to analyze the agonist-induced changes in compartmentalization of AT(1)-R, including internalization or lateral movement between plasma membrane compartments in response to stimulation using bioluminescence resonance energy transfer measurements. Our data demonstrate that angiotensin II (AngII) stimulus changes the microdomain localization of wild type or mutated (DRY → AAY or TSTS → AAAA) AT(1)-Rs co-expressed with the fluorescent probes in HEK293 cells. The comparison of the trafficking of AT(1)-R upon AngII stimulus with those of [Sar(1),Ile(8)]AngII or [Sar(1),Ile(4),Ile(8)]AngII stimulus revealed different types of changes, depending on the nature of the ligand. The observed changes in receptor compartmentalization of the AT(1)-R are strikingly different from those of <em>5HT</em>-2C and EGF receptors, which demonstrate the usefulness of the bioluminescence resonance energy transfer-based measurements in the investigation of receptor trafficking in the plasma membrane in living cell experiments.

Serotoninergic fibers densely innervate olfactory bulb glomeruli, the first sites of synaptic integration in the olfactory system. Acting through <em>5HT</em>2A receptors, serotonin (<em>5HT</em>) directly excites external tufted cells (ETCs), key excitatory glomerular neurons, and depolarizes some mitral cells (MCs), the olfactory bulb's main output neurons. We further investigated <em>5HT</em> action on MCs and determined its effects on the two major classes of glomerular interneurons: GABAergic/dopaminergic short axon cells (SACs) and GABAergic periglomerular cells (PGCs). In SACs, <em>5HT</em> evoked a depolarizing current mediated by <em>5HT</em>2C receptors but did not significantly impact spike rate. <em>5HT</em> had no measurable direct effect in PGCs. Serotonin increased spontaneous excitatory and inhibitory postsynaptic currents (sEPSCs and sIPSCs) in PGCs and SACs. Increased sEPSCs were mediated by <em>5HT</em>2A receptors, suggesting that they are primarily due to enhanced excitatory drive from ETCs. Increased sIPSCs resulted from elevated excitatory drive onto GABAergic interneurons and augmented GABA release from SACs. Serotonin-mediated GABA release from SACs was action potential independent and significantly increased miniature IPSC frequency in glomerular neurons. When focally applied to a glomerulus, <em>5HT</em> increased MC spontaneous firing greater than twofold but did not increase olfactory nerve-evoked responses. Taken together, <em>5HT</em> modulates glomerular network activity in several ways: 1) it increases ETC-mediated feed-forward excitation onto MCs, SACs, and PGCs; 2) it increases inhibition of glomerular interneurons; 3) it directly triggers action potential-independent GABA release from SACs; and 4) these network actions increase spontaneous MC firing without enhancing responses to suprathreshold sensory input. This may enhance MC sensitivity while maintaining dynamic range.

1. It has been long suggested that central <em>5HT</em>-mediated systems may be involved in modulation of anxiety and in the anxiolytic effect of benzodiazepines. However, recent evidence has questioned this hypothesis, particularly with respect to the mode of action of benzodiazepines. 2. Development of <em>5HT</em> agonists and antagonists selective for different <em>5HT</em> receptor sub-types (<em>5HT</em>1A, <em>5HT</em>1B, <em>5HT</em>2, <em>5HT</em>3) has opened a new avenue for investigation of the potential role of <em>5HT</em> in anxiety. 3. Buspirone is clinically active in the treatment of anxiety and it, and other anxiolytic candidates, gepirone and isapirone, may act as agonists (or perhaps partial agonists) on <em>5HT</em>1A receptors. 4. The prototype <em>5HT</em>1A agonist 8OH-DPAT may also have potential anxiolytic effects. <em>5HT</em>1A agonists may act to suppress the activity of <em>5HT</em> neurones as a major part of their action. 5. Although there is some supporting evidence, there is no clear indication of anxiolytic activity with agonists with some selectivity for <em>5HT</em>1B sites (RU24969, mCPP, TMPP). 6. A selective <em>5HT</em>2 antagonist, ritanserin, has anxiolytic effects in clinical studies but, like the <em>5HT</em>1A agonists, does not show a similar profile to benzodiazepines in models of anxiety. 7. This raises the question of clinical predictivity of the various models used. 8. A recently developed <em>5HT</em>3 antagonist, GR38032F, has been claimed to possess potential anxiolytic activity but its mode of action in this respect requires further elucidation.

Drug resistance in schizophrenic disorders treated with an antipsychotic medication is highly problematic, lacking sound criteria to define it, and to discriminate between drug response and clinical remission. This article reviews some neurochemical, psychoimmunological, pharmacogenetic and neuromorphological patterns which can affect drug response and determine drug-resistance phenomena in schizophrenia. Several neurochemical abnormalities have been reported to be relevant for the pathogenesis of schizophrenic disorders and have been related to clinical symptoms as well as to the quality of response to antipsychotics: most of the findings come from studies on DA and <em>5HT</em> brain metabolism, but more recently other non-dopaminergic pathways have been implicated (e.g., glutamatergic ones). Literature data suggest that schizophrenia may be associated with significant alterations of T-cell functions, showing the activation of the inflammatory response system (IRS), particularly in treatment-resistant schizophrenia, and differential effects on IRS have been reported for conventional and atypical antipsychotics. Furthermore molecular genetic approaches provide a novel method of dissecting the heterogeneity of psychotropic drug response, providing the means of determining the molecular substrates of drug efficacy and drug-induced adverse events. On the other hand, functional neuroimaging techniques, including single photon emission computed tomography (SPECT), positron emission tomography (PET) and functional magnetic resonance imaging (FMRI), providing an in vivo assessment of the expression and function of neuroreceptors, transporters and enzymes, seem to be particularly promising for a better understanding of 'real' drug resistance. Finally, a multidimensional approach taking into account all these variables in the future would likely be the more valuable strategy to optimise response, reducing relapses or resistant clinical situations.

Nerve injury, tissue damage, and inflammation all cause hyperalgesia. A factor contributing to this increased sensitivity is a long-term (>24 hr) hyperexcitability (LTH) in the sensory neurons that mediate the responses. Using the cluster of nociceptive sensory neurons in Aplysia californica as a model, we are examining how inflammation induces LTH. A general inflammatory response was induced by inserting a gauze pad into the animal Within 4 days, the gauze is enmeshed in an amorphous material that contains hemocytes, which comprise a cellular immune system. Concurrently, LTH appears in both ipsilateral and contralateral sensory neurons. The LTH is manifest as increased action potential discharge to a normalized stimulus. Immunocytochemistry revealed that hemocytes have antigens recognized by antibodies to TGFbeta1, IL-6, and <em>5HT</em>. When a localized inflammation was elicited on a nerve, hemocytes containing the TGFbeta1 antigen were present near axons within the nerve and those containing the IL-6 were on the surface. Western blots of hemocytes, or of gauze that had induced a foreign body response, contained a 28-kD polypeptide recognized by the anti-TGFbeta1 antibody. Exposure of the nervous system to recombinant human TGFbeta1 elicited increased firing of the nociceptive neurons and a decrease in threshold. The TGFbeta1 also caused an activation of protein kinase C (PKC) in axons but did not affect a kinase that is activated in axons after injury. Our findings, in conjunction with previous results, indicate that a TGFbeta1-homolog can modulate the activity of neurons that respond to noxious stimuli. This system could also contribute to interactions between the immune and nervous systems via regulation of PKC.

5-HT(6) receptor is one of the most recently cloned serotonin receptors, and it might play important roles in Alzheimer's disease, depression, and learning and memory disorders. Availability of only very few 5-HT(6) receptor agonists, however, does not allow examining their contribution in psychopharmacological processes. Therefore, a new 5-HT(6) receptor agonist, ST1936, was synthesized. ST1936 binds to human 5-HT(6) receptors with good affinity (K(i)=28.8 nM). ST1936 also exhibited some moderate binding affinity for <em>5HT</em>(2B), <em>5HT</em>(1A), <em>5HT</em>(7) receptors and adrenergic α receptors. ST1936 behaved as a full 5-HT(6) agonist on cloned cells and was able to increase Ca(2+) concentration, phosphorylation of Fyn kinase, and regulate the activation of ERK1/2 that is a downstream target of Fyn kinase. These effects were completely antagonized by two 5-HT(6) receptor antagonists, SB271046 and SB258585. The other 5-HT(6) receptor agonist, WAY181187 also increased Fyn kinase activity. These results suggest that both ST1936 and WAY181187 mediate 5-HT(6) receptor-dependent signal pathways, such as cAMP, Fyn and ERK1/2 kinase, as specific agonists.

The dependence of ADP- and epinephrine-induced platelet aggregation and secretion on extracellular divalent cations was examined by quantitating these responses in citrate-, heparin-, and hirudin-anticoagulated platelet-rich plasma. ADP-induced 14C-<em>5HT</em> secretion in heparin-PRP and hirudin-PRP was generally decreased, relative to that in citrate-PRP, without corresponding reductions in aggregation, whereas in response to epinephrine, both aggregation and secretion were decreased in heparin-PRP, and abolished in hirudin-PRP. In heparin-PRP, but not in hirudin-PRP, the degree to which these responses were altered was highly variable among normal subjects, and was dependent on the anticoagulant concentration. Addition of citrate restored the extent of ADP-induced secretion and of epinephrine-induced aggregation and secretion in heparin-PRP to that observed in citrate-PRP, and increased the extent of ADP-induced secretion in hirudin-PRP. Addition of EDTA or EGTA, however, had no effect of ADP-induced secretion in heparin-PRP. These results suggest that ADP-induced aggregation and secretion, as well as responses to ADP vs. epinephrine, have different dependencies on extracellular or surface-bound divalent cations. The variable responses observed in heparin-PRP may reflect direct interactions of heparin with platelets, and this variability may account for the conflicting results of previous studies.