Immunocytochemical double-labeling methods are important tools in cell and neurobiology. Here we describe a method which is based on double immunofluorescence and allows specific detection of two different antigens located in the same cell compartment by two primary antibodies raised in the same species. As an example, we present the double-immunolabeling method for the S-antigen (SAg), a photoreceptor-specific protein, and the indoleamine serotonin (<em>5HT</em>) in dissociated trout and rat pineal cells immobilized on coverslipped and in frozen sections of the trout pineal organ. As a first step, the preparations on the slides or coverslips were sequentially incubated with the first primary antibody (rabbit anti-SAg), the fluorescein-labeled (anti-rabbit) secondary antibody, and then with normal rabbit serum. Meanwhile, the second primary antibody (rabbit anti-<em>5HT</em>) was coupled to a Cy3-labeled secondary (anti-rabbit) antibody in a reaction tube and excess binding sites were quenched with normal rabbit serum. This complex was applied to the specimens after completion of the first (SAg) immunoreaction on the slide. For control experiments, the first (anti-SAg) or the second (anti-<em>5HT</em>) primary antibody were omitted. Most of the rat and trout pinealocytes were double immunolabeled for SAg and <em>5HT</em>. In the trout, few cells contained SAg or <em>5HT</em> immunoreaction only. This underlines the selectivity of each immunoreaction. The results show that the method can be used for the analysis of whole cells and tissue sections by means of conventional fluorescence and confocal laser scanning microscopy.

Social behavior and desire for social relationships have been independently linked to the serotonergic system, the prefrontal cortex, especially the orbitofrontal cortex (OFC), and the anterior cingulate cortex (ACC). The goal of this study was to explore the role of serotonin <em>5HT</em>(2A) receptors in these brain regions in forming and maintaining close interpersonal relationships. Twenty-four healthy subjects completed the Temperament and Character Inventory (TCI) prior to undergoing [(18)F]setoperone brain positron emission tomography (PET) to measure serotonin <em>5HT</em>(2A) receptor availability within the OFC (BA 11 and 47) and ACC (BA 32). We explored the relationship between desire for social relationships, as measured by the TCI reward dependence (RD) scale, and <em>5HT</em>(2A) receptor non-displaceable binding potential (BP(nd)) in these regions. Scores of RD were negatively correlated with <em>5HT</em>(2A) BP(nd) in the ACC (BA 32, r = -.528, p = .012) and OFC (BA 11, r = -.489, p = .021; BA 47, r = -.501, p = .017). These correlations were corroborated by a voxel-wise analysis. These results suggest that the serotonergic system may have a regulatory effect on the OFC and ACC for establishing and maintaining social relationships.

(1) Serotonin (<em>5HT</em>)-mediated calcium signaling was investigated in hypoglossal motoneurons (HGMs) in brain stem slices of neonatal mice. Electrical activity and associated calcium signaling were studied by simultaneous patch clamp recordings and high resolution calcium imaging. (2) Bath application of <em>5HT</em> (5-50 microM) depolarized membrane potential of HGMs and generated action potential discharges that were accompanied by elevations in intracellular calcium concentrations ([Ca2+]i) in the soma and dendrites. Current-evoked bursts of action potentials were more intense in the presence of <em>5HT</em>; however, the corresponding calcium signals were reduced. (3) The <em>5HT</em>2 receptor agonist alpha-Methyl-<em>5HT</em> (25, 50 microM) had effects on membrane potential, discharge properties and [Ca]i that were identical to those observed for <em>5HT</em>, whereas the <em>5HT</em>3 receptor agonist 1-(m-chlorophenyl) biguanide (50 microM) had no effect on membrane properties or intracellular calcium levels. (4) 8-OHDPAT (25, 50 microM), a <em>5HT</em>1A receptor agonist, was without effect on steady-state membrane potential or basal [Ca]i. Similar to <em>5HT</em> and alpha-Methyl-<em>5HT</em>, 8-OHDPAT depressed stimulus-evoked calcium transients in current and voltage clamp mode. (5) Our results suggest that calcium profiles in hypoglossal motoneurons are differentially regulated by <em>5HT</em>1A and <em>5HT</em>2 receptors. Activation of <em>5HT</em>1A receptors primarily reduced voltage-activated Ca2+ signals without a significant impact on basal [Ca]i. In contrast, activation of <em>5HT</em>2 receptors initiated a net inward current followed by membrane depolarization, where the resulting pattern of action potential discharges represents the essential determinant of global elevations in [Ca2+]i. Taken together, our results therefore identify <em>5HT</em>-dependent signal pathways as a versatile tool to modulate hypoglossal motoneuron excitability under various physiological and pathophysiological conditions.

Adenosine-to-inosine RNA editing events that have been demonstrated for <em>5HT</em> (2C) receptors resulted in alterations of the amino acid sequence at positions 156, 158 and 160 in the intracellular loop 2 (IL2) region. The edited receptor isoforms were shown to have reduced basal activity, but similar maximum responses to agonist binding. To identify the molecular mechanism of these pharmacological effects of editing we explored the conformational properties of the edited IL2 in comparison with the wild type. The results from conformational studies of the IL2 isoforms, using biased Monte Carlo simulations with an implicit solvent model based on a screened Coulomb potential, show that the compared loops differ in their preferred spatial orientations as a result of differences in the conformational space that is accessible to them by energy criteria. For the IL2 of the unedited (<em>5HT</em> (2C-INI) ) receptor, the preference for structures oriented towards the 7TM bundle is larger than for the <em>5HT</em> (2C-VGV) edited receptor. This difference in preferred orientation can affect the association of IL2 with other intracellular loop domains involved in G protein coupling and hence the coupling efficiency. The results illustrate the high sensitivity of the system to small changes in the interaction surface presented to other intracellular loops, and/or the G protein.

We have previously characterized a mechanism of <em>5HT</em>-stimulated extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation via the non-RNA-edited isoform of the serotonin <em>5HT</em>(2C) receptor (<em>5HT</em>(2C)R-INI) in a CHO cell line. We have now used CV1 cells, which endogenously express epidermal growth factor receptors (EGFRs), to investigate whether the mechanisms underlying ERK1/2 activation by the <em>5HT</em>(2C)R change in a time-, agonist-, and cell background-dependent manner. Interrogation of the CV1 <em>5HT</em>(2C)R-INI ERK1/2 signaling pathway, using a variety of pathway-selective inhibitors, revealed a clear time-dependence in the involvement of specific pathway components such as phosphatidylinositol 3-kinase, EGFR, matrix metalloproteases and protein kinase C. The contribution of these components to the overall response also varied with the agonist used to stimulate the receptor, providing further evidence for the ability of <em>5HT</em>(2C)R-INI to signal in an agonist-specific manner. We also investigated the impact of <em>5HT</em>(2C)R RNA editing on this phenomenon. Although we found no alteration in antagonist pharmacology, the partially edited VSV and fully edited VGV isoforms of the <em>5HT</em>(2C)R exhibited altered temporal and pharmacological characteristics, including the degree of dependence on specific effectors, in signaling to ERK1/2 in comparison to the <em>5HT</em>(2C)R-INI. In conclusion, we provide evidence for remarkable flexibility in <em>5HT</em>(2C)R-mediated ERK1/2 signaling that can be pharmacologically and mechanistically distinct depending on the agonist or edited isoform involved and on the duration of receptor activation.

BACKGROUND

Increased serotonin (5-hydroxytryptamine [<em>5HT</em>]) receptor (<em>5HT</em>R) signaling has been associated with cardiac valvulopathy. Prior cell culture studies of <em>5HT</em>R signaling in heart valve interstitial cells have provided mechanistic insights concerning only static conditions. We investigated the hypothesis that aortic valve biomechanics participate in the regulation of both <em>5HT</em>R expression and interrelated extracellular matrix remodeling events.

METHODS

The effects of cyclic stretch on aortic valve <em>5HT</em>R, expression, signaling, and extracellular matrix remodeling were investigated using a tensile stretch bioreactor in studies which also compared the effects of adding <em>5HT</em> and (or) the <em>5HT</em>-transporter inhibitor, fluoxetine.

RESULTS

Cyclic stretch alone increased both proliferation and collagen in porcine aortic valve cusp samples. However, with cyclic stretch, unlike static conditions, <em>5HT</em> plus fluoxetine caused the greatest increase in proliferation (p<0.0001), and also caused significant increases in collagen (p<0.0001) and glycosaminoglycans (p<0.0001). The DNA microarray data demonstrated upregulation of <em>5HT</em>R2A and <em>5HT</em>R2B (>4.5-fold) for cyclic stretch versus static (p<0.001), while expression of the <em>5HT</em> transporter was not changed significantly. Extracellular matrix genes (eg, collagen types I, II, III, and proteoglycans) were also upregulated by cyclic stretch.

CONCLUSIONS

Porcine aortic valve cusp samples subjected to cyclic stretch upregulate <em>5HT</em>R2A and 2B, and also initiate remodeling activity characterized by increased proliferation and collagen production. Importantly, enhanced <em>5HT</em>R responsiveness due to increased <em>5HT</em>R2A and 2B expression results in a significantly greater response in remodeling endpoints (proliferation, collagen, and GAG production) to <em>5HT</em> in the presence of <em>5HT</em> transporter blockade.

Stress-induced activation of the hypothalamic-pituitary-adrenal (HPA) axis stimulates the release of both facilitatory and inhibitory components. We proposed that the transient removal of the inhibitory component, corticosterone, during a stressor would leave the HPA axis in a state of hyper-responsiveness (facilitated state). Consistent with this expectation, we have previously observed that aminoglutethimide (AG)-induced removal of corticosterone during an immobilization stressor resulted in the hypersecretion of both ACTH and corticosterone to a subsequent stressor. In the present study we determined the effect of stressor duration on the magnitude of facilitation. AG plus a 10-min immobilization (IMM(10)) stress on day 1 resulted in facilitation of the HPA axis. This was reflected in higher ACTH and corticosterone responses to an injection stress on day 2 as compared to appropriate control rats. AG plus a 60-min immobilization (IMM(60)) stress on day 1 resulted in significantly greater facilitation as compared to the AG+IMM(10) pretreatment. It is apparent that facilitation of the HPA axis is dependent on the duration of stress. Stress can alter plasma corticosterone-binding globulin levels and AG administration can cause accumulation of the corticosterone biosynthetic precursor, adrenal cholesterol. In order to rule out these peripheral reasons for the hypersecretion of ACTH and corticosterone in our paradigm, we measured the plasma free fraction of corticosterone and adrenal mitochondrial cholesterol levels on day 2 after different pretreatments on day 1. AG+IMM(60) pretreatment caused a significant increase in the plasma free fraction of corticosterone. Hypersecretion of ACTH and corticosterone in this group, despite an enhanced feedback signal, suggests central loci for the origin of facilitation. Also, AG treatment on day 1 did not result in accumulation of free or esterified adrenal cholesterol levels on day 2, and therefore cannot account for the hypersecretion of corticosterone. In our final study we attempted to determine if serotonin released during the first stressor is partially responsible for stress-induced facilitation of the HPA axis. We administered 8-hydroxy-2-(di-n-propylamino)tetralin (DPAT), a <em>5HT</em>(1A) agonist, either alone or in conjunction with stress and examined the effects of these pretreatments on the magnitude of facilitation. Interestingly, DPAT administered in lieu of stress produced facilitation similar in magnitude to that produced by IMM(10). DPAT administered in conjunction with IMM(10) augmented stress-induced facilitation. Our results suggest that stress-induced facilitation of the HPA axis is associated with the release of serotonin during stress.

Negative energy balance inhibits reproduction by restraining GnRH secretion. Leptin is a permissive metabolic signal for reproduction, but GnRH neurons do not appear to express leptin receptors, suggesting that interneurons transmit leptin signals to these cells. Serotonin (<em>5HT</em>) has satiety effects similar to those of leptin and alters LH release, and serotonergic neurons, which have been shown to express leptin receptors, terminate on GnRH neurons. We hypothesized that serotonergic neurons convey leptin signals to the reproductive neuroendocrine axis. To test this, mice were fasted for 48 h beginning on Diestrous Day 1. While fasting, mice received saline or leptin every 12 h or the <em>5HT</em>-selective reuptake-inhibitor fluoxetine once at the start of the fast. Estrous cycles of fasted mice were longer (mean +/- SEM, 10.2 +/- 0.5 days; P < 0.0001) than those of fed mice (4.5 +/- 0.2 days). As previously reported, leptin prevented fasting-induced cycle lengthening (4.6 +/- 0.7 days). Fluoxetine also rescued estrous cycles in fasted mice (4.7 +/- 0.6 days), suggesting that <em>5HT</em> and leptin have similar positive effects on reproduction. Coadministration of the <em>5HT</em> 1/2/7 receptor-antagonist metergoline blocked rescue of cycle length by fluoxetine and by leptin. Treating leptin-deficient ob/ob and leptin receptor-deficient db/db mice with fluoxetine did not normalize body weight or rescue fertility, perhaps due to altered serotonergic tone in these animals. Together, these data demonstrate a permissive role for serotonergic systems in the metabolic control of reproduction and are consistent with the hypothesis that serotonergic neurons convey leptin signals to GnRH neurons.

Modulation of muscle acetylcholine (AcCho) receptors (AcChoRs) by serotonin [5-hydroxytryptamine (<em>5HT</em>)] and other serotonergic compounds was studied in Xenopus laevis oocytes. Various combinations of alpha, beta, gamma, and delta subunit RNAs were injected into oocytes, and membrane currents elicited by AcCho were recorded under voltage clamp. Judging by the amplitudes of AcCho currents generated, the levels of functional receptor expression were: alpha beta gamma delta>> alpha beta delta>> alpha beta gamma>> alpha gamma delta. The alpha beta gamma delta and alpha beta delta AcChoR Subtypes were strongly blocked by <em>5HT</em>, whereas the alpha beta gamma receptor was blocked only slightly. The order of blocking potency of AcChoRs by <em>5HT</em> was: alpha beta delta>> alpha beta gamma delta>> alpha beta gamma. <em>5HT</em> receptor antagonists, such as methysergide and spiperone, were even more potent blockers of AcChoRs than <em>5HT</em> but did not show much subunit selectivity. Blockage of alpha beta gamma delta and alpha beta delta receptors by <em>5HT</em> was voltage-dependent, and the voltage dependence was abolished when the delta subunit was omitted. These findings may need to be taken into consideration when trying to elucidate the mode of action of many clinically important serotonergic compounds.

Brain serotonin (<em>5HT</em>) and dopamine (DA) turnover were studied at various stages of the reproductive cycle of the female rainbow trout by simultaneous determination by HPLC of neurotransmitters and major related metabolites. An increase of <em>5HT</em> turnover in telencephalon and hypothalamus and a decrease of DA turnover in pituitary and hypothalamus were observed during the periovulatory period. Some changes also occurred during vitellogenesis: decreased <em>5HT</em> metabolite in telencephalon and preoptic area and increased DA content in preoptic area. These data suggest that physiological fluctuations of biogenic amines could be involved in both ovarian recrudescence and ovulation, with major effects on the hypothalamo-hypophysial complex during the periovulatory period.

The serotonin (<em>5HT</em>) agonist, m-chlorophenylpiperazine (MCPP), has been used as a challenge agent to assess central <em>5HT</em> receptor sensitivity in normal subjects and patients with panic disorder, obsessive-compulsive disorder, and major depression. Adrenocorticotropin, cortisol, and prolactin responses to MCPP were among the variables measured. MCPP's usefulness as a probe of <em>5HT</em> receptors, however, hinges on its <em>5HT</em> selectivity. To address MCPP's selectivity for <em>5HT</em>, this study tested whether two different <em>5HT</em> antagonists, methysergide (4 mg p.o.) and metergoline (4 mg p.o.), could block the hormonal and behavioral effects of MCPP (0.5 mg/kg p.o.) in 10 normal male subjects in comparison to placebo. Both <em>5HT</em> antagonists abolished the prolactin release to MCPP. Metergoline, the antagonist with the more potent <em>5HT</em> binding affinity, significantly blocked MCPP's effect on cortisol release as compared to placebo, and methysergide showed a nonsignificant trend to that effect. MCPP alone did not have a significant effect on behavioral variables, perhaps explaining why neither <em>5HT</em> antagonist affected these measures. The findings from this study suggest that both MCPP-induced prolactin release and cortisol release are indeed <em>5HT</em>-mediated effects.

CRF is produced in the Leydig cells and acts as a negative autocrine regulator of Leydig cell function. To clarify the hormonal control of CRF secretion by Leydig cells, we evaluated the participation of serotonin (<em>5HT</em>) and serotonin agonists in the release of CRF from Leydig cells and their effects on hCG-induced cAMP generation and steroidogenesis. Serotonin stimulated CRF secretion up to 4-fold above basal levels and inhibited basal and hCG-stimulated cAMP generation and testosterone production (ID50, 1 nM). The inhibitory action of <em>5HT</em> was prevented by a CRF antibody and the alpha-helical CRF-(9-41) antagonist. The selective <em>5HT</em>2 receptor agonist (+-)1-[2,5-dimethoxy-4-iodophyryl]2-amino propane hydrochloride (DOI) also stimulated CRF secretion and inhibited hCG-stimulated cAMP generation and testosterone production to control levels (ID50, 7 microM). Serotonergic <em>5HT</em>1A, <em>5HT</em>1B/1C, <em>5HT</em>1D, and <em>5HT</em>3/<em>5HT</em>2 agonists were less effective inhibitors of hCG-stimulated cAMP and testosterone production, while agonists for the <em>5HT</em>3 receptor had no effect. [125I]DOI binding studies in Leydig cells demonstrated two sets of receptors with Kd values in the nanomolar and micromolar range, with low and high capacities, respectively. The low affinity site differed from that of brain receptors (Kd, 4.2 nM) and displayed higher binding capacity (50-fold). The selective <em>5HT</em>2 receptor antagonist ketanserin prevented CRF stimulation and blocked the inhibitory actions of <em>5HT</em> and DOI, while the alpha 1-adrenergic antagonist prazosin had no effect. Also, treatment of cells with ketanserin increased sensitivity to hCG and raised maximal cAMP and testosterone production. <em>5HT</em> was a more effective stimulus than hCG in stimulating CRF secretion, and gonadotropin-induced CRF release was inhibited by ketanserin. Inhibitory effects of exogenous CRF were demonstrable after blockade of <em>5HT</em> action by ketanserin. The inhibitory actions of <em>5HT</em> were unaffected by pertussis and cholera toxins and were reversed by the addition of 8-bromo-cAMP. These results demonstrate that <em>5HT</em> acts on <em>5HT</em>2 receptors in Leydig cells that are distinct from those in the brain to stimulate CRF secretion through a pertussis toxin-insensitive G-protein. This action of <em>5HT</em> is predominantly mediated by the low affinity <em>5HT</em>2-binding site and requires full occupancy for maximal CRF stimulation, indicating the absence of spare receptors. <em>5HT</em>-stimulated CRF inhibits basal and hCG-induced cAMP generation and steroidogenesis. Furthermore, <em>5HT</em> mediates the stimulatory action of LH/hCG on CRF secretion from Leydig cells and, thus, participates in a negative autoregulatory loop to limit the testosterone response to the gonadotropic stimulus.

BACKGROUND

In the past decade, a strong argument has been built for the role of serotonin (<em>5HT</em>) and the serotonin transporter (SERT) in irritable bowel syndrome (IBS). However, it is still not clear how SERT contributes to this clinically heterogeneous disease. The present study addressed this issue by implementing platelet (plt) markers of SERT activity in the assessment protocol.

METHODS

Fasting blood samples of 149 (51 male/98 female) subjects with Rome II and III defined IBS subtypes, and 163 healthy control subjects (CSs; 75 male/88 female) were analyzed for platelet <em>5HT</em> concentration and <em>5HT</em> uptake activity [maximum uptake rate (V (max)) and affinity constant (K (m))].

RESULTS

Gender had a significant impact on platelet markers of SERT activity. Male IBS patients showed significantly lower median V (max) and K (m) values than the male CS (V (max) 1.706 vs. 2.148 nmol/10(9) plts x min, P < 0.001; K (m) 346 vs. 410 nmol, P = 0.008) without any significant reduction in platelet <em>5HT</em> concentration (362 vs. 394 ng/10(9) plts). On the other hand, V (max) values were not different between female IBS patients and female CS (1.642 vs. 1.741 nmol/10(9) plts x min), but platelet <em>5HT</em> concentration was significantly lower in females with diarrhea-predominant IBS (363 vs. 435 ng/10(9) plts, P < 0.05).

CONCLUSIONS

Although an absolute extrapolation from platelets to the gastrointestinal tissue does not appear to be justified, our findings demonstrated that the contribution of disturbed SERT activity to IBS is not uniform and is possibly gender-specific. The results suggest that an assessment of SERT function in platelets may help to elucidate the differences between IBS patients in response to drugs affecting the <em>5HT</em> system.

Endotoxins or lipopolysaccharides (LPS), when injected into mice, increase serotonin (5-hydroxytryptamine; <em>5HT</em>) in the liver and produce hypoglycaemia. In the present study, it was found that the cytokines produced by macrophages in response to LPS, interleukin-1 (IL-1, 0.1 microgram/kg or more) and tumour necrosis factor (TNF alpha, 100 micrograms/kg or more), can also induce an increase in liver serotonin and produce hypoglycaemia. The two responses correspond well with each other in terms of time course and dose dependency. These results suggest that the two responses induced by LPS may be mediated by IL-1 and/or TNF alpha and that the phenomenon of accumulation of <em>5HT</em> in the liver may be relevant to hypoglycaemia. The hypoglycaemic response to IL-1 was moderate at a wide dose range but was induced by smaller amounts than with insulin.

Five pairs of identified <em>5HT</em>-IR cells in the deutocerebrum of the crayfish Cherax are known to have their synaptic endings in the accessory and olfactory lobes. Two of these cells, one on each side of the brain, are significantly larger than the others. Dye fills of these "giant" cells reveal each to be an interneuron with its branches confined to, but distributed throughout, the olfactory and accessory lobes on the side of the brain ipsilateral to its cell body and with no branches to the contralateral side. Intracellular recordings from the giant cells were made while stimulating the olfactory afferents and tracts within the brain in an attempt to discover the inputs and outputs to the cells. Electrical stimulation of chemoreceptor sensilla on the outer branch of the antennule does not excite the giant <em>5HT</em> neurons. Focal extracellular electrical stimulation of the olfactory globular tract containing the axons of projection neurons from the olfactory and accessory lobes produces excitatory synaptic potentials and action potentials in the giant cells. Focal extracellular electrical stimulation of the deutocerebral commissure, the axons of which terminate in the glomeruli of the accessory lobes, also results in excitation of the giant cells. We conclude that the input to the giant cells is via axons in the deutocerebral commissure and collaterals from the projection neurons, ending in the glomeruli of the accessory lobes. The output of the giant cells is to the olfactory lobes, where it may serve to modulate olfactory signals.

Indices of disturbed serotonergic neurotransmission are the most robust biological findings in suicide. Tryptophan hydroxylase (TPH) and 5-hydroxytryptamine transporter (<em>5HT</em>t) are the main regulators of <em>5HT</em> signaling. Owing to the assumed functionality of intronic polymorphisms of TPH (218AC) and <em>5HT</em>t (VNTR-2) genes, we investigated frequencies of concurrence of the TPH and <em>5HT</em>t genotypes containing "lower activity" alleles (CC and 1010, respectively), in 192 suicide victims and 377 controls. Significant differences in frequencies of <em>5HT</em>t and TPH genotype combinations were found between suicide victims and control subjects (p = 0.0156), with a clear dose-effect of the suspected ("lower activity") genotypes (p = 0.0046). Concurrent presence of the two, allegedly transcriptionally less active, variants of these genes seems to be in some kind of relation to the increased susceptibility to suicide.

Substance P (SP), neurotensin (NT), bombesin (BB), serotonin (<em>5HT</em>), and carbamylcholine (CCH) transiently increase electrogenic anion secretion in chinchilla and chicken ileum. SP and CCH also transiently inhibit amiloride-sensitive Na/H exchange in isolated chicken enterocytes. Loperamide (LP) inhibits the short-circuit current responses caused by SP, NT, and BB, but not those caused by CCH, <em>5HT</em>, Ca ionophore, or cyclic nucleotides. Similarly, LP inhibits the effects of SP, but not those of CCH, on Na/H exchange. LP inhibition of the SP effects was further studied in isolated chicken enterocytes. CCH and SP transiently increased cytosolic Ca activity by 20-50 nmol/liter, but only the response to SP was inhibited by LP (10(-5) M) and by the absence of extracellular Ca. We conclude SP and CCH effects on intestinal electrolyte transport are mediated by increasing enterocyte Ca activity and LP specifically inhibits peptide hormone-activated Ca entry by an opiate receptor-independent mechanism.

It has been shown that the serotonergic mechanisms of the lateral parabrachial nucleus (LPBN) inhibit NaCl intake in different models of angiotensin II (ANG II)-dependent NaCl intake in rats. However, there is no information about the involvement of LPBN serotonergic mechanisms on NaCl intake in a model of NaCl intake not dependent on ANG II like deoxycorticosterone (DOCA)-induced NaCl intake. Therefore, in this study we investigated the effects of bilateral injections of serotonergic agonist and antagonist into the LPBN on DOCA-induced 1.8% NaCl intake in rats. Male Holtzman rats were treated with s.c. DOCA (10 mg/rat each every 3 days). After a period of training, in which the rats had access to 1.8% NaCl during 2 h for several days, the rats were implanted with stainless steel cannulas bilaterally into the LPBN. Bilateral injections of the serotonergic receptor antagonist methysergide (4 microg/0.2 microl each site) in the LPBN increased 1.8% NaCl intake (32.2+/-3.9 versus vehicle: 15.0+/-1.6 ml/2 h, n=10) and water intake (12.5+/-3.5 versus vehicle: 3.2+/-1.0 ml/2 h). Injections of the serotonergic <em>5HT</em>(2A/2C) receptor agonist DOI (5 microg/0,2 microl each site) in the LPBN reduced 1.8% NaCl intake (6.8+/-1.7 versus saline: 12.4+/-1. 9 ml/2 h, n=10) and water intake (2.2+/-0.8 versus saline: 4.4+/-1.0 ml/2 h). Besides the previously demonstrated importance for the control of ANG II-dependent water and NaCl intake, the data show that the serotonergic inhibitory mechanisms of the LPBN are also involved in the control of DOCA-induced NaCl intake.

Despite the availability of a wide range of effective blood pressure (BP)-lowering agents, a substantial proportion of patients with hypertension fail to achieve target BP levels. The majority of patients with hypertension need a combination of two or more drugs to achieve BP targets and choice of second-line or subsequent-line therapy is an important consideration in hypertension management. Alpha-1-adrenoreceptor antagonists (alpha-blockers) have a BP-lowering effect broadly similar to the other antihypertensive drug classes and are effective as add-on therapy in patients with inadequately controlled hypertension. Alpha-blockers may also have therapeutic benefits that go beyond BP control, including improvements in lipid profile and glucose metabolism, as well as reducing the symptoms of benign prostatic hyperplasia. Urapidil has an alpha-blocking effect but, unlike other alpha-blockers, also has a central sympatholytic effect mediated via stimulation of serotonin <em>5HT</em>(1A) receptors in the central nervous system. Several studies have suggested that oral urapidil is effective and well tolerated when used as second-line therapy in patients with BP inadequately controlled with other agents. Urapidil has also been shown to improve glucose and lipid metabolism in hypertensive patients with concomitant diabetes and/or hyperlipidemia. Intravenous urapidil is effective in the treatment of hypertensive crises, perioperative hypertension, and pre-eclampsia and may have a potential role in the management of acute stroke. In this review, the use of alpha-blockers in hypertension is discussed, with particular focus on urapidil for the lowering of BP in a variety of clinical settings.

Incomplete spinal cord injury (iSCI) often results in significant motor impairments that lead to decreased functional mobility. Loss of descending serotonergic (<em>5HT</em>) input to spinal circuits is thought to contribute to motor impairments, with enhanced motor function demonstrated through augmentation of <em>5HT</em> signaling. However, the presence of spastic motor behaviors in SCI is attributed, in part, to changes in spinal <em>5HT</em> receptors that augment their activity in the absence of <em>5HT</em>, although data demonstrating motor effects of <em>5HT</em> agents that deactivate these receptors are conflicting. The effects of enhancement or depression of <em>5HT</em> signaling on locomotor function have not been thoroughly evaluated in human iSCI. Therefore, the aim of the current study was to investigate acute effects of <em>5HT</em> medications on locomotion in 10 subjects with chronic (>1 year) iSCI. Peak overground and treadmill locomotor performance, including measures of gait kinematics, electromyographic (EMG) activity, and oxygen consumption, were assessed before and after single-dose administration of either a selective serotonin reuptake inhibitor (SSRI) or a <em>5HT</em> antagonist using a double-blinded, randomized, cross-over design. Results indicate that neither medication led to improvements in locomotion, with a significant decrease in peak overground gait speed observed after <em>5HT</em> antagonists (from 0.8±0.1 to 0.7±0.1 m/s; p=0.01). Additionally, 5-HT medications had differential effects on EMG activity, with <em>5HT</em> antagonists decreasing extensor activity and SSRIs increasing flexor activity. Our data therefore suggest that acute manipulation of <em>5HT</em> signaling, despite changes in muscle activity, does not improve locomotor performance after iSCI.

The uptake of MIBG, a scintigraphic agent widely used in the detection of APUD tumours, was studied with a pharmacological approach on an in vitro and an in vivo models. MIBG as well as norepinephrine (NE) was taken up by human blood platelets, a model for presynaptic nerve endings amine uptake, with a thermodependant mechanism. MIBG and NE uptake was inhibited by desimipramine and reserpine. However, MIBG but not NE uptake was inhibited by fluvoxamine, a serotonin (<em>5HT</em>) uptake inhibitor. This suggests that MIBG is a NE and also a <em>5HT</em> uptake tracer which involves uptake one and vesicular storage mechanisms. In rats treated with 6-hydroxydopamine to induce a chemical sympathectomy, we observed an inhibition of uptake similar for MIBG and NE in the heart, the salivary glands and the spleen, but no effect was observed in the liver. Some clinical inferences to best investigate specific monoamine uptake are drawn from these results.

OBJECTIVE

The serotonergic system has long been linked to migraine but recent studies highlight how much is still unclear about this link. And recent data add to the uncertainty of where/how triptans act and why they are headache specific.

RESULTS

Markers of <em>5HT</em> levels in the brains of migraine patients show no changes between attacks. Several recent meta-analyses show the most convincing data on genetic differences in the serotonergic system for <em>5HT</em> transporters. Findings of additional triptan actions on peripheral trigeminovascular neurons and in the hypothalamus add more fuel to the debate on where these drugs act. A growing list of studies show efficacy of multiple triptans and other <em>5HT</em>1b/1d agonists in preclinical models of nonheadache pain arguing for reevaluation of whether these drugs have efficacy in other pain states. Despite these issues, serotonergic drugs continue to be the gold standard for abortive agents with new members on the horizon (<em>5HT</em>1f agonists).

CONCLUSIONS

Given the clear efficacy of serotonergic drugs for migraine, continued study on the role of the endogenous <em>5HT</em> system may lead to more novel therapies. And with the list of studies demonstrating efficacy triptans in models of nonheadache, clinical studies should address whether these drugs work for other types of pain.

The present study was designed to investigate the potential influence of various neuronal processes including uptake, release and metabolism, on the in vivo microdialysis extraction fraction (Ed) of serotonin (<em>5HT</em>) and norepinephrine (NE). Paroxetine administration decreased the Ed of <em>5HT</em> in the nucleus accumbens from 24 +/- 3 to 18 +/- 0.2% (p < 0.05). Similarly, desipramine infusion reduced the NE Ed from 35 +/- 2 to 26 +/- 1% (p < 0.05). However, perfusion with pargyline or tetrodotoxin had no effect on the Ed of either <em>5HT</em> or NE. Perfusion with agonists for the <em>5HT</em>, alpha-adrenergic, D2 and histamine receptors had no effect on the Ed of <em>5HT</em>. In the same manner, perfusion with the alpha-adrenergic agonists, methoxamine or clonidine, did not affect the Ed of NE. These data are in agreement with experimental results obtained for dopamine in the nucleus accumbens and the theory of quantitative microdialysis which predicts that only changes in the rate of clearance will change Ed of monoamines. These results suggest that, like DA, changes in the Ed for <em>5HT</em> or NE are indicative of changes in the reuptake of these neurotransmitters. The results also indicate that pharmacological agents which do not affect uptake have no effect on the extraction fraction.

Deoxynivalenol (DON) produces two characteristic toxicological effects, decreased feed consumption (anorexia) and emesis. Both effects have been linked to increased central (CNS) serotoninergic activity. Although there has also been some indication of a peripheral involvement, the role of blood pools of serotonin and related compounds in mediating DON toxicity is not well defined. In this study, the effect of DON on plasma concentrations of serotonin (5-hydroxytryptamine, <em>5HT</em>), 5HIAA (5-hydroxyindoleacetic acid) and tryptophan (TRP), as a reflection of an induced peripheral serotoninergic system, was investigated in swine. Typical values for the plasma concentrations of <em>5HT</em>, 5HIAA, and TRP were established in pigs. Following administration of DON, either intragastrically or intravenously, concentration changes in these substances were measured over an eight hour period. The effect of low and high toxin doses were also compared. Analyses showed no effect on plasma levels of the compounds of interest, even at sufficient toxin doses to invoke emesis in the test animals. Any variation over the course of the study remained within acceptable control limits. These results indicated no peripheral effect by DON which could account for the increased serotoninergic activity associated with altered feeding behaviour or emesis.