We have shown that the development of cutaneous allodynia (exaggerated skin sensitivity) during migraine is detrimental to the anti-migraine action of the <em>5HT</em>(IB/ID) receptor agonists known is triptans. Because cutaneous allodynia is a manifestation of sensitization of central trigeminovascular neurons, we examined whether triptan treatment can intercept such sensitization before its initiation or after its establishment in our rat model for cutaneous allodynia induced by intracranial pain. Single-unit recordings were obtained from spinal trigeminal neurons that proved to received convergent inputs from the dura and facial skin. The effects of sumatriptan (300 microg/kg i.v.) on central sensitization induced by topical application of inflammatory soup (IS) on the dura were determined when the drug was administered either 2 h after IS (late intervention) or at the same time as IS (early intervention). Late sumatriptan intervention counteracted two aspects of central sensitization: dural receptive fields, which initially expanded by IS, shrunk back after treatment; neuronal response threshold to dural indentation, which initially decreased after IS, increased after sumatriptan. On the other hand, late sumatriptan intervention did not reverse other aspects of central sensitization: spontaneous firing rate and neuronal response magnitude to skin brushing which initially increased after IS, remained elevated after sumatriptan; response threshold to heating of the skin, which initially dropped after IS, remained low after sumatriptan. Early sumatriptan intervention effectively blocked the development of all aspects of central sensitization expected to be induced 2 h after IS application: dural receptive fields did not expand; neuronal response threshold to dural indentation and skin stimulation did not decrease; spontaneous firing rate did not increase. The early treatment results suggest that triptan action provides a powerful means of preventing the initiation of central sensitization triggered by chemical stimulation of meningeal nociceptors. The late treatment results suggest that triptan action is insufficient to counteract an already established central sensitization. Thus, triptan action appears to be exerted directly on peripheral rather than central trigeminovascular neurons.

Developmental exposure to chlorpyrifos (CPF) causes persistent changes in serotonergic (<em>5HT</em>) systems. We administered 1 mg/kg/day CPF to rats on postnatal days 1-4, a regimen below the threshold for systemic toxicity. When tested in adulthood, CPF-exposed animals showed abnormalities in behavioral tests that involve <em>5HT</em> mechanisms. In the elevated plus maze, males treated with CPF spent more time in the open arms, an effect seen with <em>5HT</em> deficiencies in animal models of depression. Similarly, in an anhedonia test, the CPF-exposed group showed a decreased preference for chocolate milk versus water. Developmental CPF exposure also has lasting effects on cognitive function. We replicated our earlier finding that developmental CPF exposure ablates the normal sex differences in 16-arm radial maze learning and memory: during acquisition training, control male rats typically perform more accurately than do control females, but CPF treatment eliminated this normal sex difference. Females exposed to CPF showed a reduction in working and reference memory errors down to the rate of control males. Conversely, CPF-exposed males exhibited an increase in working and reference memory errors. After radial-arm acquisition training, we assessed the role of <em>5HT</em> by challenging the animals with the <em>5HT</em>2 receptor antagonist ketanserin. Ketanserin did not affect performance in controls but elicited dose-dependent increases in working and reference memory errors in the CPF group, indicating an abnormal dependence on <em>5HT</em> systems. Our results indicate that neonatal CPF exposures, classically thought to be subtoxic, produce lasting changes in <em>5HT</em>-related behaviors that resemble animal models of depression.

Male rats housed in mixed-sex groups in a visible burrow system (VBS) form a dominance hierarchy in which subordinate animals show stress-related changes in behavior, endocrine function and neurochemistry. Dominants also appear to be moderately stressed compared to controls, although these animals do not develop the more pronounced behavioral and physiological deficits seen in the subordinates. In the present study, we examined the effects of chronic psychosocial stress on the morphology of Golgi-impregnated CA3 pyramidal neurons. In addition, since serotonin has been implicated in the mechanisms mediating the dendritic remodeling seen with other chronic stress regimens, we used quantitative autoradiography to measure binding to the serotonin transporter (<em>5HT</em>T) in hippocampus and dorsal and median raphe. Chronic social stress led to a decrease in the number of branch points and total dendritic length in the apical dendritic trees of CA3 pyramidal neurons in dominant animals compared to unstressed controls; subordinates also had a decreased number of dendritic branch points. [(3)H]paroxetine binding to the <em>5HT</em>T was decreased in Ammon's horn in both dominants and subordinates compared to controls, while <em>5HT</em>T binding remained unchanged in dentate gyrus and raphe. The similarity of the changes in <em>5HT</em>T binding and dendritic arborization between both groups of VBS animals, despite apparent differences in stressor severity, suggests that these changes may be part of the normal adaptive response to chronic social stress. The mechanisms underlying dendritic remodeling in CA3 pyramidal neurons are likely to involve stress-induced changes in glucocorticoids and in <em>5HT</em> and other transmitters.

The dorsal raphe nucleus (DR) has a topographic neuroanatomy consistent with the idea that different parts of this nucleus subserve different functions. Here we use dual in situ hybridization to describe the rostral-caudal neurochemical distribution of three major cell groups, serotonin (5-hydroxytryptamine; 5-HT), gamma-aminobutyric acid (GABA), and catecholamine, and their relative colocalization with each other and mRNA encoding four different receptor subtypes that have been described to influence DR responses, namely, <em>5HT</em>-1A, alpha(1b) adrenergic (alpha(1b) ADR), and corticotropin-releasing factor type 1 (CRF-R1) and 2 (CRF-R2) receptors. Serotonergic and GABAergic neurons were distributed throughout the rostral-caudal extent of the DR, whereas catecholaminergic neurons were generally restricted to the rostral half of the nucleus. These phenotypes essentially represent distinct cell populations, because the neurochemical markers were rarely colocalized. Both <em>5HT</em>-1A and alpha(1b) ADR mRNA were highly expressed throughout the DR, and the vast majority of serotonergic neurons expressed both receptors. A smaller percentage of GABAergic neurons also expressed <em>5HT</em>-1A or alpha(1b) ADR mRNA. Very few catecholaminergic cells expressed either <em>5HT</em>-1A or alpha(1b) ADR mRNA. CRF-R1 mRNA was detected only at very low levels within the DR, and quantitative colocalization studies were not technically feasible. CRF-R2 mRNA was mainly expressed at the middle and caudal levels of the DR. At midlevels, CRF-R2 mRNA was expressed exclusively in serotonin neurons, whereas, at caudal levels, approximately half the CRF-R2 mRNA was expressed in GABAergic neurons. The differential distribution of distinct neurochemical phenotypes lends support to the idea of functional differentiation of the DR.

Biogenic amines are important neuroactive molecules of the central nervous system (CNS) of several insect species. Serotonin (<em>5HT</em>), dopamine (DA), histamine (HA), and octopamine (OA) are the amines which have been extensively studied in Drosophila melanogaster. Each one of the four aminergic neuronal systems exhibits a stereotypic pattern of a small number of neurons that are widely distributed in the fly CNS. In this review, histochemical and immunocytochemical data on the distribution of the amine neurons in the larval and adult nervous system, are summarized. The majority of DA and <em>5HT</em> neurons are interneurons, most of which are found in bilateral clusters. <em>5HT</em> innervation is found in the feeding apparatus as well as in the endocrine organ of the larva, the ring gland. The octopaminergic neuronal population consists of both interneurons and efferent neurons. In the larval CNS all OA immunoreactive somata are localized in the midline of the ventral ganglion while in the adult CNS both unpaired neurons and bilateral clusters of immunoreactive cells are observed. One target of OA innervation is the abdominal muscles of the larval body wall where OA immunoreactivity is associated with the type II boutons in the axonal terminals. Histamine is mainly found in all photoreceptor cells where it is considered to be the major neurotransmitter molecule, and in specific mechanosensory neurons of the peripheral nervous system. Similarities between specific aminergic neurons and innervation sites in Drosophila and in other insect species are discussed. In addition, studies on the development and differentiation of <em>5HT</em> and DA neurons are reviewed and data on the localization of <em>5HT</em>, DA, and OA receptors are included as well. Finally, an overview on the isolation of the genes and the mutations in the amine biosynthetic pathways is presented and the implications of the molecular genetic approach in Drosophila are discussed.

Dopamine (DA) and serotonin (<em>5HT</em>) are reported to serve important roles in aggression in a wide variety of animals. Previous investigations of <em>5HT</em> function in adult Drosophila behavior have relied on pharmacological manipulations, or on combinations of genetic tools that simultaneously target both DA and <em>5HT</em> neurons. Here, we generated a transgenic line that allows selective, direct manipulation of serotonergic neurons and asked whether DA and <em>5HT</em> have separable effects on aggression. Quantitative morphological examination demonstrated that our newly generated tryptophan hydroxylase (TRH)-Gal4 driver line was highly selective for <em>5HT</em>-containing neurons. This line was used in conjunction with already available Gal4 driver lines that target DA or both DA and <em>5HT</em> neurons to acutely alter the function of aminergic systems. First, we showed that acute impairment of DA and <em>5HT</em> neurotransmission using expression of a temperature sensitive form of dynamin completely abolished mid- and high-level aggression. These flies did not escalate fights beyond brief low-intensity interactions and therefore did not yield dominance relationships. We showed next that manipulation of either <em>5HT</em> or DA neurotransmission failed to duplicate this phenotype. Selective disruption of <em>5HT</em> neurotransmission yielded flies that fought, but with reduced ability to escalate fights, leading to fewer dominance relationships. Acute activation of <em>5HT</em> neurons using temperature sensitive dTrpA1 channel expression, in contrast, resulted in flies that escalated fights faster and that fought at higher intensities. Finally, acute disruption of DA neurotransmission produced hyperactive flies that moved faster than controls, and rarely engaged in any social interactions. By separately manipulating <em>5HT</em>- and DA- neuron systems, we collected evidence demonstrating a direct role for <em>5HT</em> in the escalation of aggression in Drosophila.

Exposure to chlorpyrifos (CPF) alters neuronal development of serotonin (<em>5HT</em>) and dopamine systems, and we recently found long-term alterations in behaviors related to <em>5HT</em> function. To characterize the synaptic mechanisms underlying these effects, we exposed developing rats to CPF regimens below the threshold for systemic toxicity, in three treatment windows: gestational days (GD) 17-20, postnatal days (PN) 1-4, or PN11-14. In early adulthood (PN60), we assessed basal neurotransmitter content and synaptic activity (turnover) in brain regions containing the major <em>5HT</em> and dopamine projections. CPF exposure on GD17-20 or PN1-4 evoked long-term increases in <em>5HT</em> turnover across multiple regions; the effects were not secondary to changes in neurotransmitter content, which was unaffected or even decreased. When the treatment window was shifted to PN11-14, there were no long-term effects. Dopamine turnover also showed significant increases after CPF exposure on GD17-20, but only when the dose was raised above the threshold for overt toxicity; however, hippocampal dopamine content was profoundly subnormal after exposures below or above the acute, toxic threshold, suggesting outright neurotoxicity. These results indicate that, in a critical developmental period, apparently nontoxic exposures to CPF produce lasting activation of <em>5HT</em> systems in association with <em>5HT</em>-associated behavioral anomalies.

Because all three components of lower urinary tract control (parasympathetic, sympathetic and somatic) are intimately associated with serotonin (5-hydroxytryptamine [<em>5HT</em>])- and norepinephrine (NE)- containing terminals and receptors, in the present study, we examined the effects of increasing extracellular levels of <em>5HT</em> and NE with duloxetine, a <em>5HT</em> and NE reuptake inhibitor, on lower urinary tract function under "normal" or nonirritated conditions (transvesical infusion of saline) and in a model of bladder irritation (i.e., transvesical infusion of 0.5% acetic acid) in chloralose-anesthetized cats. Irritation reduced bladder capacity (to 20% of control) and produced insignificant increases in periurethral electromyographic (EMG) activity compared with nonirritated control animals. Duloxetine produced insignificant increases in bladder capacity and sphincter EMG activity when administered under nonirritated bladder conditions. However, this duloxetine "pretreatment" did prevent the typical acetic acid-induced reductions in bladder capacity and unmasked a marked activation of sphincter EMG activity on acetic acid infusion (by 8-fold). Furthermore, when administered initially under irritated bladder conditions, duloxetine produced dose-dependent increases in bladder capacity (by 5-fold) and increased periurethral striated muscle EMG activity (by 8-fold). The effects on bladder activity were due to central mechanisms since bladder contractions evoked by direct electrical stimulation of efferent fibers in the pelvic nerve were not effected by duloxetine. The effects of duloxetine on bladder capacity were antagonized by methiothepin, a non-selective <em>5HT</em> receptor antagonist, but not by the other <em>5HT</em> and NE receptor antagonists examined: LY53857, a <em>5HT</em>2 antagonist; prazosin, an alpha-1-adrenergic receptor antagonist; idazoxan, an alpha-2-adrenergic receptor antagonist; or propranolol, a beta-adrenergic receptor antagonist. The facilitatory effects of duloxetine on periurethral sphincter EMG were significantly antagonized to various degrees by methiothepin, LY53857 and prazosin but not by idazoxan or propranolol. These results indicate that duloxetine, through inhibition of <em>5HT</em> and NE reuptake, has weak effects under normal conditions. However, under conditions of bladder irritation, duloxetine suppresses bladder activity through <em>5HT</em> receptor mechanisms and enhances external urethral sphincter activity through <em>5HT</em>2 and alpha-1-adrenergic mechanisms.

The interaction between depression and stroke is highly complex. Post-stroke depression (PSD) is among the most frequent neuropsychiatric consequences of stroke. Depression also negatively impacts stroke outcome with increased morbidity, mortality and poorer functional recovery. Antidepressants such as the commonly prescribed selective serotonin reuptake inhibitors improve stroke outcome, an effect that may extend far beyond depression, e.g., to motor recovery. The main biological theory of PSD is the amine hypothesis. Conceivably, ischaemic lesions interrupt the projections ascending from midbrain and brainstem, leading to a decreased bioavailability of the biogenic amines--serotonin (<em>5HT</em>), dopamine (DA) and norepinephrine (NE). Acetylcholine would also be involved. So far, preclinical and translational research on PSD is largely lacking. The implementation and characterization of suitable animal models is clearly a major prerequisite for deeper insights into the biological basis of post-stroke mood disturbances. Equally importantly, experimental models may also pave the way for the discovery of novel therapeutic targets. If we cannot prevent stroke, we shall try to limit its long-term consequences. This review therefore presents animal models of PSD and summarizes potential underlying mechanisms including genomic signatures, neurotransmitter and neurotrophin signalling, hippocampal neurogenesis, cellular plasticity in the ischaemic lesion, secondary degenerative changes, activation of the hypothalamo-pituitary-adrenal (HPA) axis and neuroinflammation. As stroke is a disease of the elderly, great clinical benefit may especially accrue from deciphering and targeting basic mechanisms underlying PSD in aged animals.

During brain development, serotonin (<em>5HT</em>) provides essential neurotrophic signals, and in earlier work, we found that developmental exposure to chlorpyrifos (CPF) elicits short-term changes in <em>5HT</em> systems. In the present study, we evaluated the effects in adulthood after CPF exposures from the neural tube stage [gestational days (GD) 9-12] and the late gestational period (GD17-20) through postnatal neuronal differentiation and synaptogenesis [postnatal days (PN) 1-4 and 11-14], using treatments below the threshold for systemic toxicity. With exposure on GD9-12, CPF elicited global elevations in <em>5HT</em>1A and <em>5HT</em>2 receptors and in the <em>5HT</em> presynaptic transporter. The GD17-20 treatment elicited larger effects that displayed selectivity for regions with <em>5HT</em> nerve terminals and that were preferential for males. Although similar receptor up-regulation was seen after PN1-4 exposure, the effects were larger in regions with <em>5HT</em> cell bodies; in addition, the presynaptic transporter was down-regulated in the nerve terminal zones of females. The PN11-14 exposure had much smaller effects on receptors but still elicited transporter suppression with the same regional and sex selectivity. Although CPF exposure on GD17-20, PN1-4, or PN11-14 altered the ability of <em>5HT</em> to modulate adenylyl cyclase, this change did not correspond with the effects on <em>5HT</em> receptors, suggesting an additional set of effects on proteins that transduce the <em>5HT</em> signal. Our results indicate that CPF elicits long-lasting changes in <em>5HT</em> receptors, the presynaptic <em>5HT</em> transporter, and <em>5HT</em>-mediated signal transduction after exposure in discrete developmental windows that range from the neural tube stage through synaptogenesis. These effects are likely to contribute to neurobehavioral teratology of CPF.

The link between dysregulated serotonergic activity and depression and anxiety disorders is well established, yet the molecular mechanisms underlying these psychopathologies are not fully understood. Here, we explore the role of microRNAs in regulating serotonergic (<em>5HT</em>) neuron activity. To this end, we determined the specific microRNA "fingerprint" of <em>5HT</em> neurons and identified a strong microRNA-target interaction between microRNA 135 (miR135), and both serotonin transporter and serotonin receptor-1a transcripts. Intriguingly, miR135a levels were upregulated after administration of antidepressants. Genetically modified mouse models, expressing higher or lower levels of miR135, demonstrated major alterations in anxiety- and depression-like behaviors, <em>5HT</em> levels, and behavioral response to antidepressant treatment. Finally, miR135a levels in blood and brain of depressed human patients were significantly lower. The current results suggest a potential role for miR135 as an endogenous antidepressant and provide a venue for potential treatment and insights into the onset, susceptibility, and heterogeneity of stress-related psychopathologies.

The expression of aggressiveness, which constitutes many facets of behavior, is influenced by a complex interaction of biologic, psychologic, and social variables. Even though individual differences in impulsivity and the behavioral consequences, such as aggression, addiction, and suicidality, are substantially heritable, they ultimately result from an interplay between genetic variations and environmental factors. While formation and integration of multiple neural networks is dependent on the actions of neurotransmitters, such as serotonin (<em>5HT</em>), converging lines of evidence indicate that genetically determined variability in serotonergic gene expression influences complex traits including that of inappropriately aggressive behavior. This contribution reviews studies of major gene effects in inbred and knockout strains of mice with increased aggression-related behavior and discusses the relevance of several serotonergic gene variations in humans which include high aggressiveness as part of the phenotype. Although special emphasis is given to the molecular psychobiology of <em>5HT</em> in aggression-related behavior in rodents, nonhuman primates, and humans, relevant conceptual and methodological issues in the search for candidate genes for impulsivity and aggressiveness and for the development of mouse models of aggressive and antisocial behavior in humans are also considered.

A complementary DNA (cDNA) encoding a serotonin receptor with 51% sequence identity to the <em>5HT</em>-1C subtype was isolated from a rat brain cDNA library by homology screening. Transient expression of the cloned cDNA in mammalian cells was used to establish the pharmacological profile of the encoded receptor polypeptide. Membranes from transfected cells showed high-affinity binding of the serotonin antagonists spiperone, ketanserin and mianserin, low affinity for haloperidol (a dopamine D2 receptor antagonist), 8-OH-DPAT as well as MDL-72222 and no detectable binding of [3H]serotonin. This profile is consonant with the <em>5HT</em>-2 subtype of serotonin receptors. In agreement with this assignment, serotonin increased the intracellular Ca2+ concentration and activated phosphoinositide hydrolysis in transfected mammalian cells. The agonist also elicited a current flow, blocked by spiperone, in Xenopus oocytes injected with in vitro synthesized RNA containing the cloned nucleotide sequences.

Serotonin transporters (SERTs), sites of psychostimulant action, display multiple conducting states in expression systems. These include a substrate-independent transient conductance, two separate substrate-independent leak conductances associated with Na(+) and H(+), and a substrate-dependent conductance of variable stoichiometry, which exceeds that predicted from electroneutral substrate transport. The present data show that the SNARE protein syntaxin 1A binds the N-terminal tail of SERT, and this interaction regulates two SERT-conducting states. First, substrate-induced currents are absent because Na(+) flux becomes strictly coupled to <em>5HT</em> transport. Second, Na(+)-mediated leak currents are eliminated. These two SERT-conducting states are present endogenously in thalamocortical neurons, act to depolarize the membrane potential, and are modulated by molecules that disrupt SERT and syntaxin 1A interactions. These data show that protein interactions govern SERT activity and suggest that both cell excitability and psychostimulant-mediated effects will be dependent upon the state of association among SERT and its interacting partners.

Antidepressant-sensitive serotonin (5-hydroxytryptamine, <em>5HT</em>) transporters (SERTs) are responsible for efficient synaptic clearance of extracellular <em>5HT</em>. Previously (Qian, Y., Galli, A., Ramamoorthy, S., Risso, S., DeFelice, L. J., and Blakely, R. D. (1997) J. Neurosci. 17, 45-47), we demonstrated that protein kinase (PKC)-linked pathways in transfected HEK-293 cells lead to the internalization of cell-surface human (h) SERT protein and a reduction in <em>5HT</em> uptake capacity. In the present study, we report that PKC activators rapidly, and in a concentration-dependent manner, elevate the basal level of hSERT phosphorylation 5-6-fold. Similarly, protein phosphatase (PP1/PP2A) inhibitors down-regulate <em>5HT</em> transport and significantly elevate hSERT 32P incorporation, effects that are additive with those of PKC activators. Moreover, hSERT phosphorylation induced by beta-phorbol 12-myristate 13-acetate is abolished selectively by the PKC inhibitors staurosporine and bisindolylmaleimide I, whereas hSERT phosphorylation induced by phosphatase inhibitors is insensitive to these agents at comparable concentrations. Protein kinase A and protein kinase G activators fail to acutely down-regulate <em>5HT</em> uptake but significantly enhance hSERT phosphorylation. Basal hSERT and okadaic acid-induced phosphorylation were insensitive to chelation of intracellular calcium and Ca2+/calmodulin-dependent protein kinase inhibitors. Together these results reveal hSERT to be a phosphoprotein whose phosphorylation state is likely to be tightly controlled by multiple kinase and phosphatase pathways that may also influence the transporter's regulated trafficking.

BACKGROUND

Loss-of-function mutations in the ATP-binding cassette (ABCA)-1 gene locus are the underlying cause for familial hypoalphalipoproteinemia, providing a human isolated low-HDL model. In these familial hypoalphalipoproteinemia subjects, we evaluated the impact of isolated low HDL on endothelial function and the vascular effects of an acute increase in HDL.

RESULTS

In 9 ABCA1 heterozygotes and 9 control subjects, vascular function was assessed by venous occlusion plethysmography. Forearm blood flow responses to the endothelium-dependent and -independent vasodilators serotonin (<em>5HT</em>) and sodium nitroprusside, respectively, and the inhibitor of nitric oxide synthase NG-monomethyl-l-arginine (L-NMMA) were measured. Dose-response curves were repeated after systemic infusion of apolipoprotein A-I/phosphatidylcholine (apoA-I/PC) disks. At baseline, ABCA1 heterozygotes had decreased HDL levels (0.4+/-0.2 mmol/L; P<0.05), and their forearm blood flow responses to both <em>5HT</em> (maximum, 49.0+/-10.4%) and L-NMMA (maximum, -22.8+/-22.9%) were blunted compared with control subjects (both P< or =0.005). Infusion of apoA-I/PC disks increased plasma HDL to 1.3+/-0.4 mmol/L in ABCA1 heterozygotes, which resulted in complete restoration of vasomotor responses to both <em>5HT</em> and L-NMMA (both P</=0.001). Endothelium-independent vasodilation remained unaltered throughout the protocol.

CONCLUSIONS

In ABCA1 heterozygotes, isolated low HDL is associated with endothelial dysfunction, attested to by impaired basal and stimulated NO bioactivity. Strikingly, both parameters were completely restored after a single, rapid infusion of apoA-I/PC. These findings indicate that in addition to its long-term role within reverse cholesterol transport, HDL per se also exerts direct beneficial effects on the arterial wall.

The process of RNA editing involves the modification of mRNA at specific sites by adenosine deaminases that act on RNA (ADAR) enzymes. By catalyzing the conversion of adenosine to inosine, these enzymes alter the way in which the mRNA is translated, and consequently alter the primary structure of the resultant proteins. The serotonin (<em>5HT</em>) 2C receptor (<em>5HT</em>2CR) is currently the only known member of the superfamily of seven transmembrane domain receptors (7TMRs) to undergo this modification, and provides a fascinating case study in the effects of such changes. Here we review the current state of knowledge surrounding the editing of the <em>5HT</em>2CR, the stark differences in signalling arising due to this process, and the potential for (and difficulties in) exploiting the phenomenon for improved therapeutic intervention in various neurological disorders.

Psychological stress is widely believed to play a major role in functional gastrointestinal (GI) disorders, especially irritable bowel syndrome (IBS), by precipitating exacerbation of symptoms. The available data clearly demonstrate that inhibition of gastric emptying and stimulation of colonic transit is the most consistent pattern in the motility response of the GI tract to acute or short-term stress. Thus, one might propose that these alterations might play a pathophysiological role in dyspeptic symptoms and alterations in stool frequency and consistency in patients with stress-related functional GI disorders. Taken together, the above-mentioned studies suggest that the colonic motor response to stress is exaggerated in IBS. There is evidence that an increased emotional response is associated with this difference in colonic, and perhaps also gastric motor responses to certain stressors. However, almost no valid data are available so far from human studies addressing the question if differences in motility responses to stress between patients with functional GI disorders and healthy subjects are due to an altered stress response associated with an imbalance of the autonomic nervous system or increased stress susceptibility. We can summarize that in experimental animals the most consistent pattern of GI motor alterations induced by various psychological and physical stressors is that of delaying gastric emptying and accelerating colonic transit. Endogenous corticotropin-releasing factor (CRF) in the brain plays a significant role in the central nervous system mediation of stress-induced inhibition of upper GI and stimulation of lower GI motor function through activation of brain CRF receptors. The inhibition of gastric emptying by CRF may be mediated by interaction with the CRF-2 receptor, while CRF-1 receptors are involved in the colonic and anxiogenic responses to stress. Endogenous serotonin, peripherally released in response to stress, seems to be involved in stress- and central CRF-induced stimulation of colonic motility by acting on <em>5HT</em>-3 receptors. Taken together, the limited data available from investigations in healthy subjects and patients with functional GI disorders provide some evidence that stress affects visceral sensitivity in humans. Acute psychological stress seems to facilitate increased sensitivity to experimental visceral stimuli, if the stressor induces a significant emotional change. In summary, studies in experimental animals suggest that stress-induced visceral hypersensitivity is centrally mediated by endogenous CRF and involvement of structures of the emotional motor system, e.g. the amygdala. Stress-induced activation or sensitization of mucosal mast cells in the GI tract seem to be involved in stress-associated alterations of visceral sensitivity.

We have previously reported that exposure of rats to aerosolized endotoxin (LPS) causes a transient, dose-dependent increase in bronchial responsiveness (BR) to 5 hydroxytryptamine (<em>5HT</em>), 90 min after exposure. In the present study we examined whether LPS induces the release of tumor necrosis factor (TNF) in the airways and whether TNF contributes to the increase in BR. After 90 min following exposure to aerosolized LPS, at a concentration of 1 or 10 micrograms/ml, TNF concentrations in bronchoalveolar lavage (BAL) fluid were 17.9 +/- 6.9 and 80.5 +/- 7.8 U/ml, respectively. No TNF was detected in BAL fluid of saline-exposed animals. At 90 min after exposure to aerosolized recombinant human TNF (rhTNF) (1 microgram/ml) an increase in BR was observed: the provocative concentration of <em>5HT</em> causing a 50% increase in lung resistance (PC50RL <em>5HT</em>) was 2.7 +/- 0.4 versus 4.4 +/- 0.3 microgram/kg in saline-exposed animals (p less than 0.01). Pretreatment with anti-TNF antibodies 30 min before LPS exposure significantly diminished the increase in BR: PC50RL <em>5HT</em> was 2.3 +/- 0.4 versus 1.2 +/- 0.5 microgram/kg in control pretreated LPS-exposed rats (p less than 0.01). Exposure to aerosolized TNF also induced a significant influx of neutrophils in BAL fluid (12.1 +/- 3.7 versus 1.7 +/- 0.4% in saline-exposed animals) (p less than 0.01). The LPS-induced neutrophil influx in BAL fluid was partly inhibited by pretreatment with anti-TNF antibodies (55.2 +/- 5.1 versus 76.0 +/- 3.9%) (p less than 0.01). We conclude that TNF causes bronchial hyperresponsiveness and airway inflammation.(ABSTRACT TRUNCATED AT 250 WORDS)

Nicotine is a neuroteratogen that targets synaptic function during critical developmental stages and recent studies indicate that CNS vulnerability extends into adolescence, the time that smoking typically commences. We administered nicotine to pregnant or adolescent rats via continuous minipump infusions, using dose rates that replicate the plasma nicotine levels found in smokers. Fetal nicotine exposure (gestational days 4-21) decreased the cerebrocortical binding of paroxetine (PXT), a marker for the serotonin (<em>5HT</em>) transporter, likely indicative of a decrease in nerve terminals in that region; the effect lasted into adulthood. There was a corresponding increase in PXT binding in the midbrain/brainstem, the region containing the <em>5HT</em> cell bodies that project to the cerebral cortex, a pattern typical of reactive sprouting in response to nerve terminal damage. After adolescent nicotine treatment (postnatal days 30-47), PXT binding was reduced in the hippocampus and striatum instead of the cerebral cortex, again accompanied by increased binding in the midbrain and brainstem; the patterns of effects within each region were gender-selective, although both males and females displayed abnormalities. Superimposed on this overall effect, there were transient increases in PXT binding, likely due to acute stimulant effects of nicotine. We also assessed <em>5HT</em> presynaptic activity (5HIAA/<em>5HT</em> ratio). Withdrawal from adolescent nicotine treatment led to suppression of activity in the cerebral cortex and activation in the midbrain. These results indicate that both fetal and adolescent nicotine exposure elicit apparent damage to <em>5HT</em> projections with reactive increases in regions containing <em>5HT</em> cell bodies. Long-term changes in <em>5HT</em> innervation and/or synaptic activity may play a role in the subsequent development of depression in the offspring of women who smoke during pregnancy or in adolescent smokers.

Not all neuropathic pain patients gain relief from current therapies that include the anticonvulsant, gabapentin, thought to modulate calcium channel function. We report a neural circuit that is permissive for the effectiveness of gabapentin. Substance P-saporin (SP-SAP) was used to selectively ablate superficial dorsal horn neurons expressing the neurokinin-1 receptor for substance P. These neurons project to the brain as shown by retrograde labelling and engage descending brainstem serotonergic influences that enhance spinal excitability via a facilitatory action on <em>5HT</em>(3) receptors. We show the integrity of this pathway following nerve injury contributes to the behavioural allodynia, neuronal plasticity of deep dorsal horn neurons and the injury-specific actions of gabapentin. Thus SP-SAP attenuated the tactile and cold hypersensitivity and abnormal neuronal coding (including spontaneous activity, expansion of receptive field size) seen after spinal nerve ligation. Furthermore the powerful actions of gabapentin after neuropathy were blocked by either ablation of NK-1 expressing neurones or <em>5HT</em>(3) receptor antagonism using ondansetron. Remarkably, <em>5HT</em>(3) receptor activation provided a state-dependency (independent of that produced by neuropathy) allowing GBP to powerfully inhibit in normal uninjured animals. This circuit is therefore a crucial determinant of the abnormal neuronal and behavioural manifestations of neuropathy and importantly, the efficacy of gabapentin. As this spino-bulbo-spinal circuit contacts areas of the brain implicated in the affective components of pain, this loop may represent a route by which emotions can influence the degree of pain in a patient, as well as the effectiveness of the drug treatment. These hypotheses are testable in patients.

Triptans are <em>5HT</em>(1B/1D) receptor agonists commonly prescribed for migraine headache. Although originally designed to constrict dilated intracranial blood vessels, the mechanism and site of action by which triptans abort the migraine pain remain unknown. We showed recently that sensitization of peripheral and central trigeminovascular neurons plays an important role in the pathophysiology of migraine pain. Here we examined whether the drug sumatriptan can prevent and/or suppress peripheral and central sensitization by using single-unit recording in our animal model of intracranial pain. We found that sumatriptan effectively prevented the induction of sensitization (i.e., increased spontaneous firing; increased neuronal sensitivity to intracranial mechanical stimuli) in central trigeminovascular neurons (recorded in the dorsal horn), but not in peripheral trigeminovascular neurons (recorded in the trigeminal ganglion). After sensitization was established in both types of neuron, sumatriptan effectively normalized intracranial mechanical sensitivity of central neurons, but failed to reverse such hypersensitivity in peripheral neurons. In both the peripheral and central neurons, the drug failed to attenuate the increased spontaneous activity established during sensitization. These results suggest that neither peripheral nor central trigeminovascular neurons are directly inhibited by sumatriptan. Rather, triptan action appears to be exerted through presynaptic <em>5HT</em>(1B/1D) receptors in the dorsal horn to block synaptic transmission between axon terminals of the peripheral trigeminovascular neurons and cell bodies of their central counterparts. We therefore suggest that the analgesic action of triptan can be attained specifically in the absence, but not in the presence, of central sensitization.

We tested the expression of genes coding receptors of a cutaneous serotoninergic/melatoninergic system in whole human skin and in normal and pathologic cultured skin cells. Evaluation of serotonin (<em>5HT</em>), melatonin (MT), and melatonin-related receptors (MRR) showed expression of the isoforms <em>5HT</em>2B, <em>5HT</em>7, and MT1 genes in almost all the tested samples. Expression of other isoforms was less prevalent; <em>5HT</em>2C, MRR, and MT2 were rarely detected. We also found novel isoforms for MT2, MRR, and <em>5HT</em>2B and documented the process of RNA editing for <em>5HT</em>2C. Testing for functional activity of these receptors with serotonin and melatonin (10(-14) to 10(-10) M) showed variable effects depending on cell type and culture conditions. Thus, serotonin stimulated proliferation of melanocytes in medium deprived of growth factors, while inhibiting cell growth in the presence of growth factors. Melatonin inhibited both apoptosis of HaCaT keratinocytes incubated in serum-free media, and proliferation of cells cultured in medium supplemented with serum. Melatonin also increased the numbers of viable fibroblasts incubated in serum free medium. N-acetylserotonin (NAS) and 5 methoxytryptamine (5MTT) were generally without effect on cell proliferation, with the exception of an inhibition of melanocyte proliferation at the higher 5MTT concentration of 10(-10) M. Thus, skin cells represent a true target for the products of the serotoninergic/melatoninergic cutaneous pathway with their actions modulating cell proliferation or viability.

RNA transcripts encoding the 2C-subtype of serotonin (<em>5HT</em>(2C)) receptor undergo up to five adenosine-to-inosine editing events to encode twenty-four protein isoforms. To examine the effects of altered <em>5HT</em>(2C) editing in vivo, we generated mutant mice solely expressing the fully-edited (VGV) isoform of the receptor. Mutant animals present phenotypic characteristics of Prader-Willi syndrome (PWS) including a failure to thrive, decreased somatic growth, neonatal muscular hypotonia, and reduced food consumption followed by post-weaning hyperphagia. Though previous studies have identified alterations in both <em>5HT</em>(2C) receptor expression and <em>5HT</em>(2C)-mediated behaviors in both PWS patients and mouse models of this disorder, to our knowledge the <em>5HT</em>(2C) gene is the first locus outside the PWS imprinted region in which mutations can phenocopy numerous aspects of this syndrome. These results not only strengthen the link between the molecular etiology of PWS and altered <em>5HT</em>(2C) expression, but also demonstrate the importance of normal patterns of <em>5HT</em>(2C) RNA editing in vivo.