Aminergic signaling pathways have been implicated in a variety of neuropsychiatric illnesses, but the mechanisms by which these pathways influence complex behavior remain obscure. Vesicular monoamine transporters (VMATs) have been shown to regulate the amount of monoamine neurotransmitter that is stored and released from synaptic vesicles in mammalian systems, and an increase in their expression has been observed in bipolar patients. The model organism Drosophila melanogaster provides a powerful, but underutilized genetic system for studying how dopamine (DA) and serotonin (<em>5HT</em>) may influence behavior. We show that a Drosophila isoform of VMAT (DVMAT-A) is expressed in both dopaminergic and serotonergic neurons in the adult Drosophila brain. Overexpression of DVMAT-A in these cells potentiates stereotypic grooming behaviors and locomotion and can be reversed by reserpine, which blocks DVMAT activity, and haloperidol, a DA receptor antagonist. We also observe a prolongation of courtship behavior, a decrease in successful mating and a decrease in fertility, suggesting a role for aminergic circuits in the modulation of sexual behaviors. Finally, we find that DMVAT-A overexpression decreases the fly's sensitivity to cocaine, suggesting that the synaptic machinery responsible for this behavior may be downregulated. DVMAT transgenes may be targeted to additional neuronal pathways using standard Drosophila techniques, and our results provide a novel paradigm to study the mechanisms by which monoamines regulate complex behaviors relevant to neuropsychiatric illness.

It has become increasingly apparent that Parkinson's disease involves many transmitter systems other than dopamine. This nondopaminergic involvement impacts on the generation of symptoms, on the neurodegenerative process, but, most tellingly, in the generation of side effects of current treatments, in particular, levodopa-induced dyskinesia (LID). Such mechanisms contribute not only to the expression of LID once it has been established but also to the mechanisms responsible for the development, or priming, of the dyskinetic state and the subsequent maintenance of the brain in that primed state. Within the basal ganglia, abnormalities in different nondopaminergic components of the circuitry have been defined in LID. In particular, a role for enhanced inhibition of basal ganglia outputs by the GABAergic direct pathway has been suggested as a basic mechanism generating LID. We speculate that the external globus pallidus and subthalamic nucleus may play distinct roles in different forms of dyskinesia, e.g., chorea/dystonia; peak/diphasic/off. At the cellular level, an appreciation of abnormal signaling by, among others, glutamatergic (NMDA and AMPA receptors in particular), alpha2 adrenergic, serotonergic (<em>5HT</em>), cannabinoid and opioid mechanisms in both priming and expression of LID has begun to emerge over the last decade. This is being consolidated, though in many cases questions remain regarding the specific sites of such abnormality within the circuitry. Very recently, at the molecular level, mechanisms controlling neurotransmitter release and impacting on the ability of neurons to maintain particular forms of firing patterning and synchronization, e.g., SV2A, have been identified. This increased understanding has already delivered and will continue to define novel approaches to treatment that target both pre- and postsynaptic signaling molecules throughout the basal ganglia circuitry.

Drugs that target the serotonergic system are the most commonly prescribed therapeutic agents and are used for treatment of a wide range of behavioral and neurological disorders. However, the mechanism of the drug action remain a conjecture. Here, we dissect the genetic targets of serotonin (<em>5HT</em>), the selective <em>5HT</em> reuptake inhibitor (SSRI) fluoxetine (Prozac), the tricyclic antidepressant imipramine, and dopamine. Using the well-established serotonergic response in C. elegans egg-laying behavior as a paradigm, we show that action of fluoxetine and imipramine at the <em>5HT</em> reuptake transporter (SERT) and at <em>5HT</em> receptors are separable mechanisms. Even mutants completely lacking <em>5HT</em> or SERT can partially respond to fluoxetine and imipramine. Furthermore, distinct mechanisms for each drug can be recognized to mediate these responses. Deletion of SER-1, a <em>5HT</em>1 receptor, abolishes the response to <em>5HT</em> but has only a minor effect on the response to imipramine and no effect on the response to fluoxetine. In contrast, deletion of SER-4, a <em>5HT</em>2 receptor, confers significant resistance to imipramine while leaving the responses to <em>5HT</em> or fluoxetine intact. Further, fluoxetine can stimulate egg laying via the Gq protein EGL-30, independent of SER-1, SER-4, or <em>5HT</em>. We also show that dopamine antagonizes the <em>5HT</em> action via the <em>5HT</em>-gated ion channel MOD-1 signaling, suggesting that this channel activity couples <em>5HT</em> and dopamine signaling. These results suggest that the actions of these drugs at specific receptor subtypes could determine their therapeutic efficacy. SSRIs and tricyclic antidepressants may regulate <em>5HT</em> outputs independently of synaptic levels of <em>5HT</em>.

Meta-chlorophenylpiperazine inhibited serotonin and noradrenaline uptake by synaptosomes to the same extent with IC50 of 1.3 x 10(-6) M and 5.8 x 10(-6) M respectively. Dopamine uptake was less affected by meta-chlorophenylpiperazine (IC50 of 2.2 x 10(-5) M). Unlike d-amphetamine and d-fenfluramine, the drug did not significantly increase monoamine release in synaptosomal preparations. On the other hand, metachlorophenylpiperazine showed an IC50 of 620 nM in displacing 3H-<em>5HT</em> binding to brain membranes. Meta-chlorophenylpiperazine produced a dose-dependent reduction of food intake and this effect was prevented by a pretreatment with methergoline, a serotonin antagonist. The effect of metachlorophenylpiperazine was not modified by an intraventricular injection of 6-hydroxydopamine, electrolytic lesions of nucleus medianus raphe or ventral noradrenergic bundle, nor by a pretreatment with penfluridol, propranolol or phentolamine. The data suggest that the decrease of food intake induced by metachlorophenylpiperazine depends on its ability to act as a serotonin agonist is the brain. The specificity of the effects on serotonin suggests that this compound could prove an important tool for studies aimed at elucidating the functional role of serotonin in the central nervous system.

Schizophrenia is a disorder of a neurodevelopmental origin manifested symptomatically after puberty. Structural neuroimaging studies show that neuroanatomical aberrations precede onset of symptoms, raising a question of whether schizophrenia can be prevented. Early treatment with atypical antipsychotics may reduce the risk of transition to psychosis, but it remains unknown whether neuroanatomical abnormalities can be prevented. We have recently shown, using in vivo structural magnetic resonance imaging, that treatment with the atypical antipsychotic clozapine during an asymptomatic period of adolescence prevents the emergence of schizophrenia-like brain structural abnormalities in adult rats exposed to prenatal immune challenge, in parallel to preventing behavioral abnormalities. Here we assessed the preventive efficacy of the atypical antipsychotic risperidone (RIS). Pregnant rats were injected on gestational day 15 with the viral mimic polyriboinosinic-polyribocytidylic acid (poly I:C) or saline. Their male offspring received daily RIS (0.045 or 1.2 mg/kg) or vehicle injection in peri-adolescence (postnatal days [PND] 34-47). Structural brain changes and behavior were assessed at adulthood (from PND 90). Adult offspring of poly I:C-treated dams exhibited hallmark structural abnormalities associated with schizophrenia, enlarged lateral ventricles and smaller hippocampus. Both of these abnormalities were absent in the offspring of poly I:C dams that received RIS at peri-adolescence. This was paralleled by prevention of schizophrenia-like behavioral abnormalities, attentional deficit, and hypersensitivity to amphetamine in these offspring. We conclude that pharmacological intervention during peri-adolescence can prevent the emergence of behavioral abnormalities and brain structural pathology resulting from in utero insult. Furthermore, highly selective <em>5HT</em>(2A) receptor antagonists may be promising targets for psychosis prevention.

Patients with postinfective irritable bowel syndrome and Trichinella spiralis-infected mice share many features including visceral hypersensitivity and disordered motility. We assessed enterochromaffin (EC) numbers and serotonin transporter (SERT) using National Institute of Health (NIH) female mice studied for up to 56 days post-T. spiralis infection. The effects of steroid treatment and the T-cell dependence of the observed responses were assessed by infection of hydrocortisone-treated or T-cell receptor knock out [TCR (betaxdelta) KO] animals. Enterochromaffin cell density in uninfected animals increased from duodenum 10.0 cells mm-2 (5.9-41.0) to colon 61.8. (46.3-162) cells mm-2 P<0.0001. Infection increased duodenal and jejunal counts which rose to 37.3 (22-57.7) cells mm-2 and 50.6 (7-110.8) cells mm-2, respectively, at day 14. Infection significantly reduced jejunal SERT expression, with luminance values falling from 61.0 (45.1-98.3) to a nadir of 11.6 (0-36.0) units at day 9, P<0.001. Specific deficiencies in all T cells reduced EC hyperplasia and abrogated infection-induced mastocytosis. Thus infection induced inflammation increases EC numbers, as has been reported in PI-IBS, and reduces SERT. This may increase mucosal <em>5HT</em> availability and contribute to the clinical presentation of PI-IBS.

The rostroventromedial medulla (RVM) is an important source of descending modulatory systems that both inhibit and facilitate pain at the level of the spinal cord. Noxious stimuli can activate serotonergic neurons in the RVM and accelerate the turnover of 5-HT in the spinal cord. While numerous studies suggest a bidirectional role for serotonergic transmission at the spinal level, the subtypes of the 5-HT receptors that are associated with descending facilitation or inhibition have not been clearly determined. Here, we explore the relative contribution of spinal 5-HT7 and 5-HT3 receptors to antinociception or hyperalgesia associated with states of enhanced net descending inhibition or facilitation from the RVM. In uninjured rats, RVM microinjection of morphine produced dose-dependent antinociception in the noxious thermal paw flick test while RVM microinjection of CCK produced thermal hyperalgesia and tactile allodynia. Spinal administration of the 5-HT7 antagonist SB-269970, but not of the 5-HT3 antagonist ondansetron, blocked the antinociceptive effects of RVM morphine. In contrast, hyperalgesia induced by RVM-CCK was blocked by spinal ondansetron, but not by SB-269970. The antinociceptive effects of systemic morphine were also blocked by spinal SB-269970 but not ondansetron while hyperalgesia and allodynia resulting from SNL injury were blocked by spinal ondansetron, but not SB-269970. These studies suggest that descending pain inhibitory or facilitatory pathways from RVM act ultimately in the spinal cord in acute and chronic pain states through activation of 5-HT7 and 5-HT3 receptors, respectively.

People with schizophrenia often have substantial cognitive impairments, which may be related to nicotinic receptor deficits, (alpha7 and alpha4beta2), documented in the brains of people with schizophrenia. The large majority of people with schizophrenia smoke cigarettes. Thus, nicotinic interactions with antipsychotic drugs are widespread. Complementary co-therapies of novel nicotinic ligands are being developed to add to antipsychotic therapy to treat the cognitive impairment of schizophrenia. Thus, it is critical to understand the interaction between nicotinic treatments and antipsychotic drugs. Nicotinic interactions with antipsychotic drugs, are complex since both nicotine and antipsychotics have complex actions. Nicotine stimulates and desensitizes nicotinic receptors of various subtypes and potentiates the release of different neurotransmitters. Antipsychotics also act on a verity of receptor systems. For example, clozapine acts as an antagonist at a variety of neurotransmitter receptors such as those for dopamine, serotonin, norepinepherine and histamine. In a series of studies, we have found that in normally functioning rats, moderate doses of clozapine impair working memory and that clozapine blocks nicotine-induced memory and attentional improvement. Clozapine and nicotine can attenuate each other's beneficial effects in reversing the memory impairment caused by the psychototmimetic drug dizocilpine. A key to the clozapine-induced attenuation of nicotine-induced cognitive improvement appears to be its <em>5HT</em>(2) antagonist properties. The selective <em>5HT</em>(2) antagonist ketanserin has a similar action of blocking nicotine-induced memory and attentional improvements. It is important to consider the interactions between nicotinic and antipsychotic drugs to develop the most efficacious treatment for cognitive improvement in people with schizophrenia.

BACKGROUND

Inflammation undermines the stability of atherosclerotic plaques, rendering them susceptible to acute rupture, the cataclysmic event that underlies clinical expression of this disease. Myeloperoxidase is a central inflammatory enzyme secreted by activated macrophages and is involved in multiple stages of plaque destabilization and patient outcome. We report here that a unique functional in vivo magnetic resonance agent can visualize myeloperoxidase activity in atherosclerotic plaques in a rabbit model.

RESULTS

We performed magnetic resonance imaging of the thoracic aorta of New Zealand White rabbits fed a cholesterol (n=14) or normal (n=4) diet up to 2 hours after injection of the myeloperoxidase sensor bis-<em>5HT</em>-DTPA(Gd) [MPO(Gd)], the conventional agent DTPA(Gd), or an MPO(Gd) analog, bis-tyr-DTPA(Gd), as controls. Delayed MPO(Gd) images (2 hours after injection) showed focal areas of increased contrast (>2-fold) in diseased wall but not in normal wall (P=0.84) compared with both DTPA(Gd) (n=11; P<0.001) and bis-tyr-DTPA(Gd) (n=3; P<0.05). Biochemical assays confirmed that diseased wall possessed 3-fold elevated myeloperoxidase activity compared with normal wall (P<0.01). Areas detected by MPO(Gd) imaging colocalized and correlated with myeloperoxidase-rich areas infiltrated by macrophages on histopathological evaluations (r=0.91, P<0.0001). Although macrophages were the main source of myeloperoxidase, not all macrophages secreted myeloperoxidase, which suggests that distinct subpopulations contribute differently to atherogenesis and supports our functional approach.

CONCLUSIONS

The present study represents a unique approach in the detection of inflammation in atherosclerotic plaques by examining macrophage function and the activity of an effector enzyme to noninvasively provide both anatomic and functional information in vivo.

The neurotoxic actions of methamphetamine (METH) may be mediated in part by reactive oxygen species (ROS). Methamphetamine administration leads to increases in ROS formation and lipid peroxidation in rodent brain; however, the extent to which proteins may be modified or whether affected brain regions exhibit similar elevations of lipid and protein oxidative markers have not been investigated. In this study we measured concentrations of TBARs, protein carbonyls and monoamines in various mouse brain regions at 4 h and 24 h after the last of four injections of METH (10 mg/kg/injection q 2 h). Substantial increases in TBARs and protein carbonyls were observed in the striatum and hippocampus but not the frontal cortex nor the cerebellum of METH-treated mice. Furthermore, lipid and protein oxidative markers were highly correlated within each brain region. In the hippocampus and striatum elevations in oxidative markers were significantly greater at 24 h than at 4 h. Monoamine levels were maximally reduced within 4 h (striatal dopamine [DA] by 95% and serotonin [5-HT] in striatum, cortex and hippocampus by 60-90%). These decrements persisted for 7 days after METH, indicating effects reflective of nerve terminal damage. Interestingly, NE was only transiently depleted in the brain regions investigated (hippocampus and cortex), suggesting a pharmacological and non-toxic action of METH on the noradrenergic nerve terminals. This study provides the first evidence for concurrent formation of lipid and protein markers of oxidative stress in several brain regions of mice that are severely affected by large neurotoxic doses of METH. Moreover, the differential time course for monoamine depletion and the elevations in oxidative markers indicate that the source of oxidative stress is not derived directly from DA or <em>5HT</em> oxidation.

The mechanisms underlying the neuroprotective effects of cannabidiol (CBD) were studied in vivo using a hypoxic-ischemic (HI) brain injury model in newborn pigs. One- to two-day-old piglets were exposed to HI for 30 min by interrupting carotid blood flow and reducing the fraction of inspired oxygen to 10%. Thirty minutes after HI, the piglets were treated with vehicle (HV) or 1 mg/kg CBD, alone (HC) or in combination with 1 mg/kg of a CB₂ receptor antagonist (AM630) or a serotonin <em>5HT</em>(1A) receptor antagonist (WAY100635). HI decreased the number of viable neurons and affected the amplitude-integrated EEG background activity as well as different prognostic proton-magnetic-resonance-spectroscopy (H(±)-MRS)-detectable biomarkers (lactate/N-acetylaspartate and N-acetylaspartate/choline ratios). HI brain damage was also associated with increases in excitotoxicity (increased glutamate/N-acetylaspartate ratio), oxidative stress (decreased glutathione/creatine ratio and increased protein carbonylation) and inflammation (increased brain IL-1 levels). CBD administration after HI prevented all these alterations, although this CBD-mediated neuroprotection was reversed by co-administration of either WAY100635 or AM630, suggesting the involvement of CB₂ and <em>5HT</em>(1A) receptors. The involvement of CB₂ receptors was not dependent on a CBD-mediated increase in endocannabinoids. Finally, bioluminescence resonance energy transfer studies indicated that CB₂ and <em>5HT</em>(1A) receptors may form heteromers in living HEK-293T cells. In conclusion, our findings demonstrate that CBD exerts robust neuroprotective effects in vivo in HI piglets, modulating excitotoxicity, oxidative stress and inflammation, and that both CB₂ and <em>5HT</em>(1A) receptors are implicated in these effects.

For many populations of estrogen-sensitive neurons it remains unknown how they are associated with central nervous system circuitries that mediate estrogen-induced modulation of behavioral components. With the use of double-labeling immunohistochemistry and tracing techniques, the relationships of estrogen receptor (ER)-alpha- and ER-beta-immunoreactive (IR) neurons in the mouse brainstem and spinal cord to monoaminergic, cholinergic, and spinal projection systems are explored. Similar distributions of ER-IR neurons were present in females and males, with differences in labeling intensity of ER-alpha immunoreactivity among males and estrogen-, and oil-treated females. Barrington's nucleus, the ventrolateral medulla, and the nucleus of the solitary tract contained spinal-projecting ER-alpha-IR neurons, whereas ER-alpha-IR neurons in the periaqueductal gray, parabrachial nucleus, and catecholaminergic A1 cell group received spinal input. Numerous tyrosine hydroxylase (TH)-IR ER-alpha-IR neurons were present in the ventral periaqueductal gray, nucleus of the solitary tract, A1 cell group, and lumbosacral cord. The dorsal raphe nucleus contained ER-alpha-IR and ER-beta-IR neurons that colocalized with serotonin (<em>5HT</em>), and the reticulotegmental nucleus contained <em>5HT</em>-IR ER-alpha-IR neurons. Fibers IR for vesicular acetylcholine transporter (VAChT), TH, and <em>5HT</em> were located among ER-alpha-IR neurons in the dorsal horn and spinal autonomic regions. Robust staining for TH and VAChT, but not <em>5HT</em>, was present among ER-alpha-IR neurons in the lumbosacral lateral collateral pathway. Possible modulatory actions of estrogen on each of these ER-IR populations are discussed in the context of their specific function, including micturition, sexual behavior, ejaculation, cardiovascular and respiratory control, tactile and nociceptive sensory processing, anti-nociception, endocrine regulation, and feeding.

Reactive oxygen species decrease dopamine transporter (DAT) function in vitro. Because of this, and the finding that METH administration causes oxygen radical formation in vivo, the effects of METH administration on DAT activity in rat striatum were investigated. A single METH injection caused a dose-dependent (0-15 mg/kg) decrease in [3H]dopamine uptake into striatal synaptosomes prepared 1 h after METH administration; an effect attributable to a decreased Vmax of [3H]dopamine uptake. Similarly, multiple high-dose administrations of METH (10 mg/kg/dose; four doses at 2-h intervals) decreased DAT function. The decreases in DAT activity after either single or multiple METH administrations were reversed 24 h after treatment. [3H]<em>5HT</em> transport into striatal synaptosomes was also affected by METH treatment. Taken together, these data suggest that METH decreases DAT activity, perhaps through a reactive oxygen species-mediated mechanism. These findings may have important implications regarding the role of oxidative events in the physiological regulation of monoaminergic systems.

To explore the potential for use of ligand-conjugated nanocrystals to target cell surface receptors, ion channels, and transporters, we explored the ability of serotonin-labeled CdSe nanocrystals (SNACs) to interact with antidepressant-sensitive, human and Drosophila serotonin transporters (hSERT, dSERT) expressed in HeLa and HEK-293 cells. Unlike unconjugated nanocrystals, SNACs were found to dose-dependently inhibit transport of radiolabeled serotonin by hSERT and dSERT, with an estimated half-maximal activity (EC(50)) of 33 (dSERT) and 99 microM (hSERT). When serotonin was conjugated to the nanocrystal through a linker arm (LSNACs), the EC(50) for hSERT was determined to be 115 microM. Electrophysiology measurements indicated that LSNACs did not elicit currents from the serotonin-3 (<em>5HT</em>(3)) receptor but did produce currents when exposed to the transporter, which are similar to those elicited by antagonists. Moreover, fluorescent LSNACs were found to label SERT-transfected cells but did not label either nontransfected cells or transfected cells coincubated with the high-affinity SERT antagonist paroxetine. These findings support further consideration of ligand-conjugated nanocrystals as versatile probes of membrane proteins in living cells.

BACKGROUND

Menstrually associated migraine (MAM) is often prolonged and difficult to manage with conventional therapies. Frovatriptan is a new selective <em>5HT</em>(1B/1D) receptor agonist indicated for short-term management of migraine. It has a long half-life and good tolerability. These characteristics suggest that frovatriptan may be useful for the intermittent prevention of MAM.

METHODS

The study was a randomized, double-blind, placebo-controlled, three-way crossover design. Patients treated each of three perimenstrual periods (PMPs) with placebo, frovatriptan 2.5 mg QD, and frovatriptan 2.5 mg BID. The 6-day treatment started 2 days before the anticipated start of MAM headache. The primary efficacy endpoint was incidence of MAM headache during the 6-day PMP.

RESULTS

The population comprised 546 women (mean age, 37.6 years). Use of frovatriptan reduced the occurrence of MAM headache. The incidence of MAM headache during the 6-day PMP was 67% for placebo, 52% for frovatriptan 2.5 mg QD, and 41% for frovatriptan 2.5 mg BID. Both frovatriptan regimens were superior to placebo (p < 0.0001), and the BID regimen was superior to the QD regimen (p < 0.001). Both frovatriptan regimens also reduced MAM severity (p < 0.0001), duration (p < 0.0001), and the use of rescue medication (p < 0.01 QD; p < 0.0001 BID) in a dose-dependent manner. The incidence and type of adverse events for both regimens were similar to placebo and consistent with those reported for short-term migraine management.

CONCLUSIONS

Frovatriptan given prophylactically for 6 days was effective in reducing the incidence of menstrually associated migraine. More than half of patients who used frovatriptan 2.5 mg BID had no menstrually associated migraine headache during the 6-day perimenstrual period. The findings are consistent with the long duration of action and good tolerability of frovatriptan observed in short-term migraine management.

Methylenedioxymethamphetamine (MDMA, Ecstasy) is a popular recreationally used drug among young people in Europe and North America. The recent surge in use of MDMA and increasing concerns about possible toxic effects of the drug have inspired a great deal of research into the mechanisms by which the drug may affect the central nervous system. This paper reviews studies on the neurochemical, behavioral and neurophysiological effects of MDMA, with emphasis on MDMA effects in regions of the brain that have been implicated in reward. Experiments in awake, behaving laboratory animals have demonstrated that single injections of MDMA increase extracellular levels of the neurotransmitters dopamine (DA) and serotonin (<em>5HT</em>) in the nucleus accumbens and in several other brain regions that are important for reward. Most of the behavioral and electrophysiological changes that have been reported to date for single doses of MDMA appear to be mediated by this MDMA-induced increase in extracellular DA and <em>5HT</em>. As an example, MDMA-induced hyperthermia and locomotor hyperactivity in laboratory animals can be blocked by administering drugs that prevent MDMA-induced <em>5HT</em> release and can be attenuated by administering <em>5HT</em> receptor antagonists, whereas effects of MDMA on delayed reinforcement tasks appear to be mediated by MDMA-induced increases in extracellular DA. Similarly, the effects of MDMA on neuronal excitability in the nucleus accumbens and in several other brain regions can be prevented by administering drugs that block MDMA-induced <em>5HT</em> release and can be attenuated by depleting brain DA levels or by administering either DA D1 receptor antagonists or <em>5HT</em> receptor antagonists. In addition to the acute effects of MDMA, it is now well established that repeated or high-dose administration of MDMA is neurotoxic to a subpopulation of <em>5HT</em>-containing axons that project to the forebrain in laboratory animals. Recent studies have shown that this neurotoxic effect of MDMA is associated with long-duration changes in both DA and <em>5HT</em> neurotransmission in the nucleus accumbens. Whether these long-duration changes in neurotransmission might be related to reports of depression and other psychopathologies by some frequent users of MDMA remains to be determined. Methylene-dioxymethamphetamine has been found to increase extracellular levels of norepinephrine and to alter brain levels of several neuropeptides as well as altering levels of DA and <em>5HT</em>. Much additional research is required to understand the multiple ways in which this complex drug may alter neurotransmission in the brain, both acutely and in the long term.

Bimatoprost (Lumigan) is a pharmacologically unique and highly efficacious ocular hypotensive agent. It appears to mimic the activity of a newly discovered family of fatty acid amides, termed prostamides. One biosynthetic route to the prostamides involves anandamide as the precursor. Bimatoprost pharmacology has been extensively characterized by binding and functional studies at more than 100 drug targets, which comprise a diverse variety of receptors, ion channels, and transporters. Bimatoprost exhibited no meaningful activity at receptors known to include antiglaucoma drug targets as follows: adenosine (A(1-3)), adrenergic (alpha(1), alpha(2), beta(1), beta(2)), cannabinoid (CB(1), CB(2)), dopamine (D(1-5)), muscarinic (M(1-5)), prostanoid (DP, EP(1-4), FP, IP, TP), and serotonin (<em>5HT</em>(1-7)). Bimatoprost does, however, exhibit potent inherent pharmacological activity in the feline iris sphincter preparation, which is prostamide-sensitive. Bimatoprost also resembles the prostamides in that it is a potent and highly efficacious ocular hypotensive agent. A single dose of bimatoprost markedly reduces intraocular pressure in dogs and laser-induced ocular hypertensive monkeys. Decreases in intraocular pressure are well maintained for at least 24 hr post-dose. Human studies have demonstrated that systemic exposure to bimatoprost is low and that accumulation does not occur. The sclera is the preferred route of accession to the eye. The high scleral permeability coefficient Papp is a likely contributing factor to the rapid onset and long-acting ocular hypotensive profile of bimatoprost.

Stress response is a fundamental form of behavioral and physiological plasticity. Here we describe how serotonin (<em>5HT</em>) governs stress behavior by regulating DAF-2 insulin/IGF-1 receptor signaling to the DAF-16/FOXO transcription factor at the nexus of development, metabolism, immunity, and stress responses in C. elegans. Serotonin-deficient tph-1 mutants, like daf-2 mutants, exhibit DAF-16 nuclear accumulation and constitutive physiological stress states. Exogenous <em>5HT</em> and fluoxetine (Prozac) prevented DAF-16 nuclear accumulation in wild-type animals under stresses. Genetic analyses imply that DAF-2 is a downstream target of <em>5HT</em> signaling and that distinct serotonergic neurons act through distinct <em>5HT</em> receptors to influence distinct DAF-16-mediated stress responses. We suggest that modulation of FOXO by <em>5HT</em> represents an ancient feature of stress physiology and that the C. elegans is a genetically tractable model that can be used to delineate the molecular mechanisms and drug actions linking <em>5HT</em>, neuroendocrine signaling, immunity, and mitochondrial function.

We have utilized RN46A cells, an immortalized neuronal cell line derived from E13 brainstem raphe, as a model for transplant of bioengineered serotonergic cells. RN46A cells require brain-derived neurotrophic factor (BDNF) for increased survival and serotonin (<em>5HT</em>) synthesis in vitro and in vivo. RN46A cells were transfected with the rat BDNF gene, and the 46A-B14 cell line was subcloned. These cells survive longer than 7 weeks after transplantation into the subarachnoid space of the lumbar spinal cord and synthesize <em>5HT</em> and BDNF. Chronic constriction injury (CCI) of the sciatic nerve was used to induce chronic neuropathic pain in the affected hindpaw in rats. Transplants of 46A-B14 cells placed 1 week after CCI alleviated chronic neuropathic pain, while transplants of 46A-V1 control cells, negative for <em>5HT</em> and without the BDNF gene, had no effect on the induction of thermal and tactile nociception. When endogenous cells of the dorsal horn which contain the neurotransmitter gamma-aminobutyric acid (GABA) and its synthetic enzyme glutamate decarboxylase (GAD) were immunohistochemically quantified in the lumbar spinal cord 3 days and 1-8 weeks after CCI, the number of GABA- and GAD-immunoreactive (ir) cells decreased bilateral to the nerve injury as soon as 3 days after CCI. At 1 week after CCI, the number of GABA-ir cells continued to significantly decline bilaterally, returning to near normal numbers on the side contralateral to the nerve injury by 8 weeks after the nerve injury. The number of GAD-ir cells began to increase bilaterally to the nerve injury at 1 week after CCI and continued to significantly increase in numbers over normal values by 8 weeks after the nerve injury. When examined 2 and 8 weeks after CCI plus cell transplants, the transplants of 46A-B14 cells reversed the increase in GAD-ir cell numbers and the decrease in GABA-ir cells by 1 week after transplantation, while 46A-V1 control cell transplants after CCI had no effect on the changes in numbers of GAD-ir or GABA-ir cells. Collectively, these data suggest that altered <em>5HT</em> levels, and perhaps BDNF secretion, related to the transplants ameliorate chronic pain and reverse the induction and maintenance of an endogenous pain mechanism in the dorsal horn. This induction mechanism is likely dependent on altered GAD regulation and GABA synthesis, initiated by CCI.

Mutation of a conserved Asp (D98) in the rat serotonin (<em>5HT</em>) transporter (rSERT) to Glu (D98E) led to decreased <em>5HT</em> transport capacity, diminished coupling to extracellular Na+ and Cl-, and a selective loss of antagonist potencies (cocaine, imipramine, and citalopram but not paroxetine or mazindol) with no change in <em>5HT</em> Km value. D98E, which extends the acidic side chain by one carbon, affected the rank-order potency of substrate analogs for inhibition of <em>5HT</em> transport, selectively increasing the potency of two analogs with shorter alkylamine side chains, gramine, and dihydroxybenzylamine. D98E also increased the efficacy of gramine relative to <em>5HT</em> for inducing substrate-activated currents in Xenopus laevis oocytes, but these currents were noticeably dependent on extracellular medium acidification. I-V profiles for substrate-independent and -dependent currents indicated that the mutation selectively impacts ion permeation coupled to <em>5HT</em> occupancy. The ability of the D98E mutant to modulate selective aspects of substrate recognition, to perturb ion dependence as well as modify substrate-induced currents, suggests that transmembrane domain I plays a critical role in defining the permeation pathway of biogenic amine transporters.

BACKGROUND

Descending facilitation, from the brainstem, promotes spinal neuronal hyperexcitability and behavioural hypersensitivity in many chronic pain states. We have previously demonstrated enhanced descending facilitation onto dorsal horn neurones in a neuropathic pain model, and shown this to enable the analgesic effectiveness of gabapentin. Here we have tested if this hypothesis applies to other pain states by using a combination of approaches in a rat model of osteoarthritis (OA) to ascertain if 1) a role for descending <em>5HT</em> mediated facilitation exists, and 2) if pregabalin (a newer analogue of gabapentin) is an effective antinociceptive agent in this model. Further, quantitative-PCR experiments were undertaken to analyse the alpha 2 delta-1 and 5-HT3A subunit mRNA levels in L3-6 DRG in order to assess whether changes in these molecular substrates have a bearing on the pharmacological effects of ondansetron and pregabalin in OA.

RESULTS

Osteoarthritis was induced via intra-articular injection of monosodium iodoacetate (MIA) into the knee joint. Control animals were injected with 0.9% saline. Two weeks later in vivo electrophysiology was performed, comparing the effects of spinal ondansetron (10-100 microg/50 microl) or systemic pregabalin (0.3 - 10 mg/kg) on evoked responses of dorsal horn neurones to electrical, mechanical and thermal stimuli in MIA or control rats. In MIA rats, ondansetron significantly inhibited the evoked responses to both innocuous and noxious natural evoked neuronal responses, whereas only inhibition of noxious evoked responses was seen in controls. Pregabalin significantly inhibited neuronal responses in the MIA rats only; this effect was blocked by a pre-administration of spinal ondansetron. Analysis of alpha 2 delta-1 and 5-HT3A subunit mRNA levels in L3-6 DRG revealed a significant increase in alpha 2 delta-1 levels in ipsilateral L3&4 DRG in MIA rats. 5-HT3A subunit mRNA levels were unchanged.

CONCLUSIONS

These data suggest descending serotonergic facilitation plays a role in mediating the brush and innocuous mechanical punctate evoked neuronal responses in MIA rats, suggesting an adaptive change in the excitatory serotonergic drive modulating low threshold evoked neuronal responses in MIA-induced OA pain. This alteration in excitatory serotonergic drive, alongside an increase in alpha 2 delta-1 mRNA levels, may underlie pregabalin's state dependent effects in this model of chronic pain.

The serotonin transporter (SERT) on platelets is a primary mechanism for serotonin (<em>5HT</em>) uptake from the blood plasma. Alteration in plasma <em>5HT</em> level is associated with a number of cardiovascular diseases and disorders. Therefore, the regulation of the transporter's activity represents a key mechanism to stabilize the concentration of plasma <em>5HT</em>. There is a biphasic relationship between plasma <em>5HT</em> elevation, loss of surface SERT, and depletion of platelet <em>5HT</em>. Specifically, in platelets, plasma membrane SERT levels and platelet <em>5HT</em> uptake initially rise as plasma <em>5HT</em> levels are increased but then fall below normal as the plasma <em>5HT</em> level continues to rise. Therefore, we propose that elevated plasma <em>5HT</em> limits its own uptake in platelets by down-regulating SERT as well as modifying the characteristics of SERT partners in the membrane trafficking pathway. This review will summarize current findings regarding the biochemical mechanisms by which elevated <em>5HT</em> downregulates the expression of SERT on the platelet membrane. Intriguing aspects of this regulation include the intracellular interplay of SERT with the small G protein Rab4 and the concerted <em>5HT</em>-mediated phosphorylation of vimentin.

Incubation of platelet-rich rabbit plasma with E. coli endotoxin at 37 degrees C results in platelet aggregation and transfer of platelet 5-hydroxytryptamine to plasma. Release of <em>5HT</em> is influenced by dose of endotoxin, type of anticoagulant, and temperature of incubation. A heat-labile plasma factor is necessary for the platelet-endotoxin interaction. Additional studies have shown that incubation of endotoxin with platelet-rich rabbit plasma also results in release of platelet phospholipid and bactericidins active against B. subtilis.

The abuse of volatile organic solvents (inhalants) leads to diverse sequelae at levels ranging from the cell to the whole organism. This paper reviews findings from the last 10 years of animal models investigating the behavioral and mechanistic effects of solvent abuse. In research with animal models of inhalant abuse, NMDA, GABA(A), glycine, nicotine, and <em>5HT</em>(3) receptors appear to be important targets of action for several abused solvents with emerging evidence suggesting that other receptor subtypes and nerve membrane ion channels may be involved as well. The behavioral effects vary in magnitude and duration among the solvents investigated. The behavioral effects of acute and chronic inhalant abuse include motor impairment, alterations in spontaneous motor activity, anticonvulsant effects, anxiolytic effects, sensory effects, and effects on learning, memory and operant behavior (e.g., response rates and discriminative stimulus effects). In addition, repeated exposure to these solvents may produce tolerance, dependence and/or sensitization to these effects.