BACKGROUND

Reconstructing the evolutionary history of nervous systems requires an understanding of their architecture and development across diverse taxa. The spiralians encompass diverse body plans and organ systems, and within the spiralians, annelids exhibit a variety of morphologies, life histories, feeding modes and associated nervous systems, making them an ideal group for studying evolution of nervous systems.

RESULTS

We describe nervous system development in the annelid Capitella teleta (Blake JA, Grassle JP, Eckelbarger KJ. Capitella teleta, a new species designation for the opportunistic and experimental Capitella sp. I, with a review of the literature for confirmed records. Zoosymposia. 2009;2:25-53) using whole-mount in situ hybridization for a synaptotagmin 1 homolog, nuclear stains, and cross-reactive antibodies against acetylated α-tubulin, 5-HT and FMRFamide. Capitella teleta is member of the Sedentaria (Struck TH, Paul C, Hill N, Hartmann S, Hosel C, Kube M, et al. Phylogenomic analyses unravel annelid evolution. Nature. 2011;471:95-8) and has an indirectly-developing, lecithotrophic larva. The nervous system of C. teleta shares many features with other annelids, including a brain and a ladder-like ventral nerve cord with five connectives, reiterated commissures, and pairs of peripheral nerves. Development of the nervous system begins with the first neurons differentiating in the brain, and follows a temporal order from central to peripheral and from anterior to posterior. Similar to other annelids, neurons with serotonin-like-immunoreactivity (<em>5HT</em>-LIR) and FMRFamide-like-immunoreactivity (FMRF-LIR) are found throughout the brain and ventral nerve cord. A small number of larval-specific neurons and neurites are present, but are visible only after the central nervous system begins to form. These larval neurons are not visible after metamorphosis while the rest of the nervous system is largely unchanged in juveniles.

CONCLUSIONS

Most of the nervous system that forms during larvogenesis in C. teleta persists into the juvenile stage. The first neurons differentiate in the brain, which contrasts with the early formation of peripheral, larval-specific neurons found in some spiralian taxa with planktotrophic larvae. Our study provides a clear indication that certain shared features among annelids - e.g., five connectives in the ventral nerve cord - are only visible during larval stages in particular species, emphasizing the need to include developmental data in ancestral character state reconstructions. The data provided in this paper will serve as an important comparative reference for understanding evolution of nervous systems, and as a framework for future molecular studies of development.

Histological and morphological studies indicate that approximately 5% of striatal neurons are cholinergic or γ-aminobutyric acidergic (GABAergic) interneurons (gINs). However, the number of striatal neurons expressing known interneuron markers is too small to account for the entire interneuron population. We therefore studied the serotonin (<em>5HT</em>) receptor 3a-enhanced green fluorescent protein (<em>5HT</em>3a(EGFP)) mouse, in which we found that a large number of striatal gINs are labeled. Roughly 20% of <em>5HT</em>3a(EGFP)-positive cells co-express parvalbumin and exhibit fast-spiking (FS) electrophysiological properties. However, the majority of labeled neurons do not overlap with known molecular interneuron markers. Intrinsic electrical properties reveal at least 2 distinct novel subtypes: a late-spiking (LS) neuropeptide-Y (NPY)-negative neurogliaform (NGF) interneuron, and a large heterogeneous population with several features resembling low-threshold-spiking (LTS) interneurons that do not express somatostatin, NPY, or neuronal nitric oxide synthase. Although the <em>5HT</em>3a(EGFP) NGF and LTS-like interneurons have electrophysiological properties similar to previously described populations, they are pharmacologically distinct. In direct contrast to previously described NPY(+) LTS and NGF cells, LTS-like <em>5HT</em>3a(EGFP) cells show robust responses to nicotine administration, while the <em>5HT</em>3a(EGFP) NGF cell type shows little or no response. By constructing a molecular map of the overlap between these novel populations and existing interneuron populations, we are able to reconcile the morphological and molecular estimates of striatal interneuron numbers.

Peripheral serotonin (<em>5HT</em>) has been implicated in migraine and temporomandibular pain disorders in humans and animal models and yet the mechanism(s) by which <em>5HT</em> evokes pain remains unclear. Trigeminal pain can be triggered by activation of the transient receptor potential V1 channel (TRPV1), expressed by a subset of nociceptive trigeminal ganglia (TG) neurons and gated by capsaicin, noxious heat, and other noxious stimuli. As <em>5HT</em> is released in the periphery during inflammation and evokes thermal hyperalgesia, and TRPV1 is essential for thermal hyperalgesia, we hypothesized that <em>5HT</em> increases the activity of capsaicin-sensitive trigeminal neurons and that this increase can be attenuated by pharmacologically targeting peripheral <em>5HT</em> receptors. TG cultures were pretreated with <em>5HT</em> (10 nM-100 μM), sumatriptan (<em>5HT</em>(1B/1D) agonist), ketanserin (<em>5HT</em>(2A) antagonist), granisetron (<em>5HT</em>(3) antagonist), or vehicle prior to capsaicin (30-50 nM). Single-cell accumulation of intracellular calcium was recorded or calcitonin gene-related peptide (CGRP) release was measured following each treatment. In addition, using in situ hybridization and immunohistochemistry, we detected the colocalization of <em>5HT</em>(1B), <em>5HT</em>(1D), <em>5HT</em>(2A), and <em>5HT</em>(3A), but not <em>5HT</em>(2C) mRNA with TRPV1 in TG cells. <em>5HT</em> pretreatment evoked a significant increase in calcium accumulation in capsaicin-sensitive trigeminal neurons and enhanced capsaicin-evoked CGRP release, but had no significant effect when given alone. Sumatriptan, ketanserin, and granisetron treatment attenuated calcium accumulation and <em>5HT</em> enhancement of capsaicin-evoked CGRP release. Together these results indicate that <em>5HT</em> increases the activity of capsaicin-sensitive peripheral nociceptors, which can be attenuated by pharmacologically targeting peripheral <em>5HT</em> receptors, thereby providing a mechanistic basis for peripheral craniofacial pain therapy.

BACKGROUND

3,4-Methylenedioxymethamphetamine (MDMA) is an amphetamine derivative, which is neurotoxic to both serotonin (<em>5HT</em>) and dopamine (DA) nerve terminals. Previous reports, carried out in rodents and non-human primates, demonstrated neurotoxicity to monoamine axon terminals, although no study has analyzed nigral and striatal cell bodies at the sub-cellular level.

OBJECTIVE

In this study, we examined intrinsic nigral and striatal cells, and PC12 cell cultures to evaluate whether, in mice, MDMA might affect nigral and striatal cell bodies.

METHODS

After administering MDMA, we analyzed effects induced in vivo and in vitro using high-performance liquid chromatography (HPLC) analysis, light- and electron microscopy with immunocytochemistry, and DNA comet assay.

RESULTS

We found that MDMA (5 mg/kg x4, 2 h apart), besides a decrease of nigrostriatal DA innervation and <em>5HT</em> loss, produces neuronal inclusions within nigral and intrinsic striatal neurons consisting of multi-layer ubiquitin-positive whorls extending to the nucleus of the cell. These fine morphological changes are associated with clustering of heat shock protein (HSP)-70 in the nucleus, very close to chromatin filaments. In the same experimental conditions, we could detect oxidation of DNA bases followed by DNA damage. The nature of inclusions was further investigated using PC12 cell cultures.

CONCLUSIONS

The present findings lead to re-consideration of the neurotoxic consequences of MDMA administration. In fact, occurrence of ubiquitin-positive neuronal inclusions and DNA damage both in nigral and striatal cells sheds new light into the fine alterations induced by MDMA, also suggesting the involvement of nuclear and cytoplasmic components of the ubiquitin-proteasome pathway in MDMA toxicity.

OBJECTIVE

The efficacy and tolerability profiles of sumatriptan and other <em>5HT</em>(1B/1D) agonists (triptans) have been well established. However, the determinants for optimal response to sumatriptan are unknown. The Sumatriptan Naratriptan Aggregate Patient (SNAP) database contains data from 128 clinical trials including 28,407 migraine sufferers treating over 130,000 attacks. The authors analyzed these data to identify factors predicting response (headache relief and pain-free response) to sumatriptan.

METHODS

The authors assessed 24 possible univariate predictors of headache response in 3,706 patients (18 years and older) receiving sumatriptan tablets 100 mg or placebo in a double-blind study using recursive partitioning and logistic regression techniques.

RESULTS

The authors found seven predictors of headache relief 2 hours postdose. Moderate pain at baseline was the strongest predictor (adjusted p = 3.32 x 10(-35)), followed by absence of a disability requiring bedrest (adjusted p = 3.11 x 10(-18)). Other predictors included absence at baseline of vomiting, pulsating pain, nausea, or photophobia/phonophobia, and onset of headache during daytime hours. Logistic regression confirmed that treatment with sumatriptan was the strongest predictor of headache relief, with significant baseline covariates being pain severity, level of disability, and presence or absence of vomiting. A similar pattern of results was reported for predictors of pain-free response 2 hours after taking sumatriptan.

CONCLUSIONS

Pretreatment pain severity is the most important predicting factor for response to sumatriptan in migraine attacks: the lower baseline severity, the better.

OBJECTIVE

To determine the relative density and tissue localization of mRNAs for serotonin (5-hydroxytryptamine; <em>5HT</em>) receptor subtypes in human ocular tissues and correlate with their possible functions in the eye.

METHODS

Total RNA was extracted from human ocular tissues samples from multiple donors and transcribed into cDNA. An optimized reverse transcriptase polymerase chain reaction (RT-PCR) procedure was then used to amplify the signals using primers designed against human <em>5HT</em> receptor cDNAs. The PCR products were analyzed by gel electrophoresis and confirmed by endonuclease digestion.

RESULTS

Variable amounts of total RNA were extracted from different tissues with the least amount from ciliary epithelium and most amount from retina. <em>5HT</em>2 receptor subtype mRNAs were the most abundant with <em>5HT</em>2A and <em>5HT</em>2B being the most predominant in the retina, ciliary body, ciliary epithelium, choroid, conjunctiva, and iris. Ciliary body, choroid, and conjunctiva were most enriched in <em>5HT</em>3 receptor mRNA, with relatively lower levels in the iris. <em>5HT</em>4 receptor mRNA was most enriched in the retina, ciliary body, choroid, conjunctiva, and somewhat detectable in the iris. <em>5HT</em>5 receptor mRNA was abundant in the retina, ciliary body, and iris. <em>5HT</em>6 receptor mRNA was the least abundant of all subtypes studied and could only be detected in the iris. <em>5HT</em>7 receptor mRNA was enriched in the ciliary body, choroid, conjunctiva, and iris, with much lower levels in the retina and ciliary epithelium. Optic nerve tissue of 1-2 donors exhibited the presence of <em>5HT</em>2B, <em>5HT</em>5, and <em>5HT</em>7 receptor mRNAs. Data for human trabecular meshwork cells indicated a high density of mRNAs for <em>5HT</em>2A and <em>5HT</em>2B, with much lower levels of <em>5HT</em>2C, <em>5HT</em>5, and <em>5HT</em>7 receptor mRNAs.

CONCLUSIONS

Human ocular tissues differentially expressed mRNAs for the various <em>5HT</em> receptor subtypes. These studies suggest a diverse range of possible physiological and pharmacological functions of <em>5HT</em> receptors in these human ocular tissues.

erection is regulated by several neurotransmitters and neuropeptides at penile tissue and central nervous system levels. At penile level, the key event is the relaxation of corpora cavernosa smooth muscles. Here, three kinds of neural autonomic control have been characterized in detail. one adrenergic stimulatory, that under normal conditions maintains the corpora cavernosa contracted (that is a flaccid penis), a second cholinergic inhibitory that is believed to cooperate with a third, nonadrenergic-noncholinergic control also inhibitory, possibly mediated by nitric oxide (NO), to reduce the adrenergic tone favouring the relaxation of corpora cavernosa, as during a sexual stimulus. However, the complex interactions between these neurotransmitters that determine the final condition of the corpora cavernosa, e.g. the presence or the absence of penile erection, are still a matter of controversy. This is further complicated by the presence of several neuropeptides in nervous penile vascular and smooth muscle tissues such as vasoactive intestinal polypeptide, peptide histidine- isoleucine, peptide histidine-methionine, neuropeptide Y and endothelins,that often exert very potent (relaxant or contractant) effects in penile tissues. Also at the central level, several neurotransmitters and neuropeptides that influence penile erection have been identified. Among neurotransmitters, the most studied are dopamine (DA), serotonin (SHT), acetylcholine (ACh), glutamic acid and NO. DA, ACh. glutamic acid and NO seem to have a facilitatory role, while <em>5HT</em> may be either facilitatory or inhibitory, depending on the receptor subtype involved. Among neuropeptides, the best known are oxytocin, adrenocorticotropin (ACTH)-cc-melanocyte stimulating hormone (r-MSH)-related peptides and opioid peptides. Interestingly DA, glutamic acid and NO seem to facilitate while opioid peptides inhibit penile erection by increasing and decreasing, respectively, central oxytocinergic transmission by acting in the paraventricular nucleus of the hypothalamus. ACTH-MSH peptides also facilitate penile erection, although with a mechanism(s) different from those recalled above. Despite some recent progress, more has still to be done to clarify the role played by neurotransmitters and neuropeptides at peripheral and central levels in the control of this primary sexual function.

We have investigated if treatment with two different PAHs such as naphthalene (NAP) and benzo(a)pyrene (BaP), and the PAH-like compound beta-naphthoflavone (BNF), may modify the stress responses elicited in rainbow trout by acute or prolonged stress stimuli, and the possible involvement of brain monoamines in those responses. Two experiments (acute and prolonged stress) were performed. In the acute stress experiment, fish were i.p. injected with vegetable oil alone (control) or oil containing NAP, BNF or BaP (10 mg kg(-1)), and 72 h after injection fish were acutely stressed by chasing for 15 min. In the prolonged stress experiment, a similar group-design and injection protocol were followed, but fish were submitted to severe confinement stress by maintaining fish under high stock density (70 kg fish mass m(-3)) for 72 h. The levels of cortisol, glucose and lactate were assayed in plasma. In addition, the contents of dopamine (DA), noradrenaline (NA) and serotonin (<em>5HT</em>), as well as their oxidized amine metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxy-3-indoleacetic acid (5HIAA) were assayed in telencephalon, hypothalamus, preoptic region, optic tectum and brain stem, as well as the pituitary. Both acute and prolonged stress stimuli increased plasma levels of cortisol, which further increase with NAP and BNF treatments after acute stress. In contrast, cortisol levels of fish exposed to prolonged stress showed a clear tendency to decrease after the treatment with BNF and BaP. Stress stimuli also increased plasma glucose levels, which were not affected by PAHs in acute stressed fish but decreased in fish exposed to prolonged stress. Increased plasma levels of lactate in fish exposed to stress decreased after PAHs treatment in acute stress but not in prolonged stress. With respect to monoaminergic systems, major changes induced by both acute and prolonged stress were increases of the metabolites DOPAC and 5HIAA and DOPAC/DA or 5HIAA/<em>5HT</em> ratios in several brain regions. PAHs induced alterations in the normal responses of monoaminergic systems to stress, with dopaminergic system being the most affected after acute stress, and serotonergic system after prolonged stress. Those alterations, especially after prolonged stress, showed certain parallelism with alterations of plasma cortisol levels. Thus, results suggest that in stressed fish PAH effects on plasma cortisol levels (and its derived metabolic actions) could be in part mediated by alterations on the monoaminergic systems at the CNS of rainbow trout.

Patients with bone cancer commonly experience bone pain that is severe, intolerable, and difficult to manage. The rostral ventromedial medulla (RVM) plays an important role in the development of chronic pain via descending facilitation of spinal nociception. The compelling evidence shows that glial P2X7 receptor (P2X7R) is involved in the induction and maintenance of chronic pain syndromes. The present study explored the mechanism of glial activation and P2X7R expression underlying the induction of bone cancer pain. The results demonstrated that microglia and astrocytes in the RVM were markedly activated in bone cancer rats, and the expression of P2X7R was significantly upregulated. Injection of Brilliant Blue G (BBG), an inhibitor of P2X7R, into the RVM significantly alleviated pain behaviors of cancer rats, which was supported by intra-RVM injection of RNA interference targeting the P2X7R in the RVM. It is suggested that activation of microglia-expressed P2X7R in the RVM contributes to bone cancer pain. Given that 5-HT in the RVM is involved in modulating spinal nociception, changes in 5-HT and Fos expression were addressed in the spinal cord. Inhibition of P2X7R by BBG or small-interference RNA targeting P2X7 in the RVM markedly reduced 5-HT level and Fos expression in the spinal cord. The data clearly suggest that the activation of microglial P2X7R in the RVM contributes to the development of bone cancer pain via upregulation of spinal <em>5HT</em> levels by the descending pain facilitatory system.

BACKGROUND

Serotonin (5-hydroytryptamine or <em>5HT</em>) is associated with numerous behavioral and psychological factors and is a biochemical marker of mood. <em>5HT</em> is involved in the hypothalamic regulation of energy consumption. <em>5HT</em> controls appetite in the central nerve system (CNS) and stimulates intestinal mobility. There are few studies looking at the role of <em>5HT</em> and the relationship between peripheral circulating serotonin and obesity. The aim of this study was to find any differences in leptin, adiponectin, and <em>5HT</em> between lean and obese dogs and to identify correlations among these factors.

RESULTS

Leptin, triglyceride (TG) and cholesterol levels were higher in the obese group (all p < 0.01). Adiponectin and <em>5HT</em> levels were higher in the lean group compared to the obese group (p < 0.01). Leptin (r = 0.628, p < 0.01), TG (r = 0.491, p < 0.01) and cholesterol (r = 0.419, p < 0.01) were positively correlated with body condition score (BCS), and adiponectin (r = -0.446, p < 0.01) and <em>5HT</em> (r = -0.490, p < 0.01) were negatively correlated with BCS. Leptin was negatively correlated with adiponectin (r = -0.294, p < 0.01) and <em>5HT</em> (r = -0.343, p < 0.01). <em>5HT</em> was negatively correlated with leptin (r = -0.343, p < 0.01), TG (r = -0.268, p < 0.05) and cholesterol (r = -0.357, p < 0.05).

CONCLUSIONS

<em>5HT</em> is an important appetite control neurotransmitter, but there are limited studies for <em>5HT</em> levels related to obesity in dogs. To the best of our knowledge, this is the first study to evaluate peripheral <em>5HT</em> levels in obese dogs. From this research, we can assume that <em>5HT</em> may be correlated with canine obesity. Further studies will be needed to further elucidate the role of low serum <em>5HT</em> levels in canine obesity.

OBJECTIVE

Disturbances of the enteric serotonergic system have been implicated in several intestinal motility disorders. Patients with diverticular disease (DD) have been reported to exhibit abnormal intestinal motility and innervation patterns. Gene expression profiles of the serotonergic system and distribution of the serotonin type 4 receptor (<em>5HT</em>-4R) were thus studied in patients with DD.

METHODS

Colonic specimens from patients with DD and controls were subjected to quantitative PCR for serotonin receptors 2B, 3A, 4, serotonin transporter and synthesising enzyme tryptophan hydroxylase. Localisation of <em>5HT</em>-4R was determined by dual-label immunocytochemistry using smooth muscle actin (α-SMA) and pan-neuronal markers (PGP 9.5) and quantitative analysis was carried out. Site-specific gene expression analysis of <em>5HT</em>-4R was assessed within myenteric ganglia and muscle layers. Correlation of <em>5HT</em>-4R with muscarinic receptors 2 and 3 (M2R, M3R) messenger RNA expression was determined.

RESULTS

<em>5HT</em>-4R mRNA expression was downregulated in the tunica muscularis and upregulated in the mucosa of patients with DD, whereas the other components of the serotonergic system remained unchanged. <em>5HT</em>-4R was detected in ganglia and muscle layers, but was decreased in the circular muscle layer and myenteric ganglia of patients with DD. <em>5HT</em>-4R mRNA expression correlated with M2R/M3R mRNA expression in controls, but not in patients with DD.

CONCLUSIONS

The serotonergic system is compromised in DD. Altered expression of <em>5HT</em>-4R at mRNA and protein levels may contribute to intestinal motor disturbances reported in patients with DD. The findings support the hypothesis that DD is associated and possibly promoted by an enteric neuromuscular pathology.

Forepaw treading induced in rats by the <em>5HT</em>1A agonist 8-OH-DPAT, and head shakes caused by the administration of the <em>5HT</em>2A receptor against DOI, and by the <em>5HT</em> precursor (-)<em>5HT</em>P, were significantly increased by pretreatment with the non-competitive N-methyl-D-aspartate (NMDA) antagonist dizocilpine. Dizocilpine administration also significantly increased the locomotor activity induced by the serotonin agonists. The competitive NMDA receptor antagonist CGP 43487 increased only the head shakes induced by DOI, but did not alter the behavior elicited by 8-OH-DPAT, or (-)<em>5HT</em>P, and did not modify locomotor responses to any of the agonists used. The dizocilpine-induced potentiation of head shakes elicited by DOI and (-)<em>5HT</em>P was inhibited by the <em>5HT</em>2 agonist ketanserin, but was not modified by the selective dopamine D1 and D2 receptor blockers SCH 23390 and (-)sulpiride. The dopamine receptor antagonists did, however, counteract the dizocilpine facilitation of both forepaw treading induced by 8-OH-DPAT, and the locomotor response to all the serotonergic agonists. The results indicate that, unlike competitive NMDA receptor antagonists, the non-competitive antagonists enhanced the expression of serotonergic stimulation, and suggest that a glutamate deficiency could contribute to the pathogenesis of schizophrenia, not only through dopaminergic, but also through serotonergic, hyperactivity.

Zolmitriptan (Zomig; formerly 311C90) is a novel 5-hydroxytryptamine (<em>5HT</em>)1B/1D receptor agonist with proven efficacy in the acute treatment of migraine with or without preceding aura. The drug differs from presently available members of this drug class in that it combines <em>5HT</em>1B/1D receptor partial agonist activity with robust oral pharmacokinetics and an ability to inhibit trigeminovascular activation centrally as well as peripherally in preclinical studies. Consistent with its selectivity for <em>5HT</em>1B/1D receptors, zolmitriptan produces constriction of various isolated blood vessels, most notably cranial arteries. In anaesthetized animals, these vascular effects manifest as a selective constriction of cranial arterio-venous anastomoses resulting in a redistribution of carotid arterial blood flow. This effect is produced without significant effects on heart rate, blood pressure or blood flow to the brain, heart or lungs. Zolmitriptan also inhibits trigeminal-evoked increases in cerebral blood flow in anaesthetized cats and blocks trigeminal-evoked plasma protein extravasation in the dura of guinea-pigs. These actions are consistent with a pre-junctional inhibition of neuropeptide release from perivascular afferents of the trigeminal nerve, as confirmed by independent studies showing that zolmitriptan blocks elevations of calcitonin-gene-related peptide in jugular venous blood during electrical stimulation of the trigeminal ganglion. In all of these effects, zolmitriptan is three to four times more potent than sumatriptan, but produces the same maximum response. Zolmitriptan crosses the intact blood-brain barrier to inhibit trigeminovascular activation in the brainstem. This was shown initially by the ability of the drug to block a brainstem reflex provoking vasoactive intestinal peptide release from the VIIth cranial (facial) nerve during trigeminal stimulation. Subsequent ex vivo autoradiography confirmed that intravenously injected [3H]zolmitriptan labels a discrete population of cells in the trigeminal nucleus caudalis (TNC) and nucleus tractus solitarius. Direct evidence for a central neuromodulatory effect of zolmitriptan was provided by electrophysiological experiments which clearly demonstrated that the drug inhibits the excitability of cells in the TNC after systemic administration. This novel pre-clinical profile not only distinguishes zolmitriptan from sumatriptan, but raises intriguing questions about the clinical relevance of a dual action. Studies to date show that zolmitriptan indeed modulates cranial sensory processing in humans, yet central side-effects are no different from sumatriptan. This property may account for the remarkable consistency in clinical efficacy observed in clinical trials.

The available data suggest that circulating endogenous pyrogens (EPs) probably do not penetrate the brain, but interact with sensory elements in the organum vasculosum laminae terminalis (OVLT) which may involve <em>5HT</em> and SP as neurotransmitters. It is proposed that substance P (SP) may affect thermo-regulatory neurons in the preoptic area (POA) directly or induce the local synthesis of cytokines that secondarily act on these neurons. Recent evidence indicates that endothelial cells in the OVLT bind circulating cytokines to receptors on their luminal surface. This may result in the release of putative neuroregulators which then process the original signals inwardly to the POA, where they then affect neuronal functions leading to fever production. Thus, trans-BBB passage of cytokines is prevented, but the brain site mediating their pyrogenic effect is informed and the appropriate responses are activated. It is emphasized, however, that this suggested mechanism is still speculative.

In animals, repeated administration of 3,4-methylenedioxymethamphetamine (MDMA) reduces markers of serotonergic activity and studies show similar serotonergic deficits in human MDMA users. Using proton-magnetic resonance spectroscopy ((1)H-MRS) at 11.7Tesla, we measured the metabolic neurochemical profile in intact, discrete tissue punches taken from prefrontal cortex, anterior striatum, and hippocampus of rats administered MDMA (5mg/kg IP, 4× q 2h) or saline and euthanized 7 days after the last injection. Monoamine content was measured with HPLC in contralateral punches from striatum and hippocampus to compare the MDMA-induced loss of <em>5HT</em> innervation with constituents in the (1)H-MRS profile. When assessed 7 days after the last MDMA injection, levels of hippocampal and striatal serotonin (<em>5HT</em>) were significantly reduced, consistent with published animal studies. N-Acetylaspartate (NAA) levels were significantly increased in prefrontal cortex and not affected in anterior striatum or hippocampus; myo-inositol (INS) levels were increased in prefrontal cortex and hippocampus but not anterior striatum. Glutamate levels were increased in prefrontal cortex and decreased in hippocampus, while GABA levels were decreased only in hippocampus. The data suggest that NAA may not reliably reflect MDMA-induced <em>5HT</em> neurotoxicity. However, the collective pattern of changes in <em>5HT</em>, INS, glutamate and GABA is consistent with persistent hippocampal neuroadaptations caused by MDMA.

As part of the NIH "Facilities of Research Excellence-Spinal Cord Injury" project to support independent replication, we repeated key parts of a study reporting robust engraftment of neural stem cells (NSCs) treated with growth factors after complete spinal cord transection in rats. Rats (n=20) received complete transections at thoracic level 3 (T3) and 2weeks later received NSC transplants in a fibrin matrix with a growth factor cocktail using 2 different transplantation methods (with and without removal of scar tissue). Control rats (n=9) received transections only. Hindlimb locomotor function was assessed with the BBB scale. Nine weeks post injury, reticulospinal tract axons were traced in 6 rats by injecting BDA into the reticular formation. Transplants grew to fill the lesion cavity in most rats although grafts made with scar tissue removal had large central cavities. Grafts blended extensively with host tissue obliterating the astroglial boundary at the cut ends, but in most cases there was a well-defined partition within the graft that separated rostral and caudal parts of the graft. In some cases, the partition contained non-neuronal scar tissue. There was extensive outgrowth of GFP labeled axons from the graft, but there was minimal ingrowth of host axons into the graft revealed by tract tracing and immunocytochemistry for <em>5HT</em>. There were no statistically significant differences between transplant and control groups in the degree of locomotor recovery. Our results confirm the previous report that NSC transplants can fill lesion cavities and robustly extend axons, but reveal that most grafts do not create a continuous bridge of neural tissue between rostral and caudal segments.

During development, neural networks progress through important functional changes such as the generation of spontaneous activity, the expression of a depolarizing chloride gradient, and the appearance of neuromodulation. Little is known about how these processes are integrated to yield mature behaviors. We showed previously that, during the maturation of the locomotor network of the zebrafish, endogenous serotonin (<em>5HT</em>) increased motor activity by reducing intervals of inactivity, without affecting the active swim periods that are the target of <em>5HT</em> in other and more mature preparations. Because membrane properties were constant during the rest intervals, we examined here whether <em>5HT</em> modulates chloride homeostasis. We compared the effects of blocking (inward) chloride cotransport with bumetanide to the effects of <em>5HT</em> and its antagonists, both behaviorally by video imaging and cellularly by whole-cell and gramicidin-perforated patch recordings. Bumetanide mimicked the effects of <em>5HT</em> antagonists, by prolonging rest intervals without affecting the properties of swim episodes (duration; frequency; extent of depolarization) either behaviorally or during fictive swimming. Furthermore, bumetanide and <em>5HT</em> antagonists suppressed the amplitude of depolarizing responses evoked by ionophoresis of glycine onto spinal neurons in the presence of tetrodotoxin and transiently suppressed the amplitude of responses to glycine measured after fictive swimming. The effects of bumetanide contrasted with and occluded the effects of <em>5HT</em>. We suggest that, during development, endogenous <em>5HT</em> modulates chloride homeostasis during the quiescent intervals and thereby offsets the long periods of quiescence commonly observed in developing networks to allow expression of sustained and behaviorally relevant activity.

BACKGROUND

Despite advances in schizophrenia treatment, nearly 30% of patients do not respond to atypical antipsychotic agents, such as olanzapine. Furthermore, 30-60% of patients will gain significant weight during the course of olanzapine therapy. Little research has been done to investigate the relationship between antipsychotic treatment outcomes and genetic variability in second messengers coupled to serotonin (<em>5HT</em>) receptors. The purpose of this investigation was examine associations between the second messenger G-Protein Beta3 Subunit Gene (GNB3) C825T polymorphism and olanzapine response and weight gain treatment.

METHODS

We conducted a pharmacogenetic association study to examine GNB3 genotypes in relation to olanzapine clinical response (as measured by the Brief Psychiatric Rating Scale or Scale for the Assessment of Negative Symptoms) or weight gain. Subjects included forty-two individuals meeting DSM-IV criteria for schizophrenia that started olanzapine, were titrated to a fixed dose for 6 weeks, and subsequently genotyped for this investigation.

RESULTS

No statistically significant associations existed between our outcome variables and GNB3 genotypes. However we did observe trends suggesting a potential relationship between the TT genotype, response, and weight gain that warrant further investigation.

CONCLUSIONS

Preliminary results showed no statistical relationship between the C825T polymorphism and olanzapine response or weight gain. Numerical differences in outcome measures between the TT vs. CT/CC genotype groups indicate that G-protein second messenger systems variability coupled to primary targets of atypical antipsychotics may relate to clinical outcomes in persons with schizophrenia and that future research in this area is warranted.

Paliperidone palmitate is a new long-acting antipsychotic injection for the treatment of acute and maintenance therapy in schizophrenia. Paliperidone (9-hydroxyrisperidone) is the major active metabolite of risperidone and acts at dopamine D(2) and serotonin <em>5HT</em>(2A) receptors. As with other atypical antipsychotics, it exhibits a high <em>5HT</em>(2A):D(2) affinity ratio. It also has binding activity as an antagonist at α(1)-and α(2) adrenergic receptors and H(1) histaminergic receptors, but has virtually no affinity for cholinergic receptors. Paliperidone palmitate has been shown to be effective in reducing Positive and Negative Syndrome Scale total scores in four short-term trials in acute schizophrenia. It was also effective as maintenance therapy in a long-term trial in which time to recurrence of symptoms was significantly longer in paliperidone-treated patients compared with placebo. In addition, paliperidone was shown to be noninferior to risperidone long-acting injection in one study, but this noninferiority was not established in another longer study comparing the two drugs. Treatment should be initiated with 234 mg on day 1 and 156 mg on day 8, followed by a recommended monthly maintenance dose of 39-234 mg based on efficacy and tolerability. Paliperidone palmitate is generally well tolerated, although it can cause weight gain and a rise in prolactin levels, which is generally greater in women than in men. Overall, paliperidone palmitate may have advantages over other currently available long-acting injections, and therefore may be a useful alternative for the treatment of schizophrenia, although further long-term trials comparing it with active treatments are warranted.

The existence of DA receptors in the rat hippocampus was demonstrated with an in vivo [3H]spiperone radioreceptor assay. Kinetic studies revealed that maximum binding of [3H]spiperone in hippocampus was much smaller than in striatum and frontal cortex but much higher than in cerebellum. In inhibition studies of [3H]spiperone binding, all neuroleptics tested were active in hippocampus as well as in striatum. In contrast, <em>5HT</em> antagonists were definitely less potent in these two brain regions than in frontal cortex. Finally, even when <em>5HT</em> receptors were blocked, dipropyl-ATN and haloperidol remained fully effective in hippocampus, striatum, but also in frontal cortex although to a lesser degree. From these results it was concluded that [3H]spiperone binds mainly to DA receptors in hippocampus as well as in striatum, whereas both <em>5HT</em> and DA receptors are present in frontal cortex.

Serotonin (5-hydroxytryptamine; <em>5HT</em>) uptake sites have been measured using the selective high affinity uptake inhibitor 3H-citalopram in post-mortem frontal cortex from depressed and matched control subjects. The lateralization of these sites was assessed in neurologically normal brain. A lower concentration of 3H-citalopram binding was found in brains from depressed subjects. A nonsignificant trend toward a greater attenuation of <em>5HT</em> uptake sites was observed in brains of bipolar cases in the depressed state. No effect of antidepressant treatment or of the age at onset of illness was noted. No difference in the binding capacity of the <em>5HT</em> uptake site was noted between hemispheres of normal brains.

1. In the mollusc Tritonia escape swimming is produced by a network of central pattern generator (CPG) neurons. The purpose of this study was to determine which neurotransmitters might be involved in the swim system. 2. Injection of serotonin (<em>5HT</em>) into whole animals elicited swimming followed by a long-lasting inhibition of swimming. In isolated brain preparations, bath-applied <em>5HT</em> elicited a swim pattern at short latency and also caused a long-lasting inhibition of the swim pattern. The activation of swimming by <em>5HT</em> was associated with a tonic depolarization of cerebral cell 2 (C2) and the dorsal swim interneurons (DSI) which form part of the swim CPG network. 3. In isolated brain preparations, bath applied glycine, histamine, proctolin, and FMFRamide had no effect on the swim motor pattern elicited by electrical stimulation of a peripheral nerve. Aspartate, carbacol, dopamine, glutamate, octopamine, pilocarpine, and small cardioactive peptide-B (SCPB) inhibited the activation of swimming by nerve stimulation. 4. The <em>5HT</em> antagonists cyproheptidine, tryptamine, and 7-methyltryptamine had no effect on swimming, but methysergide and fenfluramine inhibited swimming to both normal sensory stimuli and exogenously applied <em>5HT</em>. 5. Staining with a polyclonal antibody indicated that one class of CPG neurons, the dorsal swim interneurons (DSI), was immunoreactive for <em>5HT</em>.

Quetiapine was developed in 1985 by scientists at AstraZeneca (formerly Zeneca) Pharmaceuticals. It received official US Food and Drug Administration approval in September 1997 and approval in Germany in 2000. Since then, quetiapine has been used in the treatment of severe mental illness in approximately 70 countries including Canada, most Western European countries, and Japan. Quetiapine is a dibenzothiazepine derivative with a relatively broad receptor binding profile. It has major affinity to cerebral serotonergic (<em>5HT</em>(2A)), histaminergic (H1), and dopaminergic D(1) and D(2) receptors, moderate affinity to alpha(1)- und alpha(2)-adrenergic receptors, and minor affinity to muscarinergic M1 receptors; it demonstrates a substantial selectivity for the limbic system. This receptor occupancy profile with relatively higher affinity for the <em>5HT</em>(2A) receptor compared with the D(2) receptor is in part responsible for the antipsychotic characteristics and low incidence of extrapyramidal side-effects of quetiapine. The efficacy of quetiapine in reducing positive and negative symptoms of schizophrenia has been proven in several clinical trials with placebo-controlled comparators. Quetiapine has also demonstrated robust efficacy for treatment of cognitive, anxious-depressive, and aggressive symptoms in schizophrenia. Long-term trials show sustained tolerability for a broad spectrum of symptoms. Quetiapine has also proven efficacy and tolerability in the treatment of moderate to severe manic episodes, and in the treatment of juveniles with oppositional-defiant or conduct disorders, and in the geriatric dementia population. Recent data indicate that quetiapine may also be effective in the treatment of bipolar depressive symptoms without increasing the risk of triggering manic episodes, and in borderline personality disorder. In comparison with other antipsychotics, quetiapine has a favorable side-effect profile. In clinical trials only small insignificant prolongations of the QT interval were observed. Weight-gain liabilities and new-onset metabolic side-effects occupy a middle-ground among newer antipsychotics. As a result of its good efficacy and tolerability profile quetiapine has become well established in the treatment of schizophrenia and manic episodes.

The pathophysiological basis for the pain of migraine has been the subject of substantial attention and must include activation of elements of the trigeminal innervation of the cranial vessels, the trigeminovascular system. Recently, consideration of trigeminal-evoked neurogenic plasma protein extravasation (PPE) as a model for the pain has driven the search for compounds with specific anti-extravasation properties. Calcitonin gene-related peptide (CGRP) is a marker for trigeminovascular activation and is released during the headache phase of migraine and cluster headache. CGRP may have a role in migraine through its potent cranial vasodilator effects or by an action on trigeminal nerve activity, both of which are targeted by <em>5HT</em>(1B/1D)agonist drugs but does not itself produce PPE. It has been suggested that <em>5HT</em>(1B/1D)agonists may have an anti-migraine effect via inhibition of PPE in the dura mater. Avitriptan and CP122,288 both have strong binding affinities for <em>5HT</em>(1B/1D)receptors, but only CP122,288 is a potent inhibitor of PPE. In this study we sought to compare the effects of CP122,288 and avitriptan on jugular vein CGRP release after stimulation of the superior sagittal sinus (SSS) in the cat. In eleven anaesthetized cats external jugular vein blood samples were analyzed by radioimmunoassay for CGRP levels in three settings: a) control, b) 1 min after SSS stimulation and c) 1 min after SSS stimulation in presence of drug. Stimulation of the SSS resulted in release of CGRP from the external jugular vein (77+/-1 pmol/L). At a PPE-inhibitory dose in rat (100 ng/kg intravenously) CP122, 288 had no effect on CGRP release (77+/-6 pmol/L) whereas at a clinically relevant dose (50 microgram/kg intravenously) avitriptan blocked CGRP release. This study demonstrates that the potent inhibitor of PPE, CP122, 288, which has been shown in clinical trials to be ineffective in treating acute migraine attacks, had no effect on CGRP release, whereas the effective anti-migraine drug and relatively impotent inhibitor of PPE, avitriptan, blocked CGRP release. These data emphasize the importance of CGRP release and its possible independence from PPE in migraine and more importantly suggest that other non-<em>5HT</em>-based pharmacological targets may account for PPE blockade in animal studies.