Cholinesterase inhibitors including donepezil, rivastigmine, and galantamine and the N-methyl-D-aspartate (NMDA) antagonist, memantine are the medications currently approved for the treatment of Alzheimer's disease (AD). In addition to their beneficial effects on cognitive and functional domains typically disrupted in AD, these agents have also been shown to slow down the emergence of behavioral and psychotic symptoms associated with this disease. However, the underlying mechanisms for these therapeutic effects remain poorly understood and could involve effects of these medications on non-cholinergic or non-glutamatergic neurotransmitter systems respectively. These considerations prompted us to initiate a series of investigations to examine the acute and chronic effects of donepezil (Aricept (+/-)-2,3-dihydro-5,6-dimethoxy-2-[[1-(phenylmethyl)-4-piperidinyl]methyl]-1H-inden-1-1 hydrochloride and memantine (1-amino-3,5-dimethyladamantane hydrochloride C12H21N.HCl)). The present study focuses on the acute effects of donepezil and memantine on brain extracellular levels of acetylcholine, dopamine, serotonin, norepinephrine and their metabolites. We assayed changes in the ventral and dorsal hippocampus and the prefrontal and medial temporal cortex by microdialysis. Memantine resulted in significant increases in extracellular dopamine (DA), norepinephrine (NE), and their metabolites, in the cortical regions, and in a reduction of DA in the hippocampus. Donepezil produced an increase in extracellular DA in the cortex and in the dorsal hippocampus. Norepinephrine increased in the cortex; with donepezil it increased in the dorsal hippocampus and the medial temporal cortex, and decreased in the ventral hippocampus. Interestingly both compounds decreased extracellular serotonin (<em>5HT</em>) levels. The metabolites of the neurotransmitters were increased in most areas. We also found an increase in extracellular acetylcholine (ACh) by memantine in the nucleus accumbens and the ventral tegmental area. Our results suggest both region and drug specific neurotransmitter effects of these agents as well as some similarities. We conclude that drugs influencing cognitive mechanisms induce changes in a number of neurotransmitters with the changes being both region and drug specific. Release and metabolism are altered and extracellular neurotransmitter levels can be increased or decreased by the drugs. Other studies are in progress to determine the pharmacological effects associated with chronic treatment with these compounds, which may be more pertinent to the clinical situation in which patients take these medications for months or years.

The atypical antipsychotics (AAPs) have been associated with increased risk of type-2 diabetes. Evidence suggests direct, drug-related effects independent of weight gain and although mechanisms underlying this phenomenon are unclear, it has been suggested that the heterogeneous receptor binding profile of the AAPs may influence receptors implicated in glucose metabolism. This study aimed to clarify weight gain-independent mechanisms of AAP-induced changes in insulin secretion by deconstructing their binding profile with representative antagonists. Healthy rats were pretreated with a single subcutaneous dose of darifenacin 6 mg/kg (n=10), a selective M(3) muscarinic antagonist; ketanserin 2mg/kg (n=10), a <em>5HT</em>(2A) antagonist; raclopride 0.3mg/kg (n=11) a selective D(2)/D(3) antagonist; terfenadine 20mg/kg (n=9) a selective H(1) antagonist; or, vehicle (n=11). Hyperglycemic clamps were employed following injection, providing an index of secretory capacity of pancreatic β-cells. Acute treatment with darifenacin and ketanserin significantly decreased insulin response to glucose challenge as compared to controls, which was confirmed in the darifenacin group by reduced C-peptide levels. Treatment with raclopride resulted in an increased insulin response and a strong tendency to increased C-peptide levels. H(1) blockade did not result in effects on insulin or C-peptide. Results suggest that the effects of antipsychotics on glucose dysregulation may be related to direct inhibitory effects of muscarinic (M(3)) and serotonergic (<em>5HT</em>(2)) antagonism on insulin secretion. Based on the expression of D(2)-like receptors in β-cells, which mediate inhibition of insulin secretion, we propose that prolonged D(2) blockade with antipsychotics may predispose to depletion of insulin stores and an eventual defect in pancreatic compensation.

Chronic visceral pain affects millions of individuals worldwide and remains poorly understood, with current therapeutic options constrained by gastrointestinal adverse effects. Visceral pain is strongly associated with inflammation and distension of the gut. Here we report that the voltage-gated sodium channel subtype NaV1.9 is expressed in half of gut-projecting rodent dorsal root ganglia sensory neurons. We show that NaV1.9 is required for normal mechanosensation, for direct excitation and for sensitization of mouse colonic afferents by mediators from inflammatory bowel disease tissues, and by noxious inflammatory mediators individually. Excitatory responses to ATP or PGE2 were substantially reduced in NaV1.9(-/-) mice. Deletion of NaV1.9 substantially attenuates excitation and subsequent mechanical hypersensitivity after application of inflammatory soup (IS) (bradykinin, ATP, histamine, PGE2, and <em>5HT</em>) to visceral nociceptors located in the serosa and mesentery. Responses to mechanical stimulation of mesenteric afferents were also reduced by loss of NaV1.9, and there was a rightward shift in stimulus-response function to ramp colonic distension. By contrast, responses to rapid, high-intensity phasic distension of the colon are initially unaffected; however, run-down of responses to repeat phasic distension were exacerbated in NaV1.9(-/-) afferents. Finally colonic afferent activation by supernatants derived from inflamed human tissue was greatly reduced in NaV1.9(-/-) mice. These results demonstrate that NaV1.9 is required for persistence of responses to intense mechanical stimulation, contributes to inflammatory mechanical hypersensitivity, and is essential for activation by noxious inflammatory mediators, including those from diseased human bowel. These observations indicate that NaV1.9 represents a high-value target for development of visceral analgesics.

Subject numbers in neuroreceptor imaging studies of antipsychotic treatment in schizophrenia are generally insufficient to directly test the relationship of regional D(2)/D(3) and <em>5HT</em>(2A) receptor binding to clinical efficacy. We selected positron emission tomography (PET) and single photon emission computed tomography (SPECT) studies of antipsychotic dose vs occupancy at both temporal cortex and striatal D(2)/D(3) receptors. We selected corresponding SPECT and PET studies of <em>5HT</em>(2A) receptor occupancy. We also selected randomized double-blind clinical trials of antipsychotics, where patients were treated with randomly assigned fixed doses. For each antipsychotic drug, we compared the optimum effective antipsychotic dose with the dose inducing maximal occupancy of D(2)/D(3) receptors in striatum and in temporal cortex as well as at <em>5HT</em>(2A) receptors. Both first- and second-generation antipsychotic (FGA, SGA) drugs produced high temporal cortex D(2)/D(3) occupancy. Only FGA produced high striatal D(2)/D(3) receptor occupancy. The clinically effective dose showed correlation with doses inducing maximal dopamine D(2)/D(3) receptor occupancy both in striatum and in temporal cortex, the strongest correlation being with temporal cortex binding. Extrapyramidal side effects (EPSE) were primarily related to striatal D(2)/D(3) receptor occupancy. There was no correlation between <em>5HT</em>(2A) occupancy and clinically effective dose. We conclude that cortical dopamine D(2)/D(3) receptor occupancy is involved in antipsychotic efficacy, with striatal D(2)/D(3) occupancy having a likely therapeutic role while also inducing EPSE. We found no evidence for <em>5HT</em>(2A) blockade involvement in antipsychotic action, although we cannot exclude this possibility.

Precise anatomical distribution of <em>5HT</em>1 binding sites has been investigated in the nuclei raphe dorsalis, raphe centralis and locus caeruleus of the rat brain. An original pattern of distribution was observed in the raphe nuclei, closely correlated to the already known distribution of <em>5HT</em> containing elements. This pattern, more pronounced when <em>5HT</em>1A sites were labelled, completely disappeared after lesioning by 5, 7DHT indicating the presence of this subtype of <em>5HT</em>1 binding sites on <em>5HT</em> containing neurons. It is postulated that these <em>5HT</em>1A sites correspond in these raphe nuclei to <em>5HT</em> autoreceptors.

The suprachiasmatic nucleus (SCN) is a circadian oscillator and a critical component of the mammalian circadian system. It receives afferents from the retina and the mesencephalic raphe. Retinal afferents mediate photic entrainment of the SCN, whereas the serotonergic afferents originating from the midbrain modulate photic responses in the SCN; however, the serotonin (<em>5HT</em>) receptor subtypes in the SCN responsible for these modulatory effects are not well characterized. In this study, we tested the hypothesis that <em>5HT</em>1B receptors are located presynaptically on retinal axon terminals in the SCN and that activation of these receptors inhibits retinal input. The <em>5HT</em>1B receptor agonists TFMPP and CGS 12066A, administered systemically, inhibited light-induced phase shifts of the circadian activity rhythm in a dose-dependent manner at phase delay and phase advance time points. This inhibition was not affected by previous systemic application of either the selective <em>5HT</em>1A receptor antagonist (+)WAY 100135 or by the <em>5HT</em>2 receptor antagonist mesulergine, whereas pretreatment with the nonselective <em>5HT</em>1 antagonist methiothepin significantly attenuated the effect of TFMPP. TFMPP also produced a dose-dependent reduction in light-stimulated Fos expression in the SCN, although a small subset of cells in the dorsolateral aspect of the caudal SCN were TFMPP-insensitive. TFMPP (1 mM) infused into the SCN produced complete inhibition of light-induced phase advances. Finally, bilateral orbital enucleation reduced the density of SCN <em>5HT</em>1B receptors as determined using [125I]-iodocyanopindolol to define <em>5HT</em>1B binding sites. These results are consistent with the interpretation that <em>5HT</em>1B receptors are localized presynaptically on retinal terminals in the SCN and that activation of these receptors by <em>5HT</em>1B agonists inhibits retinohypothalamic input.

OBJECTIVE

ZBP-89 (also ZNF148 or Zfp148) is a butyrate-inducible zinc finger transcription factor that binds to GC-rich DNA elements. Deletion of the N-terminal domain is sufficient to increase mucosal susceptibility to chemical injury and inflammation. We investigated whether conditional deletion of ZBP-89 from the intestinal and colonic epithelium of mice increases their susceptibility to pathogens such as Salmonella typhimurium.

METHODS

We generated mice with a conditional null allele of Zfp148 (ZBP-89(FL/FL)) using homologous recombination to flank Zfp148 with LoxP sites (ZBP-89(FL/FL)), and then bred the resulting mice with those that express VillinCre. We used microarray analysis to compare gene expression patterns in colonic mucosa between ZBP-89(ΔInt) and C57BL/6 wild-type mice (controls). Mice were gavaged with 2 isogenic strains of S. typhimurium after administration of streptomycin.

RESULTS

Microarray analysis revealed that the colonic mucosa of ZBP-89(ΔInt) mice had reduced levels of tryptophan hydroxylase 1 (Tph1) messenger RNA, encoding the rate-limiting enzyme in enterochromaffin cell serotonin (5-hydroxytryptamine [<em>5HT</em>]) biosynthesis. DNA affinity precipitation demonstrated direct binding of ZBP-89 to the mouse Tph1 promoter, which was required for its basal and butyrate-inducible expression. ZBP-89(ΔInt) mice did not increase mucosal levels of <em>5HT</em> in response to S. typhimurium infection, and succumbed to the infection 2 days before control mice. The ΔhilA isogenic mutant of S. typhimurium lacks this butyrate-regulated locus and stimulated, rather than suppressed, expression of Tph1 approximately 50-fold in control, but not ZBP-89(ΔInt), mice, correlating with fecal levels of butyrate.

CONCLUSIONS

ZBP-89 is required for butyrate-induced expression of the Tph1 gene and subsequent production of <em>5HT</em> in response to bacterial infection in mice. Reductions in epithelial ZBP-89 increase susceptibility to colitis and sepsis after infection with S. typhimurium, partly because of reduced induction of <em>5HT</em> production in response to butyrate and decreased secretion of antimicrobial peptides.

OBJECTIVE

To use statistical parametric mapping to determine the extent of previously reported serotonin type 2A (<em>5HT</em>(2A)) receptor binding potential (BP) increases in postmenopausal women following hormone therapy.

METHODS

Repeated measures positron emission tomography (PET) study.

METHODS

Academic research environment.

METHODS

Five healthy, postmenopausal women.

METHODS

Serial PET images of [(18)F]altanserin uptake were acquired to measure <em>5HT</em>(2A) receptor BP at menopausal baseline, following estradiol (E(2)), and following combined E(2) + micronized progesterone (P(4)).

METHODS

<em>5HT</em>(2A) receptor BP.

RESULTS

Combined E(2) + P(4) treatment was associated with significant increases in the <em>5HT</em>(2A) receptor BP increases in widespread areas of cerebral cortex. Treatment with E(2) alone was also associated with widespread cortical BP increases, although these changes reached statistical significance in fewer regions. The rate of [(18)F]altanserin metabolism was significantly decreased in the E(2) + P(4) condition relative to menopausal baseline, but this difference did not appear to correlate with changes in <em>5HT</em>(2A) receptor BP.

CONCLUSIONS

Estradiol priming followed by combined E(2) + P(4) is associated with widespread increases in <em>5HT</em>(2A) receptor BP in the cerebral cortex, consistent with the E(2)-associated increases in <em>5HT</em>(2A) receptor density previously observed in experimental animals.

Spinal cord stimulation (SCS) may alleviate certain forms of neuropathic pain; its mechanisms of action are, however, not fully understood. Previous studies have mainly been focused onto segmental spinal mechanisms, though there is evidence indicating a supraspinal involvement. This study aims to evaluate the relative importance of segmental and supraspinal mechanisms related to the activation of the dorsal columns (DCs). Rats were used to induce the spared nerve injury neuropathy and simultaneously subjected to chronic bilateral DC lesions at the C6-C8 level. Two pairs of miniature electrodes were implanted in each animal, with a monopolar system placed in the dorsal epidural space at a low thoracic level (below lesion) and a bipolar system placed onto the dorsal column nuclei (above lesion). Stimulation (50 Hz, 0.2 ms, 2-4V, 5 min) was applied via either type of electrodes, and tests for sensitivity to tactile and thermal stimuli were used to assess its inhibitory effects. Various receptor antagonists {bicuculline (GABA(A)), saclofen (GABA(B)), ketanserine (<em>5HT</em>(2)), methysergide (<em>5HT</em>(1-2)), phentolamine (α-adrenergic), propranolol (β-adrenergic), sulpiride (D(2)/D(3) dopamine) or saline were injected prior to the SCS. Rostral and caudal stimulations produced a comparable inhibition of neuropathic manifestations, and these effects were attenuated by about 50% after DC lesions. Pretreatment with the various receptor antagonists differentially influenced the effects of rostral and caudal stimulation. Our findings suggest that both supraspinal and segmental mechanisms are activated by SCS, and that in this model with DC lesions, rostral and caudal stimulations may activate different synaptic circuitries and transmitter systems.

BACKGROUND

Areca catechu, commonly known as betel nut, is very famous for its medicinal use in multiple disorders. It is also popular as a remedy against inflammatory disorders in the Unani (Greco-Arab) system of medicine.

OBJECTIVE

This study was aimed at investigating the anti-inflammatory and analgesic activities of the crude extract of Areca catechu and its respective fractions.

METHODS

Paw edema, formalin-induced nociception and acetic acid-induced writhing assays were carried out in vivo. Free radical scavenging activity of the plant extract was performed in vitro.

RESULTS

Preliminary experiments using a single dose (100 mg/kg) of Areca catechu and its respective fractions demonstrated an anti-inflammatory effect on carrageenan-induced edema in mice and rats, the aqueous fraction being distinctly more effective. When studied on prostaglandin E₂ (PGE₂), arachidonic acid, histamine, or serotonin (<em>5HT</em>)-induced edema in rats, Areca catechu and its aqueous fraction markedly repressed only the PGE₂ and arachidonic acid-induced inflammation. When studied for analgesic activity, the crude extract and its aqueous fraction produced a dose-dependent (10-100 mg/kg) inhibitory effect on formalin-induced nociception in mice and acetic acid-induced writhing in rats, similar to aspirin. In DPPH assay, Areca catechu and its aqueous fraction exhibited free radical scavenging activity with respective IC(50) values of 5.34 μg/ml (4.93-5.78, CI; 95%, n=5) and 7.28 μg/ml (6.04-7.95, n=4), like that of rutin with IC(50) value of 4.75 μg/ml (3.89-5.42, n=4).

CONCLUSIONS

These results indicate the anti-inflammatory and analgesic effects of Areca catechu and provide a rationale for its medicinal use in inflammatory disorders.

Group A β-hemolytic streptococcal (GAS) infection is associated with a spectrum of neuropsychiatric disorders. The leading hypothesis regarding this association proposes that a GAS infection induces the production of auto-antibodies, which cross-react with neuronal determinants in the brain through the process of molecular mimicry. We have recently shown that exposure of rats to GAS antigen leads to the production of anti-neuronal antibodies concomitant with the development of behavioral alterations. The present study tested the causal role of the antibodies by assessing the behavior of naïve rats following passive transfer of purified antibodies from GAS-exposed rats. Immunoglobulin G (IgG) purified from the sera of GAS-exposed rats was infused directly into the striatum of naïve rats over a 21-day period. Their behavior in the induced-grooming, marble burying, food manipulation and beam walking assays was compared to that of naïve rats infused with IgG purified from adjuvant-exposed rats as well as of naïve rats. The pattern of in vivo antibody deposition in rat brain was evaluated using immunofluorescence and colocalization. Infusion of IgG from GAS-exposed rats to naïve rats led to behavioral and motor alterations partially mimicking those seen in GAS-exposed rats. IgG from GAS-exposed rats reacted with D1 and D2 dopamine receptors and <em>5HT</em>-2A and <em>5HT</em>-2C serotonin receptors in vitro. In vivo, IgG deposits in the striatum of infused rats colocalized with specific brain proteins such as dopamine receptors, the serotonin transporter and other neuronal proteins. Our results demonstrate the potential pathogenic role of autoantibodies produced following exposure to GAS in the induction of behavioral and motor alterations, and support a causal role for autoantibodies in GAS-related neuropsychiatric disorders.

Taste buds, the taste sensory organs, are conserved in vertebrates and composed of distinct cell types, including taste receptor, basal/presynaptic and support cells. Here, we characterize zebrafish taste bud development and show that compromised Fgf signaling in the larva results in taste bud reduction and disorganization. We determine that Fgf activity is required within pharyngeal endoderm for formation of Calb2b(+) cells and reveal miR-200 and Delta-Notch signaling as key factors in this process. miR-200 knock down shows that miR-200 activity is required for taste bud formation and in particular for Calb2b(+) cell formation. Compromised delta activity in mib(-/-) dramatically reduces the number of Calb2b(+) cells and increases the number of <em>5HT</em>(+) cells. Conversely, larvae with increased Notch activity and ascl1a(-/-) mutants are devoid of <em>5HT</em>(+) cells, but have maintained and increased Calb2b(+) cells, respectively. These results show that Delta-Notch signaling is required for intact taste bud organ formation. Consistent with this, Notch activity restores Calb2b(+) cell formation in pharyngeal endoderm with compromised Fgf signaling, but fails to restore the formation of these cells after miR-200 knock down. Altogether, this study provides genetic evidence that supports a novel model where Fgf regulates Delta-Notch signaling, and subsequently miR-200 activity, in order to promote taste bud cell type differentiation.

We report here a study of the effects of alprazolam on in vivo pituitary-adrenal function in jacketed nonrestrained nonhuman primates and on in vitro CRH release from rat hypothalami and ACTH release from rat dispersed anterior pituicytes. We undertook this study because alprazolam is the only benzodiazepine effective in treating both major depressive and anxiety disorders, and recent data suggest that the hypercortisolism of major depression reflects hypersecretion of CRH. Moreover, the intracerebroventricular administration of CRH can reproduce many of the components of the symptom complex of major depression, including not only hypercortisolism, but also hypothalamic hypogonadism, decreased libido, anorexia, and intense anxiety. As a comparison, we also assessed the effects of diazepam on in vitro CRH release, because in contrast to alprazolam, diazepam is effective in anxiety states but not in depression. Alprazolam (0.01-0.3 mg/kg, iv) produced a dose-dependent inhibition of both plasma ACTH and cortisol secretion in non-restrained adult male rhesus monkeys. Our in vitro studies showed that alprazolam significantly inhibited serotonin (<em>5HT</em>)-induced CRH release in a dose-dependent fashion (10(-10)-10(-5) M). Diazepam also inhibited <em>5HT</em>-induced CRH release, but was 40 times less potent than alprazolam. Alprazolam was ineffective in blocking basal or CRH-induced ACTH release from rat dispersed anterior pituicytes, suggesting that its in vivo effects are through inhibition of CRH secretion. As expected, the inactive benzodiazepine ligand Ro 15-1788 inhibited the effects of alprazolam on <em>5HT</em>-induced CRH release, but this occurred only at doses below 10(-7) M. Interestingly, when incubated alone in higher doses with our rat hypothalamic organ culture, Ro 15-1788, like alprazolam, produced a dose-dependent inhibition of <em>5HT</em>-induced CRH release (10(-7)-10(-5) M), suggesting an agonistic action of Ro 15-1788 at the benzodiazepine receptor at higher concentrations. We conclude that alprazolam is capable of suppressing the primate pituitary-adrenal axis, and that this suppression most likely reflects suppression of the CRH neuron rather than of the pituitary corticotroph cell. We speculate that the enhanced capacity of alprazolam to suppress the CRH neuron relative to other benzodiazepines may contribute to its unique efficacy among this class of drugs in the treatment of major depression. The capacity of Ro 15-1788 to reverse alprazolam-induced suppression of the CRH neuron indicates that the effects of alprazolam are mediated at least in part via its interaction with the benzodiazepine component of the gamma-aminobutyric acidA macromolecular complex.

It is well documented that atypical antipsychotics have an influence on cognitive function in patients with schizophrenia, although the neurochemical basis for this effect is not well understood. One suggestion is that the effects are exerted through action on <em>5HT</em>-2A receptors, which leads to changes in the level of dopamine in the prefrontal cortex. The following study explored this hypothesis by comparing the cognitive effects of the atypical antipsychotics which have a high affinity for <em>5HT</em>-2A receptors, with those that have little or no affinity to these receptors. Forty-four patients with a DSM-IV diagnosis of schizophrenia were recruited within 6 weeks of starting one of the atypical antipsychotics: clozapine, olanzapine, risperidone, quetiapine, or amisulpride. The patients were divided into two groups according to the <em>5HT</em>-2A affinity of the individual medications (high <em>5HT</em>-2A affinity--clozapine, olanzapine, risperidone vs. low <em>5HT</em>-2A affinity--quetiapine, amisulpride). Patients were tested on a broad range of neuropsychological measures after 9 months and 18 months of treatment. The high <em>5HT</em>-2A affinity group showed a decrement in performance on tests of visual recognition memory and planning ability. In contrast, the low-<em>5HT</em>-2A affinity group showed improvements on these measures in addition to others. The <em>5HT</em>-2A affinity of the atypical antipsychotics is an important determinant of their cognitive effects.

The attention deficit/hyperactivity disorder (ADHD) can affect human infants and adolescents. One important feature of this disorder is behavioural impulsivity. This study assessed the ability of chronic acetyl-L-carnitine (ALC, saline or 100 mg/kg SC, plus 50 mg/kg orally) to reduce impulsivity in a validated animal model for ADHD. Food-restricted rats were tested during adolescence (postnatal days, pnd, 30-45) in operant chambers with two nose-poking holes, one delivering one food pellet immediately, and the other five pellets after a delay. Delay length was increased over days (from 0 to 80 s). Individual differences in the preference-delay curve emerged, with the identification of two distinct subpopulations, i.e. one with a nearly horizontal curve and another with a very steep ("impulsive") slope. The impulsivity profile was slightly but consistently reduced by chronic ALC administration. Consistent results were also obtained with methylphenidate (MPH, saline or 3 mg/kg IP twice daily). Impulsive rats exhibited a lower metabolite/serotonin (5HIAA/<em>5HT</em>) ratio in the medial frontal cortex (MFC) and lower noradrenaline (NA) levels in the MFC and cingulate cortex (CC) when compared with the other subgroup. The ALC treatment increased NA levels in the CC and the 5HIAA/<em>5HT</em> ratio in both CC and MFC. Present data suggest that ALC, a drug devoid of psychostimulant properties, may have some beneficial effects in the treatment of ADHD children.

In the mollusk Aplysia the neurotransmitter serotonin (<em>5HT</em>) has a fundamental modulatory role in several forms of learning and memory that involve an increase in the efficacy of synaptic transmission between tail sensory neurons (SNs) and motor neurons. The classical <em>5HT</em> antagonist cyproheptadine (CYP) permits dissociation of three forms of serotonergic modulation in these SNs: (i) CYP reversibly blocks spike-broadening induced either by exogenous application of <em>5HT</em> or by sensitizing stimulation of a tail nerve. (ii) CYP does not block <em>5HT</em>-induced or tail input-induced increases in SN somatic excitability. (iii) Concomitant with its block of spike-broadening, CYP reversibly blocks <em>5HT</em>-induced facilitation of synaptic transmission from SNs. These results suggest that endogenously released <em>5HT</em> may act at different receptor subtypes that are coupled to different forms of neuromodulation in tail SNs of Aplysia.

To date, several studies have demonstrated that phospholipase C-coupled receptors stimulate the production of endocannabinoids, particularly 2-arachidonoylglycerol. There is now evidence that endocannabinoids are involved in phospholipase C-coupled serotonin 5-HT(2A) receptor-mediated behavioral effects in both rats and mice. The main objective of this study was to determine whether activation of the 5-HT(2A) receptor leads to the production and release of the endocannabinoid 2-arachidonoylglycerol. NIH3T3 cells stably expressing the rat 5-HT(2A) receptor were first incubated with [(3)H]-arachidonic acid for 24 h. Following stimulation with 10 mum serotonin, lipids were extracted from the assay medium, separated by thin layer chromatography, and analyzed by liquid scintillation counting. Our results indicate that 5-HT(2A) receptor activation stimulates the formation and release of 2-arachidonoylglycerol. The 5-HT(2A) receptor-dependent release of 2-arachidonoylglycerol was partially dependent on phosphatidylinositol-specific phospholipase C activation. Diacylglycerol produced downstream of 5-HT(2A) receptor-mediated phospholipase D or phosphatidylcholine-specific phospholipase C activation did not appear to contribute to 2-arachidonoylglycerol formation in NIH3T3-<em>5HT</em>(2A) cells. In conclusion, our results support a functional model where neuromodulatory neurotransmitters such as serotonin may act as regulators of endocannabinoid tone at excitatory synapses through the activation of phospholipase C-coupled G-protein coupled receptors.

We have initiated a study of the CNS of mutant Drosophila melanogaster larvae carrying a genetic deletion of the gene Ddc that encodes the enzyme dopa decarboxylase (DDC). The two major objectives of this study were (1) to ascertain that the DDC encoded by the gene Ddc was the only decarboxylase utilized in serotonin (<em>5HT</em>)-containing neurons and (2) to determine the effect of DDC deficiency on the development of <em>5HT</em>-immunoreactive neurons. CNSs of wild-type larvae and of larvae genetically deficient for the gene Ddc were processed for serotonin immunocytochemistry using a monoclonal antibody against <em>5HT</em>. The pattern of <em>5HT</em> immunoreactivity in the wild-type and the Ddc-deficient CNS is compared. In contrast to the wild-type, <em>5HT</em> immunoreactivity is absent in the Ddc-deficient CNSs. The lack of immunocytochemically detectable <em>5HT</em> in the mutant CNSs is consistent with the idea that the DDC encoded by the gene Ddc is utilized in <em>5HT</em>-containing neurons. To study the development of neurons committed to the <em>5HT</em> differentiation pathway in the absence of <em>5HT</em>, we used a second biochemical property characteristic of <em>5HT</em>-containing neurons, the ability to take up <em>5HT</em>. CNSs from mutant animals were incubated in exogenous <em>5HT</em> and the accumulated <em>5HT</em> detected immunocytochemically. Neurons capable of selective <em>5HT</em> uptake were present in the mutant CNSs in the same pattern as the <em>5HT</em>-immunoreactive neurons in the wild-type CNS. This result suggests that the presumed inability to synthesize <em>5HT</em> does not preclude differentiation of other normal biochemical properties of <em>5HT</em>-containing neurons.

Serotonin <em>5HT</em>(1A)-binding sites can be detected in living human brain with the positron-emitting antagonist [(11)C]WAY-100635. Previous measurements of the availability of [(11)C]WAY-binding sites in normal aging are equivocal, in part because of the greatly variable binding of this ligand. To test the null hypothesis that the binding potential (pB) of <em>5HT</em>(1A) sites remains constant with age; 19 healthy volunteers aged 23-73 years (8 women, 11 men) underwent positron emission tomography. To determine pBs, we applied a novel tissue reference method of analysis, Estimation of Reversible Ligand Binding and Receptor Density (ERLiBIiRD) (Gjedde, 2003; Rosa-Neto et al, 2004), which extrapolates measures of specific binding to an estimated steady-state. We compared these estimates in the two age groups with results obtained with the conventional Logan Plot and Simplified Reference Tissue Method (SRTM) applied to both regions of interest-based as parametric analyses. The regional distribution of specific binding of free sites [(11)C]WAY-100635 was similar to that reported in previous studies, with the highest pBs in limbic structures and the raphé nuclei. Although the results of the three methods differed, pBs in the elderly subjects consistently were lower than those of young subjects. Thus, the correlation between pB and age applied to regions-of-interest revealed significant decline of pB at the rate of 3 or 4% per decade, and a 10% decline of the global mean <em>5HT</em>(1A) receptor-pB in elderly relative to young subjects. The results demonstrate that the number of available <em>5HT</em>(1A)-binding sites declines with age.

Increasing evidence indicates that modulation of Na(+)/K(+) ATPase activity is involved in forms of neuronal and synaptic plasticity. In tactile (T) neurons of the leech Hirudo medicinalis, Na(+)/K(+) ATPase is the main determinant of the afterhyperpolarization (AHP), which characterizes the firing of these mechanosensory neurons. Previously, it has been reported that cAMP (3',5'-cyclic adenosine monophosphate), which mediates the effects of serotonin (<em>5HT</em>) in some forms of learning in the leech, negatively modulates Na(+)/K(+) ATPase activity, thereby reducing the AHP amplitude in T neurons. Here, we show that a transient inhibition of Na(+)/K(+) ATPase can affect the synaptic connection between two ipsilateral T neurons. Bath application of 10 nm dihydroouabain (DHO), an ouabain analogue, causes an increase in the amplitude of the synaptic potential (SP) recorded in the postsynaptic element when a test stimulus is applied in the presynaptic neuron. Iontophoretic injection of cAMP into the presynaptic T neuron also produces an increase of SP. Simulations carried out by using a computational model of the T neuron suggest that a reduction of the pump rate and a consequent depression of the AHP might facilitate the conduction of action potentials to the synaptic terminals. Moreover, nearly intact leeches injected with 10 nm DHO respond with a swimming episode more quickly to an electrical stimulation, which selectively activates T neurons exhibiting sensitization of swimming induction. Collectively, our results show that inhibition of Na(+)/K(+) ATPase is critical for short-term plasticity.

In the nervous system of the obligatory endoparasite Diphyllobothrium dendriticum immunoreactivity (IR) to growth hormone-releasing factor (GRF), peptide histidine isoleucine (PHI), bovine pancreatic polypeptide (BPP), gastrin, gastrin-releasing peptide (GRP), oxytocin, FMRFamide (FMRF) and serotonin (<em>5HT</em>) was demonstrated by immunocytochemical methods. A very strong GRF-IR was observed in the CNS and PNS of larvae and of the constantly growing adult worms. GRF-IR axon terminals occur beneath the basal lamina of the tegument along the inside of the bothridia, the holdfast organ of the worm. GRF-IR fibres surround the yolk producing vitelline glands and occur in the wall of the vagina. PHI-IR was observed in the CNS and PNS of larvae and adult worms. PHI-IR terminals occur beneath the basal lamina of the tegument along the strobila, the nutrient absorbing surface of the worm. PHI-IR fibres seem to innervate the testicular follicles. FMRF-IR fibres and perikarya occur close to the vitelline glands and the uterine pore and in the male copulatory organ. Numerous large <em>5HT</em>-IR perikarya with long varicose fibres were observed in the nervous system of the worm. <em>5HT</em>-IR perikarya occur close to the genital atrium. D. dendriticum is the phylogenetically lowest organism in which IR to PHI has been demonstrated.

Neurotransmitter transporters are regulated through a variety of signal transduction mechanisms which may operate in order to maintain appropriate levels of transmitter in the synaptic cleft. GABA and glycine transporters both interact with components of the neurotransmitter release, such as the SNARE protein syntaxin 1A, suggesting that protein-protein interactions are a common method for regulating members of the neurotransmitter transporter family, and thus, linking the release of transmitter to its subsequent re-uptake. In the present report, the interaction of syntaxin 1A with endogenous serotonin transporters (SERT) expressed in developing thalamocortical neurons is examined. Incubation of thalamocortical cultures with botulinum toxin C1, which specifically cleaves syntaxin 1A, decreased SERT function. Serotonin (<em>5HT</em>) saturation analysis showed that the effect of the toxin was to decrease maximum transport capacity with little change to the affinity of the transporter for <em>5HT</em>. The <em>5HT</em> uptake data were consistent with biotinylation experiments showing a decrease in the surface expression of SERT following toxin treatment. In addition, co-immunoprecipitation experiments showed that SERT and syntaxin 1A form a protein complex in these neurons. These data show that components of the transmitter release machinery interact with endogenously expressed amine transporters, and suggest a mechanism for the control of transmitter levels in disorders related to aminergic signaling.

An elevated plasma concentration of serotonin ([5-HT]) is a common feature of cardiovascular disease often associated with enhanced platelet activation and thrombosis. Whether elevated in vivo plasma 5-HT per se represents an independent risk factor for platelet hyperreactivity or only is an epiphenomenon of cardiovascular disease is poorly understood. We examined in vitro and in vivo platelet function following a 24h elevation of plasma [5-HT] in mice. In vivo administration of 5-HT using osmotic minipumps increased plasma [5-HT] in treated mice compared to control mice instrumented with saline loaded pumps. 5-HT infusion did not increase systolic blood pressure, but markers of platelet activation including P-selectin and (PE)Jon/A staining were increased and these findings coincided with the enhanced aggregation of isolated platelets in response to type I fibrillar collagen. Tail bleeding times and the time to occlusion following chemical damage to the carotid artery were shortened in 5-HT-infused mice. 5-HT-infused mice were treated with paroxetine (Prx) to block 5-HT uptake via the serotonin transporter (SERT). Prx lowered platelet [5-HT] and attenuated platelet activation and aggregation. These results and our biochemical indices of enhanced 5-HT intracellular signaling in the platelets of 5-HT-infused mice reveal a mechanistic link between elevated plasma [5-HT], abnormal intracellular 5-HT signaling and accentuated platelet aggregation. Although a down-regulation of the serotonin transporter (SERT) on the platelet surface may counteract the pro-thrombotic influence of elevated plasma [<em>5HT</em>], this compensatory mechanism may fail to prevent the increased thrombotic risk caused by elevated plasma [5-HT].

Neurons of the medullary raphe nuclei in cats were retrogradely labelled following injection of horseradish peroxidase (HRP) into the L6 spinal cord segment. Brainstems were cut in sagittal section to facilitate examination of the rostral-caudal extent of raphe neurons projecting to the spinal cord. Large numbers of HRP-labelled neurons were found in nucleus raphe magnus, nucleus raphe pallidus, and nucleus raphe obscurus (as well as a few neurons in nucleus raphe pontis). Dorsal or ventral hemisections at the T12-L1 level restricted HRP retrograde transport to those pathways within the intact portion of spinal cord, allowing a determination of the part of the cord through which raphe neurons project to the lumbar enlargement. Neurons of nucleus raphe magnus were found to project primarily in dorsolateral fasciculus. A significant number of neurons of nucleus reticularis gigantocellularis also project in dorsolateral fasciculus. Nucleus raphe obscurus neurons were found to project primarily in ventral funiculus, while nucleus raphe pallidus neurons project in the ventrolateral fasciculi and ventral funiculus. The serotonergic (<em>5HT</em>) fibers described by Dahlström and Fuxe ('65) to terminate in the dorsal horn, intermediolateral cell column, and ventral horn are likely to coincide with the raphe-spinal projections documented in this work.