To identify residues in the neuronal alpha7 acetylcholine subunit that confer high affinity for the neuronal-specific toxin conotoxin ImI (CTx ImI), we constructed alpha7-alpha1 chimeras containing segments of the muscle alpha1 subunit inserted into equivalent positions of the neuronal alpha7 subunit. To achieve high expression in 293 human embryonic kidney cells and formation of homo-oligomers, we joined the extracellular domains of each chimera to the M1 junction of the 5-hydroxytryptamine-3 (<em>5HT</em>-3) subunit. Measurements of CTx ImI binding to the chimeric receptors reveal three pairs of residues in equivalent positions of the primary sequence that confer high affinity of CTx ImI for alpha7/<em>5HT</em>-3 over alpha1/<em>5HT</em>-3 homo-oligomers. Two of these pairs, alpha7Trp55/alpha1Arg55 and alpha7Ser59/alpha1Gln59, are within one of the four loops that contribute to the traditional non-alpha subunit face of the muscle receptor binding site. The third pair, alpha7Thr77/alpha1Lys77, is not within previously described loops of either the alpha or non-alpha faces and may represent a new loop or an allosterically coupled loop. Exchanging these residues between alpha1 and alpha7 subunits exchanges the affinities of the binding sites for CTx ImI, suggesting that the alpha7 and alpha1 subunits, despite sequence identity of only 38%, share similar protein scaffolds.

Immunohistochemistry is a key tool for analyzing target molecule localization within tissues. However, accurate results require an antibody that can distinguish between similar compounds. We present a simple immunohistochemical method that can also be used to rapidly evaluate antibodies' specificities. We demonstrate this technique with serotonin, an extremely labile compound. Serotonin (5-hydroxytryptamine, <em>5HT</em>) is an important neurotransmitter regulating normal cognition and several mental disorders, as well as tumor growth, cardiopathology, and embryogenesis. Immunohistochemical detection of serotonin is commonly used as a neuronal cell marker and to provide crucial information on serotonin's role as an embryonic morphogen. It is necessary to be able to distinguish serotonin from closely related molecules with significantly different biological activity. Using our method, we identify antibodies that are specific for serotonin and show that some commercial <em>5HT</em> antibodies often used to identify serotonergic cells in published papers are not <em>5HT</em>-specific. These data demonstrate the necessity of specifically testing antibodies (especially in areas of high clinical relevance such as <em>5HT</em>). We also illustrate detection of serotonergic cells in embryonic tissue using our technique. This method offers a number of general advantages for testing specificity of antibodies to any biological molecule, and helps avoid false positives and negatives during immunohistochemistry.

Dual action antidepressants have important therapeutic implications. Methylene blue (MB), a charged compound structurally related to tricyclic antidepressants, acts on both monoamine oxidase (MAO) and the nitric oxide (NO)-cGMP pathway, and has demonstrated antidepressant activity in rodents. We investigated the antidepressant properties of MB and selected structural analogues and whether their actions involve MAO, NO synthase (NOS) and regional brain monoamines. Acute imipramine (IMI, 15 mg/kg), saline, MB, acriflavine (ACR), methylene green (MG), methylene violet (MV), thionine (THI) and tacrine (TAC) (1-60 mg/kg i.p.) were tested for antidepressant activity in the forced swim test (FST), as well as MAO-A/B inhibitory activity. Active antidepressant compounds were subsequently studied at their most effective dose during sub-chronic treatment, followed by behavioural sampling in the FST and assay of cortico-limbic monoamines and hippocampal nitrate (for NOS activity). Only IMI, MB (15, 30, 60 mg/kg) and MG (7.5, 25, 40 mg/kg) reduced immobility in the acute FST. MB, MG and ACR were potent inhibitors of especially MAO-A. Following sub-chronic treatment, IMI (15 mg/kg) increased noradrenergic behaviour in the FST, while MB (15 mg/kg) and MG (15 mg/kg) enhanced serotonergic behaviour. MB and MG bolstered cortico-limbic serotonin (<em>5HT</em>) levels and to a lesser extent l-norepinephrine (l-NE), but did not significantly alter regional dopamine (DA) levels. MB, and to lesser degree MG, reduced hippocampal nitrate levels. MB and MG present with structure-specific antidepressant-like effects following acute and sub-chronic treatment, possibly involving NOS and MAO-A inhibition and cortico-limbic <em>5HT</em> and l-NE release. A role for MAO-B and DA appears minimal.

Significant weight gain is a side effect associated with olanzapine treatment in some patients. We investigated the efficacy of high-dose fluoxetine as a weight-reducing agent for patients who develop early weight gain with olanzapine treatment. Patients that gained>>/=3% of their baseline weight in the initial 8 weeks of olanzapine treatment (n=31) were randomized to double-blind treatment with placebo or fluoxetine (60 mg/day). Clinical, weight, and weight-related measures were assessed. Fluoxetine failed to demonstrate weight-reducing effects (fluoxetine group: baseline mean 80.5 kg, SD=19.1, last mean=83.5 kg, SD=19.8; placebo group: baseline mean=77.1 kg, SD=12.1, last mean=78.8 kg, SD=10.6; F=1.3; df=1, 18; p=0.3). There were no differential effects in psychopathology, extrapyramidal side effects or weight-related measures between the placebo and fluoxetine groups. Serotonin reuptake inhibitors are probably not a practical option to counteract weight gain induced by atypical antipsychotics. Atypical-induced weight gain may result from mechanisms other than <em>5HT</em> reuptake blockade.

Bacopa monniera is a well-known medhya-rasayana (memory enhancing and rejuvenating) plant in Indian traditional medical system of Ayurveda. The effect of a standardized extract of Bacopa monniera (BESEB CDRI-08) on serotonergic receptors and its influence on other neurotransmitters during hippocampal-dependent learning was evaluated in the present study. Wistar rat pups received a single dose of BESEB CDRI-08 during postnatal days 15-29 showed higher latency during hippocampal-dependent learning accompanied with enhanced <em>5HT</em>(3A) receptor expression, serotonin and acetylcholine levels in hippocampus. Furthermore, <em>5HT</em>(3A) receptor agonist 1-(m-chlorophenyl)-biguanide (mCPBG) impaired learning in the passive avoidance task followed by reduction of <em>5HT</em>(3A) receptor expression, <em>5HT</em> and ACh levels. Administration of BESEB CDRI-08 along with mCPBG attenuated mCPBG induced behavioral, molecular and neurochemical alterations. Our results suggest that BESEB CDRI-08 possibly acts on serotonergic system, which in turn influences the cholinergic system through 5-HT(3) receptor to improve the hippocampal-dependent task.

Twenty patients receiving a variety of emetogenic cytotoxics (including cisplatin in 5) were given a single i.v. infusion of 40 micrograms kg-1 of BRL43694 (as the hydrochloride salt) in successive groups of 3-4 patients between 0-6 hours after chemotherapy. Eleven patients were completely protected from vomiting; 9 had mild to moderate nausea and vomiting, but none severe enough to require alternative anti-emetic 'rescue'. In 4 of the patients in whom BRL43694 was delayed until 4-6 h after chemotherapy, vomiting had already begun; in each case immediate termination of vomiting occurred when BRL43694 was infused. No adverse effects attributable to the anti-emetic were observed. Mean pharmacokinetic parameters in 14 patients in whom plasma assay data are available were: Maximum observed concentration = 30.7 ng ml-1; terminal phase half-life = 8.96 h; total body clearance = 0.376 (1 h-1) kg-1; apparent volume of distribution = 2.85 l kg-1. This study shows BRL43694 to be an effective and well tolerated anti-emetic. Further studies aimed at defining an optimal dose and schedule for use against the most emetogenic cytotoxics are in progress.

The concept of hypnogenic factor(s) which could increase during sleep deprivation was initiated by Piéron in 1913. However this idea was not accepted. It is difficult indeed to differentiate a true sleep inducing factor from the numerous exogeneous or endogeneous factors which may facilitate sleep onset, slow wave sleep (SWS) and/or paradoxical sleep (PS). At the present time there is no true sleep inducing factor for which it has been proven that its inactivation would led to long lasting selective insomnia. A review of the effect of central peptides upon the sleep waking cycle of rats does not permit to isolate any true "sleep" peptide. Only vaso-intestinal peptide (VIP) has some true hypnogenic effect since its injection is able to restore sleep in insomniac PCPA pretreated rats. The serotonergic system of the raphe was considered first as a true hypnogenic system since its inactivation by lesion or inhibition of <em>5HT</em> biosynthesis with PCPA led to a total insomnia which could be reversed into physiological sleep by a secondary injection of <em>5HT</em>P (the direct precursor of <em>5HT</em>). This hypothesis has been rejected recently. Indeed, it has been shown that the activity of <em>5HT</em> neurons was higher during waking than during sleep. Moreover the release of <em>5HT</em> and 5HIAA (as measured with voltametric method) is also higher during waking. The following hypothesis may solve these contradictions: when <em>5HT</em> neurons are active during waking, they initiate through neurohormonal mechanisms the biosynthesis of sleep factor(s) which are stored until they trigger sleep. Thus the amount of waking may be correlated with subsequent sleep. Recent experiments favor the hypothesis of the <em>5HT</em> directed biosynthesis of PS factor which may trigger PS even in PCPA pretreated cats which are depleted of <em>5HT</em>. On the one hand, PCPA injection during PS instrumental deprivation is followed by a subsequent PS rebound despite <em>5HT</em> depletion. On the other hand, transfer of CSF from a normal sleep deprived donor cat induces PS in a PCPA pretreated insomniac recipient cat.

We examined the relationship between hyperalgesia and degranulation of mast cells in rats following treatment with nerve growth factor (NGF) and inflammatory mediators. Intracutaneous injection to the rat's paw of NGF (50-5000 ng, 50 microl) evoked dose-dependent thermal hyperalgesia as measured by paw withdrawal latency to noxious heat. Even more intense hyperalgesia was induced by an inflammatory mediators soup' containing BK, <em>5HT</em>, PGE2 and histamine (10 microM). Counts of mast cells in the dermis were performed on coded slides under x 1000 magnification after fixation in Carnoy's solution and staining with toluidine blue. After injection of three different concentrations of NGF (50, 500, 5000 ng in 50 microl), the proportion of mast cells that were degranulated was significantly higher (P < 0.001) than after saline injection. No significant differences were found among the three concentrations of NGF. The effect of inflammatory soup on the proportion of degranulated mast cells was similar to that of NGF. These results indicate that NGF activates cutaneous mast cells and stimulates the release of inflammatory tissue mediators in the acute phase of NGF-induced hyperalgesia in the rat's paw. However, the finding that degranulation was not dose-dependent indicates that other mechanisms, independent of mast cells, are probably operating in response to NGF.

OBJECTIVE

This study aims to clarify the relationship between blood Pb(2+) concentration as a ubiquitous environmental pollutant and plasma neurotransmitters as biochemical parameters that reflect brain function in Saudi autistic patients.

METHODS

RBC's lead content together with plasma concentration of gamma aminobutyric acid (GABA), serotonin (<em>5HT</em>) and dopamine (DA) were measured in 25 Saudi autistic patients and compared to 16 age-matching control samples.

RESULTS

The obtained data recorded that Saudi autistic patients have a remarkable higher levels of Pb(2+) and significantly elevated levels of GABA, <em>5HT</em> and DA compared to healthy subjects. ROC analysis revealed satisfactory values of specificity and sensitivity of the measured parameters.

CONCLUSIONS

This study suggests that postnatal lead toxicity in autistic patients of Saudi Arabia could represent a causative factor in the pathogenesis of autism. Elevated GABA, <em>5HT</em> and DA were discussed in relation to the chronic lead toxicity recorded in the investigated autistic samples.

1. Liver and kidney extract adrenaline and noradrenaline from the circulation by a mechanism which does not seem to be one of the classical catecholamine transporters. The hypothesis that OCT1 is involved the organic cation transporter type 1 which exists in rat kidney and liver-was tested. 2. Based on human embryonic kidney cells (293), we constructed a cell line which stably expresses OCT1r (293OCT1r cells). Transfection with OCT1 resulted in a transport activity not only for prototypical known substrates of OCT1 such as 3H-1-methyl-4-phenylpyridinium and 14C-tetraethylammonium but also for the catecholamines 3H-adrenaline, 3H-noradrenaline (3H-NA) and 3H-dopamine (3H-DA), the indoleamine 3H-5-hydroxytryptamine (3H-<em>5HT</em>) as well as the indirect sympathomimetic 14C-tyramine. 3. For 3H-DA, 3H-<em>5HT</em> and 3H-NA, at non-saturating concentrations, the rate constants for inwardly directed substrate flux (kin) were 6.9+/-0.8, 3.1+/-0.2, and 1.2+/-0.1 microl min(-1) mg protein(-1). In wild type cells (293WT) the corresponding kin's were considerably lower, being 0.94+/-0.40, 0.47+/-0.08 and 0.23+/-0.05 microl min(-1) mg protein ' (n=12). The indirectly determined half-saturating concentrations of DA, <em>5HT</em>, and NA were 1.1 (95% c.i.: 0.8, 1.4), 0.65 (0.49, 0.86), and 2.8 (2.1, 3.7) mmol l(-1) (n=3). 4. Specific 3H-DA uptake in 293OCT1r cells was resistant to cocaine (1 micromoll(-1)), 3H-<em>5HT</em> uptake was resistant to citalopram (300 nmol l(-1)) and 3H-NA uptake was resistant to desipramine (100 nmoll(-1)), corticosterone (1 micromol l(-1)), and reserpine (10 nmoll(-1)) which rules out the involvement of classical transporters for biogenic amines. 5. The findings demonstrate that OCTI efficiently transports catecholamines and other biogenic amines and support the hypothesis that OCT1 is responsible for hepatic and renal inactivation of circulating catecholamines.

The current study aimed to elucidate the role of pharmacokinetic (PK) parameters and neurotransmitter efflux in explaining variability in (±) 3, 4-methylenedioxymethamphetamine (MDMA) self-administration in rats. PK profiles of MDMA and its major metabolites were determined after the administration of 1.0 mg/kg MDMA (iv) prior to, and following, the acquisition of MDMA self-administration. Synaptic levels of 5-hydroxytryptamine (<em>5HT</em>) and dopamine (DA) in the nucleus accumbens were measured following administration of MDMA (1.0 and 3.0 mg/kg, iv) using in vivo microdialysis and compared for rats that acquired or failed to acquire MDMA self-administration. Effects of the <em>5HT</em> neurotoxin, 5,7 dihydroxytryptamine (5, 7-DHT), on the acquisition of MDMA and cocaine self-administration were also determined. In keeping with previous findings, approximately 50% of rats failed to meet a criterion for acquisition of MDMA self-administration. The PK profiles of MDMA and its metabolites did not differ between rats that acquired or failed to acquire MDMA self-administration. MDMA produced more overflow of <em>5HT</em> than DA. The MDMA-induced <em>5HT</em> overflow was lower in rats that acquired MDMA self-administration compared with those that did not acquire self-administration. In contrast, MDMA-induced DA overflow was comparable for the two groups. Prior 5,7-DHT lesions reduced tissue levels of <em>5HT</em> and markedly increased the percentage of rats that acquired MDMA self-administration and also decreased the latency to acquisition of cocaine self-administration. These data suggest that <em>5HT</em> limits the initial sensitivity to the positively reinforcing effects of MDMA and delays the acquisition of reliable self-administration.

Neuromodulators modify network output by altering neuronal firing properties and synaptic strength at multiple sites; however, the functional importance of each site is often unclear. We determined the importance of monoamine modulation of a single synapse for regulation of network cycle frequency in the oscillatory pyloric network of the lobster. The pacemaker kernel of the pyloric network receives only one chemical synaptic feedback, an inhibitory synapse from the lateral pyloric (LP) neuron to the pyloric dilator (PD) neurons, which can limit cycle frequency. We measured the effects of dopamine (DA), octopamine (Oct), and serotonin (<em>5HT</em>) on the strength of the LP→PD synapse and the ability of the modified synapse to regulate pyloric cycle frequency. DA and Oct strengthened, whereas <em>5HT</em> weakened, LP→PD inhibition. Surprisingly, the DA-strengthened LP→PD synapse lost its ability to slow the pyloric oscillations, whereas the <em>5HT</em>-weakened LP→PD synapse gained a greater influence on the oscillations. These results are explained by monoamine modulation of factors that determine the firing phase of the LP neuron in each cycle. DA acts via multiple mechanisms to phase-advance the LP neuron into the pacemaker's refractory period, where the strengthened synapse has little effect. In contrast, <em>5HT</em> phase-delays LP activity into a region of greater pacemaker sensitivity to LP synaptic input. Only Oct enhanced LP regulation of cycle period simply by enhancing LP→PD synaptic strength. These results show that modulation of the strength and timing of a synaptic input can differentially affect the synapse's efficacy in the network.

Radioligand binding studies have revealed four distinct serotonin (<em>5HT</em>) binding sites in rat brain that are thought to function as <em>5HT</em> receptors. These include the <em>5HT</em>-1a, <em>5HT</em>-1b, <em>5HT</em>-1c, and <em>5HT</em>-2 binding sites. Studies have shown that the <em>5HT</em>-2 binding site mediates a number of effects of <em>5HT</em> agonists and serves as a <em>5HT</em> receptor in neuronal and non-neuronal tissues. The <em>5HT</em>-2 site employs phosphoinositide hydrolysis for signal transduction. The <em>5HT</em>-1c binding site is also a functional receptor that is linked to phosphoinositide hydrolysis. However, the physiological role of the <em>5HT</em>-1c receptor is not yet known. Lack of appropriate pharmacological tools for probing the <em>5HT</em>-1a and <em>5HT</em>-1b binding sites has made it difficult to definitively determine whether these binding sites are coupled to biochemical effector systems or mediate any of the physiological responses to <em>5HT</em> agonists. However, there is some evidence that the <em>5HT</em>-1a site is coupled to adenylate cyclase, and a number of functional roles for the <em>5HT</em>-1a and <em>5HT</em>-1b sites have been proposed.

Serotonin (<em>5HT</em>) has significant effects on renal metabolism and glomerular function and is a potent renal vasoconstrictor. In this study we describe and localize a highly active biosynthetic pathway for serotonin in the kidney. Rat kidneys were dissected into cortical and medullary fractions; in some experiments the cortex was also separated into subfractions enriched with glomeruli or proximal tubules. Serotonin and tryptophan hydroxylase (TyOH) were measured by radioenzymatic techniques. (table; see text) Renal denervation did not alter tryptophan hydroxylase activity. In kidneys from human cadaveric donors, cortical tryptophan hydroxylase (4.13 +/- 0.68 nM/30 min/g) exceeded that in the medulla (1.96 +/- 0.86 nM/30 min/g). Aromatic L-amino acid decarboxylase, the remaining enzyme for serotonin synthesis, is present in both rat renal cortex and medulla; however, we found 15-fold greater decarboxylase activity in proximal tubular (2070 nM/30 min/g) as compared to glomerular (131 nM/30 min/g) subfractions. We were able to demonstrate that under physiological conditions, free urine serotonin reflects actual biosynthesis by the kidney. Thus, although serotonin stores retained by the kidney appear small and relatively localized to the medulla, the enzymatic activity for the synthesis of serotonin in the kidney is comparable to that in the brain, with the complete pathway localized to renal cortical proximal tubules. These data suggest that further studies of renal serotonin metabolism may contribute to our understanding of renal function in health and disease.

5,7-Dihydroxytryptamine lesions of the nucleus accumbens septi, or substantia nigra, resulted in a twofold increase in spontaneous locomotor activity. Striatal <em>5HT</em> depletion also raised basal activity levels, as well as increasing rearing behaviour in an open field. The sterotyped responses to all doses of amphetamine tested (2.5--10 mg/kg, i.p.) were enhanced by lesions of the nucleus accumbens or substantia nigra. Striatal lesions only affected the response to the lowest dose of amphetamine. Lesions of the tuberculum olfactorium were without effect on spontaneous or amphetamine induced responses. The results support the concept of a modulatory <em>5HT</em> influence on nigro-striatal function, and suggest that <em>5HT</em> in the nucleus accumbens has an antagonistic role with respect to dopamine function in this site.

The dopamine D1-receptor antagonist SCH 23390 was a potent competitive antagonist of <em>5HT</em>-induced vasoconstriction in the isolated perfused rat tail artery preparation (pA2 8.17) but a very weak antagonist of phenylephrine-induced responses (pA2 5.94). In rat brain cerebral cortex, SCH 23390 inhibited 5-HT2-sensitive [3H]spiperone binding with an IC50 of 112 nM. Binding of [3H]<em>5HT</em> to <em>5HT</em>1 receptors in the cortex was inhibited by SCH 23390 with an IC50 of 2.49 microM. SCH 23390 has significant affinity for <em>5HT</em> receptors in addition to the reported selective dopamine D1-receptor antagonist properties.

The circadian rhythm of melatonin production (high melatonin levels at night and low during the day) in the mammalian pineal gland is modified by visible portions of the electromagnetic spectrum, i.e., light, and reportedly by extremely low frequency (ELF) electromagnetic fields as well as by static magnetic field exposure. Both light and non-visible electromagnetic field exposure at night depress the conversion of serotonin (<em>5HT</em>) to melatonin within the pineal gland. Several reports over the last decade showed that the chronic exposure of rats to a 60 Hz electric field, over a range of field strengths, severely attenuated the nighttime rise in pineal melatonin production; however, more recent studies have not confirmed this initial observation. Sinusoidal magnetic field exposure also has been shown to interfere with the nocturnal melatonin forming ability of the pineal gland although the number of studies using these field exposures is small. On the other hand, static magnetic fields have been repeatedly shown to perturb the circadian melatonin rhythm. The field strengths in these studies were almost always in the geomagnetic range (0.2 to 0.7 Gauss or 20 to 70 mu tesla) and most often the experimental animals were subjected either to a partial rotation or to a total inversion of the horizontal component of the geomagnetic field. These experiments showed that several parameters in the indole cascade in the pineal gland are modified by these field exposures; thus, pineal cyclic AMP levels, N-acetyltransferase (NAT) activity (the rate limiting enzyme in pineal melatonin production), hydroxyindole-O-methyltransferase (HIOMT) activity (the melatonin forming enzyme), and pineal and blood melatonin concentrations were depressed in various studies. Likewise, increases in pineal levels of <em>5HT</em> and 5-hydroxyindole acetic acid (5HIAA) were also seen in these glands; these increases are consistent with a depressed melatonin synthesis. The mechanisms whereby non-visible electromagnetic fields influence the melatonin forming ability of the pineal gland remain unknown; however, the retinas in particular have been theorized to serve as magnetoreceptors with the altered melatonin cycle being a consequence of a disturbance in the neural biological clock, i.e., the suprachiasmatic nuclei (SCN) of the hypothalamus, which generates the circadian melatonin rhythm. The disturbances in pineal melatonin production induced by either light exposure or non-visible electromagnetic field exposure at night appear to be the same but whether the underlying mechanisms are similar remains unknown.

In a previous study we showed that selective occupation of the mineralocorticoid receptor (MR) in hippocampal slices from adrenalectomized (ADX) rats attenuates the membrane hyperpolarization and resistance decrease induced in CA1 pyramidal neurons by serotonin (<em>5HT</em>). In the present study we established responses to <em>5HT</em> in the hippocampal slice when not only MRs but also glucocorticoid receptors (GRs) were occupied, using either a combination of selective MR and GR ligands or different concentrations of the endogenous mixed agonist corticosterone. We observed that the GR agonist RU 28362 blocks the attenuating action of the MR agonist aldosterone on responses to 3, 10 and 30 microM <em>5HT</em>; RU 28362 by itself did not affect <em>5HT</em> responses. If a low concentration of the mixed agonist corticosterone (0.5 nM, close to the Kd for the MR) was continuously perfused in vitro, <em>5HT</em> responses were steadily depressed with a delay of 2 h, while high levels of corticosterone (5 nM, around Kd for GR) only temporarily reduced <em>5HT</em> responses. Finally, <em>5HT</em> responses in slices from sham-operated rats (with relatively high plasma corticosterone levels) were similar to the responses obtained in slices from ADX rats. These data suggest that the previously reported MR-mediated attenuation of <em>5HT</em> responses may be limited to conditions of low adrenocortical activity or pathophysiological conditions where the balance of MR- and GR-mediated effects is disturbed.

BACKGROUND

Kappa agonists can attenuate reinstatement of cocaine-seeking behavior induced by cocaine priming. The mechanisms underlying this effect have not been characterized fully but may have a serotonergic component as kappa agonists also increase the release of serotonin (5-hydroxytryptamine, 5-HT).

OBJECTIVE

This study investigated the role of kappa opioid receptor and 5-HT mechanisms in kappa agonist-induced attenuation of cocaine priming in monkeys.

METHODS

Squirrel monkeys were trained to self-administer cocaine (0.18-0.3 mg/kg/injection) under a second-order schedule in which drug seeking was maintained jointly by cocaine injections and a cocaine-paired visual stimulus. In extinction sessions, saline was substituted for cocaine, and the cocaine-paired stimulus was omitted. During test sessions, only saline was available for self-administration, and response-contingent presentations of the cocaine-paired stimulus were restored.

RESULTS

Priming injections of cocaine (0.1-1.0 mg/kg) induced reinstatement of drug seeking. Maximal levels of responding were similar to those maintained by active cocaine self-administration. Pretreatment with the kappa agonists enadoline (0.01 mg/kg) and spiradoline (0.3 mg/kg) or the 5-HT transport inhibitors fluoxetine (5.6 mg/kg) and citalopram (10.0 mg/kg) attenuated the priming effects of cocaine, shifting the cocaine dose-response function rightward and downward. Inhibition of cocaine-induced reinstatement of drug seeking by spiradoline and fluoxetine was reversed by R(+)8-hydroxy-2-(di-n-propylamino)tetralin (0.03 mg/kg), a <em>5HT</em>(1A) agonist that inhibits 5-HT release. The effects of spiradoline also were reversed by the kappa antagonist nor-binaltorphimine (10.0 mg/kg).

CONCLUSIONS

Results suggest that the capacity of kappa opioid agonists to increase extracellular 5-HT levels may at least partially underlie kappa agonist-induced modulation of cocaine seeking.

A full-length cDNA encoding a 5-hydroxytryptamine (5-HT) receptor from the Southern cattle tick, Boophilus microplus, was isolated using a strategy based on sequence homology among G protein-coupled receptors. The deduced amino acid sequence revealed highest identity with Drosophila melanogaster <em>5HT</em>-dro2A (Z11489, 50.8%) and <em>5HT</em>-dro2B (Z11490, 49.5%) receptors. The receptor was transiently expressed in mammalian HEK293 cells, and Western blot analysis showed the expected 43.3 kDa band. In these cells, application of 5-HT (10 microm) inhibited forskolin-induced cAMP synthesis by 26%. The results indicate that the tick receptor is an invertebrate 5-HT1-like receptor that couples to Galphai protein.

Increased platelet serotonin level (PSL) has been consistently found in a portion of autistic patients. Suggested mechanisms for hyperserotonemia in autism have been increased synthesis of serotonin (<em>5HT</em>) by tryptophan hydroxylase (TPH), increased uptake into platelets through <em>5HT</em> transporter (<em>5HT</em>t), diminished release from platelets through <em>5HT</em>2A receptor (<em>5HT</em>2Ar) and decreased metabolism by monoamine oxydase (MAOA). The allelic influence of genes, encoding the mentioned <em>5HT</em> elements, on PSL was investigated in 63 autistic subjects. Our study shows that <em>5HT</em>t-LPR and -1438AG <em>5HT</em>(2Ar) genotypes did not significantly affect PSL. However, significantly higher PSLs were observed in subjects with "cc" genotype of a218c TPH and subjects with "4" genotype of uVNTR MAOA. In addition, when TPH-cc and MAOA-4 were combined as "high <em>5HT</em>" genotypes, a correlative increase in PSL was observed with the increase in the number of "high <em>5HT</em>" genotypes. These results suggest a possible synergistic effect of genes regulating <em>5HT</em> synthesis/degradation in dysregulation of the peripheral <em>5HT</em> homeostasis of autistic patients.

The RN46A cell line was derived from embryonic day 13 rat medullary raphe cells by infection with a retrovirus encoding the temperature-sensitive mutant of SV40 large T antigen. This cell line is neuronally restricted and constitutively differentiates following a shift to non-permissive temperature. Brain-derived neurotrophic factor (BDNF) induced the serotonergic phenotype and increased the survival of RN46A cells in vitro. After transfection of the rat BDNF gene into RN46A cells, an autocrine BDNF-secreting cell line, 46A-B14, was isolated and transplanted into the rat CNS. Transplanted 46A-B14 cells had increased survival and enhanced serotonin (<em>5HT</em>) synthesis compared to 46A-V1 cells, RN46A cells transfected with vector-alone. When 46A-B14 cells were transplanted in the lumbar subarachnoid space of the spinal cord 1 week after a chronic constriction injury (CCI) of the sciatic nerve, they survived longer than 6 weeks on the pia mater. Furthermore, the tactile and cold allodynia and thermal hyperalgesia induced by CCI was significantly reduced during a 4-6- week period. The maximal effect occurred 1 week after transplantation. 46A-V1 cells, transplanted after CCI, did not survive beyond 2-3 weeks and had no effect on the allodynia and hyperalgesia induced by CCI. Acute intrathecal injection of the <em>5HT</em> receptor antagonist methysergide decreased the antinociceptive effects of the 46A-B14 cells to pre-transplant levels. These data suggest that a chronically applied, low local dose of serotonin near the dorsal horn was able to reverse the development of chronic neuropathic pain following CCI. The use of neural cell lines that are able to deliver inhibitory neurotransmitters such as serotonin, in a model of chronic pain offers a novel approach to pain management.

The stereochemical and pharmacological properties of yohimbine and some of its isomers are briefly reviewed. Several pharmacological and physical properties of a selection of the isomers have been determined with a view to elucidating which might be important in the elaboration of the known behavioral effects produced by them. Activity is not dependent upon lipid solubility or on the ease of access to the central nervous system. The isomers are weak inhibitors of rat-brain acetylcholinesterase and weak antagonists at muscarinic cholinergic receptors. In the rat brain in vitro they do not possess significant monoamine oxidase-inhibiting properties nor do they inhibit the uptake of serotonin. They are relatively potent antagonists of <em>5HT</em> on the rat isolated fundus preparation and their potency in this preparation may be related to their ability to produce behavioral and cardiovascular effects in man and dogs.

In Xenopus oocytes expressing neuronal nicotinic acetylcholine receptors (nAcChoRs), made up of alpha 2 and beta 4 subunits, acetylcholine (AcCho) elicited ionic membrane currents (AcCho currents) that were modulated by serotonergic agents. Both agonists and antagonists specific for various serotonin (5-hydroxytryptamine, <em>5HT</em>) receptor subtypes interacted directly with alpha 2 beta 4 nAcChoRs: <em>5HT</em>, (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin, methysergide, spiperone, and ketanserin reversibly reduced the amplitude of AcCho currents and accelerated their decay. The AcCho-current time course decayed with two exponential functions. In the presence of <em>5HT</em>, the fast time constant of current decay (tau f) was not greatly modified, but the slow time constant (tau s) was reduced. With AcCho and <em>5HT</em> both at 100 microM, tau s was reduced from 140 s to 85 s. The order of potency for inhibition of AcCho current amplitudes was (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin>> methysergide>> spiperone>> ketanserin>> <em>5HT</em>. The inhibition was voltage-dependent but the magnitude of the voltage dependence for the different blockers did not correspond to their blocking potency: e.g., the block with spiperone was stronger than with <em>5HT</em>, but it was less voltage-dependent. Our results suggest that serotonergic agents block neuronal nAcChoRs in a noncompetitive manner, similar to the block of muscle nAcChoR by curare and other substances. These results show that neuronal nAcChoR channels that have been activated by their specific neurotransmitter may be modulated by nonspecific neurotransmitters and their antagonists. These effects may help to better understand brain functions as well as the mode of action of the many serotonergic agents that are used in medical practice.