Sound-induced seizures in serotonin 5-HT2c receptor mutant mice

OBJECTIVES: In previous University of California, San Diego (UCSD) studies, nocturnal illumination shortened menstrual cycles that were longer than 33 days. The studies reported here extend the previous findings, confining the illumination to the sleep period. DESIGN: Two light levels (235 to 250 lux and less than 1 lux) and 2 modes of light delivery (lighted sleep mask and bedside lamp) were tested. RESULTS: 235 to 250 lux treatment cycle lengths were significantly shorter than baseline, but not significantly shorter than the less than 1 lux treatment cycle lengths. Subjective reports of sleep disturbance were greater with the 235 to 250 lux treatment, but there was no significant difference in overall quality of sleep between the two light levels. CONCLUSIONS: The current data alone do not exclude spontaneous remission or suggestion, but our previous studies demonstrated significant contrasts between 235 to 250 lux and less than 1 lux light levels. This study suggests that treatment may be effective when confined to the sleep period, and that light masks, which do not disturb bed partners, may be used in place of bedside lamps.     

Perturbed dentate gyrus function in serotonin 5-HT2C receptor mutant mice

Serotonin systems have been implicated in the regulation of hippocampal function. Serotonin 5-HT2C receptors are widely expressed throughout the hippocampal formation, and these receptors have been proposed to modulate synaptic plasticity in the visual cortex. To assess the contribution of 5-HT2C receptors to the serotonergic regulation of hippocampal function, mice with a targeted 5-HT2C-receptor gene mutation were examined. An examination of long-term potentiation at each of four principal regions of the hippocampal formation revealed a selective impairment restricted to medial perforant path-dentate gyrus synapses of mutant mice. This deficit was accompanied by abnormal performance in behavioral assays associated with dentate gyrus function. 5-HT2C receptor mutants exhibited abnormal performance in the Morris water maze assay of spatial learning and reduced aversion to a novel environment. These deficits were selective and were not associated with a generalized learning deficit or with an impairment in the discrimination of spatial context. These results indicate that a genetic perturbation of serotonin receptor function can modulate dentate gyrus plasticity and that plasticity in this structure may contribute to neural mechanisms underlying hippocampus-dependent behaviors.     

Alcohol-heightened aggression in mice: attenuation by 5-HT1A receptor agonists

One of the critical mechanisms by which alcohol heightens aggression involves forebrain serotonin (5-HT) systems, possibly via actions on 5-HT1A receptors. The present experiments tested the hypothesis that activating 5-HT1A receptors by selective agonists will block the aggression-heightening effects of ethanol. Initially, the selective antagonist WAY 100635 was used to assess whether or not the changes in aggressive behavior after treatment with 8-OH-DPAT and flesinoxan result from action at the 5-HT1A receptors. Resident male CFW mice engaged in aggressive behavior (i.e. attack bites, sideways threats, tail rattle) during 5-min confrontations with a group-housed intruder male. Quantitative analysis of the behavioral repertoire revealed systematic reductions in all salient elements of aggressive behavior after treatment with 8-OH-DPAT (0.1-0.3 mg/kg, i.p.) or flesinoxan (0.1-1.0 mg/kg, i.p.). The 5-HT1A agonists also reduced motor activities such as walking, rearing and grooming, although to a lesser degree. Pretreatment with the antagonist WAY 100635 (0.1 mg/kg, i.p.) shifted the agonist dose-effect curves for behavioral effects to the right. In a further experiment, oral ethanol (1.0 g/kg, p.o.) increased the frequency of attacks in excess of 2 SD from their mean vehicle level of attacks in 19 out of 76 resident mice. Low doses of 8-OH-DPAT (0.03-0.3 mg/kg) and flesinoxan (0.1, 0.3, 0.6 mg/kg), given before the ethanol treatment, attenuated the alcohol-heightened aggression in a dose-dependent fashion. By contrast, these low 5-HT1A agonist doses affected motor activity in ethanol-treated resident mice to a lesser degree, suggesting behavioral specificity of these anti-aggressive effects. The current results support the hypothesized significant role of 5-HT1A receptors in the aggression-heightening effects of alcohol. If these effects are in fact due to action at somatodendritic 5-HT1A autoreceptors, then the anti-aggressive effects would be associated with decreased 5-HT neurotransmission.     

Hippocampal 5-HT1A receptor binding site densities, 5-HT1A receptor messenger ribonucleic acid abundance and serotonin levels parallel the activity of the hypothalamo-pituitary-adrenal axis in rats

We have previously demonstrated that susceptibility of the Lewis rat to inflammatory disease, compared to the relatively resistant Fischer F344 rat, is related to a hyporesponsive hypothalamopituitary adrenal axis to inflammatory and other stress mediators. Since 5-HT and the 5HT1A receptor are important stimulators of this axis, we have investigated the levels of 5-HT1A receptor binding sites and encoding mRNA, 5-HT and 5-hydroxyindole acetic acid in various brain regions of Lewis, Harlan Sprague Dawley and Fischer F344 rats. Lewis rats expressed significantly less hippocampal and frontal cortical 5-HT1A receptor binding sites and mRNA than Harlan Sprague-Dawley and Fischer F344 rats. Adrenalectomy increased the number of 5HT1A receptor binding sites and mRNA expression in the hippocampus of all three strains. The levels of hippocampal 5-HT in Fischer F344 rats were significantly greater than levels detected in the same regions for the other two strains. Hypothalamic 5-HT and 5-hydroxyindole acetic acid levels in Harlan Sprague-Dawley rats were higher than the same area from the other two strains. Adrenalectomy increased the levels of 5-hydroxyindole acetic acid in the hypothalamus of all three strains. We conclude that hippocampal 5-HT1A receptor densities and 5-HT levels in the rat parallel the the activity and responsiveness of the hypthalamopituitary-adrenal axis. We have published these data in an earlier report.     

Differential effects of serotonin (5-HT) lesions and synthesis blockade on neuropeptide-Y immunoreactivity and 5-HT1A, 5-HT1B/1D and 5-HT2A/2C receptor binding sites in the rat cerebral cortex

The present study was aimed at comparing the effects of serotonin (5-HT) synthesis blockade using chronic administration of p-chlorophenylalanine (PCPA) and 5,7-dihydroxytryptamine injections of variable volume (3 vs. 6 microl) on the density of NPY immunoreactive (Ir) neurons and binding of [3H]8-OH-DPAT, S-CM-G[125I]TNH2 and [125I]DOI to 5-HT1A, 5-HT1B/1D, and 5-HT2A/2C receptors in rat cortical regions. Three weeks after large but partial (89% depletion in 5-HT tissue concentration) lesions of 5-HT neurons no changes in neither NPY immunoreactivity nor 5-HT receptor binding were detected. The complete 5,7-DHT lesions produced increases in the number of NPY-Ir neurons in the upper regions of the cingular (134%), frontal (140%) and parietal cortex (48%) and corresponding decreases in 5-HT2A/2C binding (16-26%). No changes in 5-HT1A and 5-HT1B/1D binding were observed after lesions of this kind. After PCPA treatment, decreases in NPY-Ir neurons density (22-40%) and increases in 5-HT1A and 5-HT1B/1D receptor binding sites (20-50%) were distributed in both upper and deeper cortical regions. The lack of effect of the partial lesion suggests that spared 5-HT neurons may exert compensatory mechanisms up to a large extent. The changes in NPY immunoreactivity and 5-HT2A/2C binding detected in the upper regions of the cortex after complete 5-HT lesions probably result from local cellular rearrangements, whereas blocking 5-HT synthesis has more widespread influence on NPY neurons and on 5-HT1A and 5-HT1B/1D receptor subtypes. Moreover, decreases in DOPAC concentrations detected only after complete lesions suggest that the involvement of catecholaminergic transmission may also differentiate 5,7-DHT and PCPA treatments. Altogether, these data suggest that different receptor subtypes might be involved in 5-HT-NPY relationships.     

Hypothalamic paraventricular nucleus lesions differentially affect serotonin-1A (5-HT1A) and 5-HT2 receptor agonist-induced oxytocin, prolactin, and corticosterone responses

A number of receptor subtypes mediate hormonal responses to serotonin (5-HT). To test the hypothesis that the hypothalamic paraventricular nucleus (PVN) mediates 5-HT1A and 5-HT2 receptor-mediated oxytocin, PRL, and corticosterone responses, we studied the effects of the 5-HT1A agonist ipsapirone and the 5-HT2A/2C agonist 1-(2,5-dimethoxy-4-iodophenyl)2-aminopropane (DOI) after surgical PVN lesions or sham operations. Chronically cannulated, conscious, freely moving, male Wistar rats were injected iv (1 mg/kg) shortly after (3-4 days) and 5 weeks after (35-37 days) the operations. In sham-operated rats, ipsapirone caused marked elevations in plasma PRL and corticosterone, but not oxytocin concentrations, whereas DOI increased plasma concentrations of all three hormones. Short term PVN lesions prevented ipsapirone-induced corticosterone and DOI-induced oxytocin responses. DOI-induced PRL and corticosterone responses were also markedly inhibited 3-4 days after lesioning, although small rises over the baseline values were still observed. The ipsapirone-induced PRL response was unaffected by the lesioning. Five weeks after PVN lesioning, partial recoveries were observed in ipsapirone- and DOI-induced corticosterone and DOI-induced oxytocin responses, whereas DOI-induced PRL responses remained suppressed. The present findings suggest that the PVN or neural pathways close to it mediate oxytocin, PRL, and corticosterone responses to the 5-HT2 receptor agonist DOI as well as corticosterone, but not PRL, responses to the 5-HT1A receptor agonist ipsapirone. The results after long term PVN lesioning show that the oxytocin and corticosterone responses may be partially restored with time after lesioning.     

Differential inhibition of stress-induced adrenocortical responses by 5-HT1A agonists and by 5-HT2 and 5-HT3 antagonists

The present studies were designed to determine the effects of 5-HT1A receptor agonists and 5-HT2A/2C and 5-HT3 antagonists on adrenocortical responses to a variety of stress paradigms in conscious male rats. The following stressors were examined: acoustic stress (105 dB for 2 min); foot shock (0.2 mA, five shocks over 5 min); conditioned fear (animals placed in the foot shock chamber for 5 min, 24 h after foot shock); restraint (Plexiglas restrainer for 5 min); injection of recombinant human interleukin-1 alpha (IL-1, 20 micrograms/kg, IP); injection of cocaine hydrochloride (20 mg/kg, IP). Drug treatments consisted of intracerebroventricular (ICV) or intraperitoneal (IP) injections of the 5-HT1A agonists, 8-OH-DPAT and ipsapirone (0.1 pmol, ICV), the 5-HT2A/2C antagonist, ketanserin (2 mumol/kg, IP), and the 5-HT3 antagonist, MDL-72222 (20 nmol, ICV). The plasma corticosterone (CS) responses to foot shock and restraint stress were not affected by any of the serotonergic drugs tested. The 5-HT1A agonist, 8-OH-DPAT, was able to attenuate the adrenocortical responses to acoustic stimulation, conditioned fear, IL-1 alpha, and cocaine administration, with ipsapirone also being effective in reducing the responses to acoustic stimulation and cocaine injection. The 5-HT2 antagonist, ketanserin was able to reduce the adrenocortical response in the conditioned fear paradigm and the response to IL-1 alpha injection. The 5-HT3 antagonist, MDL-72222 was only effective in reducing the response to acoustic stimulation. Thus, adrenocortical responses to each of the applied stressors were differentially affected by the 5-HT receptor ligands tested. The results of this study indicate that 5-HT1A agonists may be efficient stress response-reducing agents. However, their efficacy depends on the lack of a somatosensory component to the applied stressor and their agonist properties suggest that their action may not involve direct effects on serotonergic pathways mediating the observed responses. In contrast, the specificity of the 5-HT2 and 5-HT3 antagonists in blocking adrenocortical responses to certain stressors suggests that these drugs exert their effects by blocking serotonergic neurotransmission in pathways mediating the adrenocortical responses to specific stimuli.     

Head and whole-body jerking in guinea pigs are differentially modulated by 5-HT1A, 5-HT1B/1D and 5-HT2A receptor antagonists

The effects of a serotonin (5-HT) releasing drug, p-chloroamphetamine, on plasma glucose levels were investigated in rats. p-Chloroamphetamine elicited a significant hyperglycemia. The hyperglycemic effects of p-chloroamphetamine were completely prevented by the 5-HT synthesis inhibitor, p-chlorophenylalanine. Prior adrenodemedullation abolished the hyperglycemia elicited by p-chloroamphetamine. p-Chloroamphetamine-induced hyperglycemia was prevented by methysergide, which blocks the 5-HT1 and 5-HT2 receptor, the 5-HT1A/1B/2C receptor antagonist, (-)-propranolol, the selective 5-HT1A receptor antagonist, 4-(2'-methoxyphenyl-1-[2'-n-2"pyridinyl)-p-iodobenzamido]-ethyl-pi perazine (p-MPPI), the 5-HT2A/2B/2C receptor antagonists, ritanserin and 4-isopropyl-7-methyl-9-(2-hydroxy-1-methyl-propoxycarbonyl)-4,6A,7 ,8,9,10,10A-octahydro-indolo[4,3-FG]quinolone maleate(LY 53857). However, the 5-HT3 and 5-HT4 receptor antagonist, tropisetron, the 5-HT4 receptor antagonist, 2-methoxy-4-amino-5-chloro-benzoic acid 2-(diethylamino) ethyl ester (SDZ 205-557), and the 5-HT2A receptor antagonist, ketanserin, did not affect the p-chloroamphetamine-induced hyperglycemia. These results suggest that p-chloroamphetamine-induced hyperglycemia is elicited by an enhanced 5-HT release and facilitated adrenaline release. Moreover, our results indicate that p-chloroamphetamine-induced hyperglycemia is mediated by 5-HT1A and 5-HT2B/2C receptors.     

Linkage of antisocial alcoholism to the serotonin 5-HT1B receptor gene in 2 populations

We examined the effects of repeated administration of (S)-alpha-fluoromethylhistidine (FMH), a specific inhibitor of histidine decarboxylase (HDC), on radial maze performance and brain contents of histamine and amino acids in rats. By daily subcutaneous (s.c.) administration of FMH (100 mg/kg), rats showed significant enhancement of a radial maze performance without changes in locomotion. Six days after FMH treatment, the histamine levels both in the cerebral cortex and diencephalon decreased significantly. However, the glutamate and glycine levels significantly increased in the cerebral cortex and hippocampus. These results suggest that FMH enhances the acquisition phase of radial maze study with the increases in glutamate and glycine levels in the cerebral cortex and hippocampus of rats.     

The effects of 5-HT1A receptor agonists, 5-HT1A receptor antagonists and their interaction on the fear-potentiated startle response

Time-resolved small-angle X-ray scattering (TR-SAXS) was used to monitor the structural changes that occur upon the binding of the natural substrates to a mutant version of the allosteric enzyme aspartate transcarbamoylase from Escherichia coli, in which the creation of a critical link stabilizing the R state of the enzyme is hindered. Previously, SAXS experiments at equilibrium showed that the structures of the unligated mutant enzyme and the mutant enzyme saturated with a bisubstrate analog are indistinguishable from the T and R state structures, respectively, of the wild-type enzyme (Tauc et al., Protein Sci. 3:1998-2004, 1994). However, as opposed to the wild-type enzyme, the combination of one substrate, carbamoyl phosphate, and succinate, an analog of aspartate, did not convert the mutant enzyme into the R state. By using TR-SAXS we have been able to study the transient steady-state during catalysis using the natural substrates rather than the nonreactive substrate analogs. The steady-state in the presence of saturating amount of substrates is a mixture of 60% T and 40% R structures, which is further converted entirely to R in the additional presence of ATP. These results provide a structural explanation for the reduced cooperativity observed with the mutant enzyme as well as for the stimulation by ATP at saturating concentrations of substrates. They also illustrate the crucial role played by domain motions and quaternary-structure changes for both the homotropic and heterotropic aspects of allostery.     

Effect of a selective 5-HT reuptake inhibitor in combination with 5-HT1A and 5-HT1B receptor antagonists on extracellular 5-HT in rat frontal cortex in vivo

1. Selective 5-hydroxytryptamine (5-HT; serotonin) reuptake inhibitors (SSRIs) cause a greater increase in extracellular 5-HT in the forebrain when the somatodendritic 5-HT1A autoreceptor is blocked. Here, we investigated whether blockade of the terminal 5-HT1B autoreceptor influences a selective 5-HT reuptake inhibitor in the same way, and whether there is an additional effect of blocking both the 5-HT1A and 5-HT1B autoreceptors. 2. Extracellular 5-HT was measured in frontal cortex of the anaesthetized rat by use of brain microdialysis. In vivo extracellular recordings of 5-HT neuronal activity in the dorsal raphe nucleus (DRN) were also carried out. 3. The selective 5-HT reuptake inhibitor, paroxetine (0.8 mg kg-1, i.v.), increased extracellular 5-HT about 2 fold in rats pretreated with the 5-HT1A receptor antagonist, WAY100635. When administered alone neither paroxetine (0.8 mg kg-1, i.v.) nor WAY100635 (0.1 mg kg-1, i.v.) altered extracellular 5-HT levels. 4. Paroxetine (0.8 mg kg-1, i.v.) did not increase 5-HT in rats pretreated with the 5-HT1B/D receptor antagonist, GR127935 (1 mg kg-1, i.v.). GR127935 (1 and 5 mg kg-1, i.v.) had no effect on extracellular 5-HT when administered alone. 5. Interestingly, paroxetine (0.8 mg kg-1, i.v.) caused the greatest increase in 5-HT (up to 5 fold) when GR127935 (1 or 5 mg kg-1, i.v.) was administered in combination with WAY100635 (0.1 mg kg-1, i.v.). Administration of GR127935 (5 mg kg-1, i.v.) plus WAY100635 (0.1 mg kg-1, i.v.) without paroxetine, had no effect on extracellular 5-HT in the frontal cortex. 6. Despite the lack of effect of GR127935 on 5-HT under basal conditions, when 5-HT output was elevated about 3 fold (by adding 1 microM paroxetine to the perfusion medium), the drug caused a dose-related (1 and 5 mg kg-1, i.v.) increase in 5-HT. 7. By itself, GR127935 slightly but significantly decreased 5-HT cell firing in the DRN at higher doses (2.0-5.0 mg kg-1, i.v.), but did not prevent the inhibition of 5-HT cell firing induced by paroxetine. 8. In summary, our results suggest that selective 5-HT reuptake inhibitors may cause a large increase in 5-HT in the frontal cortex when 5-HT autoreceptors on both the somatodendrites (5-HT1A) and nerve terminals (5-HT1B) are blocked. This increase is greater than when either set of autoreceptors are blocked separately. The failure of a 5-HT1B receptor antagonist alone to enhance the effect of the selective 5-HT reuptake inhibitor in our experiments may be related to a lack of tone on the terminal 5-HT1B autoreceptor due to a continued inhibition of 5-HT cell firing. These results are discussed in relation to the use of 5-HT autoreceptor antagonists to augment the antidepressant effect of selective 5-HT reuptake inhibitors.     

5-HT1A receptor activation: short-term effects on the mRNA expression of the 5-HT1A receptor and galanin in the raphe nuclei

Systemic administration of the 5-HT1A receptor agonist 8-OH-2-(di-n-propylamino)-tetralin (8-OH-DPAT; 0.3 mg/kg, s.c.) was used to explore the effects of activation of 5-HT1A receptors on expression of mRNA coding for 5-HT1A receptor, tryptophan hydroxylase (TPH) and galanin in the ascending raphe nuclei. 8-OH-DPAT increased the hybridization signal of the 5-HT1A receptor by 105% in the dorsal raphe nucleus (B7) 30 min after the injection. No effects were seen at the later time points (2-8 h). In the median raphe nucleus (B8) and the B9 cell group in the medial lemniscus, 8-OH-DPAT induced a marked decrease in labeling 30 min after injection. At 8 h following 8-OH-DPAT injection, the effect had shifted to an increase in 5-HT1A receptor labeling by 68% in the B8 area. Importantly 8-OH-DPAT had no significant effects on the expression of mRNA coding for TPH and galanin. The results suggest an important and differential mechanism for the regulation of 5-HT1A receptor mRNA levels in the dorsal and median raphe nuclei. This regulation may be of importance for the differential control of the activity of the ascending 5-HT neurons, and hence for mood regulation. The results also indicate a dissociation between the effects mediated by 5-HT1A receptor functions and those regulating the coexisting peptide galanin in the dorsal raphe.     

Benzylimidazolines as h5-HT1B/1D serotonin receptor ligands: a structure-affinity investigation

Benzylimidazolines may represent a class of 5-HT1D ligands that has yet to be exploited. On the basis of a previous report that the 2-(substituted-benzyl)imidazoline alpha-adrenergic agonist oxymetazoline (8) binds with high affinity at calf brain 5-HT1D receptors, we explored the structure-affinity relationships of a series of related derivatives. Each of the aromatic substituents was removed and then reinstated in a systematic manner to determine the influence of the individual substituents on binding. It was found that all of the aromatic substituents of 8 act in concert to impart high affinity. However, although the 3-hydroxy group could be removed without significantly reducing affinity for h5-HT1D (i.e., human 5-HT1Dalpha) receptors, this modification reduced h5-HT1B (i.e., human 5-HT1Dbeta) receptor affinity by nearly 50-fold. The 2, 6-dimethyl groups also contribute to binding but seem to play a greater role for h5-HT1B binding than h5-HT1D binding. With the appropriate structural modifications, several compounds were identified that display 20- to >100-fold selectivity for h5-HT1D versus h5-HT1B receptors. Preliminary functional data suggest that these compounds behave as agonists. Given that 5-HT1D agonists are currently being explored for their antimigraine action and that activation of h5-HT1B receptors might be associated with cardiovascular side effects, h5-HT1D-selective agents may offer a new lead for the development of therapeutically efficacious agents.     

Animal models for studying serotonin (5-HT) receptor subtypes: relationship to 5-HT system pathologies

Despite more than 30 years of intensive research, the specific role of serotonin (5-HT) receptor subtypes in animal models of "anxiety" still remains unclear. The present study focused on the particular role of 5-HT1A receptor subtype in aversive learning, i.e., the passive avoidance (PA) task in the rat. Taken together, the data strongly suggest that: (1) 5-HT1A receptor but not 5-HT2A receptors play a crucial role in PA; (2) the postsynaptic but not presynaptic 5-HT1A receptors are mainly involved in the regulation of PA; (3) 5-HT1A receptors appear to be directly involved both in acquisition and retrieval but not in the consolidation of PA; and (4) besides the "prototypical" 5-HT1A receptor subtype, an additional and yet-unidentified 5-HT receptor subtype seems to play an important modulatory role in PA.     

5-HT1Dbeta receptor mediates the supersensitivity of isolated coronary artery to serotonin in variant angina

Although serotonin (5-hydroxytryptamine; 5-HT) is used for provocation of coronary spasm, 5-HT receptor subtypes in spastic coronary arteries remain undetermined. We demonstrated the supersensitivity of isolated coronary artery to ergonovine, 5-HT, and sumatriptan, a 5-HT1D receptor agonist, in a patient with variant angina. Furthermore, we detected gene expression of 5-HT1Dbeta and 5-HT2A receptors in spastic coronary artery using RNase protection assay. These findings suggest that the leftward shift of the dose-response curve for 5-HT, which plays an important role in the pathogenesis of coronary spasm, is mediated by activation of 5-HT1Dbeta receptor.     

Differential membrane targeting and pharmacological characterization of chimeras of rat serotonin 5-HT1A and 5-HT1B receptors expressed in epithelial LLC-PK1 cells

The serotonin 5-HT1A and 5-HT1B receptors are two structurally related but pharmacologically distinguishable 5-HT receptor types. In brain, the 5-HT1A receptor is localized on the soma and dendrites of neurons, whereas the 5-HT1B receptor is targeted to the axon terminals. We previously showed that these two receptors are targeted in different membrane compartments when stably expressed in the epithelial LLC-PK1 cell line. Further investigations on the mechanisms responsible for their differential targeting were done by constructing chimeras of 5-HT1A and 5-HT1B receptors still able to bind specifically [3H]lysergic acid diethylamide and selective agonists and antagonists. Their cellular localization examined by confocal microscopy suggests that the third intracellular domain of the 5-HT1B receptor was responsible for its Golgi-like localization in transfected LLC-PK1 cells. In contrast, the third intracellular domain of the 5-HT1A receptor apparently allowed the sorting of the chimeras to the plasma membrane. Further inclusion of the C-terminal domain of the 5-HT1A receptor in their sequence led to a basolateral localization, whereas that of the 5-HT1B receptor allowed an apical targeting, suggesting the existence of a targeting signal in this portion of the receptor(s).     

The 5-HT1A receptor antagonist p-MPPI blocks 5-HT1A autoreceptors and increases dorsal raphe unit activity in awake cats

The effects of the putative 5-HT1A receptor antagonist 4-iodo-N-[2-[4-(methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinyl-benzam ide (p-MPPI) were examined on the activity of serotonergic dorsal raphe nucleus neurons in freely moving cats. Systemic administration of p-MPPI produced a dose-dependent increase in firing rate. This stimulatory effect of p-MPPI was evident during wakefulness (when serotonergic neurons display a relatively high level of activity), but not during sleep (when serotonergic neurons display little or no spontaneous activity). p-MPPI also blocked the ability of the 5-HT1A receptor agonist 8-hydroxy-(2-di-n-propylamino)tetralin (8-OH-DPAT) to inhibit serotonergic neuronal activity. This antagonism was evident both as a reversal of the neuronal inhibition produced by prior injection of 8-OH-DPAT and as a shift in the potency of 8-OH-DPAT following p-MPPI pretreatment. Overall, these results in behaving animals indicate that p-MPPI acts as an effective 5-HT1A autoreceptor antagonist. The increase in firing rate produced by p-MPPI supports the hypothesis that autoreceptor-mediated feedback inhibition operates under physiological conditions.     

Sequence and functional analysis of cloned guinea pig and rat serotonin 5-HT1D receptors: common pharmacological features within the 5-HT1D receptor subfamily

This study was undertaken to investigate the pharmacology of cloned guinea pig and rat 5-hydroxytryptamine (serotonin; 5-HT)1D receptor sites. Guinea pig, rat, and mouse 5-HT1D receptor genes were cloned, and their amino acid sequences were compared with those of the human, dog, and rabbit. The overall amino acid sequence identity between these 5-HT1D receptors is high and varies between 86 and 99%. The sequence homology is slightly more divergent (13-27%) in the N-terminal extracellular region of these 5-HT1D receptors. Guinea pig and rat 5-HT1D receptors, stably and separately expressed in rat C6 glial cells, are negatively coupled to cyclic AMP formation upon stimulation with agonists, as previously found for cloned human 5-HT1D receptor sites. The cyclic AMP data show some common pharmacological features for the 5-HT1D receptors of guinea pig, rat, and human: an almost similar rank order of potency for the investigated 5-HT1D receptor agonists, stereoselectivity for the binding affinity and agonist potency of R(+)-8-hydroxy-2-(di-n-propylamino)tetralin, and equal 5-HT1D receptor-mediated antagonist potency for methiothepin and the 5-HT2 receptor antagonists ritanserin and ketanserin. In conclusion, the pharmacology of the cloned 5-HT1D receptor subtype seems, unlike the 5-HT1B receptor subtype, conserved among various mammal species such as the human, guinea pig, and rat.     

Dimerization of 8-OH-DPAT increases activity at serotonin 5-HT1A receptors

We have identified and partially purified two DNA polymerase activities from purified Trypanosoma brucei mitochondrial extracts. The DNA polymerase activity eluted from the single-stranded DNA agarose column at 0.15 M KCl (polymerase M1) was significantly inhibited by salt concentrations greater than 100 mM, utilized Mg2+ in preference to Mn2+ as a cofactor on deoxyribonucleotide templates with deoxyribose primers, and in the presence of Mn2+ favored a ribonucleotide template with a deoxyribose primer. A 44 kDa peptide in this fraction crossreacted with antisera against the Crithidia fasciculata beta-like mitochondrial polymerase. In activity gels the catalytic peptide migrated at an apparent molecular weight of 35 kDa. The DNA polymerase activity present in the 0.3 M KCl DNA agarose fraction (polymerase M2) exhibited optimum activity at 120-180 mM KCl, used both Mg2+ and Mn2+ as cofactors, and used deoxyribonucleotide templates primed with either deoxyribose or ribose oligomers. Activity gel assays indicate that the native catalytic peptide(s) is approximately 80 kDa in size. The two polymerases showed different sensitivities to several inhibitors: polymerase M1 shows similarities to the Crithidia fasciculata beta-like mitochondrial polymerase while polymerase M2 is a novel, salt-activated enzyme of higher molecular weight.     

Spiperone: influence of spiro ring substituents on 5-HT2A serotonin receptor binding

Spiperone (1) is a widely used pharmacological tool that acts as a potent dopamine D2, serotonin 5-HT1A, and serotonin 5-HT2A antagonist. Although spiperone also binds at 5-HT2C receptors, it is one of the very few agents that display some (ca. 1000-fold) binding selectivity for 5-HT2A versus 5-HT2C receptors and, hence, might serve as a useful template for the development of novel 5-HT2A antagonists if the impact of its various substituent groups on binding was known. In the present investigation we focused on the 1, 3,8-triazaspiro[4.5]decanone portion of spiperone and found that replacement of the N1-phenyl group with a methyl group only slightly decreased affinity for cloned rat 5-HT2A receptors. However, N1-methyl derivatives displayed significantly reduced affinity for 5-HT1A, 5-HT2C, and dopamine D2 receptors. Several representative examples were shown to behave as 5-HT2 antagonists. As such, N1-alkyl analogues of spiperone may afford entry into a novel series of 5-HT2A-selective antagonists.