Cataleptic effect of neurosteroid 3α-hydroxy-5α-pregnan-20-one in mice: modulation by serotonergic agents

The neurosteroid 3α-hydroxy-5α-pregnan-20-one (3α,5α-THP) induced catalepsy in mice is modified by dopaminergic, adenosinergic and GABAergic agents. In light of serotonergic agents being implicated in antipsychotic-induced catalepsy and their ability to increase brain neurosteroid content, the present study was undertaken to investigate the effect of various 5-HT agents on catalepsy induced by 3α,5α-THP in mice. Pretreatment with selective serotonin reuptake inhibitor, fluoxetine (5 mg/kg, i.p.), 5-HT releaser, fenfluramine (10 mg/kg, i.p.), 5-HT_1A receptor agonist, 8-OH-DPAT (0.3 mg/kg, s.c.), 5-HT_1B/1C receptor agonist, TFMPP (3 mg/kg, i.p.), 5-HT_2A/1C receptor agonist, DOI (1.5 mg/kg, s.c.) and 5-HT₃ agonist, 2-methylserotonin (5 mg/kg, i.p.) potentiated the catalepsy induced by exogenous administration of 3α,5α-THP. Furthermore, FGIN 1–27, an MDR agonist that increases endogenous content of 3α,5α-THP although per se failed to exhibit any cataleptic effect but enhanced the cataleptic response in combination with these serotonergic agents. The potentiating action of 5-HT_1A, 5-HT_2A/1C or 5-HT₃ receptor agonist on 3α,5α-THP induced catalepsy was not blocked by prior administration of sub-effective dose (1 mg/kg, s.c.) of their respective receptor antagonists pindolol, ritanserin or ondansetron or by pretreatment with serotonergic neurotoxin 5,7-DHT (100 μg/mouse, i.c.v.). However this effect of different serotonergic agents was antagonized by the GABA_A receptor antagonist, bicuculline (1 mg/kg, i.p.) or the 3α-hydroxysteroid oxidoreductase enzyme inhibitor, indomethacin (5 mg/kg, i.p.). The 5-HT agents enhance neurosteroid-induced catalepsy by increasing GABAergic tone, likely as a consequence of increased brain content of 3α,5α-THP.     

Serotonergic agents modulate antidepressant-like effect of the neurosteroid 3alpha-hydroxy-5alpha-pregnan-20-one in mice

The present study demonstrated the antidepressant-like effect of neurosteroid 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha, 5alpha THP) in mouse forced swim test of depression and its modulation by different serotonergic agents. Pretreatment with the selective serotonin reuptake inhibitor, fluoxetine (5 mg/kg, i.p.), the 5-HT releaser, fenfluramine (10 mg/kg, i.p.), the 5-HT(1A) receptor agonist, 8-OH-DPAT (0.1 mg/kg, s.c.), the 5-HT(1B/1C) receptor agonist, TFMPP (4 mg/kg, s.c.) and the 5-HT(2A/1C) receptor agonist, DOI (2 mg/kg, s.c.) potentiated the antidepressant-like effect of 3alpha, 5alpha THP. At these doses the serotonergic agents per se did not modify the duration of immobility. However, fluoxetine (20 mg/kg, i.p.), fenfluramine (20 mg/kg, i.p.) or imipramine (5 or 20 mg/kg, i.p.) not only reduced immobility but also enhanced the antidepressant-like effect of 3alpha, 5alpha THP. Such a potentiating effect of the 5-HT(1A) or the 5-HT(2A/1C) receptor agonist was not antagonized by the sub-effective dose (0.1 mg/kg, s. c.) of their respective antagonists p-MPPI or ketanserin. Pretreatment with p-CPA (300x3 mg/kg, i.p.), a depleter of 5-HT neuronal store failed to block the influence of fluoxetine and fenfluramine on antidepressant-like effect of 3alpha, 5alpha THP. The accelerated effect of 3alpha, 5alpha THP in presence of serotonergic agents was antagonized by the GABA(A) receptor antagonist, bicuculline (1 mg/kg, i.p.) or the 3alpha-hydroxysteroid oxidoreductase enzyme inhibitor, indomethacin (5 mg/kg, i.p.). These findings for the first time demonstrate that serotonergic agents potentiate the antidepressant-like action of 3alpha, 5alpha THP, by enhancing the GABAergic tone as a likely consequence of increased brain content of this neurosteroid.     

Evidence for central α2-adrenergic modulation of rat pineal melatonin synthesis

This study was performed to distinguish central and peripheral α₂-adrenoceptors in the inhibition of rat pineal melatonin synthesis. The rats received lipo- or hydrophilic α₂-adrenoceptor ligand injections at middark; after 1 or 2 h the pineal melatonin contents were measured. The lipophilic agonist medetomidine (100 μg/kg s.c.) suppressed the melatonin contents significantly, while the hydrophilic agonists ST-91 and p-aminoclonidine (10 or 100 μg/kg i.v.) did not. The suppression by medetomidine was counteracted by the lipophilic antagonist yohimbine (0.3–3.0 mg/kg i.p.) but not by the hydrophilic antagonist L-659,066 (1–10 mg/kg i.v.). In conclusion, the suppression of nocturnal melatonin synthesis by α₂-adrenoceptor agonists is mainly of central origin.     

Influence of centrally administered α- and γ2-melanocyte-stimulating hormone on hypothalamic blood flow autoregulation in the rat

The effect of intracerebroventricularly (i.c.v.) administered α-melanocyte-stimulating hormone (MSH) and γ₂-MSH on hypothalamic blood flow autoregulation was studied in anesthetized rats at different levels of standardized arterial hypotension. Autoregulation was impaired upon i.c.v. administration of 5 γ g/kg γ₂-MSH while α-MSH caused no change.. Since this effect of γ₂-MSH wa identical to that produced by i.c.v. naloxone in the same model, γ₂-MSH may be a functional antagonist of central opioid mechanisms participating in the control of cerebral blood flow autoregulation.     

Involvement of μ- and δ-opioid receptors in the ethanol-associated place preference in rats exposed to foot shock stress

The purpose of this study was to establish the ethanol-induced place preference in rats exposed to foot shock stress using the conditioned place preference paradigm. We also investigated the role of the endogenous opioid system in the development of the ethanol-induced place preference. The administration of ethanol (300 mg/kg, i.p.) with foot shock stress, but not without such stress, induced a marked and significant place preference. Naloxone (1 and 3 mg/kg, s.c.), a non-selective opioid receptor antagonist, significantly attenuated the ethanol-induced place preference. Moreover, the selective μ-opioid receptor antagonist β-funaltrexamine (3 and 10 mg/kg, i.p.) and selective δ-opioid receptor antagonist naltrindole (1 and 3 mg/kg, s.c.), but not the selective κ-opioid receptor antagonist nor-binaltorphimine (1 and 3 mg/kg, i.p.), significantly attenuated the ethanol-induced place preference. Furthermore, 150 mg/kg ethanol (which tended to produce a place preference, although not significantly) combined with each dose (that did not produce a place preference) of the μ-opioid receptor agonist morphine (0.1 mg/kg, s.c.) or selective δ-opioid receptor agonist 2-methyl-4aα-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12aα-octahydroquinolino [2,3,3-g] isoquinoline (TAN-67; 20 mg/kg, s.c.), but not the selective κ-opioid receptor agonist trans-3,4-dichloro-N-(2-(1-pyrrolidinyl)cyclohexyl)benzenacetamide methanesulfonate (U50,488H; 1 mg/kg, s.c.), produced a significant place preference. These data indicate that stress may be important for development of the rewarding effect of ethanol, and that μ- and δ-opioid receptors may be involved in the rewarding mechanism of ethanol under stressful conditions.     

The role of β- and α-adrenoceptors in the regulation of the stages of the sleep-waking cycle in the cat

The effects of β-adrenergic drugs alone and in combination with α-adrenergic drugs on the stages of the sleep-waking cycle were studied in adult cats. Polygraphic sleep recordings of 16 h showed that prenalterol (20 and 40 mg/kg i.p.), a β₁-adrenoceptor-stimulating drug increased paradoxical sleep (PS) in a dose-related manner during 4–12 h. Salbutamol (40 mg/kg), a β₂-adrenoceptor-stimulating drug, decreased PS during the first 4 h. Metoprolol (10 and 50 mg/kg), a relatively selective β₁-adrenoceptor blocking drug, increased drowsy waking during the first 4 h. The larger dose also tended to decrease PS. Already at the lower dose metoprolol partially antagonized the PS increase produced by prazosin, an α₁-adrenoceptor blocking drug. Propranolol (5 mg/kg), a β₁-andβ₂-adrenoceptor blocking drug, which alone decreases PS, antagonized the PS increase induced by phentolamine, an α₁-andα₂-adrenoceptor drug. Atenolol (5 mg/kg), a poorly lipid-soluble β-adrenoceptor blocking drug, failed to counteract phentolamine in increasing PS. Metoprolol (10 and 50 mg/kg) and propranolol (5 mg/kg) clearly potentiated the increase in drowsy waking and decrease in deep slow wave sleep and PS induced by clonidine (0.01 mg/kg), an α₂-adrenoceptor-stimulating drug.The results support the involvement of β-adrenoceptors in the regulation of the sleep-waking cycle. A high level of β-adrenergic activity may facilitate the production of PS. A low level of β-adrenergic activity, especially in combination with a high level of α₂-adrenergic activity, may facilitate the production of drowsy waking. Central α₁-andβ₁-adrenoceptors may mediate opposite functions in the regulation of PS.     

S-100β protects cultured neurons against glutamate- and staurosporine-induced damage and is involved in the antiapoptotic action of the 5 HT1A-receptor agonist, Bay x 3702

The serotonin (5-HT)_1A receptor agonists have already been shown to protect cultured neurons from excitotoxic as well as from apoptotic damage [B. Ahlemeyer, J. Krieglstein, Stimulation of 5-HT_1A receptors inhibits apoptosis induced by serum deprivation in cultured neurons from chick embryo, Brain Res. 777 (1997) 179–186.; B. Ahlemeyer, A. Glaser, C. Schaper, I. Semkova, J. Krieglstein, The 5-HT_1A receptor agonist, Bay x 3702, inhibited apoptosis induced by serum deprivation in cultured neurons, Eur. J. Pharmacol. 370 (1999) 211–216.; J.H.M. Prehn, M. Welsch, C. Backhauß, J. Nuglisch, F. Ausmeier, C. Karkoutly, J. Krieglstein, Effects of serotonergic drugs in experimental brain ischemia: evidence for a protective role of serotonin in cerebral ischemia, Brain Res. 630 (1993) 110–120.; I. Semkova, P. Wolz, J. Krieglstein, Neuroprotective effect of 5-HT_1A receptor agonist, Bay x 3702, demonstrated in vitro and in vivo, Eur. J. Pharmacol. 359 (1998) 251–260.; B. Suchanek, H. Struppeck, T. Fahrig, The 5-HT_1A receptor agonist, Bay x 3702, prevents staurosporine-induced apoptosis, Eur. J. Pharmacol. 355 (1998) 95–101.] and to increase the release of the neurotrophic protein, S-100β [P.M. Whitaker-Azmitia, R. Murphy, E.C. Azmitia, Stimulation of astroglial 5-HT_1A receptors releases the serotonergic growth factor, protein S-100, and alters astroglial morphology, Brain Res. 497 (1989) 80–86.; P.M. Whitaker-Azmitia, R. Murphy, E.C. Azmitia, S-100 protein is released from astroglial cells by stimulation of 5-HT_1A receptors, Brain Res. 528 (1990) 155–158.]. In this study, we tried to find out whether S-100β can protect cultured neurons from glutamate- and staurosporine-induced damage and whether the neuroprotective activity of the highly selective 5-HT_1A receptor agonist, Bay x 3702, is mediated by an induction of S-100β. Extracellularly added S-100β (1–10 ng/ml) reduced staurosporine-induced damage in pure neuronal cultures from chick embryo telencephalon as well as in mixed neuronal/glial cultures from neonatal rat hippocampus. In addition, S-100β (1 ng/ml) reduced neuronal death induced by exposure to glutamate (0.25 mM, 30 min) in mixed neuronal/glial cultures from neonatal rat hippocampus. In cultured rat cortical astrocytes, a 24 h-treatment with Bay x 3702 (1 nM) increased the S-100β content in the culture medium from 2.2±0.3 (controls) to 6.2±0.7 ng/ml. In the adult rat, a 4 h-infusion of 4 μg/kg Bay x 3702 (i.v.) was found to increase the S-100β content in the striatum 6 h after the beginning of the infusion to 153±37 μg/g compared with 60±20 μg/g in vehicle-treated rats. Bay x 3702 had no effect on the S-100β content in the rat hippocampus. Finally, we tried to block the protective effect of Bay x 3702 against staurosporine-induced damage in mixed neuronal/glial cultures from rat neonatal hippocampus by anti-S-100β antibodies. We found only a partial blockade, although the antibodies fully blocked the antiapoptotic effect of S-100β itself demonstrating that the antibody was effective in blocking neuroprotection by S-100β. Thus, we conclude that S-100β was able to protect cultured neurons against glutamate- and staurosporine-induced damage. Furthermore, S-100β mediated partially the protective effect of the 5-HT_1A receptor agonist, Bay x 3702, against staurosporine-induced apoptosis in mixed neuronal/glial cultures from neonatal rat hippocampus.     

The socially-isolated mouse: a model to study the putative role of allopregnanolone and 5α-dihydroprogesterone in psychiatric disorders

Allopregnanolone (3α,5α-TH PROG) and 5α-dihydroprogesterone (5α-DH PROG), the two most important neuroactive steroids synthesized in the brain, potently modulate neuronal activity by allosterically regulating GABA action at GABA_A receptors or by changing specific GABA_A receptor subunit gene expression, respectively. We recently reported [Proc. Natl. Acad. Sci. USA 95 (1998) 3239] that in patients with severe depression there is a decrease in the CSF levels of 3α,5α-TH PROG, which is normalized by treatment with drugs (i.e. fluoxetine) that improve psychopathology. The mechanism by which fluoxetine and other selective serotonin reuptake inhibitors normalize 3α,5α-TH PROG CSF levels appears to involve a direct stimulation of 3α-hydroxysteroidoxidoreductase (3α-HSD), an enzyme that catalyses the reduction of 5α-DH PROG into 3α,5α-TH PROG. Here, we propose the use of socially-isolated mice that have a downregulation of 3α,5α-TH PROG and of 5α-DH PROG expression to establish a model to study the behavioral consequences of this deficiency. After 4–6 weeks of isolation, these mice exhibit increased anxiety and aggressive behavior and also a decreased response to the administration of GABA-mimetic drugs. In these mice, the decrease in 3α,5α-TH PROG is selectively normalized by the use of fluoxetine in doses that reduce behavioral abnormalities. In addition, the expression of 5α-reductase Type I mRNA and protein was lower in socially-isolated mice than that in group-housed mice whereas 3α-HSD mRNA expression remained unchanged. The results of these studies may enable us to design drugs that specifically affect neurosteroidogenic enzymatic activities and may provide an efficacious treatment for the psychopathologies associated with psychiatric disorders.     

The antihyperalgesic activity of a selective P2X7 receptor antagonist, A-839977, is lost in IL-1αβ knockout mice

The pro-inflammatory cytokine interleukin-1β (IL-1β) has been implicated in both inflammatory processes and nociceptive neurotransmission. Activation of P2X7 receptors is the mechanism by which ATP stimulates the rapid maturation and release of IL-1β from macrophages and microglial cells. Recently, selective P2X7 receptor antagonists have been shown to reduce inflammatory and neuropathic pain in animal models. However, the mechanisms underlying these analgesic effects are unknown. The present studies characterize the pharmacology and antinociceptive effects of a structurally novel P2X7 antagonist. A-839977 potently (IC₅₀ = 20–150 nM) blocked BzATP-evoked calcium influx at recombinant human, rat and mouse P2X7 receptors. A-839977 also potently blocked agonist-evoked YO-PRO uptake and IL-1β release from differentiated human THP-1 cells. Systemic administration of A-839977 dose-dependently reduced thermal hyperalgesia produced by intraplantar administration of complete Freund's adjuvant (CFA) (ED₅₀ = 100 μmol/kg, i.p.) in rats. A-839977 also produced robust antihyperalgesia in the CFA model of inflammatory pain in wild-type mice (ED₅₀ = 40 μmol/kg, i.p.), but the antihyperalgesic effects of A-839977 were completely absent in IL-1αβ knockout mice. These data demonstrate that selective blockade of P2X7 receptors in vivo produces significant antinociception in animal models of inflammatory pain and suggest that the antihyperalgesic effects of P2X7 receptor blockade in an inflammatory pain model in mice are mediated by blocking the release of IL-1β.     

Effect of a 5-HT1A receptor agonist, flesinoxan, on the extracellular noradrenaline level in the hippocampus and on the locomotor activity of rats

We have studied effects of 5-hydroxytryptamine 1A (5-HT_1A) receptor-selective compounds on the extracellular noradrenaline (NA) level in the hippocampus of rats using microdialysis and on their locomotor activity. A selective 5-HT_1A receptor agonist, flesinoxan (5 mg/kg, i.p.) increased the extracellular NA level in the hippocampus, and increased the locomotor activity. Both responses were blocked by pretreatment with a 5-HT_1A receptor antagonist, WAY100635 (1 mg/kg, i.p.) and an α₂ adrenoceptor agonist, clonidine (50 μg/kg, i.p.). Bilateral intrahippocampal injection of flesinoxan (200 nmol in 2 μl, respectively) increased the locomotor activity of rats and the intrahippocampal perfusion of flesinoxan (1 mM, 2 μl/min) increased the extracellular NA level in the hippocampus. Bilateral intrahippocampal injections of a small amount of WAY100635 (0.1 nmol in 2 μl, respectively) blocked the flesinoxan (5 mg/kg, i.p.)-induced hyperactivity. Flesinoxan (5 mg/kg, i.p.) did not significantly influence the level of serotonin or its major metabolite in the hippocampus, or dopamine or its metabolites in the striatum. In conclusion, these behavioral as well as pharmacological results indicate that postsynaptic 5-HT_1A receptor activation by flesinoxan increase the extracellular NA level in the hippocampus, which may be the cause of the increase of the locomotor activity.     

Serotonergic influence on the potentiation of D-amphetamine and apomorphine-induced rotational behavior by the α2-adrenoceptor antagonist 2-methoxy idazoxan in hemiparkinsonian rats

Summary. The α₂-adrenoceptor antagonists potentiate both ipsilateral and contralateral rotations induced by amphetamine and apomorphine respectively in hemiparkinsonian rats. The present study investigated the role of serotonergic transmission in this potentiation in unilaterally 6-hydroxydopamine nigral lesioned rats. D-amphetamine (0.5 mg/kg, i.p.) produced ipsilateral rotations, which were decreased by the dopamine receptor antagonist haloperidol (0.2 mg/kg, i.p.) and the α₁-receptor antagonist prazosin (1 mg/kg, i.p.). The selective α₂-antagonist 2-methoxy idazoxan (0.2 mg/kg, i.p.) potentiated the amphetamine-induced ipsilateral rotations, that were attenuated by haloperidol and prazosin. The selective serotonin re-uptake inhibitor citalopram (10 mg/kg, i.p.) and selective serotonin synthesis inhibitor p-chlorophenylalanine (150 mg/kg, i.p., 3 days) decreased and increased the observed potentiation respectively. Apomorphine (0.2 mg/kg, s.c.) produced contralateral rotations, which were decreased by haloperidol but not by prazosin. 2-methoxy idazoxan potentiated these rotations which were attenuated by haloperidol but not by prazosin. Citalopram and p-chlorophenylalanine increased and decreased the observed potentiation respectively. Citalopram and p-chlorophenylalanine had no effect by per se on D-amphetamine and apomorphine-induced rotations. 2-methoxy idazoxan alone increased both ipsilateral and contralateral spontaneous rotations. Taken together, these findings indicate that an increase in noradrenergic tone by 2-methoxy idazoxan potentiates both D-amphetamine-induced ipsilateral and apomorphine induced contralateral rotations. α₁-Antagonism attenuates D-amphetamine induced ipsilateral rotations and its potentiation by 2-methoxy idazoxan but not apomorphine rotations or its potentiation. Increasing and decreasing the serotonergic transmission decreases and increases D-amphetamine potentiation, whereas increases and decreases apomorphine potentiation respectively. The possible mechanisms for these findings are discussed.     

Evidence for the involvement of the serotonergic 5-HT2A/C and 5-HT3 receptors in the antidepressant-like effect caused by oral administration of bis selenide in mice

The present study investigated a possible antidepressant-like activity of bis selenide using two predictive tests for antidepressant effect on rodents: the forced swimming test (FST) and the tail suspension test (TST). Bis selenide (0.5–5 mg/kg, p.o.) decreased the immobility time in the mouse FST and TST. The anti-immobility effect of bis selenide (1 mg/kg, p.o.) in the TST was prevented by the pretreatment of mice with p-chlorophenylalanine methyl ester (PCPA; 100 mg/kg, i.p., an inhibitor of serotonin synthesis), ketanserin (1 mg/kg, i.p., a 5-HT_2A/2C receptor antagonist), and ondasentron (1 mg/kg, i.p., a 5-HT₃ receptor antagonist). Pretreatment of mice with prazosin (1 mg/kg, i.p., an α₁-adrenoceptor antagonist), yohimbine (1 mg/kg, i.p., an α₂-adrenoceptor antagonist), propranolol (2 mg/kg, i.p., a β-adrenoceptor antagonist), SCH23390 (0.05 mg/kg, s.c., a dopamine D₁ receptor antagonist), sulpiride (50 mg/kg, i.p., a dopamine D₂ receptor antagonist), or WAY 100635 (0.1 mg/kg, s.c., a selective 5-HT_1A receptor antagonist) did not block the antidepressant-like effect of bis selenide (1 mg/kg, p.o.) in the TST. Administration of bis selenide (0.1 mg/kg, p.o.) and fluoxetine (1 mg/kg), at subeffective doses, produced an antidepressant-like effect in the TST. Bis selenide did not alter Na⁺ K⁺ ATPase, MAO-A and MAO-B activities in whole brains of mice. Bis selenide produced an antidepressant-like effect in the mouse TST and FST, which may be related to the serotonergic system (5-HT_2A/2C and 5-HT₃ receptors).     

Antidepressant-like effects of SR 57227A,a 5-HT3 receptor agonist,in rodents

Summary We have investigated the effect of SR 57227A, a selective 5-HT₃ receptor agonist which crosses the blood brain barrier, on three rodent models in which antidepressants are active. In the forced swimming test, SR 57227A dose-dependently reduced the duration of immobility in mice and rats after i.p. administration. (ED₅₀=14.2mg/kg i.p. in mice, and 7.6 mg/kg i.p. in rats.) The compound was also active in both species after oral administration. In a time-course study in mice, SR 57227A (20 mg/kg p.o.) produced a significant effect lasting 6 h. SR 57227A (1 and 3 mg/kg i.p.) reduced the elevation of the escape failures in the learned helplessness model in rats by 50–60% on the last two days of the avoidance task, and reduced isolation-induced aggressivity in mice by 50 to 85%, an effect which was antagonised by zacopride (1 mg/kg i.p.). These results suggest that the stimulation of 5-HT₃ receptors can produce antidepressant-like effects in behavioural tests in rodents.     

α2, α2A, α2B/2C-Adrenoceptor subtype antagonists prevent lipopolysaccharide-induced fever response in rabbits

Exogenous pyrogens, e.g., bacterial lipopolysaccharides (LPS), are thought to stimulate macrophages to release endogenous pyrogens, e.g., TNFα, IL-1 β, and IL-6, which act in the hypothalamus to produce fever. We studied the effect of different α₁⁻ and α₂-adrenoceptor subtype antagonists, applied intraperitoneally, on the febrile response induced by LPS in rabbits. Evidence was obtained that prazosin, an α₁⁻ and α_2B/2C-adrenoceptor antagonist; WB-4101, an α₁⁻ and α_2A-adrenoceptor antagonist; CH-38083, a highly selective α₂-adrenoceptor antagonist (α₂: α₁ > 2000); BRL-44408, an α_2A-adrenoceptor antagonist; and ARC-239, an α_2B/2C⁻ and also α₁-adrenoceptor antagonist, blocked the increase of colonic temperature of the rabbit produced by 2 μg/kg LPS administered intravenously without being able in themselves to affect colonic temperature. In addition, prazosin, WB-4101 and CH-38083 antagonized the fall in skin temperature that occurred at the time when the colonic temperature was rising in control animals injected with LPS. All these results suggest that norepinephrine, through stimulation of both α₁⁻andα₂⁻ (α_2A⁻andα_2B/2C⁻) adrenoceptor subtypes, is involved in producing fever in response to bacterial LPS.     

The peripheral nociceptive actions of intravenously administered 5-HT in the rat requires dual activation of both 5-HT2 and 5-HT3 receptor subtypes

In 16-week-old Sprague-Dawley rats lightly anesthetized with pentobarbital, 5-HT (3–96 μg/kg, i.v.;n = 6) produced distinct pseudaffective responses and a dose-dependent (slope= 17.2 ± 6.8s/log₁₀dose) inhibition of the tail-flick (TF) reflex (ED₅₀ = 32.6 ± 9.2 μg/kg). In the same rats, a 1:1 combination of α-methyl 5-HT (a 5-HT₂ receptor selective agonist) and 2-methyl 5-HT (a 5-HT₃ receptor selective agonist) (3–192 μg/kg, i.v.), produced the same profile of pseudaffective responses and also resulted in a dose-dependent (slope= 34.0± 7.0s/log₂dose) inhibition of the TF reflex (ED₅₀ = 88.4 ± 20.5 μg/kg). In contrast, administration of α-methyl 5-HT (3–192 μg/kg, i.v.) or 2-methyl 5-HT (3–192 μg/kg, i.v.) alone did not produce any pseudaffective responses or any change in TF latency from baseline. In conscious 16-week-old male Sprague-Dawley rats, administration of 5-HT (48 μg/kg, i.v.;n = 5), or a 1:1 combination of α-methyl 5-HT and 2-methyl 5-HT (total dose= 120 μg/kg, i.v.;mn = 5), resulted in a passive avoidance behavior assessed in a step-down paradigm (slopes= 139.7 ± 58.2and154.9 ± 63.9s/trial, respectively), and the same profile of distinct pseudaffective responses exhibited by the lightly pentobarbital-anesthetized rats. However, administration of either α-methyl 5-HT (96 μg/kg, i.v.;n = 4) or 2-methyl 5-HT (96 μg/kg, i.v.;n = 4), while producing significant 5-HT receptor-mediated cardiovascular responses, produced a learned behavior not different from saline (0.25 ml, i.v.;n = 6) (slopes= 7.6 ± 2.5, 6.3 ± 1.8and7.4 ± 3.6s/trial, respectively). These results are consistent with the hypothesis that the peripheral nociceptive responses to i.v. 5-HT requires dual activation of 5-HT₂ and 5-HT₃ receptor subtypes.     

Chronic ethanol administration alters the modulatory effect of 5α-pregnan-3α-ol-20-one on the binding characteristics of various radioligands of GABAA receptors

In this study, we investigated the modulatory effect of 5α-pregnan-3α-ol-20-one, a neurosteroid, on the binding characteristics of [ ]flunitrazepam (2 nM), [ ]muscimol (5 nM), and 4 nM [ ]t-butylbicyclophosphorothionate (TBPS) in cerebral cortex, cerebellum, and hippocampus of control, ethanol-dependent, and ethanol-withdrawn rats. 5α-Pregnan-3α-ol-20-one potentiated the binding of [ ]flunitrazepam and [ ]muscimol in all the rat brain regions investigated in this study. There was a significant increase in the maximal potentiation of [ ]flunitrazepam as well as [ ]muscimol binding (E_max) in the ethanol-dependent rat cerebellum as compared to control group (p<0.025). Furthermore, 5α-pregnan-3α-ol-20-one elicited a biphasic response, i.e., it potentiated the binding of [ ]TBPS at lower concentrations (100 nM) and inhibited the binding at higher concentrations (>100 nM). There was a significant higher inhibition of [ ]TBPS binding (−E_max) by 5α-pregnan-3α-ol-20-one in the hippocampus of ethanol-dependent as well as ethanol-withdrawn rats (p<0.025). These observations suggest that the neurosteroid binding site associated with the γ-aminobutyric acid_A (GABA_A) receptors in cerebellum and hippocampus plays an important role during ethanol-dependence and ethanol-withdrawal, and some of the changes following ethanol dependence and its withdrawal may be mediated through the neurosteroid binding site.     

Progesterone 5α-reductase in mouse brain

We have examined the distribution of progesterone 5α-reductase activity in minces of discrete brain areas from two strains of adult ovariectomized mice known to be differentially responsive to the behavioral effects of exogenously administered 5α-pregnane-3,20-dione (5α-DHP). Minces of freshly dissected midbrain tegmentum (TEG), hypothalamus-preoptic area (HPOA), hippocampus (HIP), and parietal cortex (CTX) were incubated with [4-¹⁴C]progesterone in a Krebs-Henseleit bicarbonate buffer solution containing 10 mM glucose for 2 h at 37°C. The [¹⁴C]5α-DHP produced was identified by comparison with known standards in paper and thin-layer chromatography systems, derivative formation, and recrystallization to a constant¹⁴C/³H ratio. We found a 6-fold difference in the extent to which various brain areas formed 5α-DHP. The amount of conversion was similar for both strains of mice. When expressed as picomoles of 5α-DHP formed per 2 h per 20 mg of tissue, the following order of activity was observed: TEG > HPOA > HIP > CTX.     

Subtype-specificity of the presynaptic α2-adrenoceptors modulating hippocampal norepinephrine release in rat

In vivo brain microdialysis and high-performance liquid chromatography with electrochemical detection were used to study the effect of different selective α₂-antagonists on hippocampal norepinephrine (NE) release in freely moving awake rat. Systemic administration (0.5 mg/kg i.p.) of either the α_2AD-antagonist BRL 44408 or the α-_2BC-antagonist ARC 239 did not significantly change the basal release of NE. At a higher dose (5 mg/kg i.p.) ARC 239 was still ineffective, whereas BRL 44408 caused a significant increase of the extracellular level of NE. Similar results were obtained from in vitro perfusion experiments. Rat hippocampal slices were loaded with [³H]NE and the electrical stimulation-evoked release of [³H]NE was determined. The α₂-antagonists were applied in a concentration range of 10⁻⁸ to 10⁻⁶ M. ARC 239 was ineffective, whereas BRL 44408 significantly increased the electrically induced release of [³H]NE. In agreement with the data of microdialysis and perfusion experiments. BRL 44408 displaced [³H]yohimbine from hippocampal and cortical membranes of rat brain with high affinity whereas ARC 239 was less effective. The pK_i values of eight different α₂-adrenergic compounds showed a very good correlation (r = 0.98, slope = 1.11 P < 0.0001) in hippocampus and frontal cortex where the α₂-adrenoceptors have been characterized as α_2D-subtype. Our data indicate that hippocampal NE release in rat is regulated by α_2D-adrenoceptors, a species variation of the human α_2A-subtype.     

Functional evidence for the rapid desensitization of 5-HT3 receptors on vagal afferents mediating the Bezold–Jarisch reflex

The aim of this study was to determine whether 5-hydroxytryptamine (5-HT)₃ receptors on cardiopulmonary afferents mediating the Bezold–Jarisch reflex (BJR) desensitize upon repeated exposure to selective agonists. BJR-mediated falls in heart rate, diastolic arterial blood pressure and cardiac output elicited by the 5-HT₃-receptor agonists, phenylbiguanide (100 μg/kg, i.v.) or 2-methyl-5-HT (100 μg/kg, i.v.), progressively diminished upon repeated injection in conscious rats. The BJR responses elicited by 5-HT (40 μg/kg, i.v.) were markedly reduced in rats which had received the above injections of phenylbiguanide or 2-methyl-5-HT whereas the BJR responses elicited by -S-nitrosocysteine (10 μmol/kg, i.v.) were similar before and after the injections of the 5-HT₃ receptor agonists. These findings suggest that tachyphylaxis to 5-HT₃ receptor agonists may be due to the desensitization of 5-HT₃ receptors on cardiopulmonary afferents rather than the impairment of the central or peripheral processing of the BJR.     

Substance P suppresses the activity of α2-adrenoceptors of the nucleus reticularis gigantocellularis involved in cardiovascular regulation in the rat

We evaluated possible interactions between substance P (SP) and the α₂-adrenoceptors in the nucleus reticularis gigantocellularis (NRGC) of the medulla oblongata involved in cardiovascular regulation. Adult, male Sprague-Dawley rats anesthetized with pentobarbital sodium (40 mg/kg, i.p., with 10 mg/kg/h i.v. supplements) were used. The circulatory suppressant efficacy of a centrally acting α₂-adrenoceptor agonist, guanabenz, was used as the experimental index. Bilateral microinjection of SP (300 or 600 pmol) into the NRGC, a medullary site that is critically involved in the cardiovascular depressive actions of guanabenz, significantly diminished the hypotensive and bradycardiac efficacy of the aminoguanidine compound (100 μg/kg, i.v.). This implied reduction in α₂-adrenoceptor activity in the NRGC by SP was antagonized by its selective receptor antagonist, [d-Pro²,d-Trp^7,9]-SP (1200 pmol). Similarly, attenuation by SP of the cardiovascular suppressant effects of guanabenz was also reversed by immunocytochemically verified depletion of dopamine-β-hydroxylase-immunoreactive nerve terminals in the NRGC, elicited by the selective noradrenergic neurotoxin, DSP4 (50 μg). These data suggest that SP may exert an inhibitory action on the α₂-adrenoceptors in the NRGC that are involved in central cardiovascular regulation, possibly via a presynaptic modulation on noradrenergic neurotransmission.