Antagonism of ethanol effects on cerebellar Purkinje neurons by the benzodiazepine inverse agonists Ro 15-4513 and FG 7142: electrophysiological studies

Ro 15-4513, a benzodiazepine inverse agonist, has been reported to antagonize the ataxic effects of ethanol. The present study investigates the Ro 15-4513 sensitivity of rat cerebellar Purkinje neurons to the depressant effects of locally applied ethanol. Local applications of ethanol by pressure ejection from multibarrel micropipettes caused reversible and dose-dependent depressions of the neuronal firing rates of single cerebellar Purkinje neurons. The ethanol-induced depressions could be antagonized by local applications of Ro 15-4513 applied from another barrel of the same micropipette. This antagonism was not competitive, suggesting that Ro 15-4513 does not interfere directly with the initial step of the ethanol mechanism of action. A beta-carboline inverse agonist, FG 7142, was more efficacious than Ro 15-4513 for antagonizing the ethanol-induced depressions, but appeared to be less potent. Recovery of ethanol-induced depressions of Purkinje neurons firing rates after Ro 15-4513 antagonism was not usually observed for 1 hr or more after the antagonist application. In contrast to ethanol effects, qualitatively similar gamma-aminobutyric acid-induced depressions of these same neurons were not antagonized by the doses of Ro 15-4513 used. We conclude that the electrophysiological depressant effects of ethanol on cerebellar neuronal activity can be antagonized by the benzodiazepine inverse agonists, Ro 15-4513 and FG 7142.     

Regional distribution of a Ro5 4864 binding site that is functionally coupled to the gamma-aminobutyric acid/benzodiazepine receptor complex in rat brain

The hypothesis that a novel drug binding site linked to a gamma-aminobutyric acid (GABA)-regulated chloride ionophore mediates the excitatory effects of the atypical benzodiazepine (BZ) Ro5 4864 is further evaluated in the present study. Dose-dependent inhibition of [3H]flunitrazepam to the central BZ receptor in rat cerebral cortex by the cage convulsant t-butylbicyclophosphorotionate (TBPS) is modulated by Ro5 4864 and the isoquinoline PK 11195 in a manner consistent with their reported pro/anticonvulsant effects. The ability of Ro5 4864 to enhance the binding of [35S]TBPS to a GABA-regulated chloride ionophore in rat cortex is unchanged after the irreversible labeling of the central BZ receptor by the photoaffinity label Ro15 4513. Together, these observations further suggest that 1) the effect of Ro5 4864 on [35S]TBPS is not mediated by the central BZ receptor and 2) the Ro5 4864 binding site is allosterically coupled to the GABA/BZ receptor-chloride ionophore complex in rat cerebral cortex. Anatomical localization of Ro5 4864-stimulated [35S]TBPS binding in rat brain by autoradiography reveals a distribution of chloride ionophore-coupled Ro5 4864 sites which is in many instances similar to that of the GABA/BZ receptor-chloride ionophore complex. These studies lend additional support to the postulate that this drug binding site represents an additional locus for the regulation of GABAergic neurotransmission in the central nervous system.     

Electrophysiological actions of cocaine on noradrenergic neurons in rat locus ceruleus

Extracellular microelectrode experiments were conducted to study the effects of cocaine HCl on the activity of spontaneously firing single locus ceruleus (LC) noradrenergic neurons in vivo. The responses of single identified noradrenergic LC neurons to the systemic (i.v.) administration of cocaine were observed over a wide range of doses (0.0625-2.0 mg/kg). The spontaneous activity of all LC neurons receiving doses greater than the threshold dose (0.0625 mg/kg) was inhibited in a dose-dependent manner. The local anesthetics, procaine and mepivacaine, did not affect LC neuronal activity, action potential amplitude or slope. The inhibitory effects of cocaine on spontaneous LC neuron activity was reversed by the subsequent i.v. administration of the specific alpha-2 adrenoceptor antagonist, piperoxone, but not the opiate receptor antagonist, naloxone. Pretreatment with another alpha-2 adrenoceptor antagonist, yohimbine (5 mg/kg i.p.), attenuated significantly the inhibition of spontaneous LC activity elicited by i.v. cocaine. Intravenous cocaine produced a brief increase in mean arterial pressure which did not appear to be correlated with the more sustained inhibition of LC neurons. Reserpine pretreatment (10 mg/kg i.p.) attenuated significantly the inhibitory effects of cocaine on LC activity. These results suggest that the inhibition of spontaneous LC neuron activity by i.v. cocaine is most likely mediated by an augmented action of catecholamines at central alpha-2 adrenoceptors and not by the local anesthetic effects of cocaine.     

Electrophysiological evidence for presynaptic actions of phencyclidine on noradrenergic transmission in rat cerebellum

The actions of the psychotomimetic drug phencyclidine (PCP) were studied using Purkinje neurons in the cerebellum of urethane-anesthetized rats. PCP, applied by micropressure ejection through multibarreled micropipettes, depressed the spontaneous activity of these neurons as recorded by extracellular electrophysiological techniques. This depressant effect was blocked by neuroleptic drugs and lithium, both of which also block the depressant effects of norepinephrine, but not those of gamma-aminobutyric acid. PCP-elicited depressions could not be obtained in rats in which the cerebellar noradrenergic terminals had been lesioned selectively by pretreatment with the neurotoxin 6-hydroxydopamine. However, PCP was still an effective depressant in animals after destruction of non-noradrenergic intrinsic excitatory and inhibitory interneurons which synapse on the Purkinje cell by neonatal X-irradiation. Further treatment of the X-irradiated animal with 6-hydroxy-dopamine resulted in Purkinje neurons which were not responsive to PCP. Administration of magnesium ions, which reduces the release of neurotransmitters from afferent terminals, also blocked the depressant effects of PCP. The results of this study suggest that PCP acts in the cerebellum by a presynaptic mechanism involving the release of norepinephrine from intact, functioning noradrenergic terminals.     

Downregulation of [3H]Ro5-4864 binding sites after exposure to peripheral-type benzodiazepines in vitro

Peripheral-type benzodiazepine (BZD) binding sites undergo a rapid and pronounced downregulation after exposure to these compounds in vitro. Friend erythroleukemia cells were incubated with micromolar concentrations of BZD after which they were washed thoroughly and the binding of the specific peripheral-type BZD radioligand [3H]Ro5-4864 was determined. Exposure to the peripheral-type BZD Ro7-3351 decreased the number of [3H]Ro5-4864 binding sites from 324 to 41 fmol/10(6) cells with no change in affinity. Downregulation appears to require active cellular processes because it is blocked when exposure to BZD is at 4 degrees C rather than at 37 degrees C. Furthermore, whereas [3H]Ro5-4864 binding is decreased substantially in membrane preparations made from downregulated cells, it is not altered when membrane preparations from control cells are exposed to BZD. The time course of downregulation is quite rapid, as it occurs within minutes. In contrast, the return of sites requires days and there is a close relationship between return of sites and growth of new cells. The ability of BZDs to downregulate correlates more closely with affinity for the peripheral-type site than with biological activity. The ability to undergo downregulation is characteristic of receptors and its occurrence suggests that peripheral-type BZD binding sites are functional receptors.     

Electrophysiological evidence for expression of glycine receptors in freshly isolated neurons from nucleus accumbens

In the course of studying N-methyl-D-aspartate (NMDA) receptors of the nucleus accumbens (NAcc), we found that 20% of freshly isolated medium spiny neurons, as well as all interneurons, responded in an unexpected way to long (5-s) coapplication of NMDA and glycine, the coagonist of NMDA receptors. Whereas the reversal potential of the peak NMDA current of this subset of neurons was still around 0 mV, the desensitizing current became outward at hyperpolarized potentials around -30 mV. A Cl(-)-free solution shifted the equilibrium potentials of the desensitized currents to around 0 mV. This outward current was not blocked by a Ca(2+)-free, Ba(2+)-containing solution, suggesting that the anionic conductance was not activated by Ca(2+) influx through NMDA receptor channels. Interestingly, glycine alone also evoked a current with a similar hyperpolarized reversal potential in this subset of neurons. The glycine current reversed around -50 mV, rectified outwardly, and inactivated strongly. Its desensitization was best fitted with a double exponential. Only the slow desensitization showed clear voltage dependence. The glycine current was not blocked by 200 microM picrotoxin and 10 microM zinc, was weakly antagonized by 1 microM strychnine, and was not enhanced by 1 microM zinc. In addition, 1 mM taurine, but not GABA, inactivated glycine currents, and 1 mM glycine occluded 10 mM taurine-mediated currents. These data indicate that a subset of nucleus accumbens neurons expresses glycine receptors and that either glycine or taurine could be an endogenous agonist for these receptors.     

Effects of Ro15-4513 and other benzodiazepine receptor inverse agonists on alcohol-induced intoxication in the rat

The ability of the imidazobenzodiazepine Ro15-4513 to antagonize the behavioral intoxication produced by ethanol and related short-chain alcohols was examined in the rat. Ro15-4513 dose dependently (0.5-10 mg/kg i.p.: IC50, 1.5 mg/kg) inhibited the intoxication induced by ethanol (2 g/kg), as well as t-amyl alcohol (0.36 g/kg) and methanol (4.66 g/kg). The effects of Ro15-4513 in blocking ethanol-induced intoxication were blocked by the benzodiazepine receptor antagonists Ro15-1788 and CGS-8216. However, Ro15-4513 was ineffective in antagonizing the intoxication observed after higher doses of ethanol (4 g/kg). In contrast, ethanol-induced intoxication was not antagonized by the benzodiazepine receptor antagonists Ro15-1788 (10 mg/kg) or CGS-8216 (20 mg/kg), nor by the inverse agonists FG-7142 (10-30 mg/kg) or beta CCE (10 mg/kg). When administered after ethanol, Ro15-4513 also reversed ethanol-induced intoxication in a dose-dependent manner (2.5-10 mg/kg i.p.: IC50, 5 mg/kg), an effect which was also blocked by Ro15-1788 and CGS-8216. However, neither beta CCE (10 mg/kg) or FG-7142 (less than or equal to 30 mg/kg) alone reversed ethanol-induced intoxication. Moreover, beta CCE (10 mg/kg), when administered just before Ro15-4513, completely antagonized the actions of Ro15-4513 in reversing ethanol-induced intoxication. These data suggest that the ability of Ro15-4513 to antagonize, and to reverse, ethanol-induced intoxication is mediated via central benzodiazepine receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neurochemical characterization of the actions of 5-amino-2,4- dihydroxy-alpha-methylphenylethylamine (5-ADMP): a selective neurotoxin to central noradrenergic neurons

The concentrations of amine neurotransmitters [dopamine (DA), norepinephrine (NE) and 5-hydroxytryptamine] and some of their deaminated metabolites [dihydroxyphenylacetic acid and 5-hydroxyindoleacetic acid] were determined in selected regions of the rat brain, including several hypothalamic nuclei, at various times after the i.c.v. injection of various doses of 5-amino-2,4-dihydroxy-alpha-methylphenylethylamine (5-ADMP). Seven days after a single i.c.v. injection of 100 micrograms of 5-ADMP the concentration of NE in all brain regions examined was reduced markedly, as was the concentration of DA in the median eminence. The concentrations of DA and dihydroxyphenylacetic acid in regions other than the median eminence, and the concentrations of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in all regions analyzed were unaltered. Multiple injections of 5-ADMP (100 micrograms i.c.v. every 48 hr X 3) did not increase the NE depletion, but caused slight decreases in DA and 5-hydroxytryptamine in some regions. The NE concentrations in hypothalamic nuclei were reduced significantly 4 hr after 100 micrograms i.c.v. administration of 5-ADMP and this depletion was maintained for at least 28 days. The 5-ADMP-induced decline of NE in all brain region, but not the decline of DA in the median eminence, was prevented partially by pretreatment with desipramine. These results indicate that 5-ADMP is a relatively specific neurotoxin for NE neurons, being particularly effective in destroying NE nerve terminals in the hypothalamus.     

Electrophysiological effects of ethanol on hippocampal and cerebellar neurons cografted with locus coeruleus in oculo: role of the noradrenergic circuitry.

Nucleus locus coeruleus (LC) was sequentially transplanted with hippocampus or cerebellum from rat fetuses to the anterior eye chamber of adult rat hosts. Histological, electrophysiological and pharmacological studies indicate that the LC neurons survive and functionally innervate neurons in hippocampal and cerebellar cografts. Ethanol, when superfused over the double transplants in urethane-anesthetized hosts, caused excitations of hippocampal neuronal activity at doses between 1 and 30 mM, whereas applications above 30 mM depressed the activity of grafted hippocampal neurons. Similar results were observed in cerebellar Purkinje neurons cografted in oculo, except that cerebellar neurons were more sensitive to both the excitatory and the depressant effects of ethanol. The excitations caused by lower ethanol doses in double grafts were prevented by the cosuperfusion of 0.5 to 1.0 microM clonidine, a treatment which effectively removed the inhibitory influence of the LC neurons from the grafted neuronal circuit by depressing the LC neuronal activity. Ethanol-induced excitations were also not observed in single grafts of hippocampus, which lack a catecholamine innervation. Furthermore, in double grafts, when the noradrenergic inhibition was blocked postsynaptically with the alpha adrenergic antagonist phentolamine, ethanol-induced excitations were prevented, although ethanol did not alter the postsynaptic actions of norepinephrine. Our data suggest that the ethanol-induced excitations in the cerebellar and hippocampal grafts appear to be disinhibitions mediated by an ethanol-induced depression of the inhibitory noradrenergic input to these target tissues from LC cografts. Indeed, the doses of ethanol that induced neuronal excitations in hippocampal transplants also elicited marked depressions of LC neurons.     

Ro 19-8022, a nonbenzodiazepine partial agonist at benzodiazepine receptors: neuropharmacological profile of a potential anxiolytic.

The partial agonist at benzodiazepine receptors, Ro 19-8022, has been characterized as a putative anxiolytic drug with an improved side effect profile. This orally active compound is a representative of a quinolizinone structure class and shows potent anticonflict activity in mice and rats. It protects rodents from convulsions induced by pentylenetetrazol, N-methyl-D-aspartic acid and maximal electroshock, as well as against audiogenic seizures, with an efficacy comparable to that of the full agonist alprazolam. No appreciable sedative or motor-impairing effects could be detected up to a very high dose (100 mg/kg) in the horizontal wire test or the rotarod performance test in mice and rats and in spontaneous behavior in monkeys. Consistent with its characterization as a partial agonist, Ro 19-8022 antagonized the motor impairment induced by the full agonists diazepam or meclonazepam measured in horizontal wire and rotarod tests in rodents, and reduced flunitrazepam-induced effects in squirrel monkeys, with an efficacy comparable to that of the benzodiazepine receptor antagonist flumazenil. After subchronic administration of Ro 19-8022 to mice, antagonist-precipitated withdrawal syndrome was dramatically weaker than after alprazolam treatment, which is indicative of a lower physical dependence liability of Ro 19-8022. Pharmacodynamic effects recorded in convulsion and reversal of motor impairment tests after i.v. administration suggest a long duration of action of this compound. Taken together, such preclinical data suggest that benzodiazepine receptor partial agonists with a neurological and behavioral profile such as that of Ro 19-8022 may offer an innovative therapeutic approach to the treatment of anxiety disorders.     

Effects of a benzodiazepine antagonist, Ro 15-1788, on hippocampal field potentials in freely moving rats.

Effects of a benzodiazepine receptor agonist (diazepam) and an antagonist (Ro 15-1788, flumazenil) administered separately or in combination on field potentials recorded from the hippocampal dentate area were examined in unanesthetized, unrestrained rats. Population excitatory postsynaptic potentials (EPSPs) evoked by stimulation of the perforant path were depressed significantly by diazepam (4 mg/kg, i.p.). However, diazepam did not affect the firing (spike) threshold of dentate granule cells. The injection of Ro 15-1788 (4 mg/kg, i.p.) alone affected neither excitatory synaptic transmission nor population spike threshold. Strength of gamma-amino butyric acid-mediated recurrent inhibition as measured by the paired-pulse technique was potentiated by diazepam but unaffected by Ro 15-1788. However, the diazepam-enhanced inhibition was reversed by a subsequent administration of Ro 15-1788. Previous studies indicate that Ro 15-1788 acts not only as a selective benzodiazepine antagonist but also as a partial agonist-antagonist or an inverse agonist depending probably on doses. The present study demonstrated that Ro 15-1788 acted as a pure antagonist at low doses. These data suggest that the clinical use of Ro 15-1788 at high doses against comas induced by unidentified drugs could worsen the conditions and that low doses are recommendable for initial treatments because of its pure antagonist action.     

Metabolism of the antimalarial endoperoxide Ro 42-1611 (arteflene) in the rat: evidence for endoperoxide bioactivation

Ro 42-1611 (arteflene) is a synthetic endoperoxide antimalarial. The antimalarial activity of endoperoxides is attributed to iron(II)-mediated generation of carbon-centered radicals. An alpha, beta-unsaturated ketone (enone; 4-[2',4' bis(trifluoromethyl)phenyl]-3-buten-2-one), obtained from arteflene by reaction with iron(II), was identified previously as the stable product of a reaction that, by inference, also yields a cyclohexyl radical. The activation of arteflene in vivo has been characterized with particular reference to enone formation. [14C]Arteflene (35 micromol/kg) was given i.v. to anesthetized and cannulated male rats: 42.2 +/- 7.0% (mean +/- S.D., n = 7) of the radiolabel was recovered in bile over 5 h. In the majority of rats, the principal biliary metabolites were 8-hydroxyarteflene glucuronide (14.2 +/- 3. 9% dose, 0-3 h) and the cis and trans isomers of the enone (13.5 +/- 4.6% dose, 0-3 h). In conscious rats, 15.3 +/- 1.6% (mean +/- S.D., n = 8) of the radiolabel was recovered in urine over 24 h. The principal urinary metabolite appeared to be a glycine conjugate of a derivative of the enone. Biliary excretion of the glucuronide, but not of the enones, was inhibited by ketoconazole. 8-Hydroxyarteflene was formed extensively by rat and human liver microsomes but no enone was found. Bioactivation is a major pathway of arteflene's metabolism in the rat. Although the mechanism of in vivo bioactivation is unclear, the reaction is not catalyzed by microsomal cytochrome P-450 enzymes.     

Paraventricular oxytocinergic hypothalamic prevention or interruption of long-term potentiation in dorsal horn nociceptive neurons: Electrophysiological and behavioral evidence

Spinal long-term potentiation (LTP) elicited by noxious stimulation enhances the responsiveness of dorsal horn nociceptive neurons to their normal input, and may represent a key mechanism of central sensitization by which acute pain could turn into a chronic pain state. This study investigated the electrophysiological and behavioral consequences of the interactions between LTP and descending oxytocinergic antinociceptive mechanisms mediated by the hypothalamic paraventricular nucleus (PVN). PVN stimulation or intrathecal oxytocin (OT) reduced or prevented the ability of spinal LTP to facilitate selectively nociceptive-evoked responses of spinal wide dynamic range (WDR) neurons recorded in anesthetized rats. In a behavioral model developed to study the effects of spinal LTP on mechanical withdrawal thresholds in freely moving rats, the long-lasting LTP-mediated mechanical hyperalgesia was transiently interrupted or prevented by either PVN stimulation or intrathecal OT. LTP mediates long-lasting pain hypersensitivity that is strongly modulated by endogenous hypothalamic oxytocinergic descending controls.     

Electrophysiologic effects of amiloride in canine Purkinje fibers: evidence for a delayed effect on repolarization

Amiloride, a potassium-sparing diuretic, inhibits Na+ transport, Na+-H+ exchange and possibly Na+-Ca++ exchange in a variety of cellular and epithelial tissues. Similar membrane ion transport mechanisms exist in cardiac tissue, yet there are little data on possible interference by amiloride with ion transport in the heart. Given recent evidence for a delay in amiloride uptake into erythroid cells, we studied the electrophysiologic effects of amiloride after prolonged drug exposure in canine Purkinje fibers using standard microelectrode techniques. Amiloride (1-10 microM) led to a progressive lengthening of action potential duration with a tau of 1.8 +/- 0.5 hr (n = 15). At long cycle lengths (greater than or equal to 2000 msec) early afterdepolarizations and oscillations around the plateau were seen. To determine the etiology of the afterdepolarizations, Purkinje fibers treated for 2 hr with 10 microM amiloride were then exposed to tetrodotoxin, manganese and nisoldipine. Tetrodotoxin (7.8 X 10(-7) M) reversed completely all amiloride effects rapidly and reversibly. MnCl2 (4 mM) increased the afterdepolarizations, and arrest occurred at the plateau potential routinely. Nisoldipine (10(-6) M), a more selective blocker of slow inward current, shortened action potential duration somewhat but did not reverse fully the effects of amiloride. We conclude that amiloride has a pronounced effect on repolarization in the canine Purkinje fiber and this effect is manifest only after prolonged exposure to the drug.     

Gambierol acts as a functional antagonist of neurotoxin site 5 on voltage-gated sodium channels in cerebellar granule neurons

The marine toxin gambierol, a polyether ladder toxin derived from the marine dinoflagellate Gambierdiscus toxicus, was evaluated for interaction with voltage-gated sodium channels (VGSCs) in cerebellar granule neuron (CGN) cultures. At concentrations ranging from 10 nM to 10 microM, gambierol alone had no effect on the intracellular Ca2+ concentration [Ca2+]i of exposed CGN cultures. Furthermore, there was no evidence of neurotoxicity in CGN cultures exposed for 2 h to gambierol (1 nM-10 microM). However, gambierol was a potent inhibitor (IC50 = 189 nM) of the elevation of [Ca2+]i that accompanies exposure of CGN cultures to the VGSC activator brevetoxin-2 (PbTx-2). To further explore the potential interaction of gambierol with VGSCs, the influence of gambierol on PbTx-2-induced neurotoxicity was assessed. Gambierol reduced the PbTx-2-induced efflux of lactate dehydrogenase in exposed CGN cultures in a concentration-dependent manner (IC50 = 471 nM). It is noteworthy that the potencies of gambierol as an inhibitor of both PbTx-2-induced Ca2+ influx and cytotoxicity were coincident. Finally, the inhibitory effects of gambierol on PbTx-2-induced elevation of [Ca2+]i were compared with those of brevenal, a natural inhibitor of the toxic effects of brevetoxin isolated from cultures of Karina brevis. Like gambierol, brevenal inhibited PbTx-2-induced elevation of [Ca2+]i in a concentration-dependent manner (IC50 = 108.6 nM). These results provide evidence for gambierol acting as a functional antagonist of neurotoxin site 5 on neuronal VGSCs.     

Homeopathy for depression: a systematic review of the research evidence

OBJECTIVE: To systematically review the research evidence on the effectiveness of homeopathy for the treatment of depression and depressive disorders. METHODS: A comprehensive search of major biomedical databases including MEDLINE, EMBASE, CINAHL, PsycINFO and the Cochrane Library was conducted. Specialist complementary and alternative medicine (CAM) databases including AMED, CISCOM and Hom-Inform were also searched. Additionally, efforts were made to identify unpublished and ongoing research using relevant sources and experts in the field. Relevant research was categorised by study type and appraised according to study design. Clinical commentaries were obtained for studies reporting clinical outcomes. RESULTS: Only two randomised controlled trials (RCTs) were identified. One of these, a feasibility study, demonstrated problems with recruitment of patients in primary care. Several uncontrolled and observational studies have reported positive results including high levels of patient satisfaction but because of the lack of a control group, it is difficult to assess the extent to which any response is due to specific effects of homeopathy. Single-case reports/studies were the most frequently encountered clinical study type. We also found surveys, but no relevant qualitative research studies were located.: Adverse effects reported appear limited to 'remedy reactions' ('aggravations') including temporary worsening of symptoms, symptom shifts and reappearance of old symptoms. These remedy reactions were generally transient but in one study, aggravation of symptoms caused withdrawal of the treatment in one patient. CONCLUSIONS: A comprehensive search for published and unpublished studies has demonstrated that the evidence for the effectiveness of homeopathy in depression is limited due to lack of clinical trials of high quality. Further research is required, and should include well-designed controlled studies with sufficient numbers of participants. Qualitative studies aimed at overcoming recruitment and other problems should precede further RCTs. Methodological options include the incorporation of preference arms or uncontrolled observational studies. The highly individualised nature of much homeopathic treatment and the specificity of response may require innovative methods of analysis of individual treatment response.