Silvia L. Cruz

Effects of volatile solvents on recombinant N‐methyl‐D‐aspartate receptors expressed in Xenopus oocytes

We have previously shown that toluene dose‐dependently inhibits recombinant N‐methyl‐D‐aspartate (NMDA) receptors at micromolar concentrations. This inhibition was rapid, almost complete and reversible. The NR1/2B combination was the most sensitive receptor subtype tested with an IC50 value for toluene of 0.17 mM. We now report on the effects of other commonly abused solvents (benzene, m‐xylene, ethylbenzene, propylbenzene, 1,1,1‐trichlorethane (TCE) and those of a convulsive solvent, 2,2,2‐trifluoroethyl ether (flurothyl), on NMDA‐induced currents measured in Xenopus oocytes expressing NR1/2A or NR1/2B receptor subtypes. All of the alkylbenzenes and TCE produced a reversible inhibition of NMDA‐induced currents that was dose‐ and subunit‐dependent. The NR1/2B receptor subtype was several times more sensitive to these compounds than the NR1/2A subtype. The convulsant solvent flurothyl had no effect on NMDA responses in oocytes but potently inhibited ion flux through recombinant GABA receptors expressed in oocytes. Overall, these results suggest that abused solvents display pharmacological selectivity and that NR1/2B NMDA receptors may be an important target for the actions of these compounds on the brain.

Evidence for the involvement of a spinal pattern generator in the control of the genital motor pattern of ejaculation

One of the hypotheses to explain the neural mechanisms underlying rhythmic behaviours suggests that the central nervous system has the intrinsic capacity to produce repetitive, rhythmic output to the muscles involved in the response by means of a neuronal circuit named central pattern generator (CPG). The occurrence of rhythmic motor patterns during ejaculatory behaviour in mammals, which includes the genital motor pattern, has been shown. A CPG might regulate the timing of the repetitive muscular responses that constitute the ejaculatory motor pattern. The objective of the present study was to evidence that a CPG at a spinal level is involved in the expression and pacing of the rhythmic motor pattern generated during ejaculation. To this purpose we used the genital reflex as a model system. Following the general principles for the study of rhythmic motor patterns, the data obtained in the present series of experiments document that: (1) a rhythmic muscular response, the genital motor pattern, is registered during the ejaculatory event (expulsion of the urethral contents); (2) this ejaculatory motor response has similar EMG characteristics in intact and in spinal urethane-anaesthetised male rats; (3) interruption of the afferent inflow (deafferentation) does not disrupt the expression of the ejaculatory motor train; (4) a change in the stimulation interval does not alter the intrinsic pacing of the ejaculatory-like response; and (5) fictive ejaculation can be induced by pharmacological means. Together, this evidence supports the notion that a CPG produces the rhythmic ejaculatory motor pattern registered during fictive ejaculation.

Inhibition of cardiac sodium currents by toluene exposure

Toluene is an industrial solvent widely used as a drug of abuse, which can produce sudden sniffing death due to cardiac arrhythmias. In this paper, we tested the hypothesis that toluene inhibits cardiac sodium channels in Xenopus laevis oocytes transfected with Nav1.5 cDNA and in isolated rat ventricular myocytes. In oocytes, toluene inhibited sodium currents (INa+) in a concentration‐dependent manner, with an IC50 of 274 μM (confidence limits: 141–407μM). The inhibition was complete, voltage‐independent, and slowly reversible. Toluene had no effect on: (i) the shape of the I–V curves; (ii) the reversal potential of Na+; and (iii) the steady‐state inactivation. The slow recovery time constant from inactivation of INa+ decreased with toluene exposure, while the fast recovery time constant remained unchanged. Block of INa+ by toluene was use‐ and frequency‐dependent. In rat cardiac myocytes, 300 μM toluene inhibited the sodium current (INa+) by 62%; this inhibition was voltage independent. These results suggest that toluene binds to cardiac Na+ channels in the open state and unbinds either when channels move between inactivated states or from an inactivated to a closed state. The use‐ and frequency‐dependent block of INa+ by toluene might be responsible, at least in part, for its arrhythmogenic effect.

Anxiolytic-like actions of toluene in the burying behavior and plus-maze tests: differences in sensitivity between 5-HT1B knockout and wild-type mice

This paper compares the anxiolytic-like actions of toluene in two anxiety paradigms, the burying behavior and plus-maze tests, in 5-HT(1B) knockout (KO) and 129/Sv-ter wild-type (WT) mice. Static exposures were conducted in 29-l gas chromatographic jars. Animals were exposed to toluene (0, 1000, 2000 or 4000 ppm; n=8-12, each) for 30 min, and immediately after, tested in one of the anxiety paradigms. Motor coordination was evaluated in the rota-rod test in independent groups of mice. Toluene produced a dose-dependent decrease in anxiety-like levels in both anxiety paradigms and in both the strains. However, toluene exerted its effects at lower concentrations in KO mice than in the WT strain. These results cannot be attributed to a decrease in motor coordination since all the animals behaved similarly in the rota-rod test, regardless of the treatment. To discard any inherent difference in the nociception threshold between strains, mice were tested in the hot plate immediately after being exposed to either air or toluene. Toluene increased nociception in a similar fashion in both the strains. Our results suggest that 5-HT(1B) KO mice are more sensitive to those of toluenes actions related to anxiety, but not to those related with motor coordination or nociception. Data are discussed in terms of toluenes mechanisms of action and on differences between WT and KO animals.

Gastrointestinal effects of 5-hydroxytryptamine and related drugs

This paper deals with the effects of 5-hydroxytryptamine (5-HT; serotonin) and related drugs on the gastrointestinal tract (GIT). The nomenclature and classification of 5-HT receptors, as well as their putative role in the GIT are updated in this review. Besides its effects on the cardiovascular system, which have been extensively described, several lines of evidence suggest a role for 5-HT in regulating gastrointestinal functions. 5-HT is present in the gastrointestinal tissues, and can elicit contraction or relaxation by activation of a wide variety of mechanisms and receptors. At least four main types of receptors (5-HT1, 5-HT2, 5-HT3 and 5-HT4) have been described and all the four types seem to influence the GIT. In this respect, the 5-HT2, and in some cases the 5-HT1 receptors, appear to be present on the gastrointestinal smooth muscle, while 5-HT3 and 5-HT4 are mainly neuronal.

Effects of inhaled toluene and 1,1,1-trichloroethane on seizures and death produced by N-methyl-d-aspartic acid in mice

Evidence exists that some abused solvents have N-methyl-D-aspartic acid (NMDA) antagonist activity, although which of their effects may be related to this mechanism is not well understood. The effects of toluene and 1,1,1-trichloroethane (TCE) on NMDA-induced seizures in mice were studied using three experimental protocols: (a) animals injected i.p. with 120 or 170 mg/kg NMDA and immediately afterwards exposed to solvent vapors or air for 30 min (co-exposure protocol); (b) mice exposed for 30 min to solvent or air, then injected with NMDA and placed in the chamber for a second 30-min exposure (pre-exposure+co-exposure protocol); and (c) mice that inhaled 4000 ppm toluene or air for 30 min twice a day, 6 h apart, for 7 days, and were injected with 120 mg/kg NMDA immediately before a 30-min toluene exposure (repeated exposure protocol). When given acutely, toluene, but not TCE, produced concentration-dependent protection against NMDA-induced seizures. Higher concentrations of toluene were also effective against the lethal effects produced by 170 mg/kg NMDA. Clearer effects were seen when the pre-exposure+co-exposure protocol was followed. Under these conditions the IC(50) for toluene was 739 ppm (653-825) against seizure occurrence and 2127 ppm (1966-2288) against lethality. Repeated exposure to toluene did not result in tolerance to its anticonvulsant effects. These results are consistent with the in vitro effects described for toluene as a noncompetitive NMDA antagonist and as a compound that enhances GABAergic transmission. The lack of protective effects of TCE is not consistent with its in vitro actions.

Classification of abused inhalants

Many hundreds of household and industrial products can be volatilized readily and are subject to abuse. Inhalant abuse research has been hampered by a lack of consensus on whether or not there are subclassifications of abused inhalants based on chemical structure, form or intended use of the product or pharmacological properties. This paper discusses strengths and weaknesses of various approaches to classification of inhalants and suggests areas for future research in this area. It is concluded that classification of inhalants by form or product types is not useful for scientific purposes; rather, subclassification of inhalants should be based on a yet-to-be-determined combination of chemical and pharmacological similarity and shared patterns of abuse. One of the ways in which we can improve our understanding of inhalant abuse is to obtain more detailed information on individual products and chemicals, their patterns of use and the geographical distribution of their use.

Comparative study of the effects of toluene, benzene, 1,1,1-trichloroethane, diethyl ether, and flurothyl on anxiety and nociception in mice

The main purpose of this study was to compare the effects of solvents from different chemical classes on anxiety and nociception. Independent groups of mice were exposed to air (control group), toluene (1000-4000 ppm), benzene (1000-4000 ppm), 1,1,1-trichloroethane (TCE, 2000-12000 ppm), diethyl ether (10,000-30,000) or flurothyl (200-600 ppm). After a 30-min exposure, animals were tested either in the anxiety paradigm conditioned defensive burying (CDB) test or in the hot plate test. All solvents but flurothyl produced anxiolytic-like actions being the order of potency toluene > benzene > TCE > diethyl ether. When tested in the hot plate paradigm, toluene and TCE increased nociception, benzene and diethyl ether had no effects, and flurothyl decreased nociception Additional groups of mice were conditioned to recognize the aversive stimulus (electrified prod) prior to toluene exposure and then tested in the CDB test. In unconditioned animals, toluene increased the number of shocks that mice received; however, when mice had previous experience in the CDB test, toluene lacked this effect. Taken together, these results show that inhalants have different effects with different potencies both in the CDB and in the hot plate tests. Additionally, data suggest that acute administration of toluene could impair learning.

Role of opioid receptors in the reduction of formalin-induced secondary allodynia and hyperalgesia in rats

This study assesses the effects of peripheral or intrathecal pre-treatment or post-treatment with micro, delta, kappa and nociceptin/orphanin FQ (NOP) opioid receptor agonists (morphine, U-50488 [trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide hydrochloride], DADLE [D-Ala2-Leu5-enkephalin] and nociceptin, respectively) on formalin-induced secondary mechanical allodynia and hyperalgesia in rats. 1% Formalin injection produced acute nociceptive behaviors (flinching and licking/lifting) followed by long-term tactile secondary allodynia and hyperalgesia. Neither peripheral (into the formalin-injected paw) nor intrathecal morphine post-treatment reversed formalin-induced secondary allodynia and hyperalgesia. In contrast, morphine pre-treatment prevented the development of these pain behaviors. Intrathecal and peripheral post- but not pre-treatment with U-50488 or DADLE significantly reduced secondary allodynia and hyperalgesia. Interestingly, nociceptin reduced both pain behaviors regardless of the administration site or treatment time. Local antinociceptive effects of morphine, DADLE, U-50488 or nociceptin were blocked by naltrexone, naltrindole, 5-guanidinonaltrindole and [Nphe(1)]nociceptin(1-13)NH(2), respectively. These results suggest that the long-term nociceptive behaviors induced by formalin are differentially modulated by selective opioid receptor agonists. In addition, data suggest that peripheral and spinal delta and kappa opioid receptors are important when nociceptive behaviors are established. In contrast, micro opioid receptors are more important at the beginning of the injury when the sensory system has not changed. NOP receptors participate diminishing both the development and maintenance of nociceptive behaviors. Results suggest that a barrage of afferent input induced by formalin injection initiates a long-term differential change in peripheral and spinal processing that affect the efficacy of opioid receptor agonists.

Morphine and dipyrone co-administration delays tolerance development and potentiates antinociception

This work analyses the time course of tolerance development and antinociceptive potentiation throughout repeated co-administration of morphine (an opioid receptor agonist) plus dipyrone (a non-steroidal anti-inflammatory drug) in the tail-flick test. Male Wistar rats were i.v. injected with morphine (3.1 mg/kg), dipyrone (600 mg/kg) or the combination morphine/dipyrone twice a day for 5 days. Dipyrone produced antinociceptive effects with a trend towards tolerance development at the end of the treatment. Morphine was initially effective, but complete tolerance developed after its fifth administration. The combination of morphine plus dipyrone produced a significant potentiation and longer duration of antinociceptive effects. The antinociceptive efficacy of morphine and dipyrone co-administration gradually decreased after the sixth injection. An additional group of rats treated with dipyrone for 11 days developed complete tolerance after the 19th administration. These data suggest that repeated co-administration of morphine plus dipyrone results in a delay of tolerance development and in a potentiation of their individual antinociceptive effects.