Richard A. Rabin

Ethanol effects on three strains of zebrafish: model system for genetic investigations

The effects of acute and chronic ethanol administration on the wild-type (WT), long-fin striped (LFS), and blue long-fin (BLF) strains of zebrafish were investigated. In the LFS strain, acute exposure to 0.25% (v/v) ethanol inhibited the startle reaction and increased both the area occupied by a group of subjects and the average distance between each fish and its nearest neighbor. Similar effects were found in the WT fish although higher concentrations of ethanol were required. No effects on the behavior of the BLF fish were observed with up to 1.0% (v/v) ethanol. Brain alcohol levels were comparable among the three strains precluding a pharmacokinetic explanation for the behavioral results. In LFS zebrafish, behavioral tolerance was observed after 1 week of continual exposure to ethanol. Conversely, chronic ethanol exposure of the WT fish for up to 2 weeks did not result in the development of tolerance, but rather appeared to increase the disruptive action of the drug. The present results suggest the observed strain differences in the effects of ethanol reflect genotypic differences in both the response of the central nervous system (CNS) to ethanol as well as the ability of the CNS to adapt to ethanol exposure. Although preliminary, the present study indicates that the zebrafish is an excellent model system to investigate the genetic determinants involved in regulating the responses to ethanol.

The role of the 5-HT2A and 5-HT2C receptors in the stimulus effects of hallucinogenic drugs I: Antagonist correlation analysis

Investigations conducted over the past 3 decades have demonstrated that serotonergic receptors, specifically the 5-HT2A and 5-HT2C subtypes, play an important role in the behavioral effects of hallucinogenic compounds. The present study was designed to determine the respective significance of these two receptors in the stimulus effects of LSD and (−)DOM in the rat. Specifically, the interactions of a series of serotonergic antagonists (risperidone, pirenpirone, metergoline, ketanserin, loxapine, LY53857, pizotyline, spiperone, cyproheptadine, mesulergine, promethazine, and thioridazine) with the LSD stimulus and the (−)DOM stimulus in LSD-trained subjects was defined. From these data, IC50 values were determined for the inhibition of the LSD-appropriate responding elicited by either 0.1 mg/kg LSD (15-min pretreatment time) or 0.4 mg/kg (−)DOM (75-min pretreatment). In addition, the affinities of these antagonists for 5-HT2A and 5-HT2C receptors were determined in radioligand competition studies. 5-HT2A affinity correlated significantly with IC50 values for the blockade of the LSD (r=+0.75,P<0.05) and (−) DOM (r=+0.95,P<0.001) stimuli in the LSD trained subjects. 5-HT2C affinity did not correlate significantly with either series of IC50 values. These data indicate that (1) the stimulus effects of LSD, and (2) the substitution of (−)DOM for the LSD stimulus are mediated by agonist activity at 5-HT2A receptors.

Serotonergic control of androgen-induced dominance

The present study investigates the role of serotonergic systems in anabolic steroid-induced aggression. An animal model of aggressive dominance was used to assess the chronic effects of testosterone propionate. When rats that had become dominant following administration of testosterone propionate received serotonergic agonists with selectivity for the 5-HT1A receptor (8-OH-DPAT, buspirone, gepirone), the 5-H1B receptor (eltoprazine, TFMPP), or the 5-HT2A/2C receptor (DOM), a dose-dependent decrease in dominance was demonstrated. Pretreatment with three serotonergic antagonists (pizotyline, pirenpirone, and pindolol) blocked agonist-induced reductions in dominance in varying degrees. Nonserotonergic agonists with CNS depressant effects were also tested in dominant animals. The benzodiazepine, chlordiazepoxide, did not reduce dominance except at doses that interfered with motor behavior. The opioid agonist, morphine, dose dependently decreased dominance, but this effect was reversible with administration of the serotonergic antagonist, pirenpirone, suggesting the antidominant effect of morphine had a serotonergic component. Biochemical experiments demonstrated that following chronic testosterone propionate, there was a decrease in levels of 5-HT and 5-HIAA in the hippocampus but not in the striatum or the frontal cortex. Chronic testosterone propionate also caused an increase in the affinity of [3H]8-OH-DPAT for the 5-HT1A receptor but no corresponding change in the density of 5-HT1A binding sites in the hippocampus. There was also no change in the properties of the 5-HT2 receptor in the frontal cortex following chronic testosterone propionate. These data suggest that serotonergic systems may play an important role in the control of anabolic steroid-induced aggressive dominance.

The role of the 5-HT2A and 5-HT2C receptors in the stimulus effects ofm-chlorophenylpiperazine

Abstractm-Chlorophenylpiperazine (mCPP), a major metabolite of the atypical antidepressant trazadone, has been observed to produce marked physiological and behavioral effects in both humans and animals. These effects have been attributed to the interaction of mCPP with serotonergic receptors. The present study was designed to characterize those interactions of mCPP with central serotonergic receptors which mediate mCPP-induced stimulus control. A series of serotonergic antagonists (mesulergine, pizotyline, ketanserin, spiperone, risperidone, ritanserin, metergoline, pirenpirone, and LY53857) was tested for the ability to block the mCPP stimulus. The affinity of these antagonists for 5-HT2A and 5-HT2C receptors was then correlated with maximal percent inhibition of the mCPP stimulus. Kd at the 5-HT2C receptor was inversely proportional (r=−0.75,P<0.05), and Kd at the 5-HT2A receptor directly proportional (r=+0.67,P<0.05) to the maximal percent inhibition of the mCPP stimulus. The 5-HT2C selectivity ratio [Kd(5-HT2A)/Kd(5-HT2C)] of the antagonists was directly proportional (r=+0.86,P<0.01) to maximal percent inhibition of the mCPP stimulus. A multiple regressions analysis indicated that 81% of the variance in the ability of a given antagonist to block the mCPP stimulus could be predicted on the basis of its affinity for 5-HT2A and 5-HT2C receptors. It is concluded that the stimulus effects of mCPP are mediated predominantly by a combination of agonist activity at 5-HT2C receptors and antagonist activity at 5-HT2A receptors.

Role of 5-HT2A and 5-HT2C receptors in the stimulus effects of hallucinogenic drugs II: reassessment of LSD false positives

In the context of animal studies of hallucinogens, an LSD-false positive is defined as a drug known to be devoid of hallucinogenic activity in humans but which nonetheless fully mimics LSD in animals. Quipazine, MK-212, lisuride, and yohimbine have all been reported to be LSD false positives. The present study was designed to determine whether these compounds also substitute for the stimulus effects of the more pharmacologically selective hallucinogen (−)DOM (0.56 mg/kg, 75-min pretreatment time). The LSD and (−)DOM stimuli fully generalized to quipazine (3.0 mg/kg) and lisuride (0.2 mg/kg), but only partially generalized to MK-212 (0.1–1.0 mg/kg) and yohimbine (2–20 mg/kg). In combination tests, pirenpirone (0.08 mg/kg), a compound with both D2 and 5-HT2A affinity, blocked the substitution of quipazine and lisuride for the (−)DOM stimulus. Ketanserin (2.5 mg/kg), an antagonist with greater than 1 order of magnitude higher affinity for 5-HT2A receptors than either 5-HT2C or D2 receptors, also fully blocked the substitution of these compounds for the (−)DOM stimulus, while the selective D2 antagonist thiothixene (0.1–1.0 mg/kg) failed to block the substitution of lisuride for the (−)DOM stimulus. These results suggest that quipazine and lisuride substitute for the stimulus properties of the phenylalkglamine hallucinogen (−)DOM via agonist activity at 5-HT2A receptors. In addition, these results suggest that 5-HT2A agonist activity may be required, but is not in itself sufficient, for indolamine and phenylalkglamine compounds to elicit hallucinations in humans. Finally, it is concluded that MK-212 and yohimbine are neither LSD nor (−)DOM false positives.

Hallucinogen-like actions of 5-methoxy-N,N-diisopropyltryptamine in mice and rats

Few studies have examined the effects of 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) in vivo. In these studies, 5-MeO-DIPT was tested in a drug-elicited head twitch assay in mice where it was compared to the structurally similar hallucinogen N,N-dimethyltryptamine (N,N-DMT) and challenged with the selective serotonin (5-HT)2A antagonist M100907, and in a lysergic acid diethylamide (LSD) discrimination assay in rats where its subjective effects were challenged with M100907 or the 5-HT 1A selective antagonist WAY-100635. Finally, the affinity of 5-MeO-DIPT for three distinct 5-HT receptors was determined in rat brain. 5-MeO-DIPT, but not N,N-DMT, induced the head twitch responses in the mouse, and this effect was potently antagonized by prior administration of M100907. In rats trained with LSD as a discriminative stimulus, there was an intermediate degree (75%) of generalization to 5-MeO-DIPT and a dose-dependent suppression of response rates. These interoceptive effects were abolished by M100907, but were not significantly attenuated by WAY-100635. Finally, 5-MeO-DIPT had micromolar affinity for 5-HT 2A and 5-HT 2C receptors, but much higher affinity for 5-HT 1A receptors. 5-MeO-DIPT is thus effective in two rodent models of 5-HT2 agonist activity, and has affinity at receptors relevant to hallucinogen effects. The effectiveness with which M100907 antagonizes the behavioral actions of this compound, coupled with the lack of significant antagonist effects of WAY-100635, strongly suggests that the 5-HT 2A receptor is an important site of action for 5-MeO-DIPT, despite its apparent in vitro selectivity for the 5-HT 1A receptor.

Ethanol enhances neurite outgrowth in primary cultures of rat cerebellar macroneurons

Effects of ethanol on neurite outgrowth and morphometry were investigated in primary cultures of rat cerebella. Cell cultures were prepared from cerebella on embryonic day 17 (E17) for treatment with a series of ethanol concentrations (50, 75, 100, 150 and 200 mM). Ethanol did not reduce neuronal survival or attachment to the substrate at any of the concentrations that were used. Treatment with 75 mM ethanol significantly enhanced neurite outgrowth. Measurements from dissociated cultures exposed to 75 mM ethanol immediately after plating showed a significant increase in the percentage of neurite-bearing cells after 8 and 24 h in vitro. Measurements of the area and perimeter of neuronal cell bodies in dissociated cell cultures showed that the cell bodies of ethanol-treated neurons were also larger than those of control neurons. Ethanol was also associated with significant increases in the total neuritic length per cell and in the length of the longest neurite in each cell. The mean number of neurite branches was also greater in the ethanol-treated neurons. Measurements from suspension cell cultures, in which dissociated cells were suspended overnight in the presence of 75 mM ethanol prior to plating, corroborated these results. These findings suggest that ethanol may have distinct effects on neurite initiation and outgrowth and branching. The cellular mechanisms involved and the functional significance of these effects are currently not known. The present results also indicated that high concentrations of ethanol (150-200 mM) and long periods of exposure (4-7 days) were required to produce toxic effects on neurons and glial cells in this system.

Lysergic acid diethylamide and [-]-2,5-dimethoxy-4-methylamphetamine increase extracellular glutamate in rat prefrontal cortex

The ability of hallucinogens to increase extracellular glutamate in the prefrontal cortex (PFC) was assessed by in vivo microdialysis. The hallucinogen lysergic acid diethylamide (LSD; 0.1 mg/kg, i.p.) caused a time-dependent increase in PFC glutamate that was blocked by the 5-HT(2A) antagonist M100907 (0.05 mg/kg, i.p.). Similarly, the 5-HT(2A/C) agonist [-]-2,5-dimethoxy-4-methylamphetamine (DOM; 0.6 mg/kg, i.p.), which is a phenethylamine hallucinogen, increased glutamate to 206% above saline-treated controls. When LSD (10 microM) was directly applied to the PFC by reverse dialysis, a rapid increase in PFC glutamate levels was observed. Glutamate levels in the PFC remained elevated after the drug infusion was discontinued. These data provide direct evidence in vivo for the hypothesis that an enhanced release of glutamate is a common mechanism in the action of hallucinogens.

Enhanced caspase activity during ethanol-induced apoptosis in rat cerebellar granule cells.

The effects of ethanol on cerebellar granule cell death were examined in cultures maintained for either 5 days in vitro (immature) or 8 and 12 days in vitro (mature). Ethanol did not alter cell survival under the usual growth conditions (i.e., 10% serum and 25 mM KCl). However, in mature cultures ethanol enhanced apoptosis induced by either serum withdrawal or incubation in non-depolarizing media. In immature cultures, serum deprivation, but not non-depolarizing media, resulted in granule cell death that was enhanced by ethanol. Serum removal increased both cleavage of the caspase-specific substrate N-acetyl-Asp-Glu-Val-Asp-7 amino-4-methylcoumarin (Ac-DEVD-amc) and the amount of active caspase-3. Inclusion of ethanol during the serum deprivation augmented Ac-DEVD-amc cleavage without further increasing the amount of active caspase-3. This study demonstrates that when neurotrophic factors are limiting, ethanol is toxic to cerebellar granule cells regardless of maturation status. The ability of ethanol to promote apoptosis involves an increase in caspase activity, but this does not entail an increase in the proteolytic activation of caspase-3.

The paradox of 5-methoxy-N,N-dimethyltryptamine: an indoleamine hallucinogen that induces stimulus control via 5-HT1A receptors.

Stimulus control was established in rats trained to discriminate either 5-methoxy-N,N-dimethyltryptamine (3 mg/kg) or (-)-2,5-dimethoxy-4-methylamphetamine (0.56 mg/kg) from saline. Tests of antagonism of stimulus control were conducted using the 5-HT1A antagonists (+/-)-pindolol and WAY-100635, and the 5-HT2 receptor antagonist pirenperone. In rats trained with 5-MeO-DMT, pindolol and WAY-100635 both produced a significant degree of antagonism of stimulus control, but pirenperone was much less effective. Likewise, the full generalization of 5-MeO-DMT to the selective 5-HT1A agonist [+/-]-8-hydroxy-dipropylaminotetralin was blocked by WAY-100635, but unaffected by pirenperone. In contrast, the partial generalization of 5-MeO-DMT to the 5-HT2 agonist DOM was completely antagonized by pirenperone, but was unaffected by WAY-100635. Similarly, in rats trained with (-)-DOM, pirenperone completely blocked stimulus control, but WAY-100635 was inactive. The results obtained in rats trained with (-)-DOM and tested with 5-MeO-DMT were more complex. Although the intraperitoneal route had been used for both training drugs, a significant degree of generalization of (-)-DOM to 5-MeO-DMT was seen only when the latter drug was administered subcutaneously. Furthermore, when the previously effective dose of pirenperone was given in combination with 5-MeO-DMT (s.c.), complete suppression of responding resulted. However, the combination of pirenperone and WAY-100635 given prior to 5-MeO-DMT restored responding in (-)-DOM-trained rats, and provided evidence of antagonism of the partial substitution of 5-MeO-DMT for (-)-DOM. The present data indicate that 5-MeO-DMT-induced stimulus control is mediated primarily by interactions with 5-HT1A receptors. In addition, however, the present findings suggest that 5-MeO-DMT induces a compound stimulus that includes an element mediated by interactions with a 5-HT2 receptors. The latter component is not essential for 5-MeO-DMT-induced stimulus control, but is revealed in animals tested or trained with a 5-HT2-selective agonist such as (-)-DOM. Based upon the present data, we conclude that 5-MeO-DMT differs from DOM with respect to the serotonergic element that mediates stimulus control in the rat, but that it shares with DOM a functionally significant interaction with 5-HT2 receptors.