R.J. Marley

Potential regulation of nicotine and ethanol actions by α4-containing nicotinic receptors

A restriction fragment length polymorphism (RFLP) associated with a major nicotinic receptor subunit (i.e., alpha4) has been identified in two mouse lines that were selectively bred for differences in sensitivity to ethanol. These mice, referred to as Long-Sleep (LS) and Short-Sleep (SS) mice, also differ in sensitivity to several effects of nicotine. The potential role of the alpha4 RFLP in regulating several responses to nicotine and ethanol was evaluated by using the LSxSS-derived recombinant inbred (RI) strains. Those RI strains that carried the LS-like alpha4 RFLP were more sensitive to the depressant effects of nicotine on Y-maze crossing and rearing activities and ethanol-induced increases in Y-maze crossing activity than were those RI strains that carry the SS-like alpha4 RFLP. The LS-like RI strains were also more sensitive to nicotine-induced hypothermia. The RFLP was not associated with strain differences in ethanol-induced body temperature or sleep time. The potential role of the RFLP in regulating ethanol and nicotine consumption was evaluated in heterogeneous stock (HS) mice. An association was found between the alpha4 RFLP and variation in ethanol consumption, but not in nicotine consumption, as measured in a four-bottle choice test. Recent studies of ethanol and tobacco abuse by human beings suggest that common genes may influence these two forms of substance abuse. The results of the studies reported here suggest that the alpha4 nicotinic receptor gene should be evaluated for its potential role in regulating ethanol and tobacco abuse in human beings.

Correlation between the enhancement of flunitrazepam binding by GABA and seizure susceptibility in mice

Various populations of mice exhibit differential sensitivity to seizure-inducing agents. The relationship of seizure susceptibility to alterations in the GABA receptor complex was investigated in six different populations of mice consisting of four inbred strains (C57BL, DBA, C3H, and BALB) and two selected lines (long sleep and short sleep). Seizure activity was induced by intraperitoneal administration of the GAD inhibitor, 3-mercaptopropionic acid, and latencies to seizure onset and tonus were measured. In naive mice of the same populations, GABA enhancement of 3H-flunitrazepam binding was measured in extensively washed whole brain membranes at several GABA concentrations. Both differential seizure sensitivity to 3-mercaptopropionic acid and differential enhancement of 3H-flunitrazepam binding by GABA were observed in these six populations of mice. Correlational analyses indicated a positive correlation between the degree of GABA enhancement of 3H-flunitrazepam binding and resistance to the seizure-inducing properties of 3-mercaptopropionic acid. These data suggest that genetic differences in sensitivity to seizure-inducing agents that disrupt the GABAergic system may be related to differences in coupling between the various receptors associated with the GABA receptor complex.

Further characterization of benzodiazepine receptor differences in long-sleep and short-sleep mice

Molecular and conformational characteristics of benzodiazepine (BZ) receptors in cortex and cerebellum from long-sleep and short-sleep mice were investigated using heat inactivation and beta-carboline competition techniques. To investigate differences in the allosteric coupling between GABA and BZ receptors, the protection of BZ receptors from heat inactivation, by GABA, was also evaluated. The two genotypes do not differ in the affinity or number of BZ receptors in the cortex or cerebellum. They do, however, appear to differ in the molecular structure and/or regulation of the conformational state of the receptor in the cortex, as indicated by a greater sensitivity of LS mice to both heat inactivation and beta-carboline competition of 3H-flunitrazepam (FNZ) binding in this region. Evidence for differences in the nature of coupling between GABA and BZ receptors is provided by the finding that in both regions, GABA protected BZ receptors from inactivation to a greater degree in LS mice. The relationship between these differences and the multiplicity of expression of BZ receptors is discussed.

Differential sensitivity to bicuculline in three inbred mouse strains

We examined the inbred mouse strains DBA/2Ibg, C57BL/6Ibg, and C3H/2Ibg for differences in susceptibility to bicuculline-induced seizures, as well as to bicuculline-induced epileptiform activity recorded in the CA1 pyramidal cell layer of hippocampal slices. For susceptibility to seizure onset the strain rank order was (most to least susceptible): C3H = DBA greater than C57. The rank order for sensitivity to effects of bicuculline on tonic seizure latency and on hippocampal epileptiform activity were identical: C3H greater than DBA = C57. It is suggested that mechanisms underlying the development of bicuculline-induced epileptiform events in the hippocampal slice may be similar to those involved in the development of tonic seizures measured in vivo.

Genetic influences on GABA-related seizures

Genetic differences in susceptibility to chemically induced seizures were examined in various populations of mice. Three inbred strains: C57BL, DBA, and C3H and a heterogenous stock (HS) of mice were tested for sensitivity to seizures induced by 3-mercaptopropionic acid (MP) and flurothyl. Dose response curves were constructed for each population of mice with each agent by quantitating latencies to specific stages of seizures. Significant strain and sex differences were observed in sensitivity to MP-induced seizures. Rank order from least sensitive to most sensitive was C57BL, HS, DBA, and C3H. Sensitivity to flurothyl-induced seizures was also strain dependent, but the rank order of sensitivity was different than for MP. The least sensitive strain was C57BL followed by HS, C3H, and DBA. Analysis of GABA receptors in seven brain regions obtained from C57BL and DBA using 3H-muscimol to measure high affinity GABA binding did not reveal significant differences in receptor number between these two strains. It thus appears that different genetic factors influence susceptibility to MP-induced seizures than to flurothyl-induced seizures. Furthermore, there is probably little correlation between the number of high affinity GABA receptors and sensitivity to seizures.

Differential response to flurazepam in long-sleep and short-sleep mice.

In addition to differing in ethanol sensitivity, long-sleep (LS) and short-sleep (SS) mice also differ in response to GABAergic agents. In the present study the sensitivity of LS and SS mice to the anesthetic, hypothermic and anticonvulsant effects of benzodiazepine, flurazepam, was determined. Flurazepam (75-300 mg/kg) induced a dose-dependent loss of righting response in both lines. The LS line displayed a two-fold greater sensitivity to the anesthetic effects of flurazepam. A dose-dependent decrease in body temperature was also observed following administration of flurazepam (25-150 mg/kg), but the two lines did not differ on this measure. Determination of the anticonvulsant effects of flurazepam (1-6 mg/kg) against seizures induced by 3-mercaptopropionic acid revealed that the SS line was more sensitive to the anticonvulsant effects of this benzodiazepine. These studies demonstrate that LS and SS mice differ in response to flurazepam, but the nature of the difference depends on the type of response measured and the dose of flurazepam employed.

Classical genetic analysis of GABA-related seizures

Differences in resistance to 3-mercaptopropionic acid (MP)-induced seizures exist between DBA/2Ibg and C57BL/6Ibg inbred strains of mice; C57BL/6Ibg mice are more resistant to MP-induced seizures. To determine the mode of inheritance for seizure resistance, a classical genetic analysis was conducted using these two parental strains, their F1, F2, and backcross generations. Latencies to seizure onset and tonus after intraperitoneal (IP) injections of MP (25-40 mg/kg) were quantified. For all populations mean latencies to onset and tonus decreased in a dose dependent manner with the hybrid generations exhibiting a seizure resistant phenotype resembling the C57BL/6Ibg strain. In general, female mice were less resistant to MP-induced seizures than males; however, a significant degree of resistance was retained by the C57BL/6Ibg females and their female progeny. A quantitative assessment of the pattern of inheritance for seizure resistance using a weighted least-squares regression approach indicated that an additive-dominance model explained latency to seizure onset data at 25, 35 and 40 mg/kg. However, at 30 mg/kg, the model required the addition of an epistatic parameter to best describe mean scores at this dose. The results of these analyses suggest that resistance to MP-induced seizures is transmitted in a dominant manner.

Genetic differences in the effects of δ-aminolevulinic acid on seizure latency in mice

Abstract δ-Aminolevulinic acid, an intermediate in heme formation, is elevated in certain human disorders including acute intermittent porphyria, tyrosinemia, and lead poisoning. It has been implicated in the central nervous system manifestations of these disorders via interactions with the GABAergic system. This potential interaction was examined by testing whether or not δ-aminolevulinic acid could alter the latency to seizure in mice. Seizures were induced in a variety of inbred strains of mice including C57BL, C3H, DBA mice and in a heterogeneous stock of mice. Flurothyl and 3-mercaptopropionic acid were used to induce seizures in the presence and absence of δ-aminolevulinic acid administered either i.p. (0.5 and 1.5 mmol/kg), or i.c.v. (4.5 and 450 nmol). δ-Aminolevulinic acid increased the latency to myoclonic and clonic seizures induced by flurothyl when administered i.p.; i.c.v. injections also delayed clonic seizures induced by flurothyl, and increased the latency to tonic seizures induced by 3-mercaptopropionic acid. The degree to which δ-aminolevulinic acid altered seizure latency in all tests was dependent on strain of mouse tested. These data support the conclusion that δ-aminolevulinic acid can act as an anticonvulsant agent, and mimic the effects of GABA. Moreover, there is genetic variation in the sensitivity of the various strains of mice to δ-aminolevulinic acid.