Alpha 4 beta 2* nicotinic acetylcholine receptors modulate the effects of ethanol and nicotine on the acoustic startle response.

BACKGROUND: Ethanol modulates the functional activity of alpha4beta2 neuronal nicotinic cholinergic receptors (nAChR) when measured in vitro, but the potential role of alpha4beta2 nAChRs in regulating behavioral effects of ethanol is unknown. Recently, Tritto et al. (Tritto T, Stitzel JA, Marks MJ, Romm E, Collins AC (2002) Variability in response to nicotine in the LSxSS RI strains: potential role of polymorphisms in alpha4 and alpha6 nicotinic receptor genes. Pharmacogenetics 12:197-208) reported that a polymorphism (A529T) in the alpha4 nAChR subunit gene is associated with variability in nicotine's effects on startle in the LSxSS recombinant inbred (RI) strains. Ethanol also alters the acoustic startle response. Thus, we evaluated the potential role of alpha4beta2 nAChRs in modulating ethanol's effects on acoustic startle. METHODS: The effects of ethanol on acoustic startle were determined in the LSxSS RI strains. In addition, the effects of ethanol and nicotine were also measured in alpha4 gain of function and beta2 null mutant mice. The beta2 mutants do not express the major variant of alpha4 nAChRs, alpha4beta2. RESULTS: An association between the alpha4 A529T polymorphism and ethanol's effects on startle was found in the LSxSS RI strains; those strains that express the A529 variant of alpha4 were more sensitive to ethanol-induced depression of startle. The alpha4 gain of function mutants were more sensitive to the effects of both nicotine and ethanol and the beta2 null mutants were less sensitive to both drugs. CONCLUSIONS: alpha4beta2-containing nAChRs may play important roles in modulating the effects of both ethanol and nicotine on the acoustic startle response. We suggest that nAChR subunit genes should be evaluated as potential contributors to both alcoholism and tobacco abuse.

A polymorphism in the alpha4 nicotinic receptor gene (Chrna4) modulates enhancement of nicotinic receptor function by ethanol.

BACKGROUND: Several studies indicate that ethanol enhances the activity of alpha4beta2 nicotinic acetylcholine receptors (nAChR). Our laboratory has identified a polymorphism in the alpha4 gene that results in the substitution of an alanine (A) for threonine (T) at amino acid position 529 in the second intracellular loop of the alpha4 protein. Mouse strains expressing the A variant have, in general, greater nAChR-mediated 86Rb+ efflux in response to nicotine than strains with the T variant. However, the possibility of the polymorphism modulating the effects of ethanol on the 86Rb+ efflux response has not been investigated. METHODS: We have used the 86Rb+ efflux method to study the acute effects of ethanol on the function of the alpha4beta2 nAChR in the thalamus in six different mouse strains. Experiments were also performed on tissue samples taken from F2 intercross animals. The F2 animals were derived from A/J mice crossed with a substrain of C57BL/6J mice that carried a null mutation for the gene encoding the beta2 nAChR subunit. RESULTS: In strains carrying the A polymorphism (A/J, AKR/J, C3H/Ibg), coapplication of ethanol (10-100 mM) with nicotine (0.03-300 microM) increased maximal ion flux when compared with nicotine alone with no effect on agonist potency. In contrast, ethanol had little effect on the nicotine concentration-response curve in tissue prepared from strains carrying the T polymorphism (Balb/Ibg, C57BL/6J, C58/J). Experiments with the F2 hybrids demonstrated that one copy of the A polymorphism was sufficient to produce a significant enhancement of nAChR function by ethanol (50 mM) in animals that were also beta2 +/+. Ethanol had no effect on nicotine concentration-response curves in T/T beta2 +/+ animals. CONCLUSIONS: The results suggest that the A/T polymorphism influences the initial sensitivity of the alpha4beta2 nAChR to ethanol.

Inbred mouse strain differences in the establishment of long-term fear memory.

Studies describing variations in fear-related memory in inbred mouse strains typically focus upon 24 h retention. As a consequence, little is known about strain differences in the establishment of longer lasting memories of aversive events. In the present study, male mice from the strains A/Ibg, AKR/J, BALB/cByJ, CBA/J, C3H/HeIbg, C57BL/6J, DBA/2J, LP/J, SJL/J and 129/SvevTac were tested 24 h, 14, or 60 days after contextual and auditory-cued fear conditioning. Consistent with previous data, 24 h after conditioning these strains exhibited substantial variation in levels of memory for the context and the auditory cue as measured by freezing scores. Sixty days after training, most strains exhibited some forgetting of the context and auditory cue, and again there was significant strain variation. Strain rankings at 60-day retention were similar to that at 24 h with a significant genetic correlation between freezing values for the two time periods. Fourteen days following training, nearly all strains exhibited generalized freezing, a behavioral phenotype originally observed in C57BL/6 but not DBA/2 mice. These data confirm that cognitive differences exist between several popular inbred mouse strains during 24 h contextual fear recall. In addition, they extend these differences into retention time frames longer than those typically used and reveal several unique learning profiles of mouse strains that may be useful in furthering our understanding of how memories are formed. Emotionally arousing situations are often recalled a great deal of time after an event. Therefore, a more complete picture of the biochemical and genetic underpinnings of learning and memory will benefit from studies using time points that assess time points beyond 24 h retention. The utility of the 14-day hyper responsiveness phenotype as a potential model for fear-related psychopathology is also discussed.

Contextual and cued fear conditioning in C57BL/6J and DBA/2J mice: context discrimination and the effects of retention interval.

Context discrimination and time course studies of contextual fear conditioning revealed strain differences between C57BL/6J (B6) and DBA/2J (D2) mice. Both strains discriminated contexts, but D2 mice exhibited less freezing in a shock-paired context. The strains did not differ immediately, or at 1 and 3 hr after contextual fear conditioning training. D2 mice showed less freezing at 15 min, 30 min, and 24 hr after training. B6 mice exhibited exaggerated generalized freezing and poor discrimination between the context and altered context 7-30 days after training. The acoustic startle response in B6 mice was also enhanced at 14 days after training. D2 mice did not show this pattern of generalized freezing. B6, but not D2, mice retained contextual memories for at least 60 days.

A polymorphism in the mouse neuronal alpha4 nicotinic receptor subunit results in an alteration in receptor function.

Nicotine-stimulated (86)Rb(+) efflux and [(3)H]cytisine binding, both of which seem to measure the nicotinic acetylcholine receptor, composed of alpha4 and beta2 subunits, were assessed in eight brain regions obtained from 14 inbred mouse strains. The potential role of a single nucleotide polymorphism (SNP) in the nicotinic receptor alpha4 subunit gene (Chrna4) on nicotinic receptor binding and function in mice was also evaluated. This SNP leads to an alanine-to-threonine variation at amino acid position 529 of the nascent alpha4 subunit polypeptide. Both nicotine-stimulated (86)Rb(+) efflux and [(3)H]cytisine binding were found to vary across brain regions and among mouse strains. Variability in nicotine-stimulated (86)Rb(+) efflux was positively correlated (r > 0.9) within each strain with the number of [(3)H]cytisine binding sites. However, the number of [(3)H]cytisine binding sites was not correlated with nicotine-stimulated (86)Rb(+) efflux across mouse strains. In contrast, the Chrna4 polymorphism was associated with receptor function across mouse strains: (86)Rb(+) efflux was greater in seven of the eight brain regions studied in those mouse strains that carry the Ala-529 variant of Chrna4. The Chrna4 SNP did not seem to influence the number of [(3)H]cytisine binding sites across mouse strains. These data indicate that inbred mouse strains exhibit differences in receptor function that cannot be attributed to variation in receptor expression but may be explained, at least in part, by the missense polymorphism in the alpha4 subunit.