Previous studies reported that the Chrna4
T529A polymorphism is associated with a wide range of behavioral and physiological responses to nicotine in mice [12
]. However, it could not be determined whether these reported associations were specifically due to the T529A polymorphism, a different polymorphism in Chrna4
or a polymorphism in a gene linked to Chrna4
. In the data reported here, Chrna4
A529 knockin mice were utilized to confirm that the T529A polymorphism does influence nicotine-induced hypothermia and free-choice oral nicotine consumption. Chrna4
A529 knockin mice were found to be more sensitive to the hypothermic effects of nicotine and to consume less nicotine by choice than wildtype controls. The A529 knockin mice also were found to differ from their control littermates for the reinforcing properties of nicotine. Mice possessing the T529 allele exhibited a clear reinforcement to a dose of 0.09 mg/kg nicotine that was absent in animals carrying the A529 allele. The positive reinforcement to nicotine seen in mice possessing the T529 allele of Chrna4 may explain why these animals consume more nicotine by choice than do T529A knockin animals. However, since only a single dose of nicotine was tested in the place preference paradigm, it remains to be determined if the A529 knockin mice completely lack positive reinforcement to nicotine or have a shifted dose-response to nicotine reinforcement.
A529 knockin mice, however, did not differ from controls in sensitivity to the locomotor depressant effects of nicotine across several doses of nicotine (only the data for saline and the 0.5 mg/kg dose are shown in ). This finding suggests that the previous reported association between the T529A polymorphism and nicotine-induced hypolocomotion in the Y-maze may be due to a different polymorphism in either Chrna4
or a linked gene. Alternatively, an effect of the T529A polymorphism on nicotine-induced hypolocomotion may be immeasurable in T529A knockin mice because α4β2* nAChRs are only one of at least three different nAChR subtypes that mediate the effects of nicotine on locomotor activity [40
]. It should be noted that locomotor activity was only assessed for three minutes in the Y-maze to allow for direct comparisons with previously published data. To more thoroughly assess the role of the Chrna4 T529A polymorphism on locomotor activity a full time course of the effect of nicotine on locomotion is required.
Results of this study demonstrated that the Chrna4
T529A polymorphism alters the function of α4β2* nAChRs in a brain region-specific manner. In the midbrain, two effects of the polymorphism were observed. First, maximal acetylcholine-stimulated 86
Rb+ efflux from α4β2* nAChRs was greater in mice possessing the T529 variant of Chrna4
relative to α4β2* nAChRs from mice carrying the A529 allele of Chrna4
. Second, the fraction of the total acetylcholine-stimulated 86
Rb+ efflux that could be attributed to the high sensitivity population of α4β2* nAChRs was significantly greater in A529 knockin mice. The observation that there is a functional effect of the polymorphism on midbrain α4β2* nAChRs as well as individual differences in oral nicotine consumption, conditioned place preference and nicotine induced hypothermia is consistent with previous studies that have shown that α4β2* nAChRs in the midbrain are essential for nicotine self-administration [25
], nicotine place preference [27
] and the effects of nicotine on body temperature [40
Rb+ efflux data from midbrain also confirmed previous reports that the Chrna4
T529A polymorphism leads to a shift in the proportion of α4β2* nAChRs with high and low sensitivity to activation by agonists. The phenomena that α4β2* nAChRs can exist in two pharmacologically distinct forms, one activated at low concentrations of agonist and the other activated only at higher agonist concentrations, has been described both in heterologous expression systems [36
] and in mouse brain synaptosomes [33
]. Moreover, it has been speculated that alterations in the ratio of high sensitivity to low sensitivity α4β2* nAChRs might be important for the addiction process [46
]. The data reported here provide the first evidence that innate differences in the ratio of high to low sensitivity α4β2* nAChRs might influence susceptibility to nicotine addiction by altering sensitivity to the reinforcing effects of nicotine.
Although an effect of the Chrna4
T529A polymorphism on α4β2* function was observed in the midbrain, no detectable affect of the polymorphism was observed in the hippocampus, striatum or thalamus. The reason for the region-specific effect of the polymorphism on receptor function is not clear. However, one possible explanation is that these regions, but not the midbrain, express functional α4β2* nAChRs that include α5 [48
] or other subunits [51
]. Inclusion of these other subunits may mask the effect of the polymorphism on receptor function. Studies that examine the effect of the polymorphism on the function of α4β2 nAChRs in mice that lack these other subunits are needed to address this possibility. In addition, assessing the function of subpopulations of α4β2* nAChRs, such as those that contribute to dopamine and GABA release, may uncover functional effects of the Chrna4
T529A polymorphism not detected by the 86
Rb+ efflux assay.
One unexpected finding is that the T529A polymorphism affected the expression of α4β2* nAChRs in the thalamus. Previous studies provided little evidence that the T529A polymorphism affects receptor expression or α4 protein levels in mouse brain [31
] or transiently transfected cells [29
]. Although the basis for the apparent effect of the T529A polymorphism on the expression of thalamic α4β2* nAChRs is not known, one possible explanation is that the level of α4β2* function in the thalamus is under tight homeostatic control. This hypothesis is based upon the observation that, despite the differences in expression of thalamic α4β2* nAChRs, there is no difference in the functional response of these receptors between T529 littermates and A529 knockin animals. Because the T529 variant is the normal allele expressed in C57BL/6J mice, these data suggest that α4β2* nAChRs made from the A529 allele of Chrna4
are down regulated in expression in the thalamus in order to maintain a functional response indistinguishable from the normally expressed α4β2 nAChRs in this mouse strain. If this is the case, then the effect of the T529A polymorphism on receptor expression in the thalamus is not a direct effect of the polymorphism, but rather an indirect effect via homeostatic regulation of receptor function.
The finding that the T529A polymorphism affects the function of α4β2* nAChRs in the A529 knockin mice confirms that the polymorphism is within a novel functional domain of the receptor that influences the ratio of high to low sensitivity α4β2* nAChRs. As described previously [29
], the polymorphism is within a region of the large cytoplasmic loop of the α4 subunit that is highly conserved in mammals. Understanding the role of this region in regulating the function of α4β2* nAChRs and how the T529A polymorphism alters this activity provides unique insight into the molecular regulation of α4β2* nAChR function.
Although the Chrna4 T529A polymorphism has not been found in human populations to date, the results of this study are, nonetheless, relevant for understanding the genetics of nicotine dependence in humans. For example, several groups have used genetic approaches to establish that α4β2* nAChRs are critical for nicotine self-administration, conditioned place preference and nicotine-induced hypothermia [26
]. However, these studies utilized knockout mice in which expression of α4β2* nAChRs was eliminated or hypersensitive knockin mice that express α4β2* nAChRs that exhibit a nearly twenty-fold increase in sensitivity to nicotine relative to native α4β2* nAChRs. Such dramatic changes in gene expression and function are not likely to be common among human genetic variants that influence nicotine dependence and other complex phenotypes. Results from the T529A knockin mice demonstrate that substantially smaller changes in the function or expression of α4β2* nAChRs, similar to those one would expect to occur with modest frequency in the human population, are sufficient to affect nicotine sensitivity in a meaningful way.
In summary, the data reported here are the first to demonstrate that a naturally-occurring polymorphism in the α4 nAChR subunit contributes to individual variability in sensitivity to several affects of nicotine, including positive reinforcement, oral consumption and hypothermia. The effect of the polymorphism on these behavioral and physiological phenotypes is accompanied by an effect of the polymorphism on the function of α4β2* nAChRs in the midbrain, a brain region that modulates these behavioral and physiological phenotypes in an α4β2* dependent manner. Moreover, results from these studies provide the first evidence that the ratio of high to low sensitivity α4β2* nAChRs may be behaviorally and physiologically relevant. However, the mechanism by which the Chrna4
T529A polymorphism affects the ratio of high to low sensitivity α4β2* nAChRs remains to be elucidated. Finally, these results along with the recently published data from Mague et al.
] demonstrate the utility of using the knockin mouse strategy in understanding the functional consequence of naturally occurring polymorphisms in complex behaviors related to drug addiction.