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LETTERS TO THE EDITOR
6 GABAA Receptor Subunit"Department of Neurosciences, University of New Mexico HSC, Albuquerque, New Mexico (C.F.V., P.B.), Department of Pharmaceutical Sciences, University of Colorado, Denver, Colorado (R.A.R.), and Department of Basic Neurosciences, University of Geneva, Geneva, Switzerland (M.M.)
Received October 7, 2007; accepted October 16, 2007.
). Moreover, differences in the experimental conditions between our studies are discussed under Discussion in Botta et al. (2007a). Dr. Otis is correct in that we had recorded large (>50 pA) tonic currents in two cerebellar granule neurons from 
6-100Q/100Q rats. We reviewed these recordings and confirmed that these were stable and that access resistances did not change more than 25%. Thus, we have no basis to consider these measurements erroneous. It is noteworthy that examination of the data shown in Fig. 4, A to B, of Botta et al. (2007a) indicates that, even if these two data points are not considered, the effect of 80 mM ethanol on tonic current amplitude is not increased in slices from 100Q/100Q rats. With regard to spontaneous inhibitory postsynaptic current frequencies, we did detect a significant increase in this parameter in slices from 100Q/100Q rats; this might have been revealed by the fact that we had recorded from a larger sample of neurons (n = 26–28) than Santhakumar et al. (2006) (n = 16). It should also be emphasized that the S.E.M. for our spontaneous inhibitory postsynaptic current frequency data were 0.06 (100R/100R) and 0.14 (100Q/100Q), whereas they were reported to be 0.25 (100R/100R) and 0.28 (100Q/100Q) in Santhakumar et al. (2006). Thus, the variability in our results is not unusually high. The possibility that genotype differences in ambient GABA could modify ethanol sensitivity of GABAergic currents is not supported by the data shown in Fig. 4 of Botta et al. (2007a) that indicate that basal sIPSC frequency and basal tonic current amplitudes are not significantly correlated with acute ethanol sensitivity in slices from either 100R/100R or 100Q/100Q rats. Finally, it is unlikely that a small difference in extracellular potassium concentrations (i.e., from 2.5 to 3 mM) can explain the discrepancies between our studies.
Dr. Otis is correct in that we have not attempted studies with recombinant receptors. However, Yamashita et al. (2006) recently had and were unable to detect enhancement of currents mediated by either 6β2
or 6β3 in the presence of 10, 30, or 100 mM ethanol. Although it would be interesting to attempt to replicate the findings of Hanchar et al. (2005) on the effect of the 6-R100Q substitution on ethanol sensitivity of 6β3 recombinant receptors, the physiological relevance of results obtained with these receptors is questionable as the precise subunit composition of native extrasynaptic GABAA receptors in cerebellar granule neurons remains to be determined (Wisden, 1995). With respect to the behavioral analysis with outbred Sprague-Dawley rats, we disagree that the findings of these studies provide direct support for the idea that the substitution increases ethanol sensitivity. Even if these findings were independently replicated, in a group of outbred rats, it is improbable that any two animals would differ only at the 6-100 locus. Differences in other loci could be responsible for the differences in ethanol sensitivity observed in these rats. Indeed, several lines of evidence strongly indicate that the enrichment of the Q allele in alcohol-nontolerant rats was coincidental and not because it contributed to genetic variance in behavioral tests of ethanol sensitivity (reviewed in Botta et al., 2007b).
We agree that evidence from independent studies supports that extrasynaptic GABAA receptors expressed in several brain regions are targets of ethanol, and we acknowledged under Materials and Methods in Botta et al. (2007a). However, it is important to recognize that the data from a number of studies are inconsistent with this conclusion; the reader is referred to Lovinger and Homanics (2007) where this matter is discussed in detail. As Dr. Otis correctly points out, ethanol has a reproducible presynaptic effect on the action potential-dependent GABA release at Golgi cell-to-cerebellar granule neuron synapses, and this effect is not observed in the dentate or thalamus (Carta et al., 2004; Wei et al., 2004; Hanchar et al., 2005; Liang et al., 2006; Botta et al., 2007a; Fleming et al., 2007; Glykys et al., 2007; Jia et al., 2007). Thus, ethanol, at least in part, modulates GABAergic tone via different mechanisms in cerebellar granule cells versus neurons present in these brain regions. Dr. Otis states that given the similarity between extrasynaptic GABAA receptors in different neuronal populations, it would be unexpected for cerebellar granule cells to be the only neuronal type that expresses tonic currents not directly modulated by ethanol. However, Dr. Otis ignores evidence indicating that cerebellar granule neurons lack protein kinase C, which was recently reported to be essential for ethanol-induced modulation of tonic GABAergic currents in dentate and thalamic neurons (Messing et al., 2007). In addition, he dismisses published reports indicating that extrasynaptic GABAA receptors in cerebellar granule cells probably have a unique subunit composition that appears to include more than one and/or β subunit subtype (i.e., 1, 6, β1, β2,or β3) (Jechlinger et al., 1998; Sigel and Baur, 2000; Poltl et al., 2003). Thus, the subunit composition, association with other proteins, and/or phosphorylation state of extrasynaptic GABAA receptors expressed in cerebellar granule neurons could explain their insensitivity to the direct modulatory actions of ethanol.
The selected rat lines to which Dr. Otis is referring are the alcohol-tolerant and nontolerant (AT, ANT), the alko-alcohol and nonalcohol (AA, ANA), and the Sardinian preferring and nonpreferring (sP, sNP) rats. The segregation of the gene's alleles in a selection experiment is consistent with a role for the gene in the selection trait. However, by itself, the evidence is not strong and certainly does not prove such an effect, especially considering that the number of breeders and offspring in these selection studies was relatively small. We have gone well beyond the simple observation of allele segregation in the selected AT and ANT lines to more fully address the role of the 6-100R/Q alleles in the trait for which they were selected, the tilting-plane test (Eriksson and Rusi, 1981). Using quantitative trait loci (QTL) and association analyses, we showed that the Q allele does not contribute to ethanol sensitivity in the AT and ANT rats for the tiltingplane test or, for a higher dose response, the loss of righting reflex (Botta et al., 2007b). This result led us to the conclusion that the segregation of the Gabra6 alleles was a random event, which is a potential outcome for the large number of genes that have no effect on ethanol sensitivity. A second possibility is that the Q allele has such a miniscule effect that it is immeasurable, even when tested with hundreds of animals, as in our QTL analysis. In ongoing mapping studies, we have identified several QTL for tilted-plane sensitivity, none of which was found at the location of Gabra6 on chromosome 10 (unpublished observations). We have to conclude that 6-100Q contributes little or nothing to the ANT tilting-plane phenotype and that the bulk of the difference between the lines is under the control of other mapped loci.
The AA/ANA and sP/sNP lines were selectively bred for differences in free-choice ethanol consumption, not "hypersensitivity", as indicated by Dr. Otis (Hilakivi et al., 1984; Colombo, 1997). In this regard, it is notable that an AT x ANT F2 study found no correlation between tilting-plane sensitivity and ethanol preference (Sarviharju and Korpi, 1993). If 6-100Q has an effect on both the tilting-plane test and ethanol preference, as Dr. Otis seems to be suggesting, the two traits should have been correlated in the F2 study. It is interesting that the allele segregation pattern appears to be similar in the AA/ANA and sP/sNP lines (Saba et al., 2001; Carr et al., 2003), but this observation is far from sufficient for the conclusion that 6-100Q, or any of the other GABAA receptor subunit polymorphisms that are linked to it, mediate genetic variance in ethanol preference or any other ethanol-related trait in which the lines differ (Congeddu et al., 2003). Our published results argue that 6-100Q does not contribute to the "hypersensitivity" of the ANT rats (Botta et al., 2007b); its role in ethanol preference in any of the selected lines remains an open question.
To answer Dr. Otis' last question, we do not hypothesize that the "unidentified genetic loci" mediating ethanol sensitivity are the same as those mediating benzodiazepine sensitivity. Such a hypothesis would go against the grain of decades of research into these drugs. There seems to be a failure by Dr. Otis to recognize two well accepted characteristics of benzodiazepines and ethanol. First, from a pharmacological perspective, their mechanisms of action are not identical. Benzodiazepines are relatively selective to the GABAA receptor, whereas ethanol influences a wide variety of signaling systems. Second, from a genetics perspective, responses to these drugs are considered to be quantitative traits, meaning that, in part, many genes are involved. The consequence of these two properties is that some of the genes will be unique to ethanol responses, whereas others will be unique to benzodiazepine responses. There is also a third subset of the genes that will be similar to the two drugs (see Crabbe et al., 1994). Our contention is that the 6-100R/Q polymorphism falls into the unique category; i.e., it is specific for benzodiazepines, and it does not contribute to genetic variance for ethanol responses, at least not for those that we have tested.
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Footnotes |
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ABBREVIATIONS: AT, alcohol-tolerant; ANT, alcohol-nontolerant; AA, alko-alcohol; ANA, alko-nonalcohol; sP, Sardinian preferring; sP, Sardinian nonpreferring; QTL, quantitative trait loci.
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References |
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