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LETTERS TO THE EDITOR
6 GABAA Receptor Subunit"Department of Neurobiology, Geffen School of Medicine at UCLA, Los Angeles, California
Received September 12, 2007; accepted October 16, 2007.
6 GABAR gene increases the ethanol-sensitivity of extrasynaptic GABARs on cerebellar granule cells. We are puzzled by their failure to reproduce our experimental findings but offer the following comments on the matter.
The authors assert that they used identical methods and that the basic properties of the tonic GABA currents in their article are in agreement with previously published work, yet substantial differences are apparent. Most notable are genotype-dependent differences in the distributions of amplitudes of tonic GABA currents and sIPSC frequencies under control conditions. As the scatter plot in Fig. 4B indicates, two granule cells from 100Q/100Q animals have greater than 55 pA of tonic GABA current. An examination of 10 published articles describing tonic GABA currents in rodent cerebellar granule cells of similar age shows no tonic current greater than 30 pA (Kaneda et al., 1995
; Brickley et al., 1996, 2001; Tia et al., 1996; Wall and Usowicz, 1997; Stell et al., 2003; Wall, 2003; Hanchar et al., 2005; Ortinski et al., 2006; Santhakumar et al., 2006); this can be observed most easily in the population data displayed in Fig. 7 from Wall and Usowicz (1997) and in Fig. 6 from Santhakumar et al. (2006). We are concerned that these extremely large tonic currents represent erroneous measurements. Figure 4, C and D, shows different distributions between genotypes of control sIPSC frequencies, and Table 1 reports significant differences between 100Q/100Q and 6–100R/100R across all conditions (Botta et al., 2007). We do not find any significant differences in tonic current amplitude or sIPSC frequency between genotypes under control conditions. The unusual variability evident in the data of Botta et al. (2007) suggests, among other things, that ambient GABA concentrations in their experiments are different (Santhakumar et al., 2006). Systematic differences in ambient GABA concentrations across genotypes could bias responsiveness to ethanol in a genotype-dependent manner. One methodological issue that could contribute to this profile is their use of a higher extracellular K+ concentration.
In addition to examining the sensitivity of native GABARs to ethanol, our prior study (Hanchar et al., 2005) reported that recombinant GABARs incorporating the R100Q substitution show an increased sensitivity to ethanol. The homologous substitution in 4 enhances ethanol sensitivity in GA-BARs including β3 and 
subunits (Breese et al., 2006). Finally, behavioral analyses showed large and dose-dependent differences in the sensitivity of cerebellar coordination in animals homozygous for each of the alleles in 6 (Hanchar et al., 2005). Although these findings provide direct support for the idea that the 6100Q polymorphism increases ethanol sensitivity, none of these straightforward experiments has been attempted by the Valenzuela group.
A central question in these studies is whether extrasynaptic isoforms of GABARs are directly enhanced by millimolar concentrations of ethanol. Valenzuela's group and ours agree that ethanol causes robust, dose-dependent increases in tonic GABA current in cerebellar granule cells (Carta et al., 2004; Hanchar et al., 2005; Botta et al., 2007a; Santhakumar et al., 2007). However, we disagree on the underlying mechanism. Our position is that increases in tonic current result in part from postsynaptic actions of ethanol on subunit-containing GABARs, a perspective supported by numerous studies in recombinant systems (Sundstrom-Poromaa et al., 2002; Wallner et al., 2003, 2006; Hanchar et al., 2005, 2006). This finding matches the extensive evidence obtained by several independent laboratories regarding ethanol sensitivity of tonic GABA current in other brain regions. Ethanol has been found to increase tonic GABA current mediated by subunit-containing GABARs in hippocampal interneurons (Glykys et al., 2007), thalamic neurons (Jia et al., 2007; Mody et al., 2007), and dentate gyrus granule cells (Wei et al., 2004; Liang et al., 2006; Fleming et al., 2007; Glykys et al., 2007). In all of these cases, with the exception of Borghese et al. (2006), ethanol increases tonic GABA current with no increase in GABA release. Such broad agreement is no surprise given the molecular similarity between extrasynaptic GA-BARs in these different cell types. Dismissing this evidence, Valenzuela and colleagues (Carta et al., 2004; Botta et al., 2007a) attribute ethanol actions on tonic GABA current entirely to presynaptic mechanisms, ruling out any postsynaptic effects. Such an absolutist position puzzles us; it seems unlikely that cerebellar granule cells would be the only neuronal type that had an ethanol-insensitive tonic GABA current.
In contrast to their interpretation, our hypothesis offers an explanation for why 6100Q and 6100R alleles are segregated by selective breeding for ethanol hypersensitivity in three independently derived lines of rats (Korpi et al., 1993; Saba et al., 2001; Carr et al., 2003). It is interesting that the R100Q substitution also changes the benzodiazepine sensitivity of 6 subunit-containing GABARs (Korpi and Seeburg, 1993). In a separate study conducted on the same groups of 6100R/R and 6100Q/Q animals, we found that the 6100Q allele increases the benzodiazepine sensitivity of synaptic signals (Santhakumar et al., 2006). Such findings nicely explain the increased behavioral sensitivity to benzodiazepines observed in selectively bred animals (Eriksson and Rusi, 1981; Hellevuo et al., 1989; Wong et al., 1996). Valenzuela and colleagues (Botta et al., 2007b) have argued that unidentified genetic loci acting through unknown mechanisms must be responsible for the different ethanol sensitivities in these animals. We wonder whether they hypothesize that such loci/mechanisms also influence the behavioral sensitivity to benzodiazepines, and if so how?
In the end, we cannot be certain about why these experiments failed but we do note that this group has been unable to replicate several related experiments (Valenzuela et al., 2005; Botta et al., 2007a,b). Despite this, we remain confident in our interpretation because our own data and the positive data collected by numerous, independent laboratories converge on a specific and straightforward explanation: that GABARs including these alleles strongly influence the sensitivity of cerebellar behaviors to benzodiazepines and alcohol.
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Footnotes |
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ABBREVIATIONS: GABAR, GABAA receptor; sIPSC, spontaneous inhibitory postsynaptic current.
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