The zolpidem-sensitive GABAA receptors localize to cerebellar Purkinje cells in PC-γ2-swap mice. Image reproduced from Nature Neuroscience.
Despite the directions in Age of Aquarius, it is difficult to let the sun shine in mammalian brains. Recently, researchers reported photoactivated ion channels that may help functionally dissect neural circuits. However, most mammalian brains are opaque and would require surgical implantation of light sources. Now Wulff et al. report functional dissection of neural circuits in mice with targeted sensitivity to a GABAA receptor agonist in a recent article in Nature Neuroscience.
Almost all neurons express GABAA receptors, which contain two 
, two 
and one 
2 subunit. Zolpidem (marketed as the sleep aid Ambien) is a GABAA receptor agonist that acts at a binding site formed by and 2 subunits. In the 2 subunit, phenylalanine 77 (F77) is necessary for zolpidem binding, and GABAA receptors with isoleucine at position 77 bind GABA but not zolpidem.
The authors generated mice insensitive to zolpidem except in cerebellar Purkinje cells. They generated zolpidem-insensitive 
I277lox mice by substituting a codon encoding isoleucine for the codon encoding phenylalanine in the GABAA receptor 2 subunit gene Gabrg2 and engineering adjacent loxP recombination sites. They crossed these mice to mice expressing Cre recombinase under the control of the L7 promoter to generate mice lacking GABAA receptors in cerebellar Purkinje cells (PC-∆2). The authors tagged the wild-type zolpidem-sensitive allele with green fluorescent protein (GFP) and targeted it to cerebellar Purkinje cells with the L7 promoter. They crossed mice expressing this allele with PC-
2 mice to generate PC-2-swap mice.
PC-2-swap mice showed 2F77 expression and zolpidem senstivity in cerebellar Purkinje cells. In the molecular layer of the cerebellum, GFP localized to GABAA receptor 1 subunit-positive cells that were apposed to glutamic acid decarboxylase-positive (GABAergic) nerve terminals. In cerebellar slices, Purkinje cells showed miniature inhibitory postsynaptic currents (mIPSCs) with similar properties in PC-2-swap, 2I77lox and wild-type mice, suggesting that the I772 subunit does not disrupt normal GABAergic transmission. Zolpidem had no effect on mIPSCs from 2I77lox mice. However, zolpidem increased the amplitude and timecourse of mIPSCs in cerebellar Purkinje cells but not stellate cells from PC-2-swap mice, suggesting that zolpidem sensitivity was restricted to cerebellar Purkinje cells.
Zolpidem quickly modified behavior in PC-2-swap mice. Cerebellar Purkinje cells are important in learning to balance on a rotating rod called a rotarod. PC-2-swap and 2I77lox mice performed similarly on the rotarod. Within 1-5 minutes of zolpidem treatment, PC-2-swap mice showed cerebellar deficits on the rotarod, falling faster than 2I77lox mice.
Therefore, zolpidem can quickly deactivate components of neural circuits expressing engineered GABAA receptors. Could a similar system activate these same circuit components? The -carbolines DMCM and -CCM are inverse GABAA receptor agonists that decrease receptor function and are ineffective in 2I77 mice. According to the authors, zolpidem and an inverse agonist could bidirectionally modulate the activity of neurons expressing engineered 2 subunits, helping to identify neuron function in neural circuits.