From synapse to behavior: rapid modulation of defined neuronal types with engineered GABAA receptors

Abstract

In mammals, identifying the contribution of specific neurons or networks to behavior is a key challenge. Here we describe an approach that facilitates this process by enabling the rapid modulation of synaptic inhibition in defined cell populations. Binding of zolpidem, a systemically active allosteric modulator that enhances the function of the GABA A receptor, requires a phenylalanine residue (Phe77) in the γ2 subunit. Mice in which this residue is changed to isoleucine are insensitive to zolpidem. By Cre recombinase-induced swapping of the γ2 subunit (that is, exchanging Ile77 for Phe77), zolpidem sensitivity can be restored to GABA A receptors in chosen cell types. We demonstrate the power of this method in the cerebellum, where zolpidem rapidly induces significant motor deficits when Purkinje cells are made uniquely sensitive to its action. This combined molecular and pharmacological technique has demonstrable advantages over targeted cell ablation and will be invaluable for investigating many neuronal circuits.A classical approach to the study of brain function is selective lesioning. Unfortunately, the interpretation of data from such studies can be confounded by compensatory changes, whereby unrelated systems are recruited to alleviate, if only partially, any deficit. A complementary method involves reversibly silencing, albeit with little or no cell-type selectivity, the activity of a pathway or nucleus through cooling or stereotaxic drug administration (for example, see refs. 1,2). Reversible approaches have advantages over permanent lesioning. First, the effects of acute regional inactivation cannot be easily overcome by compensatory changes, because the inactivated system is altered only briefly

The Genetic Analysis of Functional Connectomics in Drosophila

“…Quantification of the extent of knock down and comparisons of the effects produced by different reagents, are both precautions that come most obviously to mind, as is awareness of potential compensatory mechanisms. A potentially powerful strategy to overcome such compensation is to modify ectopically expressed channels and receptors so that they are sensitive to unique transient pharmacological modulation (Wulff et al ., 2007). This combination of molecular biology and pharmacology has yet to be applied in flies however.…”

Increased Motor-Impairing Effects of the Neuroactive Steroid Pregnanolone in Mice with Targeted Inactivation of the GABAA Receptor γ2 Subunit in the Cerebellum

“…Parvalbumin (Pv)-neuron GABA A γ2 subunit knockout mice (Pv-Δγ2) and littermate control Pv-Cre mice were generated as described (Wulff et al, 2009a), by crossing Pv-Cre mice with γ2I77 lox mice (Fuchs et al, 2007; Wulff et al, 2007). Pv-Δγ2 mice were important for the study, since cerebellar Purkinje cells and molecular layer interneurons are positive for parvalbumin (Leppa et al, 2011), and thus lose synaptic inhibition.…”

“…Gr-Δγ2 mice were used to study the effect of γ2 subunit deficiency in only one major cerebellar neuron population. Cerebellar Purkinje cell (PC) GABA A γ2 subunit knockout mice (PC-Δγ2) and littermate control γ2I77lox mice were generated as described (Wulff et al, 2007), by crossing γ2I77lox mice with L7-Cre mice (Barski et al, 2000; Wulff et al, 2007). The γ2I77lox mice are available at JAX labs, stock STOCK 021197 Gabrg2 tm1Wul /J, and have loxP sites surrounding exon 4 of the γ2 subunit gene (Wulff et al, 2007).…”

“…The cerebellum was very suitable for these experiments, since the two main targets we used (Purkinje and granule cells) are known to express the γ2 subunit and a limited number of other subunits [Purkinje cells α1, β2/3 and γ2; granule cells α1/6, β2/3, δ and γ2 (Wisden et al, 1996)]. The contributions of extrasynaptic and synaptic GABA A receptors in mediating pregnanolone effects were further validated by transgenic re-introduction of the γ2 subunit into Purkinje cells (PC-γ2-swap mice) (Wulff et al, 2007; Wisden et al, 2009). …”

Addiction as an adaptation process in the brain, a view from neurobiology

“…For example, we can regulate reward/ aversion by selective activation/inactivation of midbrain dopamine neurons or inhibitory GABA neurons (Adamantidis et al, 2011;Tan et al, 2012; van Zessen, Phillips, Budygin, & Stuber, 2012) using novel optogenetic or pharmacogenetic tools that have given for the first time the possibility to modulate brain activity at a single neuronal population level. These new methods are based on genetic modification of specific neuronal populations, often using viral vectors in the transfer of light or drugsensitive ion channels/receptors that can be activated later to stimulate or inhibit neuronal firing without disturbing the basic functions of the brain (Deisseroth & Schnitzer, 2013;Wulff et al, 2007). While this kind of detailed basic neurobiological research is still out of reach in human addiction research, more selective tools are also emerging for human research and treatment.…”