Transgenic mice expressing a pH and Cl^– sensing yellow‐fluorescent protein under the control of a potassium channel promoter

Abstract

During the last few years a variety of genetically encodable optical probes that monitor physiological parameters such as local pH, Ca2+, Cl-, or transmembrane voltage have been developed. These sensors are based on variants of green-fluorescent protein (GFP) and can be synthesized by mammalian cells after transfection with cDNA. To use these sensor proteins in intact brain tissue, specific promoters are needed that drive protein expression at a sufficiently high expression level in distinct neuronal subpopulations. Here we investigated whether the promoter sequence of a particular potassium channel may be useful for this purpose. We produced transgenic mouse lines carrying the gene for enhanced yellow-fluorescent protein (EYFP), a yellow-green pH- and Cl- sensitive variant of GFP, under control of the Kv3.1 K+ channel promoter (pKv3.1). Transgenic mouse lines displayed high levels of EYFP expression, identified by confocal microscopy, in adult cerebellar granule cells, interneurons of the cerebral cortex, and in neurons of hippocampus and thalamus. Furthermore, using living cerebellar slices we demonstrate that expression levels of EYFP are sufficient to report intracellular pH and Cl- concentration using imaging techniques and conditions analogous to those used with conventional ion-sensitive dyes. We conclude that transgenic mice expressing GFP-derived sensors under the control of cell-type specific promoters, provide a unique opportunity for functional characterization of defined subsets of neurons.

Intraglomerular Lateral Inhibition Promotes Spike Timing Variability in Principal Neurons of the Olfactory Bulb

“…Experimental protocols were approved by the National Institute of Health and Medical Research and the National Center of Scientific Research guidelines. Horizontal olfactory bulb slices were prepared from 14-to 30-d-old transgenic mice of either sex expressing the EYFP under the control of the Kv3.1 K ϩ channel promoter (Metzger et al, 2002) or the enhanced green fluorescent protein (EGFP) under the control of the calretinin (CR) promoter (Caputi et al, 2009). Note that, in these mice, the fluorescent protein expression may differ from the expression pattern of the Kv3.1 protein (Metzger et al, 2002) or the CR protein (Caputi et al, 2009).…”

“…Horizontal olfactory bulb slices were prepared from 14-to 30-d-old transgenic mice of either sex expressing the EYFP under the control of the Kv3.1 K ϩ channel promoter (Metzger et al, 2002) or the enhanced green fluorescent protein (EGFP) under the control of the calretinin (CR) promoter (Caputi et al, 2009). Note that, in these mice, the fluorescent protein expression may differ from the expression pattern of the Kv3.1 protein (Metzger et al, 2002) or the CR protein (Caputi et al, 2009). Mice were killed by decapitation, and the bulbs were dissected rapidly in ice-cold oxygenated (95% O 2 /5% CO 2 ) solution containing the following (in mM): 83 NaCl, 26.2 NaHCO 3 , 1 NaH 2 PO 4 , 2.5 KCl, 3.3 MgSO 4 0.5 CaCl 2 , 70 sucrose, and 22 D-glucose, pH 7.3 (osmolarity, 300 mOsm/L).…”

“…Granule and parvalbumin-positive (PV ϩ ) cells make reciprocal synapses with the lateral dendrites of mitral and tufted cells. Mitral and tufted cells activate these interneurons that in turn release GABA onto mitral and tufted cells (Isaacson and Strowbridge, 1998;Schoppa et al, 1998;Christie et al, 2001;Kato et al, 2013;Miyamichi et al, 2013). PV ϩ cells mediate a broad and nonselective interglomerular lateral inhibition of widely distributed mitral and tufted cells, whereas granule cells inhibit fewer narrowly confined principal neurons projecting into specific glomeruli (Kato et al, 2013;Miyamichi et al, 2013).…”

“…Mitral and tufted cells activate these interneurons that in turn release GABA onto mitral and tufted cells (Isaacson and Strowbridge, 1998;Schoppa et al, 1998;Christie et al, 2001;Kato et al, 2013;Miyamichi et al, 2013). PV ϩ cells mediate a broad and nonselective interglomerular lateral inhibition of widely distributed mitral and tufted cells, whereas granule cells inhibit fewer narrowly confined principal neurons projecting into specific glomeruli (Kato et al, 2013;Miyamichi et al, 2013). Models of olfactory bulb processing stress the importance of interglomerular lateral inhibition for sharpening odor tuning of principal neurons, enhancing contrast between glomerular units and facilitating odor discrimination (Yokoi et al, 1995;Mori et al, 1999;Abraham et al, 2010).…”

“…Thus, to get a better insight into the function of intraglomerular inhibition, we investigated the properties and the synaptic circuits made by a specific subset of PG cells expressing enhanced yellow fluorescent protein (EYFP) under the control of the Kv3.1 potassium channel promoter (Metzger et al, 2002). We determined in vivo and in vitro, using targeted patch-clamp recording, the circumstances under which labeled PG cells are activated.…”