Targeted gene expression in dopamine and serotonin neurons of the mouse brain

Abstract

We used a knock-in strategy to generate two lines of mice expressing Cre recombinase under the transcriptional control of the dopamine transporter promoter (DAT-cre mice) or the serotonin transporter promoter (SERT-cre mice). In DAT-cre mice, immunocytochemical staining of adult brains for the dopamine-synthetic enzyme tyrosine hydroxylase and for Cre recombinase revealed that virtually all dopaminergic neurons in the ventral midbrain expressed Cre. Crossing DAT-cre mice with ROSA26-stop-lacZ or ROSA26-stop-YFP reporter mice revealed a near perfect correlation between staining for tyrosine hydroxylase and β-galactosidase or YFP. YFP-labeled fluorescent dopaminergic neurons could be readily identified in live slices. Crossing SERT-cre mice with the ROSA26-stop-lacZ or ROSA26-stop-YFP reporter mice similarly revealed a near perfect correlation between staining for serotonin-synthetic enzyme tryptophan hydroxylase and β-galactosidase or YFP. Additional Cre expression in the thalamus and cortex was observed, reflecting the known pattern of transient SERT expression during early postnatal development. These findings suggest a general strategy of using neurotransmitter transporter promoters to drive selective Cre expression and thus control mutations in specific neurotransmitter systems. Crossed with fluorescent-gene reporters, this strategy tags neurons by neurotransmitter status, providing new tools for electrophysiology and imaging.

Introduction

Aberrant functioning of the ascending dopaminergic or serotonergic systems is prominent in many neuropsychiatric disorders including schizophrenia, Parkinson's disease, attention deficit hyperactivity disorder, drug addiction, depression, and anxiety. A striking feature of these systems is that they arise from just a few hundred thousand neurons concentrated in brainstem nuclei sending projections to widely distributed target areas (Cooper et al., 1996). We have taken advantage of the dopamine transporter (DAT) and serotonin transporter (SERT), known specific markers of dopaminergic and serotonergic neurons, respectively (Augood et al., 1993, Hensler et al., 1994), and used their promoters to generate cell-specific Cre recombinase expressing mice.

The Cre-loxP system has become a standard approach for performing region-specific gene inactivation in mice (Kuhn and Torres, 2002). However, this strategy has not previously been used to generate gene knockouts in specific neurotransmitter systems. Traditionally, transgenic lines are generated via pronuclear injection of the transgene that will be integrated into the genome randomly. The level and pattern of transgene expression are determined both by the promoter and by its integration site in the genome. Consequently, such transgenic mice often have transgene expression in regions or cell types not associated with the original specificity of the promoter (Bronson et al., 1996, Wallace et al., 2000). In order to direct the expression of the Cre recombinase more precisely to specific monoaminergic cell types, we used a knock-in approach to place the transgene downstream of the endogenous promoters of DAT and SERT.

Mice engineered using such an approach express Cre recombinase in dopaminergic and serotonergic neurons. Crossing these cre mice with reporter lines, demonstrated the high efficiency and specificity of this approach. Moreover, these Cre-expressing lines were used successfully to label dopamine and serotonin neurons in vivo with yellow fluorescent protein (YFP), allowing for visualization of the living neurons in brain slices. Our results represent the first example in which Cre recombinase activity is restricted to a specific monoamine neurotransmitter system and provide a general strategy that can be used to target mutations and fluorescent markers to specific neurotransmitter systems.