Analysis of dopamine transporter gene expression pattern ) generation of DAT-iCre transgenic mice Marc Turiault 1, *, Se ´ bastien Parnaudeau 1, *, Aude Milet 1 , Rosanna Parlato 2 , Jean-Denis Rouzeau 1 , Monique Lazar 1 and Franc¸ois Tronche 1 1 CNRS UMR7148, Molecular Genetics, Neurophysiology and Behavior, Colle ` ge de France, Institut de Biologie, Paris, France 2 Molecular Biology of the Cell I, German Cancer Research Center, Heidelberg, Germany Dopamine-synthesizing neurons modulate many biolo- gical functions in a dynamic manner. In the mamma- lian brain, dopamine neurons are distributed as groups of cells in the ventral midbrain area (A8, A9, A10), diencephalon (A11–A15), olfactory bulb (A16) and retina (A17). Dopaminergic systems are involved in the regulation of motor and motivational control, cardio- vascular and respiratory activities via ascending and descending projections that are widely distributed in the mammalian brain and spinal cord [1,2]. In the ventral midbrain, the A9 nucleus (substantia nigra) has several functions, including the modulation of motor control, and the A10 nucleus (ventral tegmental area) is involved in reward mechanisms. Hypothalamic Keywords Cre ⁄ loxP system; dopaminergic cells; dopamine transporter; gene expression; transgenic mice Correspondence F. Tronche, UMR7148 CNRS, 11 place Marcelin Berthelot, 75005 Paris, France Fax: +33 1 44 27 13 22 Tel: +33 1 44 27 13 08 E-mail: francois.tronche@gmail.com *These authors contributed equally to this work (Received 16 February 2007, revised 10 May 2007, accepted 16 May 2007) doi:10.1111/j.1742-4658.2007.05886.x The dopamine transporter is an essential component of the dopaminergic synapse. It is located in the presynaptic neurons and regulates extracellular dopamine levels. We generated a transgenic mouse line expressing the Cre recombinase under the control of the regulatory elements of the dopamine transporter gene, for investigations of gene function in dopaminergic neu- rons. The codon-improved Cre recombinase (iCre) gene was inserted into the dopamine transporter gene on a bacterial artificial chromosome. The pattern of expression of the bacterial artificial chromosome–dopamine transporter–iCre transgene was similar to that of the endogenous dopamine transporter gene, as shown by immunohistochemistry. Recombinase activ- ity was further studied in mice carrying both the bacterial artificial chromo- some–dopamine transporter–iCre transgene and a construct expressing the b-galactosidase gene after Cre-mediated recombination. In situ studies showed that b-galactosidase (5-bromo-4-chloroindol-3-yl b-d-galactoside staining) and the dopamine transporter (immunofluorescence) had identical distributions in the ventral midbrain. We used this animal model to study the distribution of dopamine transporter gene expression in hypothalamic nuclei in detail. The expression profile of tyrosine hydroxylase (an enzyme required for dopamine synthesis) was broader than that of b-galactosidase in A12 to A15. Thus, only a fraction of neurons synthesizing dopamine expressed the dopamine transporter gene. The bacterial artificial chromo- some–dopamine transporter–iCre transgenic line is a unique tool for target- ing Cre ⁄ loxP-mediated DNA recombination to dopamine neurons for studies of gene function or for labeling living cells, following the crossing of these mice with transgenic Cre reporter lines producing fluorescent proteins. Abbreviations BAC, bacterial artificial chromosome; DAT, dopamine transporter; GFP, green fluorescent protein; iCre, codon-improved Cre recombinase; IRES, internal ribosome entry site; TH, tyrosine hydroxylase; X-Gal, 5-bromo-4-chloroindol-3-yl b- D-galactoside staining. 3568 FEBS Journal 274 (2007) 3568–3577 ª 2007 The Authors Journal compilation ª 2007 FEBS dopamine cells form five groups (A11–A15). A11 neu- rons from the posterior hypothalamus project into the spinal cord. A13 neurons from the zona incerta project locally into the hypothalamus and are engaged in gonadotropin-releasing hormone neuron control. In the arcuate nucleus (A12 group) and the preoptic area ⁄ anterior hypothalamus (A14 group), most of the dopamine neurons are endocrine neurons, secreting dopamine into the portal blood system ) which bathes the anterior lobe of the pituitary gland ) or directly into the median pituitary lobe. These neurons control prolactin secretion and growth hormone secretion from the anterior pituitary gland and melanocyte-stimula- ting hormone secretion from the intermediate lobe. Dopaminergic neurons are able to synthesize dop- amine because they contain the rate-limiting enzyme tyrosine hydroxylase (TH) and the widely expressed 3,4-dihydroxyphenylalanine decarboxylase. Some sub- sets of dopamine neurons express other specific genes, such as that encoding the dopamine 2 receptor, which functions as an autoreceptor, or that encoding the dopamine transporter (DAT), an essential regulator of extracellular dopamine levels in the synaptic cleft. Dopamine system dysfunction is associated with sev- eral diseases of the motor and limbic systems. A loss of dopamine neurons is one of the characteristics of Parkinson’s disease, whereas changes in the activity or function of dopaminergic nuclei are associated with depression, schizophrenia and addiction to drugs of abuse [3–7]. The use of the Cre DNA recombinase allows target- ing of DNA recombination events to desired cell popu- lations. Efforts have recently been made to generate transgenic mice expressing the recombinase in restricted neuronal populations [8]. We generated a transgenic mouse line [bacterial artificial chromosome (BAC)– DATiCrefto, hereafter referred to as BAC-DATiCre] (iCre is codon-improved Cre recombinase) in which Cre is specifically expressed in DAT-containing neurons, as a molecular genetic tool for the investiga- tion of dopamine cell function. When crossed with reporter mice expressing b-galactosidase after recombi- nation (R26R mice [9]), the BAC-DATiCre line dis- played irreversible labeling of all cells that were expressing or had expressed the DAT gene. We found that some subsets of neurons from the A12 to A15 regions never coexpressed the TH and DAT genes, whereas all TH-positive neurons from the A8 to A10 regions were recombined. Used in combination with other conditional alleles containing loxP sites, the BAC-DATiCre transgenic line will be an invaluable tool with which to analyze dopamine neuron biology and dopamine presynaptic alterations in physiopathologic disorders involving dopaminergic systems. Results Generation of the DATiCre transgenic line We used a large (177 kb) DNA genomic segment from the C57BL ⁄ 6 mouse strain in a BAC clone encompas- sing the entire mouse DAT (slc6) gene to ensure that the iCre transgene [10] was correctly expressed (Fig. 1A). We generated the transgene by recombina- tion in bacteria, using the tools developed by Lee et al. [11]. The final construct encompassed 97 kb of DNA upstream from the DAT gene start codon and 38 kb of DNA downstream from the polyadenylation signal sequence of the gene. The homology arms of the BAC targeting vector used for recombination in bacteria were chosen such that the ATG of the iCre was in the same position as the ATG of the DAT gene (Fig. 1A). Following recombination and elimination of the selec- tion cassette, bacterial colonies were screened and the modified BAC was analyzed for the desired recombina- tion event by restriction enzyme analysis. A 177 kb DNA fragment was excised by digestion with PmeI and AscI restriction enzymes, purified and injected into mouse FVB ⁄ N zygotes. We obtained two transgenic lines. In both lines, the Cre recombinase protein was produced in the ventral tegmental area and the substantia nigra, as shown by immunohistochemistry with an antibody directed against Cre [12]. Immunostaining for Cre was com- pared with that for TH, on successive serial sections of the mesencephalon, and a perfect match was found. Cre expression in the ventral midbrain was restricted to dopamine neurons of the A9 and A10 nuclei (Fig. 1B). Cre-mediated recombination pattern We investigated the distribution of Cre expression and the DNA recombination pattern in the transgenic mouse line, by analyzing one transgenic line in detail (BAC-DATiCre line), and crossing this line with the R26R reporter line [9]. In transgenic animals carrying both the R26R and BAC-DATiCre transgenes, the Cre recombinase catalyzed the removal of a DNA sequence, leading to b-galactosidase production. A detailed analysis of serial sections stained with 5-bromo-4-chloroindol-3-yl b-d-galactoside (X-Gal) and for TH clearly showed that recombination was restricted, in the mesencephalon, to dopaminergic structures (Fig. 1C). M. Turiault et al. Cre-mediated recombination in dopaminergic cells FEBS Journal 274 (2007) 3568–3577 ª 2007 The Authors Journal compilation ª 2007 FEBS 3569 We investigated the pattern of Cre expression fur- ther, by carrying out double staining on sections of the ventral tegmental area and the substantia nigra. Using a combination of fluorescent secondary antibodies and primary antibodies directed against TH and Cre, we found that Cre (red) was restricted to the nuclei of TH-expressing neurons (Fig. 2A). We used TH immunostaining to identify cells con- taining dopamine, because DAT labeling of the cell body is very weak. Nevertheless, the labeling of mid- brain sections for both DAT and Cre showed that all neurons producing the Cre recombinase (red) also pro- duced DAT protein (green, Fig. 2B). Similarly, when analyzed at the cellular level, we showed that DNA recombination was restricted to DAT-expressing neu- rons. In mice carrying both the iCre and the R26R transgenes, b-galactosidase expression (green) was con- fined to the domain containing DAT neurons (red) A BC Fig. 1. Expression of Cre recombinase in the midbrain dopaminergic nuclei of BAC-DATiCre mice. (A) Schematic representation of the BAC- DATiCre construct and its structure. The position of the DAT gene within the RP23–408F13 BAC is indicated, together with those of the putative genes contained within this BAC. The exon ⁄ intron structure of the DAT gene is shown. The DAT gene has 14 exons (rectangles). The recombination targeted the second DAT exon, inserting the Cre transgene at the level of the ATG of the DAT gene. The iCre ORF is rep- resented as a white rectangle, whereas intron sequences and the polyadenylation signal of the bovine GH gene are shown as a dark gray rectangle. The ampicillin resistance gene (light gray rectangle), flanked by FLP recombinase target sites (black half-circles), was removed from the final construct by flp-mediated recombination from the final construct (lowest representation). (B) The Cre recombinase is expressed in TH-positive neurons from the mesencephalon. Serial sections labeled by immunohistochemistry, using antibodies directed against TH (left panel) and Cre (right panel) proteins. (C) DNA recombination occurs in TH-positive neurons from the mesencephalon. Serial sections showing the distribution of TH-positive neurons (left) and X-Gal staining indicating Cre-mediated recombination of a LacZ-expressing reporter gene (right). Magnification: scale bars ¼ 400 lm. Cre-mediated recombination in dopaminergic cells M. Turiault et al. 3570 FEBS Journal 274 (2007) 3568–3577 ª 2007 The Authors Journal compilation ª 2007 FEBS in the A10 region of the mesencephalon (Fig. 2C). b-galactosidase expression was clearly restricted to DAT-positive neurons (Fig. 2D). We used the same approach for systematic analyses of the DNA recombination pattern in groups of dop- aminergic cells, from the mesencephalon to olfactory bulbs and retina in serial sections labeled immunohis- tochemically for TH and stained for b-galactosidase activity with X-Gal substrate. In the retrorubral field (A8), substantia nigra (A9), ventral tegmental area (A10) and glomerular layer of the olfactory bulb (A16), the number of recombination events was similar to the number of TH-expressing cells in serial fields (Fig. 3). In contrast, in hypothalamic groups of cells, the overlap was only partial between recombination and TH expression. Whereas in the periventricular organ (A11) and the cell group of the dorsomedial and lateral arcuate nucleus (A12) the number of recom- bined cells was similar to the number of TH positive cells, we did not detect b-galactosidase activity in the ventromedial region of the arcuate nucleus. In the zona incerta (A13) and periaqueductal gray area, recombination events appeared to be restricted to a minority of TH-positive neurons. No X-Gal staining was detected in A14 and A15 nuclei. X-Gal staining of retina slices showed some recombination events (data not shown). Behavioral analysis of BAC-DATiCre mice The BAC-DATiCre line was, in part, used to study behavioral consequences of mutations targeted to dop- aminergic neurons. It was therefore essential to verify that the presence of the BAC-DATiCre transgene does not alter behavior. During the generation of the BAC-DATiCre line, we minimized the risks of any A B C D Fig. 2. In the midbrain of BAC-DATiCre mice, both Cre expression and recombination are restricted to dopamine neurons. Confocal images from multiple staining of double-transgenic mouse brain coronal sections (30 lm). (A) Immunofluorescence of TH (green) and Cre (red) in midbrain dopamine neurons. DNA was stained with TO-PRO Ò 3 iodide (TOPRO). The left panel is an overlay of the three previous panels. (B) As for (A), except that the green staining corresponds to the DAT protein. (C). Cre-mediated recombination visualized by immunofluorescence staining, using antibodies against b-galactosidase (green). (D) Low-power magnification showing that Cre-mediated recombination corresponds to DAT- expressing neurons (red), using a higher magnification. All nuclei of this region are stained with TOPRO. Magnification: scale bars ¼ 50 lm. M. Turiault et al. Cre-mediated recombination in dopaminergic cells FEBS Journal 274 (2007) 3568–3577 ª 2007 The Authors Journal compilation ª 2007 FEBS 3571 perturbations that could arise from undesired expres- sion of gene products encoded by the BAC transgene, by designing a transgene from which DAT proteins should not be produced. To rule out the existence of any problem, we performed comparative behavioral tests in BAC-DATiCre males (n ¼ 11) and their con- trol littermates (n ¼ 11) maintained on an FVB ⁄ N genetic background. The general appearance of trans- genic and control littermates was undistinguishable. The weights of animals from both groups were similar (BAC-DATiCre, 33.9 g ± 0.8; controls, 33.4 g ± 0.7), as were the levels of muscular strength (for two limbs – BAC-DATiCre, 1.2 N ± 0.1; control, 1.3 N ± 0.1; and for four limbs ) BAC-DATiCre, 2.4 N ± 0.1; and controls, 2.5 N ± 0.1) and locomo- tion (Fig. 4A). Anxiety-like behavior was not different between the two genotypes as assessed by two tests based on the natural avoidance behavior of mice: the dark–light transition test (latency to exit the dark com- partment for the first time, 15 s ± 2.9 versus 17.9 s ± 2.5, P ¼ 0.46, and time spent in the lit com- partment, 174.9 s ± 7.8 versus 186.8 s ± 5.9, P ¼ 0.24, for transgenic and control mice, respectively) and the elevated plus maze test (time spent in open arms: Fig. 3. Pattern of b-galactosidase activity in dopamine cell groups A8–A16 in DATiCre mice. For each group of dopamine cells, TH expression was detected by immunohistochemistry, in mice carrying both the BAC-DATiCre and R26R transgenes (left panels). Recombination events were detected on serial sections stained with X-Gal for the detection of b-galactosidase activity (right panels). In A8, A9, A10, A11, A12 and A16, iCre-induced recombination matches the pattern of TH expression, but some discrepancies are observed in the periaqueductal gray area, the ventromedial part of the arcuate nucleus (arrow) and A13. In the A14 and A15 group of cells, recombination events are less fre- quently observed than TH expression. The highlighted structures on these anatomic drawings from representative coronal sections of the adult mouse brain modified from Paxinos [38] contain dopaminergic cells. Magnification: scale bars ¼ 50 lm. Cre-mediated recombination in dopaminergic cells M. Turiault et al. 3572 FEBS Journal 274 (2007) 3568–3577 ª 2007 The Authors Journal compilation ª 2007 FEBS 150 s ± 16 versus 146 s ± 18 for transgenic and con- trol mice, respectively). Similarly, no differences were observed when despair-like behavior was studied using the forced swim test (Fig. 4B). Discussion Transgenesis with small DNA regions regulating tran- scription is inherently prone to problems of ectopic expression, mosaic expression or the absence of expres- sion, due to the influence of genomic sequences surrounding the integration site or the absence of essential DNA elements. The expression of a transgene under the control of an endogenous regulatory ele- ment, using knock-in approaches, can prevent these problems but disrupts one copy of the gene, generating results that may be difficult to interpret. The use of a large (100–250 kb) DNA segment that contains a gene with an interesting expression profile, including all DNA regions required for correct gene expression, alleviates this problem [13,14]. We targeted Cre recom- binase expression to dopamine neurons, by generating a mouse transgenic line expressing the iCre recombin- ase under the control of the DAT gene encompassed within a 177 kb BAC. We used the iCre ORF, an improved version of the Cre recombinase gene that is more efficiently expressed in mammalian cells [10]. In the ventral tegmental area and the substantia ni- gra, the pattern of iCre expression was similar to the pattern of expression of the endogenous DAT gene reported in previous in situ mRNA hybridization stud- ies [15–17]. When recombination events were analyzed in situ following the Cre-dependent recombination of a LacZ reporter construct in double-transgenic animals, b-galactosidase expression was found to be confined to areas of endogenous DAT gene expression. Recombi- nation was restricted exclusively to TH- and DAT- positive neurons. Discrepancies have been reported between TH and DAT expression in the ventral midbrain in rats [17], but we detected no TH-positive neurons that did not display recombination in the ventral midbrain group of cells. This suggests that the TH-positive, DAT-negative cells detected in previous studies may have been in a transient state or may have expressed DAT during development. The perfect concordance between Cre and DAT expression in the midbrain of BAC-DATiCre mice led us to use this strategy to improve the definition of DAT gene expression in other dopaminergic cell groups in which the precise distribution of DAT was unclear. In double-transgenic mice carrying the Cre and R26R (reporter) transgenes, transient or low-level DAT expression should be paralleled by Cre expres- sion, leading to permanent, irreversible LacZ expres- sion, increasing the sensitivity of DAT detection. We focused on the various cell groups of the hypothalamic region (from A11 to A15). No DAT-iCre-mediated recombination was observed in the ventromedial neurons of the arcuate nucleus in mice. Interestingly, in other mammals, this region has been shown to contain monoenzymatic neurons expres- sing TH or aromatic l-amino acid decarboxylase, but not the entire enzymatic machinery required for dop- amine synthesis. Dopamine produced in this region may result from an exchange of precursor molecules between complementary cells [18,19]. Previous studies have suggested that no DAT mRNA is produced in this region [17,20,21]. The authors detected very low levels of DAT expression in the dorsomedial part of the arcuate nucleus, and our results are consistent with the presence of very few recombined cells per section. DAT mRNA levels, which were considerably lower in A B Fig. 4. The presence of the BAC-DATiCre transgene did not affect locomotion and despair behavior. (A) The presence of the transgene had no effect on locomotor activity measured within a circular maze. The numbers of 1 ⁄ 4th turns per 5 min are indicated for trans- genic and control animals. (B) Transgenic and control littermates did not show differences in immobility time in a forced swim test. Immobility was measured for 6 min, in 2-min periods, over two con- secutive days. Results obtained with transgenic (n ¼ 11) and con- trol (n ¼ 11) mice are shown in black and gray, respectively. M. Turiault et al. Cre-mediated recombination in dopaminergic cells FEBS Journal 274 (2007) 3568–3577 ª 2007 The Authors Journal compilation ª 2007 FEBS 3573 the A13 region than in the ventral midbrain [17,21,22], may account for the smaller number of b-galactosi- dase-expressing neurons than of TH-positive neurons. Expression of DAT in the A14 nucleus of rats remains controversial, as some studies have reported the detec- tion of very low levels of DAT mRNA in scattered cells [21], whereas others detected neither DAT mRNA nor protein [22,23]. DAT-iCre-expressing mice allow us to answer this question, as the lack of recombina- tion in the A14 nucleus indicates an absence of DAT in these dopaminergic neurons in mice. The discrepancies observed between TH and X-Gal labeling in A15 are consistent with the absence of DAT expression in A15, as shown by in situ mRNA hybridization or immunohistofluorescence [17,21–23]. All previous published data on DAT expression pat- terns were obtained with rats. Our study is thus the first to confirm that the pattern of DAT expression in the brain is very similar in mice and rats. A recombination pattern restricted to dopamine cells has been previously achieved [24,25]. The approaches used involved insertion of the Cre gene, at different positions, into the DAT gene by homologous recombi- nation in embryonic stem cells. In two cases, this led to inactivation of the endogenous targeted DAT allele and, in the last case, to an alteration of its expression, prob- ably due to the presence of the Cre construct in the 3¢-UTR of DAT mRNA [26]. An important advantage of using BAC transgenesis is that it leaves both endog- enous DAT alleles intact. This is essential, as changes in DAT gene expression levels lead to atypical behaviors [26–31]. We confirmed that the presence of the BAC- DATiCre transgene had no effect on muscular strength, locomotion, or anxiety-related or despair behaviors. The BAC-DATiCre transgenic line is likely to prove a valuable tool for targeting DNA recombination events, resulting in reporter gene expression or gene inactivation, in studies of dopamine neuron biology and presynaptic alterations in physiopathologic disor- ders involving dopaminergic systems. Combined with mice expressing cre-dependent fluorescent protein, it will facilitate the localization and the study of dopam- ine cells in living tissues. Combined with conditional alleles of relevant genes, it will allow us to distinguish their function in dopamine cells from their function in other cell types. This will be particularly useful in the context of Parkinson’s disease, in which several genes potentially involved in the pathogenesis have a wide- spread expression pattern. In this respect, we have already used the BAC-DATiCre vector to inactivate the cAMP-response-element-binding (CREB) gene [32]. In combination with the D1Cre line, which allows targeting of recombination in dopaminoceptive neurons expressing dopamine 1a receptor [33,34], the BAC-DATiCre line will allow us to distinguish between presynaptic and postsynaptic gene functions. The BAC- DATiCrefto transgenic line has been deposited in the European Mouse Mutant Archives (http://www. emma.rm.cnr.it). Experimental procedures DNA construction and transgenesis Using the Ensembl genome database, we chose a 210 kb BAC (RP24 408F13) encompassing the DAT (slc6) gene from a CHORI BAC library [35]. This BAC was modified by homologous recombination [11], to insert a 2950 bp DNA cassette containing the ORF of the improved Cre recombin- ase, iCre [10], followed by a DNA sequence containing intron and polyA sequences from the bovine GH gene, and an ampicillin resistance gene flanked by two FLP recombi- nase target sites in the same orientation, which was subse- quently removed (Fig. 1). The BAC was modified as follows: a 319 bp 5¢ DNA fragment of the DAT gene was ampli- fied by PCR (using the primers 5¢-CTAGGTACCA CAAGCCGGCGTTAATGTGAA and 5¢-CTAATCGAT GGAGCCCGAGGAAGTCTGTTT), digested with ClaI and KpnI, and inserted upstream from the iCre DNA cassette in pMT1. The pMT1 plasmid was derived from the vector iCre-internal ribosome entry site (IRES)-green fluorescent protein (GFP)-polyA [36], by removing an NsiI–BsrGI DNA fragment containing the IRES and eGFP sequences, and then the 3¢ protruding NsiI end, and filling in the 5¢ BsrGI end before ligation (sequence available on request). The 3¢ homology region was a 200 bp DNA fragment of DAT ge- nomic DNA amplified by PCR (using the primers 3¢-CAT GCTAGCTAAAAGCAAATGCTCCGTGGG and 3¢-CTA GTATACGAAACCTCCAGACATTGGCCA), digested with BstZ17I and NheI, and inserted downstream from the DNA cassette. Recombination was induced as previously described [11]. Briefly, EL250 bacteria (a gift from N. G. Copeland, National Cancer Institute, Frederick, MO, USA) containing the RP24 408F13 BAC were electroporated with the targeting vector. The bacteria were incubated for 15 min at 42 °C to induce recombination. Correctly targeted BACs were identified by DNA restriction analysis of ampicillin- resistant colonies. The ampicillin resistance cassette used for selection was excised by inducing Flpe recombinase expres- sion by adding 0.1% l-arabinose to the culture medium for 1 h. Correctly recombined BACs were identified by DNA restriction analysis and pulsed-field gel electrophoresis. A 177 kb DNA fragment was excised from the BAC DNA by digestion with PmeI–AscI and purified by chromatography on Sepharose CL4b columns (Pharmacia, New York, NY, USA) [37]. This fragment was then used for pronuclear injec- tions into fertilized FVB ⁄ N oocytes. Cre-mediated recombination in dopaminergic cells M. Turiault et al. 3574 FEBS Journal 274 (2007) 3568–3577 ª 2007 The Authors Journal compilation ª 2007 FEBS Mouse genotyping and breeding Transgenic BAC-DATiCrefto animals were maintained in an FVB ⁄ N background as well as being backcrossed for five generations onto the C57BL ⁄ 6 background. These animals were genotyped using DNA obtained by tail biopsy, by dot blot DNA hybridization, with a 32 P-radio- labeled DNA fragment from the iCre ORF. Rosa 26 repor- ter animals [9] were maintained on a mixed background and genotyped by PCR amplification, using LacZ-Forward (5¢-GTCGTTTTACAACGTCGTGACT-3¢) and LacZ- Reverse (5¢-GATGGGCGATCGTAACCGTGC-3¢) prim- ers. Animals were housed under specific pathogen-free conditions at 22 °C, with a 12 h light ⁄ 12 h dark cycle and free access to water and a rodent diet. All experiments were performed in accordance with French (Ministe ´ re de l’Agri- culture 87-848) and European Union (EEC86-6091) guide- lines for care of laboratory animals. Histology Vibratome sections (30 lm) prepared from the brains of perfused mice (4% paraformaldehyde in 0.1 m NaCl ⁄ P i ) were incubated overnight with a rabbit polyclonal anti-Cre serum (1 : 3000 dilution [12]), 1 : 400 dilution) or a mono- clonal b-galactosidase antibody (Monosan, Am Uden, the Netherlands; 1 : 2000 dilution). Immunohistochemistry was carried out with the avidin–biotin system from Vector Laboratories (Vectastain, Burlingame, CA, USA). For double-immunofluorescence labeling, we used Alexa-488- coupled anti-mouse serum (Molecular Probes, Eugene, OR, USA) and Cy3-coupled anti-rabbit serum (Jackson Immu- noresearch, West Grove, PA, USA) at a 1 : 400 dilution as the secondary antibodies. Immunostaining controls were performed in the same way, but without primary antibod- ies. Nuclei were stained with 0.5 lm TO-PRO-3 iodide (Molecular Probes). b-galactosidase enzymatic activity was detected on brain cryosections (30 lm). The histologic immunofluorescence of sections was assessed with a Leica TCS SP 2 confocal laser scanning microscope (Leica Micro- systems, Heidelberg, Germany). For cryostat sectioning, brains were transferred to 15% sucrose in NaCl ⁄ P i at room temperature, and then embedded in 7% gelatin and 15% sucrose in NaCl ⁄ P i , before being frozen by immersion in isopentane at ) 140 °C. Cryosections were cut and incuba- ted at 37 °C overnight in X-Gal staining solution [4 mm potassium hexacyanoferrate(III), 4 mm potassium hexa- cyanoferrate(II), 2 mm MgCl 2 , 0.02% NP-40, 0.01% sodium deoxycholate, 5 mm EGTA, and 4 mgÆmL )1 X-Gal]. Behavioral studies Mutant (n ¼ 11) and control (n ¼ 11) littermate males matched for age (4–6 months) were housed together. One hour before each behavioral test, mice were isolated in individual cages. Muscular strength was quantified using the Grip strength test (Bioseb, Chaville, France). To meas- ure spontaneous locomotor activity, mice were placed, for 130 min, in a circular corridor (4.5 cm width, 17 cm exter- nal diameter) crossed by four infrared beams (1.5 cm above the base), equally spaced (Imetronic, Pessac, France). The locomotor activity was counted when animals interrupted two successive beams and thus had traveled at least a quar- ter of the circular corridor. Anxiety was assessed using the dark–light transition test and the elevated plus maze. The dark–light box was 45 · 20 · 25 cm, and separated into two compartments connected by a central aperture (5 · 5 cm) (Ligna, Paris, France). The dark compartment (black PVC, 15 cm) was covered, and in the lit compart- ment the intensity of light (white PVC, 30 cm) was 700 lux. Animals were tracked for 5 min using the Ethovision video tracking system from Noldus (Wageningen, The Nether- lands); both time of exit from the dark compartment and the time spent in the lit compartment were measured. The elevated plus maze consists of two elevated (1 m high) and open arms (24 · 8 cm) positioned opposite to one another and separated by a central platform and two arms of the same dimension, but enclosed by walls (20 cm high), form- ing a cross. The maze was lit by a light placed above the central platform (30 lux in each arm). The mouse was placed on the platform, and allowed to explore for 10 min, and the time spent and the number of presentations (two paws) and entries (four paws) in the open arms were recor- ded and quantified using Ethovision. To assess despair be- havior, we performed a forced swim test. We placed mice (for 6 min during two consecutive days) in a glass cylinder (height 25 cm, diameter 11 cm) containing water 8 cm deep (23 °C). Immobility time was measured during three peri- ods of 2 min. Data were expressed as means ± SEM and analyzed by Student’s t-test or ANOVA followed by the post hoc Scheffe test. Acknowledgements We are grateful to E. Casanova for the gift of piCre- IRES-GFP-polyA plasmid, and to N. G. Copeland for the gift of EL250 bacteria. We thank M. Cohen- Salmon, S. Vyas, J. P. Tassin, S. Mhaouty-Kodja, G. Schu ¨ tz and P.V. Piazza for discussions, critical reading or support. We thank H. Cambier for excellent technical assistance. This work was supported by the ‘Centre National de la Recherche’ and the Colle ` ge de France, by grants from the ‘Ministe ` re de l’Education National de la Recherche et de la Technologie’ (‘Action Concerte ´ e Incitative neurosciences’), the ‘Agence Nationale de la Recherche’ (neurosciences), the ‘Mission Interministe ´ rielle de Lutte contre la De ´ pendance et la Toxicomanie’ (MILDT), the ‘Fonda- tion pour la Recherche Me ´ dicale’ (FRM) and the M. Turiault et al. Cre-mediated recombination in dopaminergic cells FEBS Journal 274 (2007) 3568–3577 ª 2007 The Authors Journal compilation ª 2007 FEBS 3575 ‘Fondation NRJ’. M. Turiault held PhD fellowships from MILDT and FRM. 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Cre-mediated recombination in dopaminergic cells FEBS Journal 274 (2007) 3568–3577 ª 2007 The Authors Journal compilation ª 2007 FEBS 3577 . Analysis of dopamine transporter gene expression pattern ) generation of DAT-iCre transgenic mice Marc Turiault 1, *, Se ´ bastien Parnaudeau 1, *,. (iCre) gene was inserted into the dopamine transporter gene on a bacterial artificial chromosome. The pattern of expression of the bacterial artificial chromosome dopamine transporter iCre transgene. levels. We generated a transgenic mouse line expressing the Cre recombinase under the control of the regulatory elements of the dopamine transporter gene, for investigations of gene function in dopaminergic