Expression of Cre recombinase in dopaminoceptive neurons

Abstract

Background: Dopamine-activated signaling regulates locomotor and emotional responses and alterations in dopamine-signaling are responsible of several psychomotor disorders. In order to identify specific functions of these pathways, the Cre/loxP system has been used. Here, we describe the generation and the characterization of a transgenic mouse line expressing the Cre recombinase in dopaminoceptive neurons. To this purpose, we used as expression vector a 140 kb yeast artificial chromosome (YAC) containing the dopamine D1 receptor gene (Drd1a).

Results: In the chosen line, D1Cre, the spatio-temporal pattern of Cre expression closely recapitulated that of the endogenous Drd1a gene, as assessed by immunohistological approaches in embryonic and adult stages. Efficiency of recombination was confirmed by crossing D1Cre with three different loxP lines (Creb1loxP, CaMKIVloxP and GRloxP) and with the R26R reporter. In the three loxP lines studied, recombination was restricted to the area of Cre expression.

Conclusion: In view of the patterns of recombination restricted to the major dopaminoceptive regions as seen in the context of the CREB, CaMKIV and GR mutations, the D1Cre line will be a useful tool to dissect the contributions of specific genes to biological processes involving dopamine signaling.

Figure 1

Position-independent and copy-number dependent expression of the D1Cre YAC transgene. (a) Schematic representation of the modified D1Cre YAC. (b) Schematic view of the known distribution of Drd1a mRNA [29, 38].(c-e) Expression pattern of Cre as revealed by immunohistochemistry in D1Cre transgenic lines. (c) Line R, (d) line A and (e) line S. The accompanying Southern blots show the copy number dependent expression of Cre. The upper band is the endogenous Drd1a gene and the lower band is the D1Cre YAC transgene. Abbreviations: CL: centrolateral thalamic nucleus; CM: central medial thalamic nucleus; CPu: caudate putamen; DG: dentate gyrus; LD: laterodorsal thalamic nucleus; VL: ventrolateral thalamic nucleus;VM: ventromedial thalamic nucleus; VMH: ventromedial hypothalamic nucleus. Magnification: (b-e) 5×.

Figure 2

Detailed pattern of Cre expression. (a-i) Immunohistochemical localization of Cre expression. (a) Basal ganglia, (b) prefrontal cortex, (c) cortex, (d) olfactory nucleus, (e) hippocampus, (f) septum, (g) thalamus, (h) hypothalamus and (i) retina. Abbreviations: Acb: nucleus accumbens; CA2: hippocampus CA2 field; CL: centrolateral thalamic nucleus; CM: central medial thalamic nucleus; CPu: caudate putamen; DG: dentate gyrus; GCL: ganglion cell layer; INL: inner nuclear layer; ipl: inner plexiform layer; LD: laterodorsal thalamic nucleus; ONL: outer nuclear layer; opl: outer plexiform layer. Tu: olfactory tubercle; VI: layer VI of the cortex; VMH: ventromedial hypothalamic nucleus; a-b, c-h are coronal sections; c is a sagittal section. Magnification: (a-b) 25×; (c) 5×; (d-h) 50×; (i) 100×.

Figure 3

Expression of D1Cre in neurons expressing D1R mRNA. In situ hybridization with a riboprobe specific for D1R mRNA (blue) followed by immunohistochemistry with Cre specific antibody (brown) showing D1Cre and D1R mRNA coexpression in striatal neurons (a) and in the cortex (b). (a,b) 1000×.

Figure 4

Cre expression during embryonic development. DAPI staining on coronal sections of embryos at E14.5, E16.5 and E18.5 (a,d,g). The regions analyzed in striatum and cortex by immunohistochemistry with Cre antibody are boxed. No Cre positive cells at E14.5 are visible (b-c). At E16.5 Cre expressing cells are detected in the striatum (str) and a few scattered weakly expressing cells are observed in the cortex (cx) (e-f). At E18.5, more Cre positive cells are found in the striatum. Cre expression appeared in the cortical plate, in the presumptive layer VI (h-i). Magnification: (a,d,g) 100× (b,c,e,f,h,i) 200×.

Figure 5

Cre expression during postnatal development. Immunohistochemical localization of Cre. At P0 (a-c) and P3 (d-f), striatal Cre is restricted to the patch compartment (arrowheads). Expression in layer VI of the cortex is present already at birth. Transient and very weak expression is visible in the CA1 hippocampal region (a,d,g) and in layer IV of the cortex (c,f,i). At P6 (g-i), striatal Cre expression starts in the matrix compartment (asterisk), between the patches (arrowheads). Expression in CA1 and cortex layer IV disappears progressively. Magnification: (a,b,d,e,g,h) 50×; (c,f,i) 100×.

Figure 6

Cre activity in embryonic and adult brain in R26R^D1Cre mice. (a) Cre activity is analyzed by expression of beta-galactosidase activity (blue staining) at E10.5. The reporter mice are heterozygous for the R26R allele. Positive signal is present in the developing brain at this stage in the tectum above the mesencephalic fold (boxed area), as shown also in (d, arrowhead). In the adult brain, beta-galactosidase activity analyzed in coronal vibratome sections is present in striatum (b,e), cortex (b,f) and hippocampus (c). CA2: hippocampus CA2 field; DG: dentate gyrus; VI: layer VI of the cortex. Magnification: (a) 20×, (d) 40×, (b,c) 50×, (e,f) 100×.

Figure 7

Cre expression in striatal neurons. (a) Retrograde labeling of striatonigral neurons within the striatum (red fluorescence). Cre was labeled by immunofluorescence (green fluorescence). (b) Double immunofluorescence against Cre (green fluorescence) and neuron-specific class III tubulin (TujI monoclonal antibody, red fluorescence). (c) Expression of Cre protein in neurons expressing D2R mRNA by in situ hybridization with a riboprobe specific for D2R mRNA (blue) followed by immunohistochemistry with Cre specific antibody (brown). (d) Quantification of Cre positive neurons expressing D2R mRNA. Neurons expressing either Cre protein or D2R mRNA were also counted. Reported is the % of neurons per each group. The data are expressed as mean ± SEM. a-b-c are coronal sections; (a,b 400×; (b) 1000×.

Figure 8

Pattern of recombination in adult Creb1^D1Cre, CaMKIV^D1Cre and GR^D1Cre mice. Recombination is shown in striatum (a-f), hippocampus (g-l) and cortex (m-r). Recombination is followed by disappearance of CREB (a,b,g,h,m,n), CaMKIV (c,d,i,j,o,p) and GR proteins (e,f,k,l,q,r) in the respective mutants. All animals are homozygous for the respective loxP-modified allele in the case of Creb1^loxP, CaMKIV^loxP and GR^loxP mice. Control animals are devoid of the D1Cre transgene while mutant animals carry the D1Cre trangene, as indicated. The hippocampus CA2 field is indicated by arrowheads. The cortical layers are indicated. Magnification: (a-f) 200×; (g-l) 50×; (m-r) 54×.