Inducible, 5-HT neuron-specific recombination in TPH2-tTA/Ptet-Cre/R26R mice.

<p>Adult mice (P90) were sacrificed and analyzed with X-Gal staining and IHC. (A–L) X-Gal staining of coronal sections of the brain stem and midbrain. (M–X) Dual-label fluorescence IHC with βgal and 5-HT neuron-specific TPH2 antibodies. Co-labelling indicates 5-HT neuron-specific recombination. Without Dox administration (−Dox), X-Gal staining showed intense βgal activity in all areas of the raphe nuclei where 5-HT neurons are located (A,E,I). Dual-label IHC confirmed that βgal expression occurs tissue-specifically in virtually all 5-HT neurons (M,Q,U). Of note, extraserotonergic βgal expression was found in the median raphe nuclei of untreated mice (U, arrows). (B,F,J,N,R,V) With permanent Dox administration (+Dox), Cre-induced recombination and consequently βgal expression did only occur in a minor fraction of 5-HT neurons of TPH2-tTA/Ptet-Cre/R26R mice (V, arrow). To assess inducibility of Cre-mediated recombination, Dox was either administered during embryonic development until P0 (+Dox –P0; C,G,K,O,S,W) or during embryonic and postnatal development until P60 (+Dox –P60; D,H,L,P,T,X), when Dox was withdrawn until sacrifice at P90. X-Gal staining showed equally strong βgal activity in the raphe nuclei of TPH2-tTA/Ptet-Cre/R26R mice which were induced at P0 (C,G,K) or at P60 (D,H,L). As shown with IHC, recombination occurred tissue-specifically in the majority of 5-HT neurons, irrespective of the time point of induction at P0 (O,S,W) or P60 (P,T,X). The efficiency of tissue-specific recombination was indistinguishable from mice which never received Dox (M,Q,U) which proves that induction of Cre-mediated recombination after Dox suppression is equally efficient. Of note, when Cre recombination was suppressed with Dox until P0, extraserotonergic recombination in the median raphe nuclei could not be detected anymore (W,X). Grey arrowheads indicate scarce non-recombined, βgal-negative TPH2+5-HT neurons. Scale bars: 100 µm.