A genetic approach to access serotonin neurons for in vivo and in vitro studies

Abstract

Serotonin (5HT) is a critical modulator of neural circuits that support diverse behaviors and physiological processes, and multiple lines of evidence implicate abnormal serotonergic signaling in psychiatric pathogenesis. The significance of 5HT underscores the importance of elucidating the molecular pathways involved in serotonergic system development, function, and plasticity. However, these mechanisms remain poorly defined, owing largely to the difficulty of accessing 5HT neurons for experimental manipulation. To address this methodological deficiency, we present a transgenic route to selectively alter 5HT neuron gene expression. This approach is based on the ability of a Pet-1 enhancer region to direct reliable 5HT neuron-specific transgene expression in the CNS. Its versatility is illustrated with several transgenic mouse lines, each of which provides a tool for 5HT neuron studies. Two lines allow Cre-mediated recombination at different stages of 5HT neuron development. A third line in which 5HT neurons are marked with yellow fluorescent protein will have numerous applications, including their electrophysiological characterization. To demonstrate this application, we have characterized active and passive membrane properties of midbrain reticular 5HT neurons, which heretofore have not been reported to our knowledge. A fourth line in which Pet-1 loss of function is rescued by expression of a Pet-1 transgene demonstrates biologically relevant levels of transgene expression and offers a route for investigating serotonergic protein structure and function in a behaving animal. These findings establish a straightforward and reliable approach for developing an array of tools for in vivo and in vitro studies of 5HT neurons.

Fig. 1.

ePet-Cre. (A) Whole-mount X-gal stain showing LacZ expression directed by ePet-Cre in adult R26R^ePet-Cre brain. Black arrowheads, dorsal and median raphe. Asterisk, ventral medullary raphe. (B-I) ePet-Cre activity is restricted to developing and adult 5-HT neurons. Colocalization (D and G)of TPH (B and E) (FITC, anti-TPH 1:200) and β-gal (C and F) (Texas red, anti-β-gal 1:5,000) in B7 dorsal raphe nucleus (B-D) and B2 raphe obscurus nucleus (E-G). (H and I) Colocalization of Cre recombinase (FITC, anti-Cre recombinase 1:100) with 5-HT (Texas red, anti-5-HT 1:10,000) in rostral (H) and caudal (I) E12 hindbrain. (Scale bars: 800 μm, A;50 μm, B-I.)

Fig. 2.

ePet-Cre-2 directs delayed recombination in a subset of 5HT neurons. (A-F) X-gal staining with neutral red counterstain of ROSA R26R^ePet-Cre embryos in E15 rostral (A) and E15 caudal (B) or ROSA R26R^ePet-Cre-2 in E16 rostral (C), E16 caudal (D), E18 rostral (E), and E18 caudal (F) hindbrain. The asterisk in D shows small numbers of LacZ-positive cells at E16 in caudal ROSA R26R^ePet-Cre-2 hindbrain. (G) Coimmunostaining with anti-TPH (FITC, anti-TPH 1:200) and anti-β-gal (Texas red, anti-β-gal 1:5,000) shows mosaic pattern of recombination directed by ePet-Cre-2 in the P60 adult. (H) High magnification of boxed area in G. (Scale bars: 100 μm, A-F;50 μm, G; and 25 μm I.)

Fig. 3.

ePet-EYFP. (A) EYFP fluorescence in midbrain B7 dorsal raphe. (B-D) Colocalization (D) of TPH (B) and EYFP fluorescence (C) and in ventral medullary B1 raphe pallidus. (E) Colocalization of EYFP fluorescence with TPH (Texas red, anti-TPH 1:200) in lateral B9 5HT neurons. (F) Confocal image of a dissociated EYFP+ 5HT neuron in culture. (Scale bars: 250 μm, A;20 μm, B-D;50 μm, C; and 5 μm, D.)

Fig. 4.

Electrophysiological recordings of EYFP+ 5HT neurons in B9. (A) Responses of an EYFP+ B9 neuron to 40-pA depolarizing and hyperpolarizing current steps at -68 mV. The depolarizing current step evoked an overshooting action potential with a pronounced afterhyperpolarization. (B) Responses from the same B9 neuron to 60-, 80-, and 110-pA step depolarizations from -72 mV. (C) Plot of instantaneous firing frequency (reciprocal of the first interspike interval) versus current step amplitude. (D) Plot of spike frequency adaptation in a EYFP+ B9 neuron during a response to a 250-pA step depolarization from -60 mV (voltage response shown in Inset).

Fig. 5.

ePet directed rescue of Pet-1 loss of function. 5HT neurons in the B7 dorsal raphe were identified by immunostaining of coronal sections with anti-TPH antisera (diaminobenzidine, anti-TPH, 1:200) in +/+ (A), Pet-1^-/- (B), and Pet-1^{-/-,ePet-Pet/+} (C) transgenic mice. (Scale bar: 100 μm.)