Fig. 1: Generation of mice with selective disruption of Hcrtr2 in dopamine neurons.

a Homologous recombination of the Hcrtr2 gene with the targeting vector creates the Hcrtr2-flox allele. The 5’loxP site was inserted in Exon1 5’-untranslated-region. 3’loxP was inserted within Intron1. In Dat-IRES-Cre-expressing neurons, CRE/lox recombination creates the Hcrtr2^del-GFP allele, with genomic deletion of DNA encoding 74 aa, encompassing HCRTR2 signal peptide, N-terminal domain, and most transmembrane region 1, and replacement of the Hcrtr2 coding sequence with a promoterless Gfp cassette. The endogenous Hcrtr2 promoter now drives Gfp instead of Hcrtr2 in DA neurons, marking cells having lost Hcrtr2 expression with GFP. TSS, Transcription start site. pA, polyadenylation signal. Chrm9, Chromosome 9. Hcrtr2^flox is Hcrtr2^tm1.1Ava (MGI:5637402), and Hcrtr2^KO-Gfp is Hcrtr2^tm1.2Ava (MGI: 5637403). b Schematic representation of DA^OxR2-KO mice. c Evidence of tissue-specific genomic recombination. DNA was isolated from various tissues and subject to PCR. Unrecombined HcrtR2-flox diagnostic band (Flox, 2,145 bp) is observed in cortex, TMN, VTA and ear from DA^OxR2-CT and DA^OxR2-KO mice, while the knockout diagnostic band (KO; 792 bp) is only observed in VTA of DA^OxR2-KO mice. The 792 bp recombined fragment was fully sequenced, confirming exact recombination event (n = 2). d GFP and Tyrosine Hydroxylase (TH) immunostaining demonstrates efficiency and specificity of Hcrtr2 Exon1 deletion in DA cells of the ventral midbrain (−2.92 to −3.88 mm from bregma) of DA^OxR2-KO mice. (Left) Quantification in several midbrain subregions. (Right) Overall penetrance (% of TH⁺ neurons co-expressing GFP) was 87.2 ± 1.5% (n = 12 sections, 2 mice). Specificity (% of GFP⁺ cells co-expressing TH) was 73.2 ± 2.4% (n = 12 sections, 2 mice). VTA, ventral tegmental area; PBP, parabrachial pigmented nucleus; PN paranigral nucleus, SNc substantia nigra pars compacta. e Representative confocal images depicting TH and GFP co-localization in ventral midbrain of DA^OxR2-KO, but not DA^OxR2-CT, mice. Coronal 20-µm brain sections at −3.08 mm from bregma. Scale bar: low magnification, 100 µm; high magnification, 20 µm. f Electrophysiological demonstration that Hcrtr2 Cre/lox recombination inactivates HCRTR2. (Left) Voltage trace recordings from putative histaminergic neurons in TMN of C57BL/6 J, Hcrtr2^flox/flox, and Hcrtr2^del/del mice. Cells were held at −50 mV in current-clamp mode. Voltage traces represent 5-min continuous recordings, before, during (green horizontal line), and after OXB-AL (200 nM) application. OXB-AL triggers a long train of action potentials in neurons from C57BL/6J and Hcrtr2^flox/flox, but not Hcrtr2^del/del, mice. (Right) Percentage of neurons responding to different treatments in each genotype. Treatments were as follows: OXB (100 nM); OXB-AL (200 nM); TCS (5 μM) + OXB (100 nM); TCS (10 μM) + OXB (100 nM); TCS (10 μM) + OXB-AL (200 nM). Number of neurons per treatment: C57BL/6J: 19/17/17/8/5; Hcrtr2^flox/flox: 4/3/6/5/6; Hcrtr2^del/del: 10/11/7/5/5). OXB-AL: [Ala¹¹, D-Leu¹⁵]-Orexin B; TCS: TCS-OX2-029; TMN: tuberomammillary nucleus.