Volume 27, Issue 5, Pages 1327-1333.e3 (April 2019) LOVIT Is a Putative Vesicular Histamine Transporter Required in Drosophila for Vision  Ying Xu, Tao Wang  Cell Reports  Volume 27, Issue 5, Pages 1327-1333.e3 (April 2019) DOI: 10.1016/j.celrep.2019.04.024 Copyright © 2019 The Authors Terms and Conditions

Cell Reports 2019 27, 1327-1333.e3DOI: (10.1016/j.celrep.2019.04.024) Copyright © 2019 The Authors Terms and Conditions

Figure 1 CG45782 Is Required in Photoreceptors for Normal Visual Responses (A–C) ERGs recorded from flies expressing various UAS-CG45782 RNAi transgenes (CG45782RNAi1, CG45782RNAi2, and CG45782RNAi3: 38367, 40568, and 61340 from the Bloomington Stock Center, respectively) under the control of (A) GMR-Gal4 driver specific for compound eyes (GMR-Gal4/+;UAS-CG45782RNAi/+), (B) neuronal driver elav-Gal4 (elav-Gal4/+;UAS-CG45782RNAi/+), and (C) glial-specific driver repo-Gal4 (repo-Gal4/UAS-CG45782RNAi). Arrowheads indicate ON and OFF transients. Flies were dark adapted for 1 min and subsequently exposed to a 5-s pulse of orange light. (D) Phototactic behavior of flies corresponding to those in (A)–(C) compared with the control flies (w1118 and GMR-Gal4 flies). Three repeats were made for each group, and each group contained ≥20 flies. (E and F) ERG recordings (E) and phototatic behaviors (F) of GMR-Gal4, GMR > CG45782RNAi1, rdhB-lovit rescue (GMR-Gal4/+; UAS-CG45782RNAi1/rdhB-lovit) and ninaE-lovit rescue (GMR-Gal4/+; UAS-CG45782RNAi1/ninaE-lovit) flies. (G) ERG recordings from control (w1118) and lovit1 flies. (H) Phototactic behaviors of 2-day-old flies in (G). Cell Reports 2019 27, 1327-1333.e3DOI: (10.1016/j.celrep.2019.04.024) Copyright © 2019 The Authors Terms and Conditions

Figure 2 LOVIT Is a Synaptic Vesicular Transporter (A–C) Whole-mounted fly heads were labeled with anti-LOVIT (green), anti-24B10 (A and B, red; expressed in photoreceptors), and anti-Ebony (C, blue; expressed in lamina epithelial glia). A longitudinal section (B) of the distal medulla neuropil and a cross-section (C) of the lamina are shown (Br, brain; La, lamina; Me, medulla; Re, retina). (D and E) The co-localization between LOVIT (red) and (D) photoreceptor cell membrane marker Na K-ATPase (green) or (E) mitochondria marker CoVI (green). Scale bars: 200 μm in (A); 25 μm in (B); 5 μm in (C)–(E). Cell Reports 2019 27, 1327-1333.e3DOI: (10.1016/j.celrep.2019.04.024) Copyright © 2019 The Authors Terms and Conditions

Figure 3 Loss of LOVIT Affects the Histamine Distribution and Content In Vivo (A and B) Cryosections of heads were stained for histamine (HA) together with (A) CSP (synaptic vesicle marker) and (B) 24B10 (photoreceptor marker) in control (w1118) and lovit1 flies. Sections are parallel to photoreceptor axons. Scale bars: 50 μm. (C) Cryosections of lamina neuropil from w1118 and lovit1 flies were immunolabeled with CSP and histamine. Sections are across photoreceptor axons. Scale bar: 10 μm. (D) Average red fluorescence (histamine signal) intensity ratio between the entire lamina and retina immunolabeled layers. (E) Head histamine contents in 4-day-old control (w1118), lovit1, and carT1 flies. Each sample contained 50 Drosophila heads, and the mean values from three samples were calculated. Cell Reports 2019 27, 1327-1333.e3DOI: (10.1016/j.celrep.2019.04.024) Copyright © 2019 The Authors Terms and Conditions

Figure 4 The Proposed Role of LOVIT Transporter in Photoreceptor Histamine-Recycling Pathways In photoreceptor cells (PRs), primary neurotransmitter histamine is initially synthesized by histidine decarboxylase (Hdc) (Burg et al., 1993) and loaded into the synaptic vesicles by a LOVIT-dependent mechanism. Upon light stimulation, photoreceptors release vesicular histamine to activate histamine-gated chloride channels (HisClA) on postsynaptic large monopolar cells (LMCs) (Pantazis et al., 2008). Histamine in the synaptic cleft is quickly taken up into epithelial glia and inactivated to carcinine by Ebony, which catalyzes the conjugation of histamine and β-alanine (Borycz et al., 2002). Carcinine is subsequently transported back to PRs via CarT (Chaturvedi et al., 2016; Stenesen et al., 2015; Xu et al., 2015), After being deconjugated from carcinine by Tan in the PRs, histamine is repackaged into synaptic vesicles through LOVIT to maintain the neurotransmitter pool for vesicular release. Cell Reports 2019 27, 1327-1333.e3DOI: (10.1016/j.celrep.2019.04.024) Copyright © 2019 The Authors Terms and Conditions