The generation of knock-in mice expressing fluorescently tagged galanin receptors 1 and 2

Abstract

The neuropeptide galanin has diverse roles in the central and peripheral nervous systems, by activating the G protein-coupled receptors Gal1, Gal2 and the less studied Gal3 (GalR1–3 gene products). There is a wealth of data on expression of Gal1–3 at the mRNA level, but not at the protein level due to the lack of specificity of currently available antibodies. Here we report the generation of knock-in mice expressing Gal1 or Gal2 receptor fluorescently tagged at the C-terminus with, respectively, mCherry or hrGFP (humanized Renilla green fluorescent protein). In dorsal root ganglia (DRG) neurons expressing the highest levels of Gal1-mCherry, localization to the somatic cell membrane was detected by live-cell fluorescence and immunohistochemistry, and that fluorescence decreased upon addition of galanin. In spinal cord, abundant Gal1-mCherry immunoreactive processes were detected in the superficial layers of the dorsal horn, and highly expressing intrinsic neurons of the lamina III/IV border showed both somatic cell membrane localization and outward transport of receptor from the cell body, detected as puncta within cell processes. In brain, high levels of Gal1-mCherry immunofluorescence were detected within thalamus, hypothalamus and amygdala, with a high density of nerve endings in the external zone of the median eminence, and regions with lesser immunoreactivity included the dorsal raphe nucleus. Gal2-hrGFP mRNA was detected in DRG, but live-cell fluorescence was at the limits of detection, drawing attention to both the much lower mRNA expression than to Gal1 in mice and the previously unrecognized potential for translational control by upstream open reading frames (uORFs).

Localization of the cannabinoid type‐1 receptor in subcellular astrocyte compartments of mutant mouse hippocampus

“…However, no information is available to date whether the CB 1 receptor localizes in astroglial mitochondria as it does in mitochondria of hippocampal GABAergic and glutamatergic neurons (Bénard et al, ; Hebert‐Chatelain et al, a,b, ). In order to address this, we used conditional CB 1 receptor rescue mice re‐expressing the CB 1 receptor exclusively in astrocytic GFAP expressing cells (GFAP‐ CB 1 ‐RS), as well as CB 1 ‐WT and CB 1 ‐KO mice expressing hrGFP (De Francesco et al, ; Hadaczek et al, ; Kerr et al, ; Navarro‐Galve et al, ; Ward & Cormier, ) under the control of the GFAP promoter (GFAPhrGFP‐ CB 1 ‐WT and GFAPhrGFP‐ CB 1 ‐KO, respectively). As a first step, we determined the CB 1 receptor expression and distribution in the conditional mutant mice in order to draw the level of agreement with the CB 1 receptor expression pattern in the CB 1 ‐WT mice.…”

Distribution of galanin receptor 2b neurons and interaction with galanin in the zebrafish central nervous system

“…Semi-quantitative reverse transcription PCR (qRT-PCR) analysis revealed that galr2b expression was first detected at 24 hpf, and its expression was maintained in the brain and spinal cord through adulthood (data not shown). Restricted expression of galr2a and galr2b in the zebrafish CNS is very similar to mammalian galr2, indicating that zebrafish galr2a and galr2b are orthologs of mammalian galr2 [2,3,5,6,14]. In addition, widespread expression of zebrafish galr2b in the brain and spinal cord suggests that galr2b plays a role as a main receptor for galanin neuropeptides in CNS.…”

“…In mammals, galr2 is widely expressed in the brain, including the olfactory system, preoptic area, hypothalamus, hippocampus, amygdala, pituitary gland, and brainstem [2,3,5,6,14]. To investigate the expression pattern of galr2a and galr2b in zebrafish, we first performed whole-mount in situ RNA hybridization with galr2a and galr2b.…”

“…Unlike GALR1, GALR2 is widely distributed in almost all tissues, including the brain, such as the olfactory system, preoptic area, hypothalamus, hippocampus, amygdala, pituitary gland, and brainstem [2,3,5,6,14]. GALR3 showed limited expression in CNS but is relatively abundant in peripheral tissues [12].…”

Cell-type specific expression and behavioral impact of galanin and GalR1 in the locus coeruleus during opioid withdrawal

“…Brains were postfixed in 4% PFA overnight and transferred to 30% sucrose in PBS for ~48 h. Brains were frozen in chilled isopentane and sectioned on a cryostat at 40 μm increments. Sections from the LC and paraventricular nucleus of the thalamus (PVT), which strongly expresses GalR1, 27 were stained using an adapted protocol for mCherry detection in mu opioid receptor-mCherry tagged mice. 28 See supplement for protocol ( Methods S1 ).…”