Distribution and characterisation of Glucagon-like peptide-1 receptor expressing cells in the mouse brain

Abstract

Objective: Although Glucagon-like peptide 1 is a key regulator of energy metabolism and food intake, the precise location of GLP-1 receptors and the physiological relevance of certain populations is debatable. This study investigated the novel GLP-1R-Cre mouse as a functional tool to address this question.

Methods: Mice expressing Cre-recombinase under the Glp1r promoter were crossed with either a ROSA26 eYFP or tdRFP reporter strain to identify GLP-1R expressing cells. Patch-clamp recordings were performed on tdRFP-positive neurons in acute coronal brain slices from adult mice and selective targeting of GLP-1R cells in vivo was achieved using viral gene delivery.

Results: Large numbers of eYFP or tdRFP immunoreactive cells were found in the circumventricular organs, amygdala, hypothalamic nuclei and the ventrolateral medulla. Smaller numbers were observed in the nucleus of the solitary tract and the thalamic paraventricular nucleus. However, tdRFP positive neurons were also found in areas without preproglucagon-neuronal projections like hippocampus and cortex. GLP-1R cells were not immunoreactive for GFAP or parvalbumin although some were catecholaminergic. GLP-1R expression was confirmed in whole-cell recordings from BNST, hippocampus and PVN, where 100 nM GLP-1 elicited a reversible inward current or depolarisation. Additionally, a unilateral stereotaxic injection of a cre-dependent AAV into the PVN demonstrated that tdRFP-positive cells express cre-recombinase facilitating virally-mediated eYFP expression.

Conclusions: This study is a comprehensive description and phenotypic analysis of GLP-1R expression in the mouse CNS. We demonstrate the power of combining the GLP-1R-CRE mouse with a virus to generate a selective molecular handle enabling future in vivo investigation as to their physiological importance.

Keywords: AP, area postrema; BNST, bed nucleus stria terminalis; Channelrhodopsin; DMH, dorsomedial nucleus of the hypothalamus; DMV, dorsal motor nucleus of the vagus; Electrophysiology; Ex-4, Exendin-4; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; GLP-1; GLP-1, Glucagon-like peptide-1; GLP-1R, Glucagon-like peptide-1 receptor; Glucagon-like peptide-1 receptor; NAc, nucleus accumbens; NTS, nucleus of the solitary tract; PARV, parvalbumin; PPG; PPG, preproglucagon; PVN, paraventricular nucleus of the hypothalamus; Preproglucagon; TH, tyrosine hydroxylase; VTA, ventral tegmental area; YFP, yellow fluorescent protein.

Figure 1

GLP-1 receptor expressing cells in the CNS. A, B. Demonstrates the reporter expression in the dorsal vagal complex of the eYFP and tdRFP mice. Expression was equivalent in the area postrema (AP), nucleus tractus solitarius (NTS) and dorsal motor nucleus of the vagus (DMV). High levels of reporter expression were observed in the area postrema (A, B) dorsal lateral septum, subfornical organ (C), ventrolateral hypothalamus, arcuate (D), hypothalamic paraventricular nucleus (E) superior colliculus and periaqueductal grey (F). Expression was also observed, albeit at lower levels, in the nucleus tractus solitarius (A, B), dorsomedial hypothalamus (D), hippocampus, geniculate, thalamic reticular formation and mammilliary nucleus (F). Scale bars: A, B, E = 200 μm. C, F = 1 mm. D = 500 μm. Abbr: CC: central canal, AP: area postrema, NTS: nucleus tractus solitarius, LSD: dorsal lateral septum, SFO: subfornical organ, DMH: dorsomedial hypothalamus, VMH: ventromedial hypothalamus, ARC: arcuate nucleus, DG: dentate gyrus, Gen: geniculate nucleus, SC: superior colliculus, PAG: periaqueductal grey, CoA: cortical amygdala, TRt: thalamic reticular formation, Mamm: mammillary nucleus, PVN: paraventricular nucleus, 3V: 3rd ventricle.

Figure 2

Diagrams of coronal sections showing the distribution of GLP-1R expressing cell bodies in the brains of GLP-1R-Cre mice. Filled circles represent the presence of YFP- or RFP-immunoreactive somata. The density of the filled circles indicates the relative density of the RFP-positive somata in each brain region. Drawings are based on the Paxinos Mouse Brain Atlas and numerical values next to each section indicate the rostro-caudal position in relation to Bregma. Abbreviations: PrL, prelimbic cortex; FrA, frontal association cortex; MO, medial orbital cortex; EPI, external plexiform layer; LS, lateral septum; AcbC, nucleus accumbens core; AcbS, nucleus accumbens shell; MS, medial septum nucleus; Cl, claustrum; Pir, piriform cortex; Cpu, caudate putamen; BNST-D/V, bed nucleus of the stria terminalis dorsal/ventral; PO, preoptic area; MPO, medial preoptic area; MnPO, median preoptic area; SFO, subfornical organ; Re, reuniens thalamic nucleus; PVN, paraventricular nucleus; LH, lateral hypothalamus; ARC, arcuate nucleus; Rt, reticular nucleus; PVT, thalamic paraventricular nucleus; VPM, ventral posteromedial thalamic nucleus; CeA, central amygdala; BLA, basolateral amygdala; DMH, dorsomedial hypothalamus; VMH, ventromedial hypothalamus; PAG, periaqueductal grey area; PH, posterior hypothalamus; PSTh, parasubthalamic nucleus; MM, mammillary nucleus; DG, dentate gyrus; DLG, dorsolateral geniculate nucleus; PGMC, pregeniculate nucleus magnocellular part; SCol, superior colliculus; VTA, ventral tegmental area; MG, medial geniculate nucleus; AP, area postrema; NTS, nucleus tractus solitarius; XII, hypoglossal nucleus; Ro, nucleus of roller; IRt, intermediate reticular nucleus; MdD, dorsal medullary reticular nucleus; RVLM, rostral ventrolateral medulla.

Figure 3

GLP-1 receptors in the meso-limbic system. Photomicrographs of YFP-immunoreactive cells in (A) cingulate cortex, (B) the nucleus accumbens, lateral septum, claustrum and cortex, (C) the bed nucleus of the stria terminals, (D) amygdala and ventral posteromedial thalamic nuclei, and (E) the dorsal lateral septum. Abbr: Cl: claustrum, CTX: cortex, ant comm: anterior commissure, CeA: central amygdala, LS: lateral septum, MeA: medial amygdala, VPM: ventral posteromedial thalamic nuclei. Scale bars: A, C, E = 200 μm; D = 300 μm; B = 1 mm.

Figure 4

Dual immunofluorescence of GLP-1R-eYFP expressing neurons and tyrosine hydroxylase (TH) expressing neurons throughout the brain. A. Only few TH neurons in the NTS were found to co-localise with eYFP expressing neurons. Conversely, almost all TH positive neurons in the AP co-expressed GLP-1R. B. Higher magnification image of boxed region in A. C. Around half of all TH positive neurons in the RVLM co-expressed GLP-1R, however the majority of GLP-1R neurons were TH-negative. D. A few TH positive neurons in the DMH were found to have eYFP staining, however the majority did not. E. Only a few TH positive neurons in the VTA expressed GLP-1R. Indeed the VTA contained very few GLP-1R positive somata. F–I. Scale bars in A, C, D and E = 200 μm. B, F, H and I = 20 μm. G = 50 μm. Arrow heads in B, F–I represent dual labelled neurons. * = YFP positive, TH negative neurons.

Figure 5

Dual immunofluorescence of GLP-1R-tdRFP expressing neurons and parvalbumin (PARV) expressing neurons (A–C) and GLP-1R-eYFP expressing cells and GFAP immunofluorescence (D–G). PARV positive neurons were present in the hippocampus (A), cingulate (B), piriform (C) and thalamic regions (not shown). No tdRFP-positive cell was found to co-express PARV in any region, although the populations did intermix. Similarly, GFAP was expressed throughout the CNS, and although intermixed with eYFP expression, no co-expression was identified. Scale bars in A-F = 200 μm; A’–C’ = 50 μm and G = 500 μm.

Figure 6

Unilateral microinjection of a cre-dependent AAV expressing ChannelRhodopsin-2 (AAV-flexswitch-ChR2-eYFP) in the hypothalamic PVN. A. ChR2 expression (green) is confined to the boundaries of the PVN and expressed only in cells which co-express tdRFP (red). (A’) Higher magnification image of ChR2 expressing tdRFP positive neurons in the PVN. This study confirms the presence of active cre-recombinase in these cells and provides proof-of-principle evidence that these mice can be used to virally target GLP-1R expressing cells in discrete nuclei. B, C. ChR2-eYFP expressing axons of virally-targeted PVN neurons are distributed primarily in the caudal NTS up to the rostral end of the AP. Scale bar A = 100 μm. A’ = 10 μm. B, C = 200 μm. 3V = 3rd ventricle.

Figure 7

GLP-1 elicits electrical responses in GLP-1R-Cre positive cells in various brain regions. A, B. Voltage-clamp recording from an RFP-positive PVN neuron. Bath application of 100 nM GLP-1 elicited a small inward current at a holding potential of −50 mV accompanied by an increase in whole-cell conductance. These effects reversed upon washout of GLP-1 from the bath solution. C, D. Similarly, in a BNST neuron bath application of 100 nM GLP-1 elicits an inward current accompanied by an increase in conductance. E. Voltage clamp recordings in RFP-positive hippocampal neurons revealed that GLP-1 either elicits an inward current (left panel) as in PVN and BNST, or an outward current. F. Mean data from current clamp recordings (left panel) demonstrating that GLP-1 causes a 10–15 mV depolarisation in most neurons tested, and a hyperpolarisation in individual hippocampal cells. Mean data from voltage clamp recordings in the right panel demonstrate the inward current of 10–20 pA amplitude elicited by 100 nM GLP-1 underlying the depolarisation in current clamp. n-numbers are given below the bars. **: p < 0.01.