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

Cork SC, Richards JE, Holt MK, Gribble FM, Reimann F, Trapp S - Mol Metab (2015)

Bottom Line: Large numbers of eYFP or tdRFP immunoreactive cells were found in the circumventricular organs, amygdala, hypothalamic nuclei and the ventrolateral medulla.However, tdRFP positive neurons were also found in areas without preproglucagon-neuronal projections like hippocampus and cortex.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.

View Article: PubMed Central - PubMed

Affiliation: Centre for Cardiovascular and Metabolic Neuroscience, Department of Neuroscience, Physiology & Pharmacology, University College London, London, WC1E 6BT, UK.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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fig6: 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.

Mentions: Expression of cre-recombinase in cells where the GLP-1R promoter is active enabled the functional manipulation of GLP-1R neurons in vivo. We injected EF1a-double floxed-hChR2(H134R)-EYFP AAV particles into the PVN to express ChR2-eYFP selectively in PVN GLP-1R neurons only (Figure 6A). Axons expressing ChR2-eYFP were primarily found in the caudal NTS (Figure 6B, C) and to a much smaller extent in the VLM (not shown).


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

Cork SC, Richards JE, Holt MK, Gribble FM, Reimann F, Trapp S - Mol Metab (2015)

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.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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fig6: 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.
Mentions: Expression of cre-recombinase in cells where the GLP-1R promoter is active enabled the functional manipulation of GLP-1R neurons in vivo. We injected EF1a-double floxed-hChR2(H134R)-EYFP AAV particles into the PVN to express ChR2-eYFP selectively in PVN GLP-1R neurons only (Figure 6A). Axons expressing ChR2-eYFP were primarily found in the caudal NTS (Figure 6B, C) and to a much smaller extent in the VLM (not shown).

Bottom Line: Large numbers of eYFP or tdRFP immunoreactive cells were found in the circumventricular organs, amygdala, hypothalamic nuclei and the ventrolateral medulla.However, tdRFP positive neurons were also found in areas without preproglucagon-neuronal projections like hippocampus and cortex.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.

View Article: PubMed Central - PubMed

Affiliation: Centre for Cardiovascular and Metabolic Neuroscience, Department of Neuroscience, Physiology & Pharmacology, University College London, London, WC1E 6BT, UK.

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.

No MeSH data available.


Related in: MedlinePlus