Identification of neuronal subpopulations that project from hypothalamus to both liver and adipose tissue polysynaptically

Abstract

The autonomic nervous system regulates fuel availability and energy storage in the liver, adipose tissue, and other organs; however, the molecular components of this neural circuit are poorly understood. We sought to identify neural populations that project from the CNS indirectly through multisynaptic pathways to liver and epididymal white fat in mice using pseudorabies virus strains expressing different reporters together with BAC transgenesis and immunohistochemistry. Neurons common to both circuits were identified in subpopulations of the paraventricular nucleus of the hypothalamus (PVH) by double labeling with markers expressed in viruses injected in both sites. The lateral hypothalamus and arcuate nucleus of the hypothalamus and brainstem regions (nucleus of the solitary tract and A5 region) also project to both tissues but are labeled at later times. Connections from these same sites to the PVH were evident after direct injection of virus into the PVH, suggesting that these regions lie upstream of the PVH in a common pathway to liver and adipose tissue (two metabolically active organs). These common populations of brainstem and hypothalamic neurons express neuropeptide Y and proopiomelanocortin in the arcuate nucleus, melanin-concentrating hormone, and orexin in the lateral hypothalamus and in the corticotrophin-releasing hormone and oxytocin in the PVH. The delineation of this circuitry will facilitate a functional analysis of the possible role of these potential command-like neurons to modulate autonomic outflow and coordinate metabolic responses in liver and adipose tissue.

Fig. 1.

Major sites of PRV152 infection after injection into the liver. C57Bl6 mice were killed 3–8 days after injection of 1.7 × 10⁶ pfu of PRV152 into the left lobe of the liver (n = 3 per day for days 3–7; n = 7 for day 8). Viral infection was visualized by immunofluorescence against GFP. The columns show representative areas of the five phases of infection described in Table S1. Phase I infection shows GFP expression in the spinal cord. Phase II infections include GFP expression in the brainstem dorsal motor 10N, PVH, and parasubthalamic nucleus (PSTh). Phase III infections include GFP immunofluorescence in the NTS, LH, and ventral tegmental area (VTA). Phase IV infections include GFP expression in the area postrema (AP) and A5 region and the central nucleus of the amygdala (Ce). Phase V infection shows GFP immunofluorescence in the M1 region of the cortex. (Scale bar, 200 μm.)

Fig. 2.

Major neuronal populations common to the outflow tracts to liver and eWAT. PRV infection of C57Bl6 mice after dual injection with GFP-expressing PRV152 into eWAT and RFP-expressing PRV614 into liver. Dual-labeled neurons, common to both liver and eWAT innervation, are present initially in the PVH followed by the NTS, A5 region, LH, and ARC. (A) Viral infection within the PVH. (B) Viral infection within the NTS. (C) Viral infection within the A5 region of the brainstem. (D) Viral infection within the LH. (E) Viral infection within the ARC. (Scale bar, 200 μm for left panels and 50 μm for right panels.) (F) Number and average percentage ± SEM of coexpression for PRV-infected neuronal populations after eWAT and liver injection. Number of positive neurons per region: +, 1–10; ++, 11–20; +++, 21–30; ++++, 31–50; +++++, >50 (n = 4–5 for each region).

Fig. 3.

Characterization of early PVH neuronal populations infected after PRV injection into eWAT and liver. Dual-labeled neurons, common to liver and eWAT innervation, express OT and CRH. Mice were killed 4–5 days after administration of PRV152 into eWAT and PRVBaBlu into liver, the earliest point at which infected neurons were detected in the PVH, and IHC for PVH-expressed neuropeptides was performed. Neurons with dual PRV infection and peptide expression appeared white. Arrows indicate triple-labeled neurons. (A) PRV eWAT infection (green), PRV liver infection (red), and AVP immunofluorescence (blue). (Scale bar, 200 μm.) (B) PRV eWAT infection (green), PRV liver infection (red), and prothyrotrophin-releasing hormone (proTRH) immunofluorescence (blue). (Scale bar, 200 μm.) (C and D) PRV eWAT infection (green), PRV liver infection (red), and CRH immunofluorescence (blue). (Scale bar, 200 μm on the left and 50 μm on the right.) (E and F) PRV eWAT infection (green), PRV liver infection (red), and OT immunofluorescence (blue). (Scale bar, 200 μm on the left and 50 μm on the right.) (G) Number and average percentage ± SEM of coexpression for each PVH neuropeptide for PRV-infected neuronal populations after eWAT and liver injection. Number of positive neurons per region: +, 1–10; ++, 11–20; +++, 21–30; ++++, 31–50; +++++, >50 (n = 3–4 for each region).

Fig. 4.

Involvement of LH MCH and ORX neuronal populations in output pathways to liver and eWAT. Both MCH and ORX neurons in the LH are involved in the outflow circuits to liver and adipose tissue. (A) Colocalization of liver PRV infection (red) and MCH neurons (green). Merged photomicrographs show colocalization of liver PRV infection in a subpopulation of MCH neurons (yellow, indicated). (Scale bar, 50 μm.) (B) Colocalization of ORX neurons (green) and liver PRV infection (red). Liver viral infection and ORX immunoreactivity were observed in a subpopulation of ORX neurons (yellow, indicated). (Scale bar, 50 μm.) (C) Localization of MCH neurons (green) and eWAT PRV infection (red). (Scale bar, 50 μm.) (D) Localization of ORX neurons (green) and eWAT PRV infection (red). Colocalization of eWAT PRV infection and ORX immunoreactivity was observed in a subpopulation of ORX neurons. (Scale bar, 50 μm.) (E) Number and average percentage ± SEM of coexpression for each LH peptide for PRV-infected neuronal populations after eWAT and liver injection. Number of positive neurons per region: +, 1–10; ++, 11–20; +++, 21–30; ++++, 31–50; +++++, >50 (n = 4 for each region).

Fig. 5.

Involvement of NPY and POMC brainstem neuronal populations in output pathways to liver and eWAT. Brainstem NPY neurons contribute to the efferent pathways to both liver and eWAT, but POMC neurons are only involved in the outflow circuit to adipose tissue. (A) Colocalization of NPY (green) and PRV infection (red) in the NTS after injection of PRV614 into the liver of NPY–GFP mice. An overlay of the GFP and PRV immunofluorescence images indicates colocalization of GFP expression and PRV infection (yellow, marked). (Scale bar, 50 μm.) (B) Colocalization of NPY (green) and PRV infection (red) in the NTS after injection of PRV614 into the eWAT of NPY–GFP mice. An overlay of the GFP and PRV immunofluorescence images indicates colocalization of GFP expression and PRV infection (yellow, marked). (Scale bar, 50 μm.) (C) Colocalization of POMC–GFP expression (green) and eWAT PRV infection (red) is observed. (Scale bar, 50 μm.) (D) Number and average percentage ± SEM of coexpression for each brainstem peptide for PRV-infected neuronal populations after eWAT and liver injection are shown. Number of positive neurons per region: +, 1–10; ++, 11–20; +++, 21–30; ++++, 31–50; +++++, >50 (n = 4 for each region).

Fig. 6.

Involvement of ARC NPY and POMC neuronal populations in output pathways to liver and eWAT. Only ARC POMC neurons are involved in the output pathways to both liver and eWAT with no evidence of ARC NPY neuronal involvement. No evidence of colocalization of hypothalamic NPY neurons (green) and PRV infection (red) after injection of PRV614 into (A) the liver or (B) eWAT of NPY–GFP mice. (Scale bar, 200 μm.) Colocalization of ARC POMC neurons (green) and PRV infection (red) after injection of PRV614 into (C) the liver or (D) eWAT of POMC–GFP mice. (Scale bar, 50 μm.) (E) Number and percentage ± SEM of coexpression for each ARC neuropeptide for PRV-infected neuronal populations after eWAT and liver injection. Number of positive neurons per region: +, 1–10; ++, 11–20; +++, 21–30; ++++, 31–50; +++++, >50 (n = 4 for each region).