Individual arcuate nucleus proopiomelanocortin neurons project to select target sites

Abstract

Proopiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus (ARH) are a diverse group of neurons that project widely to different brain regions. It is unknown how this small population of neurons organizes its efferent projections. In this study, we hypothesized that individual ARH POMC neurons exclusively innervate select target regions. To investigate this hypothesis, we first verified that only a fraction of ARH POMC neurons innervate the lateral hypothalamus (LH), the paraventricular nucleus of the hypothalamus (PVN), the periaqueductal gray (PAG), or the ventral tegmental area (VTA) using the retrograde tracer cholera toxin B (CTB). Next, two versions of CTB conjugated to distinct fluorophores were injected bilaterally into two of the regions such that PVN and VTA, PAG and VTA, or LH and PVN received tracers simultaneously. These pairs of target sites were chosen based on function and location. Few individual ARH POMC neurons projected to two brain regions at once, suggesting that there are ARH POMC neuron subpopulations organized by their efferent projections. We also investigated whether increasing the activity of POMC neurons could increase the number of ARH POMC neurons labeled with CTB, implying an increase in new synaptic connections to downstream regions. However, chemogenetic enhancement of POMC neuron activity did not increase retrograde tracing of CTB back to ARH POMC neurons from either the LH, PVN, or VTA. Overall, subpopulations of ARH POMC neurons with distinct efferent projections may serve as a way for the POMC population to organize its many functions.

Keywords: Gq DREADD; POMC; efferent projections; hypothalamus; retrograde labeling.

Figure 1.

Retrograde tracing from target regions yields a small percentage of CTB-labeled ARH POMC neurons. Representative images of CTB injections into the VTA (A) at approximately −2.92 mm from the bregma, PVN (B) at approximately −0.46 mm from the bregma, ventrolateral PAG (C) at approximately −4.72 mm from bregma, and LH (D) at approximately −1.22 mm from the bregma. All estimates from the bregma are based on Paxinos and Franklin stereotaxic coordinates (43). All scale bars are 1 mm. E: bar graph showing the percentage of labeled POMC cell bodies containing CTB from individual target regions. F: bar graph showing the percentage of labeled POMC cell bodies containing CTB for control experiments in the VTA. 200 nL CTB data are the same data as in E. Each point represents colocalization from a single animal and data are shown as means ± SE. ARH, arcuate nucleus of the hypothalamus; CTB, cholera toxin B; FS, FluoSpheres; LH, lateral hypothalamus; PAG, periaqueductal gray; POMC, proopiomelanocortin; PVN, paraventricular nucleus of the hypothalamus; VTA, ventral tegmental area; ns, not significant.

Figure 2.

Dual retrograde labeling of CTB conjugates in ARH POMC from the target regions is negligible. A: schematic of experimental procedure depicting CTB injection of two different CTB conjugates into two different brain regions in Pomc^dsRED mice followed by 10 days of incubation. After the incubation period, brains were sliced and imaged in the area of the arcuate nucleus of the hypothalamus, where POMC neurons expressed a fluorescent tag. B: images of CTB and POMC^dsRED cells in the ARH from a brain injected with CTB-647 into the PVN and CTB-488 into the VTA. Scale bar is 50 µm. Slice is approximately −1.94 mm from the bregma based on Paxinos and Franklin stereotaxic coordinates (43). C: boxes 1 and 2 in B showing CTB-647 and CTB-488 (top) with respective merged images with Pomc^dsRED cells (bottom). White arrows, cells that contain CTB-647 and Pomc^DsRed; yellow arrows, POMC cells with CTB-488; magenta arrow, a POMC cell containing both CTB conjugates. Scale bar is 50 µm. D: bar graph showing the percentage of labeled POMC cell bodies containing two conjugates of CTB, one conjugate from each target region is indicated. Each point represents colocalization from a single animal and data are shown as means ± SE. The mouse and mouse brain slice schematics in A were modified from clipart-library.com and Motifolio with a user license, respectively. ARH, arcuate nucleus of the hypothalamus; CTB, cholera toxin B; LH, lateral hypothalamus; PAG, periaqueductal gray; POMC, proopiomelanocortin; Pomc^dsRED, POMC cells expressing Discosoma red; PVN, paraventricular nucleus of the hypothalamus; VTA, ventral tegmental area.

Figure 3.

Gq DREADDs activate POMC neurons in vitro and in vivo. A: representative trace of GCaMP6f fluorescent activity in an ARH POMC neuron virally expressing AAV-hM3Dq (Gq DREADDs) in a Pomc^Cre/+mouse after 10 µM CNO application. B: GCaMP6f fluorescence increased soon after CNO application and decreased within 10 min of continuous CNO application but remained significantly above the baseline fluorescence. Each point represents a single POMC cell. Number of animals = 2. Analyzed with one-way ANOVA with Tukey’s multiple comparisons. Cumulative food intake during a 2-h refeed after an overnight fast was decreased in POMC cre-ER^{T2 +} mice compared with POMC cre-ER^T2− female (C) and male (D) mice. Analyzed with unpaired t test, *P > 0.05, ***P > 0.001. E: immunohistochemical detection of HA-tagged Gq DREADDs (green) in ARH Pomc-cre:ER^T2+; Pomc^dsRED neurons (red). No detectable Gq DREADD expression was observed in tissue from POMC cre-ER^T2− mice (left), but Gq DREADDs were detected in tissue from Pomc^Cre/+ mice (right). Scale bar is 50 µm. Slice is approximately −1.58 mm from the bregma based on Paxinos and Franklin stereotaxic coordinates (43). AAV, adeno-associated virus; ARH, arcuate nucleus of the hypothalamus; CNO, clozapine-n-oxide; HA, hemagglutinin; POMC, proopiomelanocortin; Pomc^dsRED, POMC cells expressing Discosoma red.

Figure 4.

Activation of ARH POMC neurons with Gq DREADDs does not increase colocalization with CTB injected into target regions. A: schematic of experimental procedure depicting 5 days of tamoxifen injections to express tamoxifen-inducible cre in POMC neurons to cause the expression of Gq DREADDs in POMC neurons. At least 1 wk later, mice received CNO 90 min before CTB stereotaxic injection into two brain regions with two different conjugates of CTB. After surgery, mice received CNO twice per day for 10 days. Tissue was then collected and imaged in the region of the arcuate nucleus. Percent colocation of CTB in cell bodies of ARH POMC neurons from brains injected in either the VTA (B), LH (C), or PAG (D) was not different between Pomc-cre:ER^T2+ and Pomc-cre:ER^T2− mice treated with CNO. Each point represents colocalization from a single animal and data are shown as means ± SE. The mouse and mouse brain slice schematics in A were modified from clipart-library.com and Motifolio with a user license, respectively. ARH, arcuate nucleus of the hypothalamus; CNO, clozapine-n-oxide; CTB, cholera toxin B; POMC, proopiomelanocortin; LH, lateral hypothalamus; PAG, periaqueductal gray; POMC, proopiomelanocortin; VTA, ventral tegmental area.