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. 2021 Dec 1:478:49-64.
doi: 10.1016/j.neuroscience.2021.09.016. Epub 2021 Sep 28.

Pituitary Adenylate Cyclase Activating Polypeptide Inhibits A10 Dopamine Neurons and Suppresses the Binge-like Consumption of Palatable Food

Nikki Le  1 , Jennifer Hernandez  1 , Cassandra Gastelum  1 , Lynnea Perez  1 , Isabella Vahrson  1 , Sarah Sayers  1 , Edward J Wagner  2
Affiliations

Pituitary Adenylate Cyclase Activating Polypeptide Inhibits A10 Dopamine Neurons and Suppresses the Binge-like Consumption of Palatable Food

Nikki Le et al. Neuroscience. .

Abstract

Pituitary adenylate cyclase-activating polypeptide (PACAP) binds to PACAP-specific (PAC1) receptors in multiple hypothalamic areas, especially those regulating energy balance. PACAP neurons in the ventromedial nucleus (VMN) exert anorexigenic effects within the homeostatic energy balance circuitry. Since PACAP can also reduce the consumption of palatable food, we tested the hypothesis that VMN PACAP neurons project to the ventral tegmental area (VTA) to inhibit A10 dopamine neurons via PAC1 receptors and KATP channels, and thereby suppress binge-like consumption. We performed electrophysiological recordings in mesencephalic slices from male PACAP-Cre and tyrosine hydroxylase (TH)-Cre mice. Initially, we injected PACAP (30 pmol) into the VTA, where it suppressed binge intake in wildtype male but not female mice. Subsequent tract tracing studies uncovered projections of VMN PACAP neurons to the VTA. Optogenetic stimulation of VMN PACAP neurons in voltage clamp induced an outward current and increase in conductance in VTA neurons, and a hyperpolarization and decrease in firing in current clamp. These effects were markedly attenuated by the KATP channel blocker tolbutamide (100 μM) and PAC1 receptor antagonist PACAP6-38 (200 nM). In recordings from A10 dopamine neurons in TH-Cre mice, we replicated the outward current by perfusing PACAP1-38 (100 nM). This response was again completely blocked by tolbutamide and PACAP6-38, and associated with a hyperpolarization and decrease in firing. These findings demonstrate that PACAP activates PAC1 receptors and KATP channels to inhibit A10 dopamine neurons and sex-dependently suppress binge-like consumption. Accordingly, they advance our understanding of how PACAP regulates energy homeostasis via the hedonic energy balance circuitry.

Keywords: binge eating; dopamine; estradiol; obesity; pituitary adenylate cyclase-activating polypeptide; sex difference.

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Figures

Figure 1.
Figure 1.
Coronal schematics illustrating the accuracy of our bilateral AAV ChR2 injections in the VMN (A) and the guide cannula implantations and AAV eYFP injections in the VTA (B).
Figure 2.
Figure 2.
PACAP reduces binge feeding accompanied by decreases in meal size and bout duration. A & B, Pronounced increases in energy expenditure triggered by the binge episode. C-E, The escalation of the daily consumption is significantly dampened by PACAP (30 pmol; VTA; n = 6) as compared to saline-treated controls (n = 6). F, No changes in chow consumption over the remaining 23 hours were observed. G & H, PACAP-induced changes in meal pattern. Symbols represent means and lines 1 SEM. The bars of the box-and-whisker plot represent the median, the 25th and 75th quartiles, whereas the lines depict the 5th and the 95th percentiles. #, p < 0.05 with respect to time (A & B) or day (C), repeated measures ANOVA/LSD; *, p < 0.05; with respect to saline, repeated measures multifactorial ANOVA/LSD (C), Student’s t-test (D, F-H), Mann-Whitney U-test (E).
Figure 3.
Figure 3.
PACAP shows no effect in OVX wildtype female mice. A & B, Pronounced increases in energy expenditure triggered by the binge episode; with heat production being potentiated by EB (A). C & D, EB (20 μg/kg; s.c.; n = 6) but not PACAP (30 pmol; VTA; n = 6) decreases binge intake as well as the percent of the daily intake consumed during the binge hour as compared to their sesame oil (n = 6) and saline controls (n = 6). E, Chow consumption over the remaining 23 hours is significantly reduced in EB- and PACAP-treated animals. Bars represent means and lines 1 SEM. Box-and-whisker plots illustrate the median, 25th and 75th quartiles, as well as the 5th and 95th percentiles. #, p < 0.05 with respect to time (A & B); *, p < 0.05; with respect to saline; ^, p < 0.05 with respect to sesame oil; repeated measures, multi-factorial ANOVA/LSD (A – C, E), rank-transformed, repeated measures, multi-factorial ANOVA/LSD (D).
Figure 4.
Figure 4.
Retrograde tract tracing revealing a subpopulation of PACAP-containing, VTA-projecting VMN neurons. A, The injection site seen within the VTA (5X; denoted by the arrow). B, Fluorogold labelling in the VMN and surrounding areas (5X). C, Fluorogold labelling in the VMN (10X). D, PACAP immunostaining in the VMN as visualized with AF546. E, Composite overlay. Bars represent means and vertical lines 1 SEM. D–E were also photographed at 10X.
Figure 5.
Figure 5.
Photostimulation of VMN PACAP neurons inhibits VTA neurons in PACAP-Cre mice. A & B, eYFP ChR2 reporter signal in the VMN (4X & 40X). C & D, eYFP ChR2 reporter signal in the VTA (4X & 40X). E, DIC image (40X) of a recorded VTA neuron. F-I, Optogenetic stimulation of VMN PACAP neurons produces an outward current associated with an increased slope conductance and reversal of polarity at ~ −90 mV in the vast majority of VTA neurons (n = 15). Arrows indicate where I/Vs were conducted.
Figure 6.
Figure 6.
Optogenetic stimulation of VMN PACAP neurons in PACAP-Cre mice also hyperpolarizes VTA neurons and decreases their firing. A & D, Current clamp trace showing that photo-stimulation produces a reversible hyperpolarization (n = 12). B & D, The hyperpolarization is completely reversed in the presence of tolbutamide (100 μM; n = 6). C PACAP6–38 (200 nM; n = 6) significantly attenuated the hyperpolarization. D, Bar represents means and lines 1SEM of the change in membrane potential (ΔVm mV). *, p < 0.05 relative to PACAP alone, one-way ANOVA/LSD.
Figure 7.
Figure 7.
PACAP inhibits A10 dopamine neurons in slices from male TH-cre mice via a PAC1 receptor-mediated activation of KATP channels. A, TH immunostaining in the VTA. B, eYFP signal seen in these A10 dopamine neurons. C composite overlay. D & E, eYFP signal from A10 dopamine neurons in VTA slices seen at 4X and 40X. F, DIC image of the recorded A10 dopamine neuron seen in E. G-O, PACAP (100 nM; n = 12) produces a robust and reversible outward current in A10 dopamine neurons that is associated with an increased K+ conductance and abrogated by PACAP6–38 (n = 9) and tolbutamide (n = 6). Arrows indicate where I/Vs were conducted. Bars represent means and lines 1 SEM of the change in membrane current (M; pA) membrane slope conductance (M; nS) and current (N; pA). *p < 0.05 relative to PACAP alone, one-way ANOVA/LSD.
Figure 8.
Figure 8.
The outward current obtained in A10 dopamine neurons from TH-cre mice is also associated with a hyperpolarization and decrease in firing. A, Current clamp trace illustrating that bath application of PACAP1–38 produces reversible hyperpolarization that significantly lowers the membrane potential (Vm; B) and suppresses firing (C). B & C, Bars represent means and lines 1 SEM (n = 6). *, p < 0.05 relative to PACAP alone, Student’s t-test (B), signed rank test (C).
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