Neuroscience

POMC neurons control fertility through differential signaling of MC4R in Kisspeptin neurons

Rajae Talbi
Todd L Stincic
Kaitlin Ferrari
Choi Ji Hae
Karol Walec
Elizabeth Medve
Achi Gerutshang
Silvia León
Elizabeth A McCarthy
Oline K Rønnekleiv
Martin J Kelly author has email address
Víctor M Navarro author has email address

Harvard Medical School, Boston, MA, USA
Division of Endocrinology, Diabetes and Hypertension, Department of Medicine, Brigham and Women’s Hospital, Boston, MA, USA
Division of Neuroscience, Oregon National Primate Research Center, Beaverton, OR, USA
Department of Chemical Physiology and Biochemistry, Oregon Health & Science University, Portland, OR, USA
Harvard Program in Neuroscience, Boston, MA, USA

https://doi.org/10.7554/eLife.100722.1

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Figures and data

Mc4r expressed in Kiss1 neurons determines the timing of puberty onset.
Kiss1-MC4RKO females display advanced puberty onset, assessed by daily monitoring of vaginal opening (A) and first estrus (B), as documented by cumulative percent and mean age of animals at vaginal opening (A) and first estrus (B) in Kiss1-MC4RKO females (n=24) compared to WT littermates (n=19). *p<0.05 by Student’s t-test. Data presented as the mean± SEM. (C) Female Kiss1-MC4RKO (n=19) have normal body weight at the time of puberty onset compared to their WT littermates (n=15). (D) Ontogeny expression of melanocortin genes (Agrp, Pomc, Mc4r and Mc3r) in the ARH of WT female mice at different postnatal pre-pubertal and pubertal ages: P10, P15, P22 and P30, normalized to the housekeeping gene Hprt. (n values: females at P10 (n=6), P15 (n=6), P22 (n=6), and P30 (n=5)). Groups with different letters are significantly different (p<0.05), as determined by one-way ANOVA followed by the Student-Newman-Keuls test. Data presented as the mean± SEM.

Deletion of MC4R from Kiss1 neurons impairs fertility in Kiss1-MC4RKO females.
(A) Female Kiss1-MC4RKO (n=19) have normal body weight from weaning and until post-natal day (PND) 150 compared to their WT littermates (n=15). (B) Representative examples of estrous cycles in Kiss1-MC4RKO and control females (n=5/group) assessed by daily vaginal cytology for 15 days. (C) Kiss1-MC4RKO females displayed irregular estrous cycles with a longer time in diestrus and a shorter time in estrus compared to control females. *** P < 0.001 Two-way ANOVA. (D) Pattern of LH pulsatility was analyzed in gonad intact Kiss1-MC4RKO (n=6) and control (n=7) females. # Represents LH pulses. (E) Basal LH, (F) LH total secretory mass assessed by area under the curve (AUC), (G) LH pulse amplitude and (H) Total number of pulses /180 min were analyzed. *p<0.05 by Student’s t-test. The expression of the KNDy genes Pdyn (I), Kiss1 (J), Tac2 (K) and Tacr3 (L), was assessed in the ARH of adult Kiss1-MC4RKO (n=6) and control (n=3) females. **p<0.01 by Student’s t-test. Data presented as the mean± SEM. (M) Kiss1-MC4RKO (n=6) and control (n=5) females were subjected to an LH surge induction protocol. LH samples were collected in the morning (AM [8 a.m.]) and evening (PM [7 p.m.]) after lights off. **P < 0.01, Two-way ANOVA followed by Bonferroni post hoc test. (N) Ovarian histology shows a decrease in the number of corpora lutea (CL, O) and increase in the number of cystic follicles (CF, P) of Kiss1-MC4RKO compared to controls(n=5/group). # represents cystic follicles. Serum levels of Testosterone (Q) and AMH (R) in adult gonad intact Kiss1-MC4RKO and control females (n=5/group). Student t test for unpaired samples. Data presented as the mean± SEM. Kiss1-MC4RKO females display impaired fertility characterized by increased time to deliver pups (S) and decreased number of pups per litter (T), (n=9/group). *p<0.05 by Student’s t-test. Data presented as the mean± SEM.

Re-insertion of MC4R in Kiss1 neurons restores estrous cyclicity and LH pulsatility in Kiss1-cre:MC4R-LoxTB females.
(A) Kiss1-cre:MC4R-LoxTB (n=11) and MC4R-loxTB (MC4RKO) (n=7) displayed normal puberty onset as compared to their control littermates (n=14), as documented by cumulative percent and mean age of animals at vaginal opening and first estrus. (B) Kiss1-cre:MC4R-LoxTB and MC4R-loxTB both had significantly higher body weights at the time of puberty onset compared to their controls. **P < 0.01, *** P < 0.001 by one way ANOVA. (C) MC4R-loxTB (n=7) and Kiss1-cre:MC4R-LoxTB (n=11) females displayed significantly higher body weight than their littermates (n=10) from post-natal day 30 onwards, *p<0.05 and **P < 0.01. Data presented as the mean± (SEM). (D) Representative examples of estrous cycles of MC4R-loxTB (n=4), Kiss1-cre:MC4R-LoxTB (n=5) and control littermate mice, (n=4) assessed by daily monitoring of vaginal smears for 15 days. (D/M: diestrus/metestrus, P: proestrus, E: estrus). (E) MC4R-loxTB females displayed irregular estrous cycles, presenting longer time in diestrus and shorter time in estrus compared to control females, while Kiss1-cre:MC4R-LoxTB females displayed regular estrous cyclicity, similar to controls. *p<0.05, **P < 0.01, *** P < 0.001, ****P < 0.0001, Two-way ANOVA. Data presented as the mean± SEM. (F) Pattern of LH pulsatility analyzed in gonad intact MC4R-loxTB (n=4), Kiss1-cre:MC4R-LoxTB (n=5) and control littermates females (n=5). LH samples were collected every 10 min for 180 minutes; # represents LH pulses. (G) LH total secretory mass, (H) LH pulse amplitude, (I) basal LH, and (J) total number of pulses /180 min were assessed. *p<0.05, **P < 0.01 by one way ANOVA. (K) Representative samples of ovarian histology from MC4R-loxTB, Kiss1-cre:MC4R-LoxTB and control females (n=5/group); CL: Corpora lutea. Data are presented as the mean± SEM. (L) Ovarian histology showed a significant decrease in the number of corpora lutea in the MC4RKO and Kiss1-cre:MC4R-LoxTB compared to controls. Groups with different letters are significantly different. Serum levels of (M) Testosterone and (N) AMH in adult gonad intact females MC4R-loxTB, Kiss1-cre:MC4R-LoxTB and their control littermates. *p<0.05, ****P < 0.0001, One Way ANOVA. Data are presented as the mean± SEM.

Kiss1^ARH neurons are activated by melanocortin agonists and respond to optogenetic stimulation of POMC neurons.
(A) Slices were taken from Kiss1-cre injected (confocal image) brains to target fluorescent cells (arrowhead) for recording (white outline over electrode). (B) Whole cell voltage clamp recording of Kiss1^ARH neurons following the direct application of the high affinity melanocortin receptor agonist melanotan II (MTII, 250 nM) was added directly to the bath and the response was compared between slices from OVX and OVX+E females. (C) While the average inward current was slightly higher in the OVX+E state, this was not a significant difference (Student’s t-test for unpaired samples, p>0.05). (D) The MC4R-selective agonist THIQ (100 nM) was perfused, and excitatory inward currents were generated in Kiss1^ARH neurons. (E) Images are of AAV-driven labeling of POMC cells as seen through the confocal. (F) Using the AAV-driven expression of channelrhodopsin in adult Pomc-cre mice, high frequency stimulation elicited a slow inward current in Kiss1 neurons. (G) The identity of cells was confirmed through the presence of a persistent sodium current (see ²⁵) and/or with RT-PCR of harvested cytoplasm showing Kiss1 expression. (Gel: MM=molecular marker, TC=tissue controls). (H) A direct synaptic projection from POMC to Kiss1^ARH neuron (posthoc identified) was confirmed using the “rescue” protocol: 1, baseline glutamatergic responses were initially generated (black trace); 2, then action potentials were eliminated by blocking voltage-gated sodium channels with TTX and the postsynaptic response (red trace); 3, blockade of potassium channels facilitated calcium entry into the terminal through ChR2 to release synaptic vesicles, “rescuing” the postsynaptic glutamate response (green trace). I) Glutamatergic responses were also often observed, particularly in the ventral ARH. As seen when targeting low input resistance neurons (i.e., Kiss1^ARH) with low frequency optogenetic stimulation (5 ms pulse, 50 ms inter-spike interval), the first response in OVX + vehicle females was larger relative to the second response in OVX + E2-treated female mice. Representative traces are the average of 30 sweeps. (J) The averaged paired-pulse ratio was lower in recordings from estradiol-treated female mice, indicating an increased release probability (Students t-test p<0.05,). (K) High frequency optogenetic stimulation elicited a small inward current. (L) In a different cell, a high frequency inward current was noted before constant perfusion of SHU9119 for 15 minutes (break between traces). When the stimulation protocol was repeated, no inward current was elicited. (M) The majority of high frequency responses were inward and only twice was an inhibitory outward current recorded in identified Kiss1^ARH neurons. Perfusion of the non-selective opioid receptor antagonist naloxone reversed the current, eliminating MCR-activation as the mechanism.

Kiss1^AVPV/PeN neurons are inhibited by MC4R agonists in an E2-dependent manner.
(A) Immunohistochemistry showing robust labeling of α-MSH fibers in the AVPV/PeN region in OVX+E2 WT female mice. (B) Low and high power bright-field images taken during electrophysiology recording in OVX+E2 POMC-Cre mice expressing YFP:ChR2 in the ARH following AAV injection. (left) Low power image shows the location of recorded cells. (right) Higher power image of area from white box inset displaying POMC fibers innervating the area. (C) Fibers surrounded putative Kiss1-AVPV neurons (expressing I_NaP, IT and I_h) and optogenetic stimuli was able to elicit postsynaptic currents that were eliminated with tetrodotoxin (1 μM), but “rescued” with addition of K⁺ channel blockers (4-AP and TEA) which indicates a monosynaptic connection between POMC^ARH and Kiss1^AVPV neurons. (D) Whole-cell voltage clamp recordings were made in ChR2-YFP positive cells in brain slices taken from Kiss1-cre:Ai32 female mice. Bath application of melanotan II (MTII, 500 nM) generated an inhibitory outward current in a Kiss1^AVPV/PeN neuron from OVX female. As the cell was synaptically isolated using bath-applied tetrodotoxin (TTX, 1 μM) this represents a direct effect. Washout of MTII while still in TTX quickly led to a return to baseline RMP. (E) An IV relationship was plotted using voltage steps before and after MTII administration. The crossing at -80 mV (∼EK⁺) indicates that the opening of K+ channels underlie the MC4R inhibition of Kiss1^AVPV neurons. (F) In a subset of recordings from OVX brains slices, the selective membrane estrogen receptor (Gq-mER) agonist STX (10 nM) was added to the bath for ∼10 minutes prior to addition of MTII. STX pretreatment resulted in either a greatly attenuated outward current or even an inward current as shown in this example. (G) The mean outward current was significantly higher when recording in Kiss1^AVPV/Pen neurons from brain slices from vehicle-treated, OVX females compared to E2-treated, OVX females or acute STX-treated brain slices from OVX females (One-way ANOVA F_(2,19)=12.32, p<0.001; Holm-Sidak posthoc comparisons found significant differences between all groups; * p<0.05, *** p<0.001). (H) There was no difference in the frequency of mEPSCs after MTII, calculated as a percent of baseline between groups. (I) However, there was a significant difference in the mEPSC amplitude (calculated as a percent of baseline) between all groups: main effect F_(2,19) =10.58, p<0.001. Posthoc comparisons using Holm-Sidak found OVX+STX to be different from both OVX (p<0.001) and OVX+E (p<0.05).

Kiss1^AVPV/PeN neurons are inhibited by MC4R agonists in an E2-dependent manner.
(A) Immunohistochemistry showing robust labeling of α-MSH fibers in the AVPV/PeN region in OVX+E2 WT female mice. (B) Low and high power bright-field images taken during electrophysiology recording in OVX+E2 POMC-Cre mice expressing YFP:ChR2 in the ARH following AAV injection. (left) Low power image shows the location of recorded cells. (right) Higher power image of area from white box inset displaying POMC fibers innervating the area. (C) Fibers surrounded putative Kiss1-AVPV neurons (expressing I_NaP, IT and I_h) and optogenetic stimuli was able to elicit postsynaptic currents that were eliminated with tetrodotoxin (1 μM), but “rescued” with addition of K⁺ channel blockers (4-AP and TEA) which indicates a monosynaptic connection between POMC^ARH and Kiss1^AVPV neurons. (D) Whole-cell voltage clamp recordings were made in ChR2-YFP positive cells in brain slices taken from Kiss1-cre:Ai32 female mice. Bath application of melanotan II (MTII, 500 nM) generated an inhibitory outward current in a Kiss1^AVPV/PeN neuron from OVX female. As the cell was synaptically isolated using bath-applied tetrodotoxin (TTX, 1 μM) this represents a direct effect. Washout of MTII while still in TTX quickly led to a return to baseline RMP. (E) An IV relationship was plotted using voltage steps before and after MTII administration. The crossing at -80 mV (∼EK⁺) indicates that the opening of K+ channels underlie the MC4R inhibition of Kiss1^AVPV neurons. (F) In a subset of recordings from OVX brains slices, the selective membrane estrogen receptor (Gq-mER) agonist STX (10 nM) was added to the bath for ∼10 minutes prior to addition of MTII. STX pretreatment resulted in either a greatly attenuated outward current or even an inward current as shown in this example. (G) The mean outward current was significantly higher when recording in Kiss1^AVPV/Pen neurons from brain slices from vehicle-treated, OVX females compared to E2-treated, OVX females or acute STX-treated brain slices from OVX females (One-way ANOVA F_(2,19)=12.32, p<0.001; Holm-Sidak posthoc comparisons found significant differences between all groups; * p<0.05, *** p<0.001). (H) There was no difference in the frequency of mEPSCs after MTII, calculated as a percent of baseline between groups. (I) However, there was a significant difference in the mEPSC amplitude (calculated as a percent of baseline) between all groups: main effect F_(2,19) =10.58, p<0.001. Posthoc comparisons using Holm-Sidak found OVX+STX to be different from both OVX (p<0.001) and OVX+E (p<0.05).

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