Neuroscience

Glutamate neurotransmission from leptin receptor cells is required for typical puberty and reproductive function in female mice

Cristina Sáenz de Miera
Nicole Bellefontaine
Susan J. Allen
Martin G. Myers Jr.
Carol F. Elias author has email address

Department of Molecular and Integrative Physiology;
Elizabeth W. Caswell Diabetes Institute;
Department of Internal Medicine, Division of Metabolism, Endocrinology and Diabetes
Department of Obstetrics and Gynecology; University of Michigan, Ann Arbor, MI, 48109-5622, USA

https://doi.org/10.7554/eLife.93204.1

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Figures and data

Experimental groups in the study of chemogenetic activation of LepRb-Cre cells in the PMv.
Corresponding to figures 1-3. The number of animals in each group is indicated in brackets.

Chemogenetic activation of leptin receptor neurons in the ventral premammilary nucleus (PMv) induces luteinizing hormone (LH) release.
A-B. Representative fluorescent photomicrographs of unilateral AAV-hM3Dq PMv injection. Low (A) and high (B) magnification of an animal correctly targeted to the PMv. Magenta: mCherry-immunoreactivity (-ir); Green: cFos-ir. C. LH levels in “PMv-hit” animals unilaterally expressing hM3Dq in the PMv following an intravenous (iv.) injection of clozapine-N-oxide (CNO) at time=0 (n=15). Continuous lines: animals showing an increase in LH had maximum LH levels ranging from 0.65-3.11 ng/ml. Dashed lines: animals showing no detectable increase in LH. Light blue lines represent individual values. Dark blue lines represent averaged values. D. LH levels in Negative control animals (n=7), including “PMv-miss” (orange) and AAV-mCherry (pink) animals following an iv. injection of CNO at time=0. Light colored lines represent individual values. Dark colored lines represent average values. A gap is shown in the LH profile lines in animals that had a missing value at time 0 in C and D. E. Average LH levels at -10, 10 and 20 min for all 3 groups: “PMv-hit” with LH increase (n=8, dark blue), “PMv-hit” with no LH increase (n=7, light blue), Negative controls (including “PMv-miss” and AAV-mCherry, n=7, Orange). F. Positive area under the curve (AUC) of the values represented in E. One-sample t-tests (“PMv-hit” with LH increase: t₇=3.54, p = 0.009; “PMv-hit” with no LH increase: t₆=1.37, p = 0.22; Negative controls: t₆=1.00, p = 0.36), **p<0.01 compared to “0”. G. Number of cFos-ir neurons per section in the PMv in “PMv-hit” animals with (n=9) or without (n=7) an increase in LH and in Negative control animals, including “PMv-miss” and AAV-mCherry animals (n=7) 2h after the CNO injection (one-way ANOVA, F_2,19 = 81.66; p < 0.0001) **p<0.01 and ***p<0.001 vs. PMv-hit LH increase group; ^###p<0.001 vs. PMv-hit no LH increase group. H. Correlation between the number of cFos-ir neurons per section in the PMv and the peak LH level in the 3 groups (Pearson r = 0.67; p = 0.0007). 3V: Third ventricle. Scale bars: 100µm.

Clozapine induces cFos expression in the ventral premammillary nucleus (PMv) of animals expressing hM3Dq and luteinizing hormone (LH) release.
A. Representative fluorescent photomicrograph of a unilateral AAV-hM3Dq PMv injection in a clozapine injected animal. Magenta: mCherry-immunoreactivity (-ir); Green: cFos-ir. B. LH levels in animals unilaterally expressing hM3Dq in the PMv following an iv. injection of clozapine at time=0 (n=4). Light green lines represent individual values. Dark green line represents averaged values. C. LH levels in animals not carrying any AAV following an iv. injection of clozapine at time=0 (n=7). Light magenta lines represent individual values. Dark magenta line represents averaged values. Continuous lines: animals with LH increase after the injection. Discontinuous lines: animals with no increase in LH after the injection. D. Average LH levels at -10, 10 and 20 min for both groups injected with clozapine. hM3Dq: green; No AAV: magenta. E. Positive area under the curve (AUC) of the values represented in D. One-sample t-tests (hM3Dq: t₃=8.79, p = 0.003; No AAV: t₆=1.83, p = 0.12), **p<0.01 compared to “0”. F. Number of cFos-ir neurons per section of the PMv in AAV-hM3Dq and no AAV animals 2h after a clozapine injection (t₈ = 17.46; p < 0.0001). ***p<0.001 3V: Third ventricle. Scale bars: 100µm.

CNO-induction of luteinizing hormone (LH) secretion may have a mild contribution of POMC, but not KNDy, neurons.
Representative images of A. mCherry-immunoreactivity (-ir) and B. cFos-ir of an animal with strong AAV contamination of the posterior Arc. C. Number of cFos-ir neurons with mCherry-ir colocalization per section in the posterior Arc in the 3 groups 2h after the CNO injection. (Kruskal-Wallis test = 14.63; p < 0.0001), ***p<0.001 vs. PMv-hit LH increase group, ^#p<0.05 vs. PMv-hit no LH increase group. D. Correlation between the number of mCherry-/cFos-ir neurons observed in the posterior Arc and the peak LH level in the 3 groups (Pearson r = 0.62; p = 0.002). E. POMC-ir (green) in a mouse with viral contamination of the posterior Arc. Arrows: cFos-ir neurons (black) that coexpressed POMC and mCherry (magenta). Arrowheads: cFos-ir neurons that coexpressed POMC, but no mCherry, F. Confocal high magnification image of a cFos-ir neuron coexpressing POMC and mCherry. G. Confocal high magnification image of a cFos-ir neuron coexpressing POMC, but not mCherry. H. GFP-ir (green) in a Kiss1-hrGFP animal with viral contamination of the posterior Arc. Arrows: cFos-ir (black) neurons that coexpressed Kiss1-hrGFP and mCherry (magenta). I and J. Confocal high magnification images of a cFos-ir neuron (arrow in H.) coexpressing Kiss1-hrGFP and mCherry (magenta). Scale bars: 50 µm.

Adult LepRb^ΔVglut2 animals show an obese phenotype.
A-D. Micrographs of in situ hybridization showing colocalization between Lepr (Cyan) and Vglut2 (Magenta) gene expression in the dorsomedial region of the ventromedial hypothalamic nucleus (VMHdm) in control LepRb-Cre (A, C), but no colocalization in experimental LepRb^ΔVglut2 (B, D) females. A, B. Small magnification, scale bar: 50 µm. C, D. Large magnification, scale bar: 20 µm. E. Body mass in adult LepRb-Cre and LepRb^ΔVglut2 males (genotype main effect; F_1,10 = 13.1; p = 0.0047, post-hoc comparison: week 20: t₁₄₀ = 3.27; p = 0.019; week 26: t₁₄₀ = 5.47; p < 0.0001). F. Body mass in adult LepRb-Cre and LepRb^ΔVglut2 females (genotype main effect; F_1,11 = 4.34; p = 0.06; post-hoc comparisons: week 23: t₁₅₄ = 4.05; p = 0.0011; week 25: t₁₅₄ = 3.1; p = 0.03; week 26: t₁₅₄ = 5.59; p < 0.0001). The arrow indicates the age at which estrus cycling follow-up finished in this cohort. G. Body composition in males (fat mass: t₉ = 3.77; p = 0.004) and H. in females (fat mass: (t₁₁ = 3.7; p = 0.0035). *p<0.05, **p<0.01, ***p<0.001.

Reproductive development and estrus cycles are altered in LepRb^ΔVglut2 females.
A. Age of vaginal opening (VO; t₁₈ = 0.84; p = 0.41) and B. body mass at vaginal opening (VO) (t₁₈ = 1.47; p = 0.16) in females. C. Age of first estrus (t₁₄ = 2.45; p = 0.028) and D. body mass at first estrus (t₁₄ = 0.59; p = 0.56) in females. E. Age of complete balanopreputial separation (BPS; t₁₁ = 1.80; p = 0.1) and F. body mass at BPS (t₁₁ = 2.43; p = 0.033) in males. G. Representative estrus cycle profiles from control LepRb-Cre and H. experimental LepRb^ΔVglut2 females. (E: estrus, P: Proestrus, M/D: Metestrus/diestrus). I. Cycle duration in adult females (t₂₅ = 3.71; p = 0.001). J. Time spent in each phase of the cycle during the 30 days studied. Estrus: (t₂₆ = 1.97; p = 0.059), proestrus (t₂₆ =2.81; p = 0.009), diestrus (t₂₆ = 2.39; p = 0.024). K. Days spent from mating until the first litter was observed in females paired with proven male breeders (Mann-Whitney test; p = 0.32). *p<0.05, **p<0.01.

Gonadotropin-releasing hormone (GnRH) content in terminals and luteinizing hormone (LH) secretion are altered in LepRb^ΔVglut2 female mice.
Representative fluorescent photomicrograph of GnRH-immunoreactivity (-ir) in the arcuate nucleus (Arc) and median eminence (ME) in A. control Vglut2^flox and B. LepRb^ΔVglut2 diestrous females. Scale bar: 100µm. C. GnRH-ir integrated density in the Arc in Vglut2^flox (n = 4) and LepRb^ΔVglut2 (n = 3) females (t₅ = 8.01; p = 0.0005). D. Basal LH levels in Vglut2^flox (n = 7) and LepRb^ΔVglut2 (n = 5) females (t₁₀ = 1.91; p = 0.08). E. LH levels in LepRb-Cre (n = 7) and LepRb^ΔVglut2 (n = 6) females before and after a kisspeptin-10 (65 µ/kg) intraperitoneal (ip.) injection of kisspeptin at time=0. Light grey: individual LepRb-Cre females, Light yellow: individual LepRb^ΔVglut2 females. After the injection (time 7 to 28), LH levels were higher in control than in floxed females (Mixed effects model: main effect for “time” F_1.14, _12.19 = 15.91, p=0.001; “genotype" F_1, ₁₁ = 8.26, p=0.015; “time x genotype” interaction: F_3, ₃₂ = 5.00, p=0.006; Sidak’s post-hoc effect for 7 min (t_10.97 = 3.79, p=0.01) and for 14 min (t_9.4 = 3.22, p=0.04). F. Area under the curve (AUC) of LH levels after the kisspeptin injection, (t₁₁ = 3.14; p = 0.009). G. Gnrhr (t₁₀ = 0.86, p = 0.41), H. LHβ (t₁₀ = 1.47, p = 0.17) and I. FSHβ (t₉ = 1.63, p = 0.14) relative to Actin B (Actb) mRNA levels in the pituitary of Vglut2^flox (n = 7) and LepRb^ΔVglut2 (n = 5) females. J. Kiss1 (t₈ = 1.42, p = 0.19), K. Pdyn (t₈ = 0.02; p=0.98), L. Kiss1r (t₈ = 1.44, p = 0.19) M. Tac3r (t₈ = 0.99, p = 0.35) and N. Tac2 (t₈ = 3.39, p = 0.009), relative to β -2-microglobulin (B2m) mRNA levels in the mediobasal hypothalamus of Vglut2^flox (n = 5) and LepRb^ΔVglut2 (n = 5) females. *p<0.05; **p<0.01; ***p<0.001.

Glutamate signaling from the ventral premammillary nucleus (PMv) is required for leptin effect on pubertal development in female mouse.
A, B, I, M. Microphotographs of the PMv showing pSTAT3-ir following leptin administration. Note lack of pSTAT3-ir in LepR^loxTB mouse. C, D, J, N. Darkfield microphotographs showing Vglut2 mRNA (silver grains) in the PMv. Note decreased Vglut2 mRNA expression in LepR^loxTB;Vglut2^floxmice injected with AAV-Cre. E, F, K, O. Microphotographs of representative single uterine horns. Note lack of uterine growth in LepR^loxTBmouse and in LepR^loxTB;Vglut2^flox mouse injected with AAV-Cre. G, H, L, P. Microphotographs of representative ovary sections. Note lack of corpora lutea in LepR^loxTB mouse and in LepR^loxTB;Vglut2^floxed mouse injected with AAV-Cre. 3V: Third ventricle, Arc: Arcuate nucleus, f: fornix, CL: Corpus luteum. Scale bars: 100µm. Q. Number of pSTAT3 expressing cells in the PMv in Wild-type (n=5), LepR^loxTB(n=5), AAV-CRE injected LepR^loxTB (n=4), and AAV-CRE injected LepR^loxTB;Vglut2^flox (n=3) (F_3,13= 73.53, p<0.0001). R. Quantification of the Vglut2 hybridization signal in the PMv AAV-CRE injected LepR^loxTB (n=4), and AAV-CRE injected LepR^loxTB;Vglut2^flox(n=6) (t₈ = 4.20; p = 0.003). S. Number of pSTAT3 expressing cells in the Arc (F_3,16= 171.8, p<0.0001). T. Number of corpora lutea in the ovary (F_3,17= 31.16, p<0.0001).

Antibodies

Antibodies

Quantitative PCR primers

Quantitative PCR primers

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