The excitatory peptide kisspeptin restores the luteinizing hormone surge and modulates amino acid neurotransmission in the medial preoptic area of middle-aged rats

Abstract

Reproductive success depends on a robust and appropriately timed preovulatory LH surge. The LH surge, in turn, requires ovarian steroid modulation of GnRH neuron activation by the neuropeptide kisspeptin and glutamate and gamma-aminobutyric acid (GABA) neurotransmission in the medial preoptic area (mPOA). Middle-aged females exhibit reduced excitation of GnRH neurons and attenuated LH surges under estrogen-positive feedback conditions, in part, due to increased GABA and decreased glutamate neurotransmission in the mPOA. This study tested the hypothesis that altered kisspeptin regulation by ovarian steroids plays a role in age-related LH surge dysfunction. We demonstrate that middle-aged rats exhibiting delayed and attenuated LH surges have reduced levels of Kiss1 mRNA in the anterior hypothalamus under estrogen-positive feedback conditions. Kisspeptin application directly into the mPOA rescues total LH release and the LH surge amplitude in middle-aged rats and increases glutamate and decreases GABA release to levels seen in the mPOA of young females. Moreover, the N-methyl-D-aspartate receptor antagonist MK801 blocks kisspeptin reinstatement of the LH surge. These observations suggest that age-related LH surge dysfunction results, in part, from reduced kisspeptin drive under estrogen-positive feedback conditions and that kisspeptin regulates GnRH/LH release, in part, through modulation of mPOA glutamate and GABA release.

Figure 1

Illustration of microdialysis probe placements in the medial preoptic area. A, The diagram corresponds to a coronal section at approximately 0.0 mm relative to Bregma (plate 33) in the atlas of Paxinos and Watson (81). 3V, Third ventricle; och, optic chiasm; VMPO, ventromedial preoptic nucleus; VLPO, ventrolateral preoptic nucleus; SO, supraoptic nucleus; Al, alar nucleus; StA, strial part preoptic nucleus; MA, middle-aged rats; Y, young rats; OVLT, organum vasculosum laminae terminalis. B, Photomicrograph of thionin-stained coronal section showing a representative probe placement between plates 32 and 33 by Paxinos and Watson (81). Magnification, ×40, shows the approximate location of a microdialysis probe. The arrow indicates the site of probe tip.

Figure 2

The attenuated LH surge is rescued by Kp-10 and correlates with reduced production of Kiss1 mRNA under estradiol-positive feedback conditions. A, Kiss1 mRNA in the anterior hypothalamus, which includes the AVPV. Data are expressed as mean ± sem from OVX young (Y) rats primed with estradiol and progesterone (E2 + P; n = 4) or oil (n = 4) and OVX middle-aged (MA) rats primed with E2 + P or oil and killed at 4 h (oil; n = 4, E2 + P; N 4) or 7 h (oil; n = 4, E2 + P; n = 4) after the P or last oil injection. There was no statistical difference in Kiss1 mRNA levels in E2-primed MA rats killed at 4 (n = 4) or 7 h (n = 4) after P; therefore, these data were pooled. The same was true for MA rats primed with oil (n = 4/time point). There was a significant main effect of hormone treatment (F = 44, P < 0.0001), age (F = 5, P < 0.03), and an interaction between hormone treatment and age (F = 7, P < 0.01). a, P < 0.005 vs. all E2 + P groups; b, P < 0.05 vs. Y E2 + P. B–F, Plasma LH levels are expressed as mean ± sem from OVX and E2 + P primed young control rats (Y; n = 6) and middle-aged control rats (MA; n = 7) dialyzed with ACSF and young (Y + Kp-10; n = 6) and middle-aged rats (MA + Kp-10; n = 10) dialyzed with 10 nm Kp-10. Progesterone was injected at 0900 h (time 0). B, LH surge in control rats. C, LH surge in Kp-10-treated rats. D, Total LH (AUC); there was a main effect of age (F = 11, P < 0.01) and an interaction between age and Kp-10 (F = 12, P < 0.001). E, Peak LH; there was a main effect of age (F = 11, P < 0.005) and an interaction between age and Kp-10 (F = 5.5, P < 0.05). F, LH surge onset (h relative to P injection) (Kruskal Wallis = 21.8, P < 0.005). a, P < 0.05 vs. Y; b, P < 0.05 vs. Y + Kp-10; c, P < 0.01 vs. MA + Kp-10.

Figure 3

Kisspeptin acts on the hypothalamus to enhance LH surges in middle-aged rats. Data are means ± sem from OVX, estradiol, and progesterone replaced middle-aged control (MA; n = 4) and MA rats dialyzed with 10 nm Kp-10 alone (Kp-10; n = 4), with 10 nm Kp-10 and injected with vehicle (Veh; n = 4) or 10 nm Kp-10 and injected with 100 μg of the GnRH receptor antagonist cetrorelix both 24 h before and immediately before the progesterone injection (Kp-10 + cetrorelix; n = 4). Progesterone was injected at 0900 h (time 0). A, LH surges. B, Peak LH; F = 24.5, P < 0.0001. C, Total LH (AUC); F = 15.7, P < 0.001. D, LH surge onset (h relative to P injection). a, P < 0.001 vs. all other groups.

Figure 4

Kisspeptin differentially affects glutamate and GABA release in the mPOA of young and middle-aged rats on the day of the LH surge. Data are means ± sem and are from the same animals shown in Fig. 2. Time course of extracellular glutamate (Glu) (A and B) and GABA (E and F) levels in the mPOA of control and Kp-10-treated young (Y) and middle-aged rats (MA). C, Total Glu (AUC); there was an interaction between age and Kp-10 [F = 25.12, P < 0.0001]. (D, Peak Glu release; there was an interaction between age and Kp-10 [F = 14.4, P < 0.001]. (G, Total GABA; there were main effects of age [F = 7.7, P < 0.01] and treatment [F = 11.8, P < 0.005] and an interaction between age and Kp-10 [F = 24.9, P < 0.001]. (H, Peak GABA release; there were main effects of age [F = 8.5, P < 0.01] and treatment [F = 7, P < 0.02] and an interaction between age and Kp-10 [F = 11.8, P < 0.003]. a, P < 0.05 vs. Y control; b, P < 0.01 vs. MA + Kp-10; c, P < 0.001 vs. Y control; d, P < 0.01 vs. Y+ Kp-10 vs. MA + Kp-10. P, Progesterone.

Figure 5

Kisspeptin facilitation of LH release in middle-aged rats requires activation of NMDA receptors. Data are means ± sem. A, Time course of LH release from control (n = 4), Kp-10 (n = 7), and Kp-10 + MK801-treated (n = 6) middle-aged rats (MA). P, Progesterone. B, Total LH release (F = 13.9, P < 0.005). C, Total glutamate (Glu) release (F = 74.8, P < 0.001). D, Peak Glu (F = 43.2, P < 0.005). E, Total GABA release (F = 13.9, P < 0.01). F, Peak GABA (F = 9.2, P < 0.005). a, P < 0.01 vs. MA; b, P < 0.001 vs. MA + Kp-10 + MK801.

Figure 6

Proposed model for direct and indirect actions of kisspeptin on GnRH/LH release in young and middle-aged rats under estradiol-positive feedback conditions. A, Young adult rats: in the presence of estradiol (E2)-positive feedback environment, increased kisspeptin in AVPV neurons directly activate GnRH neurons and indirectly affects the GnRH/LH surge by increasing glutamate and decreasing GABA release in the mPOA. Kisspeptin can also inhibit GABA_B receptors (28) on GnRH neurons. GnRH neurons in young females also receive E2-regulated inputs that include but are not limited to norepinephrine neurons (for review see Ref. 14). Estradiol-positive feedback is mediated by ER-α, which is expressed in the neurons indicated. B, Middle-aged rats: we propose that middle-aged rats have age-related changes in responsiveness to E2 in all ER-α expressing neurons shown. E2 induces less Kiss1 mRNA in the AVPV of middle-aged females, which may attenuate GnRH/LH release and contribute to increased GABA and GABA-mediated inhibition, and decreased glutamate in the mPOA. The increase in GABA may reduce both glutamate and norepinephrine release (82). The altered balance of glutamate and GABA neurotransmission along with reductions in norepinephrine (83,84) and alterations of other neurotransmitters in response to E2-positive feedback reduces activation of GnRH neurons on the day of the LH surge (for review see Ref. 14). Kisspeptin infusion into the mPOA rescues LH surge amplitude by direct actions on GnRH neurons and by restoring the balance of glutamate and GABA release to levels typical of young females. Black arrows, Excitatory actions; gray arrows, inhibitory actions; large arrows, robust input; small arrows, reduced input. + and −, Relative amounts of afferent excitatory and inhibitory input, respectively.