Kisspeptin Neurons Excitability Is Not Modulate by Fasting

Kisspeptin is the most important neuromodulator of gonadotrophin releasing hormone neurons. Hypothalamic Kiss1-expressing neurons can be found in the anteroventral periventricular and rostral periventricular nuclei (AVPV/PeN) and in the arcuate nucleus of the hypothalamus (ARH). Food intake and energy balance are modulated by leptin, which acts primarily in the ARH. Leptin acts inhibiting orexigen neurotransmitters and stimulating anorectic neurotransmitters. Few ARH kisspeptin cells (10-15%) coexpress the leptin receptor and have their resting membrane potential (RMP) depolarized by leptin. Since the reproductive axis is sensitive to metabolic disorders, in the present study we used immunohistochemical and electrophysiological approach to understand the effects of fasting on kisspeptin neurons activity. To determine whether AVPV/PeN and ARH kisspeptin neurons activity are modulated by fasting, Kiss1/hrGFP female mice were fed regular chow ad libitum or fasted for 24 hours. Mice were euthanized after 24h. Fasting induced a significant increase of Fos protein on the ARH nucleus, as expected (rostral level of ARH, control: 19.3 ± 3.5 vs fasted: 81.3 ± 3.5 cells; caudal level, control: 15.7 ± 1.3 vs fasted: 103.3 ± 6.2 cells, P <0.0001, n=3/6 mice per group). Despite the significant increase of Fos-immunoreactive in the ARH nucleus, kisspeptin neurons did not co-express this neuronal marker. Next, we determined the RMP of kisspeptin cells obtained from control or fasted mice. Compared to the control group (AVPV/PeN, -56.5 ± 2.0 mV, n=13 cells from 4 animals; ARH, - 49.8 ± 2.4 mV, n= 9 cells from 6 animals), fasting was not sufficient to induce changes in RMP (AVPV/PeN, -56.1 ± 4.6 mV, n=6 cells from 3 animals, P=0.95; ARH, - 50.9 ± 2.6 mV, n=9 cells from 3 animals, P=0.11). The frequency (freq) and amplitude (amp) of the excitatory postsynaptic currents acting on kisspeptin neurons was further investigated. No significant difference was observed by comparing data obtained from control (AVPV/PeN: freq 0.76 ± 0.1 Hz, n=19; amp, 28.6 ± 1.8 pA, n=16; ARH: freq 0.36 ± 0.1 Hz, n=15; amp, 28.5 ± 2 pA, n=15) and fasted mice (AVPV/PeN: freq 0.7 ± 0.1 Hz, n=18; amp, 28.5 ± 1.1 pA; n=18; ARH: freq 0.5 ± 0.2 Hz, n=24; amp, 27.9 ± 1.3 pA; n=24; P > 0.5, 7/9 animals per group). Considering that 24hr fasting is not enough to inhibit estrous cyclicity, even though it is sufficient to induce a significant reduction of hypothalamic Kiss1 mRNA expression (unpublished data), our results suggest that prolonged periods of food restriction may be required to disturb excitatory inputs into kisspeptin cells.