Dominant Neuropeptide Cotransmission in Kisspeptin-GABA Regulation of GnRH Neuron Firing Driving Ovulation

A population of kisspeptin-GABA coexpressing neurons located in the rostral periventricular area of the third ventricle (RP3V) is believed to activate gonadotropin-releasing hormone (GnRH) neurons to generate the luteinizing hormone (LH) surge triggering ovulation. Selective optogenetic activation of RP3V kisspeptin (RP3V^KISS) neurons in female mice for >30 s and ≥10 Hz in either a continuous or bursting mode was found to reliably generate a delayed and long-lasting activation of GnRH neuron firing in brain slices. Optogenetic activation of RP3V^KISS neurons in vivo at 10 Hz generated substantial increments in LH secretion of similar amplitude to the endogenous LH surge. Studies using GABA_A receptor antagonists and optogenetic activation of RP3V GABA (RP3V^GABA) neurons in vitro revealed that low-frequency (2 Hz) stimulation generated immediate and transient GABA_A receptor-mediated increases in GnRH neuron firing, whereas higher frequencies (10 Hz) recruited the long-lasting activation observed following RP3V^KISS neuron stimulation. In vivo, 2 Hz activation of RP3V^GABA neurons did not alter LH secretion, whereas 10 Hz stimulation evoked a sustained large increase in LH identical to RP3V^KISS neuron activation. Optogenetic activation of RP3V^KISS neurons in which kisspeptin had been deleted did not alter LH secretion. These studies demonstrate the presence of parallel transmission streams from RP3V neurons to GnRH neurons that are frequency dependent and temporally distinct. This comprises a rapid and transient GABA_A receptor-mediated activation and a slower onset kisspeptin-mediated stimulation of long duration. At the time of the LH surge, GABA release appears to be functionally redundant with the neuropeptide kisspeptin being the dominant cotransmitter influencing GnRH neuron output.SIGNIFICANCE STATEMENT Miscommunication between the brain and ovaries is thought to represent a major cause of infertility in humans. Studies in rodents suggest that a population of neurons located in the rostral periventricular area of the third ventricle (RP3V) are critical for activating the gonadotropin-releasing hormone (GnRH) neurons that trigger ovulation. The present study provides evidence that an RP3V neuron population coexpressing kisspeptin and GABA provides a functionally important excitatory input to GnRH neurons at the time of ovulation. This neural input releases GABA and/or kisspeptin in the classical frequency dependent and temporally distinct nature of amino acid-neuropeptide cotransmission. Unusually, however, the neuropeptide stream is found to be functionally dominant in activating GnRH neurons at the time of ovulation.