Kisspeptin Neuron-Specific and Self-Sustained Calcium Oscillation in the Hypothalamic Arcuate Nucleus of Neonatal Mice: Regulatory Factors of its Synchronization

Abstract

Introduction: Synchronous and pulsatile neural activation of kisspeptin neurons in the arcuate nucleus (ARN) are important components of the gonadotropin-releasing hormone pulse generator, the final common pathway for central regulation of mammalian reproduction. However, whether ARN kisspeptin neurons can intrinsically generate self-sustained synchronous oscillations from the early neonatal period and how they are regulated remain unclear. Objective: This study aimed to examine the endogenous rhythmicity of ARN kisspeptin neurons and its neural regulation using a neonatal organotypic slice culture model. Methods: We monitored calcium (Ca 2+) dynamics in real-time from individual ARN kisspeptin neurons in neonatal organotypic explant cultures of Kiss1-IRES-Cre mice transduced with genetically encoded Ca 2+ indicators. Pharmacological approaches were employed to determine the regulations of kisspeptin neuronspecific Ca 2+ oscillations. A chemogenetic approach was utilized to assess the contribution of ARN kisspeptin neurons to the population dynamics. Results: ARN kisspeptin neurons in neonatal organotypic cultures exhibited a robust synchronized Ca 2+ oscillation with a period of approximately 3 min. Kisspeptin neuron-specific Ca 2+ oscillations were dependent on voltage-gated sodium channels and regulated by endoplasmic reticulum-dependent Ca 2+ homeostasis. Chemogenetic inhibition of kisspeptin neurons abolished synchronous Ca 2+ oscillations, but the autocrine actions of the neuropeptides were marginally effective. Finally, neonatal ARN kisspeptin neurons were regulated by N-methyl-D-aspartate and gamma-aminobutyric acid receptor-mediated neurotransmission. Conclusion: These data demonstrate that ARN kisspeptin neurons in organotypic cultures can generate synchronized and self-sustained Ca 2+ oscillations. These oscillations controlled by multiple regulators within the ARN are a novel ultradian rhythm generator that is active during the early neonatal period.

Deletion of Stim1 in hypothalamic arcuate nucleus Kiss1 neurons potentiates synchronous GCaMP activity and protects against diet-induced obesity

“…The co-release of the two peptide neurotransmitters coordinates the synchronous firing of Kiss1 ARH neurons that drives the pulsatile release of GnRH into the median eminence (Clarkson et al, 2017; Qiu J. et al, 2016). This mode of synchronization appears to be quite different than what has been reported for organotypic cultures of immature kisspeptin neurons where the peptides have a modest effect on Ca 2+ oscillations (Kim et al, 2020).…”

GABAergic Inhibition of Presynaptic Ca2+Transients in Respiratory PreBötzinger Neurons in Organotypic Slice Cultures

“…Thus, earlier variants of GECIs (GCaMP6 fused to synaptophysin) can report on presynaptic Ca 2+ in neurons in primary culture ( Brockhaus et al, 2019 ; Ferron et al, 2020 ), and in the calyx of Held synapse following viral injection of GCaMP6 variants three weeks prior ( Singh et al, 2018 ). Biolistic transfection, electroporation, or AAV transduction of hippocampal, hypothalamic, and cortical organotypic slice cultures with plasmids encoding GCaMP3/6 variants or RFP-based GECIs, result in strong somatodendritic labeling, but no apparent presynaptic labeling ( Podor et al, 2015 ; Shen et al, 2018 ; Gasterstädt et al, 2020 ; Kim et al, 2020 ). Interestingly, jGCaMP7 variants show labeling of presynaptic boutons of the Drosophila larval neuromuscular junction ( Dana et al, 2019 ).…”

New methods to investigate the GnRH pulse generator

“…Genetically encoded calcium indicators combined with single-cell imaging in a neonatal organotypic slice culture model has been used to examine endogenous calcium oscillations in KNDy neurons as well as the regulatory components contributing to calcium oscillation in these neurons’ ex vivo in mice ( Kim et al 2020 ). This type of ex vivo model preserves functional features of the original tissue, although ex vivo incubation can also introduce alterations in neuronal characteristics.…”