Abstract
The pulsatile pattern of gonadotropin release is critical for puberty and fertility. Kisspeptin neurons in the arcuate nucleus (ARN) are thought to play an important role in generating pulsatile gonadotropin secretion. Previous studies have shown that kisspeptin neurons within the ARN project to gonadotropin-releasing hormone (GnRH) neurons and that the synchronous activation of kisspeptin neurons generates pulsatile LH secretion in vivo. While studies indicated that ARN kisspeptin neurons co-express and are regulated by dynorphin (DYN) and neurokinin B (NKB), the neurotransmitters that control these neurons have not yet been fully explored. Many different internal and external factors, such as changes in stress and metabolic state, modulate pulsatile gonadotropin secretion. I hypothesized the neurotransmitters mediating the effects of stress and metabolism within the brain may act directly on ARN kisspeptin neurons to ultimately regulate pulsatile LH secretion. As such, I aimed to characterise the direct effects of several neurotransmitters upon the ARN kisspeptin neurons. To identify effects, I recorded calcium activity from ARN kisspeptin neurons in acute brain slices prepared from transgenic intact male and female mice expressing the calcium indicator GCaMP6f selectively in ARN kisspeptin neurons. These experiments were conducted in the presence of a voltage-gated sodium channel blocker and ionotropic amino acid receptor antagonists to ensure that only direct effects were measured. The effects of neurotransmitters on intracellular calcium levels in ARN kisspeptin neurons were determined by measuring changes in fluorescence within individual neurons.
In intact diestrous females, experiments demonstrated that NKB, corticotropin-releasing hormone, arginine vasopressin (AVP), oxytocin, noradrenaline (NA), serotonin (5-HT), dopamine, neuropeptide Y, vasoactive intestinal peptide (VIP), and pituitary adenylate cyclase-activating polypeptide (PACAP) all act directly on ARN kisspeptin neurons to increase intracellular calcium levels, whereas DYN and beta-endorphin directly inhibit the stimulatory effects of NKB on these cells. Alpha-melanocyte-stimulating hormone (α-MSH) and GnRH did not exhibit any direct effects on GCaMP6f fluorescence levels in ARN kisspeptin neurons.
I also identified substantial regional and sex differences. Whereas, AVP, 5-HT, VIP, and PACAP all activated a significantly greater percentage of kisspeptin neurons in caudal ARN slices, NA exerted its most potent effects in the middle ARN region. Furthermore, the effect of AVP, 5-HT, VIP, and NA were pronounced in intact females, but significantly reduced, or even absent, in intact males.
These results demonstrate that many of the neurotransmitters thought to be involved in mediating stress and metabolic actions within the brain also directly modulate the ARN kisspeptin neurons. As such, these neurons may provide an integration point through which different modalities regulate pulsatile LH secretion.