Mechanism of Arcuate Kisspeptin Neuron Synchronization in Acute Brain Slices From Female Mice

Abstract

The mechanism by which arcuate kisspeptin (ARNKISS) neurons co-expressing glutamate, neurokinin B, and dynorphin intermittently synchronize their activity to drive pulsatile hormone secretion remains unclear in females. In order to study spontaneous synchronization within the ARNKISS neuron network, acute brain slices were prepared from adult female Kiss1-GCaMP6 mice. Analysis of both spontaneous synchronizations and those driven by high frequency stimulation of individual ARNKISS neurons revealed that the network exhibits semi-random emergent excitation dependent upon glutamate signaling through AMPA receptors. No role for NMDA receptors was identified. In contrast to male mice, ongoing tachykinin receptor tone within the slice operated to promote spontaneous synchronizations in females. As previously observed in males, we found that ongoing dynorphin transmission in the slice did not contribute to synchronization events. These observations indicate that a very similar AMPA receptor-dependent mechanism underlies ARNKISS neuron synchronizations in the female mouse supporting the "glutamate two-transition" model for kisspeptin neuron synchronization. However, a potentially important sex difference appears to exist with a more prominent facilitatory role for tachykinin transmission in the female.

Keywords: arcuate nucleus; dynorphin; glutamate; kisspeptin; luteinizing hormone; neurokinin B; pulsatility; synchronization.

Figure 1.

Spatiotemporal analysis of ARN^KISS neuron synchronization. A & B, Two representative examples showing the temporal relationships between individual ARN^KISS neurons (14 and 12 cells) across 5 and 6 miniature synchronization events (SE1-6). The heatmap codes for order with early neurons in dark blue and late neurons in light blue. Empty squares indicate that the cell did not participate in the SE. A correlation scatterplot of the same information is provided below. Alongside is given the spatial location of individual ARN^KISS neurons in the coronal imaging plane showing their sequence of activity for the third (A) and second (B) mSE.

Figure 2.

Glutamatergic signaling is essential for ARN^KISS neuron synchronization in vitro. A, Example brain slice control experiment showing GCaMP fluorescence recorded from 14 neurons with a 15-minute vehicle application. mSEs are indicated by blue bars, with dark blue indicating mSEs consisting of more neurons and light blue indicating fewer. The key indicates exact number of cells taking part. B, Example brain slice showing the effect of 15-minute exposure to CNQX (20 μM) on GCaMP fluorescence recorded from 14 neurons. C–F, Histograms showing the individual data points and mean (+SEM) mSE rate for the pre-drug, drug-applied, and wash periods in response to vehicle, CNQX + DAP5, CNQX alone, and DAP5 alone in slices from diestrous female mice (*P < .05, **P < .01, Wilcoxon).

Figure 3.

NKB signaling also contributes to ARN^KISS neuron synchronization in female mice. A, Example brain slice showing the effect of 15-minute exposure to the tachykinin receptor antagonist cocktail (SDZ-NKT 343 1 µM, GR94800 1 µM, SB 222200 3 µM) on GCaMP fluorescence recorded from 9 neurons. The mSEs are indicated by blue bars, with dark blue indicating mSEs consisting of more neurons and light blue indicating fewer. The key indicates the exact number of cells taking part. B & C, Histograms showing the individual data points and mean (±SEM) mSE rate for the pre-drug, drug-applied, and wash periods in response to the tachykinin receptor antagonist cocktail or NorBNI (10 µM) in slices from diestrous female mice (∗P < .05, Wilcoxon).

Figure 4.

Effects of high and low frequency activation of single ARN^KISS neurons on other cells. A, Representative examples of low- and high-frequency firing evoked in a patched ARN^KISS neuron by current injection over 10-15 seconds. Note the marked spike frequency adaptation during high frequency stimulation. B, Histograms showing the percentage of trials in which coincident calcium events occurred between ARN^KISS neurons in the brain slice under unstimulated conditions and following low frequency and high frequency stimulation of a single neuron in the absence and presence of CNQX or neurokinin receptor (NKR) antagonists. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, Binomial test vs Control or as indicated; number in base gives the number of stimulation trials for each group (CNQX, 15 brain slices; NKR 11 brain slices).

Figure 5.

Direct electrical excitation of one ARN^KISS neuron increases GCaMP activity in nearby ARN^KISS neurons in an AMPA receptor–dependent manner. A, Example experiments showing GCaMP traces from 9 ARN^KISS neurons in the same slice as Neuron #10 (bottom trace) that was patched. Artificial activation of Neuron #10 at high (HF; maximal) and low (LF; mean 2.5 Hz) frequencies are highlighted in pink and green respectively, which are continued through CNQX (20 μM) application. Above-threshold GCaMP calcium transients are highlighted in blue. B, Example experiments showing GCaMP traces from 9 ARN^KISS neurons in the same slice as Neuron #10 (bottom trace) that was patched. Artificial activation of Neuron #10 at high (HF; maximal) frequency are highlighted in pink, which are continued through the application of a cocktail of neurokinin receptor (NK1-3R) antagonists. Above-threshold GCaMP calcium transients are highlighted in blue.