Optogenetic activation of GnRH neurons reveals minimal requirements for pulsatile luteinizing hormone secretion

Abstract

The mechanisms responsible for generating the pulsatile release of gonadotropins from the pituitary gland are unknown. We develop here a methodology in mice for controlling the activity of the gonadotropin-releasing hormone (GnRH) neurons in vivo to establish the minimal parameters of activation required to evoke a pulse of luteinizing hormone (LH) secretion. Injections of Cre-dependent channelrhodopsin (ChR2)-bearing adeno-associated virus into the median eminence of adult GnRH-Cre mice resulted in the selective expression of ChR2 in hypophysiotropic GnRH neurons. Acute brain slice experiments demonstrated that ChR2-expressing GnRH neurons could be driven to fire with high spike fidelity with blue-light stimulation frequencies up to 40 Hz for periods of seconds and up to 10 Hz for minutes. Anesthetized, ovariectomized mice had optical fibers implanted in the vicinity of GnRH neurons within the rostral preoptic area. Optogenetic activation of GnRH neurons for 30-s to 5-min time periods over a range of different frequencies revealed that 10 Hz stimulation for 2 min was the minimum required to generate a pulse-like increment of LH. The same result was found for optical activation of GnRH projections in the median eminence. Increases in LH secretion were compared with endogenous LH pulse parameters measured from ovariectomized mice. Driving GnRH neurons to exhibit simultaneous burst firing was ineffective at altering LH secretion. These observations provide an insight into how GnRH neurons generate pulsatile LH secretion in vivo.

Keywords: GnRH; luteinizing hormone; optogenetics; pulse.

Fig. 1.

Transfection of hypophysiotropic GnRH neurons with ChR2. (A) Indicates the strategy of injecting AAV into the region of the median eminence so that ChR2 and mCherry are expressed only in the hypophysiotropic GnRH neurons (red cells) that innervate the median eminence. MS, medial septum. rPOA, rostral preoptic area. (B) Histograms showing the mean + SEM numbers of total GnRH neurons per section (green) detected in the MS, rPOA, and anterior hypothalamic area (AHA) and also the mean numbers of GnRH neurons expressing ChR2 (yellow) overlaid on top. The numbers of ChR2-expressing cells that were not immunoreactive for GnRH are shown in red for the rPOA and AHA. No such cells were detected in the MS. (C) Photomicrographs showing GnRH immunoreactivity (green), mCherry (red), and overlay. White arrows indicate GnRH neurons expressing mCherry.

Fig. 2.

Optogenetic activation of GnRH neurons in vitro. (A) Representative cell-attached, voltage-clamp recordings of ChR2-expressing GnRH neurons activated with 5-ms blue-light pulses given at 5, 10, and 20 Hz for repeated 1-s on (blue bar) and 9-s off time periods. (Inset Right) Expanded view of 1-s stimulations at different frequencies. (B) Histogram showing the mean + SEM evoked spike fidelities at the different stimulation frequencies. A spike fidelity of 100% means that every blue-light stimulation generates an action potential. (C) Representative cell-attached, voltage-clamp recordings of ChR2-expressing GnRH neurons activated continuously for 1 min (blue bar) at 5, 10, and 30 Hz. The expanded traces shown below the 30-Hz stimulation shows the tailing off of spike fidelity over the course of the 1-min stimulation period. (D) Histogram showing the mean + SEM evoked spike fidelities at the different continuous 1-min stimulation frequencies. Different letter superscripts indicate significant differences between the 1-min stimulations in spike fidelity (P < 0.05; parametric one-way ANOVA with post hoc Tukey’s multiple comparisons test). Numbers at base of histograms indicate number of GnRH neurons.

Fig. 3.

Endogenous pulsatile LH secretion in OVX mice. (A and B) Representative examples of pulsatile LH secretion measured in two ovariectomized female C57B6 mice. Stars indicate LH pulses identified by the DynPeak algorithim.

Fig. 4.

Effects of optogenetic activation of GnRH neurons on LH secretion in vivo. (A–C) Representative examples of evoked LH secretion using different frequency blue-light stimulations of GnRH neuron cell bodies given continuously for 5 min (blue bars) in two AAV-ChR2-GnRH-Cre ovariectomized mice. (C) Histogram showing mean + SEM fold changes in LH secretion in response to different patterns and frequencies of blue-light activation of rPOA GnRH neurons. Numbers of mice stimulated with each frequency are given at the base of each histogram. (D–F) Effects of different durations of 10Hz optogenetic activation of GnRH neuron cell bodies. (D and E) Representative examples of evoked LH secretion using 10-Hz blue-light stimulations (blue bars) continuously for 30 s and 2 and 5 min in two AAV-ChR2-GnRH-Cre ovariectomized mice. (F) Histogram showing mean + SEM fold changes in LH secretion in response to different durations. Numbers of mice stimulated with each frequency are given at the base of each histogram. (G–I) Effects of different durations of 10-Hz optogenetic activation of GnRH neuron projections in the median eminence. (G and H) Representative examples of evoked LH secretion using 10-Hz blue-light stimulations (blue bars) applied continuously for 30 s and 2 and 5 min within the ME region in two AAV-ChR2-GnRH-Cre ovariectomized mice. In these experiments, both sides of the ME were stimulated in each mouse. (I) Histogram showing mean + SEM fold changes in LH secretion in response to different durations. Bars with different letter superscripts are significantly different from each other (P < 0.05; one-way ANOVA with post hoc Tukey’s multiple comparisons test). Number of stimulations with each frequency are given at the base of each histogram.

Fig. 5.

Comparison of optogenetic evoked pulse-like increments in LH secretion and endogenous LH pulses. (A) Dynamics of continuous 10-Hz 2- and 5-min evoked LH increments by stimulating the cell bodies (gray, black lines) or the distal projections (yellow, blue lines) of GnRH neurons compared with endogenous LH pulses (red) overlaid upon one another. Mean + SEM values are shown. (B) Dynamics of evoked and endogenous LH pulses after normalizing values to the peak LH responses for each response.