Secretin Regulates Excitatory GABAergic Neurotransmission to GnRH Neurons via Retrograde NO Signaling Pathway in Mice

Abstract

In mammals, reproduction is regulated by a wide range of metabolic hormones that maintain the proper energy balance. In addition to regulating feeding and energy expenditure, these metabolic messengers also modulate the functional performance of the hypothalamic-pituitary-gonadal (HPG) axis. Secretin, a member of the secretin-glucagon-vasoactive intestinal peptide hormone family, has been shown to alter reproduction centrally, although the underlying mechanisms have not been explored yet. In order to elucidate its central action in the neuroendocrine regulation of reproduction, in vitro electrophysiological slice experiments were carried out on GnRH-GFP neurons in male mice. Bath application of secretin (100 nM) significantly increased the frequency of the spontaneous postsynaptic currents (sPSCs) to 118.0 ± 2.64% compared to the control, and that of the GABAergic miniature postsynaptic currents (mPSCs) to 147.6 ± 19.19%. Resting membrane potential became depolarized by 12.74 ± 4.539 mV after secretin treatment. Frequency of evoked action potentials (APs) also increased to 144.3 ± 10.8%. The secretin-triggered elevation of the frequency of mPSCs was prevented by using either a secretin receptor antagonist (3 μM) or intracellularly applied G-protein-coupled receptor blocker (GDP-β-S; 2 mM) supporting the involvement of secretin receptor in the process. Regarding the actions downstream to secretin receptor, intracellular blockade of protein kinase A (PKA) with KT-5720 (2 μM) or intracellular inhibition of the neuronal nitric oxide synthase (nNOS) by NPLA (1 μM) abolished the stimulatory effect of secretin on mPSCs. These data suggest that secretin acts on GnRH neurons via secretin receptors whose activation triggers the cAMP/PKA/nNOS signaling pathway resulting in nitric oxide release and in the presynaptic terminals this retrograde NO machinery regulates the GABAergic input to GnRH neurons.

Keywords: GABA; GnRH neuron; metabolism; nitric oxide; reproduction; retrograde signaling; secretin.

FIGURE 1

Secretin increases the frequency of sPSC in GnRH neurons. (A) Secretin increased the frequency of the sPSCs with no change in the average amplitude and shape. Average sPSCs next to the recording represent no change in the shape of events after secretin treatment. The inserts below the 15 min recordings are 1-1 min zoomed periods from the recordings before and after secretin administration. The frequency distribution graph under the inserts also reveals that secretin application elevated the sPSC frequency. Cumulative probabilities of the interevent intervals and amplitudes are also presented. (B) The bar graph shows that effect of secretin increasing the sPSC frequency is dose dependent (Student’s t-test ^∗p < 0.05; ^∗∗∗p < 0.001).

FIGURE 2

Secretin (100 nM) increases the frequency of the evoked APs. (A) Representative recording shows that frequency of APs evoked by depolarizing current steps elevated 3 min after secretin administration. Also, the rheobase of APs decreased after secretin treatment. There was no change in the average amplitudes of APs. (B) Representative recording of the effect of secretin in the presence of picrotoxin (C) Secretin results in a significant rise in the frequency of APs after 1 and 3 min of its administration (marked by red ■) in the presence of picrotoxin there was no significant change (marked by ▲). (D) Changes in the R_in represented no significant alteration. (E) Membrane capacitance also showed no significant change. (^∗∗p < 0.01; ^∗∗∗p < 0.001).

FIGURE 3

Secretin elevates the frequency of mPSCs of GnRH neurons directly via secretin receptor. (A) Secretin (100 nM) increased the frequency of mPSCs in GnRH neurons, as shown in a representative recording, the 1 min zoomed periods, the frequency distribution and the cumulative probability of IEIs graphs. There was no change in the average amplitude or in the shape of the events representing the individual PSCs beside the recording. (B) Pretreatment of the brain slice with secretin receptor antagonist (Secretin 5–27) eliminated the effect of secretin on GnRH neurons. (C) Intracellular application of G-protein blocker, GDP-β-S also abolished the effect of secretin. (D) Bar graph shows that the effect of secretin was mediated via the G-protein coupled secretin receptor. (E) Depolarization in the resting potential is demonstrated in a 4 min period. Arrow shows application of secretin. The bottom recording, in the presence of secretin receptor antagonist shows no significant change after the administration of secretin (100 nM) (^∗p < 0.05). The inserts below the 15 min recordings are 1-1 min zoomed periods from the recordings before and after secretin administration. The frequency distribution is also presented under each recording. Average mPSCs next to the recording represent no change in the shape of events after secretin treatment. Cumulative probabilities of the interevent intervals and amplitudes are graphed next to the individual events. Arrow shows the administration of secretin (^∗p < 0.05).

FIGURE 4

Effects of various blockers on the secretin-evoked increase in the frequency of mPSCs in GnRH neurons. (A) Intracellular application of the nNOS blocker NPLA extinguished the effect of secretin. (B) The PKA inhibitor KT5720 applied intracellularly, also eliminated the effect of secretin. (C) Bar graph shows that secretin utilizes PKA- and retrograde NO-coupled signaling mechanisms. The inserts below the 15 min recordings are 1-1 min zoomed periods from the recordings before and after secretin administration. The frequency distribution is also presented under each recording Average mPSCs beside each recording showed change in the shape or amplitudes of events after secretin treatment. Cumulative probabilities of the interevent intervals are graphed next to the individual events. Arrow shows the administration of secretin (^∗p < 0.05).

FIGURE 5

Schematic illustration of secretin receptor signaling in GnRH neurons. Secretin activates cAMP/PKA/nNOS pathway and generates NO that binds to its presynaptic receptor, sGC, located in the GABAergic terminals. This signaling process increases the release of GABA, therefore, facilitates the synaptic inputs to GnRH neurons via GABA_A-receptor. AC, adenylate cyclase; cAMP, cyclic adenosine monophosphate; Gαs, Gβ, Gγ, G-protein subunits; GABA_A-R, GABA_A-receptor; PTX, picrotoxin, selective GABA_A-receptor blocker; PKA, protein kinase A; KT5720, protein kinase A inhibitor; nNOS, neuronal nitric oxide synthase; NPLA, nNOS inhibitor; GDP-β-S,G-protein inhibitor; sGC, soluble guanylyl cyclase, NO receptor. Red lines depict inhibitory actions, green arrows refer to the signal transduction pathway resulting in excitatory action of NO.