GnRH neurons play a pivotal role in the central regulation of fertility. Kisspeptin greatly increases GnRH/LH release and GnRH neuron firing activity and may be involved in estradiol feedback, but the neurobiological mechanisms for these actions are unknown. G protein-coupled receptor 54, the receptor for kisspeptin, is expressed by GnRH neurons as well as other hypothalamic neurons, suggesting both direct and indirect effects are possible. To investigate this and determine whether kisspeptin activation of GnRH neurons is estradiol sensitive, we recorded the firing rate of GnRH neurons in brain slices from adult female mice that were ovariectomized (OVX) and either treated with estradiol (E) capsules (OVX+E) or left without further treatment. Kisspeptin increased GnRH neuronal activity in a dose-dependent manner in cells from both OVX and OVX+E mice, and estradiol significantly potentiated the response. To begin to distinguish direct from indirect actions of kisspeptin, fast synaptic transmission mediated by ionotropic gamma-aminobutyric acid and glutamate receptors was pharmacologically blocked (blockade). Blockade reduced GnRH response to kisspeptin in OVX+E but not in OVX mice. Actions of kisspeptin were also assessed using whole-cell voltage- and current-clamp recording in slices from OVX animals. Kisspeptin application depolarized GnRH neurons in current-clamp and generated inward current in voltage-clamp recordings, even after blocking action potential-dependent neural communication, consistent with a direct effect. Blockers of potassium channels abolished the inward current. Together our data indicate that kisspeptin activates GnRH neurons via both direct and transsynaptic mechanisms and that transsynaptic mechanisms are either enabled and/or potentiated by estradiol.

Layer 1 of the neocortex is an important zone in which synaptic integration of inputs originating from a variety of cerebral regions is thought to take place. Layer 1 does not contain pyramidal cells, and several histochemical studies have suggested that most layer 1 neurons are GABAergic. However, although layer 1 neurons could be an important source of inhibition in this layer, the synaptic action of these neurons and the identity of their postsynaptic targets are unknown. We studied the physiological properties and synaptic interactions of a class of cells within layer 1 called late-spiking (LS) cells. The dendrites and axons of layer 1 LS cells were confined primarily to layer 1. Using paired recording, we showed that LS cells formed GABAergic connections with other LS cells as well as with non-LS cells in layer 1 and with pyramidal cells in layer 2/3. We also found that layer 2/3 pyramidal neurons provide excitatory inputs to LS cells. It has been suggested previously that GABAergic neurons belonging to the same class in the cortex are electrically coupled. In agreement with that hypothesis, we found that LS cells were interconnected by electrical coupling (83%), whereas electrical coupling between LS cells and non-LS cells was infrequent (2%). Thus, we provide evidence showing that a group of GABAergic neurons within layer 1 are specifically interconnected by electrical coupling and can provide significant inhibitory inputs to neurons in layer 1 and to distal dendrites of pyramidal cells.

Gonadotropin-releasing hormone (GnRH) neurons are critical to controlling fertility. In vivo, estradiol can inhibit or stimulate GnRH release depending on concentration and physiological state. We examined rapid, nongenomic effects of estradiol. Whole-cell recordings were made of GnRH neurons in brain slices from ovariectomized mice with ionotropic GABA and glutamate receptors blocked. Estradiol was bath applied and measurements completed within 15 min. Estradiol from high physiological (preovulatory) concentrations (100 pM) to 100 nM enhanced action potential firing, reduced afterhyperpolarizing potential (AHP) and increased slow afterdepolarization amplitudes (ADP), and reduced I AHP and enhanced I ADP . The reduction of I AHP was occluded by previous blockade of calcium-activated potassium channels. These effects were mimicked by an estrogen receptor (ER) ␤-specific agonist and were blocked by the classical receptor antagonist ICI182780. ER␣ or GPR30 agonists had no effect. The acute stimulatory effect of high physiological estradiol on firing rate was dependent on signaling via protein kinase A. In contrast, low physiological levels of estradiol (10 pM) did not affect intrinsic properties. Without blockade of ionotropic GABA and glutamate receptors, however, 10 pM estradiol reduced firing of GnRH neurons; this was mimicked by an ER␣ agonist. ER␣ agonists reduced the frequency of GABA transmission to GnRH neurons; GABA can excite to these cells. In contrast, ER␤ agonists increased GABA transmission and postsynaptic response. These data suggest rapid intrinsic and network modulation of GnRH neurons by estradiol is dependent on both dose and receptor subtype. In cooperation with genomic actions, nongenomic effects may play a role in feedback regulation of GnRH secretion.

A robust surge of gonadotropin-releasing hormone (GnRH) release triggers the luteinizing hormone surge that induces ovulation. The GnRH surge is attributable to estradiol feedback, but the mechanisms are incompletely understood. Voltage-gated calcium channels (VGCCs) regulate hormone release and neuronal excitability, and may be part of the surge-generating mechanism. We examined VGCCs of GnRH neurons in brain slices from a model exhibiting daily luteinizing hormone surges. Mice were ovariectomized (OVX), and a subset was treated with estradiol implants (OVXϩE). OVXϩE mice exhibit negative feedback in the A.M. and positive feedback in the P.M. GnRH neurons express prominent high-voltage-activated (HVA) and small low-voltage-activated (LVA) macroscopic (whole-cell) Ca currents (I Ca ). LVA-mediated currents were not altered by estradiol or time of day. In contrast, in OVXϩE mice, HVA-mediated currents varied with time of day; HVA currents in cells from OVXϩE mice were lower than those in cells from OVX mice in the A.M. but were higher in the P.M. These changes were attributable to diurnal alternations in L-and N-type components. There were no diurnal changes in any aspect of HVA-mediated I Ca in OVX mice. Acute in vitro treatment of cells from OVX and OVXϩE mice with estradiol rapidly increased HVA currents primarily through L-and R-type VGCCs by activating estrogen receptor ␤ and GPR30, respectively. These results suggest multiple mechanisms contribute to the overall feedback regulation of HVA-mediated I Ca by estradiol. In combination with changes in synaptic inputs to GnRH neurons, these intrinsic changes in GnRH neurons may play critical roles in estradiol feedback.

The brain controls fertility through release of gonadotropin-releasing hormone (GnRH), but the mechanisms underlying action potential patterning and GnRH release are not understood. We investigated whether GnRH neurons exhibit afterdepolarizing potentials (ADPs) and whether these are modified by reproductive state. Whole-cell current-clamp recordings of GnRH neurons in brain slices from ovariectomized mice revealed a slow ADP (sADP) after action potentials generated by brief current injection. Generating two or four spikes enhanced sADP amplitude and duration. sADP amplitude was not affected by blocking selected neurotransmitter/neuromodulator receptors, delayed-rectifier potassium channels, calcium-dependent cation channels, or hyperpolarization-activated cation channels but was halved by the calcium channel blocker cadmium and abolished by tetrodotoxin. Cadmium also reduced peak latency. Intrinsic mechanisms underlying the sADP were investigated using voltage-clamp protocols simulating action potential waveforms. A single action potential produced an inward current, which increased after double and quadruple stimulation. Cadmium did not affect current amplitude but reduced peak latency. Pretreatment with blockers of calcium-activated potassium currents (I KCa ) reproduced this shift and blocked subsequent cadmium-induced changes, suggesting cadmium changes latency indirectly by blocking I KCa . Tetrodotoxin abolished the inward current, suggesting that it is carried by sodium. In contrast, I KCa blockers increased the inward current, indicating that I KCa may oppose generation of the sADP. Strong sADPs were suprathreshold, generating repetitive spontaneous firing. I ADP , sADP, and excitability were enhanced by in vivo estradiol, which triggers a preovulatory surge of GnRH release. Physiological feedback modification of this inward current and resulting sADP may modulate action potential firing and subsequent GnRH release.