Estradiol directly attenuates sodium currents and depolarizing afterpotentials in isolated gonadotropin-releasing hormone neurons.

The gonadotropin-releasing hormone (GnRH) neuron is the pivotal control center in a tightly regulated reproductive axis. The release of GnRH controls estradiol production by the ovary, and estradiol acts at the hypothalamus to regulate GnRH release. However, the mechanisms of estradiol feedback are just beginning to be understood. We have previously shown that estradiol administered to the female mouse modulates sodium currents in fluorescently-labeled GnRH neurons. In the current studies, estradiol (1 nM) was applied directly, for 16-24h, to hypothalamic cultures from young or aged female ovariectomized mice. The direct application of estradiol modulated a tetrodotoxin-sensitive sodium current in isolated GnRH neurons from both young and aged animals. Estradiol, and the specific estrogen receptor-ß agonist DPN, decreased current amplitude measured in the morning (AM), but had no effect on afternoon currents. These compounds also decreased the rise and decay slope of the current response, increased the width of the current, and increased action potential width in AM recordings. In addition, estradiol decreased the amplitude of the depolarizing afterpotential (DAP); this effect was not time-of-day dependent. The ER-ß agonist DPN did not mimic the effect of estradiol on DAPs, and the modulation of DAPs by estradiol was no longer present in cells from postreproductive animals. These results indicate that estradiol can affect the physiology of GnRH neurons via multiple pathways that are differentially regulated during the transition to reproductive senescence, suggesting that estradiol regulation of GnRH neuronal output is modulated during the aging process.

Flufenamic acid modulates multiple currents in gonadotropin-releasing hormone neurons.

Reproduction in mammals is dependent upon the appropriate neurosecretion of gonadotropin-releasing hormone (GnRH), yet the endogenous generation of activity underlying GnRH secretion remains poorly understood. We have demonstrated that the depolarizing afterpotential (DAP), which modulates bursting activity, is reduced in isolated GnRH neurons from aged animals. Calcium-activated non-specific cation (CAN) channels contribute to the DAP in other vertebrate neurosecretory cells. We used the CAN channel blocker flufenamic acid (FFA) to examine the contribution of CAN channels to the DAP in GnRH neurons during aging. Recordings were performed on isolated fluorescent GnRH neurons from young, middle-aged and aged female mice. Flufenamic acid inhibited spontaneous activity, but significantly increased the DAP in neurons from young and middle-aged animals. Apamin did not significantly potentiate the DAP, but did reduce the effects of FFA, suggesting that the increased DAP is partially due to blockade of apamin-sensitive SK channels. Flufenamic acid increased the current underlying the DAP (I(ADP)) and decreased the preceding fast outward current (I(OUT)) at all ages. These current responses were not affected by apamin, but TEA evoked similar changes. Thus, a potassium current, likely mediated through BK channels, contributes to the fast AHP and appears to offset the DAP; this current is sensitive to FFA, but insensitive to age. The effect of FFA on the DAP, but not I(ADP), is diminished in aged animals, possibly reflecting an age-related modulation of the apamin-sensitive SK channel. Future studies will examine the expression of SK channels during the aging process in GnRH neurons.

Estradiol attenuates multiple tetrodotoxin-sensitive sodium currents in isolated gonadotropin-releasing hormone neurons.

Secretion from gonadotropin-releasing hormone (GnRH) neurons is necessary for the production of gametes and hormones from the gonads. Subsequently, GnRH release is regulated by steroid feedback. However, the mechanisms by which steroids, specifically estradiol, modulate GnRH secretion are poorly understood. We have previously shown that estradiol administered to the female mouse decreases inward currents in fluorescently labeled GnRH neurons. The purpose of this study was to examine the contribution of sodium currents in the negative feedback action of estradiol. Electrophysiology was performed on GnRH neurons dissociated from young, middle-aged, or old female mice. All mice were ovariectomized; half were estradiol replaced. The amplitude of the sodium current underlying the action potential was significantly decreased in GnRH neurons from young estradiol-treated animals. In addition, in vivo estradiol significantly decreased the transient sodium current amplitude, but prolonged the sodium current inactivation time constant. Estradiol decreased the persistent sodium current amplitude, and induced a significant negative shift in peak current potential. In contrast to results obtained from cells from young reproductive animals, estradiol did not significantly attenuate the sodium current underlying the action potential in cells isolated from middle-aged or old mice. Sodium channels can modulate cell threshold, latency of firing, and action potential characteristics. The reduction of sodium current amplitude by estradiol suggests a negative feedback on GnRH neurons, which could lead to a downregulation of cell excitability and hormone release. The attenuation of estradiol regulation in peripostreproductive and postreproductive animals could lead to dysregulated hormone release with advancing age.

Age affects spontaneous activity and depolarizing afterpotentials in isolated gonadotropin-releasing hormone neurons.

Neuronal activity underlying the pulsatile secretion of GnRH remains poorly understood, as does the endogenous generation of such activity. It is clear that changes at the level of the hypothalamus are taking place during reproductive aging, yet virtually nothing is known about GnRH neuronal physiology in aging and postreproductive animals. In these studies, we performed cell-attached and whole-cell recordings in GnRH-enhanced green fluorescent protein neurons dissociated from young (3 months), middle-aged (10 months), and old (15-18 months) female mice. All mice were ovariectomized; half were estradiol replaced. Neurons from all ages fired spontaneously, most in a short-burst pattern that is characteristic of GnRH neuronal firing. Membrane characteristics were not affected by age. However, firing frequency was significantly reduced in neurons from old animals, as was spike patterning. The amplitude of the depolarizing afterpotential, evoked by a 200-msec current pulse, was significantly smaller in aged animals. In addition, inward whole-cell currents were reduced in estradiol-treated animals, although they were not significantly affected by age. Because depolarizing afterpotentials have been shown to contribute to prolonged discharges of activity after a very brief excitatory input, a decreased depolarizing afterpotential could lead to attenuated pulses in older animals. In addition, decreases in frequency and pattern generation could lead to improper information coding. Therefore, changes in the GnRH neuron during aging could lead to dysregulated activity, potentially resulting in the attenuated LH pulses observed in the transition to reproductive senescence.

Circadian difference in firing rate of isolated rat suprachiasmatic nucleus neurons.

The suprachiasmatic nucleus (SCN) of the hypothalamus contains the primary circadian clock in mammals. Dissociated SCN neurons in long-term culture exhibit a circadian modulation of spontaneous electrical activity. To evaluate the presence of circadian differences in spontaneous activity of isolated SCN neurons without synaptic connections, dissociated rat SCN neurons were studied with on-cell recording 3-4 days after preparation, before the formation of dendrites, axons and synapses. A day-night difference in spontaneous electrical firing rate was found in acutely dissociated SCN neurons. During the first subjective day, the average firing rate (0.87+/-0.12 Hz) was significantly higher than during the first subjective night (0.24+/-0.06 Hz), while the firing rate on the next day (0.68+/-0.11 Hz) was significantly higher than during the preceding night. These data suggest that populations of isolated SCN neurons with no synaptic interactions contain a functioning circadian clock, and are particularly amenable to biophysical experiments.

Acute dissociation for analyses of NMDA receptor function in cortical neurons during aging.

The present study was designed to determine if functional age differences in the NMDA epsilon2 (NR2B) subunit were detectable at the level of individual cortical neurons. Neurons were acutely dissociated from the frontal and prefrontal cortices of young adult, middle-aged, or old mice, using a combination of proteinase K and trypsin followed by manual trituration. After overnight culture, patch-clamp electrophysiology and rapid perfusion were used to obtain whole-cell responses to 300 microM NMDA, with or without the potent NR2B antagonist ifenprodil. Healthy, phase-bright cortical neurons were isolated from animals of all ages. Cell diameter and capacitance was consistent between ages. We were able to perform kinetic analyses of the NMDA-evoked response, and demonstrated a significant increase in the rate of deactivation with increasing age. In addition, we observed a significant effect of high-concentration ifenprodil on the NMDA-evoked response in old animals. Thus, this method is ideal for the dissociation of neurons from the brain of both young and old animals, and offers a powerful tool for functional analysis at the level of the individual cell.

Episodic bursting activity and response to excitatory amino acids in acutely dissociated gonadotropin-releasing hormone neurons genetically targeted with green fluorescent protein.

The gonadotropin-releasing hormone (GnRH) system, considered to be the final common pathway for the control of reproduction, has been difficult to study because of a lack of distinguishing characteristics and the scattered distribution of neurons. The development of a transgenic mouse in which the GnRH promoter drives expression of enhanced green fluorescent protein (EGFP) has provided the opportunity to perform electrophysiological studies of GnRH neurons. In this study, neurons were dissociated from brain slices prepared from prepubertal female GnRH-EGFP mice. Both current- and voltage-clamp recordings were obtained from acutely dissociated GnRH neurons identified on the basis of EGFP expression. Most isolated GnRH-EGFP neurons fired spontaneous action potentials (recorded in cell-attached or whole-cell mode) that typically consisted of brief bursts (2-20 Hz) separated by 1-10 sec. At more negative resting potentials, GnRH-EGFP neurons exhibited oscillations in membrane potential, which could lead to bursting episodes lasting from seconds to minutes. These bursting episodes were often separated by minutes of inactivity. Rapid application of glutamate or NMDA increased firing activity in all neurons and usually generated small inward currents (<15 pA), although larger currents were evoked in the remaining neurons. Both AMPA and NMDA receptors mediated the glutamate-evoked inward currents. These results suggest that isolated GnRH-EGFP neurons from juvenile mice can generate episodes of repetitive burst discharges that may underlie the pulsatile secretion of GnRH, and glutamatergic inputs may contribute to the activation of endogenous bursts.