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Calcium and Small-Conductance Calcium-Activated Potassium Channels in Gonadotropin-Releasing Hormone Neurons before, during, and after Puberty

Section of Endocrinology, Department of Pediatrics, University of Chicago, Chicago, Illinois 60637-1470

Address all correspondence and requests for reprints to: Daniel J. Spergel, Section of Endocrinology, Diabetes, and Metabolism, Department of Medicine, University of Chicago, 5841 South Maryland Avenue, MC 1027, Chicago, Illinois 60637-1470. E-mail: dspergel{at}uchicago.edu.

	Abstract

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The pubertal increase in GnRH secretion resulting in sexual maturation and reproductive competence is a complex process involving kisspeptin stimulation of GnRH neurons and requiring Ca²⁺ and possibly other intracellular messengers. To determine whether the expression of Ca²⁺ channels, or small-conductance Ca²⁺-activated K⁺ (SK) channels, whose activity reflects cytoplasmic free Ca²⁺ concentration, changes at puberty in GnRH neurons, Ca²⁺ and SK currents in GnRH neurons were recorded in brain slices of juvenile [postnatal day (P) 10–21], pubertal (P28–P42), and adult (P56) male GnRH-green fluorescent protein transgenic mice using perforated-patch and whole-cell techniques. Ca²⁺ currents were inhibited by the Ca²⁺ channel blocker Cd²⁺ and showed marked heterogeneity but were on average similar in juvenile, pubertal, and adult GnRH neurons. SK currents, which were inhibited by the SK channel blocker apamin and enhanced by the SK and intermediate-conductance Ca²⁺-activated K⁺ channel activator 1-ethyl-2-benzimidazolinone, were also on average similar in juvenile, pubertal, and adult GnRH neurons. These findings suggest that whereas Ca²⁺ and SK channels may participate in the pubertal increase in GnRH secretion and there may be changes in Ca²⁺ or SK channel subtypes, overall Ca²⁺ and SK channel expression in GnRH neurons remains relatively constant across pubertal development. Hence, the expected increase in GnRH neuron cytoplasmic free Ca²⁺ concentration required for increased GnRH secretion at puberty appears to be due to mechanisms other than altered Ca²⁺ or SK channel expression, e.g. increased membrane depolarization and subsequent activation of preexisting Ca²⁺ channels after increased excitatory synaptic input.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
PUBERTY, THE PERIOD between childhood and adulthood during which sexual maturity and reproductive competence are attained, begins with an increase in the pulsatile release of the decapeptide GnRH from a network of approximately 800 mainly hypothalamic GnRH-synthesizing neurons into the portal vasculature connecting the hypothalamus and anterior pituitary (1, 2, 3). GnRH binds to GnRH receptors on pituitary gonadotrophs and stimulates the release into the general circulation of LH and FSH, which are required for gonadal steroid secretion and the production of mature gametes in males and females. The GnRH neurosecretory system is active during the neonatal period in primates and rodents but in primates enters a dormant state in the juvenile period. At puberty there is a gonadal-independent increase in the amplitude and frequency of GnRH and LH pulses to adult levels, which represents a reactivation or reawakening of the GnRH neurosecretory system in primates and a further activation in rodents (3, 4, 5, 6, 7, 8).

One approach to elucidate the mechanism of the pubertal increase in GnRH secretion is to investigate the pubertal increase in cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i) in GnRH neurons. Increased [Ca²⁺]_i, perhaps accompanied by changes in other intracellular messengers including cAMP, cGMP, and lipid-derived signaling molecules, is required for increased GnRH secretion in GnRH neurons (9, 10, 11, 12), probably for secretory vesicle or granule docking and fusion, as in other neurons and endocrine cells (13, 14). The pubertal increase in GnRH secretion depends on the stimulatory actions of the neurotransmitter kisspeptin via the G protein-coupled receptor 54 (15, 16) and presumably occurs by Ca²⁺ entry through voltage-gated Ca²⁺ channels or by Ca²⁺ release from intracellular stores (10, 11, 12, 13, 14, 17) after kisspeptin excitation of GnRH neurons. Kisspeptin (15, 16), along with other neurotransmitters and hormones (18, 19, 20, 21, 22, 23), may convey information from presynaptic neurons about age, growth, availability of metabolic fuels such as glucose and fats (perhaps through insulin and leptin), circadian rhythm, and other factors.

Two groups demonstrated that postnatal GnRH neurons express voltage-gated Ca²⁺ channels, but whether total Ca²⁺ channel expression in GnRH neurons changes at puberty remained unclear. One of the groups (24) used whole-cell recording of acutely dissociated green fluorescent protein (GFP)-labeled GnRH neurons from gonadal-intact juvenile, aged postnatal day (P) 4–10, and ovariectomized adult female GnRH-GFP mice. They found that maximum, peak Ca²⁺ current density (i.e. maximum, peak Ca²⁺ current divided by cell capacitance, which is proportional to membrane surface area) increased significantly (P < 0.025) at puberty from 21.0 ± 2.1 (n = 10 juvenile GnRH neurons) to 28.4 ± 2.2 pA/pF (n = 17 adult GnRH neurons), which suggested increased Ca²⁺ channel expression. However, the other group (25), using perforated-patch recordings of overnight cultures of GFP-labeled GnRH neurons from gonadal-intact neonatal/juvenile (P1-P7) and pubertal (P35-P40) male and female GnRH-GFP transgenic rats, found no change (P > 0.05) in maximum, peak Ca²⁺ current density.

GnRH neurons also appear to express voltage-independent Ca²⁺-activated K⁺ [K(Ca)] channels, which have no intrinsic voltage dependence but do obtain voltage dependence from the voltage dependence of Ca²⁺ entry through Ca²⁺ channels (13, 26, 27). In other cells, voltage-independent K(Ca) channels aid in the prolonged afterhyperpolarization after action potential firing and associated Ca²⁺ influx, and they participate in rhythmic electrical activity (13, 26, 27). They may regulate the frequency of action potential firing in GnRH neurons, determining the subsequent amount of Ca²⁺ influx and GnRH secretion. K(Ca) channel activity in GnRH neurons may change developmentally due to changes in Ca²⁺ channel expression/activity or K(Ca) channel expression as in some other neurons (28, 29, 30). Two types of voltage-independent K(Ca) currents may be responsible for the afterhyperpolarization, an apamin (a toxin from honeybee venom)-sensitive, medium afterhyperpolarization current with a decay time constant in the range of 100–200 msec, which is mediated by small-conductance (SK) K(Ca) channels, and an apamin-insensitive slow afterhyperpolarization current with a decay time constant ranging from hundreds of milliseconds to several seconds (26, 27). Immortalized mouse (31) and adult female guinea pig (32) GnRH neurons have been reported to express SK channels. However, because they are derived from tumors of embryonic GnRH neurons (33), immortalized GnRH neurons are immature neurons and do not permit the study of developmental changes. In the guinea pig study (32), although SK channel subunit mRNA was found to be expressed, it is unclear whether the afterhyperpolarizations recorded were mediated by SK channels or apamin-insensitive K(Ca) channels because apamin was not tested on GnRH neurons. Recently Kato et al. (34) reported that apamin inhibits a slow rather than a medium afterhyperpolarization current in overnight cultures of GnRH neurons from gonadal-intact adult female rats, providing the first evidence for the expression of functional SK channels in postnatal GnRH neurons. Nevertheless, the question of whether SK channel expression or activity in GnRH neurons changes at puberty was not addressed.

To determine whether Ca²⁺ or SK currents change at puberty and whether the discrepant results reported previously (24, 25) were due to species differences (rat vs. mouse) or recording technique (perforated patch vs. whole cell), and preserving more of the dendritic and axonal processes that may contribute to channel activity and avoiding artifacts that may arise from cell dissociation and culture, perforated-patch and whole-cell recordings of Ca²⁺ or SK currents in GnRH neurons in brain slices of juvenile, pubertal, and adult male GnRH-GFP mice were obtained. Males, rather than females, were selected to avoid possible confounding effects of estrous cycle stage on Ca²⁺ or SK currents and because neither Ca²⁺ nor SK currents in GnRH neurons of male mice had been previously characterized. No significant differences were found in Ca²⁺ or SK currents between GnRH neurons of juvenile, pubertal, and adult males, suggesting that Ca²⁺ and SK channel expression in GnRH neurons remains relatively constant across pubertal development.

	Materials and Methods

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Use and care of mice
Juvenile (P10–P21), pubertal (P28–P42), and adult (P56) male homozygous GnRH-GFP transgenic mice in which GFP is genetically targeted to GnRH neurons (35) were used for experiments. The time periods associated with the juvenile, pubertal, and adult groups are based on previously observed stages of mouse reproductive development (36) and were chosen to avoid borderline periods in which it may be difficult to distinguish between groups. To confirm pubertal development during these time periods, testes from some of the GnRH-GFP mice were removed and weighed. Testes weights (wet weights of combined testes) were 39.4 ± 4.7, 170.3 ± 10.7, and 205.8 ± 6.0 mg for P21, P42, and P88 mice (n = 3 mice at each age), respectively, the greater than 4-fold higher testicular weight at P42 compared with P21 indicating that pronounced testicular growth occurs between P21 and P42 in GnRH-GFP mice. Mice were housed in a temperature (22 C)- and light (on 0600–1800 h)-controlled room with ad libitum access to food and water. All procedures were approved by the Animal Care and Use Committee of the University of Chicago and conducted within the guidelines of the National Research Council publication Guide for Care and Use of Laboratory Animals.

Brain slice preparation for electrophysiology experiments
Mice were anesthetized with isoflurane (Baxter Healthcare, Deerfield, IL) and then decapitated. Brains were removed, dissected in ice-cold Ringer’s solution gassed with 95% O₂-5% CO₂, containing (in mM) 125 NaCl, 25 NaHCO₃, 1.25 NaH₂PO₄, 2.5 KCl, 2 CaCl₂, 1 MgCl₂, and 25 glucose, 316 mOsm (pH 7.4), and cut sagittally into 300-µm-thick slices with a vibratome (Campden Instruments Integraslice 7550MM; Lafayette Instruments, Lafayette, IN) as described (35). Slices were transferred using the back end of a Pasteur pipette to an incubation chamber with Ringer’s solution (gassed with 95% O₂-5% CO₂) for at least 30 min at 35 C and then stored at room temperature (22 C) in the same chamber until electrophysiological recording.

Fluorescence and infrared microscopy for visualizing and recording from GFP-labeled GnRH neurons
For visualizing and recording from GFP-labeled GnRH neurons, slices were transferred to a 3-ml, temperature-controlled recording chamber (Luigs and Neumann, Ratingen, Germany), fixed in place with a grid, and superfused at a rate of 1 ml/min with Ringer’s solution. Slices were viewed with an upright, motorized fluorescence microscope (Axioskop 2 FS; Zeiss Microimaging, Thornwood, NY) equipped with an infrared filter set and a Senarmont system for differential interference contrast, first in bright field with a x5 objective (Plan-Neofluar; Zeiss). To visualize GFP, the white light from the bright-field halogen lamp was blocked and a fluorescent lamp (HBO 100 W; Osram, Berlin, Germany) was switched on. The intensity of the fluorescent lamp was regulated by a FluoArc power supply (Zeiss). GFP filter set 41017, consisting of excitation filter HQ470/40, dichroic mirror Q495LP, and emission filter HQ525/50 (Chroma Technology, Brattleboro, VT), was used. Upon locating a fluorescent neuron with the x5 objective, a x40 (0.8 NA) water-immersion objective (Achroplan IR; Zeiss) was used for further imaging and to view cells for perforated-patch and whole-cell recording. Infrared differential interference contrast imaging was performed subsequent to fluorescence observation. After viewing a fluorescent neuron, magnification was increased by x2.5 with an intermediate phototube (Optovar; Zeiss), the light from the fluorescent lamp was blocked, and infrared light transmitted via the Senarmont system was directed to a near-infrared charge-coupled device camera (C7500–50; Hamamatsu Photonic Systems, Bridgewater, NJ). A neuron viewed with infrared optics was considered to be the same as that viewed with fluorescence optics when the infrared image and the fluorescent image of the neuron had the same position and orientation through the eyepiece of the microscope (fluorescent image) and with the infrared imaging system (infrared image).

Perforated-patch and whole-cell recording of Ca²⁺ and SK currents
Nystatin perforated-patch and conventional whole-cell recordings were used to assess Ca²⁺ and SK channel activity. Ca²⁺ and SK currents were recorded from GFP-labeled neurons in the diagonal band of Broca and preoptic area as described (35). Experiments were performed at 35 C.

The bath solution for recording Ca²⁺ currents consisted of (in mM) 117.5 NaCl, 25 NaHCO₃, 1.25 NaH₂PO₄, and 10 tetraethylammonium (TEA)-Cl (to block K⁺ channels), 2 CaCl₂, 1 MgCl₂, and 25 glucose, gassed with 95% O₂-5% CO₂ (pH 7.4, 307 mOsm) and supplemented with 0.5 µM tetrodotoxin (TTX; to block TTX-sensitive Na⁺ channels), 50 µM kynurenic acid (to block ionotropic glutamate receptors), and 50 µM picrotoxin (to block A-type -aminobutyric acid receptors). Substituting 2.5 mM Cs⁺, which blocks K⁺ channels as well as hyperpolarization-activated cation channels (13), for 2.5 of the 117.5 mM NaCl did not affect Ca²⁺ currents (data not shown).

The pipette solution for perforated-patch recording of Ca²⁺ currents contained (in mM) 130 Cs-MeSO₃, 5 CsCl, 10 TEA-Cl, 5 NaCl, 1 MgCl₂, and 10 HEPES (pH 7.28, 284 mOsm). Pipettes were briefly immersed in this solution and then backfilled with the same solution supplemented with 500 µg/ml nystatin. The latter was prepared fresh for each experiment by dissolving 2 mg nystatin in 2 µl dimethyl sulfoxide in a microfuge tube, adding 998 µl pipette solution to obtain a 2 mg/ml nystatin stock solution, sonicating for 1 min, placing 250 µl of the stock solution in a second tube containing 750 µl pipette solution, filtering with a syringe filter, and then storing on ice and in the dark until use. Nystatin was selected as the membrane perforating substance for perforated-patch recordings rather than amphotericin B, which had been used previously to record Ca²⁺ and SK currents in GnRH neurons (25, 34) because nystatin had been reported to perforate faster at similar concentrations, thereby reducing the time required for complete opening of the pores and for initiation of experiments (37). Complete opening in the present study, as assessed by monitoring series resistance, was achieved within 5–20 min after gigaseal formation.

The pipette solution for whole-cell recording of Ca²⁺ currents consisted of (in mM) 110 CsCl, 30 TEA-Cl, 5 NaCl, 1 CaCl₂, 2 Mg-ATP, 10 EGTA, and 10 HEPES (pH 7.25, 295 mOsm).

The bath solution for perforated-patch recording of SK currents consisted of (in mM) 125 NaCl, 25 NaHCO₃, 1.25 NaH₂PO₄, 2.5 KCl, 2 CaCl₂, 1 MgCl₂, and 25 glucose, equilibrated with 95% O₂-5% CO₂ (pH 7.4, 316 mOsm) and supplemented with 50 µM kynurenic acid and 50 µM picrotoxin.

The pipette solution for perforated-patch recording of SK currents contained (in mM) 130 K-acetate, 15 KCl, 5 NaCl, 1 MgCl₂, and 10 HEPES (pH 7.27, 293 mOsm). As with the pipette solution for perforated-patch recording of Ca²⁺ currents, pipettes were briefly immersed in this solution and then backfilled with the same solution supplemented with 500 µg/ml nystatin.

Recording pipettes were made from thick-walled borosilicate glass capillary tubes (length of 75 mm, outer diameter of 2 mm, inner diameter of 1 mm, and wall thickness of 0.5 mm; Hilgenberg, Malsfeld, Germany) using a P-97 Flaming/Brown micropipette puller (Sutter Instrument, Novato, CA) and had resistances ranging from 3 to 5 M when filled with either of the two pipette solutions. Pipettes were connected via an Ag-AgCl wire to the headstage of an EPC-10 patch clamp amplifier (HEKA Electronics, Mahone Bay, Nova Scotia, Canada). The reference electrode was an Ag-AgCl pellet (IVM, Healdsburg, CA) immersed in bath solution. The EPC-10 amplifier and PatchMaster software (HEKA) were used to acquire (10 kHz), filter (Bessel, 2.9 kHz), and analyze patch clamp data, which were stored on a Power Macintosh G4 computer. The patch clamp amplifier was also used to compensate pipette and cell capacitance. Series resistance was uncompensated but was always less than 50 M in recordings selected for analysis. Series resistance (mean ± SD) in perforated-patch recordings of Ca²⁺ and SK current was 26.2 ± 8.5 M for juvenile (n = 23), 16.7 ± 8.6 M for pubertal (n = 21), and 26.3 ± 8.9 M for adult (n = 20) GnRH neurons. Series resistance (mean ± SD) in whole-cell recordings of Ca²⁺ current was 21.5 ± 11.2 M for juvenile (n = 15), 12.7 ± 2.5 pF for pubertal (n = 14), and 22.9 ± 12.0 M for adult (n = 14) GnRH neurons. The similar values of series resistance in recordings from juvenile and adult GnRH neurons suggest that uncompensated series resistance did not mask possible changes in Ca²⁺ or SK current during pubertal development. Cell capacitance (mean ± SD) was 11.1 ± 2.3 pF for juvenile (n = 41), 12.3 ± 2.7 pF for pubertal (n = 35), and 12.6 ± 3.4 pF for adult (n = 34) GnRH neurons (values from perforated-patch and whole-cell recordings pooled). Capacitative and leak currents were subtracted using a P/4 protocol. Traces were processed for presentation using Igor Pro 4.0 (Wavemetrics, Lake Oswego, OR) and Canvas 8.0.5 software (Deneba Systems, Miami, FL).

Drug application
To inhibit Ca²⁺ or SK channel activity or enhance SK channel activity, CdCl₂ (200 µM) apamin (10 or 100 nM), or 1-ethyl-2-benzimidazolinone (1-EBIO; 200 µM) was added to the bath solution as indicated. After the establishment of a perforated-patch or whole-cell recording, brain slices were superfused (at a rate of 1 ml/min) for at least 10 min with bath solution lacking these drugs to record control Ca²⁺ or SK currents and then for 10 min with bath solution containing these drugs to record the Ca²⁺ or SK currents in their presence.

Statistics
Data are expressed as mean ± SEM unless indicated otherwise. Statistical comparisons were performed using Kruskal-Wallis one-way ANOVA for multiple independent groups (38) with the help of GB-STAT 5.06 software (Dynamic Microsystems, Silver Spring, MD). A difference between groups was significant if P obtained from the ² distribution associated with the Kruskal-Wallis one-way ANOVA was less than 0.05.

Reagents
Except for 1-EBIO and TTX, which were obtained from Tocris Bioscience (Ellisville, MO), all reagents used in this study were purchased from Sigma-Aldrich (St. Louis, MO).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ca²⁺ currents in juvenile, pubertal, and adult GnRH neurons
Ca²⁺ currents, evoked by voltage steps from –60 mV, near the resting membrane potential (16, 35, 39, 40), to membrane potentials of –50 to +20 mV in nystatin perforated-patch recordings from GnRH neurons in brain slices of male GnRH-GFP mice were inward, rapidly activating, slowly inactivating, and rapidly deactivating (Fig. 1A). Ca²⁺ channel activity was inhibited by the broad-spectrum Ca²⁺ channel blocker Cd²⁺ (Fig. 1B) and had a V-shaped peak current-voltage relationship (Fig. 1C) as in dissociated GnRH neurons (24, 25). Activity began around a membrane potential of –50 mV, was highest at membrane potentials between –20 and 0 mV, and declined at more depolarized potentials. Ca²⁺ channel activity evoked from a holding potential of –100 mV was similar to that evoked from –60 mV and slightly larger than that evoked from –40 mV (Fig. 1, D–F). At 0 mV in adult GnRH neurons, for example, averaged peak Ca²⁺ current densities were –33 ± 3, –35 ± 3, and –29 ± 2 pA/pF from holding potentials of –100, –60, and –40 mV, respectively (n = 11 GnRH neurons from four mice). The weak dependence of the channel activity on holding potential suggests that Ca²⁺ currents in GnRH neurons are carried mainly by high-voltage activated Ca²⁺ channels (13), in accord with previous findings (24, 25).

View larger version (16K): [in this window] [in a new window]   FIG. 1. GnRH neurons express Ca2+ channels. A, Ca2+ currents in an adult GnRH neuron evoked by 50-msec voltage pulses at 1 Hz from a holding potential (Vh) of –60 mV to membrane potentials (Vm) of –80, –70, –60, –50, –40, –30, –20, –10, 0, +10, and +20 mV. B, Inhibition of Ca2+ currents in the GnRH neuron in A by the broad-spectrum Ca2+ channel inhibitor Cd2+. Outward currents in B are most likely Cs+ currents. C, Current-voltage (I-V) relationship for the Ca2+ channel activity in A and B. D, Ca2+ currents in a different adult GnRH neuron from that in A, evoked from a Vh of –100 mV. E, Ca2+ currents from the same GnRH neuron as in D, evoked from a Vh of –60 mV. F, Ca2+ currents from the same neuron as in D, evoked from a Vh of –40 mV. Note that changing Vh from –100 to –60 or –40 mV had little effect on Ca2+ currents.

View larger version (13K): [in this window] [in a new window]   FIG. 2. Ca2+ currents vary markedly in amplitude among juvenile, pubertal, and adult GnRH neurons. A, Ca2+ currents in a juvenile GnRH neuron. B, Ca2+ currents in a different juvenile GnRH neuron than in A. C, Ca2+ currents in a pubertal GnRH neuron. D, Ca2+ currents in a different pubertal GnRH neuron than in C. E, Ca2+ currents in an adult GnRH neuron. F, Ca2+ currents in a different adult GnRH neuron than in E. These traces illustrate the wide range of Ca2+ current amplitudes in GnRH neurons. The voltage protocol, shown in A and used to obtain the traces in A–F, is identical with that in Fig. 1, A, B, and E.

View larger version (19K): [in this window] [in a new window]   FIG. 3. Averaged Ca2+ currents are similar in juvenile, pubertal, and adult GnRH neurons in perforated-patch recordings. A, Ca2+ currents in a juvenile GnRH neuron. B, Ca2+ currents in a pubertal GnRH neuron. C, Ca2+ currents in an adult GnRH neuron. The currents in A–C are representative of the averaged Ca2+ currents in juvenile, pubertal, and adult GnRH neurons in perforated-patch recordings, obtained using the same voltage protocol as in Figs. 1, A, B, and E, and 2. D, Averaged peak Ca2+ current density as a function of membrane potential (Vm) in juvenile, pubertal, and adult GnRH neurons. E, Averaged sustained Ca2+ current density as a function of Vm in juvenile, pubertal, and adult GnRH neurons. F, Averaged tail Ca2+ current density as a function of Vm in juvenile, pubertal, and adult GnRH neurons. P, Peak; S, sustained; T, tail.

However, for the most part, and as with the Ca²⁺ currents recorded using the perforated-patch technique, there were no significant differences in averaged Ca²⁺ currents between juvenile (Fig. 4A), pubertal (Fig. 4B), and adult (Fig. 4C) GnRH neurons in whole-cell recordings. Averaged peak (Fig. 4D), sustained (Fig. 4E), and tail (Fig. 4F) Ca²⁺ current densities were similar (P > 0.05) between juvenile (n = 15 GnRH neurons from four mice), pubertal (n = 14 GnRH neurons from four mice), and adult (n = 14 GnRH neurons from four mice) GnRH neurons from –50 to +20 mV in whole-cell recordings. Peak Ca²⁺ current densities at 0 mV, for example, were –26.3 ± 3.3, –29.4 ± 2.8, and –25.8 ± 3.5 pA/pF in juvenile, pubertal and adult GnRH neurons, respectively, in whole-cell recordings. Unlike in perforated-patch recordings, there were significant differences (P < 0.05) in peak Ca²⁺ current density between juvenile and pubertal GnRH neurons at –30 and –20 mV, at or near the steepest part of the V-shaped current-voltage relationship, although not between juvenile and adult or between pubertal and adult GnRH neurons. These differences may stem from the greater variability in peak current amplitude in whole-cell recordings. Also unlike in perforated-patch recordings, there was a small but significant pubertal leftward shift (P < 0.05) in the voltage required to elicit maximum, peak current (from –6.0 ± 1.2 mV in juvenile to –12.1 ± 1.9 mV in pubertal and –12.1 ± 1.5 mV in adult GnRH neurons). Nonetheless, taken together with the perforated-patch data, these whole-cell data suggest that Ca²⁺ channel expression in GnRH neurons remains relatively constant across pubertal development.

View larger version (20K): [in this window] [in a new window]   FIG. 4. Averaged Ca2+ currents are similar in juvenile, pubertal, and adult GnRH neurons in whole-cell recordings. A, Ca2+ currents in a juvenile GnRH neuron. B, Ca2+ currents in a pubertal GnRH neuron. C, Ca2+ currents in an adult GnRH neuron. The currents in A–C are representative of the averaged Ca2+ currents in juvenile, pubertal, and adult GnRH neurons in whole-cell recordings, obtained using the same voltage protocol as in Figs. 1, A, B, and E, 2, and 3. D, Averaged peak Ca2+ current density as a function of membrane potential (Vm) in juvenile, pubertal, and adult GnRH neurons. E, Averaged sustained Ca2+ current density as a function of Vm in juvenile, pubertal, and adult GnRH neurons. F, Averaged tail Ca2+ current density as a function of Vm in juvenile, pubertal, and adult GnRH neurons. P, Peak; S, sustained; T, tail.

View larger version (12K): [in this window] [in a new window]   FIG. 5. GnRH neurons express SK channels. A, SK current, recorded as the tail current upon repolarization to –60 mV after a 100-msec depolarizing pulse from –60 to +60 mV, in the absence and presence of 10 nM apamin, an SK channel inhibitor. B, SK current recorded from a different GnRH neuron than in A, in the absence and presence of 100 nM apamin. C, SK current, recorded from a different GnRH neuron than those in A and B in the absence and presence of the SK and IK channel activator 1-EBIO. D, Averaged SK current inhibition by apamin and activation by 1-EBIO.

However, again like the Ca²⁺ currents, there were no significant differences in averaged SK current between juvenile (Fig. 6A), pubertal (Fig. 6B), and adult (Fig. 6C) GnRH neurons in perforated-patch recordings. Averaged SK current density was similar (P > 0.05) among juvenile, pubertal, and adult GnRH neurons (Fig. 6D). Because SK channel activity in GnRH and other neurons reflects changes in [Ca²⁺]_i resulting from Ca²⁺ channel activity (27, 34), the lack of significant differences in SK current in GnRH neurons among juvenile, pubertal, and adult GnRH neurons was consistent with the lack of significant differences in total Ca²⁺ current and provided a further indication that Ca²⁺ channel expression in GnRH neurons remains relatively constant across pubertal development.

View larger version (8K): [in this window] [in a new window]   FIG. 6. Averaged SK current is similar in juvenile, pubertal, and adult GnRH neurons. A, SK current in a juvenile GnRH neuron. B, SK current in a pubertal GnRH neuron. C, SK current in an adult GnRH neuron. D, Averaged peak amplitude of SK current in juvenile, pubertal, and adult GnRH neurons. The voltage protocol, shown in A and used to obtain the traces in A–C is identical with that in Fig. 5, A–C.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The identities of the Ca²⁺ and SK currents recorded from GnRH neurons in brain slices in the present study were confirmed with pharmacological agents. Ca²⁺ currents were inhibited by the broad spectrum Ca²⁺ channel blocker Cd²⁺. SK currents were inhibited by the SK channel blocker apamin and enhanced by the SK and IK channel activator 1-EBIO.

The Ca²⁺ and SK currents in the present study varied markedly in their peak amplitude independent of the age of the GnRH neuron from which they were recorded. Variation in peak amplitude may be due to heterogeneity in channel expression, episodic changes in channel activity linked to GnRH pulsatility, or differences among GnRH neurons in the contributions of dendritic and axonal channels to channel activity, depending on the degree to which dendritic and axonal processes were retained during slicing. Previous whole-cell current-clamp recordings of evoked membrane potential changes in GnRH neurons suggested heterogeneity in Ca²⁺ channel expression in a development-specific manner (40). However, some of the neurons recorded from in that study may have been non-GnRH neurons, which would have accounted for some of the observed heterogeneity because they were identified based on location and morphology (rather than GnRH promoter-driven GFP fluorescence), and most but not all contained GnRH mRNA as determined by postrecording characterization with single-cell RT-PCR.

Despite the apparent heterogeneity, averaged Ca²⁺ and SK currents in juvenile, pubertal, and adult mouse GnRH neurons recorded with the perforated-patch or whole-cell technique in the present study were similar. The only statistically significant differences, which were observed in whole-cell recordings, were an increase in Ca²⁺ current density in pubertal, compared with juvenile, GnRH neurons at membrane potentials of –30 and –20 mV (rather than in maximum, peak Ca²⁺ current density, which occurred at membrane potentials between –20 and 0 mV), and a small leftward shift in the voltage, from –6 to –12 mV, required to evoke maximum, peak current. However, the first of these two differences was not maintained in adult GnRH neurons, and neither difference was observed in perforated-patch recordings. Nevertheless, these results contrast with the small but statistically significant increase in maximum, peak Ca²⁺ current density in adult vs. juvenile mouse GnRH neurons reported by Nunemaker et al. (24) using the whole-cell technique. On the other hand, they are in accord with the lack of a pubertal change in maximum, peak Ca²⁺ current density in rat GnRH neurons reported by Kato et al. (25) using the perforated-patch technique, which unlike the whole-cell technique allows for retention of cytoplasmic factors that could regulate Ca²⁺ or SK currents and is therefore more physiological. Hence, it appears that the discrepancy between the two previous reports on total Ca²⁺ currents in GnRH neurons (24, 25) is probably due more to a methodological than a species difference.

The similarities in Ca²⁺ and SK currents among juvenile, pubertal, and adult GnRH neurons suggest that Ca²⁺ and SK channel expression in GnRH neurons remains relatively constant across pubertal development. Total Ca²⁺ or SK current (I) at any given membrane potential equals the number of Ca²⁺ or SK channels (N) multiplied by the single-channel open probability (p) and single-channel current (i), i.e. I = Npi (13). A physiological change in i has yet to be described. Thus, for I not to change at puberty in GnRH neurons, which seems to be the case based on the present results, there would need to be equal and opposite changes in N and p. More likely is that neither N nor p changes, although there may be changes in N or p of individual Ca²⁺ or SK channel subtypes in GnRH neurons as suggested by previous studies (24, 25, 32). This implies that rather than being due to an alteration in the overall expression of Ca²⁺ or SK channels, which would affect N, or to a change in channel gating as may occur by phosphorylation, which would affect p (13), the pubertal increase in GnRH secretion is probably due to other mechanisms such as greater membrane depolarization and subsequent increased activation of preexisting Ca²⁺ channels after increased excitatory synaptic input.

	Footnotes

 
This work was supported by the Louis B. Block Fund and Children’s Research Foundation of the University of Chicago.

Disclosure Statement: The author has nothing to disclose.

First Published Online February 8, 2007

Abbreviations: [Ca²⁺]_i, Cytoplasmic free Ca²⁺ concentration; 1-EBIO, 1-ethyl-2-benzimidazolinone; GFP, green fluorescent protein; I, current; i, single-channel current; IK, intermediate-conductance Ca²⁺-activated; K(Ca), Ca²⁺-activated K⁺; N, number of Ca²⁺ or SK channels; p, open probability; P, postnatal day; SK, small-conductance Ca²⁺-activated K⁺; TEA, tetraethylammonium; TTX, tetrodotoxin.

Received December 18, 2006.

Accepted for publication January 26, 2007.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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