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BOR - Papers in Press, published online ahead of print August 6, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.019265
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BIOLOGY OF REPRODUCTION 69, 1914–1922 (2003)
DOI: 10.1095/biolreprod.103.019265
© 2003 by the Society for the Study of Reproduction, Inc.

Neuroendocrinology

Calcium Current Subtypes in GnRH Neurons1

Craig S. Nunemaker3, R. Anthony DeFazio4, and Suzanne M. Moenter2

Departments of Internal Medicine and Cell Biology, University of Virginia, Charlottesville, Virginia 22908


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Calcium plays roles in excitability, rhythm generation, and neurosecretion. Identifying channel subtypes that regulate calcium influx is thus important to understanding rhythmic GnRH secretion, which is a prerequisite for reproduction. Whole-cell voltage-clamp recordings were made from short-term dissociated GnRH adult ovariectomized (OVX) mice (n = 21) to identify channel subtypes that carry calcium current using selective channel blockers and voltage characteristics. Low-voltage activated (LVA) currents were not observed in 42 GnRH neurons tested, although most non-GnRH neurons (4/6) displayed LVA current. The L-type component of the high-voltage activated (HVA) calcium current was 25% ± 2%. The remaining HVA calcium current passed through N-type (27% ± 3%), P-type (15% ± 1%), Q-type (18% ± 3%), and R-type (15% ± 1%) channels. Because these data differ substantially from reports on cultured GnRH neurons, which may represent reproductively immature models, we also examined GnRH neurons from gonadal-intact young (Postnatal Days 4–10, n = 8 mice) mice. LVA currents were still rare (2/28) in young mice. Although the same HVA components were observed, the proportions were shifted toward significantly more L-type and less N-type current, suggesting a possible developmental shift in calcium currents in GnRH neurons. These data suggest that calcium channel subtypes in GnRH neurons prepared in the short term from brain slices differ substantially from those in long-term cultured GnRH models. These findings provide a vital foundation to examine the role of calcium channels in the secretory and rhythmic machinery of GnRH neurons.

calcium, GnRH, hypothalamic hormones, neuroendocrinology, puberty


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
GnRH neurons form the final common pathway for the central control of reproduction in that these cells integrate most cues to the reproductive system. The output of this network is the pulsatile secretion of GnRH peptide [1, 2]. Intermittent GnRH release signals the pituitary gland to promote the synthesis and secretion of the gonadotropins luteinizing hormone and follicle-stimulating hormone [1, 3], thus controlling downstream reproductive functions.

Calcium is firmly established as a necessary factor for neurosecretion, playing a critical role in vesicle docking and fusion [4, 5], as well as endocytosis [6]. Calcium has also been implicated in rhythm generation in a number of systems [7–10]. For example, modulation of calcium currents contributes to circadian membrane oscillations that alter the excitability of neurons in the suprachiasmatic nucleus [7]. Specific calcium current subtypes likewise have been shown to modulate the frequency and duration of more rapid rhythms in the burst firing patterns of locomotor neurons [8]. Secretion and rhythm generation form the core functions of the GnRH neurosecretory system, thus necessitating a thorough characterization of the types of calcium channels present to lay a foundation for future mechanistic studies.

Previous studies in long-term cultures of immortalized GnRH neurons (GT1 cells) and embryonic GnRH neurons identified T-type, N-type, and substantial L-type components to calcium currents [11–13]. Recent advances have allowed the study of GnRH neurons identified by green fluorescent protein (GFP) expression in short-term prepared cells from reproductively mature mice [14]. This avoids potential changes in channel expression that are secondary to adaptation to culture conditions [15–17]. Such changes have been observed within as little as 1 day in culture [17]. For this study, we made whole-cell voltage-clamp recordings from short-term dissociated GFP-GnRH neurons from ovariectomized (OVX) adult mice and found that calcium current components differed substantially from previous reports on cultured GnRH neurons. To determine if this reflected primarily a developmental or model system difference, we examined calcium current components from gonadal-intact prepubertal mice as well.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice

GnRH neurons from mice that express GFP under the control of the GnRH promoter [14] were used in these studies. The Animal Care and Use Committee of the University of Virginia approved all procedures used in these experiments. Our goal was to describe the functional calcium current subtypes observed at two developmental stages, before and after puberty. This presents two variables, age and steroid milieu, one of which must be controlled as much as possible. We chose to minimize changes in steroid milieu and examine the effects of age. We thus used adult OVX mice (referred to as "adult" hereafter) to remove cyclic gonadal influences that could affect calcium currents measured at different stages of the estrous cycle. Adult mice were anesthetized with Metofane (Janssen Pharmaceuticals, Ontario, Canada); postoperative analgesia was provided by a long-acting local anesthetic (0.25% bupivicaine, 4.5 µl per site; Abbott Laboratories, North Chicago, IL). Cells were prepared and recorded 7 ± 2 days after ovariectomy as a compromise between allowing sufficient time for removal of steroid influences and avoiding side effects of long-term steroid withdrawal. For the prepubertal stage, we used gonadal-intact mice at Postnatal Days 4–10 (referred to as "young" hereafter). Gonads were not removed from young mice because steroidogenesis is minimal, and they are not exposed to cyclical changes in gonadal factors. The exact composition of the steroid milieux in these two models is not known due to the minimal blood volume of young mice. As a consequence, differences in steroid composition, relative steroid levels, and neuron sensitivity to steroids between groups could each contribute to differences in calcium currents between the groups (see Discussion for additional details). These groups nonetheless represent prepubertal and postpubertal developmental stages with comparably low gonadal steroid levels.

Preparation of Short-term Dissociated Cells

All reagents were purchased from Sigma Chemical Company (St. Louis, MO) unless otherwise stated. All solutions (with the exception of normal Hepes) were bubbled with a 95% O2-5% CO2 mixture throughout the slice preparation and incubation and for at least 15 min before tissue exposure. Mice were decapitated and brains were rapidly removed and placed in ice-cold sucrose-substituted saline (SS) solution containing 250 mM sucrose, 3.5 mM KCl, 26 mM NaHCO3, 10 mM glucose, 1.3 mM Na2HPO4, 1.2 mM MgSO4, and 2.5 mM MgCl2. Coronal 200–400-µm brain slices were cut with a Vibratome (Ted Pella, Inc., Redding, CA) and then incubated for 15–30 min at room temperature in a solution of 50% SS and 50% normal saline solution, containing 130 mM NaCl, 26 mM NaHCO3, 10 mM glucose, 3.5 mM KCl, 1.25 mM Na2HPO4, 1.2 mM MgSO4, and 2.5 mM CaCl2. Slices were then transferred to a solution of 100% normal saline at 30–32°C.

After at least 1 h of incubation in normal saline, 3–5 brain slices that contained the highest density of GnRH neurons were transferred to a Petri dish containing SS at 22–24°C. Fragments that contained the medial GnRH neurons were dissected (Fig. 1A). Fragments from each slice were cut in half to aid in dissociation. All fragments were then deposited into a mesh container that was submerged in SS containing 3 mg/ml of Protease XXIII. Fragments were incubated at 30–32°C for 30–45 min. During this time, six nitric acid-cleaned coverslips were placed in a 60-mm Petri dish, and 200 µl of normal Hepes solution containing 150 mM NaCl, 10 mM Hepes, 10 mM glucose, 2.5 mM CaCl2, 1.3 mM MgCl2, and 3.5 mM KCl was deposited on each coverslip. After incubation in Protease XXIII, fragments were transferred to a 1.5-ml microcentrifuge tube and rinsed three times with 1 ml of SS. Fragments were suspended in 400 µl of SS and triturated using a series of four increasingly narrow-bore pipette tips until solution was cloudy. A total of 100 µl of this suspension was deposited onto each of two coverslips. An additional 200 µl of Hepes solution was added to the remaining suspension in the microcentrifuge tube and trituration continued until the mixture was again cloudy. This suspension was deposited onto each of the four remaining coverslips (~100 µl per coverslip). After 5 min (sufficient time for cells to descend to the surface of the coverslip), excess solution (~200 µl) and debris were removed from each coverslip. Cells were allowed to settle for an additional 30 min before covering the entire surface of the 60-mm Petri dish containing the coverslips with 3–4 ml of Hepes solution. Coverslips were transferred to the recording chamber as needed.



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  		FIG. 1. Preparation and appearance of dissociated cells. A) Area of tissue fragment dissected from coronal brain slices (dotted lines) for preparation of dissociated GFP-expressing GnRH neurons. Representative GnRH neuron with (B) and without (C) processes in both bright field and under fluorescence. Arrowheads point to the process in the bright field image. D) Spontaneous electrical activity recorded extracellularly from a representative dissociated GnRH neuron. Note that different recording method and solutions were used to record spontaneous electrical activity [26]. Magnification x80 for B and C

Electrophysiological Recordings

During recording, coverslips were superfused with a calcium recording solution containing 115 mM NaCl, 20 mM tetraethylammonium chloride (TEA-Cl), 5 mM 4-amino-pyridine (4-AP), 5 mM CsCl, 10 mM Hepes, 10 mM glucose, 10 mM CaCl2, and 2 mM MgCl2 (pH 7.4) at room temperature. Tetrodotoxin (500 nM) was included in recording solutions to minimize sodium channel contributions to the recorded current. TEA-Cl and 4-AP were likewise included to block potassium channels. Cells were observed with an Olympus BX50XI upright fluorescent microscope equipped with infrared differential interference contrast (Opelco, Dulles, VA). The surface of the coverslip was scanned using a 4x lens to locate GnRH neurons by GFP expression. We have previously observed both a population of brightly fluorescent bipolar GnRH neurons and faintly fluorescent cells with multiple processes in brain slices. To ensure recordings were only made from brightly fluorescent cells, only neurons with a fluorescent signal strong enough to be visible under the 4x lens were chosen for study. The health of these cells was then assessed using a 40x water-immersion lens. Cells with uneven surfaces under bright field or patchy appearance under fluorescence were not considered for study. Recording pipettes (3–6 M{Omega}) were made from capillary glass (type 7052, OD/ID 1.65/1.1 mm; World Precision Instruments, Sarasota, FL) using a two-stage pipette puller (Narashige, Tokyo, Japan). Patch pipettes were filled with a calcium recording internal solution containing 140 mM TEA-Cl, 10 mM 4-AP, 10 mM CsCl, 10 mM Hepes, 5 mM EGTA, 4 mM MgATP, 0.4 mM NaGTP, and 0.1 mM CaCl2 (pH 7.2) and wrapped in Parafilm (Pechiney Plastic Packaging, Chicago, IL) to reduce fast capacitive transients [10]. Pipettes were targeted to GnRH neurons viewed under the 40x lens using an MP-285 micromanipulator (Sutter Instruments, Novato, CA). Slight positive pressure was applied to the recording pipette before entering the bath and maintained until reaching the target cell.

Recordings were obtained with an EPC-8 patch clamp amplifier (HEKA, Nova Scotia, Canada) with Pulse Control (Instrutech, Port Washington, NY) and IgorPro software (Wavemetrics, Lake Oswago, OR) running on a G4 Macintosh computer (Apple Computer, Cupertino, CA) to acquire data. After G-{Omega} seal formation, fast capacitive transients were minimized, and then a whole-cell configuration was established. Capacitive transients were then eliminated to measure series resistance (Rs) and cell capacitance as dialed in on the EPC-8 amplifier. Rs was typically compensated 50%–70% and periodically monitored; only stable recordings with uncompensated Rs <20 M{Omega} were included in this study, resulting in a maximum voltage error <3.2 mV. Recordings were made in voltage-clamp mode with -60-mV holding potential, 10-µsec sampling interval, and 3-kHz filtering. Liquid junction potential was ~2 mV and not corrected in the presented data [18]. Voltage control was determined by examining tail currents after strong depolarizations [19, 20]. Recordings in which tail current did not decay rapidly or smoothly were excluded from the data set.

Drug Application

Drugs and control solutions were perfused through the recording chamber using a VC-6 Perfusion Valve Control System (Warner Instruments, Hamden, CN). Toxins for pharmacological blockade of specific calcium channel subtypes were obtained from Alomone (Jerusalem, Israel) and later from Bachem (Torrance, CA). Concentrations were based on a review of the literature [21–23]. Drug concentration and respective calcium channel targets were as follows: 20 nM {omega}-agatoxin IVA, P type; 1 µM {omega}-conotoxin GVIA, N type; and 1 µM {omega}-conotoxin MVIIC, P/Q type. Nimodipine, 10 and 50 µM, which targets L-type channels and 500 µM cadmium, which blocks all calcium channels, were obtained from Sigma-Aldrich (St. Louis, MO).

Analysis

Peak calcium current was measured as the greatest amplitude current response elicited during a 200-msec voltage step to 0 mV from a 200-msec prepulse step of -100 mV. Sustained current was measured at a point within the last 10 msec of the same 200-msec voltage step to 0 mV. Current density, which corrects peak current for cell size, was calculated by dividing the peak current by the measured cell capacitance. To determine the voltage at which half of the channels were activated (V 1/2act), both peak and sustained currents (I) measured at different voltages (Vstep) using an activation protocol (Fig. 3A) were divided by the GHK driving force [24] to generate a value proportional to the conductance (G).



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  		FIG. 3. Activation of HVA calcium currents in GnRH neurons. A) Representative currents elicited (top) in response to voltage protocol shown below. B) Normalized peak current voltage plot of the mean activation curve in GnRH neurons from adult OVX mice (closed circles, n = 9) and young mice (open circles, n = 8). C) Normalized peak conductance for each group fit to a Boltzmann equation. The voltage at which half of the current was activated (V1/2act) was more depolarized (right shifted) for GnRH neurons from young than in adult mice. Asterisks indicate differences between normalized conductances at the specific voltages (P < 0.05)

In this equation, Vrev is the reversal potential, which was calculated by averaging the x-intercepts of the linear fits of current vs. voltage of all individual cells at +20, +30, and +40 mV. Z is the charge (+2), K is the Boltzmann constant, and T is temperature in Kelvin. Conductance as a function of voltage step was fit with a Boltzmann equation

with minimum constrained to zero, leaving V1/2 act, steepness (k), and Gmax as fitting variables. For Figure 3C, the conductance-voltage relationship was normalized by Gmaxfor each cell, and mean and SEM were calculated for each group (n = 9 adult, n = 8 young).

Each subtype current was calculated by measuring the current during the drug application for each subtype and subtracting this response from the current measured during the preceding treatment, resulting in a subtraction current. For example, the L-type component was the difference between the current during nimodipine application subtracted from the control current response. The P-type current was determined by subtracting the current during {omega}-agatoxin treatment from the current during nimodipine treatment and so on. Currents were converted to fractions by dividing each subtraction current by the control current. The Mann-Whitney U-test was used to compare percentages of calcium current subtypes between OVX and young mice after first determining whether a difference existed among subtypes and between OVX and young mice (Kruskal-Wallis statistic, 45.7; P < 0.0001). All other comparisons were made using a two-tailed t-test, assuming unequal variance. All data are presented as mean ± SEM, with significance set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Dissociated GnRH Neurons

For this study of calcium currents, we chose to record from GFP-GnRH neurons dissociated in the short term from hypothalamic fragments (Fig. 1A) to allow for better voltage control than would be possible in the intact slice preparation. Typically, several strongly fluorescent GnRH neurons could be observed on any given coverslip, although only one recording was made from each coverslip and up to 4 cells per animal were recorded. Weakly fluorescent cells were also observed but only under higher magnification (40x). The weakly fluorescent cells often had multiple processes extending from the cell body in all directions; these cells were not targeted for recording. Brightly fluorescent monopolar or bipolar cells (Fig. 1B) and also strongly fluorescent cells that lacked processes (Fig. 1C) were targeted for recording. Short-term dissociated cells displayed burst firing patterns (Fig. 1D) similar to previous observations of GnRH neurons in brain slices [25–27]. Note that firing patterns were observed using techniques and solutions from previous studies [25–27].

Because calcium channel subtypes are often differentially distributed in dendrites, terminals, and somata [28, 29], we compared dissociated cells with and without processes to determine if the presence or absence of processes had any effect on observed calcium current subtypes. Among GnRH neurons from adult mice with sizable processes, cell capacitance (a measure of cell surface area) was 8.5 ± 0.2 pF (n = 5 cells). Cell capacitance was smaller (5.8 ± 0.2 pF, n = 5 cells) in GnRH neurons from adult mice with only proximal or no remaining processes. No significant difference in the proportion of any calcium current subtypes was observed between these types of cells in either young or adult mice (P > 0.20). Although this suggests that calcium channels in proximal processes and somatic regions do not differ significantly in these cells, it should be noted that capacitance measures in our coronal slice preparations typically range from 15 to 20 pF, suggesting the bulk of dendritic and axonal processes are eliminated by dissociation. Further, terminal regions were removed by preparation of coronal slices. Since terminals contain most of the secretory machinery in many cell types, including calcium channels, our results may differ from those in slice preparations or long-term cultured cells.

LVA Calcium Currents Rarely Observed in GnRH Neurons

One of the first observations that differed from previous reports was a paucity of low-voltage activated (LVA) calcium currents in GnRH neurons. A voltage step to -50 mV following a prepulse to -120 mV elicited LVA calcium currents from 4 of 6 unidentified control neurons (Fig. 2A) but not from GFP-GnRH neurons in adult OVX mice (Fig. 2B, n = 0 of 42). Altering the protocol by varying the prepulse duration (50, 100, 200 msec) or amplitude (-150, -120, -100 mV) or the test pulse amplitude (-70, -60, -50, and -40 mV) similarly failed to reveal LVA currents (n = 5 GnRH neurons each). LVA currents were observed, however, in a small fraction of GnRH neurons from young mice (2 of 28, Fig. 2C), suggesting that LVA currents may be expressed in these cells during development.



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  		FIG. 2. LVA currents are infrequently observed in GnRH neurons. A) LVA current (arrow) elicited from a non-GnRH neuron by a step from -120 mV to -50 mV. B) Representative current trace from an adult OVX GnRH neuron demonstrates no LVA current using the same voltage protocol. C) One of only two observations of LVA current (arrow) in young GnRH neurons. Voltage protocol for eliciting LVA currents is shown at bottom

GnRH Neurons Display Multiple High-Voltage Activated Calcium Currents

Previous studies have reported high-voltage activated (HVA) currents in cultures of immortalized GT1 cells or embryonic GnRH neurons [11–13]. To examine the HVA calcium currents in GnRH neurons from adult mice, we analyzed the currents elicited by a voltage step from a -100-mV prepulse to a variable test pulse (-70 to +40 mV). As shown in a representative example in Figure 3, both the currents observed (Fig. 3A) and the normalized current voltage curves (Fig. 3B, peak current illustrated) are consistent with the activation range for HVA calcium currents [30]. These data were converted to conductance to normalize for driving force and fit with a Boltzmann distribution (Fig. 3C, fit for peak current illustrated). This revealed the activation curve for both peak and sustained HVA currents from young GnRH neurons (n = 8) was depolarized from that of adult GnRH neurons (n = 9) by ~5 mV at half activation. Specifically, V1/2act was -4.8 ± 1.2 mV for peak calcium conductance in adult and 0.1 ± 1.8 mV for young (P < 0.05). Steepness of voltage activation did not differ between groups (adult, 6.4 ± 0.6 mV; young, 6.9 ± 0.9 mV; P > 0.60). For sustained current, V1/2act was also shifted by 6.2 mV (adult, -2.5 ± 1.4 mV; young, 3.7 ± 1.7 mV; P < 0.025), with no difference in slope (adult, 5.7 ± 0.7; young, 7.1 ± 0.9 mV; P > 0.20). Mean peak current density was greater in adult (28.4 ± 2.2 pA/µF, n = 17) compared with young mice (21.0 ± 2.1 pA/µF, n = 10, P < 0.025), suggesting increased density of calcium channels in adult mice. Neither the sustained nor the ratio of peak to sustained current density, however, differed significantly between groups (data not shown).

Pharmacological Analysis of Channel Subtypes Carrying HVA Current

To identify the subtypes of calcium channels that compose this HVA current, antagonists for each calcium channel subtype were given sequentially to isolate L-, P-, N-, Q-, and R-type currents by subtraction. Drugs were given additively (nimodipine to block L, then nimodipine + {omega}-agatoxin to block L and P, then nimodipine + {omega}-agatoxin + {omega}-conotoxin GVIA to block L, P, and N, and then nimodipine + {omega}-agatoxin + {omega}-conotoxin GVIA + {omega}-conotoxin MVIIC to block L, P, N, and Q) to maintain effective concentrations throughout the recording. Even after application of the above calcium channel antagonists, a residual current (R type) persisted. This residual current was eliminated by cadmium, a nonspecific calcium channel blocker, at the end of each experiment. Figure 4A shows a representative example of the course of drug effects. The stimulation protocol was run every 20 sec throughout the experiment; drug treatment was initiated at the accompanying arrow. In Figure 4B, the response from an average of three stimulation protocols is shown after application of each drug. Note that whole-cell recordings are subject to current run-down over the duration of recordings under control conditions likely due to dialysis. Typical current rundown (<3% per min based on control recordings 4–12-min long, n = 10) is illustrated by the dotted line in Figure 4A. Factoring in this run-down shifts the mean fractions for each subtype by only 2%–3%.



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  		FIG. 4. Identification of HVA current subtypes by sequential pharmacological blockade. A) Peak calcium current elicited at 20-sec intervals throughout a series of drugs to block sequentially all subtypes. Drug treatment was initiated at the arrow next to each drug and continued until administration of cadmium on its own. Dotted line shows typical rate of current run-down in untreated control neurons. B) Average of three traces for the current response for each drug effect. Voltage protocol is shown at bottom. Drug doses: 10 µM nimodipine, 20 nM {omega}-agatoxin IVA, 1 µM {omega}-conotoxin GVIA, 1 µM {omega}-conotoxin MVIIC, and 500 µM cadmium

Since previous studies had indicated that most calcium current in GT1 cells is specifically L type [11, 12], we altered the concentration and the sequence of calcium channel blockers to determine if an additional dihydropyridine-sensitive population would be revealed. Specifically, we first increased the dose of nimodipine from 10 to 50 µM (n = 7). Although this higher concentration resulted in an increase in the fraction of L-type calcium component observed (25% ± 2% vs. 34% ± 2%, P < 0.01), the L-type component in short-term prepared adult GnRH neurons is still substantially less than that reported in cultured GnRH models. In some recordings, {omega}-conotoxin MVIIC was applied first to remove most non-L-type currents, followed by 50 µM nimodipine (n = 5, Fig. 5) and then cadmium. At 1 µM, {omega}-conotoxin MVIIC should remove all P/Q current and also some N-type current. Calcium current was decreased by 49% ± 2% during 1 µM {omega}-conotoxin MVIIC treatment and by 34% ± 3% during 50 µM nimodipine treatment, with cadmium removing the remaining current (R type, 17% ± 2%). Although the effect of {omega}-conotoxin MVIIC was significantly less (P < 0.01) than the additive effects of the P-, N-, and Q-type blockers (60% ± 2%, n = 12) given sequentially, this was likely due to the lower affinity of {omega}-conotoxin MVIIC for N-type channels. These findings confirm that L-type current ranges from a quarter to a third of total calcium current, markedly less than the >70% observed in prior studies [11, 12] and that significant N, P/Q, and R components are observed.



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  		FIG. 5. Changing drug concentration and sequence does not alter the types of HVA currents detected. A) Peak calcium current elicited at 20-sec intervals using N/P/Q blocker ({Omega}-conotoxin MVIIC at 1 µM), followed by L-type blocker nimodipine at 50 µM, and then cadmium to block remaining current. Dotted line shows typical rate of current run-down in untreated control neurons. B) Average of three traces for the current response for each drug effect. Voltage protocol is shown at the bottom. Note that drug treatments were sequential, but not overlapping, for this set of experiments

Calcium Current Subtypes in GnRH Neurons From Young Mice

The HVA components observed differ markedly from reports on cultured GnRH neuron model systems [11–13], possibly due to differences in maturity of the GnRH neurons. To investigate whether or not the observed components of HVA calcium current change during development, we examined calcium currents in young mice (Postnatal Days 4–10).

Using protocols and drug sequences identical to those displayed in Figure 4, the same calcium channel subtypes were also found in GnRH neurons from young mice as from adult OVX mice, but in different proportions. Specifically, both peak and sustained L-type currents were significantly larger in young mice (P < 0.05, Fig. 6) and N-type currents were significantly smaller (P < 0.01, Fig. 6). The peak but not sustained R-type component was larger in adult GnRH neurons (P < 0.05). Because R-type current may be an aggregate of dihydropyridine- and toxin-insensitive currents [21, 31], the difference between peak and sustained R-type values could represent the presence of a current in adult GnRH neurons that is not present in young mice. Current densities also differed between young and adult mice for R-type peak current, N-type peak, and sustained current (Table 1). Current densities were similar, however, for L-type peak and sustained current, despite differing in fractional component. P- and Q-type current densities were also similar between adult and young mice. The striking observation in Table 1 is the large change in {omega}-conotoxin GVIA sensitivity related to changes in N-type channel.



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  		FIG. 6. Distribution of HVA current components in young vs. adult OVX mice. Peak (A) and sustained (B) current due to each HVA channel subtype in GnRH neurons from both adult OVX (open bars) and young (closed bars) mice. * P < 0.05


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  		TABLE 1. Current density (pA/pF) of the different subtypes in young and adult mice


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Hormone release and rhythm generation are two functions of the GnRH neurosecretory system that are crucial to reproductive capability. Because both these functions are strongly calcium dependent, identification of the types of calcium channels present in GnRH neurons is a critical base for future mechanistic studies. This study is the first characterization of calcium channel subtypes in adult GnRH neurons. Our results demonstrate that these cells express a variety of HVA calcium currents (L, P, N, Q, and R type). In young mice, the same components were observed, although the proportions were shifted toward significantly more L type and less N type, suggesting a switch in predominant HVA calcium currents. LVA currents were observed infrequently in GnRH neurons from young mice and never from adult OVX mice under the conditions tested. In addition, greater current density suggests increased calcium channel expression in GnRH neurons of sexually mature mice. Finally, the hyperpolarization of the activation curve of HVA currents indicate smaller depolarizations from resting potential would be necessary to open the channels that carry these currents in adults, perhaps increasing GnRH neuron excitability. Together, these results suggest several shifts between birth and reproductive maturity and provide the foundation for hypothesizing the role of calcium channels in the functions of GnRH neurons.

Our findings on the makeup of calcium currents in adult GnRH neurons differ from reports in the literature. Previous studies have examined calcium currents in cultures of embryonic GnRH neurons from the olfactory placode [13] and in the GT1 immortalized GnRH cell line [11, 12]. Specifically, previous work indicated L-type currents dominated in GT1 cells (>70% of total HVA current), whereas no P/Q component was observed. Although we observed a quarter to a third of HVA calcium current was L type, substantial N-, P/Q-, and R-type components are also present in GnRH neurons in both age groups examined in the present study. Furthermore, we observed LVA (T-type) calcium currents in few GnRH neurons and only in young mice, whereas previous studies identified LVA calcium currents in embryonic GnRH neurons [13] and GT1 cells [11].

These discrepancies may be explained by differences in methods. Most significantly, our cells were dissociated in the short term, whereas cells from previous studies were cultured for several weeks or more before study. Culturing cells for as little as 1 day can result in significant changes in ion channel expression patterns [17]. Furthermore, GT1 cultures can differ from one another, depending on culturing conditions or phenotypic drift. The use of short-term dissociated cells not only improves voltage control needed for measuring calcium currents, it also should minimize changes incurred by long-term culturing. One caveat to the dissociated approach is that short-term dissociated cells have reduced dendritic and axonal projections and no terminals, whereas GT1 cells and embryonic GnRH neurons in culture are intact. If certain calcium channel subtypes were localized to distal projections, we might expect these to have a reduced contribution in the dissociated preparation. In addition, artifacts from the dissociation process must also be considered. In this regard, studies in other brain regions indicate similar profiles of calcium channels are observed in slices and in short-term dissociated preparations of the same cell type [10]. Furthermore, we and others [25, 27] have observed similar patterns of burst firing in isolated GnRH neurons and those in slices, suggesting the overall function and health of these cells is similar.

Another important difference between the present study and previous work is the developmental stage of the GnRH neurons examined. Embryonic cultures are typically obtained on E11.5–12.5. GT1 cells were transformed after migration but likely before most stages of pubertal development. Although both of these model systems exhibit many characteristics of mature GnRH neurons, it is certainly possible that changes in calcium channel subtypes that accompany maturation of this system may not have taken place. The changes in both LVA and HVA calcium currents between young and adult mice suggest that some of the differences between the present data and previous reports are indeed due to differences in the developmental state of the GnRH neurons examined.

That said, it is important to point out that the developmental data in the present study must also be interpreted bearing in mind limitations to the animal models. The use of gonadal-intact rather than OVX prepubertal mice, although minimizing difference in circulating steroid levels at the time of the experiment and allowing us to identify a factor that contributes to the difference between our data and previous reports, leaves the potential for confounding ovarian influences. Because the age of our young animals (Postnatal Days 4–10) is well before the first outward signs of puberty (vaginal opening ~4–5 weeks [14, 32] fertility onset ~6–8 weeks [33]) and even before the first measurable increase in gonadotropin levels (Postnatal Day ~12 [32, 34]), the gonads are in a relatively quiescent state. Production of gonadal steroids in young mice, although present, is much lower than that in postpubertal mice [35, 36]. Of note, gonadal steroids in the circulation of OVX adult females drop to low but detectable levels [37], perhaps due to a low level of peripheral steroid synthesis.

The rationale for ovariectomizing adult mice was to produce a low, noncyclic gonadal function, which is similar to that of young mice. Gonadal steroids, and in particular estradiol, have been shown to alter ion channel expression and function [37–39]. Also, changes in sensitivity to steroid feedback, affecting either GnRH neurons themselves or their synaptic inputs, likely contribute to maturation of the GnRH neuronal system [40, 41]. In addition to steroids, the ovary produces peptide regulators, including inhibin and activins. Although activins have been implicated in the development of a neural pattern of ion channels [42], we are unaware of work investigating a role for these peptides in ion channel regulation in postnatal neurons. Activin also alters GnRH gene expression and secretion from GT1 cells [43, 44] and hypothalamic explants [45], but altering bioavailable activin with follistatin does not alter GnRH release in vivo [46]. These studies suggest that differences in the gonadal hormone milieu that could be present in the two models used in these experiments could contribute to expression or function of calcium channels. Despite the caution required in interpreting mechanism, our data suggest significant differences in calcium currents are present at two developmental stages with similar low circulating steroid levels. The role of specific gonadal hormones on GnRH neuron calcium channels will be explored in future studies.

Of interest to possible developmental changes, N-type components became more prominent in GnRH neurons from adult mice. The role of N-type currents in the central and peripheral nervous system is primarily secretion, as has been demonstrated in autonomic neurons of several species [47]. If GnRH neurons are similar, then the increase in N-type current between the neonatal period and adulthood is consistent with an up-regulation of secretory processes, perhaps contributing to the pubertal transition [47]. P/Q currents, incidentally, also have been shown to induce secretion, but typically only at higher stimulation frequencies. P/Q-type currents thus may control additional secretory machinery that is selectively activated only during phases of high-frequency action potential firing, such as what may occur during the GnRH surge.

We also observed a reduction in the percentage of HVA current carried by the L subtype from young to adult. This is consistent with the hypothesis that the high proportion of L-type current in GT1 cells occurs because these cells represent a population of young GnRH neurons, at least with regard to calcium channel complement. Our results suggest that L-type currents are larger in young animals, which is consistent with this notion. The significance of such a shift is not yet known, although studies in many endocrine systems suggest calcium influx through L-type channels causes secretion. L-type channels play a role in secretion from pancreatic ß-cells [48] and many pituitary cells [49, 50]. Extensive study of calcium components in the peripheral nervous system, however, suggests L-type channels do not contribute significantly to secretion in many neurons [51]. Other studies have implicated L-type calcium channels in regulating burst firing or pacemaker activity in the anterior pituitary [50], heart [52], and GT1 cells [11, 12, 53].

Lastly, the relative lack of LVA currents was somewhat surprising, because LVA currents have been associated with increased excitability [54] in other systems; we had thus hypothesized that GnRH neurons from adults would have an increased LVA component compared with those from younger mice. Differential expression of LVA current has been observed among other hypothalamic neuron types [55, 56], and LVA currents are transiently expressed in early development in other neuronal systems [57–60]. With specific regard to the former, neurosecretory neurons of the paraventricular nucleus of the hypothalamus typically did not exhibit LVA currents, whereas nonneurosecretory neurons did [56]; this observation is consistent with the relative lack of LVA currents observed in GnRH neurons in the present study. Another possible explanation for the lack of LVA currents in the present study is that the voltage protocol used failed to remove inactivation from these channels. Although possible, the observation of LVA currents in most non-GnRH neurons and in two GnRH neurons from young animals argues against this.

These findings suggest that GnRH neurons contain a much more diverse collection of calcium channels than previously thought. Developmental changes in the proportion of these channels suggest a functional shift between birth and adulthood. Identification of these calcium channel subtypes represents an important step in understanding the physiology of GnRH neurons and the roles of various calcium channel subtypes in generating rhythmic secretion in these cells.


   ACKNOWLEDGMENTS
 
We thank Dr. Xu-Zhi Xu for excellent technical assistance and Dr. Glenn Harris and Shannon Sullivan for editorial comments.


   FOOTNOTES
 
1 Supported by HD34860 and the National Institute of Child Health and Human Development/National Institutes of Health through cooperative agreement U54HD28934 as part of the center's program in reproductive research. Back

2 Correspondence: Suzanne M. Moenter, Department of Internal Medicine, University of Virginia, P.O. Box 800578, Charlottesville, VA 22908. FAX: 434 982 0088; smm4n{at}virginia.edu Back

3 Current address: Department of Pharmacology and Toxicology, Medical College of Virginia Campus, Virginia Commonwealth University, P.O. Box 980524, Richmond, VA 23298 Back

4 Current address: University of Miami School of Medicine, Department of Physiology and Biophysics, Miami, FL 33101 Back

Received: 15 May 2003.

First decision: 10 June 2003.

Accepted: 31 July 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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