Control of Action Potential-Driven Calcium Influx in GT1 Neurons by the Activation Status of Sodium and Calcium Channels

Fredrick Van Goor, Lazar Z. Krsmanovic, Kevin J. Catt and Stanko S. Stojilkovic

Endocrinology and Reproduction Research Branch National Institute of Child Health and Human Development National Institutes of Health Bethesda Maryland 20892


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
An analysis of the relationship between electrical membrane activity and Ca2+ influx in differentiated GnRH-secreting (GT1) neurons revealed that most cells exhibited spontaneous, extracellular Ca2+-dependent action potentials (APs). Spiking was initiated by a slow pacemaker depolarization from a baseline potential between -75 and -50 mV, and AP frequency increased with membrane depolarization. More hyperpolarized cells fired sharp APs with limited capacity to promote Ca2+ influx, whereas more depolarized cells fired broad APs with enhanced capacity for Ca2+ influx. Characterization of the inward currents in GT1 cells revealed the presence of tetrodotoxin-sensitive Na+, Ni2+-sensitive T-type Ca2+, and dihydropyridine-sensitive L-type Ca2+ components. The availability of Na+ and T-type Ca2+ channels was dependent on the baseline potential, which determined the activation/inactivation status of these channels. Whereas all three channels were involved in the generation of sharp APs, L-type channels were solely responsible for the spike depolarization in cells exhibiting broad APs. Activation of GnRH receptors led to biphasic changes in cytosolic Ca2+ concentration ([Ca2+]i), with an early, extracellular Ca2+-independent peak and a sustained, extracellular Ca2+-dependent phase. During the peak [Ca2+]i response, electrical activity was abolished due to transient hyperpolarization. This was followed by sustained depolarization of cells and resumption of firing of increased frequency with a shift from sharp to broad APs. The GnRH-induced change in firing pattern accounted for about 50% of the elevated Ca2+ influx, the remainder being independent of spiking. Basal [Ca2+]i was also dependent on Ca2+ influx through AP-driven and voltage-insensitive pathways. Thus, in both resting and agonist-stimulated GT1 cells, membrane depolarization limits the participation of Na+ and T-type channels in firing, but facilitates AP-driven Ca2+ influx.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The mammalian hypothalamus contains about 1000 GnRH neurons that are diffusely distributed within the mediobasal region, but nevertheless operate in a highly synchronized manner to release pulses of GnRH into the hypothalamo-hypophyseal portal vessels. This GnRH neuronal network has been termed the GnRH pulse generator (1). The propensity of GnRH neurons for pulsatile neuropeptide secretion is common among mammalian species and is critical for the episodic release of gonadotropins from the pituitary gland into the systemic circulation (2). In the monkey, the mediobasal hypothalamus exhibits periodic electrical activity that is highly synchronized with pulsatile LH release into the circulation. These observations are consistent with the hypothesis that simultaneous discharge of GnRH from nerve terminals in the median eminence is a consequence of action potential (AP) firing by the GnRH pulse generator (1).

Significant progress in elucidating the mechanism of basal GnRH secretion has been made by studies on GnRH-secreting immortalized neurons (GT1 cells) and cultured embryonic GnRH neurons (3). Several in vitro experiments have shown that changes in cytosolic Ca2+ concentration ([Ca2+]i) determine the secretory pattern of GnRH (4), suggesting that Ca2+ plays a central role in the signal transduction processes that lead to exocytosis. Furthermore, GnRH secretion from perifused GT1 and hypothalamic cells is reduced by L-type Ca2+ channel inhibitors and augmented by activation of voltage-gated Ca2+ channels (VGCC) (5). GT1 cells express a variety of plasma-membrane channels, including tetrodotoxin (TTX)-sensitive Na+ channels, transient and sustained Ca2+ channels, inward rectifier K+ channels, and several types of outward K+ channels (6). Embryonic GnRH neurons also express a comparable set of plasma membrane channels (7). In addition, single embryonic GnRH neurons and GT1 cells fire spontaneous action potentials (APs) and exhibit fluctuations in [Ca2+]i (8, 9). GT1 cells also express several gap junction proteins (10) that probably permit electrical coupling between these cells and transmit their synchronized intercellular Ca2+ waves (11).

To date, the mechanism of AP generation in these cells and the channels involved in spontaneous firing, as well as their relevance to the control of [Ca2+]i, have not been completely characterized. For example, the importance of TTX-sensitive Na+ channels in AP generation and their relevance to voltage-gated Ca2+ influx have not been clarified. Although several adenylyl cyclase and phospholipase C-coupled plasma membrane receptors are expressed in GnRH neurons (12, 13, 14, 15, 16, 17, 18), their effects on spontaneous electrical activity have not been addressed. Also, the mechanism for repletion of the endoplasmic reticulum Ca2+ pool after activation of intracellular Ca2+ release through inositol 1,4,5-trisphosphate-controlled Ca2+ channels has not been determined in these cells. This problem has been studied in lactotrophs (19) and other excitable cells, but the mechanism by which voltage-gated Ca2+ influx and inositol 1,4,5-trisphosphate-induced Ca2+ signaling are integrated has not been clarified. The present study was performed to analyze the spontaneous spiking activity of isolated GT1 neurons and its importance in the control of [Ca2+]i, and to characterize the nature and role of the plasma membrane Ca2+ oscillator in these cells. The major focus of these investigations was on inward Na+ and Ca2+ currents and their roles in electrical activity in spontaneously active cells and those stimulated by Ca2+-mobilizing agonists.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Patterns of AP Firing in Spontaneously Active GT1 Cells
The electrical membrane properties of GT1–7 neurons (hereafter described as GT1 cells) were monitored using perforated-patch recording techniques in the current-clamp mode. To eliminate the influences of electrical and synaptic coupling between cells, only isolated bipolar neurons with rounded perikarya and small neurites were chosen. Potential autocrine and paracrine effects due to endogenous neuropeptide and neurotransmitter release were minimized by constant bath perifusion. Under these recording conditions, 95% (n = 237) of the GT1 neurons exhibited self-sustained AP firing, a characteristic feature of single-cell oscillators (Fig. 1Go). The majority (n = 211) of these cells exhibited either regular or irregular spiking activity, whereas 14 cells showed bursts of four to eight APs separated by quiescent periods of 10–20 sec. The pattern of bursting activity in these cells was similar to that observed by others (8, 9). The remaining cells (n = 12) did not exhibit spontaneous AP spiking, but transient hyperpolarizing current injections elicited one to two rebound APs (not shown). Since GT1 cells secrete GnRH and express GnRH receptors (14), electrical activity was also analyzed in cells perifused with a GnRH antagonist. Application of 1 µM [D-pGly1,D-Phe2,D-Trp3,6]GnRH, which completely inhibits agonist binding to GT1 cells (14), did not affect the firing pattern (not shown).



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Figure 1. Spontaneous Electrical Activity in GT1 Cells

A–C, Typical examples of the range of electrical membrane activity observed in GT1 cells. D, Expanded time scale of single APs from the recordings in A–C labeled as a, b, and c. In this and the following figures, perforated-patch recording was employed if not otherwise specified.

 
In spontaneously active neurons, the mean value of the maximum levels of hyperpolarization observed (hereafter referred to as the baseline potential), was between -75 and -50 mV (Fig. 1Go, panels A–C). The interspike interval was characterized by a slow pacemaker depolarization to the spike initiation threshold, which was between -55 and -40 mV, depending on the baseline potential (Fig. 1DGo). The amplitude, duration, and frequency of APs were also dependent on the baseline potential. In cells with more depolarized baseline potentials, the frequency and duration of APs were greater, whereas AP amplitude progressively decreased with increasing depolarization (Figs. 1Go and 2AGo). A similar relationship between baseline potential and frequency, duration, and amplitude was observed in response to sequential depolarizing current injections in a single GT1 cell (Fig. 2BGo). Conversely, hyperpolarizing current injections of -2 to -5 pA reduced AP frequency and duration and increased the amplitude. Larger hyperpolarizing current injections abolished AP firing (data not shown).



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Figure 2. Characterization of APs in GT1 Cells

A, Relationship between the baseline potential and frequency, duration, and amplitude of APs in spontaneously active cells (r = coefficient of correlation). B, Modulation of spike frequency, amplitude, and duration of AP in a single cell exposed to sequential applications of depolarizing current injections. Right panel, Expanded time scale of APs from the recording shown in left panel.

 
These results indicate that AP properties differ from cell to cell, depending on the baseline potential reached during AP firing. Moreover, the pattern of firing in any given cell can be altered by changing the baseline potential with either depolarizing or hyperpolarizing current injections. Correlation analysis clearly indicates that the transition from sharp to broad APs is not an all-or-none event, but rather a graded, continuous process. The extremes of this continuum are represented by the neuronal-like (high amplitude, sharp) APs shown in Fig. 1CGo, and the endocrine-like (low amplitude, broadened) APs shown in Fig. 1AGo.

TTX-Sensitive Na+ Channels and Pattern of AP Firing
Since TTX-sensitive Na+ channels typically participate in AP generation in neurons, we characterized their involvement in GT1 cells exhibiting different patterns of spontaneous electrical activity (Fig. 3Go). In cells exhibiting a baseline potential more depolarized than -60 mV and low-amplitude, broad APs, TTX had no effect on electrical activity (Fig. 3AGo). However, in cells that exhibited a baseline potential around -60 mV, 1 µM TTX shifted the baseline potential to more depolarized levels, reduced AP amplitude, and increased AP duration (Fig. 3BGo). In cells exhibiting a baseline potential between -75 and -65 mV and high-amplitude, sharp APs, TTX transiently abolished spiking. This led to a gradual depolarization and the transition to low-amplitude, broad AP firing (Fig. 3CGo). The majority of cells exhibited the pattern of firing shown in Fig. 3BGo. These results demonstrate that, when operative, voltage-gated Na+ channels sharpened the APs in addition to increasing their amplitude. Thus, the exclusion of the underlying TTX-sensitive currents effectively transforms AP firing from the neuronal to the endocrine-like mode.



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Figure 3. Effects of the Voltage-Gated Na+ Channel Blocker, TTX, on Spontaneous Electrical Activity in GT1 Cells

A–C, left panels, Representative recordings of the effects of 1 µM TTX (arrow) on GT1 cells with varying baseline potentials. The majority of cells exhibited the pattern of firing shown in panel B. A–C, right panels, Expanded time scale of single APs in the absence (a) or presence (b) of 1 µM TTX.

 
To understand how the baseline potential level determines the AP properties, we examined the electrophysiological characteristics of the voltage-gated Na+ and Ca2+ currents generating each AP. To isolate inward Na+ currents, outward currents were blocked the intrapipette Cs+ and the cells were bathed with Ca2+-deficient Krebs-Ringer medium containing 5 mM tetraethylammonium (TEA). Under these conditions, a rapidly activating/inactivating Na+ current was observed after command potentials more depolarized than -60 mV (Fig. 4Go). In cells held at -97 mV, the time to peak current amplitude between command potentials of -37 mV and 3 mV was 4.1 ± 0.3 to 2.7 ± 0.1 msec (n = 4), and the maximum amplitude was around -20 mV (Fig. 4BGo). The mean Na+ current density at the peak of the current-voltage relation was 235 ± 70 pA/pFarad (pF) After activation, the Na+ current rapidly inactivated during the 200-msec voltage steps (Fig. 4AGo). The residual (<3%) non-inactivating current may suggest that an additional Na+ current is present in GT1 neurons, as in fish terminal nerve GnRH neurons (20). However, it may also be due to imperfect voltage control due to leak subtraction artifacts. The falling phase of the Na+ current elicited by command potentials to between -37 mV and 3 mV was fitted with a single exponential curve, and the calculated time course of inactivation was between 8 ± 0.6 and 1 ± 0.1 msec (n = 4).



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Figure 4. Voltage-Gated Na+ Current in GT1 Cells

A, Sodium current traces elicited by membrane potential steps to -87, -67, -47, -37, -27, -7, 13, 33, 53, 73 mV from a holding potential of -97 mV. B, Current-voltage relation of the voltage-gated Na+ current in GT1 neurons (mean ± SEM; n = 4). Inset, Whole-cell voltage-clamp recordings of the effects of TTX on Na+ current. From a cell hold at -97 mV, currents were elicited by a 20 msec voltage step to -17 mV. The average series resistance was 26 ± 2.7 M{Omega}, with 60–80% compensation.

 
The rapidly activating/inactivating Na+ currents in GT1 neurons were sensitive to the sodium channel blocker, TTX (Fig. 4Go, inset). Application of 1 µM TTX during voltage steps to -17 mV (holding potential = -97 mV) reduced the Na+ current amplitude from -721 ± 143 pA to -7 ± 3 pA (P < 0.05; n = 4). Higher concentrations of TTX had no further effect (data not shown). The inhibitory actions of TTX on Na+ current amplitude were observed at all membrane potentials between -57 mV and 83 mV (data not shown). Therefore, under our recording conditions, a conventional fast-activating and -inactivating TTX-sensitive Na+ current is present in GT1 neurons.

Although the rapid activation and current-voltage relation of the Na+ current suggest that it could generate the sharp upstroke of the APs in GT1 cells, TTX did not alter AP firing in a subpopulation of the cells examined. A possible explanation for this is that a large portion of the Na+ current is not available for activation in cells with relatively depolarized baseline potentials. This hypothesis was tested by examining the voltage dependence of isochronal (steady-state) inactivation of the Na+ current by using a two-pulse protocol (21). Cells held at -97 mV were subjected to prepulse potentials ranging from -127 mV to -7 mV for 100 msec, after which a test pulse to -17 mV was given (Fig. 5AGo). The normalized test current from five cells was plotted against the prepulse potentials, and the resulting curve was fitted with a single Boltzmann relation, where E1/2 and k (see Materials and Methods) were -68 mV and 9, respectively. This indicated that the amount of Na+ current available for activation ranges from 60–10%, depending on the baseline potential reached during AP activity. Moreover, relatively small changes in the level of the baseline potential can significantly alter the amount of Na+ current available for activation.



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Figure 5. Isochronal Inactivation and Recovery from Inactivation of the Na+ Current in GT1 Cells

A, Isochronal inactivation curves for the Na+ current were generated from prepulse experiments in which the Vm was stepped to between -127 mV and 3 mV for 100 msec before stepping to a command potential of -17 mV (holding potential = -97 mV). Left panel, representative traces of the remaining currents elicited during the command potential after prepulse potentials between -127 and 3 mV. Right panel, isochronal inactivation curve of the Na+ current (mean ± SEM, n = 5). The Na+ current was normalized to the maximum inward current and fitted with a single Boltzmann relation. B, Recovery from inactivation. A 100-msec command potential to -17 mV was given after a prepulse to -17 mV and the interpulse interval was varied from 10–500 msec. Left panel, Representative current traces elicited during the command potential at varying interpulse duration. Right panel, The Na+ current was normalized to the maximum inward current (mean ± SEM; n = 7) and plotted against the interpulse interval. The average series resistance for the recordings in panels A and B were 26 ± 2.7 M{Omega}, with 60–80% compensation. C, Changes in Vm in response to hyperpolarizing current injections of -10 pA in the absence or presence of 1 µM TTX. The asterisks indicate the high-amplitude, rebound AP after transient hyperpolarizing current injections. The arrow indicates the time of TTX application, which was present for the remainder of the experiment.

 
To determine the time period required to remove Na+ current inactivation, a 100-msec test pulse to -17 mV was given after a prepulse to the same amplitude, and the interpulse duration was varied (holding potential = -97 mV). The normalized test current was plotted against the interpulse duration, and the time required for reactivation of the Na+ current from inactivation was determined (Fig. 5BGo). Recovery from inactivation occurred at two different rates, 50% of the current being recovered in less than 50 msec and the remainder in more than 500 msec (Fig. 5BGo). This suggests that in cells with a sufficiently hyperpolarized baseline potential, the majority of the Na+ channels will recover from inactivation during the interspike interval, as in those cells exhibiting a baseline potential below -60 mV (Fig. 3Go, B and C).

Conversely, the inactivation and the time required for its removal account for the lack of involvement of TTX-sensitive Na+ channels in cells with relatively depolarized baseline potentials and high AP frequencies, as in cells firing endocrine-like APs (Fig. 3AGo). Accordingly, hyperpolarizing current injections in cells exhibiting endocrine-like APs generated a single, high-amplitude, sharp AP. In the presence of 1 µM TTX, hyperpolarizing current injections did not elicit high-amplitude, sharp APs (Fig. 5CGo). Thus, although transient membrane hyperpolarization could reactivate Na+ channels in cells exhibiting endocrine-like AP spiking, return to the depolarized baseline potential and high AP frequency prevented further Na+ channel participation.

Voltage-Gated Ca2+ Channels and Pattern of AP Firing
The properties of the VGCC subtypes expressed in GT1 cells were analyzed in Na+-deficient medium containing TEA and TTX, and with a pipette solution containing Cs+ and TEA. The extracellular Ca2+ concentration of 2.6 mM was the same as that used for the recordings of electrical membrane activity shown in Figs. 1–4GoGoGoGo. Under these conditions, command potentials more depolarized than -60 mV elicited a transient inward current that inactivated within 200 msec (Fig. 6Go, A and B). The inactivation rate of the transient current elicited during membrane potential (Vm) steps to between -60 and -40 mV could be fitted with a single exponential function and was between 110 ± 3 and 19 ± 2 msec (n = 7). These data indicate the existence of a low voltage-activated (LVA) Ca2+ current in GT1 neurons.



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Figure 6. Electrophysiological Characterization of Voltage-Gated Ca2+ Currents in GT1 Cells

A, Representative Ca2+ current traces elicited by 200-msec command potentials to -70, -50, -30, and -10 (holding potential of -90 mV) using conventional whole-cell recording techniques. B, Current-voltage relation of the early (open circles, 0–25 msec) and sustained (filled circles, 190–200 msec) Ca2+ currents (mean ± SEM; n = 7). Panels C, D, and E, Isochronal inactivation curves for the LVA and HVA Ca2+ current. The isochronal inactivation curve for the LVA Ca2+ current (panel C, open squares) was generated by giving a 200-msec command potential to -50 mV after 1-sec prepulse from -120 to -10 mV (holding potential = -90 mV). The isochronal inactivation curve for the HVA current (panel D, filled squares) was generated by measuring the sustained current amplitude during a 200-msec command potential to -10 mV after a 2-sec prepulse potentials from -120 to -10 mV. The continuous line for the isochronal inactivation curve of the LVA current is a fitted Boltzmann relation (E). Currents were normalized to the maximal inward current elicited during a command potential to -50 or -10 mV, after a prepulse to -120 mV (mean ± SEM; n = 7). All calcium current recordings shown in this figure were performed in the presence of 2.6 mM Ca2+ using regular whole-cell recording techniques.

 
In addition to the LVA Ca2+ current, 200-msec or 2-sec command potentials more depolarized than -50 mV (holding potential = -90 mV) elicited a slowly inactivating Ca2+ current (Fig. 6Go, A and B). To determine the inactivation rate of this high voltage-activated (HVA) Ca2+ current, 2-sec command potentials between -40 and 0 mV were applied, and the falling phase of the current was fitted with a double or single exponential curve. At these Vm values, the falling phase of the current was best described by a double exponential fit. The initial rates of inactivation were between 20 ± 2 and 36 ± 8 msec, followed by an inactivation rate between 280 ± 51 msec and 504 ± 116 msec (n = 7). The densities of the early (0–25 msec) and sustained (190–200 msec) Ca2+ currents at the peaks of their individual current-voltage relations were -9.2 ± 2.4 pA/pF (n = 7) and -3.6 ± 0.9 pA/pF (n = 7), respectively. These data are consistent with the coexistence of LVA and HVA Ca2+ currents in GT1 neurons.

The proportions of these Ca2+ currents available for activation at different Vm values were determined by analysis of their isochronal inactivation properties in seven cells, using a two-pulse protocol (21). The isochronal inactivation properties of the LVA Ca2+ current were examined by applying prepulse potentials from -120 to -10 mV for 1 sec (holding potential = -90 mV), followed by a test pulse to -50 mV to activate predominately LVA currents. The normalized test current was plotted against the prepulse potentials, and the resulting curve was fitted with a single Boltzmann relation (Fig. 6Go, C and E), where E1/2 and k were -68 mV and 12, respectively. To examine the isochronal inactivation properties of the HVA Ca2+ current, the current remaining at the end of a 200-msec test pulse to -10 mV was plotted against 2-sec prepulse potentials from -120 to -10 mV. The HVA current exhibited minimal voltage-dependent isochronal inactivation (Fig. 6Go, D and E). These data indicate that, like TTX-sensitive Na+ currents, the proportion of the LVA current available for activation depends on the baseline potential reached during AP activity. In contrast, a large proportion of the HVA current is available for activation, regardless of the baseline potential.

To further characterize the VGCC subtypes present, the effects of selective antagonists were examined. The T-type channel is a LVA Ca2+ channel and is sensitive to Ni2+ in the micromolar concentration range (22). In GT1 neurons, the LVA Ca2+ current elicited by a 200-msec voltage step to -50 mV (holding potential = -90 mV) was inhibited by Ni2+ in a concentration-dependent manner (Fig. 7AGo). Application of 100 µM Ni2+ reduced the LVA current amplitude to less than 10% of control, whereas up to 20% of HVA current was abolished during 200-msec membrane potential steps to -10 mV. Application of 50 µM and 100 µM Ni2+ reduced the voltage-gated Na+ current by 12 ± 2% and 14 ± 2% (n = 7), respectively. In cells exhibiting a baseline potential below -60 mV (four of nine cells), addition of 50 µM (Fig. 7BGo) and 100 µM Ni2+ (not shown) reduced the AP frequency but did not abolish spontaneous spiking. In cells with a baseline potential more depolarized than -60 mV, 50 or 100 µM Ni2+ did not alter spike frequency. Thus, as in experiments with TTX, the inhibitory effect of Ni2+ was observed only in spontaneously active cells with baseline potentials more negative than -60 mV. These results are consistent with the expression of T-type Ca2+ channels in GT1 neurons and their participation in pacemaker activity under certain conditions. As with Na+ channels, the inactivation properties probably account for the lack of involvement of Ni2+-sensitive Ca2+ channels in the generation of APs in cells exhibiting baseline potentials more depolarized than -60 mV.



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Figure 7. Pharmacological Characterization of Voltage-Gated Ca2+ Currents in GT1 Cells

A, Concentration-dependent effects of Ni2+, a blocker of T-type LVA Ca2+ channels, on isolated Ca2+ currents elicited by a 200-msec command potential to -40 mV (holding potential = -90 mV). Calcium current recordings were performed in the presence of 2.6 mM Ca2+ using regular whole-cell patch-clamp techniques. B, Effect of Ni2+ on the pattern of spontaneous spiking. C, Effect of nifedipine, a blocker of L-type HVA Ca2+ channels, on isolated Ba2+ currents. Voltage-clamp recording of isolated Ba2+ currents elicited by a 200-msec command potential to -10 mV (holding potential -90 mV) before and during the application of 1 µM nifedipine. D, Current-clamp recording of electrical membrane activity during the application of nifedipine (bar) and 5 min after its removal (right panel). Recordings shown in panels B, C, and D were done under perforated patch-clamp conditions.

 
HVA Ca2+ channels that are sensitive to the 1,4-dihydropyridine inhibitor, nifedipine, are frequently associated with Ca2+ entry during AP activity (22). The nifedipine sensitivity of the HVA currents in GT1 neurons was determined by measurement of the sustained Ba2+ current amplitude and electrical membrane activity in cells bathed in Ca2+-containing medium. As shown in Fig. 7Go, C and D, application of 1 µM nifedipine reversibly reduced the amplitude of the sustained current elicited by a 200 msec voltage step to -10 mV (holding potential = -90 mV), from -97 ± 20 pA to -13 ± 6 pA (P < 0.05; n = 4). This inhibition was observed during all command potentials more depolarized than -50 mV. Nifedipine did not affect voltage-gated Na+ currents elicited by voltage steps to -17 mV (holding potential = -97 mV; control = 1.22 ± 0.20 nA vs. 1 µM nifedipine = 1.21 ± 0.18 nA; P > 0.05; n = 4) or the LVA Ca2+ currents elicited by voltage steps to -50 mV (holding potential of -90 mV; control = -122 ± 21 pA vs. 1 µM nifedipine = -122 ± 27 pA; P > 0.05; n = 5). Spontaneous spiking activity was reversibly abolished by 1 µM nifedipine in TTX-sensitive and -insensitive cells (Fig. 7DGo).

Application of 10 µM Bay K 8644, an L-type Ca2+ channel agonist, during a 200-msec voltage pulse to -10 mV (holding potential = -90 mV) increased the sustained Ba2+ current amplitude from -102 ± 46 pA to -290 ± 69 pA (Fig. 8AGo; P < 0.05; n = 4). This increase was observed only during command potentials between -50 mV and -10 mV, resulting in a shift in the peak of the current-voltage relation to more hyperpolarized Vm; however, the activation threshold of the Ba2+ current was not altered (Fig. 8BGo). Application of 10 µM Bay K 8644 increased the amplitude and duration of AP spiking in GT1 neurons, consistent with its stimulatory actions on Ba2+ current amplitude (Fig. 8Go, C and D). Stronger membrane after-hyperpolarizations were also observed (Fig. 8CGo), suggesting that the threshold [Ca2+]i for activation of Ca2+-controlled K+ currents is reached. The increase in spike amplitude was abolished by 1 µM TTX or 50 µM Ni2+ (data not shown), again confirming that strong after-hyperpolarizations are sufficient to relieve a proportion of the voltage-gated Na+ and T-type Ca2+ currents from inactivation. In addition to these expected effects of Bay K 8644 on the firing pattern, its facilitation of Ca2+ influx was associated with an increase in the AP frequency (Fig. 8Go, C and D). Bay K 8644-induced increases in spike frequency were also observed in cells bathed in TTX- or Ni2+-containing medium (data not shown). These findings demonstrate that L-type Ca2+ current is the major spike depolarization current in spontaneously active GT1 neurons. They also further indicate that in cells with relatively depolarized baseline potentials, in which the TTX-sensitive and T-type channels are under inactivation, L-type Ca2+ channels contribute to both pacemaking and spike depolar-izations.



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Figure 8. Bay K 8644 Sensitivity of Spontaneous Spiking and Ba2+ Currents in GT1 Cells

A, Voltage-clamp recording of isolated Ba2+ currents elicited by a 200-msec command potential to -10 mV (holding potential = -90 mV) in the absence or presence of 10 µM Bay K 8644. B, Current-voltage relation of the Ba2+ current shown in panel A. C, Current-clamp recording of electrical membrane activity before, during (bar), and after 10 µM Bay K 8644 application to cells bathed in Ca2+-containing medium (2.6 µM). D, Expanded time scale of the recording in panel A before (solid line) and during (dotted line) Bay K 8644 application.

 
Plasma-Membrane Ca2+ Oscillator
In spontaneously active GT1 cells firing regular or irregular APs, each spike generated a discrete, transient increase in [Ca2+]i, producing an oscillatory-like pattern of [Ca2+]i signaling (Fig. 9AGo). The capacity of each AP to drive Ca2+ depended primarily on its duration, as the amplitude of the [Ca2+]i transients were larger in cells exhibiting broad APs than in those exhibiting sharp, high-amplitude APs (Fig. 9Go, C and D). In the small percentage of GT1 cells exhibiting bursting activity, the intermittent trains of AP spiking and the associated [Ca2+]i transients combined to generate larger amplitude [Ca2+]i fluctuations (Fig. 9BGo). These results indicate that the properties of each AP, as well as the overall pattern of AP spiking, can influence Ca2+ signaling in GT1 cells.



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Figure 9. AP-Driven Ca2+ Influx in GT1 Cells

A–C, Simultaneous measurements of spontaneous firing of APs and [Ca2+]i. A, AP-driven Ca2+ transients in a cell exhibiting relatively regular spiking behavior. B, High-amplitude [Ca2+]i fluctuations in a cell exhibiting bursting spiking behavior. C, Expanded time scale of APs and their associated [Ca2+]i transients in cells exhibiting narrow (left panel) and broad (right panel) spiking. D, Relationship between AP duration and the maximum amplitude of the [Ca2+]i transients (r = coefficient of correlation; n = 53).

 
Inhibition of spiking by hyperpolarizing current injections, or by addition of the nonselective Ca2+ channel blocker, Cd2+ (50 µM), or 1 µM nifedipine, abolished the [Ca2+]i transients and decreased global [Ca2+]i. Figure 10AGo illustrates the effects of nifedipine on spontaneous electrical activity and [Ca2+]i in a patch-clamped and indo-1-loaded cell. Perifusion of cells with Ca2+-deficient medium was also associated with abolition of spiking. However, unlike nifedipine and Cd2+ treatments, depletion of extracellular Ca2+ hyperpolarized the membrane (upper panel) and further reduced [Ca2+]i after addition of nifedipine (bottom panel) or Cd2+ (not shown). Similar effects of nifedipine and addition of Ca2+-deficient medium were observed in unpatched and fura-2-loaded cells (Fig. 10BGo). Conversely, AP broadening by the application of 10 µM Bay K 8644 increased the amplitude of the [Ca2+]i transients and increased total [Ca2+]i (Fig. 10CGo). Thus, voltage-gated Ca2+ entry through L-type Ca2+ channels contributes to AP generation in GT1 cells and drives changes in [Ca2+]i, consistent with the operation of a plasma membrane Ca2+ oscillator in these cells. Moreover, an increase in AP duration generates larger amplitude [Ca2+]i fluctuations, despite the decrease in AP amplitude. Finally, another Ca2+-conducting current participates in the control of baseline potential and [Ca2+]i.



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Figure 10. Effects of Nifedipine, Ca2+-Deficient Medium, and Bay K 8644 on Spontaneous Electrical Activity and [Ca2+]i in GT1 Cells

A, Simultaneous measurement of Vm and [Ca2+]i during the application of 1 µM nifedipine followed by removal of extracellular Ca2+ (-Ca2+) in indo-1-loaded cells. B, Effects of 1 µM nifedipine and extracellular Ca2+ removal on [Ca2+]i in fura-2-loaded and unpatched GT1 cells. The trace represents the mean ratio (F340/F380) from 44 separate cells. The dashed line in panels A and B illustrates the [Ca2+]i in the presence of nifedipine. C, Simultaneous measurements of APs and [Ca2+]i in the absence (upper panel) or presence (lower panel) of 10 µM Bay K 8644. Electrical membrane activity and [Ca2+]i were recorded in the presence of normal Krebs-Ringer’s medium using the perforated patch-clamp recording technique.

 
GnRH-Induced Shift in the Pattern of AP Firing
Activation of Ca2+-mobilizing GnRH receptors in GT1 cells generated biphasic changes in Vm and [Ca2+]i. GnRH initially induced a spike increase in [Ca2+]i that transiently hyperpolarized the plasma membrane (Fig. 11AGo). This was followed by sustained depolarization with a change in baseline potential from -63.4 ± 1.8 mV to -54.3 ± 2.2 mV (P < 0.001; n = 15) and a concomitant increase in spike frequency, which coincided with a sustained elevation in [Ca2+]i (Fig. 11AGo). In addition to cell depolarization and an increase in spike frequency, GnRH caused an increase in AP duration and a decrease in AP amplitude (Fig. 11BGo). The increase in AP duration was accompanied by an increase in the amplitude of the AP-driven [Ca2+]i transients (Fig. 11BGo). These effects were similar to those observed after current-induced membrane depolarization (Fig. 2BGo). This is consistent with the hypothesis that GnRH-induced depolarization increases the number of TTX-sensitive Na+ channels and T-type Ca2+ channels under inactivation. This, in turn, should reduce their participation in AP generation, shifting the AP firing pattern from neuronal-like to endocrine-like and thereby increasing the capacity of the plasma-membrane Ca2+ oscillator to drive Ca2+.



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Figure 11. GnRH-Induced Modulation of Spontaneous Electrical Activity in GT1 Cells

A Left panel, Simultaneous measurements of Vm and [Ca2+]i in response to application of 100 nM GnRH. Right panel, Increase in spike frequency (mean ± SEM; n = 15) after the application of 100 nM GnRH. B, left panel, Expanded time-scale of single APs and associated [Ca2+]i transients from the recording in panel A labeled a and b. Right panel, Effects of 100 nM GnRH on spike amplitude and duration (mean ± SEM; n = 15). Asterisks indicates significant differences compared with control values: P < 0.05, Student’s t-test.

 
To test this hypothesis, the actions of VGCC antagonists and extracellular Ca2+ depletion during GnRH stimulation were examined. To avoid the possible impact of patch clamping on GnRH-induced Ca2+ influx, and to monitor changes in [Ca2+]i from several cells simultaneously, Ca2+ imaging experiments using fura-2 AM-loaded GT1 cells were performed. As in patch-clamped and indo-1-loaded cells, application of 100 nM GnRH stimulated a biphasic increase in [Ca2+]i, composed of a transient spike phase and a sustained plateau phase (Fig. 12AGo). In the presence of 1 µM nifedipine, the GnRH-induced spike phase was not affected but the [Ca2+]i plateau was reduced by about 50% (Fig. 12Go, A and B). Similar results were observed during nonselective blockade of VGCC with Cd2+ (Fig. 12BGo). Application of 1 µM nifedipine during sustained GnRH stimulation also reduced the [Ca2+]i plateau phase by about 50% (Fig. 12CGo). Simultaneous measurement of Vm and [Ca2+]i further indicated that the GnRH-induced [Ca2+]i plateau was mediated in part by voltage-gated Ca2+ entry (Fig. 12EGo). Conversely, addition of 100 nM GnRH in the presence of Ca2+-depleted medium generated a monophasic increase in [Ca2+]i (Fig. 12AGo), since the [Ca2+]i plateau phase was completely abolished (Fig. 12BGo). Also, unlike blockade of voltage-gated Ca2+ entry, inhibition of voltage-gated Na+ channels by 1 µM TTX did not alter the GnRH-induced [Ca2+]i spike or plateau phase (Fig. 12DGo). Thus, an increase in the capacity of the plasma-membrane Ca2+ oscillator to drive Ca2+, due to increased AP frequency and duration, contributes to the GnRH-induced [Ca2+]i plateau phase. However, a VGCC-independent Ca2+ influx pathway also contributes to this phase of the GnRH-induced Ca2+ response.



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Figure 12. Contribution of Ca2+-Driven APs to GnRH-Induced Plateau [Ca2+]i Response in GT1 Cells

A, Pattern of GnRH-induced [Ca2+]i response in Ca2+-containg medium with or without 1 µM nifedipine or in Ca2+-deficient medium. B, GnRH-induced change in plateau [Ca2+]i (mean ± SEM, measured 3 min after GnRH addition) in the absence or presence of 1 µM nifedipine, 50 µM CdCl2, or in Ca2+-deficient medium. C, Effects of sustained addition of 1 µM nifedipine in GnRH-stimulated cells. D, Lack of effect of TTX on GnRH-induced [Ca2+]i response. In panels A–D, changes in [Ca2+]i (F340/F380) were simultaneously monitored in up to 30 individual and unpatched cells loaded with fura-2, and the mean [Ca2+]i responses are shown. E, Simultaneous measurement of Vm and [Ca2+]i in indo-1-loaded GT1 cells under perforated patch-clamp recording conditions. The bar and shaded area indicate the duration of 1 µM nifedipine or Ca2+-deficient medium application, respectively. GnRH (100 nM) was present from the time of application indicated by the arrow.

 
The agonist-induced shift from neuronal- to endocrine-like spiking may be due to inhibition of TTX-sensitive Na+ channels and T-type Ca2+ channels and/or augmentation of L-type Ca2+ channels. To test this, we examined the actions of GnRH on isolated Na+ and Ca2+ currents, as well as on Vm in the presence of VGCC antagonists. Application of 100 nM GnRH did not alter the Na+ current or the peak or sustained Ca2+ current (Fig. 13Go, A and B). Moreover, the GnRH-induced membrane depolarization was maintained in the presence of 1 µM nifedipine and 50 µM Ni2+ (Fig. 13CGo). These data indicate that neither inhibition of Na+ or T-type current, nor direct augmentation of L-type Ca2+ channels, mediates the actions of GnRH. This further supports the view that the removal of Na+ and T-type Ca2+ currents during GnRH action is due to their inactivation imposed by the GnRH-induced depolarization of the cell membrane.



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Figure 13. Effects of GnRH on Voltage-Gated Na+ and Ca2+ Currents in GT1 Cells

A, left panel, Superimposed Ca2+ current traces elicited during a 200-msec voltage-step to -10 mV (holding potential = -90 mV) in the absence or presence of 100 nM GnRH. Right panel, Ca2+ current amplitude during the peak (0–25 msec) and sustained (190–200 msec) current in the presence or absence of 100 nM GnRH (mean ± SEM; n = 6). Calcium current recordings were performed in the presence of 10 mM Ca2+. B, left panel, Superimposed Na+ current traces elicited by a 50-msec voltage step to -17 mV in the absence or presence of 100 nM GnRH. Right panel, Na+ current amplitude in the presence or absence of 100 nM GnRH (mean ± SEM; n = 6). C, Application of 100 nM GnRH in the presence of 1 µM nifedipine and 50 µM Ni2+ under current-clamp recording conditions. The dotted line represents the baseline potential in unstimulated cells.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Many neuronal and endocrine cells generate APs spontaneously or in response to agonist stimulation. When such firing is associated with periodic Ca2+ influx, it is referred to as a plasma membrane Ca2+ oscillator (23). For example, APs generated in conjunction with, or solely by, Ca2+ influx through VGCC transiently drive Ca2+ into the cell to cause discrete fluctuations in [Ca2+]i. This is an integral part of the cellular Ca2+ homeostatic pathway in these cells, and is intimately involved in the control of molecular and cellular function (24). Earlier studies have indicated that GT1 neurons express T-type and L-type voltage-gated Ca2+ channels, voltage-gated Na+ channels, and voltage-dependent and Ca2+-controlled K+ channels, but do not exhibit spontaneous AP firing (6). In other reports, bursting AP activity and high-amplitude [Ca2+]i fluctuations were observed in isolated and interconnected GT1 cells (9, 11). Embryonic and terminal nerve GnRH neurons express a comparable set of plasma membrane channels, but exhibit regular AP firing (7). Like native GnRH neurons, a majority of the GT1 cells examined in this study exhibited relatively regular AP firing, whereas a small percentage of the cells exhibited bursting AP firing comparable to that observed by Charles and co-workers (8, 9).

The ability of a majority of the GT1 cells examined in the present study to fire more frequent and relatively regular APs is probably related to their degree of differentiation. In our experiments, electrical membrane recordings were acquired from morphologically differentiated cells that had been cultured in defined medium without FCS for at least 7 days. In contrast, Bosma (6) and Charles and Hales (9) employed GT1 cells cultured in serum-containing medium, which facilitates cell division. This may affect the density of expressed channels and, consequently, the underlying excitability of the cells. For example, although both TTX-sensitive Na+ currents and voltage-gated Ca2+ currents were observed in differentiated ( Figs. 4–8GoGoGoGoGo) and less-differentiated cells (6), there were significant differences in the amplitudes of these inward currents. In general, the amplitudes of Na+ and Ca2+ currents in differentiated cells were 3- to 4-fold higher than in the less-differentiated cells. Furthermore, only isolated cells were used to exclude any effects of electrical or synaptic coupling between cells that could influence the baseline potentials in connected cells.

Although the presence of TTX-sensitive Na+ channels, as well as T-type and L-type Ca2+ channels, endows GT1 cells with the ability to fire spontaneous APs, the relative contribution of each channel is dependent on the baseline potential, which ultimately determines the pattern of AP firing. For example, in cells that were more hyperpolarized than -60 mV, all three currents contributed to AP firing. Due to their sharp profile, high amplitude, and TTX sensitivity, these APs were also referred to as neuronal-like. In contrast, in cells with baseline potentials more depolarized than -60 mV, a large proportion of the Na+ and T-type Ca2+ currents had undergone inactivation, so that only L-type Ca2+ channels contributed to AP activity. This resulted in broad, low-amplitude APs and an increase in firing frequency. Since many excitable endocrine cell types also exhibit TTX-insensitive firing (24), this pattern was referred to as endocrine-like. However, the transition from sharp, neuronal-like to broad, endocrine-like AP firing is a continuous, graded process that is determined by the proportion of Na+ and T-type Ca2+ currents available for activation. Regardless of the baseline potential, and the resulting composition of the currents contributing to each AP, transient activation of L-type Ca2+ channels underlies the basal fluctuations in [Ca2+]i in all cells. Thus, a plasma membrane Ca2+ oscillator is operative in cells exhibiting both sharp and broad APs. The capacity of the plasma membrane Ca2+ oscillator to drive Ca2+ into the cell is dependent on the baseline potential, which influences both AP frequency and duration.

In GT1 cells firing sharp APs, depolarization of the baseline potential by current injection or GnRH increases AP frequency and duration and facilitates Ca2+ influx. Membrane depolarization brings the baseline potential closer to the firing threshold, resulting in an increase in firing frequency and concomitant Ca2+ entry. In addition, membrane depolarization progressively inactivates TTX-sensitive Na+ currents and T-type Ca2+ currents, which decreases AP amplitude and increases AP duration. Similar effects were observed in cells after inhibition of voltage-gated Na+ currents by TTX. In resting and GnRH-stimulated GT1 cells, the increase in AP duration increases the magnitude of AP-driven [Ca2+]i transients, despite a decrease in AP amplitude. The increase in AP duration caused by the Ca2+ channel agonist, Bay K 8644, also amplified AP-driven [Ca2+]i transients. Although there is a decrease in AP amplitude in cells firing endocrine-like APs, comparison of AP amplitude and the Ca2+ current/voltage relation indicate that these APs are able to strongly activate HVA Ca2+ currents. As in GT1 cells, AP prolongation increased Ca2+ influx in smooth muscle cells (25). Similarly, although an increase in AP duration decreases the peak Ca2+ current in rat sympathetic neurons (26) and ventricular myocytes (27), there is a net increase in Ca2+ influx. In addition, comparison of high-amplitude, sharp APs and low-amplitude, broad APs in rat sympathetic neurons indicated that an increase in AP duration facilitates Ca2+ influx, despite the decrease in AP and Ca2+ current (26). Therefore, AP duration, as well as AP amplitude, is critical in determining the net Ca2+ influx per single spike.

The patterns of firing and associated Ca2+ influx are not only controlled by the voltage-gated inward currents that were the primary focus of this study. Voltage-gated K+ channels also play an important role in the pattern of AP spiking (28). The decrease in AP amplitude by inactivation of voltage-gated Na+ and T-type Ca2+ channels could result in a reduction in the net activation of voltage-gated K+ channels during spike depolarization. This would reduce the potassium current needed to repolarize the cells, prolonging the opening of voltage-gated L-type Ca2+ channels and resulting in AP broadening and enhanced Ca2+ influx. The increase in AP frequency after membrane depolarization may also induce AP broadening by frequency-dependent inactivation of voltage-gated K+ channels (29). The similar effects of GnRH and depolarizing current injections on AP duration argue against second messenger-mediated inhibition of K+ channels, as observed in other cell types (30). Thus, voltage-gated Na+ and T-type Ca2+ channels are critical for controlling AP duration, presumably by influencing the degree of voltage-gated K+ channel activation, whereas L-type Ca2+ currents are the major contributors to spike depolarization and AP-driven [Ca2+]i transients in GT1 cells.

In both unstimulated and agonist-stimulated cells, the ability of spontaneous APs to drive Ca2+ is proportional to the level of membrane depolarization. The extracellular Ca2+-dependence and nifedipine, Ni2+, Cd2+, and TTX insensitivity of the baseline potential, as well as the partial dependence of basal and GnRH-induced plateau [Ca2+]i responses on Ca2+ influx through voltage-insensitive Ca2+ channels, are consistent with the operation of an additional Ca2+-conducting channel in GT1 neurons. This current may be similar to the Ca2+-controlled nonselective cationic currents activated by Ca2+-mobilizing receptors in other cells (31, 32). It may also be related to the store-operated Ca2+ currents observed in nonexcitable cells (33). The lack of effect of GnRH on isolated Ca2+ or Na+ currents in GT1 cells further indicates that GnRH-induced membrane depolarization is not due to direct augmentation of either of these currents. In general, inhibition of inwardly rectifying K+ channels (6, 35) or M-like K+ currents (34) may mediate agonist-induced membrane depolarization, but not the increased Ca2+ influx and the dependence of baseline potential on extracellular Ca2+.

In conclusion, the present results demonstrate the physiological impact of spontaneous and receptor-mediated inactivation of two typical pacemaker currents carried by TTX-sensitive Na+ channels and T-type Ca2+ channels. In classical neurons, TTX-sensitive channels drive AP depolarization. Coexpression of T- and L-type Ca2+ channels is also frequently observed in neural and neuroendocrine cells that exhibit spontaneous activity (24), as well as in other spontaneously firing cell types (36). However, spontaneous activity frequently occurs at voltages at which T-type channels are almost completely inactivated. Both Na+ and T-type Ca2+ channels participate in pacemaking in GT1 neurons, but only in cells with baseline potential between -60 and -75 mV. Inhibition of these channels by TTX and Ni2+ does not silence the cells, but promotes Ca2+ influx by prolonging AP duration. These currents are suppressed during depolarization of cells to levels that inactivate the majority of the two channels. Such depolarization is facilitated by activation of Ca2+-mobilizing GnRH receptors, but also occurs spontaneously through a mechanism that has not yet been characterized. In both resting and GnRH-stimulated cells, the depolarizing current that controls the baseline potential also directly contributes to Ca2+ influx.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GT1 Cell Culture
All experiments were performed on the GT1–7 subtype of immortalized GnRH neurons (3), which were originally provided by Dr. Richard I. Weiner (University of California, San Francisco, CA). The cells were grown in 75-ml culture flasks containing culture medium (DMEM/F-12, 1:1, with L-glutamate, pyridoxine hydrochloride, 2.5 g/liter sodium bicarbonate, 10% heat-inactivated FBS, and 100 µg/ml gentamicin; GIBCO, Grand Island, NY). At confluence, the cells were dispersed by trypsinization (0.05% trypsin) for 10 min, resuspended in culture medium, and plated (50,000 cells/ml) in 35-mm tissue culture dishes (Corning, Corning, NY) with or without poly-L-lysine-coated (0.01%) coverslips. After incubation for 48 h, the culture medium was replaced with medium containing B-27 serum-free supplement (GIBCO) to induce morphological differentiation of the cells. All experiments were performed 3 to 5 days after serum removal.

Electrophysiological Recordings
Ionic currents were measured using the whole-cell, gigaohm-seal (37) or perforated-patch recording technique (38). All current-clamp recordings of Vm were carried out using the perforated-patch recording technique. Current- and voltage-clamp recordings were performed at room temperature using an Axopatch 200 B patch-clamp amplifier (Axon Instruments, Foster City, CA) and were low-pass filtered at 2 kHz. For perforated patch recordings, patch pipette tips (3–5 M{Omega}) were briefly immersed in amphotericin B-free solution and then backfilled with amphotericin B (240 µg/ml)-containing solution. Before seal formation, liquid junction potentials were canceled. An average series resistance of 19 ± 1 M{Omega} (n = 237) was reached 10 min after the formation of a gigaohm seal (seal resistance > 5 G{Omega}) and remained stable for up to 1 h. When necessary, series resistance compensation was optimized and current records were corrected for linear leakage and capacitance using a P/-N procedure (21). Pulse generation, data acquisition, and analysis were done with an PC equipped with a Digidata 1200 A/D interface in conjunction with pCLAMP 7 (Axon Instruments). All recordings of Vm were digitized at 2 kHz using the software package AxoScope 1.1 (Axon Instruments). In some cases, the current-voltage relations were fit with a single Boltzmann relation: I/Imax = Imax + exp[(E - E1/2)/k]; where Imax is the maximum inward current, E is the command potential, E1/2 is the Vm at which there is 50% of the maximal current, and k is the slope factor. Exponential fits were performed using clampfit. For the determination of the AP properties in individual cells, the mean baseline potential, as well as AP duration (half-amplitude) and amplitude (threshold to peak) were determined from at least five individual cells under control or experimental conditions. Action potential frequency was determined during a 1- to 2-min period. For the determination of the early (0–25 ms) and sustained (190–200 ms) Ca2+ current amplitude, the peak current amplitude during the respective time periods was determined. All values in the text are reported as mean ± SEM. Differences between groups were considered to be significant when P < 0.05 using paired t-test or ANOVA, followed by Fisher’s least significant difference test.

Simultaneous Measurement of [Ca2+]i and Vm
GT1 neurons were incubated for 30 min at 37 C in phenol red-free medium 199 containing Hanks’ salts, 20 mM sodium bicarbonate, 20 mM HEPES, and 0.5 µM indo-1 AM (Molecular Probes, Eugene, OR). The coverslips with cells were then washed twice with modified Krebs-Ringer’s solution containing (in millimolar concentrations): 120 NaCl, 4.7 KCl, 2.6 CaCl2, 2 MgCl2, 0.7 MgSO4, 10 HEPES, 10 glucose (pH adjusted to 7.4 with NaOH) and mounted on the stage of an inverted epifluorescence microscope (Nikon, Melville, NY). A photon counter system (Nikon) was used to simultaneously measure the intensity of light emitted at 405 nm and at 480 nm after excitation at 340 nm. Background intensity at each emission wavelength was corrected. Perforated patch recording techniques (see above) were used to monitor Vm. The data were digitized at 4 kHz using a PC equipped with the pCLAMP 7-software package in conjunction with a Digidata 1200 A/D converter (Axon Instruments. The [Ca2+]i was calibrated in vivo according to Kao (39). Briefly, Rmin was determined by exposing the cells to 10 µM Br-A23187 in the presence of Krebs-Ringer’s solution with 2 mM EGTA and 0 Ca2+ for 60 min; 15 mM Ca2+ was then added to determine Rmax. The values used for Rmin, Rmax, Sf,480/Sb,480, and dissociation constant (Kd) were 0.472, 3.634, 3.187, and 230 nM, respectively.

Measurement of [Ca2+]i in Unpatched Cells
To determine the Ca2+ signaling patterns in cells not under patch-clamp recording conditions, single-cell fura-2 Ca2+ imaging techniques were used. GT1 neurons were incubated for 30 min at 37 C in phenol red-free medium 199 containing Hanks’ salts, 20 mM sodium bicarbonate, 20 mM HEPES, and 0.5 µM fura-2 AM (Molecular Probes). The cells on coverslips were subsequently washed with Krebs-Ringer solution and mounted on the stage of an Axiovert 135 microscope (Carl Zeiss, Oberkochen, Germany) attached to the Attofluor Digital Fluorescence Microscopy System (Atto Instruments, Rockville, MD). Cells were examined under a 40x oil immersion objective during exposure to alternating 340- and 380-nm light beams, and the intensity of light emission at 520 nm was measured. The ratio of light intensities, F340/F380, which reflects changes in [Ca2+]i, was simultaneously measured in several single cells.

Chemicals and Solutions
Stock solutions of TTX citrate (Research Biochemicals International, Natick, MA) and GnRH (Peptides International, Louisville, KY) were prepared in double-distilled, deionized water. Stock solutions of nifedipine and S(-)-Bay K 8644 (Research Biochemicals International) were dissolved in dimethylsulfoxide and ethanol, respectively. The maximum final concentrations of dimethylsulfoxide and ethanol were 0.1% and 0.01%, respectively, neither of which altered electrical membrane activity or ionic currents. For recordings of electrical membrane activity and total inward and outward currents, the extracellular medium contained modified Krebs-Ringer salts and the pipette solution contained (in millimolar concentration): 70 KCl, 70 K-aspartate, 1 MgCl2, and 10 HEPES (pH adjusted to 7.2 with KOH). In some experiments, Krebs-Ringer salts without CaCl2 was used and is referred to as Ca2+-deficient medium since it may contain residual amounts of Ca2+ in the absence of added EGTA. To record isolated Na+ currents, the extracellular medium contained Krebs-Ringer’s solution without CaCl2 and with 20 mM TEA, 5 mM 4-AP, and 50 µM CdCl2, and the pipette contained [in millimolar concentration (mM) ]: 70 CsCl, 70 Cs-methanesulfonate, 2 MgCl2, and 10 HEPES (pH adjusted to 7.2 with CsOH). To record isolated Ca2+ currents using conventional whole-cell recording techniques, the extracellular medium contained (in mM): 100 tetramethylammonium-Cl, 20 TEA, 2.6 or 10 CaCl2, 1 MgCl2, 1 µM TTX and 10 HEPES (pH adjusted to 7.4 with tetramethylammonium-OH). The pipette solution contained (in mM): 120 CsCl, 20 TEA-Cl, 4 MgCl2, 10 EGTA, 9 glucose, 20 HEPES, and 0.3 Tris-GTP, 4 Mg-ATP, 14 creatine-PO4, and 50 U/ml creatine phosphokinase (pH adjusted to 7.2 with Tris base). To record isolated Ba2+ currents using perforated-patch techniques, the extracellular solution contained (mM): 120 TEA-Cl, 30 BaCl2, 2 MgCl2, 10 glucose, and 10 HEPES (pH adjusted to 7.2 with TEA-OH), and the pipette solution was the same as that used for isolated Na+ current recordings. All reported Vm values under total current and isolated Na+ current recording conditions were corrected for a liquid junction potential between the pipette and bath solution of +10 mV and +7 mV, respectively, calculated using the JPCalc program (Axon Instruments, Ref. 40). The junction potentials for isolated Ca2+ and Ba2+ current recordings were less than 3 mV and were not corrected for. The bath contained less than 500 µl of saline and was continuously perfused at a rate of 2 ml/min using a gravity-driven superfusion system. The outflow was placed near the cell, resulting in complete solution exchange around the cell within 2 sec. A solid Ag/AgCl reference electrode was connected to the bath via a 3 M KCl agar bridge.


    FOOTNOTES
 
Address requests for reprints to: Dr. Stanko Stojilkovic, National Institute of Child Health and Human Development, Endocrinology and Reproduction Research Branch, Building 49, Room 6A-36, 49 Convent Drive, Bethesda, Maryland 20892-4510. E-mail: stankos{at}helix.nih.gov

Received for publication August 31, 1998. Revision received December 14, 1998. Accepted for publication December 16, 1998.


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 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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