| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Centre for Neuroendocrinology and Department of Physiology, University of Otago School of Medical Sciences, Dunedin 9054, New Zealand
Address all correspondence and requests for reprints to: Allan E. Herbison, Centre for Neuroendocrinology, Department of Physiology, University of Otago School of Medical Sciences, P.O. Box 913, Dunedin 9054, New Zealand. E-mail: allan.herbison{at}stonebow.otago.ac.nz.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Two recent studies have demonstrated that afterdepolarizing potentials (ADPs) are important in enabling GnRH neurons to fire in a bursting manner (5, 6). The sequential summation of small ADPs, generated after each action potential, results in a depolarized plateau that facilitates repetitive firing by GnRH neurons. The ADP results from sodium influx through tetrodotoxin (TTX)-sensitive, voltage-gated sodium channels in GnRH neurons (6). The other potentials believed to play a major role in shaping burst firing are the medium (
200 msec) and slow (seconds) after hyperpolarization potentials (AHPs). These currents result from activation of Ca2+-sensitive K+ channels after calcium entry through voltage-dependent calcium channels during the action potential or calcium released from intracellular calcium stores (7, 8, 9, 10). The AHP typically reduces the probability of subsequent action potentials and controls the frequency and patterning of repetitive firing in neurons (9, 10). Small-conductance calcium-activated potassium (SK) channels underlie the medium AHP, whereas the molecular identity of the channels responsible for the slow AHP remain to be determined (8, 9). Recent investigations have shown that GnRH neurons express SK mRNA (11, 12) and that apamin, a highly selective SK channel antagonist, inhibits the AHP in rat and mouse GnRH neurons (12, 13). However, the role of SK channels and AHPs in shaping endogenous firing in GnRH neurons remains unexplored.
In the present series of experiments, we tested the hypothesis that SK channels are tonically active in GnRH neurons and that they play a role in controlling excitability and burst firing through generation of the AHP. Using a GnRH-green fluorescent protein (GFP) mouse model, current- and voltage-clamp perforated-patch recordings were used to: 1) establish a protocol for full activation of the AHP in GnRH neurons, 2) determine the sensitivity of the AHP to the SK antagonist apamin, and 3) examine the effects of SK blockade on the excitability and patterning of burst firing in GnRH neurons.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Electrophysiology
Gramicidin-perforated recordings of GnRH neurons were undertaken on 200-µm-thick coronal brain slices containing the rostral preoptic area cut with a vibratome (VT1000S; Leica, Québec, Canada). After cutting with high (6 mM) MgCl2 and low CaCl2 (0.5 mM) artificial cerebrospinal fluid (aCSF), slices were incubated at room temperature in aCSF (in mM: 118 NaCl, 3 KCl, 2.5 CaCl2, 1.2 MgCl2, 11 D-glucose, 10 HEPES, 25 NaHCO3) equilibrated with 95% O2-5% CO2 for at least 1 h before being transferred to a submerged recording chamber, in which the slices were viewed under a fixed-stage upright microscope (BX51WI; Olympus, Tokyo, Japan). Fluorescent GnRH neurons were identified under brief fluorescence illumination and patched under Nomarski differential interference contrast optics. Apamin (Sigma, St. Louis, MO) was bath applied for 4 min by inclusion in the aCSF at concentrations of 0.1, 1, 10, 100, 200, and 400 nM. Patch pipettes with resistances between 3 and 5.5 MOhms were pulled from glass capillaries (inner diameter 1.18 mm; outer diameter 1.5 mm) with a microelectrode puller (Sutter Instruments, Novato, CA). Gramicidin (Sigma) was dissolved in methanol to a concentration of 10 mg/ml, which was diluted in the pipette solution (in mM: 135 CH3KSO4, 4.5 NaCl, 0.1 CaCl2, 2 MgATP, 0.2 Na2GTP, 0.2 Na2ATP, 10 HEPES, 1.1 EGTA, 7 phosphocreatine-Tris, 0.1 leuptine (pH 7.3), 285 mOsmol) to a final concentration of 75 µg/ml just before use and then sonicated for 1–2 min. After a giga-seal was established, access resistance was monitored, and experiments in current clamp were begun when access resistance (Ra) reduced to less than 100 MOhms (mean ± SEM of 49 ± 3 MOhms).
All afterhyperpolarization current (IAHP) data were collected when Ra was between 15 and 35 M
. Signals (voltage and current) were amplified with a Multiclamp 700B (CV7B; Molecular Devices, Foster City, CA) and sampled online with the use of a Digidata 1440A interface (Molecular Devices) connected to a personal computer. Signals were filtered (3 or 10 kHz; Built-in Bessel filter of Multiclamp 700B) before being digitized at a rate of 1 or 10 kHz. Acquisition and subsequent analysis of the acquired data were performed with the Clampex suite software 10 (Molecular Devices) and Origin pro 7.5 (OriginLab Corp., Northampton, MA). The resting membrane potentials were the potentials recorded in current clamp without any holding current after whole-cell recordings established. The calculated liquid junction potentials was approximately 12 mV, with the resting membrane potential not corrected. The input resistances were determined by fitting the linear part of the current-voltage curve (in current clamp between –60 and –90 mV). During experiments, Ra was monitored, and if it changed by more than 15%, the cell was discarded. The decay time constant (two thirds from maximal amplitude) of IAHP was determined by exponential fitting to the descending phase of IAHP (complete return to baseline within 20–40 sec), evoked by depolarizing the cell to 40 mV for 600 msec from a holding potential of –60 mV (in voltage clamp). Burst analysis was performed with the Clampfit program (15). The Surprise Factor is an index of deviation from Poisson distribution such that a large Surprise Factor represents data farther away from a Poisson distribution. A significant membrane depolarization was defined as being greater than 1.5 mV change from the baseline. The amplitude of IAHP was calculated as the difference of the baseline and peak of IAHP.
Statistical analysis
Data are expressed as mean ± SEM for the number of samples indicated. Unless otherwise indicated, data were analyzed using Student’s t tests.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(n = 50). At RMP, 50% of GnRH neurons were silent, 29% exhibited bursting patterns of action potentials, with the remainder (21%) being spontaneously active but not firing in bursts.
Characterization of SK channel-mediated currents in GnRH neurons
A voltage-clamp protocol to fully activate SK channels in GnRH neurons was first established. The SK channel is voltage independent but regulated by intracellular calcium levels. Hence, the depolarization of a GnRH neuron will result in calcium influx through voltage-dependent calcium channels and subsequent activation of SK channels to generate the AHP (12). Initially, five GnRH neurons were held at –60 mV and depolarized to 40 mV for different durations ranging from 100 to 700 msec in 100-msec increments (Fig. 1A
). The amplitude of the IAHP generated by this procedure was dependent on the voltage step with the mean half-maximal activation achieved at 244 ± 17 msec (n = 5; Fig. 1A). Next, GnRH neurons were depolarized for 600 msec but with the voltage step changed in 10-mV increments from –40 to 40 mV. This showed that depolarization to 30–40 mV was required to fully activate the IAHP in GnRH neurons (Fig. 1B). In subsequent experiments a 600-ms, –60 to 40 mV depolarizing step was used to ensure maximal activation of the GnRH neuron IAHP. No sex differences were found in evoked IAHP amplitude (diestrous and estrous females, 35.0 ± 5.4 pA, n = 10; males, 35.9 ± 3.2 pA, n = 40) or decay time constant (females, 1342.5 ± 26.3 msec; males, 2479.7 ± 453.6 msec). In the male group, five of the 40 GnRH neurons exhibited exceptionally long decay constants between 9,000 and 10,000 msec. After excluding these cells, the time constant for males was 1607.6 ± 207.4 (n = 35). The amplitude (30.9 ± 5.1 pA) and decay time constant (1791.2 ± 435.9 msec) of the IAHP were found to be similar in whole-cell recordings of GnRH neurons (Ra = 7–18 M; n = 9).
|
). An apamin-sensitive current was recorded in all 50 GnRH neurons and found to have an average peak amplitude of 35.7 ± 2.7 pA (range 9.4–86.9 pA) and decay time constant of 2167.0 ± 356.7 msec (range 665.9–10705.7 msec, n = 50). In most cells, the IAHP had an onset of approximately 10 msec (time from termination of stimulation pulse). The apamin-sensitive current had a reversal potential of –92 ± 2 mV (n = 4; Fig. 1D), consistent with SK channels being selective for potassium ions. After treatment with 100 nM apamin, a residual (10%) IAHP was found in 42% of GnRH neurons (amplitude 4.0 ± 0.5 pA, range 1.4–8.5 pA, decay time constant of 9495 ± 1145 msec, n = 21). These small residual currents, which remained even in the presence of 400 nM apamin (n = 5), may represent apamin-insensitive SK channels or unidentified channels. Together these experiments showed that all GnRH neurons express the SK channel and that it is responsible for the great majority of IAHP in these cells.
Apamin-sensitive SK channels determine firing patterns of GnRH neurons
Current-clamp recordings were made from 50 GnRH neurons. Treatment with apamin (100 nM) was found to depolarize 31 of 42 of GnRH neurons (74%) by 6.8 ± 0.6 mV with the remaining 11 cells not showing any change in RMP. The effect of apamin on the patterning of GnRH neuron firing was dependent on the cell’s pretest firing pattern.
Silent GnRH neurons (RMP –58.8 ± 1.1 mV) responded to apamin by depolarization (13 of 21; 62%) with accompanying action potentials in eight neurons (Fig. 2A), three of which occurred in a bursting manner. Current injection protocols in silent GnRH neurons showed that approximately 3-fold more action potentials were evoked by 5- to 30-pA currents in the presence of apamin, compared with the control period (n = 8; Fig. 2B).
|
, C and D).
Thirteen of 13 GnRH neurons exhibiting burst firing (RMP –58.4 ± 1.0 mV) responded to apamin with depolarization accompanied by an increase in burst duration and frequency (Figs. 2E and 3) and an increase in input resistance (Fig. 2F). The effect of apamin on burst firing was clearly observed by holding GnRH neurons back to their control membrane potential in the presence of apamin (Fig. 3, A and B). The blockade of SK channels significantly increased the number of action potentials per burst (P < 0.01;Fig. 3C), duration of each burst (P < 0.01; Fig. 3D), and the normality of firing within the burst, as determined by the Surprise Factor (P < 0.01; Fig. 3E), while decreasing the intraburst interval (P < 0.05; Fig. 3F). However, apamin had no effect on mean firing frequency within the burst (Fig. 3G; control bursts, n = 78; apamin bursts, n = 115; Student’s t test).
|
The effects of apamin remained in the absence of action potentials and synaptic transmission. Six of eight GnRH neurons (75%) pretreated with 0.5 µM TTX, 1 mM kynurenic acid, and 0.1 mM picrotoxin were depolarized (8.4 ± 1.1 mV) by 100 nM apamin (Fig. 2G) with the remaining two cells not showing any change in RMP. The magnitude of the apamin-induced depolarization was the same with and without synaptic transmission (Fig. 2H, P > 0.2, Mann-Whitney tests). These observations indicate that apamin acts directly on SK channels expressed by GnRH neurons to evoke their effects and that action potentials are not necessary for the tonic SK current.
SK channels influence firing by regulating spike frequency adaptation and ADPs
To examine the ways in which SK channels/AHP determine GnRH neuron excitability, perforated-patch, current-clamp experiments were undertaken in which a train of action potentials was initiated by depolarizing current injections (amplitude of 5–30 pA in 5-pA increments, duration 2 sec) in the absence and presence of apamin.
Current injections evoked trains of action potentials that exhibited spike frequency adaptation (SFA), in which the interspike interval of action potentials increases as the action potential train proceeds (Fig. 4A). Typically the interspike interval for the first four to five action potentials is similar but then widens markedly for subsequent action potentials (Fig. 4C). Apamin almost completely abolished SFA in all GnRH neurons tested (n = 20; Fig. 4, B and C) and resulted in more action potentials per train with the increase in frequency of action potentials being proportional to the amplitude of the injected current (Fig. 4D).
|
, A and B). In contrast, the ADP associated with the action potential train became more obvious in the presence of apamin (Fig. 4, A and B, arrows). The effect of apamin on ADPs was further examined using a depolarizing current of 50–80 pA for 20 msec that typically evokes only a single action potential (Fig. 5A). In this situation, the ADP was enhanced after apamin treatment (Fig. 5A). The ADP was also found to be voltage dependent, and apamin resulted in a consistent increase in ADP amplitude across –70 to –90 mV holding potentials (Fig. 5B). When held at –70 mV, the ADP amplitude was approximately 35% larger in the presence of apamin (P < 0.001; n = 10; Fig. 5C).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Two previous electrophysiological studies have shown that GnRH neurons express SK channels. A study by Kato et al. (12) reported that cultured rat GnRH neurons expressed apamin-sensitive SK channels, and a similar finding was found recently by Spergel (13) in the mouse. Our present study confirms these findings and extends them by determining the functional role of SK channels in modulating the firing patterns of GnRH neurons in situ. The results from all three electrophysiological studies to date indicate that the channels responsible for the AHP in GnRH neurons are somewhat atypical. Elsewhere in the brain, AHP currents of approximately 200 msec duration (medium AHPs) are mediated by SK channels, whereas longer-lasting AHP currents, occurring over seconds (slow AHP), are driven by as-yet-unidentified channels (8, 9). The GnRH neurons exhibit slow AHPs that, despite being activated within milliseconds of the action potential, last for several seconds and are very clearly dependent on SK channels. How the millisecond increase in intracellular calcium accompanying the action potential can activate SK channels in GnRH neurons for seconds is unclear. It may, for example, depend on calcium pump dynamics or represent a particular spatial arrangement between the SK channels and calcium binding proteins (10), as has also been suggested recently for the slow AHP (16), or even the slow dynamics of calcium release from stores in these cells (17).
The identity of the SK channel subunits underlying the AHP in mouse GnRH neurons is unknown. The different SK subunits show differential sensitivity to apamin in the order of SK2 greater than SK3 greater than SK1 (9). Although apamin blocks nearly all [90%, here; 93% (13)] of the AHP current in mouse GnRH neurons, it has a relatively high IC50 (9.6 nM), suggesting that the SK2 subunit is not dominant and that SK channels may even be heteromeric in these cells (9). In contrast, cultured rat GnRH neurons exhibit a lower IC50 (0.1 nM) for apamin and express transcripts for all three SK subunits (12). However, in the rat, only approximately 70% of the AHP current is blocked by apamin, suggesting the existence of another major current or apamin-insensitive SK channel-generating AHPs.
It is apparent from the present study that SK channels exert a powerful regulatory influence on the excitability of GnRH neurons. The channel is tonically active in the great majority of GnRH neurons in which it contributes to both the resting membrane potential and the frequency/patterning of firing. Intriguingly, more than half of silent GnRH neurons (62%) were depolarized by apamin treatment, suggesting the existence of a tonic potassium conductance through the SK channel in these cells. Similarly, apamin continued to depolarize GnRH neurons in the presence of TTX. Thus, it is apparent that an action potential-independent mechanism exists for the calcium activation of SK channels in these cells. In this light, it may be important that more than half of adult GnRH neurons have been reported recently to exhibit spontaneous calcium transients originating from store mechanisms (17). In 85% of GnRH neurons exhibiting spontaneous activity, apamin resulted in a marked increase in firing frequency. In addition, apamin treatment resulted in GnRH neurons exhibiting more action potentials to current injection. This input-output relationship indicates that the SK channel will also play a significant role in determining the ability of synaptic inputs to activate GnRH neurons.
The powerful effect of the SK channel on GnRH neuron excitability likely results from its role underlying the great majority of the AHP in GnRH neurons and the effect of the AHP on ADP amplitude. As noted previously (6), the size of the ADP is dependent on the AHP and the two currents likely counteract each other in terms of determining postspike excitability. Chu et al. (6) found that blockade of calcium-activated potassium channels resulted in a faster-peaking ADP in GnRH neurons, and we show here that blockade of the SK channel, specifically, results in a 35% increase in ADP amplitude in these cells. The absence of the majority of the AHP, and enhancement of the ADP, after SK channel blockade would provide a strong drive toward enhanced burst firing. This is clearly observed in both spontaneously active and stimulated bursting GnRH neurons, in which apamin results in much longer duration trains of action potentials within each burst. In stimulated bursts, it is also evident that the SK channel underlies SFA, although this phenomenon was not observed in spontaneously active bursting GnRH neurons in which typically only about four spikes make up each burst. Studies in other neuronal phenotypes have suggested a role for the SK channel and AHP in the termination of burst firing (8, 9). Although other scenarios exist (18), one possibility for the GnRH neurons is that individual bursts are evoked by the ADP and terminated by the AHP.
Together these observations indicate that the SK channel exerts an important suppressive influence on GnRH neuron excitability by: 1) tonically hyperpolarizing RMP, 2) limiting the duration of action potential bursts, and 3) reducing the efficacy of excitatory synaptic inputs to these cells. Taking insight from other neuronal cell types (10), it is likely that the net activity of SK channels is regulated not only directly by calcium but also indirectly by alterations in calmodulin phosphorylation status (19) as well as SK channel abundance. In this light it is interesting to note that SK3 mRNA levels in the guinea pig hypothalamus are increased by estrogen (11), although apamin-sensitive current charge density was not found to change across the estrous cycle in overnight cultured rat GnRH neurons (12). Future experiments will now need to address the physiological factors modulating this important calcium-activated ion channel in GnRH neurons.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Disclosure Statement: The authors have nothing to disclose.
First Published Online March 27, 2008
Abbreviations: aCSF, Artificial cerebrospinal fluid; ADP, afterdepolarizing potential; AHP, after hyperpolarization potential; GFP, green fluorescent protein; IAHP, afterhyperpolarization current; Ra, access resistance; RMP, resting membrane potential; SFA, spike frequency adaptation; SK, small-conductance calcium-activated potassium; TTX, tetrodotoxin.
Received November 27, 2007.
Accepted for publication March 17, 2008.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  M. D. C. Belle, C. O. Diekman, D. B. Forger, and H. D. Piggins Daily Electrical Silencing in the Mammalian Circadian Clock Science, October 9, 2009; 326(5950): 281 - 284. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. E. Oakley, D. K. Clifton, and R. A. Steiner Kisspeptin Signaling in the Brain Endocr. Rev., October 1, 2009; 30(6): 713 - 743. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Q.-H. Chen and G. M. Toney Excitability of paraventricular nucleus neurones that project to the rostral ventrolateral medulla is regulated by small-conductance Ca2+-activated K+ channels J. Physiol., September 1, 2009; 587(17): 4235 - 4247. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. W. Okaty, M. N. Miller, K. Sugino, C. M. Hempel, and S. B. Nelson Transcriptional and Electrophysiological Maturation of Neocortical Fast-Spiking GABAergic Interneurons J. Neurosci., May 27, 2009; 29(21): 7040 - 7052. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Liu, K. Lee, and A. E. Herbison Kisspeptin Excites Gonadotropin-Releasing Hormone Neurons through a Phospholipase C/Calcium-Dependent Pathway Regulating Multiple Ion Channels Endocrinology, September 1, 2008; 149(9): 4605 - 4614. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
