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Wisconsin National Primate Research Center (H.A., E.T.) and Department of Pediatrics (E.T.), University of Wisconsin, Madison, Wisconsin 53715-1261
Address all correspondence and requests for reprints to: Ei Terasawa, Wisconsin National Primate Research Center, University of Wisconsin, 1223 Capitol Court, Madison, Wisconsin 53715-1299. E-mail: terasawa{at}primate.wisc.edu.
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Introduction |
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Studies elucidating the physiological properties of LHRH-1 neurons in vivo using electrophysiological approaches are difficult because of their diffuse distribution pattern and paucity in number in the hypothalamus-preoptic area (9). To overcome this obstruction, LHRH-1 neurons in mice and rats were genetically labeled by promoter-driven transgenic manipulation, so that LHRH-1 neurons can be visualized for identification before electrical recording. In fact, this approach has been highly successful in mice and rats (10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). However, LHRH-1 neurons labeled with green fluorescent protein (GFP) in nonhuman primates are not yet available.
We have established previously a primary cell culture system for LHRH-1 neurons derived from the olfactory placode region of the rhesus monkey at embryonic d 35 (E35) to E37 (21) and have shown that this culture system is useful for cellular and molecular studies. In the present study, we recorded activity of primate LHRH-1 neurons using the cell-attached patch-clamp method. First, we determined firing patterns of electrical activity, and, second, we examined whether estrogen rapidly alters the electrical activity of primate LHRH-1 neurons. The results suggest that 1) primate LHRH-1 neurons exhibit complex firing patterns composed of activities with different time domains, and 2) estrogen causes rapid stimulatory action in the firing activity of primate LHRH-1 neurons via an ICI182,780 (ICI)-sensitive pathway.
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Materials and Methods |
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The methods for culture of LHRH-1 neurons derived from embryonic tissue have been described in detail previously (6, 7, 21). Briefly, the olfactory placode region and ventral LHRH-1 neuron migratory pathway (terminal nerve region) were dissected out, cut into small (<1 mm3) pieces, and plated on glass coverslips (n = 24–32 individual coverslips per embryo). Cultures were grown in a growth medium (Medium 199; Sigma, St. Louis, MO) supplemented with L-glutamine (Sigma), 10% FBS (Hyclone, Logan, UT), 0.6% glucose, and 50 µg/ml gentamicin (Sigma) under 1.5% CO2 and 98.5% air at 37 C for at least 2 wk before experiments. Because of concerns regarding unknown effects of serum, half of the cultures from each embryo were grown in a modified growth medium (based on DMEM/F-12; Sigma) that contains 2% B27serum-free supplement (50x; Invitrogen, Carlsbad, CA), L-glutamine (Sigma), and gentamicin (Sigma) but neither serum nor phenol red. The medium was replaced every 1–3 d. Recording experiments were conducted in cells cultured for 3–5 wk. A total of 62 cultures (10–15 cultures from each embryo) were used for examination of the firing pattern (22 cultures), the effects of high K+ and tetrodotoxin (TTX) (six cultures), and the effects of estrogen (34 cultures). In all experiments, approximately half of the cultures were grown in serum-free medium.
Materials
For treatment of cultured LHRH-1 neurons, 17ß-estradiol (E2) was purchased from Schering (Bloomfield, NJ). The pure antagonist for estrogen receptor (ER), ICI, was purchased from Tocris Cookson (Bristol, UK). The voltage-dependent sodium channel blocker TTX was purchased from Sigma.
Electrophysiological recording and data analysis
We recorded action currents generated by action potentials from primary LHRH-1 neurons in the cell-attached patch configuration of patch-clamp technique using a PC-501A patch-clamp amplifier (Harvard Apparatus, Holliston, MA). During recordings, cultures were continuously perfused with oxygenated artificial cerebrospinal fluid [in mM: 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 5 HEPES, and 10 glucose (pH 7.4 with NaOH)] at a flow rate of 200–300 µl/min at room temperature (22–25 C). Cells were observed with a Nikon (Tokyo, Japan) TE-300 inverted microscope equipped with differential interference contrast optics. Recording electrode (3–6 M
) was made from borosilicate capillary glass (type 8520; outer diameter, 1.5 mm; inner diameter, 1.16 mm; Harvard Apparatus) using a Flaming/Brown Micropipette Puller (model P-87; Sutter Instruments, Novato, CA). Patch pipettes were filled with artificial cerebrospinal fluid and were targeted to LHRH-1 neurons (see below) using an NMN-21 three-dimensional mechanical micromanipulator (Narishige, Tokyo, Japan). Slight positive pressure was applied to the pipette before entering bath and maintained until attaching to the LHRH-1 neuron. Subsequently, gentle negative pressure was applied to the pipette to achieve the cell-attached patch configuration (seal resistances were 
1 G, except for one case that was 0.3 G). The firing activity recording was done in voltage-clamp mode with a holding potential of 0 mV, filtering at 2 kHz. The recording was continued for up to 45 min and digitized by a combination of Digidata1322A data acquisition interface and pClamp9 data acquisition and analysis software package (Axon Instruments, Union City, CA).
The effects of TTX (0.5 µM) were examined by direct application of the drug to the bath for 2 min. The effects of E2 (1 nM) were similarly examined by infusion for 10 min. Before E2 application, firing activity was recorded at least 10 min, and recording was continued for at least 10 min after E2. The blockage of the E2-induced firing activity was examined by the following protocol. After the recording of the basal firing rate for at least 10 min, ICI (0.1 µM) infusion was started 10 min before the 10-min infusion of E2 or vehicle. ICI infusion was continued at least 10 min after E2.
Individual spikes and bursts of action currents were detected using Minianalysis software (Synaptosoft, Decatur, GA). To identify clusters (slower time-course changes) of increased electrical activity within each cell-attached recording, we used the Cluster7 computer algorithm (22). Cluster7 compares clusters of points by pooled t test to look for peaks and nadirs over time. For this analysis, we detected timing of action currents and binned the number of action currents at 10-sec intervals as a dataset for Cluster7. Using six-point (equivalent to 1 min) settings of peak and nadir periods each, Cluster7 identified the location of clusters, which were composed of both multiple bursts in rapid succession and irregular beatings of action currents, and calculated the durations and intervals for recordings with multiple identified clusters. Cluster7 sometimes missed clusters of firing very near the beginning or end of the data series, because it requires a string of data flanking a peak for positive identification. Therefore, for cluster analysis, we chose recordings that have a sufficient duration, at least 20 min (n = 19 for basic firing pattern examination; n = 9 for E2; n = 7 for vehicle control). Moreover, during the course of analysis, we noticed that a cluster was composed of periodical bursting of action currents. We defined a "burst" as the events comprising at least three consecutive action currents that occur at intervals less than 500 msec.
Identification of LHRH-1 neurons and immunocytochemistry
Our cultures contain numerous epithelial cells, fibroblasts, and other types of unidentified cells in addition to primary LHRH-1 neurons. However, LHRH-1 neurons were easily identifiable during patch-clamp recording based on their morphology, such as ovoid in shape and large soma (12 µm in size) with neurites forming neuronal bundles, and their migratory pattern. To identify LHRH-1 neurons, we used the procedure detailed previously (6, 23). Briefly, we took a photomicrograph of a tested LHRH-1 neuron and recorded a reference grid number on the coverslip after the completion of each patch-clamp recording to facilitate locating the cells after immunostaining. After the completion of recording, cultures were fixed with 2% paraformaldehyde in PBS (pH 7.4) for immunocytochemistry.
LHRH-1 neurons were identified using standard immunocytochemical procedures with the antisera cocktail GF-6 and LR-1 [gifts from Dr. N. M. Sherwood (University of Victoria, British Columbia, Canada) at 1:9000 dilution and Dr. R. A. Benoit (University of Montreal, Montreal, Canada) at 1:15,000, respectively], Vectastain ABC peroxidase system (Vector Laboratories, Burlingame, CA), and 3,3'-diaminobenzidine as the chromagen, as described previously (6, 23). After staining, tested LHRH-1-immunopositive neurons were matched to the photomicrographic images. LHRH-1 immunonegative neurons in cultures were rare but were excluded from analysis when they were found.
Statistical analysis
The results from cells grown in medium with or without serum were combined for statistical analysis, because they were not different. For quantitative analysis of the estrogen effects on the firing activity, we obtained the frequency during 10-min periods before and after the initiation of the E2 treatment. Because there were considerable variations in the firing frequency among individual neurons, before statistical analysis, we normalized the data with the following equation:
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The effects of E2 and ICI (between-group comparisons) on the firing pattern of LHRH-1 neurons were examined using one-tailed alternate Welch’s t test. All data are presented as means ± SEM. Significance was attained at P < 0.05.
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Results |
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) recorded from two LHRH-1 neurons, the action currents exhibited either monophasic (Fig. 1Aa) or biphasic (Fig. 1Ab) profiles. To confirm whether these currents reflect the action potentials of LHRH-1 neurons, the effects of the sodium channel blocker TTX were examined. Bath application of TTX (0.5 µM; 2 min) blocked the generation of action currents (Fig. 1C) for approximately 10 min. This TTX effect was reversible, because spontaneous action currents returned by 10–11 min after TTX removal (n = 3 in 3 cultures). Depolarization with an increase in extracellular K+ (15 or 30 mM) also evoked an increase in frequency of action currents (n = 3 in 3 cultures) (Fig. 1B).
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). This firing pattern was disrupted by short periods (<2 min) of quiescence, but there were no long quiescence periods (10–30 min), unlike reports in mice (12, 19, 24). The firing frequency of primate LHRH-1 neurons was normally between less than 0.001 and 10 Hz (mean of 2.7 ± 0.1 Hz; calculated from 11,300 events from 22 neurons), but the instantaneous firing frequency reached as high as 100 Hz. Detailed analysis indicated that the firing pattern was composed of slower changes in firing activity, which was named a "cluster" (Fig. 2B). A single cluster was further composed of multiple rapid bursts (Fig. 2C) and irregular beatings of action currents. A single burst was further composed of multiple spikes of action currents (Fig. 2D). In the example shown in Fig. 2C, five action currents formed a burst.
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). The mean duration of a cluster was 161.4 ± 8.1 s (ranging from 60–420 sec; n = 97 events from 19 neurons), which was separated by a 104.7 ± 23.3-sec mean interval (ranging from 10–1420 sec; n = 78 events from 19 neurons), i.e. clusters occurred at approximately 1- to 25-min intervals. Three of 22 neurons did not show any clusters. The frequency distribution histogram indicated that both duration (Fig. 3Ba) and interval (Fig. 3Bb) of clusters showed long-tailed distribution. The median values of the duration and interval of a cluster were 140 and 60 sec, respectively (Table 1A).
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). For example, one LHRH-1 neuron showed bursts with a relatively low firing frequency and long duration (Fig. 3Ca), whereas another LHRH-1 neuron showed a high firing frequency and short duration (Fig. 3Cb). A small number of LHRH-1 neurons (n = 2 of 20) exhibited a stereotype pattern of rhythmic bursts with two to seven spikes occurring at 5- to 90-sec intervals with a duration less than 0.1 sec (Fig. 3Cc). In these neurons, the amplitude of the first spike was highest and followed by spikes with amplitudes gradually decreasing (Fig. 3Cc, inset). The frequency and duration were also variable from event to event within a single neuron. Reflecting these variations, the distribution histograms for the spikes per burst (panel Ca), burst duration (panel Cb), and interburst interval (panel Cc) from 597 pooled burst events from 20 neurons indicated that these parameters exhibit long-tailed distribution (Fig. 3D). The mean ± SEM spikes per burst, burst duration, and interburst interval were 5.7 ± 0.3 (ranging from 3–72; n = 597 events from 20 neurons), 1.104 ± 0.048 sec (ranging from 0.025–15.95 sec; n = 597 events from 20 neurons), and 39.548 ± 5.105 sec (ranging from 0.53–1525.823 sec; n = 577 from 20 neurons), respectively (Table 1B). The median values were 4 for spike number, 0.79 sec for burst duration, and 6.518 sec for interburst interval (Table 1B).
Effect of estrogen on the firing pattern of primate LHRH-1 neurons
To examine whether E2 alters firing activity of LHRH-1 neurons, we applied E2 (1 nM) in the bath for 10 min. This duration was chosen based on the observations that 2-min exposure of LHRH-1 neurons to TTX caused an expected response for approximately 10 min (Fig. 1C) and the estrogen application for 10 min alters the pattern of intracellular calcium oscillations in our cultured LHRH-1 neurons. Before E2 application, LHRH-1 neurons exhibited a random firing pattern, which was composed of a mixture of rapid bursts and irregular single spikes (Fig. 4, Ba, Ca, Da, and Ea). E2 application resulted in an increase in the firing activity of LHRH-1 neurons (Fig. 4, Ba, Cb, Da, and Eb). The time course of changes by E2 was clearly observed with an integrated plot of spike number per 10 sec (Fig. 4, Bb and Db). The latency of the estrogen-induced frequency increase was 60–120 sec. This E2-induced facilitation was continued as long as the seal integrity of the patch electrode was maintained (up to 25 min). Quantitative analysis suggested that E2 (1 nM) stimulated seven of nine (78%) LHRH-1 neurons. The magnitude of the firing frequency increase was 249.7 ± 109.4% (n = 9 neurons) from the basal firing rate before E2, and this was significantly higher (P < 0.05) (Fig. 5A) than that in vehicle controls (9.3 ± 11.7%; n = 7 neurons). In contrast, vehicle application for 10 min did not cause any significant changes in the firing pattern (Fig. 4A).
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) and burst duration (1.97 ± 0.06 sec in E2 vs. 1.49 ± 0.08 sec in vehicle; P < 0.001) (Fig. 5Cb) but did not alter the interburst interval (7.79 ± 1.05 sec in E2 vs. 11.44 ± 2.62 sec in vehicle; P > 0.05) (Fig. 5Cc). In contrast, E2 did not induce significant changes in the cluster pattern: the duration (198.3 ± 12.9 sec in E2 vs. 198.8 ± 24.9 sec in vehicle; n = 60 events from nine neurons and 26 events from seven neurons, respectively; P > 0.05) and interval (74.5 ± 13.9 sec in E2 vs. 82.7 ± 13.9 sec in vehicle; P > 0.05) of clusters (Fig. 5B) were not significantly different. These data suggested that E2 increased firing activity of LHRH-1 neurons by increasing firing frequency and burst duration but not cluster pattern.
The effects of ICI on the estrogen-induced firing activity
To determine whether the E2-induced increase in firing activity is mediated by classical ER, we tested the effects of ICI, a pure antagonist for both ER
and ERß. ICI was infused for 10 min before E2 and continued through the 10-min E2 application until the end of recording. ICI (0.1 µM) clearly blocked the E2-induced increase in the firing activity of LHRH-1 neurons (Fig. 6). Firing activity during the ICI (0.1 µM) plus E2 (1 nM) treatment (Fig. 6Bc) was not different from that during the ICI alone (0.1 µM) (Fig. 6Bb) or the control period (Fig. 6Ba).
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), spike number per burst (Fig. 7Ba), and burst duration (Fig. 7Bb) of the E2 plus ICI group were significantly lower than the E2 group (for all P < 0.01). Moreover, there were no significant differences between the ICI and ICI plus E2 group in all parameters examined: firing frequency (2.49 ± 23.7% in ICI plus E2 vs. –0.47 ± 13.78% in ICI alone; n = 7 and 11 neurons, respectively; P > 0.05) (Fig. 7A), spikes per burst (4.9 ± 0.2 in ICI plus E2 vs. 4.8 ± 0.1 in ICI alone; n = 478 events from seven neurons and 377 events from 11 neurons; P > 0.05) (Fig. 7Ba), and burst duration (1.208 ± 0.049 sec in ICI plus E2 vs. 0.997 ± 0.034 sec in ICI alone; P > 0.05) (Fig. 7Bb). Application of ICI alone did not induce significant changes in firing frequency (–0.47 ± 13.78% in ICI alone vs. 9.3 ± 11.7% in vehicle; n = 11 and 7 neurons, respectively; P > 0.05) (Fig. 7A). Interestingly, ICI alone decreased the spikes per burst (4.8 ± 0.1 in ICI alone vs. 7.82 ± 0.38 in vehicle controls; n = 377 events from 11 neurons and 474 events from seven neurons, respectively; P < 0.01) (Fig. 7Ba) and burst duration (0.997 ± 0.034 sec in ICI alone vs. 1.49 ± 0.08 sec in vehicle controls, respectively; P < 0.01) (Fig. 7Bb) when compared with vehicle controls.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We were successful in recording the firing activity of single LHRH-1 neurons in the rhesus monkey. To our knowledge, this is the first description on electrophysiological characteristics of primate LHRH-1 neurons. Because in the present study recording experiments were conducted at room temperature, some of our observations may not fully represent physiological conditions. The frequency (
3 Hz) of spontaneous activity in primate neurons is similar to that described in transgenic mouse LHRH-1 neurons (11, 12) and cultured LHRH-1 neurons from mouse olfactory placodes (25). Firing activity of primate LHRH-1 neurons was increased by depolarization with a high extracellular K+ and eliminated by TTX. Activation or suppression of neural activity by high K+ or TTX in LHRH-1 neurons and GT1 cells has been reported previously (12, 26, 27). Similarly, an increase in LHRH-1 release and [Ca2+]i oscillations induced by high K+ and a decrease in LHRH-1 release and [Ca2+]i oscillations induced by TTX in LHRH-1 neurons and GT1 cells have been extensively reported (6, 26, 28, 29). These reports together with the observations in this study suggest that membrane depolarization is essential for neurosecretion in LHRH-1 neurons.
Electrophysiological recording studies indicate that firing activity of GFP-labeled mouse LHRH-1 neurons and GT1 cells exhibit an episodic pattern separated by a 10–30 min quiescence period (13, 19, 30, 31). Moenter and colleagues (16, 19) further describe that mouse LHRH-1 neurons exhibit a firing pattern consisting of bursts (trains of multiple action currents) that occur at less than 20-sec intervals, clusters (trains of multiple bursts) that occur at 6- to 8-min intervals, and episodes (trains of multiple clusters) that occur at 20–30 min. These authors categorize burst, cluster, and episodes as high-, intermediate-, and low-frequency rhythms, respectively, and suggest that episodic, not burst or cluster, firing rhythms may correspond to neurosecretion in mouse LHRH-1 neurons (16, 19). The pulse interval of LH release in mice, rats, and GT1 cells is approximately 30 min (29, 32, 33, 34, 35). In the present study, we found that primate LHRH-1 neurons exhibit a firing pattern consisting of bursts (40-sec mean interburst interval with 1-sec mean burst duration) and irregular single spikes, which further form cluster firing activity (trains of multiple bursts lasting 3–7 min, occurring at 1- to 25- min intervals). However, we did not observe a long quiescence of firing activity or the episodic rhythm of firings with a longer time domain described in mouse LHRH-1 neurons and GT1 cells. These characteristics in monkey LHRH-1 neurons differ from those in mouse LHRH neurons and GT1 cells. If the episodic rhythm represents a secretory interval as suggested by Moenter et al. (16), it is possible that the episodic rhythm of an activity pattern at 50- to 60-min intervals may be seen in primate LHRH-1 neurons, when several hours of continuous recording is conducted. In this study, we only recorded action currents for up to 45 min. Alternatively, it is possible that there may be no long quiescence in primate LHRH-1 neurons. Hourly pulse intervals of LH/LHRH-1 release in rhesus monkeys have been reported extensively (1, 6, 36, 37). We do not believe, however, that the absence of long quiescence in primate LHRH-1 neurons is due to a lack of normal afferent inputs to LHRH-1 neurons, because these neurons release the decapeptide hormone at 60-min intervals (7).
Burst firing activity appears to be a common feature in LHRH-1 neurons. In the present study, we observed burst firing in primate LHRH-1 neurons at approximately 40-sec intervals. The burst firing pattern has also been described in GT1 cells (26, 31, 38) and GFP-labeled mouse LHRH-1 neurons (13, 19, 24, 39). Comparing our data with those reported in publications, the burst frequency in monkey LHRH-1 neurons appears to be slightly slower than in mouse LHRH-1 neurons and GT1 cells. Moreover, burst firing patterns, such as spike number per burst and interburst intervals, in primate LHRH-1 neurons were highly irregular within a single recording, in contrast to the regular firing pattern reported in mouse LHRH-1 neurons and GT1 cells. Interestingly, two of 20 primate LHRH-1 neurons showed a stereotyped pattern of rhythmic bursts, in which the first action current with the highest amplitude was followed by two to seven action currents with progressively decreasing amplitudes in a short duration (<0.1 sec) (Fig. 3Cc). A similar stereotyped burst firing activity has been observed in GT1 cells (26, 40). In addition, in GT1 cells each stereotyped burst activity precedes an increase in intracellular calcium, when simultaneous measurement of action currents and intracellular calcium oscillations is conducted (40). The progressively declining amplitude of action currents during bursts suggests that there is an underlying periodical fluctuation in baseline membrane potentials at approximately 40-sec intervals in monkey LHRH-1 neurons. Nonetheless, unlike GT1 cells, each burst firing in primate LHRH-1 neurons may not lead to an increase in intracellular calcium, because the time domain of periodical bursts in primate LHRH-1 neurons is seconds, whereas the interval of calcium oscillations is several minutes (6). Perhaps an irregular firing pattern, especially the irregular frequency and burst interval, in primate LHRH-1 neurons may account for the difference in the mechanism of burst firing activity and the opening of voltage-dependent calcium channels that leads to an elevation of intracellular calcium concentrations. Whether a slower burst frequency in monkey LHRH-1 neurons than mouse LHRH-1 neurons represents the difference in slower pulse frequency of LHRH-1 release in primates (60 min) than in rodents (30 min) is unknown.
In the present study, cluster firing with multiple bursts (ranging from 60–420 sec) was detected using Cluster7 algorithm. Cluster firing patterns with a similar time domain have also been reported in GT1–7 cells (31) and GFP-labeled mouse LHRH-1 neurons (19). Cluster rhythm appears to be endogenous in LHRH-1 neurons, because cluster firing with similar periodicity occurs in acutely isolated GFP-labeled LHRH-1 neurons (24). In a previous study, we have reported that individual LHRH-1 neurons exhibit intracellular calcium oscillations with an average interval of several minutes (6). Therefore, in primate LHRH-1 neurons, a cluster firing may correspond to an increase in intracellular calcium oscillations. Simultaneous measurement of firing patterns and intracellular calcium changes in single LHRH-1 neurons would clarify this issue.
It has been shown that a coordinated high-frequency burst firing activity in multiple numbers of vasopressin and oxytocin neurons is correlated with, and is a prerequisite for, neurosecretion in their posterior pituitary neuroterminals (41, 42, 43, 44, 45). Whether each high-frequency burst activity in individual LHRH-1 neurons leads to a quantum of neurosecretion at their neuroterminals is yet to be determined. Nonetheless, the following question arises: what is the cellular mechanism of LHRH-1 release, which occurs at approximately 60-min intervals? Our previous study suggests that individual primate LHRH-1 neurons and nonneuronal cells exhibit [Ca2+]i oscillations with independent rhythms, but [Ca2+]i oscillations in a number of neurons synchronize periodically at an interval similar to that observed for neurosecretion (6, 23). Studies with multiple microelectrode arrays suggest that, although individual GT1 cells are rhythmic, not every cell participates in each firing episode, and the sum of activities in multiple cells within a network appear to correlate with the interval of LHRH-1 neurosecretion (31, 38). Therefore, overall firing activity as a consequence of synchronization among LHRH-1 neurons (23) would determine pulsatile LHRH-1 release. In fact, the synchronization of spontaneous oscillations in [Ca2+]i among cells observed in our cultures is similar to the pattern of electrical activity that emerges in simulated networks of cells in which sparse, random activity in individual cells leads to intercellular activity waves (46). It is, therefore, hypothesized that pulsatile LHRH-1 release at 60 min is a network property.
E2 infusion rapidly increased the firing frequency, spikes per burst, and burst duration of primate LHRH-1 neurons. Our preliminary study indicates that the membrane-impermeable analog of E2, E2-BSA, also induced similar rapid E2 effects. These results complement the effects of E2 on [Ca2+]i oscillations (47). Both E2 and E2-BSA rapidly accelerated the frequency of [Ca2+]i oscillations in a subset of LHRH-1 neurons and resulted in more frequent synchronization among LHRH-1 neurons (47). These results suggest that the rapid effect of estrogen on firing activity of primate LHRH-1 neurons does not require E2 entry into the cell, perhaps occurring at the cell membrane level. In fact, a recent preliminary study in our laboratory shows that the membrane of LHRH-1 neurons was labeled with E2-BSA-fluorescein isothiocyanate but not BSA-fluorescein isothiocyanate, suggesting the presence of estrogen binding sites on the membrane (48).
The E2-induced firing increase was blocked by the estrogen receptor blocker ICI. The results from the calcium imaging study also indicate that the E2-induced frequency increase in [Ca2+]i oscillations is ICI sensitive, whereas the E2-induced synchronization frequency is ICI insensitive (47). As we discussed above, although the precise relationship between firing pattern with patch-clamp recording and [Ca2+]i oscillations with the calcium imaging is unclear, direct stimulatory action of E2 on LHRH-1 neurons with a short latency is consistent in both studies, and a part of the stimulatory effects of estrogen is ICI sensitive. A similar ICI-sensitive membrane-mediated stimulatory estrogen action on [Ca2+]i oscillations in cultured LHRH-1 neurons from mouse olfactory region has also been reported (49).
Several studies have shown the presence of ER in LHRH-1 neurons. LHRH-1 neurons express transcripts and peptides for ERß (50, 51, 52, 53, 54). GT1–7 cells also express both ER and ERß (55, 56). Recently, we have observed that primate LHRH-1 neurons express ERß (48). In addition, rapid action of E2 through membrane-associated ER and/or ERß in both LHRH-1 neurons and nonneuronal cells has been reported (49, 57, 58, 59). Thus, it is possible that rapid E2 action in LHRH-1 neurons is mediated by classical ERs, perhaps ERß. Recently, rapid action of estrogen through ERß has been shown in mice (60). Alternatively, it is also plausible that novel membrane E2 receptors, which are pharmacologically different from classical nuclear ERs but sensitive to ICI (61), may mediate rapid E2 action.
Nunemaker et al. (39) have examined the negative feedback effect of estrogen on firing activity of GFP-labeled LHRH-1 neurons by comparing ovariectomized mice bearing an E2 capsule for 1 wk and ovariectomized controls. Results indicate that E2 stimulated the firing frequency but also altered the episodic rhythm (low-frequency rhythm) by increasing the duration of the quiescence period in a subset (50%) of LHRH-1 neurons. Because a longer interpulse interval of pulsatile LH release with a larger pulse amplitude occurs in ovariectomized animals when compared with intact animals (62), the episodic rhythm altered by estrogen appears to represent the negative feedback effect in mouse LHRH-1 neurons (39). The question arises as to whether the stimulated firing activity by estrogen in this study is correlated with LHRH-1 neurosecretory activity. The results from a preliminary study indicate that E2 infusion for a short period induces several fold increase in LHRH-1 release for short time in our primary culture system (48).
In summary, we revealed that primate LHRH-1 neurons exhibit complex firing patterns composed of activities with different time domains and estrogen rapidly alters firing pattern by increasing burst firing activity through a membrane-associated ICI-sensitive mechanism, most likely through classical ER. The mechanism of estrogen action remains to be determined.
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Acknowledgments |
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Footnotes |
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Present address for H.A.: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Tokyo 113-0033, Japan.
First Published Online June 23, 2005.
Abbreviations: [Ca2+]i, Intracellular calcium; E, embryonic day; E2, 17ß-estradiol; ER, estrogen receptor; GFP, green fluorescent protein; ICI, ICI182,780; TTX, tetrodotoxin.
Received April 15, 2005.
Accepted for publication June 16, 2005.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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I. Nishimura, K. Ui-Tei, K. Saigo, H. Ishii, Y. Sakuma, and M. Kato 17{beta}-Estradiol at Physiological Concentrations Augments Ca2+-Activated K+ Currents via Estrogen Receptor {beta} in the Gonadotropin-Releasing Hormone Neuronal Cell Line GT1-7 Endocrinology, February 1, 2008; 149(2): 774 - 782. [Abstract] [Full Text] [PDF]
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 A. Arroyo, B. Kim, R. L. Rasmusson, G. Bett, and J. Yeh Hyperpolarization-Activated Cation Channels Are Expressed in Rat Hypothalamic Gonadotropin-Releasing Hormone (GnRH) Neurons and Immortalized GnRH Neurons Reproductive Sciences, September 1, 2006; 13(6): 442 - 450. [Abstract] [PDF]
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, P < 0.05 vs. E2; **, P < 0.01 vs. vehicle; ***, P < 0.001 vs. vehicle;