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Research Article |
Department of Physiology,4 University of California at Los Angeles School of Medicine, Los Angeles, California 90095
Department of Aquatic Bioscience,5 Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo, Tokyo 113-8657, Japan
Laboratory of Reproductive Biology,6 National Institute for Basic Biology, Okazaki, Aichi 444-8585, Japan
ABSTRACT
Gonadotropin-releasing hormone (GnRH) controls reproduction in vertebrates. Most studies have focused on the population of GnRH neurons in the hypothalamus that ultimately controls gonadal function. However, all vertebrates studied to date have two to three anatomically distinct populations of GnRH neurons that express different forms of this hormone. The purpose of the present study was to develop a new model for studying the population of GnRH neurons in the terminal nerve (TN) associated with the olfactory bulb and then to characterize their pattern of action potential firing to provide a foundation for understanding the role of these neurons in regulating reproduction. A stable line of transgenic medaka (Oryzias latipes) was generated in which a DNA construct containing the salmon GnRH (Gnrh3) promoter linked to green fluorescent protein (GFP) was expressed in TN-GnRH3 neurons. This population of GnRH neurons is located at or near the ventral surface of the brain, making them ideally situated for electrophysiological analysis. Whole-cell and loose-patch recordings in current-clamp mode were performed on these neurons from excised, intact brains of adult males in which afferent and efferent neural connections remained intact. All TN-GnRH3-GFP neurons that we recorded showed a beating pattern of spontaneous action potential firing. Action potentials were blocked by tetrodotoxin, indicating they are generated by a voltage-sensitive Na+ current; however, an oscillation in subthreshold membrane potential persisted. The present results indicate that this transgenic fish will provide an excellent model for studying the cell physiology of an extrahypothalamic population of GnRH neurons.
gonadotropin-releasing hormone, neuroendocrinology
INTRODUCTION
Gonadotropin-releasing hormone (GnRH) provides the command signal from specific populations of neurons in the central nervous system to control reproductive development and fertility in all vertebrates studied to date [1]. Most of the research concerning GnRH neurons has focused on the population of cells in the preoptic area and hypothalamus that regulates the pituitary-gonadal axis. However, recent work has shown at least two other populations of GnRH neurons in the brain: one in the terminal nerve (TN) associated with the olfactory bulb, and another in the midbrain [2]. The TN-GnRH neurons have been implicated in playing a neuromodulatory role in olfaction in axolotls (Ambystoma mexicanum) [3] and in the sexual behavior of dwarf gourami (Colisa lalia) [4], whereas the midbrain population that expresses the highly conserved chicken GnRH-II (GnRH2) has been suggested to play a role as a facilitator of sexual behavior under energy-restricted conditions in musk shrews and mice [5, 6]. However, the number and scope of studies regarding TN and midbrain GnRH neurons have been very limited. The TN-GnRH neurons are especially interesting, because many vertebrate species possess this population. Although the TN-GnRH neurons are most commonly associated with fishes [2] and amphibians [7], GnRH-containing neurons also have been described in the TN of mammalian species, such as guinea pig [8], bat [9], and dolphin [10].
The purpose of the present study was to develop a new model for studying the population of TN-GnRH neurons and then to characterize their pattern of action potential firing to provide a foundation for understanding the role of these neurons in regulating reproduction. The teleost medaka (Oryzias latipes) was chosen for this research because of previous and ongoing work on the molecular biology and genetics of its reproductive development and sex determination, the availability of large numbers of mutants and inbred strains, and its short generation time [11–14]. Furthermore, the molecular forms of GnRH in medaka have been isolated and characterized, and their expression patterns in brain have been described [15]: salmon GnRH (GnRH3) in the TN, chicken GnRH-II (GnRH2) in the midbrain, and medaka GnRH (GnRH1) in the preoptic area. In the present study, we used this molecular information to develop a stable line of transgenic medaka in which TN-GnRH3 neurons express a construct containing the promoter region of Gnrh3 linked to enhanced green fluorescent protein (GFP). The TN-GnRH3 neurons expressing GFP were then targeted for electrophysiological analysis, and the pattern of spontaneous action potential firing was characterized.
MATERIALS AND METHODS
Animals
Medaka were maintained on a 14L:10D photoperiod at a temperature of 28°C. Fish were fed twice daily with flake food and live brine shrimp. Medaka of the d-rR strain were used for the generation of transgenic fish. All procedures were carried out in accordance with the Animal Care and Use Committees of the University of California at Los Angeles and the National Institute for Basic Biology in Japan.
Generation of Construct
The medaka Gnrh3 loci were isolated by screening a bacterial artificial chromosome library as described previously [16]. A fragment of the 5'-flanking region (~5.8 kb) and exon 1 of Gnrh3 was amplified. Exon 1 coded the 5'-untranslated region, and the initiation methionine codon was present in the 5'-end of exon 2. The Gnrh3 fragment was fused with the GFP-coding sequence followed by the polyadenylation signal of SV40 T-antigen gene, which was excised from phrGFP-Nuc (Stratagene) and subcloned into the cloning vector pcR-XL-TOPO (Invitrogen). These constructs were purified and linearized with XhoI.
Generation of Transgenic Line
The DNA construct (see above) was injected into the cytoplasm of 1- or 2-cell embryos. The GFP fluorescence, under control of the Gnrh3 promoter, was monitored at 3 days postfertilization, and only embryos displaying fluorescence were grown to adulthood. Pairs of sibling adults grown from injected embryos were incrossed to identify germ line founders. Individual adults from positive pairs were then outcrossed to identify the individual founder fish. Heterozygous transgene carriers of the F1 generation were identified by analyzing fluorescence. To obtain homozygous transgenic offspring, carriers were crossed to each other. The F3 and F4 homozygous progeny were used in the present study.
Immunofluorescence and Immunocytochemistry
Unless otherwise noted, chemicals were purchased from Sigma Chemical Co. To determine the pattern of GnRH3-GFP transgene expression, four adult male medaka (age, 7 mo) were anesthetized by immersion in MS-222 (150 mg/L) and decapitated. Brains were removed and fixed for 3 days in 0.1 M PBS (pH 7.4) containing 4% paraformaldehyde, followed by cryoprotection with 20% sucrose in PBS at 4°C overnight. Brains were then placed individually in small wells filled with agar to improve physical manipulation for sectioning. Coronal sections (thickness, 40 µm) were cut on a cryostat from the anterior end of the olfactory bulbs to the posterior end of the telencephalon. To block nonspecific binding, sections were incubated with a blocking solution containing normal goat serum (2%) and Triton X-100 (0.3%) in PBS for 1 h. Sections were incubated with the monoclonal primary antibody LRH13 GnRH antisera [17] (gift of Dr. Hisae Kobayashi, Gunma University, Japan) diluted 1:3000 with PBS containing the blocking solution overnight at 4°C. The primary antibody was then removed, and sections were washed in PBS plus 0.3% Triton X-100. Sections were incubated with a goat anti-mouse immunoglobulin G secondary antibody conjugated to red-fluorescent Alexa Fluor 594 (Molecular Probes, Invitrogen Corp.) diluted 1:100 in PBS plus 0.3% Triton X-100 for 1 h at room temperature. Sections were placed on glass slides and then washed with PBS, washed in Tris buffer, air-dried, and coverslipped with Vectashield (Vector Laboratories) to reduce photobleaching. Sections were viewed with a Zeiss Axioskop 2 equipped with epifluorescent illumination (Zeiss USA). Images were captured with a computerized digital video image-analysis system (Zeiss Axiocam CCD camera and Pentium III-equipped computer).
Electrophysiology
Adult male medaka (age, 4–9 mo) were anesthetized by immersion in MS-222 (150 mg/L) and decapitated. The entire brain, from brainstem to olfactory bulb, was glued ventral-side up to a glass coverslip at the bottom of a flow-through recording chamber (P1; Warner Instrument Corp.). Temperature in the recording chamber was maintained at 21–22°C throughout the experiments. Following transfer to the recording chamber, the meninges covering the ventral side of the olfactory bulbs and telencephalon was peeled carefully away, allowing access to the cells for electrophysiology. Aerated fish saline continuously bathed the brain and was perfused through the chamber at a rate of approximately 200 µl/min. Fish saline [18] contained 134 mM NaCl, 2.9 mM KCl, 2.1 mM CaCl2, 1.2 mM MgCl2, and 10 mM Hepes. Osmolarity was adjusted to 290 mOsm with glucose, and pH was adjusted to 7.8 with NaOH. The internal solution for the whole-cell patch pipette [19] contained 112.5 mM potassium gluconate, 4 mM NaCl, 17.5 mM KCl, 0.5 mM CaCl2, 1 mM MgCl2, 5 mM MgATP, 1 mM EGTA, 10 mM Hepes, 1 mM GTP, 0.1 mM leupeptin, and 10 mM phosphocreatine. Osmolarity was adjusted to 290 mOsm by titrating the final volume of water, and pH was adjusted to 7.2 with KOH. Fura-PE3 (TEF Labs, Inc.) was added to the internal solution at a working concentration of 200 µM. The solution for the loose-patch pipette [20] contained 150 mM NaCl, 3.5 mM KCl, 2.5 mM CaCl2, 1.3 mM MgCl2, 10 mM Hepes, and 10 mM glucose. Osmolarity was adjusted to 290 mOsm, and pH was adjusted to 7.4 with NaOH.
The recording chamber was placed under an upright microscope (BX50W; Olympus) equipped with a 40x water-immersion objective (0.8 numerical aperture; Olympus). Infrared (IR) differential contrast optics and an IR camera (OL-1500; Olympus) allowed the visual selection of GFP-expressing neurons in the presence of both ultraviolet and bright-field illumination. Patch pipettes were guided to the cell of interest with a micromanipulator (MP-285; Sutter Instrument Co.). The GFP-expressing neurons also were imaged using a cooled CCD camera (Sensicam; PCO Computer Optics) controlled by PC-based imaging and analysis software (Slide-Book; Intelligent Imaging Innovations). Filter sets for GFP and Fura (Chroma Technologies) were used to excite GFP (wavelength, 450–490 nm) and Fura-PE3 (wavelength, 340 nm) fluorescence using a rotating filter wheel (Lambda 10–2; Sutter Instrument Co.). Any optical overlap between GFP and Fura was minimal to negligible under our imaging conditions, because no bleed-through occurred between GFP and Fura images (Fig. 1, F and G). Emitted light was collected through a dichroic filter, and optical images were acquired.
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Electrical activity was monitored with either a whole-cell patch pipette (resistance, 7–10 M
) or a loose-patch pipette (resistance, ~3 M) pulled from borosilicate glass (diameter, 1.5 mm; World Precision Instuments; P87 puller, Sutter Instruments). Whole-cell recordings of membrane potential (Vm) and action potentials and extracellular loose-patch recordings of action potentials were obtained using an Axopatch 200B amplifier (Axon Instruments) in current-clamp mode and digitized with an ITC-18 computer interface (Instrutech Corp.). Recordings were monitored online using both AxoGraph software (Axon Instruments) and PowerLab data acquisition and analysis instrumentation and software (ADInstruments, Inc.) and stored offline for subsequent AxoGraph data analysis of interspike Vm, spike frequency, interspike interval, action potential and after-hyperpolarization amplitude, and half-maximal spike width. The GFP-expressing neurons were approached with slight positive pressure, and offset potentials were corrected. For whole-cell recordings, after forming a high-resistance seal (>10 G) by applying negative pressure, a second pulse of negative pressure was used to rupture the membrane. Data were collected if series resistance was less than 35 M and if interspike Vm was at least –40 mV under control conditions. For loose-patch recordings, a low-resistance seal (<100 M) was obtained following release from positive pressure. Data were collected once the baseline recording stabilized (usually within 5 min from seal formation). Bath application of tetrodotoxin (TTX; 0.5 µM) was used to determine the role of voltage-gated Na+ channels in spontaneous action potential firing.
Data Analysis
Values are shown as the mean ± SEM. Long-term recordings were analyzed by repeated-measures analysis of variance followed by Tukey post-hoc analysis (Instat Statistical Analysis; GraphPad Software). Comparisons between whole-cell and loose-patch recordings, and between regular and irregular beating patterns, were performed by t-test. Correlation analysis for the current-injection experiment was performed using the Pearson linear-correlation test. Values were considered to be significantly different at P< 0.05. Variability of the beating pattern of action potential firing was determined by the coefficient of variability [%CV = 100·(mean/SD)] of the interspike interval at 10 min after the start of recording for 60 sec. Cells with an interspike interval coefficient of variation of less than 20% were labeled arbitrarily as having a regular beating pattern; those with a coefficient of variation of greater than 20% were labeled as having an irregular beating pattern.
RESULTS
Transgenic Medaka and Expression of the GnRH3-GFP Transgene
In an effort to establish an in vivo model system for GnRH3 neuronal development and cell physiology, we generated a transgenic medaka line that expressed GFP under the control of the Gnrh3 promoter. Among approximately 40 injected embryos that grew to adulthood, three F0 founders were identified through screening of their F1 progeny by monitoring GFP fluorescence. Embryos from the founder fish that exhibited the highest level of GFP expression were maintained and bred to homozygosity.
A bilateral cluster of GFP-expressing neurons (typically six neurons per cluster) were found in the TN associated with the olfactory bulbs, all at or near the ventral surface of the brain (Fig. 1, D and E). These neurons were much larger than the surrounding cells (15–20 µm compared to <8 µm). Immunocytochemical verification of expression of the transgene was performed on four GnRH3-GFP adult male medaka. All GFP-expressing neurons (n = 26 cells) (Fig. 1, A–C) in TN colocalized with immunodetectable GnRH peptide, and 84% of immunoreactive-GnRH neurons (26 of 31) expressed detectable levels of GFP. Control brain sections that were not incubated with primary antibody showed no staining of neural structures (data not shown).
Electrophysiology
We routinely found that the intact brain remained alive in aerated fish saline and that GnRH neurons were healthy based on electrophysiological properties (e.g., Vm of at least –40 mV, robust action potentials) for at least 9 h after removal from the animal. The GFP-containing GnRH neurons were easily distinguished from non-GFP-expressing neurons in the intact, living brain. Because of their location at or near the surface of the brain, they were easily targeted for electrophysiology. As the patch pipette made contact with the surface of the GFP-expressing neurons, the tip of the pipette acted as a fiber-optic, and a faint green fluorescent light was emitted into the pipette tip. This allowed us to confirm visually that each of our recordings was properly targeted to only GFP-expressing neurons. Further verification is illustrated in Figure 1, F and G. One GFP-expressing neuron from a cluster containing six such neurons (Fig. 1F) was targeted for electrophysiology (Fig. 1H) using a patch electrode filled with the fluorescent dye Fura-PE3. This dye diffused into the patched cell (Fig. 1G), demonstrating our ability to target successfully GFP-expressing neurons for electrophysiology.
We performed whole-cell, current-clamp recordings from 50 GnRH3-GFP neurons (from 35 adult male transgenic medaka) for at least 10 min. All these neurons showed a spontaneous beating pattern of action potential firing. Ninety percent of these cells showed an irregular beating pattern of spontaneous action potential firing (mean CV of interspike interval, 42%), and 10% showed a highly regular beating pattern of firing (mean CV of interspike interval, 13%) (Figs. 2 and 3). Overall, the interspike Vm was –48 ± 0.9 mV (range, –67 to –40 mV). The spike frequency was 1.57 ± 0.12 Hz (range, 4.45–0.63 Hz). The spike amplitude was 96 ± 2 mV (range, 73–118 mV), and the half-maximal width was 2.7 ± 0.1 msec (range, 1.9–6 msec). The amplitude of the after-hyperpolarization was –7.5 ± 0.6 mV (range, –11 to –4 mV). No significant differences were found in these characteristics of electrical activity between neurons showing regular beating patterns and neurons showing irregular beating patterns (Table 1). Of this population of recorded cells, we had maintained recordings for at least 60 min from six GnRH3-GFP neurons from four fish, and we found that electrical properties of these cells were stable throughout the duration of recording (Fig. 2). Data were analyzed during 1-min blocks at 10, 30, and 60 min after breaking through into whole-cell mode (Table 2). No significant change was observed in any of the measured characteristics of electrical activity.
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We performed extracellular loose-patch recordings on seven GnRH3-GFP neurons from three fish. The firing frequency was 1.59 ± 0.26 Hz, which was not significantly different from that obtained using the whole-cell recording method. Six neurons showed an irregular beating pattern of action potential firing (mean CV of interspike interval, 41.3%; not significantly different compared to that obtained using the whole-cell recording method). One of the seven neurons showed a regular beating pattern of action potential firing (CV of interspike interval, 14.0%). The proportion of cells showing a regular beating pattern of firing, as defined by a CV for the interspike interval of less than 20%, was similar between the whole-cell (10% of recorded neurons) and loose-patch (14% of recorded neurons) methods of recording. Representative examples of irregular and regular beating patterns of firing from whole-cell and loose-patch recordings are shown in Figure 3.
Bath application of 0.5 µM TTX, which blocks voltage-sensitive Na+ channels, inhibited spontaneous action potential firing; but the underlying oscillation in subthreshold Vm remained (Fig. 4). To address the voltage dependence of action potential frequency, current injections were performed in the absence and presence of TTX. Hyperpolarizing current injection decreased spike frequency, whereas depolarizing current injection increased spike frequency (Fig. 5A). The correlation between magnitude of the current injected and spike frequency was linear and highly significant (r = 0.876, P < 0.0001) (Fig. 5B). Current injection likewise altered the frequency of subthreshold oscillations of Vm in the presence of TTX (Fig. 5C), indicating that voltage-dependence of the oscillations is intrinsic to the neurons.
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DISCUSSION
In the present study, we describe the development of a novel transgenic animal in which GFP is genetically targeted to a specific population of GnRH neurons located in the TN associated with the olfactory bulb. This is an important development for studies that require identification of specific cells for analysis, and it is especially critical for experimental approaches, such as electrophysiology, that necessitate cellular identification in living tissue. Fish brains have the unusual and favorable characteristic, compared to mammalian brains, of remaining alive and healthy for many hours after removal from the animal [21]. The intact-brain preparation has the distinct advantage compared to brain-slice preparations of allowing afferent and efferent connections to remain intact. Furthermore, it avoids the inevitable severing of axons that occurs in a slice preparation, so the neurons of interest should be healthier and in a more natural state within the intact-brain preparation. The GnRH3 neurons in medaka are located at or near the ventral surface of the olfactory bulb within the TN, which allows easy access for electrophysiological recordings within the intact brain. Genetically targeting GFP to this small cluster of neurons makes it easy to identify them in the intact, living brain; it is our experience that TN-GnRH3 neurons are difficult to find in wild-type medaka. We took advantage of this combination of features of the transgenic medaka—readily identifiable neurons located near the surface of the intact brain—to conduct stable recordings for many tens of minutes. The ability to conduct relatively long-term, whole-cell patch recordings in an intact neural system will have many advantages for experiments in which the expression of changes in cellular activities takes tens of minutes to manifest. This new transgenic model system also provides a foundation for future studies in which neurons expressing different GnRH peptides can be studied for their potential interaction and in which GnRH neurons can be accurately tracked and studied at different times during embryonic development in the intact brain. This type of work is not feasible in an ordinary brain preparation.
Although to our knowledge no information has been published regarding the physiological role of the TN-GnRH3 neurons in medaka, studies in the freshwater teleost dwarf gourami suggest that this population of cells plays a role in modulating reproductive behavior. Notably, immunocytochemical analysis of biocytin-filled TN-GnRH3 neurons showed that they project widely in brain—throughout the olfactory bulb and within the telencephalon, inferior lobe of the hypothalamus, optic tectum, medulla, and spinal cord [22]. This suggests that TN-GnRH3 neurons play an important role in neuromodulation of many different neural systems. Evidence in dwarf gourami also indicates that GnRH3 peptide modulates the beat frequency of TN-GnRH3 neurons [23], suggesting that this population of neurons is self-regulated through autocrine/paracrine feedback. Regarding a specific role in reproduction, bilateral electrolytic lesions of the TN-GnRH3 neurons in male dwarf gourami led to a high level of failure to build nests [4], which is a critical behavior in this species for a successful reproductive outcome. Given the wide projections of TN-GnRH3 neurons, it is not surprising that their elimination would result in altered behaviors. Importantly, of the three reproductive behaviors tested, nest building was the only behavior to be affected by the lesions [4], suggesting that TN-GnRH3 neurons are involved in very specific behaviors rather than in global modulation of sexual activities.
The morphological and electrical properties of the GnRH3 neurons in transgenic medaka show some similarities to that of TN-GnRH3 neurons in dwarf gourami [22]. In both species, the TN-GnRH3 neurons are located as a tight cluster of cells at or near the ventral surface of the brain, and they are noticeably larger than the surrounding non-GnRH neurons. In dwarf gourami, as in the transgenic medaka, all TN-GnRH3 neurons showed spontaneous action potential firing. However, in dwarf gourami, approximately 80% of GnRH3 neurons showed a regular beating pattern, and 11% showed a more irregular beating pattern, of spontaneous action potential firing, whereas the remainder showed a bursting pattern of firing. In contrast, during the present study, only 10% of TN-GnRH3-GFP neurons recorded in medaka showed a regular beating pattern over the course of many minutes of recording. The remainder showed an irregular beating pattern, which closely resembled the irregular discharges from dwarf gourami TN-GnRH3 neurons. This was the case regardless of the method of electrophysiological recording; the majority of both whole-cell and extracellular loose-patch recordings showed an irregular beating pattern. Therefore, cytosolic washout that occurs with prolonged whole-cell recordings, but not with loose-patch recordings, could not be the cause of the irregularity. It also is unlikely that GFP expression is disrupting electrical activity, thus leading to irregularity, because all recorded neurons expressed abundant GFP, including the 10% that showed a regular beating pattern. The most likely explanation is that as with dwarf gourami, an intrinsic variability exists in the beating pattern of action potential firing from TN-GnRH3 neurons, but unlike with dwarf gourami, the irregular beating pattern dominates.
Medaka TN-GnRH3 neurons showed a similar response to TTX and current injection as the TN neurons of dwarf gourami [22]. In both cases, TTX blocked Na+-dependent action potentials, but oscillations in the subthreshold Vm persisted. This indicates that the spontaneous action potential firing of these neurons is not being driven by synaptic input from other pacemaker cells, because TTX synaptically isolates neurons. Rather, the pattern of action potential firing is an intrinsic property of these GnRH neurons. The remaining subthreshold oscillation in Vm most likely results from a TTX-resistant persistent Na+ current, as is the case with the dwarf gourami TN-GnRH neurons [24, 25]. Hyperpolarizing and depolarizing current injections led to changes in the frequency of action potential firing and in the frequency of subthreshold Vm oscillations in synaptically-isolated neurons. This finding demonstrates that TN-GnRH3 neurons show voltage dependence of firing frequency that is an intrinsic property of the cell.
In addition to studies characterizing the pattern and regulation of membrane excitability of the TN-GnRH3 neuronal population, experiments performed in several species have described the cell physiology of the preoptic area/hypothalamic population of GnRH neurons. Studies using rodent and primate model systems have all shown spontaneous cellular activity of these GnRH neurons that express the mammalian form of GnRH, be it oscillations in intracellular calcium concentrations or action potential firing [26–28]. This spontaneous activity can be much more complex than that observed from the TN-GnRH3 neurons in teleosts. For example, with a transgenic mouse model in which preoptic area/hypothalamic GnRH neurons express GFP, targeted electrophysiology revealed three different patterns of intrinsic oscillatory electrical activity over different time domains: high frequency bursts of action potentials, slower frequency clusters of action potential bursts, and very slow frequency episodes of action potential bursts [29]. The duration of the present experiments with TN-GnRH3 neurons was at least as long as that of the mouse preoptic area/hypothalamic studies, but slower frequency oscillations or bursts of action potential firing were not noted in the medaka brain preparation. In contrast to the mammalian work, recent work in the cichlid fish (Astatotilapia burtoni) showed that 86% of GnRH neurons located in the preoptic area/telencephalon, which innervates the pituitary and, ultimately, controls gonadal function, exhibit a pattern of spontaneous firing that more closely resembles the irregular beating pattern of action potential firing of the medaka TN-GnRH3 neurons [30]. Fifteen percent of these preoptic area/telencephalon neurons showed a phasic or bursting pattern of firing that more closely resembled that of the mammalian model.
Why do most of the cichlid preoptic/telencephalon GnRH neurons show a pattern of firing that more closely resembles that of the medaka TN population? One possibility is that teleost GnRH neurons, regardless of their embryonic origin and adult localization, share similar patterns of action potential firing, which differs significantly from that of the preoptic area/hypothalamic population of GnRH neurons in mammalian species. Alternatively, the smaller subpopulation of preoptic area/telencephalon GnRH neurons in cichlid that shows more of a bursting pattern of action potential firing, similar to that of the mammalian population, could be the relevant subset of neurons regulating putative pulsatile secretion of GnRH peptide that would drive pulsatile secretion of the pituitary gonadotropins. Regardless of the reason for these species and regional differences in the patterns of action potential firing, an expansion in the number of model systems available to study GnRH neuronal physiology will aid our understanding of the cellular mechanisms that control and modulate reproductive functions.
ACKNOWLEDGMENTS
We thank Janelle Asai and Dr. Kevin Sinchak for technical assistance with immunocytochemistry and image analysis as well as Evelyn Ascencio and Han Wang for animal care.
FOOTNOTES
1 Supported by seed grants from the University of California at Los Angeles and National Institutes of Health DA05010 (N.L.W.) and a grant from the National Science Foundation IOB-0414493 (N.L.W.). 
2 Correspondence: Nancy L. Wayne, Department of Physiology, Room 231, Center for Health Sciences, University of California at Los Angeles School of Medicine, Los Angeles, CA 90095. FAX: 310 206 5661; nwayne{at}mednet.ucla.edu
3 For requests regarding the transgenic fish: Kataaki Okubo, Laboratory of Reproductive Biology, National Institute for Basic Biology, Okazaki, Aichi 444-8585, Japan. FAX: 81 564 55 7556; email: okubo{at}nibb.ac.jp.
Received: 11 April 2005.
First decision: 29 April 2005.
Accepted: 15 August 2005.
REFERENCES
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  N. G. Hatcher and J. V. Sweedler Aplysia Bag Cells Function as a Distributed Neurosecretory Network J Neurophysiol, January 1, 2008; 99(1): 333 - 343. [Abstract] [Full Text] [PDF]
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