| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Department of Anatomy and Neurobiology (K.J.S., T.L.S., J.-P.W., F.E.D.), Animal Reproduction and Biotechnology Laboratory (K.J.S.), Colorado State University, Fort Collins, Colorado 80523; and Departments of Internal Medicine and Cell Biology (W.J.S., J.T.S., S.M.M.), National Science Foundation Center for Biological Timing, University of Virginia, Charlottesville, Virginia 22908
Address all correspondence and requests for reprints to: Suzanne M. Moenter, Department of Medicine, Box 578 HSC, University of Virginia, Charlottesville, Virginia 22908. E-mail: smm4n{at}virginia.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Despite the importance of modulations in GnRH-pulse characteristics to normal reproductive function, little is known about the cellular and molecular mechanisms underlying episodic GnRH release. Study of these neurons has been hampered by their rarity (600–2500 in the mammalian brain) and by their scattered distribution from the diagonal band of Broca through the medial basal hypothalamus (11). These factors have made isolation and/or identification of living GnRH neurons nearly impossible. To facilitate the study of living GnRH neurons, we generated transgenic mice in which the jellyfish reporter, green fluorescent protein (GFP, 12–14), is genetically targeted to GnRH neurons. We report here the initial characterization of this mouse model, with regard to fidelity of transgene expression, morphological assessment of intracellularly labeled GnRH neurons, and basic electrophysiological properties of these neurons.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Animals
Mice were maintained on a 14-h light, 10-h dark photoperiod with
ad lib access to standard chow (Harlan 7012,
Bartonsville, IL) diet and water. Transgenic mice were generated
through standard pronuclear injection of fertilized ova obtained from
CBB6/F1 females (15). Transgenic offspring were identified through
Southern dot blot analysis of DNA harvested from tail snips using a
32P-labeled GFP probe. All procedures were
carried out in accordance with the Animal Care and Use Committees of
the University of Virginia and Colorado State University.
Immunofluorescence and immunocytochemistry
To determine the pattern of transgene expression, six F1 male
and female mice (age, 21–60 days) were anesthetized with sodium
pentobarbital (0.1–0.2 mg ip) and perfused through the left
ventricle with 10 ml 0.1 M PBS (pH 7.4) containing 0.1%
wt/vol NaNO3, followed by 40 ml 4%
paraformaldehyde in the same buffer. Brains were removed and postfixed
in the same fixative 6–18 h, then infiltrated with 30% sucrose in PBS
with 0.1% NaN3 for cryoprotection. Coronal
sections (40 µm) were cut on a cryostat from the base of the
olfactory bulbs through the caudal extent of the mammillary bodies.
Every sixth section was subjected to immunodetection to locate GnRH
peptide using slight modifications of a previously described procedure
(16). All reagents were diluted in PBS (pH7.4) containing 0.4–1.0%
vol/vol Triton X-100. Sections were blocked 1 h at room
temperature in 4% vol/vol normal donkey serum (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), then
incubated with LR1 anti-GnRH antibody (a generous gift of Dr. Robert
Benoit, McGill University, 1:20,000 in block, 48–96 h, 4 C). Sections
were then incubated in biotinylated donkey antirabbit IgG (1:200,
1 h, Jackson ImmunoResearch Laboratories, Inc.)
followed by avidin-Texas red (1:100, 1 h, Vector Laboratories, Inc., Burlingame, CA). Sections were viewed
with a Nikon (Garden City, NY) Microphot SA
with epifluorescence to determine overlap of GFP signal with GnRH
immunoreactivity. Images were captured with a Hamamatsu CCD camera and
Open Lab software. For morphology, additional sections were prepared as
above, with GnRH visualized by standard avidin-biotin-horseradish
peroxidase complex using nickel-enhanced diaminobenzidine
(Vector Laboratories, Inc.) as the chromogen.
Slices (200 µm) from electrophysiological work and control nonrecorded slices were fixed with 4% paraformaldehyde overnight. Recorded slices were incubated with strepavidin-Cy3 (1:1,000, Jackson ImmunoResearch Laboratories, Inc.) overnight at 4 C to confirm the identity of recorded cells. Immunodetection of GnRH was performed using the procedure detailed above, with an overnight extension of secondary antibody and avidin-biotin-horseradish peroxidase complex incubation at 4 C. For fluorescence detection of GnRH in recorded sections, Cy5 conjugated secondary antibody (1:200, Jackson ImmunoResearch Laboratories, Inc.) was used. For morphology, peroxidase detection was performed as described above.
Morphological analyses
Morphological analysis was performed on dendrites defined by
immunoperoxidase labeling of 40-µm sections and of control
(unrecorded) 200-µm sections. Additionally, morphological analyses
were performed on dendrites defined by intracellular injection of
biocytin into GnRH neurons from which electrophysiological recordings
were obtained, with biocytin detected by peroxidase as described above.
Sections were mounted, dried overnight, dehydrated in increasing
concentrations of ethanol (70%–100%), cleared in xylene, and
coverslipped with Permount. Biocytin- and immunocytochemically-labeled
neurons were traced digitally using Neurolucida 2.1 (MicroBrightField,
Inc., Colchester, VT). Morphological data obtained from each
section were averaged and counted as a single observation for
statistical purposes. Average cell body area, average dendrite length,
and number of dendrites per cell were compared among groups by ANOVA
and, if significant differences were found, between groups with a
two-tailed t test. Significance was set at the
P < 0.05 level.
Electrophysiology
Mice (21–68 days of age) were anesthetized with halothane and
decapitated. The brain was quickly removed and immersed in cold (1-2
C), oxygenated (95% O2-5%
CO2) artificial cerebrospinal fluid (ACSF)
containing (in mM): NaCl (125),
NaHCO3 (26),
NaH2PO4 (1.25),
MgCl2 (1), KCl (2.5), glucose (10), and
CaCl2 (3), pH 7.3–7.4. The hypothalamus was
blocked with a razor blade, and 200-µm hypothalamic slices were cut
in the coronal plane using a vibrating microtome (Lancer 1000). Slices
were transferred to a warm incubator (32-35 C) and continuously
incubated with oxygenated ACSF. Slices were incubated for at least
2 h before recording. All slices used in these studies were from
the preoptic area and anterior portion of the hypothalamus, rostral to
or at the level of the anterior commissure. GFP-identified and
non-GFP-expressing neurons were recorded throughout this region; no
regional differences have thus far been discernible in the behavior of
GnRH neurons.
Patch pipettes with resistances between 2.5–5 M
were pulled from
glass capillaries (id, 1.20 mm; od, 1.65 mm) with a Flaming Brown
microelectrode puller (Sutter Instruments, Novato, CA). Pipettes
were filled with a solution containing (in mM): K-gluconate
(130), HEPES (10), NaCl (1), MgCl2 (1),
CaCl2 (1), EGTA (5), biocytin (5), adenosine
triphosphate (2), and brought to pH 7.2–7.4 with KOH.
For recording, slices were transferred to a chamber mounted on the stage of an upright microscope and continuously perifused with ACSF. Cells were visualized using a 40x water-immersion objective. Recording electrodes were visualized and guided to the cells of interest using an automated microdrive (Sutter MP-285). To identify GFP-expressing neurons, the slice was briefly illuminated with epifluorescence using fluorescein filters. Current-clamp recordings were obtained using an Axopatch 1D amplifier (Axon Instruments, Foster City, CA) with filtering at 5 kHz. Data were digitized on-line with a Neuro-corder (DR-484, Neurodata, Inc.) and stored on video cassettes for off-line analysis. During electrophysiological recording, cells were filled with biocytin for subsequent morphological analysis. To determine the role of voltage-dependent sodium channels in action potential firing, GFP-identified GnRH neurons were injected with 20 pA current pulses (1 Hz) in ACSF and after a 5–10 min bath application of 1 µM tetrodotoxin (TTX in ACSF; Sigma, St. Louis, MO ). The effect of 1 µM TTX on spontaneous firing was also determined.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
).
Reproductive characteristics of this line were normal, in terms of age
of vaginal opening (28 ± 2 days), onset of fertile cycles in
females housed with immature males (55.8 ± 3.3 days for
heterozygotes, 60.8 ± 6.4 days for homozygotes), and litter size
(7.8 ± 0.6 pups, all values mean ± SEM).
|
). The majority of GnRH cell bodies were in the
preoptic area just rostral to the crossing of the anterior commissure
(Fig. 1A). Likewise, GnRH terminal fields in both the organum
vasculosum of the lamina terminalis (Fig. 1A) and the median eminence
(not shown) appeared normal. Of the several hundred GnRH neurons
examined from each mouse, 84–94% could be identified by their
expression of the GFP transgene. The remaining 6–16% did not express
sufficiently detectable amounts of GFP (e.g. Fig. 1B).
Although GFP was observed in the vast majority of GnRH cell bodies, its
presence in processes was variable, i.e. considerably more
GnRH-immunopositive fibers than GFP-positive fibers were visible (Fig. 1, B–D). Thus far, we have observed only one cell that exhibited GFP
fluorescence in the absence of immunodetectable GnRH. This cell had a
neuronal morphology with helical processes and beaded varicosities that
are characteristic of GnRH neurons; thus, it likely represents a
failure of our immunofluorescent technique, rather than ectopic
expression. The fidelity of GFP identification of GnRH neurons is thus
greater than 99.5% in this model system. Further, little
autofluorescence was noted in brain regions containing GnRH neurons
that would lead to mistaken identification of a non-GnRH neuron as a
GnRH neuron. No ectopic expression of GnRH-GFP was detected in the
lateral septum, basal ganglia, hippocampus, and cortex in the sections
examined.
In living 200-µm hypothalamic slices, GFP-containing GnRH neurons
were readily distinguishable from non-GFP-containing cells. In these
slices, GFP was present throughout the full extent of the perikarya,
allowing the clear delineation of cell boundaries, an important issue
for sighted patch-clamp recording. Enhanced-GFP is modified to have
greater expression and stability in mammalian cells. This has resulted
in greater amounts of protein and hence greater visibility of cells
expressing GFP for longer durations (14). In our model, this provided
adequate time for cell localization, establishment of the whole-cell
recording configuration and multiple recording attempts from GnRH
neurons in the same slice. Following electrophysiological recording,
slices were fixed and the identity of the recorded cell confirmed by
detection of biocytin. In addition, the peptide phenotype of the cell
was determined by immunofluorescence for GnRH. Cells which were
identified by the GFP signal in slices and confirmed as recorded with
biocytin were invariably immunopositive for GnRH (Fig. 2, n = 18).
|
. No difference was observed in cell
body area using these 3 methods (Table 1). Intracellular labeling with biocytin
revealed a modestly increased, but significantly higher, number of
dendrites on GnRH neurons than found in immunolabeled material (Table 1). In addition, differences were observed in mean dendritic length of
intracellularly labeled (biocytin) GnRH neurons. The majority of this
difference in length, however, could be accounted for by the difference
in section thickness, because dendritic length in 200-µm
immunoperoxidase labeled sections was similar to that of directly
labeled cells. The longest 2 processes measured from biocytin-labeled
GnRH neurons were 436.3 and 272.3 µm. In contrast, the longest
processes from immunolabeled material were 119.2 and 83.3 µm in
40-µm sections and 138.8 and 124.8 µm in 200-µm sections. This
suggests that direct labeling can reveal longer dendrites than
immunoperoxidase in 200-µm sections; but in the plane of section
available for work, this difference was not significant. Despite an
increased number of dendritic processes, biocytin-filled GnRH neurons
displayed the previously described simple morphology, with few
dendrites and little or no secondary branching (Refs. 17, 18, 19 ; Figs. 2 and 3).
|
|
), which was not different from
non-GFP-expressing neurons (range, -79 to -50 mV; n = 18).
Likewise, input resistance, an index of cell integrity, was not
different between GnRH (i.e. GFP positive) and
non-GFP-positive neurons (1.60 ± 0.16 vs. 1.52 ±
0.25 G, Table 2). All GnRH neurons studied fired spontaneous action
potentials with an amplitude of >60 mV (82.8 ± 4.9
vs. 76.9 ± 5.5 mV, Table 2). GnRH neurons did not,
however, fire continuously. Most current-clamp recordings from
GFP-identified GnRH neurons revealed quiescent neurons with
intermittent action potentials (Fig. 4).
Application of 1 µM TTX, which blocks
voltage-dependent sodium channels, completely eliminated spontaneous
action potentials (Fig. 5A). Further, in
control medium (ACSF), GnRH neurons fired repeated action potentials in
response to depolarizing current injection, whereas 1
µM TTX blocked induction of action potentials
in GnRH neurons by current injection (Fig. 5B).
|
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Ours is the second report of transgenic mice in which GFP has been targeted to GnRH neurons. The data we present here support those of Spergel and co-workers (20) but also extend and differ from this previous report in several important ways. First, the form of GFP used in the present study, enhanced GFP, has been demonstrated to be synthesized at higher levels in mammalian systems and thus produces a brighter and longer-lasting fluorescent signal than the form used previously (14, 22). This is likely attributable both to a mutation in enhanced GFP (rendering it more stable at the body temperature of the mouse) and to the greater extinction coefficient of enhanced GFP (14). Although direct comparisons have not been made between these mice, the remarkably stable and bright GFP signal in our mice makes identification and subsequent manipulations of identified cells straightforward. Second, our animals are the first GnRH-GFP mice for which the reproductive characteristics have been reported. This is a critical point, because the goal of this model is to study the function of GnRH neurons. If GnRH neurons were compromised by expression of the transgene, the fertility of the animals should be less than normal. Despite the advantage of being able to identify GnRH neurons with GFP, if these neurons are not functioning normally, one must call into question the properties derived from these cells. The normal reproductive parameters observed in our GnRH-GFP transgenic line attest to the proper functioning of GnRH neurons in which GFP is expressed. A third difference is that all of the cells in the present report were studied in whole-cell mode. Although Spergel and co-workers performed some whole-cell recordings, most of their findings were from nucleated patches, pulled from GnRH cell bodies and removed from the slice to be studied independently. This is a common way of examining the pharmacological properties of cell membranes. Our primary interest, however, is the function of the GnRH neurons in situ. Our goal, therefore, was to use an approach that provides the best opportunity to study the characteristics of individual GnRH neurons in as physiological a setting as possible, the hypothalamic slice.
Identification of living GnRH neurons in hypothalamic slice preparations offers different approaches than can be taken using existing model systems for studying GnRH neurons. The GT1 and GN cell lines, developed by genetically targeted tumorigenesis (23, 24), have been very useful particularly for biochemical approaches (reviewed in Ref. 25), while having the further advantage of studying GnRH-derived cells in the absence of other cell types. Despite their utility, the GT1 and GN model systems have some limitations. First, studies of developmental changes are precluded using these models because they seem to maintain characteristics of immature GnRH neurons (26, 27). Second, the ongoing expression of the Simian virus 40 tumor antigen may interfere with some differentiated functions. For example, GnRH neurons expressing tumor antigen are unable to target axons to the external layer of the median eminence as normal GnRH neurons do (28). Third, study of mediators that affect the afferent inputs to the GnRH neuron, such as from steroid-receptor-containing neurons, cannot be accomplished in a clonal cell line. Fourth, because of their clonal nature, GT1 and GN cell studies represent the behavior of one GnRH neuron. Thus, if (as hypothesized; reviewed in Ref. 29) subpopulations of GnRH neurons serve different functions, such as the episodic and surge modes of release, this could not be delineated with these model systems.
These limitations are avoided or minimized in the GFP-identified cell, because multiple nontransformed GnRH neurons can be studied in their local environment from animals at different ages. Because most GnRH neurons can be identified through the GFP signal, experiments to determine whether different subpopulations exist are possible. The GnRH-GFP mouse is limited, however, as a model system for biochemical investigations of the type facilitated by the GT1 and GN cell lines. In this regard, approaches such as single-cell RT-PCR and protein methodologies in GFP-identified GnRH neurons are being developed. Further, application of fluorescent-activated cell-sorting to enrich the GnRH-GFP neuronal population in primary cultures may assist with these approaches (14, 30).
We have begun to characterize the GnRH-GFP transgenic model system in two ways: morphologically and electrophysiologically. Morphological analyses of GnRH neurons thus far have relied on immunoperoxidase labeling. Direct filling of these neurons with biocytin should provide a more accurate picture of neuronal morphology, because immunoperoxidase relies on the presence of the peptide antigen for delineation of cell boundaries. Thus, areas of the neuron that exclude GnRH or contain low levels of the peptide (such as, for example, dendrites) may not be visualized. Our data are both the first characterization of GnRH neuronal morphology based on direct, intracellular labeling with biocytin and the first comparison of these different labeling techniques within a population of neurosecretory neurons with a defined peptide phenotype. When labeled with intracellular biocytin, a small, but significantly greater, number of dendrites per cell was evident than in immunolabeled material, although the well-documented simple morphology of GnRH neurons was observed. Directly labeled GnRH neurons had processes that were, on average, double the length of those of immunolabeled material from standard-thickness sections (40 µm). A substantial portion of this difference in dendrite length may be accounted for by the thickness of the section, because the average dendrite length of immunolabeled GnRH neurons in 200-µm sections was comparable with that of directly labeled cells. Although the ability to track a dendrite over greater distances is, in part, attributable to the thicker sections, it should be noted that the longest dendrites traced from directly labeled neurons were three to four times the length of immunolabeled material of the same thickness. Previous studies of immunoperoxidase-labeled material in rats (31), sheep (32), and hamster (33) indicate that GnRH neurons receive little synaptic input. The present data in biocytin-labeled neurons suggest a more extensive neurobiological substrate for synaptic impingement. It is unclear, however, whether synaptic input at such extended distances from the cell body would impact on neuronal firing because of electrotonic decay.
With regard to function, live hypothalamic slices derived from GnRH-GFP transgenic mice provide an excellent model for studying single GnRH neuronal physiology. Patch-clamp studies of GFP-identified GnRH neurons in hypothalamic slices revealed spontaneous action potentials with amplitudes (>60 mV) consistent with those of other hypothalamic neurons (33). Likewise, resting potentials in these neurons were comparable with those for nonfluorescent neurons in the same slice (-75 to -50 mV) and were within the physiological range previously reported for preoptic area neurons (34, 35) and other hypothalamic neurosecretory cells in the arcuate, supraoptic, and paraventricular nuclei (36, 37, 38). These findings suggest neither expression of GFP in GnRH neurons nor the illumination required to identify them adversely affects their viability. Study of GFP-containing GnRH neurons and non-GFP-expressing GnRH neurons would be most useful for comparison of the presently observed electrophysiological characteristics, but such recordings are exceedingly difficult to obtain. This consideration notwithstanding, the GnRH neurons identified by GFP expression seem to be similar to other neurons in the hypothalamus.
In the present study, spontaneous action potentials in GnRH neurons, as well as those initiated in response to depolarizing current injection, were eliminated in the presence of tetrodotoxin. These data indicate that action potential firing in GnRH neurons involves sodium influx through TTX-sensitive voltage-gated channels. Similar results have been obtained in GT1–7 cells. Interestingly, these model GnRH neurons also express calcium-dependent action potentials mediated by low voltage-activated and high voltage-activated calcium channels (39). In GnRH neurons derived from the olfactory placode, however, more limited expression of such calcium-dependent currents was detected (40). Further work is required to reconcile these findings and to determine the role of voltage-activated calcium channels in hypothalamic GnRH neurons.
The pattern of firing observed in thin-slice preparations is different from that observed in two different models of cultured GnRH neurons. GFP-identified GnRH neurons in the present study did not fire continuously. Similar findings were reported in another GnRH-GFP mouse model using the hypothalamic thin-slice preparation (20). In contrast, cells in two different cultured preparations fired continuously, with only brief intervals (i.e. 10–20 sec) or complete absence of quiescence (39, 40). Of interest in this regard, pacemaker neurons from lobster stomatogastric ganglion in the intact ganglion exhibit brief episodic firing or fire action potentials when depolarized by their normal inputs (41). In contrast, after 3–4 days in culture, most of these neurons fire bursts of action potentials not seen in the intact ganglion. It is possible that similar changes in electrophysiological characteristics are induced in cultured GnRH neurons. If this were the case, cultured GnRH neurons may not accurately reflect all firing properties of hypothalamic GnRH neurons.
Our understanding of putative mechanisms linking electrophysiological activity and GnRH release is minimal. Whole-animal experiments have demonstrated GnRH release is episodic (1, 2, 3, 4, 5). Further, pulsatile LH release is well correlated with extracellular electrical discharges (volleys of multiunit activity) within the arcuate region of the hypothalamus (42, 43, 44). The model system we describe here will allow investigations of whether or not the pattern of action potential firing at the single GnRH neuron level reflects the intervals between multiunit activity, GnRH, and LH events. In summary, the GnRH-GFP transgenic mouse provides a model system for the high-fidelity identification and study of GnRH neurons. Use of this model in tandem with contemporary electrophysiological and biochemical approaches will greatly enhance our ability to investigate and understand this critical neuroendocrine system.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received July 28, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  J. C. Gill, B. Wadas, P. Chen, W. Portillo, A. Reyna, E. Jorgensen, S. Mani, G. A. Schwarting, S. M. Moenter, S. Tobet, et al. The Gonadotropin-Releasing Hormone (GnRH) Neuronal Population Is Normal in Size and Distribution in GnRH-Deficient and GnRH Receptor-Mutant Hypogonadal Mice Endocrinology, September 1, 2008; 149(9): 4596 - 4604. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. J. Suter and L. O'Farrell Impaired episodic LH secretion in female mice with GFP in GnRH neurons Am J Physiol Endocrinol Metab, July 1, 2008; 295(1): E130 - E136. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Christian and S. M. Moenter Vasoactive Intestinal Polypeptide Can Excite Gonadotropin-Releasing Hormone Neurons in a Manner Dependent on Estradiol and Gated by Time of Day Endocrinology, June 1, 2008; 149(6): 3130 - 3136. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Xu, T. A. Roepke, C. Zhang, O. K. Ronnekleiv, and M. J. Kelly Gonadotropin-Releasing Hormone (GnRH) Activates the M-Current in GnRH Neurons: An Autoregulatory Negative Feedback Mechanism? Endocrinology, May 1, 2008; 149(5): 2459 - 2466. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Zhang, T. A. Roepke, M. J. Kelly, and O. K. Ronnekleiv Kisspeptin Depolarizes Gonadotropin-Releasing Hormone Neurons through Activation of TRPC-Like Cationic Channels J. Neurosci., April 23, 2008; 28(17): 4423 - 4434. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Pielecka-Fortuna, Z. Chu, and S. M. Moenter Kisspeptin Acts Directly and Indirectly to Increase Gonadotropin-Releasing Hormone Neuron Activity and Its Effects Are Modulated by Estradiol Endocrinology, April 1, 2008; 149(4): 1979 - 1986. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Zhang, M. A. Bosch, J. E. Levine, O. K. Ronnekleiv, and M. J. Kelly Gonadotropin-Releasing Hormone Neurons Express KATP Channels That Are Regulated by Estrogen and Responsive to Glucose and Metabolic Inhibition J. Neurosci., September 19, 2007; 27(38): 10153 - 10164. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. J. Spergel Calcium and Small-Conductance Calcium-Activated Potassium Channels in Gonadotropin-Releasing Hormone Neurons before, during, and after Puberty Endocrinology, May 1, 2007; 148(5): 2383 - 2390. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Cariboni, J. Hickok, S. Rakic, W. Andrews, R. Maggi, S. Tischkau, and J. G. Parnavelas Neuropilins and Their Ligands Are Important in the Migration of Gonadotropin-Releasing Hormone Neurons J. Neurosci., February 28, 2007; 27(9): 2387 - 2395. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Christian and S. M. Moenter Estradiol Induces Diurnal Shifts in GABA Transmission to Gonadotropin-Releasing Hormone Neurons to Provide a Neural Signal for Ovulation J. Neurosci., February 21, 2007; 27(8): 1913 - 1921. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. L. Jasoni, M. G. Todman, M. M. Strumia, and A. E. Herbison Cell Type-Specific Expression of a Genetically Encoded Calcium Indicator Reveals Intrinsic Calcium Oscillations in Adult Gonadotropin-Releasing Hormone Neurons J. Neurosci., January 24, 2007; 27(4): 860 - 867. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Chu and S. M. Moenter Physiologic Regulation of a Tetrodotoxin-Sensitive Sodium Influx That Mediates a Slow Afterdepolarization Potential in Gonadotropin-Releasing Hormone Neurons: Possible Implications for the Central Regulation of Fertility. J. Neurosci., November 15, 2006; 26(46): 11961 - 11973. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
 |
|
 B. L. Jones, P. J. Whiting, and L. P. Henderson Mechanisms of anabolic androgenic steroid inhibition of mammalian {varepsilon}-subunit-containing GABAA receptors J. Physiol., June 15, 2006; 573(3): 571 - 593. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. El-Etr, Y. Akwa, E.-E. Baulieu, and M. Schumacher The Neuroactive Steroid Pregnenolone Sulfate Stimulates the Release of Gonadotropin-Releasing Hormone from GT1-7 Hypothalamic Neurons, through N-Methyl-D-Aspartate Receptors Endocrinology, June 1, 2006; 147(6): 2737 - 2743. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Pielecka and S. M. Moenter Effect of Steroid Milieu on Gonadotropin-Releasing Hormone-1 Neuron Firing Pattern and Luteinizing Hormone Levels in Male Mice Biol Reprod, May 1, 2006; 74(5): 931 - 937. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. R. Pak, W. C. J. Chung, J. L. Roberts, and R. J. Handa Ligand-Independent Effects of Estrogen Receptor {beta} on Mouse Gonadotropin-Releasing Hormone Promoter Activity Endocrinology, April 1, 2006; 147(4): 1924 - 1931. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. B. Schwartz and J. E. Levine Ontogeny of gonadotropin-releasing hormone neurons: fishing for clues in medaka. Endocrinology, March 1, 2006; 147(3): 1074 - 1075. [Full Text] [PDF]
|
|
|
|
|
|
K. Okubo, F. Sakai, E. L. Lau, G. Yoshizaki, Y. Takeuchi, K. Naruse, K. Aida, and Y. Nagahama Forebrain Gonadotropin-Releasing Hormone Neuronal Development: Insights from Transgenic Medaka and the Relevance to X-Linked Kallmann Syndrome Endocrinology, March 1, 2006; 147(3): 1076 - 1084. [Abstract] [Full Text] [PDF]
|
|
|
|
|
