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Cellular and Developmental Neurobiology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892
Address all correspondence and requests for reprints to: Susan Wray, Chief, Cellular and Developmental Neurobiology Section, NINDS, NIH Building 36, Room 5A-25, Bethesda, Maryland 20895-4156. E-mail: swray{at}codon.nih.gov
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Studies using immortalized neurons that synthesize and secrete LHRH in vitro (5, 6) have been used for identifying factors that regulate neuropeptide synthesis and secretion. Unfortunately, corroborating studies on primary LHRH neurons, whether in situ or in slice cultures, have been less productive. Postnatally, the distribution, small number, and small size of LHRH neurons hamper such studies. However, it is imperative that the mechanisms responsible for LHRH secretion identified in immortalized cell lines be evaluated in primary LHRH neurons. Without this information, the complexities imposed by interneuronal circuitry (as well as changes in hormonal and/or cytokine levels) remain difficult to decipher, and as such, the mechanism(s) underlying reproductive maturation elusive. Tissue cell culture systems of LHRH neurons derived from prenatal nasal regions (7, 8) provide an alternative model for physiological, pharmacological, and molecular studies designed to evaluate the inter and intracellular mechanisms regulating LHRH release. Similar to the developing embryo in vivo, cell cultures containing LHRH neurons derived from prenatal nasal regions turn on LHRH gene expression, peptide synthesis, and processing (8). Importantly, such cultures grown in serum-free media maintain large numbers of LHRH neurons in a concentrated area and allow for in situ identification of the cells due to their migrational behavior (9). These features allow one to characterize and perturb a relatively large number of primary LHRH neurons in a single experiment. In addition, the non-central nervous system (CNS) environment in which these LHRH neurons are located enables investigators to more clearly focus on the intrinsic properties of the neurons while controlling for extracellular influences. Recent work indicates that when maintained for long periods of time in vitro, LHRH cells in nasal cultures exhibit pulsatile-like secretion (10, 11). Thus, in the absence of CNS cues, prenatal LHRH cells continue to differentiate with respect to secretory profiles.
Of the many research interests in our laboratory, one component is the identification of specific mechanisms that regulate the onset and pattern of LHRH production and secretion in primary LHRH neurons. Toward this end, we have initiated a series of studies using the nasal explant model to investigate regulation of LHRH neuropeptide expression. In the present study, we examined mechanisms involved in the production and secretion of LHRH from primary LHRH neurons in nasal cultures maintained in serum-free media for only 6–10 days in vitro (div).
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Materials and Methods |
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For the enzyme-linked immunosorbant assay (ELISA), LHRH acetate salt, D-[lys6]-LHRH, and p-nitrophenyl phosphate tablets were purchased from Sigma, Co. (St. Louis, MO). Pierce Chemical Co. (Rockford, IL) and Amersham Pharmacia Biotech (Arlington Heights, IL) supplied F(ab)2 fragment goat antirabbit IgG and streptavidin-alkaline phosphatase, respectively. MaxiSorp microwell plates were supplied by Nunc, Inc. (Naperville, IL). Rabbit antisera directed against conjugated rat LHRH, in equal volumes from three separate bleeds (SW1, SW2, and SW3), was affinity purified by Lofstrand Labs Limited (Gaithersburg, MD); these antisera immunocytochemically stain LHRH in perikarya and fibers (12). Biotinylated-LHRH was generated by reacting D-[Lys6]-LHRH in a 1:2 molar ratio with sulfosuccinimidyl-6-(biotinamido) hexanoate sodium salt (Vector Laboratories, Inc. Burlingame, CA) in 0.1 M NaHCO3, pH 8.5, and purified using HPLC. The stock concentration of LHRH peptide standard was quantitatively analyzed by Harvard Microchemistry Facility (Cambridge, MA).
Nasal explant preparations
Olfactory pits were cultured as tissue explants as previously
described (8). Briefly, embryos were obtained from timed
pregnant animals in accordance with NIH guidelines. Olfactory pits of
E11.5 staged NIH-Swiss mice were isolated under aseptic conditions and
refrigerated for 1 h in Gey’s Balanced Salt Solution enriched
with glucose. The nasal tissues were adhered onto coverslips by a
plasma/thrombin clot. Nasal explants were maintained in a defined
serum-free medium (SFM) at 37 C in a culture chamber with a humidified
atmosphere with 5% CO2 (13). On
culture day 3, a dose of FuDR (8 x
10-5 M) was
given for 3 days to inhibit proliferation of dividing olfactory neurons
and nonneuronal explant tissue. Depending on the experimental group, on
culture day 6 and 8 the media was changed to fresh SFM. The explants
were used for experiments on culture day 6–10.
Tissue extraction for ELISA
For in vivo LHRH content analysis, the rostrum halves
of brains from E12.5 and E14.5 embryos, and the POA/hypothalamus from
1, 4, and 8 day-old as well as 2-month- old mice were extracted into
1.7 ml siliconized microtubes on ice containing 300–500 µl of 0.1
N HCl containing CPI (5 mg/ml). The tissue was
homogenized for 10 sec, frozen on dry ice, and stored at -80 C until
assay. LHRH standards were also prepared to which were added embryonic
and postnatal tissue that consisted of the caudal half of embryonic
brains and cerebellar tissue blocks from postnatal brains. Standards
and unknowns, before assay, were thawed, and microfuged for 5 min to
remove precipitate. On the day of assay, standards and unknowns were
neutralized and diluted (1.5x) in 1 M phosphate
buffer to a final pH of 6.2–6.4 before use in the ELISA.
For extraction of the explants, the coverslips on which the explants were maintained were first inverted into Teflon chambers containing 75 µl droplets of HCL/CPI. The chambers were then placed on dry ice until the fluid containing the explant cultures was frozen. The frozen droplets containing the cultures were then removed from the coverslips and placed into 1.7 ml siliconized microtubes, in groups of 2, on ice until the droplets thawed. Next, the cultures were homogenized for 10 sec then centrifuged for 10 min at 4 C, and the supernatants were collected, lyophilized to 75 µl, frozen on dry ice, and stored at -80 C until assay. Nasal tissue trimmed from the explants during culture preparation was used for preparation of LHRH standards. On the day of assay, standards and unknowns were neutralized and diluted (1.5x) in 1 M phosphate buffer to a final pH of 6.2–6.4 before use in the ELISA.
Analysis of LHRH secretion from explants
On culture day 7, the coverslips on which the explants were
maintained were inverted onto Teflon chambers containing 50 µl
droplets of SFM alone or SFM with 40 mM KCl. The explants
were placed on a slide warmer (39 C) and remained inverted in the media
droplets for 15 min. The coverslips containing the explants were then
moved to Teflon chambers containing 75 µl droplets of HCl/CPI and
processed for extraction as previously described. The chambers
containing the SFM droplets were moved onto dry ice until the droplets
were frozen. The frozen droplets were then transferred into 1.7 ml
siliconized microtubes on ice where they were pooled, 10 secretion
droplets per tube. The pooled droplets were then stored at -80 C until
assay. LHRH standards were also produced in SFM for use in the ELISA of
the explant secretion samples. On the day of assay, the standards and
samples were lyophilized and reconstituted with 66.6 µl of sterile
water and 33.4 µl 1 M phosphate buffer for assay in
duplicate. Changes in secretion due to altered osmolarity of the media
were tested for by application of 40 mM NaCl in the same
manner as KCl application.
ELISA
The ELISA was performed as previously described
(14). Briefly, microwell plates were coated with 100 µl
goat antirabbit IgG (1:500, overnight at 4 C) in 563 mM
NaCO3 and 215 mM
Na2CO3, pH 9.6. The
following day all procedures were performed at room temperature. After
washing (0.2% Tween 20 in PBS, pH 7.4), 50 µl affinity purified
anti-LHRH (1:600) in standard diluent (0.1% BSA in PBS, pH 7.4) was
incubated in the wells for 2 h. After washing, 50 µl of
neutralized and diluted standards and unknowns were incubated in wells
for 2 h. Then, 50 µl of biotinylated-LHRH (1:1,000,000) was
added to all wells and allowed to compete for anti-LHRH binding sites
for 30 min. Wells were washed and 100 µl streptavidin-alkaline
phosphatase (1:1000) was added for 30 min. After washing, 100 µl
p-nitrophenyl phosphate (1 mg/ml) in 1
mM MgCl2, 16.2
mM NaHCO3, and 17
mM
Na2CO3, pH 10, was
aliquoted into each well. The samples were allowed to react for 40–60
min in the dark, and read at 405 nm in a MRX Microplate Reader
(Dynatech Corp., Chantilly, VA). All samples were measured
in duplicate or triplicate. Unknowns were calculated from standards
plotted as a sigmoidal concentration-response curve by a nonlinear
least squares fit (Revelation Software version 2, Dynatech Corp.). The minimal and maximal detectable amounts were 10 and
1000 pg/well, respectively.
Calcium imaging
At 6–9 div, explant cultures were exposed to the calcium
indicator Calcium Green-1 AM (Molecular Probes, Inc.
Eugene, OR) for 20 min in a CO2 humidified
incubator. The dye was diluted to 2.7 mM concentration in
80% DMSO/20% pluronic F-127 solution. This solution was diluted 1:200
with SFM to a final Calcium Green concentration of 13.5
µM. The coverslips containing the explants were then
washed with media twice, 10 min each, and loaded into a heated
perfusion chamber (Warner Instruments, Hamden CT). Medium was perfused
across the cultures at a rate of approximately 100 µl/min using a
variable speed peristaltic pump (Spectra Hardware Inc., Westmoreland
City, PA). The medium was oxygenated by effervescence of the solution
at the delivery point of the perfusion chamber. Temperature control was
accomplished using a voltage regulator that controlled the temperature
of an in line heater, used to warm the medium, as well as heating the
lower stage of the perfusion chamber. Calcium Green was visualized
using an inverted Nikon microscope equipped with a 20x
fluorescence objective and an ICCD camera (Video Scope International,
Sterling VA) linked with a Power Macintosh 7300 series computer
equipped with imaging software (Ip Lab Spectrum, Signal Analytics
Corp., Vienna, VA). Excitation wavelengths were 450–490 and emission
was monitored at 520–560 nm.
Immunocytochemistry
Cultures on coverslips were fixed with 4% formaldehyde in PBS
for 1 h. After fixation, samples were washed with PBS, incubated
for 1 h in 10% NGS/0.3% Triton X-100, washed several times in
PBS, and incubated in LHRH antibody (1:2500, SW1; 12) in PBS overnight
at 4 C. The next day, the cultures were washed several times in PBS and
incubated in biotinylated secondary antibody (1:500) in PBS with 0.3%
Triton X-100 for 1 h. The cultures were washed with PBS several
times and processed for avidin-biotin-horseradish
peroxidase/3'3-diaminobenzidine (DAB) histochemistry as described
previously (12). LHRH neurons in 45 cultures were
quantified, and an average number per culture was determined. This
value was used for data normalization in subsequent analyses. For
identification of LHRH neurons after calcium imaging, a goat antirabbit
antibody conjugated to Texas red fluorophore (1:200, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was used as a
directly conjugated secondary antibody.
Statistical comparisons of data were calculated with the statistical software GB-STAT version 6.5 (Dynamic Microsystems, Inc., Silver Spring, MD). One-way ANOVA was used for statistical comparisons of the data. A P value less than 0.05 was considered statistically significant.
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800)
are detectable within nasal regions as early as day E12 in the
embryonic mouse (15). Measurement of LHRH content within
extracts of whole mouse cranium revealed a developmental increase in
the total amount of LHRH peptide (Fig 1
).
LHRH neurons in E12.5-staged embryos express low levels of LHRH. A
significant increase in total LHRH content was observed between E14.5
and birth and continued through postnatal day 8 and into adulthood.
Total LHRH peptide content in adult mice was determined to be 2020
± 375 pg per animal, consistent with that previously reported
(16, 17). Thus, during prenatal development there is over
a 2-fold increase in the amount of LHRH detected between E12.5 (LHRH
content = 89.7 ± 27 pg) and PN 1 (LHRH content =
238.6 ± 68.4 pg). Postnatally, the amount of detected LHRH
peptide continues to increase, rising approximately 10-fold from PN1 to
adulthood.
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is a
photomicrograph of a representative nasal explant immunocytochemically
labeled for LHRH. These explant cultures contain a mean of 166.9
± 19.34 (n = 45 cultures) LHRH immunoreactive cells,
approximately 20% of the total number of LHRH cells within the
NIH-Swiss mouse brain (15). If one takes the number of
LHRH cells per culture into account, then the amount of LHRH
peptide per cell in 7 and 10 div cultures is 0.16 ± 0.05 and
0.25 ± 0.12 pg, respectively. These values are greater than that
observed in vivo at E12.5 but similar to those detected at
PN1 (Fig. 3). These results demonstrate
that LHRH neurons within nasal explants undergo maturational changes in
LHRH content similar to those seen in vivo.
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LHRH content.To assess the effect of static incubations on
LHRH stores, the nasal explants themselves were measured for LHRH
content following their respective secretion paradigms. Fifteen-minute
treatment of nasal explants with medium containing a 40 mM
increase in KCl resulted in a significant increase in LHRH content
compared with explants treated with SFM alone (Fig. 4). To determine whether this phenomenon
was due to the length of time that the explant was exposed to KCl,
cultures were quickly dipped into KCl supplemented SFM and then moved
into SFM alone for 15 min. Rapid exposure to KCl produced the same
increase in LHRH peptide as seen in the 15-min exposure paradigm,
indicating that rapid peptide synthesis is initiated upon
depolarization.
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, A and B). Similar
ionic perturbations were performed as described above while LHRH cells
loaded with the dye were monitored. As expected, KCl stimulation for 15
min resulted in a dramatic rise in cytosolic calcium in LHRH neurons
(Fig. 5, C–E). The sharp rise in intracellular free calcium was
followed by a gradual decline that persisted for 8–10 min. To evaluate
the capacity of calcium imaging techniques for monitoring paradigms
known to attenuate LHRH secretion in nasal cultures (10),
we exposed the nasal explants to a calcium-depleted medium. The initial
response by LHRH neurons to a depletion of extracellular calcium is a
rapid rise in intracellular calcium levels that returns to baseline
levels within five minutes (Fig. 6A).
However, once the values returned to baseline, the LHRH neurons
appeared to be quiescent, i.e. the neurons no longer
exhibited the spontaneous spikes of increased intracellular calcium
observed in basal, SFM conditions (Fig. 6B).
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Cultures exposed to
10-4 M
picrotoxin for 24 h exhibited a significant decrease in LHRH
content after a 15-min static incubation in fresh medium containing
picrotoxin (Fig. 7A). We further tested
the stimulatory role of GABA by applying the GABA agonist
muscimol to cultures. Fifteen-minute static incubations of cultures, in
10-4 M
muscimol, resulted in a significant increase in explant LHRH peptide
content (Fig. 7A). We next examined the calcium response of LHRH
neurons to the same 15-min exposure to muscimol. Perfusion of medium
containing 10-4
M muscimol resulted in a sharp and sustained rise in the
basal levels of intracellular free calcium in LHRH neurons (Fig. 7B).
The levels of intracellular calcium remained elevated and only
decreased after muscimol was flushed from the perfusion chamber.
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). However, the
LHRH peptide increase in response to muscimol was greatly attenuated
(P = 0.4) in the presence of TTX. Subsequent
examination of calcium mobilization in LHRH neurons revealed that TTX
partially blocked the calcium response to muscimol (Fig. 8B, note
response in no longer prolonged, see Fig. 7B
). In contrast, the robust
mobilization of calcium in response to increased extracellular KCl
remained intact despite the blockage of sodium conductivity with TTX
(Fig. 8D). Consistent with this finding, the magnitude of change in
LHRH peptide content in response to KCl in the presence of TTX was
proportional to that observed in SFM-treated cultures (data not
shown).
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). The stimulatory effect of muscimol
on LHRH synthesis was significantly attenuated in the absence of
extracellular calcium (Fig. 9C); however, the response was not
completely blocked. Subsequent monitoring of intracellular calcium
levels in LHRH neurons in response to depolarizing stimuli in the
absence of extracellular calcium revealed that the KCl response was
blocked (Fig. 9B), whereas muscimol still stimulated a rise in
intracellular free calcium (Fig. 9D). The rise was similar in magnitude
compared with muscimol stimulation in SFM conditions. Calcium levels
appeared to remain elevated for approximately 7 min. However, an
obvious return to baseline was not apparent as was observed in cells
exposed to muscimol in SFM alone. Instead, a rebound effect was
observed after infusion of a normal, calcium-enriched medium under both
conditions.
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).
Thirty-minute pretreatment of cultures with DRB had no effect on either
KCl or muscimol stimulation of LHRH peptide accumulation (Fig. 10, B
and C, respectively). However, pretreatment with cycloheximide
abolished the stimulatory effect of KCl (Fig. 10B) or muscimol (Fig. 10C) on LHRH content within explant cultures during 15-min static
incubations.
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Developmental increase in LHRH peptide content
In vivo LHRH levels were measured as early as E12.5,
the first embryonic stage in the mouse when the full compliment
(800; Ref. 21) of neuroendocrine LHRH cells can be
detected. The LHRH content was 89.7 ± 27 pg. By PN1, the LHRH
content had increased over 2-fold, now measuring 238.6 ± 68.4 pg.
Total LHRH peptide content in adult mice was determined to be 2020
± 375 pg., over a 20-fold increase from that measured at E12.5. At all
of these ages, approximately 800 LHRH cells are present in the mouse;
thus, the LHRH content per cell (assuming equal production) would be
0.11 pg at E12.5, 0.30 pg at PN1, and 2.53 pg in the adult. In
vitro, the amount of LHRH peptide per cell in 7 and 10 div
cultures was 0.16 ± 0.05 and 0.25 ± 0.12 pg, respectively.
These values are greater than that observed in vivo at E12.5
and by 10 div similar to those detected at PN1. Thus, LHRH neurons
within nasal explants undergo maturational changes in LHRH content
similar to those seen in vivo. The nasal explants are
removed at E11.5. NIH-Swiss mice normally give birth at E18.5,
i.e. 7 days later. At 7 div, LHRH content/cell was not
equivalent to PN1 values detected in vivo. However, by 10
div levels were similar. The apparent maturational delay in LHRH
content/cell in vitro may be due to an initial slowing of
normal processes when the explants are made. The mechanism(s)
underlying the increase in LHRH content could be regulated at the
transcriptional, translational, and/or peptide storage/decay level.
Copy levels of LHRH mRNA have been found to be remarkably similar in
LHRH cells in PN1 rats (22), adult rats (23),
and nasal explants (24). In all three cases, LHRH was
found to be a high copy number message, suggesting that the
developmental rise in LHRH content is not regulated at the
transcription level of peptide processing. Further investigations are
necessary to examine the relative input of translational and peptide
decay mechanisms on overall LHRH peptide levels as a function of
age.
Secretion of LHRH peptide
Our investigations revealed spontaneous, basal release of LHRH
peptide from cells maintained in serum-free conditions for only 6–7
div. In addition, these LHRH neurons responded to depolarizing
stimulation by secreting significantly more LHRH. Attempts were made to
directly measure patterns of LHRH secretion in the explants. However,
because the total content of LHRH at 7 and 10 div was 25.52 ±
8.52 pg and 40.1 ± 19 pg, respectively, even if 10% of the total
content was released, only 2–4 pg would be secreted. Such values were
below the lower limit of the assay sensitivity and therefore basal
secretion was measured by combining the collected media from several
explant preparations. Because secretion patterns would be lost in this
grouping procedure, we chose an alternative approach, calcium imaging,
to analyze the dynamics of LHRH secretion.
It is known that LHRH secretion requires Ca2+ entry through voltage-gated Ca2+ channels (10). Depolarization of GT1 cells has also been shown to induce a hundred-fold increase in intracellular free calcium (25, 26, 27) as well as a 2-fold increase in LHRH secretion (26). Therefore, we used calcium-imaging techniques to monitor secretion-related calcium mobilization events. LHRH cells showed a dramatic increase in calcium in response to KCl, the same depolarizing stimulation that caused a significant increase in LHRH peptide release from the cultures. We also observed a significant decline in activity in the calcium-free condition, a condition that is known to inhibit LHRH secretion (10). Thus, there was a direct correlation between the changes in calcium mobilization observed in these studies and known affects on LHRH secretion.
In vivo, GABAergic neurons are present in the olfactory pit at a time when LHRH neurons are migrating toward the forebrain (18). Previously it was shown that GABAergic neurons are present in mouse nasal explants and that the LHRH neurons in these cultures contain functional GABAA receptors (9, 18). Electrophysiological examinations of LHRH neurons within these cultures revealed that stimulation of GABAA receptors resulted in membrane depolarization. Consistent with these findings, exposure of explants to muscimol resulted in a dramatic increase in cytosolic calcium within LHRH neurons. Certainly there is evidence that GABA is inhibitory to LHRH secretion in the adult brain (28). However, an excitatory role for GABA has also been reported (28). Thus, it is unclear whether LHRH neurons within nasal explants express their mature complement of receptor types and/or whether there is a shift in the chloride potential. Further investigations are necessary to address these issues, and examination of LHRH neurons maintained in explants for longer time periods may be informative. In primate olfactory explants, there is a significant decline in LHRH secretion after 21 div (7). This decline could represent a switch in LHRH neuron responsiveness to GABAergic signaling.
Restoration of peptide content by protein
synthesis-dependent/transcription-independent mechanisms
This investigation also evaluated the effects of acute
depolarization on stored levels of LHRH peptide. After KCl
depolarization, it was expected that a decline in total amount of LHRH
peptide in the explants would be measured due to release of peptide
into the media. However, the opposite effect was observed: the total
amount of LHRH peptide in the explant increased dramatically. This
result illustrates an important aspect of systems in which robust
secretion occurs in a pulsatile pattern: replenishment of stores.
Because the interval between pulses of LHRH release is relatively short
(20–40 min; Refs. 6, 29), the neurons must be capable of
rapid restoration of releasable pools of LHRH. Thus, we hypothesize
that the increase in LHRH content in the explant observed after
depolarization is an intrinsic mechanism of LHRH cells to ensure a
continuous supply of releasable peptide.
In our experimental paradigms, the amplitude of the increase in total LHRH content was probably an exaggerated response due to the supraphysiological level of depolarization. Supporting this idea is the fact that proportional increases in LHRH peptide were observed with treatments with KCl or muscimol alone, KCl after 24-h TTX, and muscimol after 24-h picrotoxin, possibly representing a maximal stimulatory response. However, the rapid rate of the increase in LHRH peptide levels under all conditions suggests the existence of compensatory mechanisms for immediate restoration of peptide pools for a subsequent secretory event.
Consistent with requirements for rapid secretion-coupled peptide synthesis, it has been previously shown that LHRH mRNA is a high copy number message (22) and that LHRH mRNA stability can be physiologically regulated (30). Therefore, the materials necessary for protein synthesis are abundant and it may not be necessary for LHRH neurons to perform de novo transcription of LHRH mRNA. We tested this hypothesis and found that inhibiting transcription of mRNA did not affect the rapid synthesis of LHRH peptide while inhibiting protein synthesis did. Therefore, it appears that rapid secretion-coupled peptide synthesis in LHRH neurons is a transcription-independent and translation-dependent process. Interestingly, chronic GABAergic signaling has previously been shown to significantly decrease LHRH mRNA within nasal explants (31). This result may, in part, be the result of a compensatory mechanism used by the neurons to down-regulate peptide synthesis in response to long-term exposure to a depolarizing agent.
Although depolarization of LHRH neurons with GABA and KCl resulted in LHRH secretion, the effects of these agents on LHRH peptide synthesis appear to be the result of different yet partially overlapping pathways. Both paradigms caused depolarization and an immediate and significant rise in intracellular calcium in LHRH neurons. However, whereas KCl induced a rapid rise in intracellular calcium followed by a gradual return to baseline levels, GABAergic signaling induced a sustained rise in intracellular calcium that was lower than the initial response to exposure, yet persisted until removal of the stimulating agent. Furthermore, depletion of extracellular calcium did not block the initial calcium response to GABA and only attenuated the effect on LHRH peptide synthesis. In contrast, depleting the extracellular calcium concentration completely blocked the KCl affect on intracellular free calcium and LHRH peptide synthesis. Previously, muscimol has been shown to influence protein synthesis-dependent events, such as long-term depression in hippocampal neurons (32) in the absence of extracellular calcium. In a similar vein, we propose that GABAergic signaling, in part, uses intracellular signaling cascades to stimulate LHRH peptide synthesis.
Extracellular calcium is required for muscimol-stimulated depolarization in GT1 cells (25). In addition, it is widely accepted that extracellular calcium is pivotal for secretion at nerve terminals and that removal of extracellular calcium attenuates secretion events in LHRH neurons (5, 10, 33). The portion of the peptide accumulation response lost in the calcium depleted media paradigms may therefore be correlated to a loss of the secretory response. In support of this theory, we observed complete inhibition of KCl stimulated LHRH accumulation and increased cytosolic free calcium response in calcium-depleted media. The lost signaling mechanism appears to be a component of secretion-coupled peptide synthesis. This mechanism does not require a sustained depolarization with KCl as depolarization with short KCl exposure produced the same increase in LHRH peptide accumulation.
To further dissect the mechanism by which depolarization caused rapid protein synthesis, membrane propagation was blocked with the sodium channel blocker tetrodotoxin. These experiments clearly showed that both peptide synthesis and calcium responses to KCl were not dependent on electrochemical signal propagation in LHRH neurons. These data correlate with recent investigations that revealed that TTX was unable to block KCl stimulated LHRH secretion in primate nasal cultures (10). It also demonstrates that the mechanisms for secretion-coupled peptide synthesis are not dependent upon electrochemical signal propagation. In contrast, TTX did significantly, though not entirely, attenuate peptide synthesis and calcium responses to GABAergic signaling. A similar, partial response to GABA in the presence of TTX was seen in GT1 cells (25). These data suggest either dual receptor sites for GABA on LHRH neurons, one at the terminals influencing LHRH secretion and one upstream that is dependent on membrane depolarization for signaling and/or the presence of GABAergic interneurons connecting to LHRH neurons. To date, GABAergic neurons have been identified in nasal cultures, and GABAA receptors have been shown to be present on/proximal to LHRH cell soma (18). Certainly, further investigations in which secretion of LHRH peptide is blocked by methods not affecting ionic signaling are necessary to substantiate the secretion-coupled peptide synthesis hypothesis and dissect out the molecules/signals that communicate these events, and thereby ensure that LHRH cells continually possess a releasable pool of LHRH peptide.
In summary, this investigation has shown that primary LHRH neurons undergo developmental maturation in nasal explants. Independent of cues from the CNS and devoid of serum supplements, LHRH neurons have the capacity to synthesize and secrete LHRH peptide after only 7 div corresponding to embryonic day 18.5. Furthermore, embryonic LHRH neurons are influenced by GABAergic signaling and display a biphasic increase in intracellular calcium mobilization as well as an increase in LHRH peptide synthesis in response to an exogenous GABA agonist. In addition, LHRH neurons were shown to possess the capacity for transcription-independent, rapid (15 min) peptide synthesis in response to depolarizing stimuli. These data indicate that mechanisms necessary for neuroendocrine secretory profiles are inherent in LHRH neurons and become functional during embryonic development.
Received June 8, 2000.
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J. L. Temple and S. Wray Bovine Serum Albumin-Estrogen Compounds Differentially Alter Gonadotropin-Releasing Hormone-1 Neuronal Activity Endocrinology, February 1, 2005; 146(2): 558 - 563. [Abstract] [Full Text] [PDF]
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J. L. Temple, E. Laing, A. Sunder, and S. Wray Direct Action of Estradiol on Gonadotropin-Releasing Hormone-1 Neuronal Activity via a Transcription-Dependent Mechanism J. Neurosci., July 14, 2004; 24(28): 6326 - 6333. [Abstract] [Full Text] [PDF]
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J. P. Moore Jr, E. Shang, and S. Wray In Situ GABAergic Modulation of Synchronous Gonadotropin Releasing Hormone-1 Neuronal Activity J. Neurosci., October 15, 2002; 22(20): 8932 - 8941. [Abstract] [Full Text] [PDF]
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N. Sharifi, A. E. Reuss, and S. Wray Prenatal LHRH Neurons in Nasal Explant Cultures Express Estrogen Receptor {beta} Transcript Endocrinology, July 1, 2002; 143(7): 2503 - 2507. [Abstract] [Full Text] [PDF]
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