Luteinizing Hormone-Releasing Hormone (LHRH) Biosynthesis and ...

0013-7227/00/$03.00/0 Endocrinology Copyright © 2000 by The Endocrine Society

Vol. 141, No. 12 Printed in U.S.A.

Luteinizing Hormone-Releasing Hormone (LHRH) Biosynthesis and Secretion in Embryonic LHRH Neurons J. P. MOORE, JR.

AND

S. WRAY

Cellular and Developmental Neurobiology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892 ABSTRACT Evidence indicates that LH-releasing hormone (LHRH) neurons can exhibit neuroendocrine secretory properties before entrance into the central nervous system. In this study, we evaluated LHRH biosynthesis and secretion in embryonic LHRH neurons maintained in nasal explants. Using ELISA and calcium imaging techniques, peptide content and single neuron activities were examined. LHRH neurons maintained for 7–10 days in vitro were found to possess a similar amount of LHRH/cell as the equivalent aged LHRH cells in vivo (postnatal day 1). LHRH peptide was measured in the medium of these relatively young cultures, and 40 mM KCl stimulated a 4-fold increase in LHRH secretion. KCl enhanced medium also resulted in

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HE LH-RELEASING hormone (LHRH) neuronal system is essential for initiation and maintenance of reproductive function in vertebrates. Numbering from 800 cells in mice (1) to 2000 in primates (2), LHRH neurons are primarily localized within the septal-preoptic nuclei and hypothalamus. The predominant termination point of LHRH neurons are fenestrated capillaries of the hypothalmo-pituitary portal circulation system. Pulsatile release of LHRH into the portal circulation of the pituitary stalk affects the synthesis and secretion of gonadotropins and consequently activation of gonadal function (3, 4). The mechanisms that precisely regulate LHRH secretion are poorly defined. 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 Received June 8, 2000. 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: [email protected].

a significant increase in LHRH content per culture (24.5 pg vs. 32.3). A similar effect was observed after muscimol-enhanced media (32.2 pg). Both agents also stimulated a substantial rise in intracellular calcium. Pretreatment of cultures with tetrodotoxin partially blocked the affects of muscimol on both peptide content and calcium activity, but not KCl. Calcium-depleted medium blocked the effects of KCl yet only attenuated the effects of muscimol. Treatment of cultures with cycloheximide blocked the effects of both muscimol and KCl. These results indicate that developing LHRH neurons are capable of synthesizing, secreting, and rapidly replenishing stores of LHRH peptide. (Endocrinology 141: 4486 – 4495, 2000)

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 noncentral 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). Materials and Methods Materials For tissue preparation and culturing, d-glucose, apo-transferrin, putrescine, sodium selenite, bovine insulin, l-ascorbic acid and fluorodeoxyuridine (FuDR) and thrombin were purchased from Sigma (St. Louis, MO). The supplier of Gey’s Balanced Salt Solution, Eagle Basal Medium,

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LHRH BIOSYNTHESIS AND SECRETION Ham’s F-12 Nutrient Mixture, l-glutamine, and PSN Antibiotic Mixture was Life Technologies, Inc. (Grand Island, NY). BSA and chicken plasma were purchased from Roche Molecular Biochemicals (Indianapolis, IN) and Cocalico Biologicals, Inc. (Reamstown, PA), respectively. Complete protease inhibitor cocktail mini-tablets (CPI) were purchased from Roche Molecular Biochemicals (Indianapolis, IN). 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 NIHSwiss 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 ⫻ 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 ␮l 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 (1.5⫻) 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 ␮l 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 ␮l, 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 (1.5⫻) 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 ␮l 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.

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The coverslips containing the explants were then moved to Teflon chambers containing 75 ␮l 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 ␮l of sterile water and 33.4 ␮l 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 ␮l 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 ␮l 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 ␮l of neutralized and diluted standards and unknowns were incubated in wells for 2 h. Then, 50 ␮l 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 ␮l streptavidin-alkaline phosphatase (1:1000) was added for 30 min. After washing, 100 ␮l 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 ␮m. 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 ␮l/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 20⫻ 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 anal-

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yses. 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.

Results Increase of LHRH content in early mouse development

Previous investigations have demonstrated that all of the neuroendocrine LHRH neurons present in the adult mouse brain (⬃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. Increase of LHRH content within nasal explants

To determine whether LHRH neurons in vitro undergo similar developmental changes observed in vivo, LHRH content within nasal explants was measured. Previously, it was shown that LHRH neurons, within mouse nasal explant cultures process the LHRH prohormone and form varicosities

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containing LHRH immunoreactive product (8). At 7 div, nasal explant cultures contain 25.52 ⫾ 8.52 pg LHRH (n ⫽ 41). After 10 div, the explants contain a significantly greater amount of LHRH peptide (40.1 ⫾ 19 pg, n ⫽ 10). Figure 2 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. Depolarization of nasal cultures

LHRH secretion. Recent work has shown that LHRH cells maintained for several weeks in nasal explants exhibit pulsatile-like release of LHRH (10, 11). To determine whether LHRH neurons maintained in SFM for only 6 –7 div (potentially equivalent to in vivo neurons at PN1) could release LHRH, media from nasal explants were measured for the presence of LHRH using an ELISA. Static incubation of nasal explants (n ⫽ 8 groups of 10 cultures) for 15 min resulted in the release of 3.31 pg (44.13 pg/ml) of LHRH into the incubation medium. Basal release of LHRH was inhibited by static incubation in calcium-free medium, with LHRH levels falling below the limit of detection. Incubation of the nasal explants in media containing an additional 40 mm concentration of KCl resulted in a 2.5-fold increase in LHRH release into the incubation medium compared with SFM alone (n ⫽ 5 groups of 10 cultures). Addition of 40 mm NaCl to SFM had no effect on the basal release of LHRH from the nasal explants during static incubation. These results indicate that as early as 7 div and in the absence of serum-derived factors, LHRH neurons are capable of releasing LHRH peptide. 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.

FIG. 1. LHRH peptide content undergoes developmental increases during pre- and postnatal development. Total LHRH peptide content in embryonic, early postnatal, and adult mice were determined by ELISA and are expressed as pg/animal. Each value represents the mean ⫾ SEM of 10 –18 animals/age. *, Significantly (P ⬍ 0.05) greater than previous age group.

Calcium imaging of LHRH neuronal activity

To further evaluate the mechanisms underlying changes in LHRH secretion, calcium imaging was employed. The majority of cells that load with Calcium Green dye within the nasal explants are LHRH immunopositive (Fig. 5, A and B).


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FIG. 2. Large numbers of LHRH cells are present in nasal explants. Photomicrograph of a nasal explant immunocytochemically labeled for LHRH, after 7 div. Numerous LHRH-positive perikarya and fibers were visualized within and outside the main nasal tissue. On average, 20% of the total LHRH population is accounted for in nasal explants at 7 div [166.9 ⫾ 19.34 LHRH immunoreactive cells (n ⫽ 45 cultures)]. Arrowheads indicate LHRH neurons located along the midline nasal cartilage, and as they exit the main nasal tissue mass onto the surrounding milieu. Arrows indicate LHRH neurons in the periphery of the explant. Bar, 1 mm.

FIG. 3. LHRH neurons in vitro undergo similar changes in LHRH peptide content as observed in vivo. Total LHRH content in cultures (n ⫽ 10 – 40, hatched bars) and pre- and postnatal mice (n ⫽ 10 –18/ age, solid bars) were determined by ELISA. In vitro values were divided by the average number of immunopositive LHRH neurons as determined from 45 immunocytochemically stained cultures. In vivo values were divided by 800. Each value is expressed as the mean ⫾ SEM. *, Significantly greater than preceding chronological group. 22, Significantly less than 7 and 10 div. 2, Significantly less than 10 div.

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 neu-

FIG. 4. Depolarization induces rapid increases in LHRH peptide content. Explants were treated in a static incubation for 15 min in SFM alone or SFM ⫹ 40 mM KCl. A, Explant content (mean pg LHRH ⫾ SEM) of 50 and 40 groups, respectively. Each group contained two nasal explants. *, Significantly greater than control value. The photomicrographs are of LHRH cells following 15-minute incubations in SFM (B) and KCl-enhanced medium (C). Bar, 10 microns.

rons 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). GABAergic signaling stimulates LHRH peptide accumulation in nasal explant cultures

To determine whether the rapid de novo protein synthesis of LHRH observed in nasal explants after KCl stimulation mimics an in situ depolarizing neuronal interaction, we perturbed a synaptic interaction known to exist in these cultures.

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FIG. 5. Depolarization increases cytosolic free calcium in LHRH neurons. A–D, Digitized images of neurons in nasal explants. A, Neurons loaded with Calcium Green. B, Fluorescent immunolabeling of LHRH cells in same region as A. Arrows in panels A and B indicate LHRH positive neurons that labeled with Calcium Green dye. Arrowhead in panels A and B indicate a non-LHRH neuron that loaded with the dye. Boxed area in panel A is magnified area in panels C and D. LHRH neurons loaded with Calcium Green before (C) and during (D) depolarization with 40 mM KCl. E, Trace of Calcium Green fluorescent intensity in LHRH neurons before, during, and after a 15-minute exposure to SFM with an addition of 40 mM KCl. The data in panel E is the mean ⫾ SEM of 3 cultures, 8 LHRH neurons/culture.

FIG. 6. Depletion of extracellular calcium induces a transient rise in cytosolic free-calcium in LHRH neurons and decreases spontaneous activity. Nasal explants were monitored by calcium imaging techniques in SFM for 15 min followed by infusion of a calcium-depleted SFM for 15 min. A, Mean ⫾ SEM of the intracellular calcium-fluorescence recordings of 10 LHRH neurons. B, Intracellular calcium-fluorescence of three of the individual LHRH neurons that were averaged in 6A. The traces in panel B have been stacked for aid in visualization. Under these conditions, there is an initial, transient increase in intracellular calcium levels, followed by basal levels accompanied by a dramatic decrease in the spontaneous fluctuations observed (arrows) before depletion of calcium within the medium.

GABAergic neurons have been identified within nasal explants (18), and a robust GABAergic synaptic input to LHRH neurons previously recorded (9). In contrast to its inhibitory role in the adult CNS, GABA can be an excitatory signal that depolarizes receptive neurons during development (19, 20). Such depolarization by GABA of LHRH cells via GABAA receptors was observed in nasal explants maintained for 7 div (9). To evaluate a possible influence of GABAergic signaling on LHRH production, cultures were treated with the GABAA receptor antagonist picrotoxin. 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 incu-

bations 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. GABAergic stimulation of LHRH peptide synthesis in nasal explants is activity dependent

To examine the influence of spontaneous and synaptic activity on changes in nasal explant LHRH content in response to stimulation, explant cultures were treated with


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FIG. 7. GABAergic signaling stimulates rapid LHRH peptide synthesis and calcium mobilization in LHRH neurons. Explants were treated for 24 h, with SFM alone or SFM containing 10⫺4 M picrotoxin. Afterward, 15-min static incubations were performed. The 24 h SFM group was incubated in SFM alone or SFM ⫹ 10⫺4 M muscimol, whereas the explants exposed to picrotoxin for 24 h were incubated in SFM ⫹ 10⫺4 M picrotoxin. * Significantly less than SFM. ** Significantly greater than SFM. Values in A ⫽ mean ⫾ SEM. B, Calcium Green fluorescent intensity trace in LHRH neurons before, during, and after exposure to SFM ⫹ 10⫺4 M muscimol. The data in B ⫽ mean ⫾ SEM of 3 cultures, 8 LHRH neurons/culture. Note that the change in calcium signal in response to muscimol is a rapid, relatively large increase followed by a partial decline that maintains calcium levels above those observed in SFM alone, for the extent of muscimol exposure.

F IG . 8. GABAergic modulation of LHRH content is partially activity dependent. Panels A and C show the effects of 10⫺6 M tetrodotoxin on peptide synthesis in response to GABAergic and potassium stimulation. LHRH content in A and C ⫽ mean ⫾ SEM of 20 –50 groups of two cultures. *, Significantly greater than control value. B and D, Calcium Green fluorescent intensity traces from LHRH neurons in SFM containing 10⫺6 M TTX, before, during, and after a 15-min exposure to SFM with an additional 10⫺4 M muscimol or 40 mM KCl. Data in B and D ⫽ mean ⫾ SEM of 3 cultures, 8 LHRH neurons/ culture. Although an initial response to muscimol was detected, the prolonged effect was abolished (see Fig. 7).

10⫺6 m TTX 24 h before static incubations. Fifteen-minute static incubations were then performed in SFM containing 10⫺6 m TTX alone, TTX with additional 40 mm KCL, or TTX with 10⫺4 m muscimol. The 24-h exposure to TTX significantly (P ⬍ 0.01) decreased (22.07 ⫾ 8.347 vs. 17.36 ⫾ 8.87) the explant LHRH peptide content (n ⫽ 52) compared with explants incubated in SFM alone (n ⫽ 45). In the presence of TTX, 40 mm KCl still induced a significant (P ⬍ 0.01), and rapid increase in LHRH peptide content in nasal explants (Fig. 8). 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). Requirement of extracellular calcium for stimulated rapid LHRH synthesis

It is known that depletion of extracellular calcium attenuates the secretion of LHRH peptide in nasal cultures (10). To determine whether extracellular calcium is also required for the effect of depolarization on LHRH synthesis, we measured LHRH levels in nasal explants after KCl or muscimol exposure in a calcium-depleted medium. Depletion of extracellular calcium completely blocked the stimulatory effect of KCl on LHRH peptide synthesis (Fig. 9A). 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),

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FIG. 9. Extracellular calcium is not required for rapid peptide synthesis in response to GABAergic signaling. Effects of depleted extracellular calcium on peptide synthesis in response to potassium (A) and GABAergic (C) stimulation. LHRH content in A and C ⫽ mean ⫾ SEM of 8 –12 groups of two cultures. *, Significantly greater than control value. The effect of muscimol in low calcium is not significantly greater than control, but it is also not significantly different from SFM⫹M. Thus, although attenuated, low calcium did not exclude a partial response to muscimol. B and D, Calcium Green fluorescent intensity traces of LHRH neurons, in calcium-free SFM, before, during, and after a 15-min exposure to SFM with an additional 40 mM KCl or 10⫺4 M muscimol. Data in panels B and D ⫽ mean ⫾ SEM of 3 cultures, 8 LHRH neurons/culture. In contrast to KCl, a response to muscimol was observed during low calcium perfusion, consistent with the partial effect observed in LHRH peptide synthesis under the same conditions.

FIG. 10. Stimulation of rapid increases in LHRH content is a transcription-independent translation-dependent event. A, LHRH content in nasal explants with or without 30 min pretreatment and 15 min static incubations in 150 ␮M DRB or 35 ␮M cycloheximide. No changes were detected. Effect on LHRH peptide content of 15 min static incubation with 40 mM KCl (B) or 10⫺4 M muscimol (C) with or without a 30-min pretreatment and 15 min cotreatment with DRB or cycloheximide. Cyclohexamide inhibited increases in LHRH peptide content under both conditions. Values in all panels ⫽ mean ⫾ SEM of 10 –50 groups of two cultures. *, Significantly less than SFM ⫹ stimulant.

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. Stimulation of rapid increases in LHRH content is a translation-dependent and transcription-independent event

To further evaluate the effect(s) of depolarization on LHRH content, messenger RNA (mRNA) transcription or protein synthesis was blocked before and during KCl or

muscimol exposure. Treatment with the classic transcription inhibitor DRB (150 ␮m) or the protein synthesis inhibitor cycloheximide (35 ␮m) had no effect on LHRH peptide content when not paired with a depolarizing stimulus (Fig. 10A). 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. Discussion

The present studies evaluated the developmental capacity of primary LHRH neurons to synthesize and secrete LHRH


peptide. We report here that LHRH neurons maintained in serum-free conditions within nasal explants undergo an appropriate developmental increase in LHRH peptide content, secrete LHRH peptide under basal conditions, increase secreted peptide after stimulation, and show rapid restoration of peptide content by protein synthesis-dependent/transcription-independent mechanisms. These data, together with the observations that at 7 div LHRH neurons exhibit spontaneous electrical activity, possess highly differentiated electrophysiological properties as well as voltage-and ligand-gated ion channels (9), strengthen the argument that LHRH neurons become functionally mature during embryonic development and that cues from the developing brain and periphery are not essential for this aspect of LHRH neuronal development. 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

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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 (25–27) 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 synthesisdependent/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 in-

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terval 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 longterm 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

Endo • 2000 Vol. 141 • No. 12

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.

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