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Endocrinology Vol. 148, No. 1 63-71
Copyright © 2007 by The Endocrine Society

Cholecystokinin Directly Inhibits Neuronal Activity of Primary Gonadotropin-Releasing Hormone Cells through Cholecystokinin-1 Receptor

Paolo Giacobini and Susan Wray

Cellular and Developmental Neurobiology Section (P.G., S.W.), National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892-3714; and Department of Human and Animal Biology (P.G.), Laboratory of Neurobiology, University of Turin, 10126 Turin, Italy

Address all correspondence and requests for reprints to: Dr. Susan Wray, Building 35, Room 3A-1012, Cellular and Developmental Neurobiology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892-3714. E-mail: wrays{at}ninds.nih.gov.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Pulsatile secretion of GnRH-1 regulates gonadotropin release from anterior pituitary and thus is essential for reproduction. The present study focused on the role of cholecystokinin (CCK) in the GnRH-1 system. CCK is a neuropeptide abundantly expressed in the brain, which is implicated in activation of female reproductive behaviors and release of anterior pituitary hormones. Using dual-label immunocytochemistry coupled to confocal analysis, GnRH-1 neurons in adult mouse brain were found to express CCK-1 receptors (CCK-1R), and CCK fibers were detected contacting GnRH-1 axons. To address the function of CCK on GnRH-1 neurons, calcium imaging was used to monitor patterns of activity of GnRH-1 neurons maintained in an in vitro system known to retain many characteristics of GnRH-1 cells in vivo. Endogenous receptors for CCK (CCK-1R and CCK-2R) were blocked with selective antagonists. Results indicate that CCK-1R but not CCK-2R antagonist treatment increased the number of calcium peaks/GnRH-1 cell, mean peak amplitude, and percentage of GnRH-1 cells displaying high activity. The increased activity in GnRH-1 neurons observed after application of CCK-1R antagonist was blocked by coincubation with exogenous CCK. This study provides evidence that CCK acts directly on GnRH-1 neurons to attenuate GnRH-1 neuronal activity via CCK-1R activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PULSATILE RELEASE OF GnRH-1 into the portal vascular system regulates secretion of anterior pituitary gonadotropins and as such is essential for reproduction in mammals (1). In addition, initial activation of the GnRH-1 system is key to reproduction and is developmentally controlled, with inappropriate stimulation or inhibition resulting in precocious puberty or delayed puberty respectively. Recently we showed that prenatal GnRH-1 neurons express a cholecystokinin(CCK)-8 G protein-coupled receptor (2). CCK is a neuropeptide abundantly expressed in the brain (3), which is implicated in activation of female reproductive behaviors and release of anterior pituitary hormones (4, 5), and CCK levels in several brain nuclei (medial preoptic nucleus, bed nucleus of the stria terminalis and medial nucleus of the amygdala) appear to be regulated by circulating gonadal steroids (6). A variety of in vivo studies have been conducted to elucidate the role of CCK on GnRH-1 release; however, the central actions of CCK on this system have remained controversial (5, 7, 8).

CCK interacts with two G protein-coupled receptors. The CCK-2R was thought to be the major CCK receptor (CCKR) in brain (9, 10, 11), with the CCK-1R abundant in peripheral organs (10). However, CCK-1R has now been reported in numerous central nervous system regions (9, 10, 12, 13, 14, 15) and has been identified in orexin-producing hypothalamic neurons (16). Like orexin-producing neurons of the hypothalamus, CCK-1R but not CCK-2R was found in prenatal GnRH-1 cells (2). In the present study, we show that in adult mice, GnRH-1 neurons continue to coexpress CCK-1R, CCK is present in the anteroventral periventricular nucleus (AVPV), and CCK immunoreactive terminals contact GnRH-1 neuronal processes. In the hypothalamic orexin-producing neurons, CCK was found to stimulate activity (16). Similar findings were obtained in the dorsal motor nucleus of the vagus (17). To examine the role of CCK on GnRH-1 neuronal activity, we used an in vitro model of postmitotic, primary GnRH-1 cells in nasal explants (18, 19, 20). Similar to the developing embryo in vivo, GnRH-1 neurons in nasal explants turn on GnRH-1 expression, exhibiting pulsatile GnRH-1 neuronal activity and secretion (20, 21, 22, 23). In vitro analysis allows one to monitor groups of GnRH-1 neurons. Such sampling is difficult in vivo because GnRH-1 neurons are diffusely distributed from olfactory bulbs to caudal hypothalamus (24). Sampling across the GnRH-1 neuronal population is critical to elucidate mechanisms underlying activation of the GnRH-1 system as well as molecules that may modulate GnRH-1 pulsatile activity and secretion. Using calcium imaging, we show that CCK can directly attenuate GnRH-1 neuronal activity via CCK-1R signal transduction pathways.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult mice and colchicine treatment.
Experiments were conducted in accordance with current European Union and Italian law under authorization of the Italian Ministry of Health (no. 66/99-A). Six adult CD-1 female mice (2–6 months old in random stages of the estrous cycle) and two CD-1 adult males (3 months old; Charles River, Milan, Italy) were used. Mice were anesthetized with an ip injection of ketamine (200 mg/kg), and perfused with 4% paraformaldehyde. Three females were treated with colchicine to enhance visualization of CCK immunoreactivity in cell bodies (25). Colchicine treatment was performed as follows: 10 µg colchicine (Sigma, St. Louis, MO) in 1.5 µl saline was injected into the lateral ventricle (stereotaxic coordinates: 0 mm bregma line, 1 mm lateral to sagittal sinus, and 2 mm depth) by means of a glass micropipette and a pneumatic pressure injection apparatus (Picospritzer II; General Valve, Fairfield, IL) under xylazine/ketamine anesthesia (5 mg/kg xylazine + 75 mg/kg ketamine). After the micropipette was removed, the skin was sutured and the mice quickly revived under a warm lamp. Seventy-two hours after the treatment, these mice were anesthetized with an ip injection of ketamine (200 mg/kg) and perfused with 4% paraformaldehyde.

The brains were dissected and postfixed (4% paraformaldehyde) overnight at 4 C. Brains were then cryoprotected [15% sucrose solution in 0.1 M phosphate buffer (pH 7.4), overnight followed by a 30% sucrose solution], frozen, and cryostat sectioned (30 µm thick free-floating sections). Coronal sections were cut from the olfactory bulbs to the level of the median eminence.

Nasal explants
Nasal regions were cultured as previously described (20). Briefly, embryos were obtained from timed pregnant animals in accordance with National Institutes of Health/National Institute of Neurological Disorders and Stroke guidelines and Animal Care and Use Committee approval. Nasal pits of embryonic d 11.5 staged NIH-swiss mice were isolated under aseptic conditions in Gey’s balanced salt solution (Life Technologies, Inc., Grand Island, NY) enriched with glucose (Sigma). Nasal explants were adhered onto coverslips by a plasma (Cocalico Biologicals, Inc., Reamstown, PA)/thrombin (Sigma) clot. The explants were maintained in defined serum-free medium (SFM) (20) at 37 C with 5% CO2. On culture d 3, fresh media containing FuDR (8 x 10–5 M; Sigma) was given to inhibit proliferation of dividing olfactory neurons and nonneuronal explant tissue. The media were changed to fresh SFM on culture d 6 and 8.

Cell isolation and PCR analysis
At three time points [7, 14, and 28 d in vitro (div)], single GnRH-1 cells (n = 10 for each in vitro stage) were isolated from nasal explants and cDNA produced and PCR amplification performed as previously described (26, 27). All cDNA libraries were screened for GnRH-1 (assuring cell phenotype), ßIII-tubulin, and L19 (two housekeeping genes, microtubule and ribosomal) using PCR. cDNA libraries that were positive for all three transcripts were used in this study. Based on the technique used to generate the cDNA pools, 3' untranslated region-biased primers are necessary. Primers were designed with the 5' primer being less than 500 bases from the polyA site and the 3' primer close to, but not into, the polyA region. All designed primers were screened using BLAST to ensure specificity of binding. Primers used for screening the cellular phenotype were: GnRH-1 (5'-ACTGGTCCTATGGGTTGCGCCCTG-3', 5'-CGGGGCCAGTGGACAGTACATTCG-3'), ßIII-tubulin (5'-GAGGACAGAGCCAAGTGGAC-3', 5'-CAGGGCCAAGACAAGCAG-3'), and L19 (5'-CCTGAAGGTCAAAGGGAATGTGTTC-3', 5'-GGACAG-AGTCTTGATGATCTCCTCC-3'). For each reaction, 30.5 µl of nuclease-free H2O, 5 µl of 10 x PCR GOLD buffer (Applied Biosystem), 4 µl of 25 mM MgCl2, 5 µl dNTP mix (2.5 mM), 0.5 µl Amplitaq Gold (Applied Biosystems, Foster City, CA) were mixed. Primers (250 nmol) and template cDNA (1 µl) were added to the mixture. The PCR program was: 10 min, 94 C, before run; 30 sec, 94 C; 30 sec, 55 or 65 C (depending on primers); 2 min, 72 C for 40 cycles; and 10 min, 72 C, after run.

The same PCR profile was used for subsequent screening with the following primers: CCK-1R forward primer (5'-ATGCGAGGCCAGTAGCTAGA-3'), CCK-1R reverse primer (5'-CTTCTTACCCGGAGGCATTT-3'). Specific bands were observed in total brain lanes, whereas no bands were seen in water or brain RNA– reverse transcription lanes.

Calcium imaging
Calcium imaging was conducted as previously described (23). Briefly, nasal explants were exposed to the indicator dye calcium green-1 acetoxymethyl (2.7 mM in 80% dimethylsulfoxide/20% pluronic F-127, diluted 1:200 with SFM) for 20 min in a humidified chamber. Explants were then washed with fresh SFM (2 x 10 min) and transferred to a heated perfusion chamber (37.5 C; Warner Instruments, Hamden, CT). Medium was perfused across the explant at 100 µl/min via a peristaltic pump (Spectra Hardware, Inc., Westmoreland City, PA). Relative intracellular calcium levels were visualized using a Nikon microscope equipped with a x20 fluorescence objective and an intensified charge-coupled device camera (Video Scope International, Sterling, VA). The camera shutter was controlled by a Macintosh computer via imaging software (IP Labs, Scanalytics Corp., Vienna, VA). Excitation wavelengths were 450–490 nm and emission was monitored at 520–560 nm.

Analysis of calcium imaging data.
Unless otherwise indicated, cells were imaged every 20 sec for 100 min. At the end of the 100-min period, explants were exposed to an acute dose of KCl (20 mM, to cause an abrupt increase in intracellular calcium to confirm the viability of the cells). Using IP Labs, relative ODs were measured within each cell, and background values were subtracted; thus, the corrected OD values represent only intracellular events. The traces of intracellular calcium were then analyzed by PULSAR (28) to determine when calcium peaks occurred (OD readings 2 SD above baseline). Time points when peaks were detected in each cell were compiled into a single file for the entire neuronal population within the imaged field. The MATLAB program divided these data into 1-min intervals and assigned either a 0 (no calcium peaks detected during that 1 min period) or a 1 (≥1 significant calcium peak detected). These data were then transformed by the WAVELET analysis program to determine when calcium pulses occurred. A pulse was defined as a period of time when multiple GnRH-1 neurons displayed synchronous calcium oscillations. For each pulse, the peak amplitude and the number of cells contributing to the pulse were determined.

Experimental groups.
Experimental groups consisted of two to four explants/treatment with an average of 13.8 ± 0.98 (range 8–31) cells visualized per explant. In the groups below, drugs were perfused continuously across the explant for the duration of the experiments. The following treatments were given: the sodium channel blocker tetrodotoxin (10–6 M in acetate buffer), CCK (10–7 M, Research Plus Inc., NJ), CCK-1R antagonist (lorglumide; 10–7 M; Sigma-RBI; Natick, MA), CCK-2R antagonist (L-365,260; 10–7 M; ML Laboratories, Liverpool, UK), proglumide, a receptor antagonist effective for both CCK-1R and CCK-2R (10–4 M; Sigma-RBI). These same concentrations of CCK and CCKR antagonists were used in previous electrophysiological and calcium imaging studies (29).

To determine the specificity of CCK-1R antagonist effects on calcium oscillations, acute experiments were conducted in which cells were cotreated with CCK1R antagonist and CCK (see Fig. 5Go, A and B). In these experiments, explants (6–9 div) were imaged during treatment with CCK-1R antagonist for 5 min. The antagonist was then washed out for 30 min to allow calcium oscillations to return to baseline. Administration of CCK-1R antagonist in combination with CCK was preceded by one (n = 6 GnRH-1 cells recorded; see Fig. 5AGo) or two applications of CCK-1R antagonist alone (n = 11 GnRH-1 cells recorded, see Fig. 5BGo). This approach ensured that attenuation of calcium signal in the presence of the antagonist plus CCK was not the result of desensitization.


Figure 5
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  		FIG. 5. CCK inhibits GnRH-1 neuronal activity through CCK/CCK-1R signaling pathway. Left panels, Two representative traces of GnRH-1 cells exposed to different experimental paradigms. CCK-1R selective antagonist (10–7 M) induced an increase in Ca2+ in GnRH-1 neurons (A, 75% response, six of eight cells; B, 65% response, 11 of 17 cells). When the antagonist was removed, after 5 min exposure, the CCK-1R antagonist-induced Ca2+ increase returned to basal level 5 min after washout (A and B). A second application of exogenous antagonist was still able to induce an intracellular calcium increase (A), whereas coapplication of CCK (10–7 M) and CCK-1R antagonist abolished such an increase (A and B). Right panels, CCK-1R antagonism unmasked GABAergic stimulatory signals. WAVELET analysis (C, top panel) of the calcium activity of 12 post hoc identified GnRH-1 neurons (one of which is shown in bottom panel) from an 8 div nasal explant exposed to the pharmacological treatments as indicated (45-min recording time). Note that the exposure to CCK-1R antagonist increased the number of peaks in GnRH-1 cells, compared with SFM, whereas coapplication of CCK-1R antagonist and Pic eliminated the calcium oscillations of GnRH-1 cells. Asterisks indicate significant pulses of synchronized activity in the population of GnRH-1 cells (WAVELET analysis). Hatch marks at the top of the calcium green trace (bottom panel) demonstrate significant peaks as detected by the PULSAR algorithm.

 
Finally, the effects of {gamma}-aminobutyric acid (GABA)ergic and CCKergic signals on GnRH-1 neuronal activity were examined by coapplication of GABAA and CCK-1R antagonists, picrotoxin [Pic; 10–4 M (30, 31)] and lorglumide (10–7 M), respectively, to 6–9 div nasal explants. Administration of CCK-1R antagonist in combination with Pic was preceded by one application of CCK-1R antagonist alone (see Fig. 5CGo).

Statistical comparisons of data were calculated using ANOVA, followed by a Fisher’s least significant differences post hoc test. All analyses were conducted using Statview Statistical Software (Abacus Concepts, Inc., Berkeley, CA). {chi}2 data on the percent of cells exhibiting high activity were considered significantly different if P < 0.05. ANOVA data were considered significantly different if P < 0.05. All data are expressed as mean ± SEM.

Immunocytochemistry
Primary antisera used were against: GnRH-1 [SW1 (preprohormone) 1:1000 (32); SMI-41 (GnRH-1 amidated decapeptide) 1:2000; Sternberger Monoclonals Inc., Baltimore, MD], CCK (1:1000; ImmunoStar Inc., Hudson, WI), and CCK-1R [1:2000; kindly provided by Prof. P. Beart, Howard Florey Institute, Victoria, Australia (12)]. Methods and antibody specificity were assessed by elimination of the primary antibody and inclusion of control tissues. Duodenal tissue and cerebral cortex, known to contain CCK and CCKRs (9, 10, 33, 34, 35), were used as positive controls (data not shown). Antibodies were diluted in 0.01 M PBS/0.3% Triton X-100 and normal goat serum (1:100).

In vivo.
Standard double-immunofluorescence methods were used on cryostat free-floating sections. SMI-41 (a monoclonal) was coincubated (48 h, 4 C) with either anti-CCK (rabbit polyclonal) or anti-CCK-1R (rabbit polyclonal). After PBS washes, sections were incubated (1 h, room temperature) with goat antirabbit Alexa-Fluor 488 (1:500; Molecular Probes, Eugene, OR) and goat antimouse CY-3 (1:800; Jackson ImmunoResearch, West Grove, PA). Sections were mounted in 1,4-diazabicyclo[2.2.2]octane (Sigma) and observed with a laser-scanning Olympus Fluoview confocal system (Olympus, New Hyde Park, NY).

In vitro.
After calcium-imaging experiments, explants were fixed (4% formalin, 1 h), rinsed (PBS), and blocked (1 h, 10% normal goat serum/0.1% sodium azide/0.3%Triton X-100). After blocking, explants were rinsed (PBS) and then incubated overnight (4 C) in GnRH-1 antibody (SW1). The next day, explants were washed with PBS and incubated in goat antirabbit-conjugated Cy3 (1:800; Jackson ImmunoResearch). Immunopositive GnRH-1 cells were then compared with the calcium dye labeling to verify the phenotype of the calcium-labeled cells. Any cells that were not GnRH-1 positive were analyzed separately (<5%).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CCK-1R and CCK expression in adult neuroendocrine GnRH-1 neurons
Prominent expression of CCK-1R has been previously reported in adult hypothalamic areas in which GnRH-1 neurons are located (10, 12, 15, 36). We recently showed that prenatally, GnRH-1 neuroendocrine cells located within the nasal compartment expressed CCK-1R (2). To determine whether CCK-1R expression in GnRH-1 neurons is maintained throughout development in vivo, the expression of CCK-1R and GnRH-1 in the adult hypothalamus was examined (Fig. 1Go). Neurons in the paraventricular hypothalamic lateral magnocellular nucleus have been described to robustly express CCK-1R (12). We detected a strong signal in neurons in the paraventricular hypothalamic lateral magnocellular nucleus as well, confirming the specificity of the antibody (data not shown). Confocal microscopic analysis of double-labeled sections [GnRH-1 (Fig. 1AGo) and CCK-1R (Fig. 1BGo)] confirmed the presence CCK-1R in GnRH-1 cells. Colocalization of these markers is demonstrated by three-dimensional reconstructions of single cells (Fig. 1CGo). Light microscopic analysis revealed that approximately 40% of GnRH-1 neurons express CCK-1R, independent of anatomical location.


Figure 1
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  		FIG. 1. CCK-1R is expressed by GnRH-1 neurons in adult hypothalamus. A and B, Fluorescent double labeling for GnRH-1 (A) and CCK-1R (B) in coronal sections at the level of the organum vasculosum lamina terminalis (OVLT) revealed CCK-1R is expressed in cells throughout this region; however, arrows (A and B) point to cells expressing both molecules. Three-dimensional-reconstructed cells are shown in boxed area (C). GnRH-1-positive cells (red) are colabeled with CCK-1R (green). Reconstructed orthogonal projections are presented as viewed in the x-z (bottom) and y-z (right) planes. Scale bars 20 µm (A and B), 8 µm (C).

 
Differential and sexually dimorphic expression of CCK in a subset of hypothalamic GnRH-1 neurons has been previously documented in postpubertal rats with its expression in GnRH-1 cells being higher in females (~37% colocalization) than males (only 1% colocalization) (25). To determine whether CCK is coexpressed in GnRH-1 cells in mouse as well, double immunohistochemistry for GnRH-1 and CCK was performed on brains from adult female and male mice. Coexpression between the two peptides was not observed in any of the areas containing GnRH-1 perikarya (Fig. 2AGo); however, CCK fibers were detected contacting GnRH-1 immunoreactive somata and processes (Fig. 2Go, B and C). GnRH-1 neurons located in the preoptic area of adult females (14 GnRH-1 cells; n = 2) and adult males (20 GnRH-1 cells; n = 2) were evaluated using confocal microscopy. In females, 50% of these neurons possessed CCK fiber appositions. In contrast, only 25% of the GnRH-1 population assayed in the preoptic area of adult males were contacted by CCK fibers. Because colchicine is often required to detect CCK peptide in cell soma in vivo (37), intracerebroventricular (i.c.v.) colchicine injections were performed (Fig. 2Go, F–H). In untreated mice, CCK staining was seen only in axon varicosities, being widely distributed throughout hypothalamic areas including the periventricular and paraventricular nucleus (PVN; Fig. 2DGo), AVPV, organum vasculosum lamina terminalis, and medial preoptic nucleus (data not shown). However, after colchicine treatment, strongly labeled CCK-immunoreactive cell bodies were detected in the PVN (Fig. 2FGo), suprachiasmatic nucleus, and AVPV (Fig. 2HGo). This distribution is similar to that reported for rat (38, 39). The lack of GnRH-1-immunoreactive varicosities in the median eminence (ME) in colchicine-treated animals (Fig. 2GGo), compared with untreated mice (Fig. 2EGo), further confirms the effectiveness of the treatment. It should be noted that after colchicine treatment, colocalization of CCK in GnRH-1 cells were not detected, even in areas in which double-labeled GnRH-1/CCK cells were found in rats (25).


Figure 2
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  		FIG. 2. CCKergic fibers contact GnRH-1 axons. A–C, Fluorescent double labeling for GnRH-1 (red, arrows) and CCK (green, arrowheads) in brain sections combined with confocal microscopy analysis showed GnRH-1-positive cells were CCK negative. However, intense CCK-positive varicosities and fibers (arrowheads in A–C and inset in C) were evident along GnRH-1 cell proximal processes (arrows, B and C). Colchicine treatment induced accumulation of CCK peptide in cell bodies located in different areas of the brain (compare D, arrowheads; and F, arrows). Double labeling for GnRH-1 and CCK in these mice showed intense CCK immunoreactivity in cells in the PVN; however, no GnRH-1 staining is detectable in these neurons (F). A robust CCK staining was also detected in cell bodies located in the AVPV (H, arrows). E and G show GnRH-1 distribution in the ME of untreated (E) and colchicine-treated (G) mice. In normal animals, GnRH-1 immunoreactive terminals are widely distributed in the ME (E), whereas in the treated mice, little GnRH-1 staining is evident (G), showing the effectiveness of the treatment. och, Optic chiasm; III, third ventricle. Scale bars, 10 µm (A); 4 µm (B); 8 µm (C); 2 µm (inset in C); 20 µm (D and F); 30 µm (E and G) and 80 µm (H); 10 µm (inset in H).

 
CCK-1R expression in primary GnRH-1 neurons
Nasal explants represent a valuable tool to dissect apart spatial from temporal cues and focus on the properties of GnRH-1 neurons by controlling extracellular influences. Thus, to determine whether CCK could influence GnRH-1 neuronal activity, nasal explants were used. We previously showed that GnRH-1 neurons, maintained in this in vitro system, express CCK-1R but not CCK-2R (2) at a time when synchronous neuronal activity is present (23). To identify the in vitro temporal window for conducting these experiments, cDNA pools from 10 single GnRH-1 cells at three different in vitro ages, 7, 14, and 28 div, were examined for CCK-1R transcript. Negative controls included omission of the reverse transcriptase enzyme or template (water; Fig. 3AGo). Amplicons were detected in GnRH-1 cells (Fig. 3AGo) at 7 div (10 of 10 cells), 14 div (seven of 10 cells), and 28 div (six of 10 cells). Based on the fact that GnRH-1 neurons maintained for 7 div showed robust expression levels of both CCK-1R transcript (Fig. 3AGo) and protein (2) and that by 6 div GnRH-1 neurons maintained in SFM conditions exhibit synchronized pulses of intracellular calcium (40), explants from 6 to 9 div were used for subsequent analysis.


Figure 3
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  		FIG. 3. GnRH-1 neurons express functional CCK-1R in vitro. A, Representative gel of PCR products from single-cell RT-PCR performed on GnRH-1 cells (7, 14, and 28 div) extracted from the periphery of nasal explants. Products produced by PCR amplification using CCK-1R-specific primers. CCK-1R transcript (181 bp) was detected in primary GnRH-1 neurons at 7 div (100%), 14 div (70%), and 28 div (60%); no specific band was detected in water (W). MW, Molecular weight; Br, adult brain. B–G, Perturbation of the CCK/CCK-1R system alters GnRH-1 neuronal activity. Calcium imaging (B) and GnRH-1 immunofluorescence (C) in explants at 7 div are shown. Neurons in the periphery that label with calcium green are predominantly GnRH-1 neurons (compare B and C). Arrows indicate clusters of calcium green-labeled cells that were also GnRH-1 positive, whereas small arrowheads indicate single cells double labeled. Oversized arrowheads (A) indicate non-GnRH-1 cells loaded with calcium green. D–F, Representative calcium green traces (for a 90-min recording time) from post hoc identified GnRH-1 cells in SFM- (D), CCK- (E), and CCK-1R antagonist-treated explants (F). Hatch marks at the top of the graphs demonstrate significant peaks as detected by the PULSAR algorithm. Note that the exposure to CCK-1R antagonist (10–7 M) increases the number of peaks in the GnRH-1 cell. The activity trace shown in G is an example of non-GnRH-1 cell from an explant-treated with CCK-1R antagonist. Values represent mean OD, after background corrections, of calcium green fluorescence within the cell soma. Scale bar, 20 µm.

 
Endogenous CCK inhibits intracellular calcium levels in GnRH-1 neurons
Nasal explants were treated with CCK and CCKR-specific antagonists and intracellular calcium levels monitored in GnRH-1 cells in the periphery of the explants (Fig. 3Go, B and C). It should be noted that previous work identified endogenous CCK present in olfactory components maintained in nasal explants (2).

Calcium imaging experiments performed by application of only exogenous CCK did not induce any significant effect, compared with SFM-treated groups (Figs. 3Go, D and E; and 4Go). Three different doses of CCK (10–5, 10–7, 10–9 M) were tested. None of the concentrations significantly altered the calcium oscillations of the GnRH-1 neurons [SFM peaks/cell = 7.2 ± 0.6 (n = 53); CCK 10–5 M peaks/cell = 8 ± 0.8 (n = 12); CCK 10–7 M peaks/cell = 5.3 ± 0.7 (n = 45); CCK 10–9 M peaks/cell = 7 ± 1.7 (n = 9)]. In contrast to exogenous CCK and in agreement with earlier studies (23), tetrodotoxin reduced GnRH-1 neuronal activity to a basal level (Fig. 4Go). To uncover a potential effect of endogenous CCK [present in the olfactory system in vivo and in vitro (2)] on GnRH-1 neuronal activity, calcium imaging experiments were next performed in the presence of CCK-1R (lorglumide) and CCK-2R (L-365,260) antagonists. Lorglumide dramatically increased the number of calcium peaks detected in GnRH-1 neurons, compared with GnRH-1 neurons in SFM only (Figs. 3FGo and 4Go). Few calcium peaks were observed in non-GnRH-1 cells, which received the same treatment (Fig. 3GGo). PULSAR analysis identified a significant increase in the number of calcium peaks/cell and the peak amplitude as well as the percentage of cells with more than 10 peaks, compared with the SFM group (Fig. 4Go). In addition, WAVELET analysis revealed a significant decrease in the interpulse interval (IPI = 10.1 ± 1.1 min) of synchronized calcium pulses in CCK-1R antagonist-treated cultures as compared with untreated controls (IPI = 18.9 ± 1.1 min), but whether these are synchronized events or the result of overt increases in activity is unclear.


Figure 4
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  		FIG. 4. CCK-1R antagonist increases GnRH-1 neuronal activity in 6–9 div explants. Upper table summarizes data collected from calcium imaging experiments. Note that only exposure to CCK-1R antagonist or to proglumide, a receptor antagonist effective for both CCK-1R and CCK-2R, significantly increased all of the parameters examined. No variation in GnRH-1 neuronal activity was detected when explants were treated with CCK-2R antagonist. Bottom panels depict representative WAVELET analysis of numerous GnRH-1 cells in a single explant treated with SFM (left panel) or CCK-1R antagonist (right panel). PULSAR detected calcium peaks are plotted in the black and white panels under the WAVELET analysis. In these panels, the X-axis is time (minutes). Each row is data from an individual cell (denoted in the left panel by black arrows) throughout the time of recording with the black hatch marks representing the time at which a significant peak in intracellular calcium occurred. The top panels represent the one-dimensional continuous sum of plotted cell activity (from bottom panel) in each preparation and depict synchronized calcium oscillations across multiple cells (denoted with an asterisk). CCK-1R antagonist application increased the calcium oscillations of the monitored population of GnRH-1 neurons.

 
The lorglumide-induced response returned to basal level after washout (Fig. 5AGo). A second application of the antagonist was still able to induce an intracellular calcium increase (Fig. 5AGo), whereas this effect was abolished when CCK was applied in the presence of the antagonist (Fig. 5Go, A and B), indicating specificity of the observed response [note: 10–7 M was used for these experiments, a concentration used in previous calcium imaging studies (16, 29)]. Differences in these parameters were not detected after treatment with a CCK-2R antagonist (Fig. 4Go). In a manner similar to lorglumide, proglumide treatment (a receptor antagonist effective for both CCK-1R and CCK-2R) significantly increased GnRH-1 neuronal activity and the frequency of synchronized calcium events as compared with the SFM-treated group (Fig. 4Go). These findings indicate that CCK is acting through CCK-1R to inhibit GnRH-1 neuronal activity.

In nasal explants, muscimol, a GABAA agonist, causes depolarization of GnRH-1 neurons, which results in a significant rise in intracellular calcium as well as increased synthesis of GnRH-1 peptide (30, 31). To determine whether CCK-1R antagonism unmasked GABAergic stimulatory signals to GnRH-1 neurons, Pic was used to selectively inhibit GABAA receptors. At 1 wk in vitro, Pic treatment attenuates GnRH-1 neuronal activity and synchronization (23). Lorglumide induced a significant increase in intracellular calcium oscillations in GnRH-1 neurons, compared with SFM, whereas coapplication of the CCK-1R antagonist and Pic abolished calcium peaks (Fig. 5CGo; n = 18).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
CCK-containing neurons are widely expressed in brain areas involved in stress response, fear, anxiety, cognition, and sexual behavior (41, 42, 43). In monkey, i.c.v. administration of CCK elicits the release of GnRH-1 as measured by changes in circulating LH concentrations (7). In rat administration of CCK resulted in a drop in plasma LH concentrations (5, 44), whereas in goats, CCK accelerated the activity of the GnRH-1 pulse generator and occurrence of LH pulses in plasma (8). Although all of the studies are based on indirect measurements of GnRH-1 neuronal activity, they support a central action of CCK on the GnRH-1 system, albeit the manner of action remained unclear. Recently CCK-1Rs were identified on prenatal GnRH-1 neurons in mice (2), providing a potential signal transduction pathway for the action of CCK. In addition, using immunocytochemistry, sexually dimorphic expression of CCK in postpubertal rat hypothalamic GnRH-1 neurons has been reported, being higher in females than males (25). Thus, CCK might act on GnRH-1 neurons in a paracrine or autocrine manner. To address this issue, we mapped the expression of CCK and CCK-1Rs in adult mice in relation to GnRH-1 neurons and evaluated the action of CCK on GnRH-1 neurons maintained in nasal explants.

CCK expression was not detected in GnRH-1 neurons even when mice received i.c.v. administration of colchicine. Differences between mice and rats could certainly represent species-specific expression patterns, and alternative pathways may be used by these two animals to modulate GnRH-1 neuronal activity. However, in mice, GnRH-1 containing axons were often found apposed by CCK-positive fibers, suggesting that CCKergic input on GnRH-1 neurons might modulate this system via interneurons. In fact, immunohistochemistry combined with laser-scanning confocal microscopy analysis indicated that GnRH-1 neurons located in adult mouse hypothalamic areas express CCK-1R, suggesting that GnRH-1 neurons maintain CCK-1R expression throughout development in vivo. To determine the potential source of these CCK fibers, we screened candidate regions. In particular CCK expressing neurons have been identified in the bed nucleus of the stria terminalis and AVPV of the rat (39, 45). These two regions, together with other limbic structures, integrate hormonal and sensory information associated with reproduction and transmit this information to hypothalamic regions and specifically GnRH-1 neurons (46). Robust CCK expression was identified in the AVPV region in mice. Thus, a potential neural circuit exists for CCKergic neurons in the AVPV-modulating GnRH-1 neuronal activity.

Sampling across the GnRH-1 neuronal population is difficult in vivo because GnRH-1 neurons are diffusely distributed from olfactory bulbs to caudal hypothalamus (24). Therefore, several groups have established an in vitro model of postmitotic, primary GnRH-1 cells in nasal explants. Individual GnRH-1 neurons in nasal explants exhibit electrical and synaptic properties similar to GnRH-1 neurons in vivo, including responses to GABAergic and glutamatergic stimulation (30). In addition to depolarized-induced GnRH-1 release (21, 31), GnRH-1 neurons in nasal explants release GnRH-1 in a pulsatile manner (21, 22) with the IPI being species dependent.

Previous data indicated that GnRH-1 cells maintained in nasal explants express CCK-1R but not CCK-2R (2). Several lines of evidence indicate that in GnRH-1 neurons in nasal explants, rises in intracellular calcium reflect changes in neuronal activity and correlate with pulses of GnRH-1 secretion (21, 22, 23, 31). Thus, changes in intracellular calcium were monitored during different pharmacological treatments. Acute CCK exposure did not induce significant changes, compared with SFM-treated groups at any of the concentrations used, although a consistent decrease (albeit small) in peaks/GnRH-1 was observed. We previously showed that endogenous CCK is present and functional in nasal explants and that the source of CCK is likely to be the developing olfactory system (2). Recently it was shown that the CCK-induced response of orexin neurons peaks 1 min after CCK exposure (16). In the present study, we recorded intracellular calcium fluctuations every 20 sec. However, no effect could be detected, even when images were captured every 2 sec [to be sure not to underestimate the number of peaks/cell (data not shown)], suggesting that endogenous concentrations of the octapeptide might be sufficient to saturate the receptors for the duration of the experiment. It is noteworthy that chronic exogenous application of CCK to nasal explants was able to inhibit GnRH-1 neuronal migration, whereas acute exposure did not change GnRH-1 neuronal activity.

To unmask the effect of endogenous CCK, selective CCK-receptor antagonists were used. In agreement with the expression pattern of the receptors in GnRH-1 cells, CCK-1R but not CCK-2R antagonist treatment altered neuronal activity, dramatically increasing the number of calcium peaks/cell, the mean amplitude of the peaks as well as the percentage of cells displaying more than 10 calcium peaks. Moreover, synchronous activity in the GnRH-1 neuronal population exposed to CCK-1R antagonist treatment occurred more frequently. However, this is most likely an indirect consequence of an overall increase in GnRH-1 neuronal activity rather than a direct effect on the IPI.

To our knowledge, this is the first report of CCK acting via the CCK-1R to inhibit neuronal activity. CCK interneurons in the hippocampus (47) have recently been shown to generate long-lasting inhibition, but this response is via release of GABA from the CCK cells. In our study, CCK itself appears to be inhibitory. However, GABA may be responsible for the observed increased activity. In 6–9 div nasal explants, GABAergic neurons are present, and GABAA receptor activation on GnRH-1 neurons results in depolarization (23). We showed that blockage of CCK-1R and GABAA receptors resulted in a reduced GnRH-1 neuronal activity. Thus, CCK-1R signaling may counterbalance GABAA receptor activation. Whether this is an age-related effect of CCK on the activity of GnRH-1 cells, which is active only before the developmental transition of GABAergic signaling from excitatory to inhibitory (48, 49), is presently unclear. However, acting as a modulator prenatally, CCK might prevent precocious activation of the GnRH-1 system. One would then predict that in the absence of CCK-1R signaling, GnRH-1 neurons could become hyperreactive to the depolarizing influence of GABAA receptor activation. Once the maturity of the GnRH-1 system is established and GABA signaling becomes inhibitory (49), it is unknown whether CCK signaling would remain inhibitory or switch to excitatory, as is observed in other mature hypothalamic neurons (16). CCK, acting through CCK-1R, is known to activate a variety of intracellular signaling mechanisms in pancreatic acinar cells (50), and switching of receptor coupling to different G proteins has been observed (51). Future studies on the transduction pathways coupled to the CCK-1R in prenatal vs. postnatal GnRH-1 neurons might help elucidate whether such a switch is possible in this system.

In summary, this work demonstrates that CCK acts directly on GnRH-1 neurons to inhibit their neuronal activity through CCK-1R and that a relation exists, most likely in an age-dependent manner, between CCK modulation of, and GABAergic signals to, GnRH-1 neurons. Because CCK-1R is widely expressed in central nervous system areas and is implicated in a variety of functions (10, 12), additional studies using conditional knockout mice that lack CCK-1R targeted in GnRH-1 neurons are needed to clarify the physiological role of CCK on these cells.


   Acknowledgments
 
We thank Andree Reuss for her technical skills in generating nasal explants and Dr. Silvia De Marchis for great help in performing i.c.v. colchicine treatments. We are thankful to Professor P. Beart for the polyclonal antiserum to CCK-1R.


   Footnotes
 
This work was supported in part by Compagnia di San Paolo (to P.G.), Consiglio Nazionale delle Ricerche (Short Term Mobility Program 2005) (to P.G.), and Ricerca Scientifica Applicata CIPE-A23 Reg. Piemonte, Italy (to P.G.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online October 5, 2006

Abbreviations: AVPV, Anteroventral periventricular nucleus; CCK, cholecystokinin; CCKR, CCK receptor; div, days in vitro; GABA, {gamma}-aminobutyric acid; i.c.v., intracerebroventricular; IPI, interpulse interval; ME, median eminence; PVN, paraventricular nucleus; Pic, picrotoxin; SFM, serum-free medium.

Received June 7, 2006.

Accepted for publication September 25, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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