Abstract
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Hormone secretion in transgenic rats and electrophysiological activity in their gonadotropin releasing-hormone neurons
2,5,*Abstract
Expression of GFP in GnRH neurons has allowed for studies of individual GnRH neurons. We have demonstrated previously the preservation of physiological function in male GnRH-GFP mice. In the present study, we confirm using biocytin-filled GFP-positive neurons in the hypothalamic slice preparation that GFP-expressing somata, axons, and dendrites in hypothalamic slices from GnRH-GFP rats are GnRH1 peptide positive. Second, we used repetitive sampling to study hormone secretion from GnRH-GFP transgenic rats in the homozygous, heterozygous, and wild-type state and between transgenic and Wistar males after ~4 yr of backcrossing. Parameters of hormone secretion were not different between the three genetic groups or between transgenic males and Wistar controls. Finally, we performed long-term recording in as many GFP-identified GnRH neurons as possible in hypothalamic slices to determine their patterns of discharge. In some cases, we obtained GnRH neuronal recordings from individual males in which blood samples had been collected the previous day. Activity in individual GnRH neurons was expressed as total quiescence, a continuous pattern of firing of either low or relatively high frequencies or an intermittent pattern of firing. In males with both intensive blood sampling (at 6-min intervals) and recordings from their GnRH neurons, we analyzed the activity of GnRH neurons with intermittent activity above 2 Hz using cluster analysis on both data sets. The average number of pulses was 3.9 ± 0.6/h. The average number of episodes of firing was 4.0 ± 0.6/h. Therefore, the GnRH pulse generator may be maintained in the sagittal hypothalamic slice preparation.
ever since the initial observation that LH secretion is intermittent (8), a major goal has been to understand the neuronal substrate regulating this episodic pattern of reproductive hormone secretion. We have known for some time that in several species the episodes of pulsatile LH secretion have invariably been preceded by concurrent pulses of gonadotropin-releasing hormone (GnRH) (2). Additionally, electrical activity within the neural complex that results in pulsatile GnRH release (the so-called GnRH “pulse generator”) is manifested in extracellular multiunit recordings from the hypothalamus (10, 11, 14). However, the precise neuronal mechanisms that initiate this sequence of neural activation and periodic GnRH secretion continue to be obscured.
The anatomic issues surrounding the GnRH system have hindered our understanding of the composition of the GnRH pulse generator. First, GnRH neurons are relatively few in number, with the total number of neurons varying from 800 to 2,500, depending on the species. Additionally, GnRH neurons are distributed diffusely throughout the hypothalamus, rendering manipulations such as discrete and selective lesions untenable (see Ref. 29 for review of anatomy). Because of these anatomic constraints and the lack of techniques for identifying the GnRH neurons in living tissue, electrical recordings in the wild-type (WT) GnRH neurons have been, and will likely continue to be, very rare (17).
The genetic manipulation that causes GnRH neurons to express green fluorescent protein (GFP) has made the acquisition of electrical recordings from single GnRH neurons tractable. This has led to a profound expansion of our knowledge of the activity of individual GnRH neurons in hypothalamic slices. Mice were the earliest models in which GFP was expressed in GnRH neurons (30, 34). However, transgenic rats with GFP-expressing GnRH neurons are also available (16).
The expression of GFP in the GnRH neurons of male mice does not interfere profoundly with GnRH secretion (33). However, in female mice, the pulsatile pattern of LH release was disrupted when the GnRH neurons were modified to express GFP, although other indices of reproductive function appeared to be normal (33). Thus, whether the GnRH-GFP rat model (16) exhibits the anticipated pattern of LH secretion requires explicit demonstration.
In the present study, we utilized the GnRH-GFP rat model to identify GFP-containing neurons in living hypothalamic slices to determine whether these neurons contain the GnRH1 peptide. Second, we assessed the function of the GnRH pulse generator in GnRH-GFP rats by monitoring peripheral LH and testosterone release. Finally, we obtained electrical recordings from single GnRH neurons in brain slices obtained from transgenic rats, some of which were sampled intensively for hormone release on the day prior to recordings.
METHODS
Animals
All studies used male rats where GnRH neurons express GFP (16). Animals carried Clontech's GFP construct with expression under the regulation of 3.0 kB of the GnRH1 peptide promoter. Animals were maintained on a 12:12-h light-dark cycle (lights on 0700) with ad libitum access to standard rodent chow and water. All procedures performed on animals were reviewed and approved by the Institutional Animal Care and Use Committees at Emory University and the University of Texas San Antonio and were in accord with the National Institutes of Health Guide for Care and Use of Laboratory Animals.
Experiment 1
Are GFP neurons in hypothalamic slices from GnRH-GFP rats GnRH neurons? For this objective, we filled putative GnRH-containing neurons which were visualized in live hypothalamic slices using GFP with the cellular marker biocytin. The average age (days ± SE) of animals from which biocytin-filled neurons were obtained was 73.9 ± 2.8 (range 54–100 days).
Tissue preparation.
Live hypothalamic slices (300 μm) were prepared using a vibrating microtome (HM 650V; Sigmann Elektronik). All slices were prepared in sagittal orientation. Following decapitation under isoflurane anesthesia, brains were removed and immediately placed in cold (1–2°C) artificial cerebrospinal fluid (ACSF) solution containing (in mM) 124 NaCl, 26 NaHCO3, 1.25 NaH2PO4, 2.5 KCl, 2 CaCl2, 2 MgCl2, and 10 glucose equilibrated with 95% O2-5% CO2, pH 7.3–7.4. After incubation in ACSF for 1–2 h at 32°C, slices were transferred to a recording chamber mounted on the stage of an upright microscope (Axioskop 2 FS plus; Carl Zeiss Microimaging, Thornwood, NY) and perfused continuously with ACSF (32°C). GnRH neurons were identified through their GFP expression using epifluorescent excitation at 470 nm with a ×60 water immersion Olympus objective lens.
Pipettes (9–12 MΩ) were made from borosilicate glass capillary tubes (AM Systems, Carlsborg, WA), using a pipette puller (PC-10; Narishige, Tokyo, Japan), and coated with Sylgard 184 (Dow Corning, Midland, MI) to minimize pipette capacitance. Pipettes were filled with (in mM) 140 K-gluconate, 10 HEPES, 0.2 EGTA, 6 NaCl, 2 MgCl2, 4 NaATP, 0.4 NaGTP, 0.05 spermine, and 5 glutathione with 0.5% biocytin.
Live cell filling with biocytin.
After the whole cell recording configuration was achieved, GnRH neurons were filled with biocytin via diffusion of biocytin from the pipette solution. Neurons were filled for 15–20 min. Slices containing biocytin-filled GnRH neurons were placed immediately in 4% paraformaldehyde. Twenty-four hours later, biocytin-containing neurons were identified with Cascade Blue Conjugate antibody (A-2663, 1:100; Invitrogen, Carlsbad, CA). Slices were then washed (3 times for 10 min each) with PBS on a shaker at room temperature (RT). Slices were incubated in a blocking solution composed of 600 μl of 5% BSA, 2% Triton X-100, and 6% normal goat serum in PBS for 1.5 h with shaking at RT or overnight at 4°C. Incubated slices were immersed in 250 μl of fresh block and 2.5 μl of the Cascade Blue Conjugate antibody. Slices were incubated in this solution for 5–7 days at 4°C. Slices were washed (3 times for 10 min each) with PBS on a shaker at RT. The presence of Cascade Blue-labeled cells as well as the continued presence of endogenous GFP expression were verified by epifluorescence microscopy using a standard 4,6-diamidino-2-phenylindole and FITC filter set, respectively.
Slices with Cascade Blue-labeled and GFP-expressing cells were then reincubated in 250 μl of the fresh block, as described above, along with RU11B GnRH antibody (1:12.5 antibody; gift from H. Urbanski, Beaverton, OR). Alternatively, anti-GnRH1 (1:100) from Sigma (St. Louis, MO) was used. In both cases, standard controls for immuncytochemistry were also performed (omission of the primary antibody and separately omission of the secondary antibody). Regardless of the primary antibody, slices were incubated for 5–7 days. After incubation in the primary antibody, slices were washed three times for 10 min each with PBS on a shaker at RT. The rhodamine goat anti-rabbit secondary antibody (1:100, A11036; Invitrogen) was applied with fresh, modified blocking solution with 0.2% Triton X-100, replacing the normal 2% Triton X-100 used previously, as described above. Slices were incubated for 2 h at RT in a light-protected shaker.
Confocal acquisition.
A Zeiss LSM 510 meta laser scanning confocal microscope with Revision 4 of the acquisition software (Carl Zeiss, Heidelberg, Germany) was used to collect images. The GFP was excited with the system's Argon gas laser equipped with a 488-nm line. The rhodamine secondary was excited using a Helium-Neon laser 543-nm line. Cascade Blue was localized using a 405-nm laser. Endogenous GFP expression was visualized using an emission filter of 505–560 nm. The rhodamine secondary was visualized with an LP 560 emission filter. Cascade Blue was visualized with a LP420-nm emission filter. The location of the neurons was then mapped to schematics (7).
Experiment 2
Is episodic hormone secretion maintained in GnRH-GFP rats? For this objective, we catheterized castrated male rats at three levels of genetic expression of GFP [homozygous (HO), heterozygous (HT), and WT litter mates; n = 4 for each group]. Additionally, we examined hormone secretion in intact HO GnRH-GFP rats (n = 7) and Wistar controls (n = 4) after ~4 yr of backcrossing of the transgenic line. Hormone secretion was assessed in both objectives using sequential blood sample collection.
Surgical procedures.
Bilateral orchidectomy and catheterization were performed aseptically using isoflurane anesthesia. The tip of a catheter [a 35-mm segment of Silastic tubing; inner diameter (id) 0.3 mm, outer diameter (od) 0.64 mm] was positioned at the junction of the right atrium via the external jugular vein. The free end of the catheter was channeled under the skin and exited between the scapulae. Catheters were maintained by filling and flushing daily with heparinized saline (100 U heparin/ml saline).
Repetitive sampling.
Three to five days after catheterization, animals were attached to extension tubing to minimize disruption during sampling. Sequential blood samples were taken at 12- or 6-min intervals between 1300 and 1600, an interval during which episodic LH secretion has been demonstrated in male rats (4, 5). Sampling intervals were selected on the basis of insight from earlier studies in rats (6). Following withdrawal of each 300- to 350-μl sample, plasma was obtained by centrifugation and then dispensed into PCR tubes and frozen. Red blood cells were returned to animals through the catheter following suspension in a volume of Plasmamate (Dublin Medical, San Diego, CA) that approximated the volume of harvested plasma. At the termination of a sampling sequence, the catheter was flushed, filled with heparinized saline, and plugged with a pin.
Sampling protocols.
First, we examined pulsatile LH secretion in HO, HT, and WT males. Given the temporal relationship between GnRH and LH pulses (20), we used repetitive sampling of venous blood in male GnRH-GFP rats to determine the pattern of LH secretion as an index of episodic GnRH release. These animals were bred at Charles Rivers Laboratory during the process of backcrossing to achieve homozygosity in the animal line. Tail biopsies were subjected to quantitative PCR at Charles River Laboratories to distinguish HO and HT males.
Males of each phenotype (n = 4) were acclimated for ≥1 mo after being shipped to Emory University. Animals were derived from two separate litters. The average age (days ± SE) of HO males was 107 ± 9.1 at the time of sampling. HT males were 109.5 ± 7.3, and WT littermates were 108 ± 6.8 days of age at the time of sampling. HO, HT, and WT males had been castrated at 38 ± 4.2, 43.8 ± 2.1, and 43.8 ± 2.1 days, respectively, prior to sampling. These males were sampled at 12-min intervals.
Second, we examined episodic hormone secretion [LH and testosterone (T)] in intact HO males (n = 7) after the animal line had been bred for ~4 yr and compared their hormone secretion with that of normal Wistar controls (n = 4), the background strain of the transgenic line (16). Average age of animals was 121.5 ± 8.0 and 112.7 ± 7.8 in control and transgenic animals, respectively. Samples were taken at 12-min intervals. Finally, we examined hormone secretion in males following short-term castration. Males (n = 9) had been castrated and catheterized 3.9 ± 1.2 days earlier and were 97.4.4 ± 13.2 days of age (range: 70–120 days of age). Surgical and sampling procedures were performed as described above. However, samples were taken at 6-min intervals to capture the initial increase in LH pulses that occurs following short-term castration (5).
Serum was collected and stored at −20°C until assay. LH and T levels were assayed by the Ligand Assay and Analysis Core Laboratory at the University of Virginia. The LH assays used the RP-3 standard. Minimum detection was at 0.07 ng/ml with 2.8 and 8.0% intra- and interassay coefficients of variation, respectively. The total T assays (Siemens Medical Solutions Diagnostics, Los Angeles, CA) had minimum detection at 10 ng/dl. Intra- and interassay coefficients of variation were 3.5 and 8.3%, respectively.
Pulse analysis.
Statistically significant episodes of hormone secretion were defined using PULSAR (21). The following G values were used for analysis of LH pulses: G1 = 4.4, G2 = 2.6, G3 = 1.92, G4 = 1.46, and G5 = 1.13. The pattern of T secretion was analyzed using the same program, with the following G values: G1 = 4.2, G2 = 2.56, G3 = 1.82, G4 = 1.36, and G5 = 1.05 (14). Cluster analysis was also performed on hormone secretion using Cluster8 (Pulse XP Software, Version 2.0) (15, 35). The presence of a peak is defined as a significant increase followed by a significant decrease. A nadir is defined as a significant decrease followed by a significant increase. We tested 1 × 2 clusters (i.e., the no. of points used in testing peaks against nadirs).
Electrophysiological Recordings
Long-term recordings were performed in slices derived from 15 males. In seven of these animals, repetitive blood sampling had been performed the preceding day. Recordings were performed between 1200 and 1700, an interval that approximated the times of day of the repetitive blood sampling. We used the cell-attached mode with low-resistance seals (~30–50 MΩ). We have previously validated our long-term recording approach using Axon Instrument's 2B Axoclamp (Axon Instruments, Union City, CA) in current clamp mode as the primary amplifier used in series with a second amplifier (AM Systems 3000) to increase signal detection and allow for detection of action potentials (12). Data were acquired at 10 kHz using p-Clamp software (version 10.0; Molecular Devices, Union City, CA). At the time of recording, anatomic locations of neurons were visually mapped to schematics (7).
Analysis of electrophysiological recordings.
Spike discrimination was performed in Matlab version 7.12.0 (R2011a) using custom scripts. Action potential times were used to compute frequency. Frequencies were then reported in 30-s and in some cases 5-s time bins. Coefficients of variation on frequency were also determined.
Cluster analysis.
Event detection was performed using Cluster8 (15, 35). We tested 3 × 1 clusters (i.e., the no. of points used in testing peaks against nadirs) using a coefficient of variation of 1 for each data series and symmetrical t-statistics (t = 5) to identify both a significant increase and a significant decrease in the data. The threshold value for a peak was 2 Hz so that peaks at or <2 Hz were not included in the analysis.
RESULTS
Figure 1 shows GFP-positive neurons (A and D), biocytin detection using avidin-Cascade blue (B and E), and GnRH peptide immunocytochemistry (C and F). All biocytin-filled neurons (n = 23; 16 animals) with GFP expression were positive for GnRH. Somata and dendrites (Fig. 1C) and somata and axons (Fig. 1F) were immunopositive for the GnRH peptide. Biocytin-filled neurons were distributed throughout the rostral hypothalamus (Fig. 1, G–I).
Verification of gonadotropin-releasing hormone (GnRH)1 peptide content in GnRH neurons from GnRH-green fluorescent protein (GFP) transgenic rats. A and D: endogenous GFP fluorescence in hypothalamic slices. B and E: dectection of biocytin subsequent to cell filling in the same neurons shown in A and D. C and F: immunocytochemistry for the GnRH1 peptide in the above neurons. Panels G–I: location of biocytin-filled GFP neurons. Locations of 3 filled neurons were not noted after biocytin filling. AC, anterior commissure; OX, optic chiasm; ARC, arcuate nucleus; VB, vertical band.
Figure 2 shows representative profiles of LH release in castrated GnRH-GFP-positive male rats (HO and HT) and their WT littermates. All animals exhibited intermittent LH secretion, including HO (Fig. 2, A and B) and HT animals (Fig. 2, C and D) and WT littermates (Fig. 2, E and F). Parameters of hormone secretion did not differ between genetic groups (Fig. 2G).
Pulsatile release of LH in castrated male GnRH-GFP rats based on 12-min sampling. A and B: secretion in homozygous (HO) males. C and D: secretion in heterozygous (HT) males. E and F: secretion in wild-type (WT) males. G: characteristics of pulsatile hormone secretion between HO, HT, and WT males.
Robust intermittent secretion of LH was observed in both Wistar males and GnRH-GFP transgenics generated on the Wistar background (Fig. 3, A and B) after 4 yr of backcrossing. Transgenic males exhibited larger LH pulses than Wistar males (Fig. 3C). In both animal lines, pulses of T were detected (Fig. 3, D and E). Groups did not differ in any quantitative aspect (Fig. 3F).
Pulsatile release of LH and testosterone (T) in male GnRH-GFP rats. A: secretion of LH in transgenic (TG; HO) males. B: secretion of LH in a Wistar (CN) male. C: characteristics of pulsatile LH secretion in TG and CN males. D: T secretion in a TG male. E: T secretion in a CN male. F: characteristics of pulsatile T secretion between TG and CN males. *Significant pulses.
Representative profiles of LH secretion based on intensive sampling (q = 6 min) from castrated HO GnRH-GFP males are shown in Fig. 4, A–D. Mean LH levels following castration were 2.70 ± 0.20 ng/ml (Fig. 4E). Pulse frequency was 11.9 ± 0.14 pulses/3 h (range: 11–12 pulses/3 h; Fig. 4E). Average pulse amplitude was 1.6 ± 0.94 ng/ml.
A–D: rapid sampling (at 6-min intervals) reveals robust pulsatile hormone secretion in acutely castrated (~4 days postcastration) males. E: characteristics of pulsatile LH secretion in acutely castrated TG males.
Electrophysiological recordings were obtained from 79 GnRH neurons in 15 castrated males. Since we utilized two electrophysiological stations operating at the same time and some of the neurons were recorded simultaneously as pairs in the same slice, some of the neurons were recorded from the same clock times.
Figure 5A shows locations of all GnRH neurons (indicated as dots) from which electrophysiological recordings were obtained; location of paired recordings is indicated within squares. On average, 5.3 ± 1.8 neurons/animal were recorded. In Fig. 5B, each bar indicates the average action potential frequency for individual animals. The overall average action potential frequency in GnRH neurons was 1.70 ± 0.02 Hz. Of the 79 GnRH neurons from which we obtained recordings, 51 neurons had average frequencies that were <2 Hz. Some of these neurons never had frequencies >2 Hz (n = 20). Additionally, the firing pattern from 15 of these 79 total neurons exhibited relatively continuous patterns of firing based on coefficients of variation. In seven animals, we obtained both intensive sampling (at 6-min intervals) and recordings from their GnRH neurons the next day. Based on cluster analysis, the average number of pulses was 3.9 ± 0.6/h. The average number of episodes of firing was 4.0 ± 0.6/h.
A: anatomic location of GnRH neurons from which long-term electrophysiological recordings were obtained. GnRH neurons are indicated by dots. Pairs of GnRH neurons are enclosed in squares. Schematic is reproduced from Ref. 7 with permission. B: mean firing rates (in Hz) of recorded GnRH neurons. Each bar indicates the frequency of an individual neuron. C: raw data from a cell-attached recording of a GnRH neuron.
Figure 5C shows a portion of an electrophysiological recording of 1-h duration. The average frequency in this neuron was 1.4 ± 0.09 Hz. However, this GnRH neuron exhibited an intermittent profile of activity during which periods of high-frequency action potentials were interrupted by brief periods of quiescence.
To facilitate viewing the entire recording period, the frequency of action potentials was plotted as histograms of 30-s duration. Figure 6, A and D, shows frequency histograms of all neurons recorded from two animals. In the histogram, each neuron is numbered. When the same number is indicated on two plots, this means the neuron exhibited intermittent activity. In the first animal, recordings were obtained from four GnRH neurons. Because we used two recording stations, three of these four cells were recorded with overlapping clock times. One neuron exhibited a continuous pattern of firing. Two of the three remaining neurons exhibited episodes with action potential frequencies of >2 Hz (indicated by the lines in Fig. 6A). Activity of these neurons is plotted with an expanded time course in Fig. 6, B and C. Each neuron had an initial period of activity followed by a period of relative quiescence and then by a second increase in discharge frequency.
Spike frequency histograms (30-s bins) of all neurons from 2 animals. A and D: all recorded neurons from representative animals. Each neuron in A and D is indicated by the number on the plot of that cell's activity. When a number appears twice, this indicates that the activity of the cell decreased for a period of time and then resumed spiking activity. B, C, E, and F: expanded spike histograms (5-s bins) from neurons indicated below lines in A and D. Different color histograms (dark gray, black, white, light gray) indicate different neurons. In all graphs, open sides of histograms indicate initiation or termination of recordings.
In a second animal, recordings were obtained from five GnRH neurons and included two paired recordings (Fig. 6D). One GnRH neuron exhibited a continuous firing pattern. Two additional neurons had only short intervals with frequencies >2 Hz. One neuron exhibited intermittent activity (see Fig. 6E for expanded time scale). The remaining neuron whose activity was >2 Hz is plotted on an expanded time scale in Fig. 6F. This neuron exhibited only one time period of activity >2 Hz.
DISCUSSION
Transgenic animals with GFP-expressing GnRH neurons represent a major advance in studying the mechanisms underlying pulsatile GnRH release. However, their value depends on preservation of normal physiological function in the genetically altered GnRH neuron and in the in vitro preparations in which these neurons are studied. The present study took an integrative approach to answering this question regarding the function of GnRH neurons in a transgenic rat model where GnRH neurons express GFP.
Our results indicate that GFP-expressing neurons in live slices from GnRH-GFP rats are GnRH-containing neurons. Kato et al. (16) reported immunostaining for GnRH following fixation of hypothalamic sections and of GnRH somata in culture from these animals. Another study indicated that 94% of soma (and axons) were GnRH1-immunopositive cells but that only 82% of GnRH1-immunopositive cells were GFP positive (27). Finally, Yin et al. (36) indicated that these GFP-containing neurons are positive for GnRH mRNA.
Our findings confirm that, in hypothalamic slices prepared for electrophysiological recordings, neurons selected on the basis of GFP expression are subsequently immunopositive for GnRH. Most of our biocytin-filled neurons resided in the locations that were identified earlier as containing GFP neurons that were positive for GnRH (27). The GFP fluorescence in these neurons is comparable with that in GnRH-GFP transgenic mouse models. Thus, GFP provides a reasonable beacon for locating GnRH neurons in live slices prepared from these rats.
As indicated by LH pulses, episodic GnRH release appears to be maintained in GnRH-GFP male rats. This finding is consistent with our earlier findings (33) in the males of one strain (30) of GnRH-GFP mice. Moreover, our assessments of pulsatile LH secretion suggest that the magnitude of GFP expression (i.e., HO, HT, or WT) does not alter hormone secretion. To the best of our knowledge, this is the first time that such an examination has been performed in any of the rodent GFP-GnRH models. Furthermore, these experiments indicate that intermittent LH and T release in transgenic male rats is maintained over several generations of backcrossing of homozygous animals. Finally, based on 6-min blood samples, these transgenic males exhibit pulsatile LH frequencies that are similar to those in other short-term castrated male rats that do not express GFP in their GnRH neurons (5). Taken together, it seems that GnRH-GFP rats possess a fully functional GnRH pulse generator despite the presence of GFP in their GnRH neurons.
Until the advent of animals with GnRH-GFP neurons, we relied extensively if not exclusively on hypothalamic multiunit activity (MUA) for insight into the relationship between neuronal activity and reproductive hormone secretion. It is evident that there are neuronal elements in the hypothalamus that exhibit activity coincident with LH secretion (34, 35). Alterations in MUA are consistent with changes in pulsatile LH secretion in response to several physiological cues (24–26). Moreover, so-called positive electrodes (those with increasing MUA during LH secretion) were either adjacent to GnRH somata or enmeshed in GnRH fibers (28).
In the present study, we detected high-frequency discharges from single GnRH neurons in hypothalamic slices derived from castrated male rats. At times, the frequency of action potentials reached as high as 15 Hz. This frequency is consistent with neuropeptide release (19). However, the average frequency in single GnRH neurons was considerably lower than 15 Hz (<2 Hz). The discrepancy in action potential frequencies reflects the intermittent nature of activity in some GnRH neurons. An earlier study in ovariectomized mice reported similar cyclic activity in GnRH neurons in hypothalamic slices (22). However, the pattern of activity of GnRH neurons in wild-type rats is unknown, reflecting the difficulty of locating GnRH neurons without the presence of a marker for idenification.
The above consideration notwithstanding, the intermittent nature of activity in GnRH neurons from transgenic rats in the present study tempts one to speculate that if activity similar to these patterns occurs in vivo, there would be hormone release. This notion is based on findings in other neurosecretory neurons where intermittent activity is responsible for hormone release (19). However, the pattern of electrical activity that results in GnRH secretion remains a matter of speculation. Moreover, it should be noted that many of the recorded GnRH neurons exhibited patterns of action potential discharges that were not intermittent. Instead, in these GnRH neurons, activity was continuous throughout the duration of recordings. Cardenas et al. (1) found that, in rhesus monkeys, single units within the MUA volley exhibit increases (60% of single units), decreases (10%), or no change (30% of single units) in firing rates associated with LH secretion. They concluded that it was unlikely that single units whose activity was unchanged during MUA/LH secretion were related to GnRH pulse generator activity. Finally, many GnRH neurons exhibited only low-frequency discharges that are unlikely to result in GnRH secretion. Given the observation of continuous activity in a substantial portion of single GnRH neurons and weak activity in others, it seems possible that these cells may underlie some functions other than reproductive hormone secretion.
It is possible that, in our data set of 79 recordings, we failed to record from a GnRH neuron that was actively involved in GnRH secretion during the moments of recording. Hypogonadal mice, which fail to produce biologically active GnRH, experience restoration of reproductive hormone secretion even if the transplanted neurons that are positive for GnRH are remarkably sparse (9, 18). More recent evidence using a molecular approach has suggested that as few as 20 GnRH neurons can sustain reproductive function in mice (13). Such studies indicate that relatively few GnRH neurons can support hormone secretion, but it is unknown how many GnRH neurons actually do participate in endogenously driven hormone secretion. These considerations preclude investigators from knowing whether any given recording was obtained from a GnRH neuron that had been involved in hormone release in the living animal.
The degree to which the GnRH pulse-generating mechanism is maintained in hypothalamic slices is unknown. In the present study, we used the same analysis paradigm (cluster) on both blood sampling data and electrophysiological data from the same animal. In this group of animals from which both hormone and electrophysiological data sets were obtained, the cluster algorithm detected pulses of hormone in living animals at roughly the same frequency as episodes of electrical activity in remaining neurons exhibiting intermittent activity. Thus, it is tempting to speculate that the mechanisms that comprise the GnRH pulse generator are maintained in the sagittal hypothalamic slice preparation.
The present data notwithstanding, a majority of postsynaptic currents in GnRH neurons in hypothalamic slices are action potential independent (2). This form of neurotransmitter release differs from the action potential-dependent release in the intact nervous system. The nature of this release most likely reflects the severing of afferent fibers that control GnRH neurons and their synaptic boutons, which remain apposed to GnRH somata (3). Some of these severed inputs may have contributed to the pulse-generating mechanism. Nonetheless, it seems reasonable to suggest that some local circuitry is preserved in hypothalamic slices and that this circuitry retains the ability to modulate critical aspects of the activity of GnRH neurons (2, 31, 32).
GRANTS
This work was supported by National Institute of Child Health and Human Development Grants HD-045436 and HD-049664 and funds from the Arthur C. Guyton Award from the American Physiological Society, the Mathematical Biosciences Institute, and the National Science Foundation (NSF) under Grant DMS-0931642 (J. Best and D. Schmidt) as well as NSF Career Grant DMS-0956057 (J. Best). J. Best is an Alfred P. Sloan Research Foundation Fellow. We thank M. Kato for providing founders from their group's GnRH-GFP rat line. We thank H. Urbanski for generous provisions of HU 11B (GnRH primary antibody). Assays for LH and T were performed at University of Virginia, Center for Research in Reproduction, Ligand Assay and Analysis Core [NICHD (SCCPRR) Grant U54-HD-28934].
AUTHOR CONTRIBUTIONS
V.L.G., D.S., J.A.B., L.O., and K.J.S. did the conception and design of the research; V.L.G., P.J.H., O.P.O., L.O., and K.J.S. performed the experiments; V.L.G., O.P.O., and K.J.S. interpreted the results of the experiments; V.L.G., D.S., O.P.O., Z.H.Z.H., L.O., and K.J.S. edited and revised the manuscript; V.L.G., P.J.H., D.S., O.P.O., Z.H.Z.H., J.A.B., L.O., and K.J.S. approved the final version of the manuscript; P.J.H., D.S., O.P.O., Z.H.Z.H., J.A.B., and K.J.S. analyzed the data; D.S., O.P.O., Z.H.Z.H., and K.J.S. prepared the figures; O.P.O., L.O., and K.J.S. drafted the manuscript.
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Gonadotropin-releasing hormone neuronal cell lines.
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Cited by: 57 articles | PMID: 1468602
Review
Funding
Funders who supported this work.
NICHD NIH HHS (4)
Grant ID: U54-HD-28934
Grant ID: R01 HD045436
Grant ID: HD-045436
Grant ID: HD-049664