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-Aminobutyric AcidA Receptor Manipulation on Migrating Gonadotropin-Releasing Hormone Neurons through the Entire Migratory Route in Vivo and in Vitro1
Eunice Kennedy Shriver Center, Division of Biomedical Sciences, Waltham, Massachusetts 02452
Address all correspondence and requests for reprints to: Dr. Elizabeth Bless, Eunice Kennedy Shriver Center, 200 Trapelo Road, Waltham, Massachusetts 02452. E-mail: ebless{at}shriver.org
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-aminobutyric acid (GABA) present in GnRH
neurons during development. To clarify the influence of GABA across the
entire GnRH migration route, we examined the effects of muscimol and
bicuculline (GABAA agonist and antagonist) in
vivo and in vitro, maintaining the integrity of
the nasal-forebrain connection. For in vivo experiments,
mice were administered muscimol, bicuculline, or vehicle on days 10–15
of pregnancy and were killed on embryonic day 15 (E15). For in
vitro experiments, 250-µm parasagittal slices of whole heads
of E13 mice were incubated with muscimol, bicuculline, or vehicle for 2
days. Muscimol inhibited GnRH cell migration and decreased extension of
GnRH fibers. Bicuculline treatment led to a disorganized distribution
of GnRH cells in the forebrain and a concomitant dissociation of GnRH
cells from fibers of guidance. These results suggest that GABA’s
influence on GnRH development changes as the cells move out of the
nasal compartment and extend processes toward the median eminence. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In adults the neurotransmitter -aminobutyric acid (GABA) may play an
important role in GnRH synthesis and release (5). In development, GABA
is present in cells and fibers along the GnRH migratory route
throughout the nasal compartment (4, 6, 7). In embryonic olfactory
explant cultures, endogenous GABAergic input to GnRH neurons was
demonstrated (8). In adult and juvenile rats, the messenger RNA for
GABAA receptor subunits has been colocalized with
GnRH messenger RNA (9, 10) and was also found in the GT1–7 cell line,
which secretes GnRH in response to GABA receptor agonists (11).
GABA may play a role in neuronal migration (12, 13, 14, 15). Specific agonists for GABAA, GABAB, and GABAC receptors increased the rate of cortical cell movement, whereas antagonists of GABA receptors inhibited the increased migratory rate induced by GABA (14). An exception was bicuculline, a GABAA antagonist that had a tendency to increase the rate of cell motility. Therefore, although activation of GABAB and/or GABAC receptors may increase cell motility, activation of GABAA receptors may actually inhibit cell migration (14, 15).
Preliminary data from our laboratory using an in vitro slice preparation that maintains the connectivity of the entire migration route (16) showed that GABA inhibited the rate of GnRH neuronal migration through the nasal compartment and was reversed by coadministration of a GABAA/GABAC receptor antagonist (17). Recently, Fueshko et al. (18) showed that the rate of migration of embryonic GnRH neurons from olfactory placode explants was decreased after muscimol (GABAA agonist) treatment compared with that after bicuculline treatment. A key element excluded from previous findings is an understanding of GABA’s role across the diverse span of the entire GnRH migratory route. The present study was conducted to address this issue by analyzing the effects of GABAergic drugs both in vivo and in vitro.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An additional experiment was conducted to investigate the influence of a GABAA antagonist on the association of GnRH neurons and the caudoventral extension of the VNN in the forebrain using high resolution confocal microscopy. Four additional litters were injected sc (two control and two bicuculline) as described above to obtain E15 embryos for this experiment.
In vitro studies
Timed pregnant C57BL/6J mice were anesthetized as described
above on E13 (crown-rump length, 
10 mm) to investigate the in
vitro influence of a GABAA agonist and an
antagonist on GnRH neuronal migration. In vitro procedures
were described previously (16). Briefly, embryonic heads were embedded
in 8% low gelling temperature agarose (type VIIa, Sigma,
St. Louis, MO), and parasagittal sections were cut at 250 µm using a
Vibratome and placed in cold sterile-filtered Krebs buffer containing
HEPES buffer (0.01 M), penicillin (100 U/ml),
streptomycin (0.1 mg/ml), and gentamicin (0.1 mg/ml). The
sterile-filtered Krebs buffer was then replaced with Eagle’s MEM
containing 10% FCS (HyClone Laboratories, Inc., Logan,
UT), 0.5% glucose, penicillin (133 U/ml), streptomycin (0.13 mg/ml),
and glutamine (1.32 mM) and subsequently placed
for 35 min in an incubator set at 5% CO2 and 36
C. Slices were then washed with the medium described above and placed
onto coverslips (24 x 60 mm) that had previously been coated with
poly-L-lysine (0.5 mg/ml) and Vitrogen (1.5
mg/ml; Cohesion, Palo Alto, CA) and were maintained in 100-mm
culture dishes. Excess medium was drawn off coverslips with a 26-gauge
syringe, and slices were placed in the incubator for 1 h. Each
slice was then coated with approximately 25 µl of a mixture made from
1 ml vitrogen (3 mg/ml), with 125 µl 10 x MEM, 23 µl
penicillin (10,000 U/ml)/streptomycin (10 mg/ml), and 33 µl 1
M sodium carbonate. After 1.5 h, 5 ml
neurobasal medium (Life Technologies, Inc., Gaithersburg,
MD) containing 2% B-27 supplement (Life Technologies, Inc.), 0.5% glucose, penicillin (133 U/ml), streptomycin (0.13
mg/ml), and glutamine (1.32 mM) were added to
each culture dish.
Treatments in vitro
Day 0. For each experiment the slices from at least one
animal were killed on the day of slicing (E13). These slices went
through all of the steps described above until the point of plating on
the glass coverslips. At this point, the tissue was fixed in 4%
formaldehyde (made from 10% methanol-free stock solution;
Polysciences, Inc., Warrington, PA).
Day 2. All slices that were administered drugs were maintained in culture dishes for 2 days. To manipulate the degree of GABAA stimulation in vitro, stock solutions of muscimol (500 µM) and bicuculline (1 mM) were constituted in sterile-filtered distilled water. Culture dishes containing 5 ml medium were given 50 µl muscimol (for 5 µM), bicuculline (for 10 µM), or water vehicle. All medium was changed and fresh drug treatments given on the afternoon of day 1 of incubation. On the afternoon of day 2 all slices were fixed for 15 min with 4% formaldehyde (methanol-free) and then placed into 0.1 M PB until processing for immunocytochemistry.
Immunocytochemistry
To detect immunoreactive GnRH (GnRH-ir) a rabbit polyclonal
antisera (LR-1, provided by Dr. Robert Benoit) was used at a
concentration of 1:10,000. Immunocytochemical procedures were
previously described (6, 16). Briefly, sections or slices (at 4 C) were
pretreated with 0.1 M glycine in 0.05 M PBS (pH
7.5; in vivo sections only) followed by 0.5% sodium
borohydride in 0.05 M PBS and 5% normal goat
serum with 0.3% Triton X-100 (Tx)/PBS and 1% hydrogen peroxide.
Washes with PBS separated each step, and times were extended for
250-µm thick slices vs. 50-µm thick sections. Tissue was
then incubated with the LR-1 antiserum for 2 nights (in vivo
sections) or 6 nights (in vitro slices). For secondary
antibody processing, tissue was washed with PBS/1% normal goat serum
with 0.02% Tx before incubation with goat antirabbit biotinylated
secondary antibody (Vector Laboratories, Inc., Burlingame,
CA) for 2 h at room temperature (in vivo sections) or
overnight at 4 C (in vitro slices). After washes in
PBS/0.02% Tx (all at room temperature), tissue was incubated with
Vectastain ABC reagent (Vector Laboratories, Inc.). Black reaction product was produced in 50-µm tissue
sections using 0.25% 3,3'-diaminobenzidine (DAB; dissolved in
Tris-buffered saline) with 0.2% nickel ammonium and 0.02% hydrogen
peroxide. Brown reaction product was produced in 250-µm slices using
DAB in PBS without nickel.
The 250-µm slices were double labeled to reveal peripherin-containing fibers by incubating DAB-reacted slices in a rabbit polyclonal antiserum directed against peripherin (Chemicon, Temecula, CA) at a concentration of 1:5000 for 3 nights. This was used for labeling of olfactory axons, which demarcates a large portion of the migratory pathway for GnRH neurons (3). The fluorophore cy3 conjugated to a donkey antirabbit secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was used for visualization.
Double fluorescent immunocytochemistry was performed for tissue collected for use with confocal microscopy. To detect immunoreactive GnRH, a mouse monoclonal antibody (QED Bioscience, San Diego, CA) was used at a concentration of 1:5000. Fluorescein isothiocyanate conjugated to an antimouse secondary antibody (Jackson ImmunoResearch Laboratories, Inc.) was used for visualization. To detect immunoreactive peripherin, the same primary and cy3-coupled secondary antibodies listed above were used.
All tissue was mounted onto gelatin-coated slides. The tissue sections from the in vivo experiments in which DAB was used for visualization were dehydrated and coverslipped using Permount (Fisher Scientific, Suwanee, GA). The fluorescent tissue sections from the in vivo experiment were coverslipped using Vectashield reagent (Vector Laboratories, Inc.), and the fluorescent 250-µm thick slices from the in vitro experiment were coverslipped using an aqueous mounting media (Accurate Chemical and Scientific Corp., Westbury, NY).
Data analysis
In vivo: cell and fiber distributions. The number of
cells containing GnRH-ir was counted manually at x40 magnification
using an Olympus Corp. BH-2 brightfield microscope (New
Hyde Park, NY). Cells were counted as being within the nasal
compartment if they were located in the region from the rostral tip of
the tissue to the cribriform plate. Cells were counted as being within
the olfactory bulb if they were located in the region between the
cribriform plate and the caudal extension of the VNN before its
defasciculation. Cells were counted as being within the brain if they
were located in any region beyond the cribriform plate and not in the
olfactory bulb. All counts were performed with the experimenter blinded
to drug treatment. Counts for each subject represent half the total
number of GnRH neurons, as the sections were placed in alternating
boats. A subjective rating of the organization of cells in the brain
was also made blind to treatment and was based on an analysis of
photomicrographs. Sections for subjective ratings were chosen on the
basis of consistent angle and a large number of cells. Subjective
ratings ranged from 4 (excellent organization: clear, strong pathway
with uniform orientation of cells and fibers present) to 1 (poor
organization: scattered cells, disoriented fibers with no apparent
pathway). A rating of 2 was assigned for a disturbed cell and fiber
pattern and little apparent pathway, and a rating of 3 was assigned
when there were minor deviations from a uniform pattern of cells and
fibers.
An analysis of the locations of fibers containing GnRH-ir was also performed. The percentage of sections containing pituitaries that had GnRH-ir in fibers in the adjacent brain tissue was determined (number of sections with fibers present at the pituitary/total number of sections with pituitaries).
High resolution laser scanning confocal microscopy
The association of GnRH-ir cells with peripherin-ir fibers was
analyzed using high resolution laser scanning confocal microscopy. The
area of GnRH neuron migration into the brain, as shown in Fig. 3
, was
identified and divided into 2 fields (67 x 67 µm each). The
first field was the area where VNN fibers begin to defasciculate on
their descent into the forebrain. Field 2 was an adjacent field 67 µm
deeper into the forebrain. Each field contained a minimum of 3 GnRH
neurons. Individual scans through a field were a maximum of 2 µm
thick, and each field typically required 16 scans for a total depth of
32 µm. Without knowledge of treatment, 1 investigator went through
each scan and determined for each GnRH-ir cell whether it was touching
a peripherin-ir fiber. The percentage of cells in each field that were
in association with a peripherin-ir fiber was calculated. Two sections
from each animal were analyzed, and the mean percentage was taken as
data for each animal.
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Slices that met the above criteria were mapped and counted by outlining the slice and marking GnRH-ir cells using a neuron tracing system (SunTechnologies, Inc., Raleigh, NC) attached to an Olympus Corp. BH-2 microscope. The boundaries between the nasal compartment and olfactory bulb and between the olfactory bulb and brain were the same as those for in vivo sections (see above).
Statistical analyses
All data were analyzed using the JMP3.2.2 computer package
(SAS Institute, Inc., Cary, NC). Data from the three
experiments were analyzed separately. A one-way ANOVA was run for each
measure in each of the experiments (except the confocal microscopy
experiment, see below). For the in vivo experiment this
consisted of three levels (vehicle, muscimol, and bicuculline
treatment), and for the in vitro experiment this consisted
of four levels (day 0 and day 2 control, muscimol and bicuculline). The
data from the confocal microscopy experiment were run as a mixed
repeated measures/between-subject design, with field as the
within-subject variable and treatment as the between-subject variable.
Where a significant overall ANOVA was found, orthogonal contrasts and
post-hoc comparisons using Tukey-Kramer highest significant
difference test or Dunnett’s method were performed where appropriate
to further clarify significant differences between individual treatment
groups.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The mean numbers of
cells that were immunoreactive for GnRH per subject (representative of
half of the total head; ±SEM) were 675.21
± 22.37, 679.29 ± 27.28, and 701.82 ± 31.18 for control
(n = 19), bicuculline-treated (n = 14), and muscimol-treated
(n = 17) animals, respectively, and did not differ significantly
(Fig. 2A).
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). This inhibition was
measured by an increase in the percentage of GnRH-ir cells in the nasal
compartment of animals treated with muscimol [F(2,47) = 6.00;
P < 0.005; by Dunnett’s test, P <
0.05, control compared with muscimol; Fig. 2B]. There was a tendency
for muscimol treatment to decrease the percentage of GnRH-ir cells in
the brain compartment, although this did not reach significance. The
percentage of GnRH-ir cells in the AOB and OB did not differ
significantly in any treatment group (data not shown).
GABAA receptor manipulation influences
GnRH-ir cell distribution and fiber association in the forebrain
A measure of organization of GnRH-ir cells beyond the cribriform
plate and into the forebrain was strongly affected by
GABAA antagonism (Fig. 3), even though the percentage of cells
in different compartments along the migratory route was relatively
unaffected. Bicuculline treatment resulted in a reliably higher rating
of poor organization, as measured by the appearance of cells that were
scattered in many directions and with processes oriented in multiple
directions [F(2,47) = 8.11; P < 0.001; by
Dunnett’s test, P < 0.05, bicuculline compared with
control].
To investigate potential bases for the disorganized appearance of
GnRH-ir cells in the forebrain of bicuculline-treated subjects, an
additional study using confocal microscopic analysis was carried out
(Fig. 4). As expected, the percentage of
GnRH-ir cells in close association with peripherin-ir fibers
(olfactory-derived fibers constituting the caudoventral extension of
the VNN) decreased as they were found further away from the OB and into
the basal forebrain [for association of cells with fibers between
fields 1 and 2; F(1, 12) = 6.14; P < 0.005].
Importantly, inhibiting GABAA receptors (with
bicuculline) significantly decreased the association of GnRH-ir cells
with the caudoventral extension of the VNN. The decreased association
was quantified by a significant reduction in the percentage of GnRH-ir
cells associated with peripherin-ir fibers across fields [F(1, 12)
= 7.89; P < 0.05]. Post-hoc comparisons
revealed that the difference between the two treatment groups was
greatest at field 2 (P < 0.05).
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, A and B].
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), and immunoreactive cell bodies and
fibers were similar in morphology to in vivo sections (Fig. 1). The mean numbers of cells that were GnRH-ir per slice
(±SEM) were 600.12 ± 45.39 (day 0; n
= 8), 552.5 ± 26.58 (control; n = 8), 498.67 ± 43.54
(bicuculline; n = 6), and 463.14 ± 39.75 (muscimol; n =
7), with no significant difference among groups (P >
0.05; Fig. 7A).
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and 7, B and D). The migration
of GnRH-ir cells over the 2 days in vitro was indicated by a
significant decrease in the percentage of GnRH-ir cells in the nasal
compartment [F(3, 25) = 5.38; P < 0.005].
Concordantly, there was an increased percentage of cells in the brain
compartment [F(3, 25) = 6.37; P < 0.005] between
day 0 and day 2 control treated slices (by Dunnett’s test,
P < 0.05).
Muscimol treatment decreased GnRH-ir neuron migration from the
nasal compartment to the brain over 2 days in vitro (Fig. 7B). The percentage of cells in the nasal compartment of
muscimol-treated slices was similar to that in slices killed on day 0,
indicating little migration. By contrast, control and
bicuculline-treated slices had a significantly smaller percentage of
cells in the nasal compartment compared with day 0 slices, indicating
significant migration (P < 0.05, by Dunnett’s test).
There was a difference in the percentage of neurons in the OB between
treatments as well [F(3, 25) = 4.53; P < 0.05],
which was due to a smaller percentage in muscimol-treated slices than
in day 0 slices (P < 0.05, orthogonal contrast).
Lastly, all day 2 treatment groups differed from day 0 slices in the
increased percentage of cells in the brain (P < 0.05,
by Dunnett’s test).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The ability to examine GnRH neuronal migration in vitro
provides significant advantages for controlling the neuronal
environment (16, 22, 23, 24). Interpretation of previous in
vitro studies is difficult when low numbers of GnRH neurons are
accounted for (18, 25, 26). However, the slice preparation used in the
current experiments maintains the majority of the GnRH-ir cell
population (16). Thus, the number of immunoreactive cells observed
in vitro was similar on the initial day of plating (E13),
after 2 days in vitro, and after perfusion on E15
(600/slice or half-brain). Changing locations of GnRH-ir cells in
the current in vitro experiments probably represent total
net migration because cells were not unaccounted for due to loss of
detectable GnRH or death. Furthermore, as the slices contained an
intact region around the cribriform plate, the interactions and
differences between the nasal and brain compartments could be
evaluated.
In the current study the GABAA receptor agonist muscimol inhibited GnRH cell migration out of the nasal compartment after either in vivo or in vitro administration, as evidenced by an increase in nasal compartment neurons and a tendency to decrease the percentage of GnRH-ir cells in areas outside of the nasal compartment. This agrees with others, who found inhibitory effects of GABAA receptor activation on cell motility (15, 18). The muscimol-dependent decrease in cells beyond the cribriform plate was seen as a decrease in OB cells in vitro and a significant decrease in the brain compartment in vivo. This result is consistent with a shorter length of treatment (2 days in vitro compared with 6 days in vivo) decreasing the percentage of cells closer to the area actually affected (nasal compartment). The inhibition of migration due to muscimol treatment was on the order of 25% both in vivo and in vitro. This effect could be due to a small general inhibition of all GnRH neurons or to a greater inhibition of a subset of GnRH neurons that are particularly sensitive to GABA receptor manipulation. In our previous studies of cells containing GABA-ir in the nasal compartment, we found a subset of cells that contained both GABA-ir and GnRH (17), and the approximate percentage in mice was similar to the inhibition seen in the current study. The presence of GABA within neurons has been proposed to alter their sensitivity to the migratory influences of GABA (15). It may be, therefore, that this subpopulation of cells in the nasal compartment that contains both GnRH and GABA is especially sensitive to the inhibitory influence of muscimol.
The extension of fibers to the median eminence, adjacent to the pituitary (the major destination of forebrain GnRH fibers), was also decreased after maternal muscimol treatment. This effect could be due to the decrease in cell migration into the brain, which would agree with the approximately 20–25% inhibition of fiber extension that was observed. We may not have seen an influence of bicuculline if the fibers reaching the median eminence on E15 in control animals were already at a relative maximum.
Bicuculline treatment in vivo revealed a significant influence of GABAA receptor inhibition on GnRH neurons in the brain. A disturbance of the distribution of GnRH neurons beyond the cribriform plate after bicuculline treatment was striking. High resolution confocal microscopy further revealed a significant effect of bicuculline on the association of GnRH neurons with the caudoventral extension of the VNN. The dissociation of GnRH neurons from caudally directed VNN fibers that occurs normally as migration proceeds was augmented significantly after treatment with the GABAA antagonist bicuculline. As peripherin-labeled fibers appeared similar in control and bicuculline-treated tissue, it is not likely that bicuculline disrupted the VNN fibers themselves. Therefore, the activation of GABAA receptors may promote the maintenance of the association of GnRH neurons with their fibers of guidance in the brain compartment. One possibility is that GABA influences the expression of specific molecules important for cell adhesion. For example, previous studies have linked GABA action to the polysialic acid (PSA) content of the neural cell adhesion molecule (PSA-NCAM) (27). Other studies have linked PSA-NCAM to GnRH neuronal migration (28). If GABA influences the PSA content of NCAM on VNN fibers, effects on GnRH neurons could be mediated via a PSA-NCAM-dependent mechanism(s). Another possible explanation for the disorganization of GnRH neurons in the brain after bicuculline treatment can be seen from the tendency for their migration to be increased. This was shown by a small decrease in the percentage of GnRH neurons in the nasal compartment after bicuculline treatment compared with that in controls both in vivo and in vitro. It may be, therefore, that GnRH neurons arrive in the brain compartment at an early stage after bicuculline treatment when signals on the fibers of guidance or the GnRH neurons themselves have not yet fully developed. This, in turn, may lead to a disorganization of GnRH neurons.
In summary, the current experiments demonstrate that GABA influences GnRH neurons from the time of earliest migration in the nasal compartment through their settling in positions in the basal forebrain. Previous studies (6, 7) had shown that GABAergic elements are in position to influence GnRH neuron migration in multiple species, including humans. The additional evidence of migratory influence (the current study and Ref. 18) suggest the need for an examination of the effects of prenatal exposure to GABAergic drugs on the reproductive axis of treated offspring. These observations underscore the importance of understanding the specific influences of GABA during the differentiation of the neural network that regulates gonadotropin secretion and reproductive capacity.
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Acknowledgments |
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Footnotes |
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Received August 25, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-aminobutyric
acid and gonadotropin-releasing hormone during neuronal migration
through the olfactory system. Endocrinology 137:5415–5420[Abstract]
-aminobutyric acid receptors in immortalized
luteinizing hormone-releasing hormone neurons. Endocrinology 133:1983–1989[Abstract]
-aminobutyric
acid and gonadotropin-releasing hormone in neurons during migration.
78th Annual Meeting of The Endocrine Society, San Francisco, CA,
1996
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, Significantly different from day 0, P <
0.05, by post-hoc orthogonal contrast.