194 A
GABA inhibition may be a key factor for the activation of Jackson laboratory Bar Harbor, ME. Animals were group
the GnRH pulse generator and, thus, the onset of puberty. housed n54 cage in polypropylene cages and had access
The previously discussed animal model established by to food Agway Lab Chow 3000 and water ad libitum. All
this laboratory, which mimics the clinical neuroendocrine animals were maintained from birth in a colony room
abnormalities frequently associated with VPA treatment, equipped with a controlled photoperiod 12 L:12 D, with
was recently utilized in a preliminary morphological study lights off at 18:00 h. All procedures were approved by the
exploring the effects of VPA on the normal development of Institutional Animal Care and Use Committee at the
the GnRH cell population within the mPOA [36]. Interest- University at Buffalo prior to implementation.
ingly, Snyder and Badura [36] found that chronic adminis- tration of VPA in DBA 2J mice delays the typical pattern
2.2. Drug administration of GnRH cell morphological changes occurring during
reproductive maturation. As the effects of VPA on GnRH Juvenile male DBA 2J mice were weaned at 14 days of
cell morphology are correlated with a slowing of gonadal age and were placed either on oral administration of VPA
development, it is likely that this delayed maturation of 17 mg kg day or control vehicle solution CON. Since
GnRH cells within the mPOA may lead to diminished VPA is highly water-soluble and is rapidly absorbed from
anterior pituitary activity. Such a mechanism might explain the digestive tract [3], it was administered via the drinking
the reproductive maturational delays that are observed in water to which 1 sucrose was added to increase
some epileptic patients. palatability. This dosage of VPA was calculated to corre-
Thus, if it is assumed that these morphological changes late with therapeutically relevant serum concentrations of
are functional in nature, it appears that VPA may exert at VPA when used as an anticonvulsant in the clinical
least some of its inhibitory effects on the HPG axis at the population [3]. Where appropriate, drug dosage was in-
level of the hypothalamic GnRH cell population. Taking creased in 2-week steps in order to account for develop-
into consideration both the inhibitory role of GABA as a mental differences in body weight, as well as tolerance
neurochemical regulator of GnRH cells, as well as VPA’s effects. Previous work from this laboratory has revealed
proposed GABAergic mechanism of action, it is possible that, under these conditions, individual mice consume 6–8
that VPA may mimic and or elevate the activity of the ml of fluid day and there are no differences in fluid
GABAergic system to slow normal morphological matura- consumption between the VPA and CON groups [35].
tional changes occurring within the GnRH neuronal popu- lation.
2.3. Tissue preparation The goal of the present study was to expand upon the
preliminary findings of Snyder and Badura [36] by ex- Subsets of animals n55–9 age group receiving VPA
amining the putative effects of VPA on GnRH–GABA or control solution were sacrificed via anesthetic overdose
interactions within the mPOA across pubertal develop- with sodium pentobarbital 100 mg kg. Animals were
ment, as well as in reproductively mature adult animals. As either sacrificed at 21, 24, 28, or 32 days of age for the
these mice typically become reproductively mature around developmental portion of the study experiment 1, or
30–45 days of age, the addition of adult animals to this following 6 weeks of drug administration for the adult 8
study allows for an examination of any residual effects of weeks of age portion of the study experiment 2. The
VPA treatment on the GnRH cell population after, under animals were then administered 0.1 ml of heparin and
normal circumstances, reproductive maturity should have perfused transcardially with 0.87 physiological saline,
been reached. followed by 4 paraformaldehyde in 0.1 M NaPO buffer
4
This investigation was accomplished via a dual-labeling pH 7.3 using a perfusion pump. Brains were removed
immunocytochemical technique for both GnRH and and post-fixed in the same fixative overnight. Tissue was
GABA. By using light level microscopy with immuno- then cryoprotected with 4 paraformaldehyde, 20 suc-
histochemical visualization techniques, this study allowed rose pH 7.3 for 2 days prior to frozen-sectioning 40 mm
any significant gross morphological changes occurring in through the preoptic area on a sliding microtome with a
the relationship between these two cell groups as a result freezing stage. All sections were stored in 0.1 M NaPO
4
of VPA administration to be detected. buffer pH 7.3 until immunocytochemical staining for
GnRH and GABA.
2. Methods
2.4. Dual-labeling immunocytochemistry 2.1. Animals and housing
All reactions were carried out with free-floating sections Male inbred DBA 2J mice were obtained from a
on an orbital shaker table. Sections were treated for sites of breeding colony at the University at Buffalo, which was
GnRH and GABA immunoreactivity in sequential order. In originally derived from progenitors acquired from the
between each incubation step, sections were rinsed three
A .M. Illig et al. Brain Research 885 2000 192 –200
195
times for 5 min each in 0.1 M phosphate-buffered saline data obtained in experiment 1, a two-way drug3age
with 0.2 Triton PBS-TX, pH 7.3. Firstly, sections were analysis of variance ANOVA was utilized. For the adult
incubated for 30 min in 0.3 H O . This was followed by data obtained in experiment 2, a one-way ANOVA for drug
2 2
a 30-min blocking step in 1 normal goat serum. Next, effects was used. Significant interactions were broken
sections were incubated for 24 h at 48C in rabbit anti- down using analysis of simple main effects, with Fisher’s
GnRH Incstar, LOT [ 635479, dilution51:4000, which LSD post-hoc tests where appropriate. All results were
served as the primary antibody for the first labeling considered significant if P,0.05.
sequence. The following day, there was a 2-h incubation in biotinylated goat anti-rabbit second antibody dilution5
1:100. This was followed by a 1-h incubation in Vectas- tain avidin-biotin complex ABC, Vector laboratories.
3. Results
Sections were developed using 0.1 diaminobenzidine DAB and 0.2 glucose-oxidase GOD, which produced
The total number of GnRH-immunoreactive neurons did a brownish-red reaction product.
not differ significantly among drug or age groups in either The second labeling sequence began with a re-blocking
experiment 1 or experiment 2. Stained neurons were step in 1 normal goat serum. This was followed by a
divided into unipolar and bipolar groups based upon their 24-h
incubation at
48C in
guinea pig
anti-GABA morphology. The processes of these neurons were fol-
Chemicon, LOT[ 18060181, dilution51:2000, which lowed through several planes of focus in order to ensure
served as the primary antibody for the second labeling that they extended out from the cell bodies of the neurons
sequence. The following day, there was a 2-h incubation in of interest and were not part of other GnRH neurons
biotinylated goat
anti-guinea pig
second antibody
located in proximity to them. dilution51:100. This was followed by a 1-h incubation
in Vectastain avidin-biotin complex ABC, Vector labora- tories. Sections were developed using 0.1 DAB and 8
3.1. Experiment 1: effects of VPA on GnRH–GABA nickel chloride, which produced a bluish-black reaction
interactions across development product.
There was a significant main effect of drug F 5
1,37
32.972, P,0.001, but no main effect of age on the total 2.5. Histological evaluation
number of bipolar neurons among groups. Follow-up analyses revealed that the VPA-treated group had more
Two representative sections from each animal were bipolar neurons than the CON group at every age except
subjected to histological examination with an Olympus the 24-day sample P,0.001; Fig. 1. However, for the
BH-40 microscope. The sections chosen for analysis total number of unipolar neurons, there were no significant
extended in 40-mm increments beginning rostrally at the differences between groups at any time point Fig. 2.
decussation of the anterior commissure. The total number There were also no significant differences at any time
of neurons immunoreactive for GnRH was counted for point for the percentage of total of bipolar neurons
each section, and the percentages of total of unipolar and among groups Fig. 3. However, for the percentage of
bipolar neurons were determined for the analyses. Addi- total of unipolar neurons, there was a significant main
tionally, the total number of unipolar and bipolar neurons effect of drug F
553.958, P,0.001, but no main
1,37
having GABA associations was determined. For the pur- effect of age. Follow-up comparisons revealed that the
pose of this study, a GnRH-immunoreactive neuron was percentage of unipolar neurons was greater for the CON
defined as having a GABA association if at least one group at all time points except for the 24-day period
GABA-immunoreactive process was found to cross over P,0.001; Fig. 4.
its cell body and or one of its processes within the same There was a significant main effect of drug F
5
1,37
plane of section. All ratings were conducted by two 34.758, P,0.001, but no main effect of age, on the mean
experimenters who were blind to the treatment condition of number of bipolar neurons with GABA associations.
each animal inter-rater reliability was high, r50.92. Follow-up analyses revealed that the VPA-treated group
Ratings from the two experimenters were combined and had more bipolar neurons with GABA associations at
averaged for all statistical analyses. every sampling age P,0.001; Fig. 5. Furthermore, for
the mean number of unipolar neurons with GABA associa- tions, there was also a significant main effect of drug
2.6. Statistical analyses F
550.362, P,0.001, but no main effect of age.
1,37
Follow-up comparisons indicated that the mean number of The immunocytochemical data for the developmental
unipolar neurons with GABA associations was signifi- experiment 1 and adult portions experiment 2 of the
cantly greater for the VPA-treated group at every sampling study were analyzed separately. For the developmental
age P,0.001; Fig. 6.
196 A
Fig. 1. Mean6S.E.M. number of bipolar neurons at each age group. Significantly greater then CON group for all sampling times except 24 days, P,0.001.
3.2. Experiment 2: effects of VPA on GnRH–GABA groups Table 1. Likewise, there was no significant effect
interactions in adulthood of drug on the total number of unipolar neurons Table 1.
Additionally, there were no significant differences between There was no significant effect of drug on the total
the two treatment groups on the mean number of bipolar or number of bipolar neurons between the two adult treatment
unipolar neurons with GABA associations Table 2.
Fig. 2. Mean6S.E.M. number of unipolar neurons at each age group.
A .M. Illig et al. Brain Research 885 2000 192 –200
197
Fig. 3. Mean6S.E.M. percentage of total of bipolar neurons at each age group.
4. Discussion ment of the GnRH neurosecretory system during the
