P . Aggarwal, R.B. Gibbs Brain Research 882 2000 75 –85
79
as previously described [12,17,25]. Briefly, the average treated animals. Significant interactions between Surgical
number of ChAT-positive profiles on each side of the Condition3Side F2,255112.7, P,0.001, and Hor-
MS section was determined for each animal by counting mone Treatment3Surgical Condition3Side F2,255
the number of labeled profiles with an identifiable nucleus 7.01, P,0.005 were also detected.
in each of three matched sections animal. Group means Subsequent post hoc analyses revealed no significant
were calculated and the effects of hormone treatment and interaction between Hormone Treatment3Surgical Con-
fimbria transection were compared using analysis of dition on either the intact F2,2552.60, P50.10 or the
variance with repeated measures on ‘side’. lesioned F2,2550.26, P50.78 side, contrary to the
three-way interaction detected in the ANOVA. Additional post hoc analyses revealed a significant effect of Surgical
3. Results Condition on both the intact F2,2557.30, P,0.005 and
the lesioned F2,25528.62, P,0.005 side. 3.1. Serum estradiol levels
A one-way ANOVA comparing the effects of ibotenate in animals killed 2 weeks and 3 months following injection
Blood was collected from two E-treated animals and the relative to controls revealed a significant effect of surgical
serum levels of 17b-E2 were determined by radioim- condition on the number of ChAT-positive profiles de-
munoassay. The serum estradiol levels in these animals tected in the NBM on the lesioned side F3,37530.40,
were 85.6 and 115.0 pg ml, which is consistent with our P,0.001. A significant effect of ibotenate on the number
recent studies which show that these capsules produce of ChAT-positive cells detected in the NBM contralateral
serum levels of estradiol in the range of 60–120 pg ml to the lesion was also detected F3,3754.6, P,0.01.
[21]; R.B. Gibbs, unpublished observations. Post hoc analysis revealed a significant decrease in the
number of ChAT-positive cells ipsilateral to the lesion at 3.2. Neurochemical lesions
both 2 weeks and 3 months following surgery relative to the sham P,0.001 and cannula P,0.001 controls Fig.
3.2.1. Effects on ChAT-positive profiles in the NBM 3. No significant difference between the sham and cannula
The number of ChAT-positive cells detected in the controls with respect to the number of ChAT-positive cells
NBM on each side of the brain in the ibotenate-injected detected in the NBM ipsilateral to the cannula placement
animals and controls is summarized in Table 1. A signifi- was detected P51.0.
cant loss approximately 68 of ChAT positive profiles Post hoc analysis also revealed a significant increase in
was detected in the NBM ipsilateral to the ibotenate the number of ChAT-positive cells detected in the NBM
injection at 2 weeks post-injection. A similar loss approxi- contralateral to the lesion in animals killed 2 weeks P,
mately 63 was likewise detected at 3 months post- 0.01, but not 3 months P50.52 post-surgery. More
injection. Analysis of the data from animals killed 2 weeks ChAT-positive cells were also detected in the NBM
post-surgery revealed a significant main effect of ‘Side’ contralateral to the cannula placement in the cannula
F1,255297.9, P,0.001, but no significant effect of controls than in the sham controls; however, this difference
Hormone Treatment F1,2552.6, P50.12 or Surgical was not statistically significant P50.37.
Condition F2,2551.6, P50.22. No significant inter- action between Hormone Treatment3Surgical Condition
3.2.2. Effects on chat-positive profiles in the ms was detected F2,2550.96, P50.40; however, a signifi-
The number of ChAT-positive cells detected in the MS cant
interaction between
Hormone Treatment3Side
on each side of the brain in the ibotenate-injected animals F1,25510.8, P,0.005 was detected, reflecting the
and controls is summarized in Table 2. Significantly more presence of significantly more ChAT-positive cells in the
ChAT-positive cells were detected in the MS both ipsila- NBM contralateral to the lesion in E-treated vs. non-E-
teral and contralateral to the NBM lesion at 2 weeks
Table 1
a
Mean numbers of ChAT positive profiles in the nucleus basalis magnocellularis No estrogen
Estrogen Contralateral
Ipsilateral Contralateral
Ipsilateral Sham: 2 weeks
8886 84.3 8226 69.0
9286 79.7 7986 46.4
Cannula injury: 2 weeks 10386 99.6
7656100.9 11376 91.5
8586108.5
[
Ibotenate: 2 weeks 10686121.0
2526 28.9 15576154.0
2676118.6
[
Ibotenate: 12 weeks 9846 79.6
2676 64.0 11376 37.8
3246 92.2
a
Values indicate mean numbers of profiles6S.E.M. ipsilateral and contralateral to the NBM infusions. n55 in each group with the exception of the estrogen-treated shams where n56. P,0.001 relative to the intact side of the cannula lesioned and sham controls and to the intact side of estrogen-treated,
[
ibotenate-treated animals. P,0.001 relative to the intact side of the cannula controls and sham controls and to the non-estrogen-treated, ibotenate-treated animals.
80 P
tralateral F3,3753.3, P50.03 to the NBM lesion. A Tukey test revealed significantly more ChAT-positive cells
detected in the MS ipsilateral to the NBM lesion in the 2-week ibotenate group relative to the cannula P,0.001
and sham P,0.001 controls. Significantly more ChAT- positive profiles were also detected in the MS ipsilateral to
the injection in animals killed 2 weeks vs. 3 months following injection P,0.001 Contralateral to the NBM
lesion, significantly more ChAT-positive cells were de- tected in the MS of animals killed 2 weeks following
ibotenate injection relative to the sham controls P,0.05. Notably, more ChAT-positive cells were detected in the
MS both ipsilateral and contralateral to the cannula place- ment in the cannula controls than in the sham controls;
however, these differences were not statistically signifi- cant.
Fig. 3. Graph illustrating the effects of surgical condition on the number
3.3. Mechanical lesions
of ChAT-positive profiles in the NBM detected ipsilateral and contralater- al to the ibotenate lesion. Bars represent the marginal means of ChAT1
profiles6S.E.M. n510 in cannula, ibo-2 and ibo-12 groups; n511 in the
An example of AChE staining in the ipsilateral and
sham-operated group. P,0.001 relative to the contralateral side of the
contralateral hippocampus of an animal with a complete
[
sham-treated controls. P,0.001 relative to the ipsilateral side of the
unilateral transection of the fimbria fornix is shown in Fig.
cannula lesion and sham-treated controls.
4. Of the 30 animals that received unilateral transection of post-injection. Analysis of the data from animals killed 2
the fimbria fornix, five were excluded from the data weeks post-surgery revealed a significant main effect of
analysis three killed 2 weeks following injury and two Side F1,25515.8, P,0.001, a significant main effect
killed 3 months following injury due to incomplete of Surgical Condition F2,25532.2, P,0.001 and a
transections. The number of ChAT-positive cells detected significant Surgical Condition3Side interaction F2,255
in the MS of the remaining animals ipsilateral and con- 8.5, P50.001. Post hoc analysis revealed that the effect of
tralateral to the lesion is summarized in Table 3. Fimbria Surgical Condition on the number of ChAT-positive
fornix transection resulted in a significant decrease profiles detected in the MS was significant both ipsilateral
41.166.7 in E-treated animals, 52.367.9 in non-E- F2,25531.80, P,0.001 and contralateral F2,255
treated animals in the number of ChAT-positive cells 5.47, P50.01 to the ibotenate injection. No significant
detected in the MS ipsilateral to the lesion in animals main
effect of
Hormone Treatment
was detected
killed 2 weeks following injury P,0.01. At 3 months F1,2551.3, P50.28; however, the interaction between
following injury, no significant decrease in the number of Hormone Treatment3Side was very close to statistical
ChAT-positive cells was detected in the MS on the significance F1,2554.0, P50.06. No significant inter-
lesioned vs. the unlesioned side in either E-treated or actions between Hormone Treatment3Surgical Condition
non-E-treated animals. Analysis of variance revealed a F2,2550.84, P50.45 or Hormone Treatment3Surgical
significant effect of Side F1,20538.1, P,0.001, a Treatment3Side F2,2550.23, P50.80 were detected.
significant effect of Time F1,20514.6, P,0.005, and a A one-way ANOVA comparing the effects of ibotenate
significant interaction between Side3Time F1,20523.8, in animals killed 2 weeks and 3 months following injection
P,0.001. No significant effect of Hormone Treatment relative to controls revealed a significant effect of surgical
F1,2053.1, P50.09 and no significant interactions condition ipsilateral F3,37525.9, P,0.001 and con-
between Hormone Treatment3Side F1,2052.1, P5
Table 2
a
Mean numbers of ChAT-positive profiles in the medial septum No estrogen
Estrogen Contralateral
Ipsilateral Contralateral
Ipsilateral Sham: 2 weeks
7266 38.4 6486 52.5
7956 66.1 8606100.9
Cannula injury: 2 weeks 7926 51.6
7926 67.5 7806 49.3
10986166.1 Ibotenate: 2 weeks
11946187.4 16686160.9
9726156.1 17886176.9
Ibotenate: 12 weeks 8106109.4
7206100.0 9966159.2
8106121.1
a
Values indicate mean numbers of profiles6S.E.M. in the MS ipsilateral and contralateral to the NBM lesion. n55 in each group with the exception of the estrogen-treated shams where n56. P,0.001 relative to the cannula and sham-operated controls.
P . Aggarwal, R.B. Gibbs Brain Research 882 2000 75 –85
81
cell loss. In addition, no recovery in the number of ChAT- IR cells was detected on the injected side at 3 months
following injection in either E-treated or non-E-treated animals, suggesting that estrogen treatment did not reduce
cell death or enhance recovery of the cholinergic pheno- type following ibotenic acid injection.
The data also demonstrate that estrogen replacement did not prevent the loss of ChAT-positive cells in the MS
following mechanical injury to the fimbria fornix. This is demonstrated by the fact that unilateral transection of the
fimbria fornix produced a significant decrease in the number of ChAT-positive cells detected in the MS on the
injured side at 2 weeks post-injury, and that estrogen treatment had no significant effect on the magnitude of the
cell loss. Unlike the effects of ibotenate on ChAT-positive cells in the NBM, the number of ChAT-positive cells in
the MS recovered to control levels within 3 months following fimbria fornix transection. This is consistent
with previous reports [31,42,49] and suggests that the decrease in ChAT-positive cells in the MS following
fimbria fornix transection is due to an injury-related decrease in ChAT expression as opposed to a loss of cells.
ChAT expression then recovers over time resulting in an increase in the number of ChAT-positive cells to control
levels. Estrogen replacement had no apparent effect on the
Fig. 4. Photomicrographs showing AchE staining in the hippocampus
number of ChAT-positive cells detected in the MS on the
contralateral A and ipsilateral B to the fimbria fornix transection
injured side at 3 months following injury. Therefore,
found in an animal killed 2 weeks following transection. Note the
despite the evidence that estrogen replacement can enhance
presence of AchE-positive staining through the hippocampus contralateral
the functional status of cholinergic neurons in the MS and
to the lesion, compared with the absence of staining ipsilateral to the
NBM [14], these data suggest that hormone replacement
lesion. Scale bar51 mm in both.
does not prevent the loss of cholinergic neurons or the loss of ChAT expression following either neurochemical or
0.16 or Hormone Treatment3Side3Time F1,2050.05, mechanical injury. It should be noted that this does not
P50.82 were detected. preclude the possibility that estrogen enhances other
measures of cholinergic function within the remaining cholinergic neurons such as increasing high affinity choline
4. Discussion uptake or acetylcholine production and release.
