Directory UMM :Data Elmu:jurnal:B:Brain Research:Vol882.Issue1-2.2000:

(1)

www.elsevier.com / locate / bres

Research report

Estrogen replacement does not prevent the loss of choline

acetyltransferase-positive cells in the basal forebrain following either

neurochemical or mechanical lesions

*

Payal Aggarwal, Robert B. Gibbs

Department of Pharmaceutical Sciences, University of Pittsburgh School of Pharmacy, 1004 Salk Hall, Pittsburgh, PA 15261, USA Accepted 8 August 2000

Abstract

Recent studies have shown that estrogen replacement can enhance the functional status of basal forebrain cholinergic neurons. Studies have also shown that estrogen has neuroprotective effects both in vitro and in vivo on a variety of cells and against a variety of insults. The present study examined the ability of estrogen replacement to protect basal forebrain cholinergic neurons from the effects of neurochemical and mechanical injury. Ovariectomized Sprague–Dawley rats received either estrogen replacement or sham surgery, and then received either a unilateral injection of ibotenic acid into the nucleus basalis magnocellularis, or unilateral transection of the fimbria fornix. Cholinergic neurons in the medial septum and nucleus basalis were detected and quantified using immunohistochemical techniques. The data show that neither 3 weeks nor 13 weeks of continuous estrogen replacement prevented the loss of choline acetyltransferase (ChAT)-containing cells in the nucleus basalis following a unilateral injection of ibotenic acid. Likewise, estrogen replacement did not prevent a decrease in ChAT-positive cells detected in the medial septum following unilateral transection of the fimbria fornix. Notably, increased numbers of ChAT-positive cells were detected in the contralateral nucleus basalis, and in the ipsilateral and contralateral medial septum, at 2 weeks following a unilateral injection of ibotenic acid into the nucleus basalis; however, these effects were not related to hormone treatment. These data suggest that estrogen replacement does not protect cholinergic neurons in the medial septum and nucleus basalis from the effects of excitotoxic or mechanical injury.  2000 Elsevier Science B.V. All rights reserved.

Theme: Disorders of the nervous system Topic: Trauma

Keywords: Hormone replacement; Cholinergic neuron; Neuroprotection; Ibotenic acid

1. Introduction cognitive processes continues to unfold (see Ref. [11] for review).

Several recent prospective studies suggest that es- Recent studies have show that estrogen has significant trogen replacement can help to reduce the risk of Alz- neuroprotective effects in vitro on a wide variety of heimer’s-related dementia in postmenopausal women cultured cells exposed to a variety of toxic insults (see Ref. [3,34,62]. The mechanisms for this effect are still largely [39]). For example, estrogen has been shown to reduce cell unknown, although evidence for significant estrogen ef- death associated with serum deprivation, growth factor fects on neuronal anatomy, biochemistry, and function deprivation, b-amyloid toxicity, excitatory amino acid within regions of the brain that play an important role in toxicity, and various inducers of oxidative stress, in cells ranging from primary neuronal cultures to neuroblastoma and non-neuronal cell lines. In some cases estrogen-me-diated neuroprotection in culture is blocked with selective estrogen receptor antagonists [5,55], suggesting that es-trogen supports survival via the activation of eses-trogen

*Corresponding author. Fax:11-412-624-1850.

E-mail address: gibbsr1@pitt.edu (R.B. Gibbs). receptor regulated genes (e.g. bcl-2 [10]). In other cases,

(2)

neuroprotection in culture is related to the structure of the 2. Materials and methods estrogen molecule and not to any specific estrogenicity of

the molecule or to the activation of estrogen receptors 2.1. Animals [4,27–29]. Neuroprotective effects of estrogen in vivo

have also been described. Specifically, several studies have Young female Sprague–Dawley rats were purchased reported the ability of estrogen replacement to reduce from Hilltop Laboratories and housed in pairs on a 12:12-h infarct size and neuronal damage following experimentally light / dark cycle with food and water available ad libitum. induced transient forebrain ischemia [7,53,61,64]. In one All animals were housed for a minimum of 2 weeks before study, the reduction in infarct size was associated with the use. All animals then underwent bilateral ovariectomy and prevention of injury-induced down-regulation of bcl-2 and 2 weeks later, received either 3-mm silastic capsules estrogen receptor beta gene expression [7]. Ischemic injury containing 17b-estradiol crystals (implanted s.c.) or blank was also shown to dramatically upregulate estrogen re- capsules. Studies have shown that these estrogen capsules ceptor alpha gene expression on the injured side. Estrogen produce mean circulating levels of estradiol in the range of has also been shown to reduce kainic acid-induced cell loss 60–120 pg / ml serum.

in the dentate gyrus [2]. These studies suggest that at least

some neuroprotective effects of estrogen in vivo may 2.2. Neurochemical lesions involve the selective regulation of estrogen receptors and

estrogen receptor target genes. Seven to ten days following capsule implantation, One of the consistent biochemical features of Alzheim- animals (n541) were anesthetized with a combination of er’s disease is the loss of cholinergic neurons in the medial ketamine (14 mg / 100 g body wt.) and xylazine (2.8 septum, the diagonal band of Broca, and the nucleus mg / 100 g body wt.) and placed into a standard stereotaxic basalis magnocellularis (see Ref. [33] for review). These device. Twenty animals (ten estrogen (E)-treated, ten non-neurons are the major source of cholinergic innervation to E-treated) received unilateral lesions of the nucleus basalis the hippocampus and neocortex [69] and play an important magnocellularis (NBM) by intracerebroventricular infusion role in learning, memory, and attentional processes of ibotenic acid. Briefly, the skull was exposed and a hole [6,8,36,47,63,66]. Recent studies have shown that estrogen was drilled 1.3 mm posterior to Bregma and 2.4 mm to the replacement can enhance the functional status of basal left of the midline. A stainless steel cannula (28 gauge; forebrain cholinergic projections as indicated by increases Small Parts, Inc.) containing ibotenic acid (10 mg / ml in in choline acetyltransferase mRNA and protein in the sterile filtered saline) was lowered 7.5 mm from the skull medial septum and nucleus basalis magnocellularis into the NBM. One microliter (10 mg) of ibotenic acid [16,12,38], and by increases in high affinity choline uptake solution was infused into the NBM at a rate of 12 ml / h. [46,56] and acetylcholine release [24] in the hippocampus Following infusion, the cannula was left in place for 5 min and overlying cortex. Treatment with estrogen and with and then withdrawn very slowly. The skin overlying the estrogen plus progesterone have also been shown to skull was sutured shut and the animals were placed onto a increase the expression of trkA (nerve growth factor warm heating pad during recovery. Two separate control receptor) mRNA [18,40] in the medial septum and to groups were prepared. Cannula controls (consisting of five increase relative levels of brain-derived neurotrophic factor E-treated and five non-E-treated animals) had an empty mRNA in the hippocampus [18,20,57], suggesting that infusion cannula lowered into the NBM to control for estrogen may enhance basal forebrain cholinergic projec- damage produced by lowering the cannula. Sham controls tions in part by increasing trophic support for the choliner- (consisting of six E-treated and five non-E-treated animals) gic neurons. were anesthetized and placed into the stereotaxic ap-These findings suggest that one mechanism by which paratus, the skull was exposed and then the skin was estrogen replacement can help to reduce the risk of sutured shut. Following surgery, all animals were given Alzheimer’s-related dementia in postmenopausal women is Torbugesic (0.02 cc / 100 g) twice a day for 3 days to by increasing the functional status of cholinergic projec- reduce discomfort associated with the surgical procedures. tions to the hippocampus and neocortex. Whether estrogen

replacement also reduces the loss of cholinergic neurons 2.3. Fimbria /fornix transections associated with Alzheimer’s disease, and / or protects the

cholinergic neurons from neurotoxic and mechanical in- Seven to ten days following capsule implantation, jury, is currently unknown. The purpose of this study was animals (n530) were anesthetized with a combination of to examine potential neuroprotective effects of estrogen ketamine (14 mg / 100 g body wt.) and xylazine (2.8 replacement on basal forebrain cholinergic neurons by mg / 100 g body wt.) and placed into a standard stereotaxic examining the ability of estrogen replacement to reduce device. Unilateral transections of the fimbria / fornix were losses of ChAT positive neurons following ibotenic acid performed as previously described [22]. Briefly, the skull injection into the nucleus basalis magnocellularis and was exposed and a trench was drilled extending laterally following transection of the fimbria fornix. from midline 1.0 mm posterior to the coronal suture. An

(3)

incision was made through the dura to expose the brain. method as previously described [23] to verify that the An ultrathin microknife (Tieman) was lowered 6.0 mm unilateral transections were complete.

into the brain at midline. Care was taken to avoid the

midsagittal sinus which was gently pushed to the side. The 2.5. ChAT immunostaining knife was moved laterally along the trench to bone and

then removed. The trench was packed with gelfoam soaked ChAT was detected as previously described [12] using with sterile saline. The skin was sutured shut and the an affinity purified goat polyclonal antibody raised against animals were placed onto a warm heating pad during rat ChAT (Chemicon Pharmaceuticals, AP144P; diluted

recovery. 1:3500 in 50 mM PBS (pH 7.2) containing 0.05% Triton

X-100 and 1% normal rabbit serum). Briefly, sections were 2.4. Tissue preparation placed in the primary antibody solution for 3 days at 48C. The sections were rinsed well with PBS and then incubated Ten of the ibotenate-treated animals (five E-treated and with a biotinylated rabbit-anti-goat antibody (Vector Lab-five non-E-treated), 18 of the fimbria / fornix transected oratories, diluted 1:1000) for 1 h at room temperature. animals (nine E-treated, nine non-E-treated), and all of the Sections were rinsed again with PBS and then incubated controls were killed 2 weeks post-surgery. The remaining with an avidin–HRP complex (Vectastain Elite kit; Vector ten ibotenate-treated animals (five E-treated and five non- Laboratories) for 1 h at room temperature. Sections were E-treated) and 12 fimbria / fornix transected animals (six rinsed with PBS, placed in 50 mM Tris–HCl (pH 7.6) E-treated, six non-E-treated) were killed 12 weeks post- containing 3,39-diaminobenzidine (DAB; 0.5 mg / ml), and surgery. For euthanasia, the animals were anesthetized then reacted with 50 mM Tris containing DAB (0.5 mg / with pentobarbital (100 mg / kg) and perfused with saline ml), H O (0.01%), and NiCl (0.032%) for 7–10 min.2 2 2

followed by 4% paraformaldehyde in 50 mM sodium The sections were then rinsed with PBS, mounted onto acetate (pH 6.5), and then 4% paraformaldehyde in 50 mM Superfrost plus slides (Fisher Scientific), counterstained Tris (pH 9.0). Blood was collected by cardiac puncture with cresyl violet (Sigma), dehydrated, and coverslipped. from two E-treated animals killed after 3 weeks of

estradiol treatment to verify that the estrogen capsules 2.6. Data analysis were producing the expected levels of circulating estradiol.

All capsules were inspected and the presence of estradiol 2.6.1. Ibotenate-treated animals and related controls crystals in the E-capsules was verified. Note that these Sections through the MS and NBM of ibotenate-treated capsules have been shown to continue releasing estradiol animals and related controls were analyzed using unbiased even after long-term implantation [19]. The brains were stereological techniques. Briefly, the total number of removed and postfixed for 3–4 h with 4% paraformal- ChAT-positive profiles in the NBM and MS on each side dehyde in phosphate buffer (50 mM, pH 7.2) at 48C and of the brain was estimated using the Optical Fractionator then stored overnight in 15% sucrose in 50 mM phosphate- method [30]. The analysis was performed using a Leitz buffered saline (PBS; pH 7.2) at 48C. Laborlux-S microscope equipped with a Ludle Biopoint For the ibotenate-treated animals and controls, 40-mm motorized x-y-z-stage connected to a Power Macintosh G3 coronal sections were cut through the MS (including plates computer running the Neurozoom software [43]. Three 15–17 of Paxinos and Watson [48]) and the NBM (includ- equivalently spaced sections through the MS (120 mm ing plates 24–25 of Paxinos and Watson [48]) of each apart, beginning at approximately plate 15 of Paxinos and brain and every third section was processed for immuno- Watson [48]) and six equivalently spaced sections through cytochemical detection of choline acetyltransferase the NBM (120mm apart, beginning at approximately plate (ChAT) as described below. Unbiased stereological pro- 23 of Paxinos and Watson [48]) were analyzed from each cedures were then used to quantify the number of ChAT- brain. The dorsal and ventral boundaries of the septum positive cells detected in the MS and NBM of each animal were defined at low magnification by the corpus callosum (see below). Brains from the fimbria / fornix transected and by a line connecting the anterior commisures. The animals were processed prior to the use of stereological medial boundary was defined by drawing a line down the procedures in our laboratory. For these animals, 25-mm middle of the septum (Fig. 1). The boundaries of the NBM coronal sections through the MS were cut and every third were defined at low magnification by drawing lines section was processed for immunocytochemical detection connecting the globus pallidus to the reticular nucleus of of ChAT. These sections were analyzed by counting the the thalamus and the substantia innominata (Fig. 2). Once number of ChAT-positive profiles in the MS of three the borders of each region were outlined, 20% of the total matched sections / animal and then comparing the number area was analyzed in each tissue section on each side. The of immunoreactive cells / section (see below). Sections dissector, which consists of two planes separated by a through the hippocampus of the fimbria / fornix-transected specified distance, was set at 10 mm (less than the animals were stained for the detection of acetylcholinester- minimum diameter of a ChAT-positive cell). A counting ase using a modified copper thiocholine identification box was set at 10.37mm by 7.44mm and consisted of two

(4)

Fig. 1. Bright-field photomicrographs showing ChAT-positive profiles detected in the MS at low (A) and high (B) power. The borders used for stereology are also outlined in A. Scale bars: A51 mm; B562.5mm.

forbidden lines and two inclusive lines. Objects completely inside the counting frame were counted whereas objects completely outside the counting frame were excluded. Objects which crossed through any of the three forbidden planes (bottom, front, and left sides of the frame) were excluded. Objects which crossed through any of the remaining planes (top, right, and back sides of the frame) were included if they either came into focus or went out of focus when focusing through the dissector [68]. The nucleus of each labeled cell was used as the reference point for counting. An estimate of the total number of

Fig. 2. Bright-field photomicrographs showing ChAT staining detected in

ChAT-positive neurons in each region was then calculated _{the NBM contralateral (A) and ipsilateral (B) to the ibotenate infusion.} with the following formula: Total ChAT-positive cells / Arrows in A and B indicate the borders used for stereology. (C)

region5Total profiles counted35(1 / percentage of total High-power magnification of ChAT-positive profiles in the NBM con-tralateral to the lesion. Scale bars: A,B51 mm; C562.5mm.

area)3separation factor (3 for the NBM and 2 for the MS). Statistical analyses were performed using SYSTAT 5.2

for Macintosh. The effects of surgical manipulation (ibote- relative to the controls was analyzed by one-way ANOVA. nate vs. cannula vs. sham) and hormone treatment (E vs. Individual group comparisons were made using the Tukey no E) on the number of ChAT-positive cells in the MS and test.

NBM at 2 weeks post-surgery were analyzed by

three-factor (Hormone Treatment3Surgical Condition3Side) 2.6.2. Fimbria /fornix transected animals

ANOVA with repeated measures on ‘Side’. The effects of Sections through the medial septum of animals with a ibotenic acid 2 weeks and 3 months following injury unilateral transection of the fimbria / fornix were analyzed

(5)

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 (F(2,25)5112.7, P,0.001), and Hor-MS / section was determined for each animal by counting mone Treatment3Surgical Condition3Side (F(2,25)5

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 (F(2,25)52.60, P50.10) or the variance with repeated measures on ‘side’. lesioned (F(2,25)50.26, 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 (F(2,25)57.30, P,0.005) and

the lesioned (F(2,25)528.62, 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 (F(3,37)530.40, 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 (F(3,37)54.6, 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 (F(1,25)5297.9, P,0.001), but no significant effect of controls than in the sham controls; however, this difference Hormone Treatment (F(1,25)52.6, P50.12) or Surgical was not statistically significant (P50.37).

Condition (F(2,25)51.6, P50.22). No significant

inter-action between Hormone Treatment3Surgical Condition 3.2.2. Effects on chat-positive profiles in the ms

was detected (F(2,25)50.96, 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 (F(1,25)510.8, 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*

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.

(6)

tralateral (F(3,37)53.3, 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 margincontralater-al 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 (F(1,25)515.8, P,0.001), a significant main effect killed 3 months following injury) due to incomplete of Surgical Condition (F(2,25)532.2, P,0.001) and a transections. The number of ChAT-positive cells detected significant Surgical Condition3Side interaction (F(2,25)5 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-(F(2,25)531.80, P,0.001) and contralateral (F(2,25)5 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 (F(1,25)51.3, 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 (F(1,25)54.0, 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 (F(2,25)50.84, P50.45) or Hormone Treatment3Surgical significant effect of Side (F(1,20)538.1, P,0.001), a Treatment3Side (F(2,25)50.23, P50.80) were detected. significant effect of Time (F(1,20)514.6, P,0.005), and a A one-way ANOVA comparing the effects of ibotenate significant interaction between Side3Time (F(1,20)523.8, 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 (F(1,20)53.1, P50.09) and no significant interactions condition ipsilateral (F(3,37)525.9, P,0.001) and con- between Hormone Treatment3Side (F(1,20)52.1, 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

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.

(7)

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 (F(1,20)50.05, 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.

One possibility is that estrogen failed to reduce the toxic The data demonstrate that estrogen replacement did not effects of ibotenic acid because either the dose of estrogen prevent the loss of cholinergic neurons in the NBM was too low, or the dose of ibotenic acid was too high. following injections of ibotenic acid. This is demonstrated Previous studies, including additional data collected from by the fact that unilateral injection of ibotenic acid into the over one hundred animals (unpublished observations), have NBM produced a significant decrease in the number of shown that the estrogen capsules that were used produce ChAT-positive cells detected in the NBM on the injected mean circulating levels of estradiol in the high physiologi-side within 2 weeks post-injection, and that estrogen cal range (60–120 pg / ml). This was confirmed in two treatment had no significant effect on the magnitude of the E-treated animals that had serum levels of estradiol of 85.6

Table 3

a Mean numbers of ChAT-positive profiles in the medial septum following fimbria / fornix transection

Survival time No estrogen Estrogen

following transection

Contralateral Ipsilateral Contralateral Ipsilateral

2 weeks 55.7611.3 29.9614.2* 54.76 7.7 22.569.9*

12 weeks 64.6614.3 65.6621.7 56.4611.2 48.6613.4

Values indicate mean numbers of profiles6S.E.M. ipsilateral and contralateral to a unilateral fimbria / fornix transection. n57 for each treatment group killed 2 weeks following transection and n55 for each treatment group killed 12 weeks following transection. *P,0.05 relative to the contralateral side.

(8)

and 115.0 pg / ml. This suggests that estrogen levels were direct estrogen effects on the cholinergic neurons mediated sufficient to elicit any neuroprotective effect that would be by the activation of classical estrogen receptors. Recent associated with physiological levels of circulating es- studies have shown that a significant percentage of the tradiol. Furthermore, a preliminary study (unpublished cholinergic neurons in the MS contain estrogen receptor observations) showed that half the dose of ibotenic acid alpha mRNA and protein and bind 17b-estradiol produced much less cell loss overall, and much less of a [15,41,52], suggesting that estrogen may directly effect loss of ChAT-positive cells (less than half the degree of ChAT expression via ligand-mediated activation of the cell loss) than was obtained in the present study. This estrogen receptor. It is possible that mechanical injury suggests that the extent to which the dose–response curve induces cellular responses that prevent estrogen receptor-would need to be affected in order to elicit a significant mediated activation of the ChAT gene. One interesting neuroprotective effect was not so great as to account for study would be to administer estrogen 3 months following the lack of an effect of estrogen. In addition, Singer et al. transection of the fimbria / fornix when the injury response [55] reported that estrogen significantly protected cortical has subsided and the number of ChAT-positive cells in the neurons from glutamate-induced cell death in culture using MS has recovered. An estrogen-mediated induction of glutamate levels as high as 0.1 mM. Azcoitia et al. [2] ChAT expression at this time could indicate that the reported that estrogen reduced cell loss in the dentate gyrus inability to induce ChAT expression 2 weeks following following systemic injection of 7 mg / kg kainic acid. These injury is due to an injury-induced response which blocks data suggest that the concentration of ibotenate used in the estrogen-mediated transcriptional activation as opposed to present study was not so overwhelming as to preclude the disconnection from the hippocampus.

ability to detect a neuroprotective effect of estrogen. Estrogen has also been shown to exert neuroprotective Nevertheless, the possibility that estrogen has neuroprotec- effects via non-classical mechanisms, possibly involving tive effects on the cholinergic neurons when administered activation of specific second messenger systems such as at different doses or for different periods of time, or that ERK and MEK. Several studies have documented rapid estrogen protects the cholinergic neurons from other types membrane effects of estrogen in cells which lack estrogen of neurotoxic or mechanical injury, cannot be excluded. receptors, resulting in neuroprotection or in the activation Notably, estrogen treatment did increase the number of of ERK and MEK [54,58,59,65]. Whether estrogen acts on ChAT-positive cells detected in the NBM contralateral to basal forebrain cholinergic neurons via non-classical path-the ibotenate injection, and in path-the MS ipsilateral to path-the ways and whether those effects are altered following NBM injection, at 2 weeks post-injury. We interpret these neurochemical or mechanical injury are currently un-increases as un-increases in ChAT protein within cholinergic known.

cells that were previously undetectable. These data are Another interesting observation was the fact that unila-consistent with previous studies showing that estrogen teral injection of ibotenic acid into the NBM produced a replacement increases the number of ChAT-positive cells significant increase in the number of ChAT-positive cells [12,25], as well as relative levels of ChAT mRNA [16], in detected in the contralateral NBM, and in both the the MS and NBM and that the effects depend on both the ipsilateral and contralateral MS, at 2 weeks following dose and duration of estrogen treatment [12]. The fact that injury relative to controls. Increased numbers of ChAT-estrogen replacement did not diminish the loss of ChAT positive cells were also observed in the cannula controls positive cells in the MS following fimbria / fornix transec- although the effects were not statistically significant. This tion suggests that the injury interferes with the mecha- suggests that the magnitude of the effect increases with an nism(s) by which estrogen increases ChAT. This could be increased amount of injury. In contrast, no significant due to the disconnection of the cholinergic neurons from increases in the numbers of ChAT-positive cells were their targets, thereby isolating the cells from target-derived detected in animals killed 3 months following ibotenic acid growth factors. Estrogen and progesterone replacement injection, suggesting that the effect is an acute response to have been shown to increase the levels of brain derived injury in the NBM. One likely possibility is that the neurotrophic factor (BDNF) mRNA in the hippocampus increases in ChAT-postive cells detected in the MS and the [18,20,57] and BDNF protein in the septum [20]. BDNF is contralateral NBM are due to injury-induced growth a nerve growth factor-related polypeptide which is pro- factors which accumulate around the site of injury and in duced in the hippocampus and cortex, and which has been the targets of the injured cholinergic neurons. Recent shown to exert trophic effects on basal forebrain choliner- studies have shown that astrocytes can produce nerve gic neurons [45,67]. Transection of the fimbria / fornix growth factor both in vitro [37,50,60] and in vivo [1], and would prevent any estrogen-mediated induction of BDNF that NGF production by astrocytes can increase dramatical-in the hippocampus from affectdramatical-ing the dramatical-injured choldramatical-inergic ly in response to brain injury [1,26]. NGF levels have also cells, which could explain the inability of estrogen replace- been shown to increase in the hippocampus and cortex ment to reduce the loss of cholinergic neurons in the MS following the loss of basal forebrain cholinergic inputs following fimbria / fornix transection. [44,51]. Numerous studies have shown that NGF has Another possibility is that the injury may interfere with trophic effects on basal forebrain cholinergic neurons (see

(9)

growth factor expression after chronic neurotoxic injury — I.

Ref. [13]). Therefore, it is reasonable to suspect that

Degeneration and plastic response of basal forebrain neurons,

injury-induced increases in NGF contribute at least in part

Neuroscience 65 (1995) 633–645.

to the increases in ChAT-positive cells detected in the MS _{[2] I. Azcoitia, A. Sierra, L.M. Garcia-Segura, Estradiol prevents kainic} and in the contralateral NBM. _{acid-induced neuronal loss in the rat dentate gyrus, Neuroreport 9}

Even so, the increases in the numbers of ChAT-positive (1998) 3075–3079.

[3] M. Baldereschi, A. Di Carlo, V. Lepore, L. Bracco, S. Maggi, F.

cells detected in the ipsilateral MS of E-treated and

non-E-Grigoletto, G. Scarlato, L. Amaducci, Estrogen-replacement therapy

treated animals 2 weeks following an injection of ibotenate

and Alzheimer’s disease in the Italian Longitudinal Study on Aging,

into the NBM were quite striking (2–3 fold). This

indi-Neurology 50 (1998) 996–1002.

cates a transient, but powerful, effect of the ibotenate _{[4] C. Behl, T. Skutella, F. Lezoualch, A. Post, M. Widmann, C.J.} injections on cholinergic neurons in the ipsilateral MS. _{Newton, F. Holsboer, Neuroprotection against oxidative stress by} This may be caused by the leakage of some ibotenate up estrogens: structure–activity relationship, Mol. Pharmacol. 51

(1997) 535–541.

the cannula tract into the hippocampus, resulting in

[5] J.A. Chowen, I. Torres-Aleman, L.M. Garcia-Segura, Trophic

excitation of the hippocampal neurons. Previous studies

effects of estradiol on fetal rat hypothalamic neurons,

Neuro-have shown that increased electrical activity in the

hip-endocrinology 56 (1992) 895–901.

pocampus results in the up-regulation of both NGF and _{[6] J.A.M. Dekker, D.J. Connor, L.J. Thal, The role of cholinergic} BDNF within hippocampal neurons [9,32,35,70]. Any _{projections from the nucleus basalis in memory, Neurosci.} leakage of ibotenate up the cannula tract and into the Biobehav. Rev. 15 (1991) 299–317.

[7] D.B. Dubal, P.J. Shughrue, M.E. Wilson, I. Merchenthaler, P.M.

hippocampus would undoubtedly produce increased

neuro-Wise, Estradiol modulates bcl-2 in cerebral ischemia: a potential

nal activity within the hippocampus, which could result in

role for estrogen receptors, J. Neurosci. 19 (1999) 6385–6393.

increased NGF and BDNF expression and increased

[8] H.C. Fibiger, Cholinergic mechanisms in learning, memory and

trophic support for cholinergic neurons in the MS. This _{dementia: a review of recent evidence, Trends Neurosci. 14 (1991)} could account for the dramatic and transient increase in the _220–223.

number of ChAT-positive cells detected in the MS ipsila- [9] C.M. Gall, P.J. Isackson, Limbic seizures increase neuronal pro-duction of messenger RNA for nerve growth factor, Science 245

teral to the ibotenate injections. Additional studies which

(1989) 758–761.

examine changes in hippocampal and cortical levels of

[10] L.M. Garcia-Segura, P. Cardona-Gomez, F. Naftolin, J.A. Chowen,

NGF and BDNF following unilateral ibotenic acid

in-Estradiol upregulates Bcl-2 expression in adult brain neurons,

jections into the NBM would reveal the extent to which _{NeuroReport 9 (1998) 593–597.}

changes in neurotrophin expression may contribute to the _{[11] R.B. Gibbs, Effects of estrogen on basal forebrain cholinergic} increased numbers of ChAT-positive cells detected in the neurons and cognition: Implications for brain aging and dementia in women, in: M. Morrison (Ed.), Hormones, Aging, and Mental

MS.

Disorders, Cambridge University Press, Cambridge, 2000, pp. 183–

In conclusion, the present studies failed to provide

222.

evidence for an ability of estrogen replacement to prevent

[12] R.B. Gibbs, Effects of estrogen on basal forebrain cholinergic

the loss of ChAT-positive neurons in the MS and NBM _{neurons vary as a function of dose and duration of treatment, Brain} following excitatory amino acid toxicity or mechanical _{Res. 757 (1997) 10–16.}

injury. Whether estrogen is neuroprotective for the [13] R.B. Gibbs, Estrogen and nerve growth factor-related systems in brain: effects on basal forebrain cholinergic neurons and

implica-cholinergic cells against other types of injury such as

tions for learning and memory processes and aging, in: V.N. Luine,

b-amyloid-induced toxicity or cortical impact injury, and

C.F. Harding (Eds.), Hormonal Restructuring of the Adult Brain,

whether estrogen can reduce the loss of cholinergic

Basic and Clinical Perspectives, Vol. 743, New York Academy of

neurons associated with normal aging, are still unknown. _{Science, New York, 1994, pp. 165–199.}

Hence, the possibility that neuroprotective effects of [14] R.B. Gibbs, Oestrogen and the cholinergic hypothesis: Implications

estrogen on basal forebrain cholinergic neurons contribute for oestrogen replacement therapy in postmenopausal women, in: J. Goode (Ed.), Neuronal and Cognitive Effects of Oestrogens,

Novar-to the ability of estrogen replacement Novar-to reduce the risk

tis Found. Symposium 230, Wiley, Chichester, 2000, pp. 94–111.

for developing Alzheimer’s-related dementia in

post-[15] R.B. Gibbs, Expression of estrogen receptor-like immunoreactivity

menopausal women cannot be excluded. _{by different subgroups of basal forebrain cholinergic neurons in}

gonadectomized male and female rats, Brain Res. 720 (1996) 61–68.

[16] R.B. Gibbs, Fluctuations in relative levels of choline

acetyltrans-Acknowledgements

ferase mRNA in different regions of the rat basal forebrain across the estrous cycle: effects of estrogen and progesterone, J. Neurosci.

Estradiol radioimmunoassays were performed by the _{16 (1996) 1049–1055.}

Assay Core of the Center for Reproductive Physiology. [17] R.B. Gibbs, Impairment of basal forebrain cholinergic neurons

This work was supported by NIH Grant NS28896. associated with aging and long-term loss of ovarian function, Exp. Neurol. 151 (1998) 289–302.

[18] R.B. Gibbs, Levels of trkA and BDNF mRNA, but not NGF mRNA, fluctuate across the estrous cycle and increase in response to acute

References _{hormone replacement, Brain Res. 787 (1998) 259–268.}

[19] R.B. Gibbs, Long-term treatment with estrogen and progesterone ¨

[1] T. Arendt, M.K. Bruckner, S. Pagliusi, T. Krell, Degeneration of rat enhances acquisition of a spatial memory task by ovariectomized cholinergic basal forebrain neurons and reactive changes in nerve aged rats, Neurobiol. Aging 21 (2000) 107–116.

(10)

[20] R.B. Gibbs, Treatment with estrogen and progesterone affects [39] B.S. McEwen, S.E. Alves, Estrogen actions in the central nervous relative levels of brain-derived neurotrophic factor mRNA and system, Endocr. Rev. 20 (1999) 279–307.

protein in different regions of the adult rat brain, Brain Res. 844 [40] P.J. McMillan, C.A. Singer, D.M. Dorsa, The effects of ovariectomy (1999) 20–27. and estrogen replacement on trkA and choline acetyltransferase [21] R.B. Gibbs, A.M. Burke, D.A. Johnson, Estrogen replacement mRNA expression in the basal forebrain of adult female Sprague–

attenuates effects of scopolamine and lorazepam on memory acqui- Dawley rat, J. Neurosci. 16 (1996) 1860–1865.

sition and retention, Horm. Behav. 34 (1998) 112–125. _{[41] E.J. Mufson, W.J. Cai, S. Jaffar, E.Y. Chen, G. Stebbins, T. Sendera,} [22] R.B. Gibbs, M.V. Chao, D.W. Pfaff, Effects of fimbria-fornix and _{J.H. Kordower, Estrogen receptor immunoreactivity within}

subreg-NGFR

angular bundle transection on expression of the p75 mRNA by _{ions of the rat forebrain: neuronal distribution and association with} cells in the medial septum and diagonal band of Broca: correlations _{perikarya containing choline acetyltransferase, Brain Res. 849} with cell survival, synaptic reorganization and sprouting, Mol. Brain _{(1999) 253–274.}

Res. 11 (1991) 207–219. _{[42] T. Naumann, G.M. Peterson, M. Frotscher, Fine structure of rat} [23] R.B. Gibbs, E.W. Harris, C.W. Cotman, Replacement of damaged _{septohippocampal neurons: II. A time course analysis following}

cortical projections by homotypic transplants of entorhinal cortex, J. _{axotomy, J. Comp. Neurol. 325 (1992) 219–242.}

Comp. Neurol. 237 (1985) 47–64. _{[43] E.A. Nimchinsky, P.R. Hof, W.G. Young, F.E. Bloom, J.H.} Mor-[24] R.B. Gibbs, A. Hashash, D.A. Johnson, Effects of estrogen on

rison, NeuroZoom software: development, validation, and neuro-potassium-evoked acetylcholine release in the hippocampus and

biological application, FASEB J. 12 (1998) A628. overlying cortex of adult rats, Brain Res. 749 (1997) 143–146.

[44] A. Nitta, Y. Furukawa, K. Hayashi, T. Hasegawa, T. Nabeshima, [25] R.B. Gibbs, D.W. Pfaff, Effects of estrogen and fimbria / fornix

NGFR Nerve growth factor content of rat brain increases following basal transection on p75 and ChAT expression in the medial septum

forebrain lesions induced by ibotenic acid, but not by electrolysis, and diagonal band of Broca, Exp. Neurol. 116 (1992) 23–39.

Biol. Pharm. Bull. 17 (1994) 34–38. [26] J.R. Goss, M.E. O’Malley, L. Zou, S.D. Styren, P.M. Kochanek, S.T.

[45] T. Nonomura, H. Hatanaka, Neurotrophic effect of brain-derived DeKosky, Astrocytes are the major source of nerve growth factor

neurotrophic factor on basal forebrain cholinergic neurons in culture upregulation following traumatic brain injury in the rat, Exp. Neurol.

from postnatal rats, Neurosci. Res. 14 (1992) 226–233. 149 (1998) 301–309.

[46] C.A. O’Malley, R.D. Hautamaki, M. Kelley, E.M. Meyer, Effects of [27] P.S. Green, J. Bishop, J.W. Simpkins, 17-alpha-estradiol exerts

ovariectomy and estradiol benzoate on high affinity choline uptake, neuroprotective effects on SK-N-SH cells, J. Neurosci. 17 (1997)

ACh synthesis, and release from rat cerebral cortical synaptosomes, 511–515.

Brain Res. 403 (1987) 389–392. [28] P.S. Green, K. Gordon, J.W. Simpkins, Phenolic A ring requirement

[47] D.S. Olton, Dementia: animal models of the cognitive impairments for the neuroprotective effects of steroids, J. Steroid Biochem. Mol.

following damage to the basal forebrain cholinergic system, Brain Biol. 63 (1997) 229–235.

Res. Bull. 25 (1990) 499–502. [29] P.S. Green, K.E. Gridley, J.W. Simpkins, Nuclear estrogen

receptor-independent neuroprotection by estratrienes — a novel interaction [48] G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, with glutathione, Neuroscience 84 (1998) 7–10. 2nd ed., Academic Press, London, 1986.

[30] H.J. Gundersen, P. Bagger, T.F. Bendtsen, S.M. Evans, L. Korbo, N. [49] G.M. Peterson, T. Naumann, M. Frotscher, Identified septohip-Marcussen, A. Moller, K. Nielsen, J.R. Nyengaard, B. Pakkenberg et pocampal neurons survive axotomy: a fine-structural analysis in the al., The new stereological tools: disector, fractionator, nucleator and rat, Neurosci. Lett. 138 (1992) 81–85.

point sampled intercepts and their use in pathological research and [50] J.P. Schwartz, N. Nishiyama, Neurotrophic factor gene expression in diagnosis, APMIS 96 (1988) 857–881. astrocytes during development and following injury, Brain Res. [31] C.A. Haas, T. Deller, T. Naumann, M. Frotscher, Selective expres- Bull. 35 (1994) 403–407.

sion of the immediate early gene c-jun in axotomized rat medial [51] L.W. Shaughnessy, S.J. Barone, Damage to the NBM leads to a septal neurons is not related to neuronal degeneration, J. Neurosci. sustained lesion-induced increase in functional NGF in the cortex,

16 (1996) 1894–1903. NeuroReport 8 (1997) 2767–2774.

[32] P.J. Isackson, M.M. Huntsman, K.D. Murray, C.M. Gall, BDNF [52] P.J. Shughrue, P.J. Scrimo, I. Merchenthaler, Estrogen bonding and mRNA expression is increased in adult rat forebrain after limbic estrogen receptor characterization (ERaand ERb) in the cholinergic seizures: temporal patterns of induction distinct from NGF, Neuron neurons of the rat basal forebrain, Neuroscience 96 (2000) 41–49. 6 (1991) 937–948. [53] J.W. Simpkins, G. Rajakamar, Y.Q. Zhang, C.E. Simpkins, D. [33] P. Kasa, Z. Rakonczay, K. Gulya, The cholinergic system in Greenwald, C.J. Yu, N. Bodor, A.L. Day, Estrogens may reduce Alzheimer’s disease, Prog. Neurobiol. 52 (1997) 511–535. mortality and ischemic damage caused by middle cerebral artery [34] C. Kawas, S. Resnick, A. Morrison, R. Brookmeyer, M. Corrada, A. occlusion in the female rat, J. Neurosurg. 87 (1997) 724–730.

Zonderman, C. Bacal, D.L. Lingle, E. Metter, A prospective study of [54] C.A. Singer, X.A. Figueroa-Masot, R.H. Batchelor, D.M. Dorsa, The estrogen replacement therapy and the risk of developing Alzheim- mitogen-activated protein kinase pathway mediates estrogen neuro-er’s disease: The Baltimore Longitudinal Study of Aging, Neurology protection after glutamate toxicity in primary cortical neurons, J.

48 (1997) 1517–1521. Neurosci. 19 (1999) 2455–2463.

[35] J.C. Lauterborn, P.J. Isackson, C.M. Gall, Seizure-induced increases [55] C.A. Singer, K.L. Rogers, T.M. Strickland, D.M. Dorsa, Estrogen in NGF mRNA exhibit different time-courses across forebrain protects primary cortical neurons from glutamate toxicity, Neurosci. regions and are biphasic in hippocampus, Exp. Neurol. 125 (1994) Lett. 212 (1996) 13–16.

22–40. [56] M. Singh, E.M. Meyer, W.J. Millard, J.W. Simpkins, Ovarian steroid [36] A.D. Lawrence, B.J. Sahakian, Alzheimer disease, attention, and the deprivation results in a reversible learning impairment and compro-cholinergic system, Alzheimer Dis. Assoc. Disord. 9 (Suppl. 2) mised cholinergic function in female Sprague–Dawley rats, Brain

(1995) 43–49. Res. 644 (1994) 305–312.

[37] R.M. Lindsay, P.C. Barber, M.R. Sherwood, J. Zimmer, G. Raisman, [57] M. Singh, E.M. Meyer, J.W. Simpkins, The effects of ovariectomy Astrocyte cultures from adult rat brain. Derivation, characterization and estradiol replacement on brain-derived neurotrophic factor and neurotrophic properties of pure astroglial cells from corpus messenger ribonucleic acid expression in cortical and hippocampal callosum, Brain Res. 243 (1982) 329–343. brain regions of female Sprague–Dawley rats, Endocrinology 136 [38] V.N. Luine, Estradiol increases choline acetyltransferase activity in (1995) 2320–2324.

specific basal forebrain nuclei and projection areas of female rats, [58] M. Singh, G. Setalo Jr., X. Guan, M. Warren, C.D. Toran-Allerand, Exp. Neurol. 89 (1985) 484–490. Estrogen-induced activation of mitogen-activated protein kinase in

(11)

cerebral cortical explants: convergence of estrogen and neurotrophin perfusion-independent mechanisms in rats, Stroke 30 (1998) 630– signaling pathways, J. Neurosci. 19 (1999) 1179–1188. 637.

[59] M. Singh, G. Setalo, X.P. Guan, D.E. Frail, C.D. ToranAllerand, [65] J.J. Watters, J.S. Campbell, M.J. Cunningham, E.G. Krebs, D.M. Estrogen-induced activation of the mitogen-activated protein kinase Dorsa, Rapid membrane effects of steroids in neuroblastoma cells: cascade in the cerebral cortex of estrogen receptor-alpha knock-out effects of estrogen on mitogen activated protein signaling cascade mice, J. Neurosci. 20 (2000) 1694–1700. and c-fos immediate early gene transcription, Endocrinology 138 [60] S. Strauss, U. Otten, B. Joggerst, K. Pluss, B. Volk, Increased levels (1997) 4030–4033.

of nerve growth factor (NGF) protein and mRNA and reactive [66] G.L. Wenk, The nucleus basalis magnocellularis cholinergic system: gliosis following kainic acid injection into the rat striatum, Neurosci. one hundred years of progress, Neurobiol. Learn. Mem. 67 (1997)

Lett. 168 (1994) 193–196. 85–95.

[61] S. Sudo, T.C. Wen, J. Desaki, S. Matsuda, J. Tanaka, T. Arai, N. [67] H.R. Widmer, B. Knusel, F. Hefti, BDNF protection of basal Maeda, M. Sakanaka, Beta-estradiol protects hippocampal CA1 forebrain cholinergic neurons after axotomy: complete protection of neurons against transient forebrain ischemia in gerbil, Neurosci. Res. p75NGFR-positive cells, NeuroReport 4 (1993) 363–366. 29 (1997) 345–354. [68] R.W. Williams, P. Rakic, Three-dimensional counting: an accurate [62] M.-X. Tang, D. Jacobs, Y. Stern, K. Marder, P. Schofield, B. and direct method to estimate numbers of cells in sectioned material, Gurland, H. Andrews, R. Mayeux, Effect of oestrogen during J. Comp. Neurol. 278 (1988) 344–352, published erratum appears in menopause on risk and age at onset of Alzheimer’s disease, Lancet J. Comp. Neurol. 281 (1989) 335).

348 (1996) 429–432. [69] N.J. Woolf, Cholinergic systems in mammalian brain and spinal [63] M.L. Voytko, Cognitive functions of the basal forebrain cholinergic cord, Prog. Neurobiol. 37 (1991) 475–524.

system in monkeys: memory or attention? Behav. Brain Res. 75 [70] F. Zafra, B. Hengerer, J. Leibrock, H. Thoenen, D. Lindholm, (1996) 13–25. Activity dependent regulation of BDNF and NGF mRNAs in the rat [64] Q. Wang, R. Santizo, V.L. Baughman, D.A. Pelligrino, Estrogen hippocampus is mediated by non-NMDA glutamate receptors,

(1)

tralateral (F(3,37)53.3, 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 margincontralater-al 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 (F(1,25)515.8, P,0.001), a significant main effect

killed 3 months following injury) due to incomplete

of Surgical Condition (F(2,25)532.2, P,0.001) and a

transections. The number of ChAT-positive cells detected

significant Surgical Condition3Side interaction (F(2,25)5

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-(F(2,25)531.80, P,0.001) and contralateral (F(2,25)5

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

(F(1,25)51.3, 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 (F(1,25)54.0, 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

(F(2,25)50.84, P50.45) or Hormone Treatment3Surgical

significant effect of Side (F(1,20)538.1, P,0.001), a

Treatment3Side (F(2,25)50.23, P50.80) were detected.

significant effect of Time (F(1,20)514.6, P,0.005), and a

A one-way ANOVA comparing the effects of ibotenate

significant interaction between Side3Time (F(1,20)523.8,

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

(F(1,20)53.1, P50.09) and no significant interactions

condition ipsilateral (F(3,37)525.9, P,0.001) and con-

between Hormone Treatment3Side (F(1,20)52.1, P5

Table 2

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

(2)

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 (F(1,20)50.05,

mechanical injury. It should be noted that this does not

P

50.82) 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.

One possibility is that estrogen failed to reduce the toxic

The data demonstrate that estrogen replacement did not

effects of ibotenic acid because either the dose of estrogen

prevent the loss of cholinergic neurons in the NBM

was too low, or the dose of ibotenic acid was too high.

following injections of ibotenic acid. This is demonstrated

Previous studies, including additional data collected from

by the fact that unilateral injection of ibotenic acid into the

over one hundred animals (unpublished observations), have

NBM produced a significant decrease in the number of

shown that the estrogen capsules that were used produce

ChAT-positive cells detected in the NBM on the injected

mean circulating levels of estradiol in the high

physiologi-side within 2 weeks post-injection, and that estrogen

cal range (60–120 pg / ml). This was confirmed in two

treatment had no significant effect on the magnitude of the

E-treated animals that had serum levels of estradiol of 85.6

Table 3

Mean numbers of ChAT-positive profiles in the medial septum following fimbria / fornix transection

Survival time No estrogen Estrogen

following transection

Contralateral Ipsilateral Contralateral Ipsilateral

2 weeks 55.7611.3 29.9614.2* 54.76 7.7 22.569.9*

12 weeks 64.6614.3 65.6621.7 56.4611.2 48.6613.4

(3)

and 115.0 pg / ml. This suggests that estrogen levels were

direct estrogen effects on the cholinergic neurons mediated

sufficient to elicit any neuroprotective effect that would be

by the activation of classical estrogen receptors. Recent

associated with physiological levels of circulating es-

studies have shown that a significant percentage of the

tradiol. Furthermore, a preliminary study (unpublished

cholinergic neurons in the MS contain estrogen receptor

observations) showed that half the dose of ibotenic acid

alpha

mRNA

and

protein

and

bind

17b-estradiol

produced much less cell loss overall, and much less of a

[15,41,52], suggesting that estrogen may directly effect

loss of ChAT-positive cells (less than half the degree of

ChAT expression via ligand-mediated activation of the

cell loss) than was obtained in the present study. This

estrogen receptor. It is possible that mechanical injury

suggests that the extent to which the dose–response curve

induces cellular responses that prevent estrogen

receptor-would need to be affected in order to elicit a significant

mediated activation of the ChAT gene. One interesting

neuroprotective effect was not so great as to account for

study would be to administer estrogen 3 months following

the lack of an effect of estrogen. In addition, Singer et al.

transection of the fimbria / fornix when the injury response

[55] reported that estrogen significantly protected cortical

has subsided and the number of ChAT-positive cells in the

neurons from glutamate-induced cell death in culture using

MS has recovered. An estrogen-mediated induction of

glutamate levels as high as 0.1 mM. Azcoitia et al. [2]

ChAT expression at this time could indicate that the

reported that estrogen reduced cell loss in the dentate gyrus

inability to induce ChAT expression 2 weeks following

following systemic injection of 7 mg / kg kainic acid. These

injury is due to an injury-induced response which blocks

data suggest that the concentration of ibotenate used in the

estrogen-mediated transcriptional activation as opposed to

present study was not so overwhelming as to preclude the

disconnection from the hippocampus.

ability to detect a neuroprotective effect of estrogen.

Estrogen has also been shown to exert neuroprotective

Nevertheless, the possibility that estrogen has neuroprotec-

effects via non-classical mechanisms, possibly involving

tive effects on the cholinergic neurons when administered

activation of specific second messenger systems such as

at different doses or for different periods of time, or that

ERK and MEK. Several studies have documented rapid

estrogen protects the cholinergic neurons from other types

membrane effects of estrogen in cells which lack estrogen

of neurotoxic or mechanical injury, cannot be excluded.

receptors, resulting in neuroprotection or in the activation

Notably, estrogen treatment did increase the number of

of ERK and MEK [54,58,59,65]. Whether estrogen acts on

ChAT-positive cells detected in the NBM contralateral to

basal forebrain cholinergic neurons via non-classical

path-the ibotenate injection, and in path-the MS ipsilateral to path-the

ways and whether those effects are altered following

NBM injection, at 2 weeks post-injury. We interpret these

neurochemical or mechanical injury are currently

un-increases as un-increases in ChAT protein within cholinergic

known.

cells that were previously undetectable. These data are

Another interesting observation was the fact that

unila-consistent with previous studies showing that estrogen

teral injection of ibotenic acid into the NBM produced a

replacement increases the number of ChAT-positive cells

significant increase in the number of ChAT-positive cells

[12,25], as well as relative levels of ChAT mRNA [16], in

detected in the contralateral NBM, and in both the

the MS and NBM and that the effects depend on both the

ipsilateral and contralateral MS, at 2 weeks following

dose and duration of estrogen treatment [12]. The fact that

injury relative to controls. Increased numbers of

ChAT-estrogen replacement did not diminish the loss of ChAT

positive cells were also observed in the cannula controls

positive cells in the MS following fimbria / fornix transec-

although the effects were not statistically significant. This

tion suggests that the injury interferes with the mecha-

suggests that the magnitude of the effect increases with an

nism(s) by which estrogen increases ChAT. This could be

increased amount of injury. In contrast, no significant

due to the disconnection of the cholinergic neurons from

increases in the numbers of ChAT-positive cells were

their targets, thereby isolating the cells from target-derived

detected in animals killed 3 months following ibotenic acid

growth factors. Estrogen and progesterone replacement

injection, suggesting that the effect is an acute response to

have been shown to increase the levels of brain derived

injury in the NBM. One likely possibility is that the

neurotrophic factor (BDNF) mRNA in the hippocampus

increases in ChAT-postive cells detected in the MS and the

[18,20,57] and BDNF protein in the septum [20]. BDNF is

contralateral NBM are due to injury-induced growth

a nerve growth factor-related polypeptide which is pro-

factors which accumulate around the site of injury and in

duced in the hippocampus and cortex, and which has been

the targets of the injured cholinergic neurons. Recent

shown to exert trophic effects on basal forebrain choliner-

studies have shown that astrocytes can produce nerve

gic neurons [45,67]. Transection of the fimbria / fornix

growth factor both in vitro [37,50,60] and in vivo [1], and

would prevent any estrogen-mediated induction of BDNF

that NGF production by astrocytes can increase

dramatical-in the hippocampus from affectdramatical-ing the dramatical-injured choldramatical-inergic

ly in response to brain injury [1,26]. NGF levels have also

cells, which could explain the inability of estrogen replace-

been shown to increase in the hippocampus and cortex

ment to reduce the loss of cholinergic neurons in the MS

following the loss of basal forebrain cholinergic inputs

following fimbria / fornix transection.

[44,51]. Numerous studies have shown that NGF has

Another possibility is that the injury may interfere with

trophic effects on basal forebrain cholinergic neurons (see

(4)

growth factor expression after chronic neurotoxic injury — I.

Ref. [13]). Therefore, it is reasonable to suspect that

Degeneration and plastic response of basal forebrain neurons,

injury-induced increases in NGF contribute at least in part

Neuroscience 65 (1995) 633–645.

to the increases in ChAT-positive cells detected in the MS

_{[2] I. Azcoitia, A. Sierra, L.M. Garcia-Segura, Estradiol prevents kainic}

and in the contralateral NBM.

_{acid-induced neuronal loss in the rat dentate gyrus, Neuroreport 9}

Even so, the increases in the numbers of ChAT-positive

(1998) 3075–3079.

[3] M. Baldereschi, A. Di Carlo, V. Lepore, L. Bracco, S. Maggi, F.

cells detected in the ipsilateral MS of E-treated and

non-E-Grigoletto, G. Scarlato, L. Amaducci, Estrogen-replacement therapy

treated animals 2 weeks following an injection of ibotenate

and Alzheimer’s disease in the Italian Longitudinal Study on Aging,

into the NBM were quite striking (2–3 fold). This

indi-Neurology 50 (1998) 996–1002.

cates a transient, but powerful, effect of the ibotenate

_{[4] C. Behl, T. Skutella, F. Lezoualch, A. Post, M. Widmann, C.J.}

injections on cholinergic neurons in the ipsilateral MS.

_{Newton, F. Holsboer, Neuroprotection against oxidative stress by}

This may be caused by the leakage of some ibotenate up

estrogens: structure–activity relationship, Mol. Pharmacol. 51

(1997) 535–541.

the cannula tract into the hippocampus, resulting in

[5] J.A. Chowen, I. Torres-Aleman, L.M. Garcia-Segura, Trophic

excitation of the hippocampal neurons. Previous studies

effects of estradiol on fetal rat hypothalamic neurons,

Neuro-have shown that increased electrical activity in the

hip-endocrinology 56 (1992) 895–901.

pocampus results in the up-regulation of both NGF and

_{[6] J.A.M. Dekker, D.J. Connor, L.J. Thal, The role of cholinergic}

BDNF within hippocampal neurons [9,32,35,70]. Any

_{projections from the nucleus basalis in memory, Neurosci.}

leakage of ibotenate up the cannula tract and into the

Biobehav. Rev. 15 (1991) 299–317.

[7] D.B. Dubal, P.J. Shughrue, M.E. Wilson, I. Merchenthaler, P.M.

hippocampus would undoubtedly produce increased

neuro-Wise, Estradiol modulates bcl-2 in cerebral ischemia: a potential

nal activity within the hippocampus, which could result in

role for estrogen receptors, J. Neurosci. 19 (1999) 6385–6393.

increased NGF and BDNF expression and increased

[8] H.C. Fibiger, Cholinergic mechanisms in learning, memory and

trophic support for cholinergic neurons in the MS. This

_{dementia: a review of recent evidence, Trends Neurosci. 14 (1991)}

could account for the dramatic and transient increase in the

_220–223.

number of ChAT-positive cells detected in the MS ipsila-

[9] C.M. Gall, P.J. Isackson, Limbic seizures increase neuronal pro-duction of messenger RNA for nerve growth factor, Science 245

teral to the ibotenate injections. Additional studies which

(1989) 758–761.

examine changes in hippocampal and cortical levels of

[10] L.M. Garcia-Segura, P. Cardona-Gomez, F. Naftolin, J.A. Chowen,

NGF and BDNF following unilateral ibotenic acid

in-Estradiol upregulates Bcl-2 expression in adult brain neurons,

jections into the NBM would reveal the extent to which

_{NeuroReport 9 (1998) 593–597.}

changes in neurotrophin expression may contribute to the

_{[11] R.B. Gibbs, Effects of estrogen on basal forebrain cholinergic}

increased numbers of ChAT-positive cells detected in the

neurons and cognition: Implications for brain aging and dementia in women, in: M. Morrison (Ed.), Hormones, Aging, and Mental

MS.

Disorders, Cambridge University Press, Cambridge, 2000, pp. 183–

In conclusion, the present studies failed to provide

222.

evidence for an ability of estrogen replacement to prevent

[12] R.B. Gibbs, Effects of estrogen on basal forebrain cholinergic

the loss of ChAT-positive neurons in the MS and NBM

_{neurons vary as a function of dose and duration of treatment, Brain}

following excitatory amino acid toxicity or mechanical

_{Res. 757 (1997) 10–16.}

injury. Whether estrogen is neuroprotective for the

[13] R.B. Gibbs, Estrogen and nerve growth factor-related systems in brain: effects on basal forebrain cholinergic neurons and

implica-cholinergic cells against other types of injury such as

tions for learning and memory processes and aging, in: V.N. Luine,

b-amyloid-induced toxicity or cortical impact injury, and

C.F. Harding (Eds.), Hormonal Restructuring of the Adult Brain,

whether estrogen can reduce the loss of cholinergic

Basic and Clinical Perspectives, Vol. 743, New York Academy of

neurons associated with normal aging, are still unknown.

_{Science, New York, 1994, pp. 165–199.}

Hence, the possibility that neuroprotective effects of

[14] R.B. Gibbs, Oestrogen and the cholinergic hypothesis: Implications

estrogen on basal forebrain cholinergic neurons contribute

for oestrogen replacement therapy in postmenopausal women, in: J. Goode (Ed.), Neuronal and Cognitive Effects of Oestrogens,

Novar-to the ability of estrogen replacement Novar-to reduce the risk

tis Found. Symposium 230, Wiley, Chichester, 2000, pp. 94–111.

for developing Alzheimer’s-related dementia in

post-[15] R.B. Gibbs, Expression of estrogen receptor-like immunoreactivity

menopausal women cannot be excluded.

_{by different subgroups of basal forebrain cholinergic neurons in}

gonadectomized male and female rats, Brain Res. 720 (1996) 61–68.

[16] R.B. Gibbs, Fluctuations in relative levels of choline

acetyltrans-Acknowledgements

ferase mRNA in different regions of the rat basal forebrain across the estrous cycle: effects of estrogen and progesterone, J. Neurosci.

Estradiol radioimmunoassays were performed by the

_{16 (1996) 1049–1055.}

Assay Core of the Center for Reproductive Physiology.

[17] R.B. Gibbs, Impairment of basal forebrain cholinergic neurons

This work was supported by NIH Grant NS28896.

associated with aging and long-term loss of ovarian function, Exp. Neurol. 151 (1998) 289–302.

[18] R.B. Gibbs, Levels of trkA and BDNF mRNA, but not NGF mRNA, fluctuate across the estrous cycle and increase in response to acute

References

_{hormone replacement, Brain Res. 787 (1998) 259–268.}

[19] R.B. Gibbs, Long-term treatment with estrogen and progesterone ¨

(5)

protein in different regions of the adult rat brain, Brain Res. 844 [40] P.J. McMillan, C.A. Singer, D.M. Dorsa, The effects of ovariectomy

(1999) 20–27. and estrogen replacement on trkA and choline acetyltransferase

[21] R.B. Gibbs, A.M. Burke, D.A. Johnson, Estrogen replacement mRNA expression in the basal forebrain of adult female Sprague– attenuates effects of scopolamine and lorazepam on memory acqui- Dawley rat, J. Neurosci. 16 (1996) 1860–1865.

subreg-NGFR

cortical projections by homotypic transplants of entorhinal cortex, J. _{axotomy, J. Comp. Neurol. 325 (1992) 219–242.}

Comp. Neurol. 237 (1985) 47–64. _{[43] E.A. Nimchinsky, P.R. Hof, W.G. Young, F.E. Bloom, J.H.} Mor-[24] R.B. Gibbs, A. Hashash, D.A. Johnson, Effects of estrogen on

rison, NeuroZoom software: development, validation, and neuro-potassium-evoked acetylcholine release in the hippocampus and

biological application, FASEB J. 12 (1998) A628. overlying cortex of adult rats, Brain Res. 749 (1997) 143–146.

[44] A. Nitta, Y. Furukawa, K. Hayashi, T. Hasegawa, T. Nabeshima, [25] R.B. Gibbs, D.W. Pfaff, Effects of estrogen and fimbria / fornix

NGFR Nerve growth factor content of rat brain increases following basal

transection on p75 and ChAT expression in the medial septum