estrogen replacement does not prevent the loss of choline acetyltransferase-positive cells in the...

Brain Research 882 (2000) 75–85www.elsevier.com/ locate /bres

Research report

Estrogen replacement does not prevent the loss of cholineacetyltransferase-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. Studieshave 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 ofneurochemical and mechanical injury. Ovariectomized Sprague–Dawley rats received either estrogen replacement or sham surgery, andthen received either a unilateral injection of ibotenic acid into the nucleus basalis magnocellularis, or unilateral transection of the fimbriafornix. Cholinergic neurons in the medial septum and nucleus basalis were detected and quantified using immunohistochemicaltechniques. The data show that neither 3 weeks nor 13 weeks of continuous estrogen replacement prevented the loss of cholineacetyltransferase (ChAT)-containing cells in the nucleus basalis following a unilateral injection of ibotenic acid. Likewise, estrogenreplacement did not prevent a decrease in ChAT-positive cells detected in the medial septum following unilateral transection of thefimbria fornix. Notably, increased numbers of ChAT-positive cells were detected in the contralateral nucleus basalis, and in the ipsilateraland contralateral medial septum, at 2 weeks following a unilateral injection of ibotenic acid into the nucleus basalis; however, theseeffects were not related to hormone treatment. These data suggest that estrogen replacement does not protect cholinergic neurons in themedial septum and nucleus basalis from the effects of excitotoxic or mechanical injury. 2000 Elsevier Science B.V. All rightsreserved.

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] forreview).

Several recent prospective studies suggest that es- Recent studies have show that estrogen has significanttrogen replacement can help to reduce the risk of Alz- neuroprotective effects in vitro on a wide variety ofheimer’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 cellunknown, although evidence for significant estrogen ef- death associated with serum deprivation, growth factorfects on neuronal anatomy, biochemistry, and function deprivation, b-amyloid toxicity, excitatory amino acidwithin 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 neuroblastomaand non-neuronal cell lines. In some cases estrogen-me-diated neuroprotection in culture is blocked with selectiveestrogen receptor antagonists [5,55], suggesting that es-trogen supports survival via the activation of estrogen*Corresponding author. Fax: 11-412-624-1850.

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

0006-8993/00/$ – see front matter 2000 Elsevier Science B.V. All rights reserved.PI I : S0006-8993( 00 )02832-8

76 P. Aggarwal, R.B. Gibbs / Brain Research 882 (2000) 75 –85

neuroprotection in culture is related to the structure of the 2. Materials and methodsestrogen molecule and not to any specific estrogenicity ofthe molecule or to the activation of estrogen receptors 2.1. Animals[4,27–29]. Neuroprotective effects of estrogen in vivohave also been described. Specifically, several studies have Young female Sprague–Dawley rats were purchasedreported the ability of estrogen replacement to reduce from Hilltop Laboratories and housed in pairs on a 12:12-hinfarct 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 beforestudy, the reduction in infarct size was associated with the use. All animals then underwent bilateral ovariectomy andprevention of injury-induced down-regulation of bcl-2 and 2 weeks later, received either 3-mm silastic capsulesestrogen receptor beta gene expression [7]. Ischemic injury containing 17b-estradiol crystals (implanted s.c.) or blankwas also shown to dramatically upregulate estrogen re- capsules. Studies have shown that these estrogen capsulesceptor alpha gene expression on the injured side. Estrogen produce mean circulating levels of estradiol in the range ofhas 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 leastsome neuroprotective effects of estrogen in vivo may 2.2. Neurochemical lesionsinvolve the selective regulation of estrogen receptors andestrogen 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 ofer’s disease is the loss of cholinergic neurons in the medial ketamine (14 mg/100 g body wt.) and xylazine (2.8septum, the diagonal band of Broca, and the nucleus mg/100 g body wt.) and placed into a standard stereotaxicbasalis 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 basalisthe hippocampus and neocortex [69] and play an important magnocellularis (NBM) by intracerebroventricular infusionrole 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 thereplacement 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 inin choline acetyltransferase mRNA and protein in the sterile filtered saline) was lowered 7.5 mm from the skullmedial 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 minand overlying cortex. Treatment with estrogen and with and then withdrawn very slowly. The skin overlying theestrogen plus progesterone have also been shown to skull was sutured shut and the animals were placed onto aincrease the expression of trkA (nerve growth factor warm heating pad during recovery. Two separate controlreceptor) mRNA [18,40] in the medial septum and to groups were prepared. Cannula controls (consisting of fiveincrease relative levels of brain-derived neurotrophic factor E-treated and five non-E-treated animals) had an emptymRNA in the hippocampus [18,20,57], suggesting that infusion cannula lowered into the NBM to control forestrogen may enhance basal forebrain cholinergic projec- damage produced by lowering the cannula. Sham controlstions 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 wasestrogen replacement can help to reduce the risk of sutured shut. Following surgery, all animals were givenAlzheimer’s-related dementia in postmenopausal women is Torbugesic (0.02 cc /100 g) twice a day for 3 days toby increasing the functional status of cholinergic projec- reduce discomfort associated with the surgical procedures.tions to the hippocampus and neocortex. Whether estrogenreplacement also reduces the loss of cholinergic neurons 2.3. Fimbria /fornix transectionsassociated with Alzheimer’s disease, and/or protects thecholinergic 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 ofto examine potential neuroprotective effects of estrogen ketamine (14 mg/100 g body wt.) and xylazine (2.8replacement on basal forebrain cholinergic neurons by mg/100 g body wt.) and placed into a standard stereotaxicexamining the ability of estrogen replacement to reduce device. Unilateral transections of the fimbria / fornix werelosses of ChAT positive neurons following ibotenic acid performed as previously described [22]. Briefly, the skullinjection into the nucleus basalis magnocellularis and was exposed and a trench was drilled extending laterallyfollowing transection of the fimbria fornix. from midline 1.0 mm posterior to the coronal suture. An

P. Aggarwal, R.B. Gibbs / Brain Research 882 (2000) 75 –85 77

incision was made through the dura to expose the brain. method as previously described [23] to verify that theAn ultrathin microknife (Tieman) was lowered 6.0 mm unilateral transections were complete.into the brain at midline. Care was taken to avoid themidsagittal sinus which was gently pushed to the side. The 2.5. ChAT immunostainingknife was moved laterally along the trench to bone andthen removed. The trench was packed with gelfoam soaked ChAT was detected as previously described [12] usingwith sterile saline. The skin was sutured shut and the an affinity purified goat polyclonal antibody raised againstanimals were placed onto a warm heating pad during rat ChAT (Chemicon Pharmaceuticals, AP144P; dilutedrecovery. 1:3500 in 50 mM PBS (pH 7.2) containing 0.05% Triton

X-100 and 1% normal rabbit serum). Briefly, sections were2.4. Tissue preparation placed in the primary antibody solution for 3 days at 48C.

The sections were rinsed well with PBS and then incubatedTen 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 incubatedcontrols were killed 2 weeks post-surgery. The remaining with an avidin–HRP complex (Vectastain Elite kit; Vectorten ibotenate-treated animals (five E-treated and five non- Laboratories) for 1 h at room temperature. Sections wereE-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), andsurgery. 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 ontoacetate (pH 6.5), and then 4% paraformaldehyde in 50 mM Superfrost plus slides (Fisher Scientific), counterstainedTris (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 ofestradiol treatment to verify that the estrogen capsules 2.6. Data analysiswere producing the expected levels of circulating estradiol.All capsules were inspected and the presence of estradiol 2.6.1. Ibotenate-treated animals and related controlscrystals in the E-capsules was verified. Note that these Sections through the MS and NBM of ibotenate-treatedcapsules have been shown to continue releasing estradiol animals and related controls were analyzed using unbiasedeven after long-term implantation [19]. The brains were stereological techniques. Briefly, the total number ofremoved and postfixed for 3–4 h with 4% paraformal- ChAT-positive profiles in the NBM and MS on each sidedehyde in phosphate buffer (50 mM, pH 7.2) at 48C and of the brain was estimated using the Optical Fractionatorthen stored overnight in 15% sucrose in 50 mM phosphate- method [30]. The analysis was performed using a Leitzbuffered 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 G3coronal sections were cut through the MS (including plates computer running the Neurozoom software [43]. Three15–17 of Paxinos and Watson [48]) and the NBM (includ- equivalently spaced sections through the MS (120 mming plates 24–25 of Paxinos and Watson [48]) of each apart, beginning at approximately plate 15 of Paxinos andbrain and every third section was processed for immuno- Watson [48]) and six equivalently spaced sections throughcytochemical detection of choline acetyltransferase the NBM (120 mm apart, beginning at approximately plate(ChAT) as described below. Unbiased stereological pro- 23 of Paxinos and Watson [48]) were analyzed from eachcedures were then used to quantify the number of ChAT- brain. The dorsal and ventral boundaries of the septumpositive 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. Theanimals were processed prior to the use of stereological medial boundary was defined by drawing a line down theprocedures in our laboratory. For these animals, 25-mm middle of the septum (Fig. 1). The boundaries of the NBMcoronal sections through the MS were cut and every third were defined at low magnification by drawing linessection was processed for immunocytochemical detection connecting the globus pallidus to the reticular nucleus ofof ChAT. These sections were analyzed by counting the the thalamus and the substantia innominata (Fig. 2). Oncenumber of ChAT-positive profiles in the MS of three the borders of each region were outlined, 20% of the totalmatched sections /animal and then comparing the number area was analyzed in each tissue section on each side. Theof immunoreactive cells / section (see below). Sections dissector, which consists of two planes separated by athrough the hippocampus of the fimbria / fornix-transected specified distance, was set at 10 mm (less than theanimals were stained for the detection of acetylcholinester- minimum diameter of a ChAT-positive cell). A countingase using a modified copper thiocholine identification box was set at 10.37 mm by 7.44 mm and consisted of two


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

forbidden lines and two inclusive lines. Objects completelyinside the counting frame were counted whereas objectscompletely outside the counting frame were excluded.Objects which crossed through any of the three forbiddenplanes (bottom, front, and left sides of the frame) wereexcluded. Objects which crossed through any of theremaining planes (top, right, and back sides of the frame)were included if they either came into focus or went out offocus when focusing through the dissector [68]. Thenucleus of each labeled cell was used as the referencepoint for counting. An estimate of the total number of

Fig. 2. Bright-field photomicrographs showing ChAT staining detected inChAT-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; C562.5 mm.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 Tukeyno 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 animalsANOVA with repeated measures on ‘Side’. The effects of Sections through the medial septum of animals with aibotenic acid 2 weeks and 3 months following injury unilateral transection of the fimbria / fornix were analyzed


as previously described [12,17,25]. Briefly, the average treated animals. Significant interactions between Surgicalnumber 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)5the 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 significantwere 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 thevariance with repeated measures on ‘side’. lesioned (F(2,25)50.26, P50.78) side, contrary to the

three-way interaction detected in the ANOVA. Additionalpost hoc analyses revealed a significant effect of Surgical

3. Results Condition on both the intact (F(2,25)57.30, P,0.005) andthe lesioned (F(2,25)528.62, P,0.005) side.

3.1. Serum estradiol levels A one-way ANOVA comparing the effects of ibotenatein 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 surgicalserum 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 numberrecent studies which show that these capsules produce of ChAT-positive cells detected in the NBM contralateralserum 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 at3.2. 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 cellsNBM on each side of the brain in the ibotenate-injected detected in the NBM ipsilateral to the cannula placementanimals 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 inwas detected in the NBM ipsilateral to the ibotenate the number of ChAT-positive cells detected in the NBMinjection 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. Moreinjection. Analysis of the data from animals killed 2 weeks ChAT-positive cells were also detected in the NBMpost-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 differenceHormone 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 mswas detected (F(2,25)50.96, P50.40); however, a signifi- The number of ChAT-positive cells detected in the MScant 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 morepresence 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 1aMean 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 46.4Cannula 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 theestrogen-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-treatedanimals.


tralateral (F(3,37)53.3, P50.03) to the NBM lesion. ATukey test revealed significantly more ChAT-positive cellsdetected in the MS ipsilateral to the NBM lesion in the2-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 tothe injection in animals killed 2 weeks vs. 3 monthsfollowing injection (P,0.001) Contralateral to the NBMlesion, significantly more ChAT-positive cells were de-tected in the MS of animals killed 2 weeks followingibotenate injection relative to the sham controls (P,0.05).Notably, more ChAT-positive cells were detected in theMS 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 number3.3. Mechanical lesionsof 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 andsham-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 theunilateral 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 dataweeks post-surgery revealed a significant main effect of analysis (three killed 2 weeks following injury and twoSide (F(1,25)515.8, P,0.001), a significant main effect killed 3 months following injury) due to incompleteof Surgical Condition (F(2,25)532.2, P,0.001) and a transections. The number of ChAT-positive cells detectedsignificant 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 decreaseprofiles 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 cells5.47, P50.01) to the ibotenate injection. No significant detected in the MS ipsilateral to the lesion in animalsmain 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 ofHormone Treatment3Side was very close to statistical ChAT-positive cells was detected in the MS on thesignificance (F(1,25)54.0, P50.06). No significant inter- lesioned vs. the unlesioned side in either E-treated oractions 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), aTreatment3Side (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 Treatmentrelative to controls revealed a significant effect of surgical (F(1,20)53.1, P50.09) and no significant interactionscondition ipsilateral (F(3,37)525.9, P,0.001) and con- between Hormone Treatment3Side (F(1,20)52.1, P5

Table 2aMean numbers of ChAT-positive profiles in the medial septum

No estrogen Estrogen


Sham: 2 weeks 7266 38.4 6486 52.5 7956 66.1 8606100.9Cannula injury: 2 weeks 7926 51.6 7926 67.5 7806 49.3 10986166.1Ibotenate: 2 weeks 11946187.4* 16686160.9* 9726156.1* 17886176.9*Ibotenate: 12 weeks 8106109.4 7206100.0 9966159.2 8106121.1a 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 theestrogen-treated shams where n56. *P,0.001 relative to the cannula and sham-operated controls.


cell loss. In addition, no recovery in the number of ChAT-IR cells was detected on the injected side at 3 monthsfollowing injection in either E-treated or non-E-treatedanimals, suggesting that estrogen treatment did not reducecell death or enhance recovery of the cholinergic pheno-type following ibotenic acid injection.

The data also demonstrate that estrogen replacement didnot prevent the loss of ChAT-positive cells in the MSfollowing mechanical injury to the fimbria / fornix. This isdemonstrated by the fact that unilateral transection of thefimbria / fornix produced a significant decrease in thenumber of ChAT-positive cells detected in the MS on theinjured side at 2 weeks post-injury, and that estrogentreatment had no significant effect on the magnitude of thecell loss. Unlike the effects of ibotenate on ChAT-positivecells in the NBM, the number of ChAT-positive cells inthe MS recovered to control levels within 3 monthsfollowing fimbria / fornix transection. This is consistentwith previous reports [31,42,49] and suggests that thedecrease in ChAT-positive cells in the MS followingfimbria / fornix transection is due to an injury-relateddecrease in ChAT expression as opposed to a loss of cells.ChAT expression then recovers over time resulting in anincrease in the number of ChAT-positive cells to controllevels. 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 thecontralateral (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 enhancepresence of AchE-positive staining through the hippocampus contralateral

the functional status of cholinergic neurons in the MS andto the lesion, compared with the absence of staining ipsilateral to theNBM [14], these data suggest that hormone replacementlesion. Scale bar51 mm in both.does not prevent the loss of cholinergic neurons or the lossof 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 notP50.82) were detected. preclude the possibility that estrogen enhances other

measures of cholinergic function within the remainingcholinergic 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 estrogenprevent 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 fromby the fact that unilateral injection of ibotenic acid into the over one hundred animals (unpublished observations), haveNBM produced a significant decrease in the number of shown that the estrogen capsules that were used produceChAT-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 twotreatment had no significant effect on the magnitude of the E-treated animals that had serum levels of estradiol of 85.6

Table 3aMean numbers of ChAT-positive profiles in the medial septum following fimbria / fornix transection

Survival time No estrogen Estrogenfollowing transection


2 weeks 55.7611.3 29.9614.2* 54.76 7.7 22.56 9.9*12 weeks 64.6614.3 65.6621.7 56.4611.2 48.6613.4a Values indicate mean numbers of profiles6S.E.M. ipsilateral and contralateral to a unilateral fimbria / fornix transection. n57 for each treatment groupkilled 2 weeks following transection and n55 for each treatment group killed 12 weeks following transection. *P,0.05 relative to the contralateral side.


and 115.0 pg/ml. This suggests that estrogen levels were direct estrogen effects on the cholinergic neurons mediatedsufficient to elicit any neuroprotective effect that would be by the activation of classical estrogen receptors. Recentassociated with physiological levels of circulating es- studies have shown that a significant percentage of thetradiol. Furthermore, a preliminary study (unpublished cholinergic neurons in the MS contain estrogen receptorobservations) showed that half the dose of ibotenic acid alpha mRNA and protein and bind 17b-estradiolproduced much less cell loss overall, and much less of a [15,41,52], suggesting that estrogen may directly effectloss of ChAT-positive cells (less than half the degree of ChAT expression via ligand-mediated activation of thecell loss) than was obtained in the present study. This estrogen receptor. It is possible that mechanical injurysuggests 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 interestingneuroprotective effect was not so great as to account for study would be to administer estrogen 3 months followingthe 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 theneurons from glutamate-induced cell death in culture using MS has recovered. An estrogen-mediated induction ofglutamate levels as high as 0.1 mM. Azcoitia et al. [2] ChAT expression at this time could indicate that thereported that estrogen reduced cell loss in the dentate gyrus inability to induce ChAT expression 2 weeks followingfollowing systemic injection of 7 mg/kg kainic acid. These injury is due to an injury-induced response which blocksdata suggest that the concentration of ibotenate used in the estrogen-mediated transcriptional activation as opposed topresent 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 neuroprotectiveNevertheless, the possibility that estrogen has neuroprotec- effects via non-classical mechanisms, possibly involvingtive effects on the cholinergic neurons when administered activation of specific second messenger systems such asat different doses or for different periods of time, or that ERK and MEK. Several studies have documented rapidestrogen protects the cholinergic neurons from other types membrane effects of estrogen in cells which lack estrogenof 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 onChAT-positive cells detected in the NBM contralateral to basal forebrain cholinergic neurons via non-classical path-the ibotenate injection, and in the MS ipsilateral to the ways and whether those effects are altered followingNBM injection, at 2 weeks post-injury. We interpret these neurochemical or mechanical injury are currently un-increases as 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 areplacement 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 thethe MS and NBM and that the effects depend on both the ipsilateral and contralateral MS, at 2 weeks followingdose 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 controlspositive cells in the MS following fimbria / fornix transec- although the effects were not statistically significant. Thistion suggests that the injury interferes with the mecha- suggests that the magnitude of the effect increases with annism(s) by which estrogen increases ChAT. This could be increased amount of injury. In contrast, no significantdue to the disconnection of the cholinergic neurons from increases in the numbers of ChAT-positive cells weretheir targets, thereby isolating the cells from target-derived detected in animals killed 3 months following ibotenic acidgrowth factors. Estrogen and progesterone replacement injection, suggesting that the effect is an acute response tohave been shown to increase the levels of brain derived injury in the NBM. One likely possibility is that theneurotrophic 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 growtha nerve growth factor-related polypeptide which is pro- factors which accumulate around the site of injury and induced in the hippocampus and cortex, and which has been the targets of the injured cholinergic neurons. Recentshown to exert trophic effects on basal forebrain choliner- studies have shown that astrocytes can produce nervegic neurons [45,67]. Transection of the fimbria / fornix growth factor both in vitro [37,50,60] and in vivo [1], andwould prevent any estrogen-mediated induction of BDNF that NGF production by astrocytes can increase dramatical-in the hippocampus from affecting the injured cholinergic ly in response to brain injury [1,26]. NGF levels have alsocells, which could explain the inability of estrogen replace- been shown to increase in the hippocampus and cortexment to reduce the loss of cholinergic neurons in the MS following the loss of basal forebrain cholinergic inputsfollowing 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


growth factor expression after chronic neurotoxic injury — I.Ref. [13]). Therefore, it is reasonable to suspect thatDegeneration and plastic response of basal forebrain neurons,injury-induced increases in NGF contribute at least in partNeuroscience 65 (1995) 633–645.to the increases in ChAT-positive cells detected in the MS

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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 therapytreated animals 2 weeks following an injection of ibotenateand 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

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