Tha Journal of Nauroaciancn, May 1992, 12(5): 1936-1944

Cholinergic Cell Loss and Hypertrophy in the of the Behaviorally Characterized Aged Rhesus Monkey

Heidi M. Stroessner-Johnson, la2 Peter R. Rapp,’ and David G. Amarall ‘The Salk Institute, and ZThe Group in Neurosciences, The University of California at San Diego, La Jolla, California 92037

chemical, and physiological changesthat occur during scnes- Quantitative studies were conducted to determine the num- cence. A valuable experimental strategy emerging from these ber and size of cholinergic neurons in the medial septal investigations involves combined behavioral and neurobiol- nucleus of four aged (23-25 years old) and four young (1 O- ogical assessmentin the samesubjects as a meansof identifying 12 years old) rhesus monkeys. All of the animals had been those neural alterations in the aged brain that are associated tested on an extensive battery of learning and memory tasks with senescentmemory dysfunction. prior to these experiments. Two of the aged monkeys dis- Multiple lines of evidence indicate that the played a pattern of recognition memory deficits that resem- cholinergic system is significantly affected as a consequenceof bled the effects of medial temporal lobe damage. The post- normal aging. Numerous studies have reported a substantial mortem anatomical data were analyzed in relation to both degreeof cholinergic cell lossand/or atrophy in the septal nuclei the age and behavioral status of the animals. Across all of aged rodents (Hombcrgcr et al., 1985; Fischer et al., 1987, rostrocaudal levels of the medial septal nucleus, there was 1989; Gilad et al., 1987; Mesulam et al., 1987; Altavista et al., a 19.3% decrease in the number of cholinergic neurons in 1990; Markram and Segal, 1990). Interestingly, these morpho- the aged monkeys. The loss was regionally selective, how- metric changesare most pronounced among aged subjectsthat ever, and ranged from a low of 6.2% rostrally to 40.9% exhibit robust deficits on learning and memory tasks that are caudally. The degree of cell loss was similar in both memory- dependent on the functional integrity of the hippocampal for- impaired and memory-unimpaired aged animals. Morpho- mation (Fischer et al., 1989; Koh et al., 1989). In the spatial logical analysis also revealed that the mean cross-sectional version ofthe Morris water maze, for example, age-relatedleam- area of cholinergic neurons was significantly larger in the ing and memory deficits are correlated with both the number aged animals. At caudal levels, the increase in average cell and size ofcholinergic cells in the medial septal nucleus(Fischer size was at least partly due to a disproportionate loss of et al., 1989). While the role of the cholinergic system in normal small to medium size neurons. At rostra1 levels of the medial memory function remains controversial (Dunnett et al., 1991; septal nucleus, however, where there was minimal cell loss, Fibigcr, 199 l), data from pharmacological and lesion studies a clear hypertrophy of cholinergic neurons was evident. In- demonstrating that disruption of cholinergic function can pro- terestingly, the cell hypertrophy observed at these rostra1 foundly affect memory in young rats and humans(reviewed in levels was present only in brains from the behaviorally im- Drachman and Sahakian, 1979; Deutsch, 1983) support the paired aged monkeys. These findings represent the first view that cholinergic abnormalities may contribute to cognitive morphological demonstration of alterations in cholinergic dysfunction in both normal agingand Alzheimer’s disease(Bar- neurons in the aged nonhuman primate. The results empha- tus et al., 1982). size the utility of combined behavioral and neurobiological Recent biochemical evidence indicates that cholinergic sys- assessment in the same subjects in efforts to evaluate the tems are also affected in the nonhuman primate during aging functional significance of neural alterations in the aged pri- (Wenk et al., 1989; Wagster et al., 1990). To date, however, mate brain. there have been no morphometric analysesof forebrain cholin- ergic cells in the aged monkey. Moreover, earlier neurochemical Experimental animal studies and human clinical observations and anatomical studiesin nonhuman primates have compared support the view that deficits in memory and other cognitive brains from young and aged monkeys basedon their chrono- functions are a common consequenceof normal aging.A prom- logical age alone, independent of the functional status of the inent focus of modern researchon the neurobiology of aging is aged subjects.While theseinvestigations have proven valuable to determine which brain regions or systemsare most suscep- in identifying a variety of age-relatedneural alterations (Brizzee tible to aging, and to define the specific morphological, neuro- et al., 1980; Cupp and Uemura, 1980; Uemura, 1980; Wenk et al., 1989, 1991; Price et al., 1990; Wagster et al., 1990) com- Received Aug. 20, 1991; revised Dec. 16, 1991; accepted Dec. 27, 1991. bining material from behaviorally impaired and cognitively in- This work was supported in part by NIH Grants PSO AD-OS 13 1 (a component tact aged subjectsmay provide a misleading representation of of the UCSD Alzheimer’s Disease Research Center), NS-16980, and AG09973. the “average” degreeof neurobiological changethat can be ex- Additional funding was provided by a grant from the Fritz Bums Foundation. We greatly appreciate the expert technical assistance of Ms. Rem% Wall. We thank pccted to emerge as a consequenceof normal aging. This ap- Dr. Ricardo Insausti for assistance in preparing material analyzed in this study. proach also fails to addressthe important possibility that neural Correspondence should be addressed to Dr. David G. Amaral, Laboratory of Neuronal Structure and Function, The Salk Institute, P.O. Box 85800, San Diego, alterations contributing to senescentmemory impairment may CA 92186-5800. be present only in functionally compromised individuals. Copyright 0 1992 Society for Neuroscience 0270-6474/92/121Y36-09$05.00/O Our laboratory has recently initiated a program of studiesin The Journal of Neuroscience. May 1992. 12(5) 1937 which young and aged monkeys are tested on an extensive bat- fixative was removed from the brain by perfusing a solution of 5% tery of learning and memory tasks that has been widely used to sucrose in PB for 20 min. The brains were blocked stereotaxically, removed from the skull, and cryoprotected in a solution of 10% glycerol examine the effects ofexpcrimental medial temporal lobe lesions in PB containing 2% dimethyl sulfoxide (DMSO) for I d, followed by in young monkeys (Rapp and Amaral, 1989, 199 1; Rapp, 1990). 20% glycerol in PB with 2% DMSO for 3 d. The brains were then rapidly Parallel to findings in aged rodents and humans (reviewed in frozen in isopentane (Rosene et al., 1986) and stored at -70°C until Gallagher and Pelleymounter, 1988; Shimamura, 1990), one histologically processed. The brains were sectioned at 30 pm on a freezing sliding microtome important result to emerge from these investigations is that only in the coronal plane and stored as serial adjacent series in a cryopro- a subpopulation of aged monkeys is impaired on memory tasks tectant solution at - 70°C. A l-in- IO series of sections through the septal that are dependent on intact hippocampal function (Presty et nuclei was processed immunohistochemically for the demonstration of al., 1987; Moss et al., 1988; Bachevalier et al., 1991; Rapp and choline acetyltransferase (ChAT) using a monoclonal antibody (ABS) Amaral, 199 1). Approximately 65% of aged monkeys tested in kindly provided by Dr. Bruce Wainer. To prevent variability in staining due to slight modifications in immunohistochemical processing, tissue their mid twenties exhibit a pattern of memory impairment on from the same anatomical level of all eight subjects was processed in the delayed nonmatchingto sample (DNMS) task that resembles the same vessels, using the same reagents, throughout all stages of the the effects of experimental medial temporal lobe damage in procedure. A closely adjacent I -in- IO series of Nissl preparations (thion- young subjects (Rapp and Amaral, 199 1). Recognition memory in) from each brain was used for anatomical reference. Sections were processed using the peroxidase-antiperoxidase (PAP) in other monkeys of the same chronological age remains intact. method (Stemberger, 1986) as previously described (Amaral and Bas- The significance ofthis individual variability is that postmortem sctt, 1989). Briefly, free-floating sections were incubated for 48 hr at analyses can be directed toward identifying those neurobiol- 4°C in a I :500 solution of primary antibody AB8 in Tris-buffered saline ogical markers of aging that are specifically associated with se- (TBS; pH 7.4) containing 2% BSA, 0.5% Triton X-100 (TX- 100) and nescent memory impairment. 20% normal rabbit serum (NRS). Sections were then placed in rabbit anti-rat IgG secondary antiserum (2Ab) (Rappel Labs) diluted I:50 in The present study represents the first in a series of investi- TBS containing 2% BSA, 0.2% TX-100, 20% NRS, and 10% normal gations with the long-range goal of defining the neural basis of monkey serum at room temperature. The tissue was subsequently washed senescent memory dysfunction in the nonhuman primate. A and placed in rat PAP diluted I:50 in the same solution as the 2Ab. A more immediate aim of this research program, however, is to double bridging procedure was employed; sections were incubated for I hr in 2Ab, 2 hr in PAP, I additional hr in the 2Ab, followed by 1.5 define the neuroanatomical effects of aging in memory-related hr in PAP. The tissue was reacted for 20 min in a 0.05% solution of brain regions. As a starting point, we have examined immu- diaminobenzidine in TBS containing 0.015% H,O,. Sections were sub- nohistochemical preparations from behaviorally characterized sequently mounted on gelatin-coated slides, air dried for 3-l d at 37”c, young and aged monkeys, and quantified the number and size and osmium intensified as described previously (Amaral and Bassett, 1989). Following intensification, the sections were dehydrated in as- of medial septal nucleus cells that provide the major cholinergic cending alcohols to xylene and coverslipped with DPX. input to the (Amaral and Cowan, 1980; Quuntitutive analysis. Nissl preparations were used as a basis for Mesulam et al., 1983). The results provide the first demonstra- selecting neuroanatomically matched sections through the medial septal tion in the nonhuman primate that choline& cells undergo a nucleus from each brain. This is an important step since substantial variety of morphometric alterations during aging. In addition, variability in brain size and shape precludes the use ofstereotaxic criteria alone. Only those histological sections in which the medial septal nucleus these findings from relatively small groups of subjects raise the was clearly separated from the subjacent vertical limb of the nucleus of intriguing possibility that the behavioral capacities ofaged mon- the diagonal band were selected for analysis. The baseline section from keys may distinguish a subpopulation of aged animals that is each case was chosen as one section rostra1 to the first appearance of differentially affected by the neurobiological consequences of the descending columns of the fomix. The four sections rostra1 to this (spaced at 300 pm) were also included in the analysis. Thus, the five senescence. sections studied from each brain spanned a rostrocaudal distance of approximately I .2 mm. Materials and Methods In order to count the number of ChAT-positive cells in the medial Subjech and behavioral testing history. Histologicalpreparations used septal nucleus, the positions of immunoreactive profiles were plotted for these studies were obtained from four aged (23-25 years old) and using a computer-aided plotting system. The histological preparations four young (10-l 2 years old) female rhesus monkeys (Mucacu muluffa). were viewed with a 40 x objective, and all cells within the confines of All eight subjects had been tested on a standardized battery of learning the classically defined medial septal nucleus on both sides of the brain and memory tasks prior to death (Rapp and Amaral, 1989, I99 I ; Rapp, were plotted. The number of ChATpositive cells was counted directly 1990). The aged monkeys were divided into impaired (n = 2) and from the plots. Because our intention was to compare the relative num- unimpaired (n = 2) subgroups based on their performance on a DNMS ber of labeled neurons in young and aged brains, rather than to derive recognition memory task that has been widely used to examine the an accurate estimate of the total number of cholinergic medial septal elfecis ofexperimental medial temporal lobe damage in young monkeys. cells in the monkey brain, stereological correction factors were not em- Brieflv. subiects were tested on DNMS according to standard procedures ployed. Cells were classified into three categories: (I) those containing using reteniion intervals ranging from I5 set to IO min. A performance a clearly defined nucleus, (2) those containing a nucleus but with nu- score for each monkey was then calculated as the average percentage merous vacuoles, and (3) neuronal profiles without a definable nucleus. correct across all delays. Aged monkeys with performance scores below The majority of cells were of the nucleated, nonvacuolated variety, and the range of the young group were operationally defined as impaired, the results described below are based on the analysis of this category and the remaining aged monkeys were defined as unimpaired. The alone. Essentially the same pattern of findings was observed, however, impaired subgroup displayed the same delay-dependent pattern of DNMS when other cell categories were included in the analysis. impairment that characterizes performance in young subjects with me- To quantify cross-sectional area, the somata of all nucleated ChAT- dial temporal lobe damage. The unimpaired aged monkeys, in contrast, positive cells without vacuoles were drawn using a camera lucida at- performed as accurately as young controls. These results are described tachment on a Leitz Dialux microscope. Sections were viewed using a in detail elsewhere (Rapp and Amaral, I99 I). 100 x oil immersion lens, and the outlines of immunoreactive somata Histological processing. Following the completion of behavioral as- were traced while focusing through the tissue as needed to avoid in- sessment, animals were deeply anesthetized and transcardially perfused cluding the proximal dendritic trees. In cases where two or more ChAT- with aldehyde fixatives. Perfusion was initiated with a solution of 1% positive cells were overlapping, only those with clearly definable somal paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) (PB) for 2 min outlines were traced and analyzed. The areas of all drawn profiles were (250 ml/min) followed by 4% paraformaldehyde in 0.1 M PB for I hr then measured using a digitizing tablet and microcomputer-based mor- (IO min at 250 ml/mitt, followed by 50 mir at 100 ml/min). Excess phometry software (SIGMASCAN). 1939 StroessnerJohneon et al. - Morphological Alterations in Aged Monkey Medial Septal Nucleus

Figure 1. Photomicrographsof ChAT-labeledneurons in the medialseptal nucleus. Sections are from a youngcontrol animal(left panels), an aged-unimpairedanimal (middle panels), and an aged-impairedanimal (right panels). For eachanimal, the top panel illustratesa rostra1section throughthe medialseptal nucleus (level 2), and the bottom panel representsa more caudal section (level 4). Scalebar, 300 pm.

All measurementswere conductedby one of the authors,who was In particular, the density of reaction product in labeledcells was blind with respectto the ageand behavioral status of the donor animals. comparable in all of the experimental preparations. The pop- Data from both the cell numberand cell sizemeasurements were sta- tistically analyzedusing the STATVIEW II softwarepackage. For docu- ulation of ChAT-positive neurons in the medial septal nucleus mentationof the analyzedsections, 35 mm photomicrographsof the comprised a variety of morphologically distinct cell types. In medialseptal nucleus were taken using a Lcitz Dialux microscopeand a Wild MPS 55 camerasystem. quantifying the number and size of labeled neurons, however, no attempt was made to distinguish between stellate, fusifortn, Results or ovoid cells. The results describedbelow are basedon a total The general staining characteristics of ChAT-immunoreactive sample, acrossall eight brains, of 5265 neurons with a clearly neuronswere similar in the young and aged brains (Figs. 1, 2). visible nucleus. The Journal of Neuroscience, May 1992, f.?(5) 1939

Figure 2. Higher-magnification photomicrographs of ChAT-immunoreactive cells shown in Figure 1; the cells are located in the dorsal half of the medial septal nucleus. Note that the staining characteristics of these cells are similar in all three groups of animals (young, left; aged-unimpaired, middle;aged-impaired, right) and the somal outline can be clearly distinguished from the unstained background. Scale bar, 20 pm.

spectively; t[6] = 3.0, p < 0.05; two-tailed). Interestingly, the Analysis of cell number increasein cell size in the agedgroup was most pronounced in For eachbrain, the total number of ChAT-imunoreactive cells brains from subjectsthat were impaired on the DNMS task (Fig. wasdetermined in five anatomically matched sectionsthrough 4B). Average ChAT-positive cell size was 11.1%greater in the the medial septalnucleus (Fig. 3). Across all of the rostrocaudal aged-impairedmonkeys [mean (*SE), 223.0 (2.5) prn2]relative levelsanalyzed, there wasa 19.3%decrease in the averagenum- to the young subjects[mean (&SE), 200.8 (1.9) pm2]. An overall ber of labeledcells in the agedgroup relative to young animals ANOVA revealed a significant group effect (F[2,5] = 28.5; p < [means(*SE), 588.0 (26.6) and 728.3 (99.4) respectively]. Al- 0.01). Subsequentcomparisons between groups (Scheffe meth- though the data for the two groupswere largely nonoverlapping, od) confirmed that medial septal cholinergic cells were signifi- the effect of agefailed to reach statistical significancedue to a cantly larger in the aged-impairedmonkeys than in either young single young monkey in which the total number of ChAT-pos- subjects(F, = 28.5; p < 0.05) or aged-unimpaired animals (F, itive cells fell below the range of values for the agedgroup. = 9.7; p < 0.05). The 3.6% difference in the cross-sectionalarea Analysis of the number of labeledcells alongthe rostrocaudal extent of the medial septalnucleus revealed that ChAT-positive cell loss in the aged animals was regionally selective (Figs. 1, 3). Across the rostra1three levels, the mean number of immu- noreactive cells in the young and agedgroups differed by only 6.2% [means(*SE), 454.3 (44.8) and 426.3 (30.5), respectively; p > 0.1, one-tailed t test]. In contrast, an averageof 40.9% fewer ChAT-positive neuronswere observed in the two most caudal levels of the nucleusin the agedmonkeys [means(f SE): aged, 161.8(5.8); young, 274.0 (55.5); t[6] = 2.0,~ < 0.05, one-tailed]. Among the aged animals, there was no obvious relationship betweenthe number of immunoreactive cells and performance on the DNMS task. Unimpaired and impaired aged subjects exhibited a comparable degreeof cholinergic cell lossin both caudal and rostra1aspects of the medial septal nucleus (Table 1; Fig. 3). 60. I I I Analysis of cell size 1 2 3 4 5 ROSTRAL + CAUDAL Across the five levels of the medial septal nucleus, the mean LEVEL cross-sectionalarea of ChAT-immunoreactive cells was signif- Figure3. Mean number of ChAT-positive cells across the five rostro- icantly greater in the agedmonkeys relative to young animals caudal levels (1-5) analyzed for the young, aged-unimpaired, and aged- [Fig. 4A; means (&SE), 215.5 (4.4) and 200.8 (1.9) pmZ, re- impaired groups. 1940 StrcessnerJohnson et al. - Morphological Alterations in Agad Monkey Medial Saptal Nucleus

2301 2301

Young Aged Young AgedUnimpaired Aged Impaired C ROSTRAL THREE LEVELS CAUDAL TWO LEVELS 235 235 1 230 230-

Young AgedUnimpaired AgedImpaired Young AgedUnimpaired AgedImpaired

Figure 4. Meancross-sectional area (pm2) of ChAT-positiveneurons in the medialseptal nucleus. A, Comparisonof the youngand aged groups averagedacross all five rostrocaudallevels. B, Comparisonof cell sizein the young,aged-unimpaired, and aged-impairedgroups averaged across all five rostrocaudallevels. C, Comparisonof cell sizein the threegroups of monkeysacross rostra1 (lefl) and caudal(right) levels of the medial septalnucleus. Note that at rostra1levels, neurons in the aged-impairedgroup are largerthan in eitherthe youngor aged-unimpairedgroups. At caudallevels, cells in both of the agedgroups are larger than in the younggroup. of labeledneurons in young [mean (*SE), 200.8 (1.9) pm>]and hypertrophy of cholinergic neuronsin the agedbrain. The cross- aged-unimpaired monkeys [mean (*SE), 208.0 (0.4) pm21was sectional area of labeled cells was therefore analyzed separately not statistically reliable. for the rostra1three sectionsof the medial septalnucleus, where The observed increasein average cell size in the aged brain the number ofChAT-positive cellswas comparable across groups, could be accounted for by either of the following alternatives: and for the caudal two levels of the nucleus, where substantial (I) a selective lossof relatively small cells with a resulting in- cc11loss had occurred. Across the rostra1sections, mean ccl1 size creasein the mean of the cell size distribution or (2) an actual in the aged group as a whole was 7.5% greater than in young subjects[mean (*SE), 2 10.4 (6.2) and 195.7 (2.6) prnZ,respec- tively]. This effect, however, wasalmost entirely attributable to Table 1. Mean number of ChAT-positive cells an increasein the cross-sectionalarea of ChAT-positive cells in brains from aged subjects that were impaired on the DNMS Number(*SE) task [Fig. 4C, means,in pm* (?SE): young, 195.7 (2.6); aged- Rostra1three Caudaltwo unimpaired, 200.0 (1.O); aged-impaired = 220.9 (3.7)]. Labeled Grouo sections sections cells in the aged-impaired animals were 12.9% larger than in Young 454.3 (44.8) 274.0 (55.5) young subjects.Between-group comparisons demonstrated that Agedunimpaired 423.0 (34.0) 169.0(5.0) the overall group effect (F[2,5] = 19.5;p < 0.0 1)was attributable Agedimpaired 429.5 (66.5) 154.5 (8.5) to a significant cell size increasein the aged-impaired subjects relative to either young (F, = 18.9;p < 0.05) or aged-unimpaired The Journal of Neuroscbnce, May 1992, f2(5) 1941


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CELL SIZE @n2) CELL SIZE (pm2) Figure5. Frequency distribution of ChAT-positive cells in 40 @cm2size bins. Histograms for young, aged-unimpaired, and aged-impaired subjects are illustrated for rostra1 (A) and caudal (E) portions of the medial septal nucleus. Note that at rostra1 levels, aged-impaired animals have more cells in the larger size bins and fewercells in the smallersize bins.At caudallevels, both agedgroups appear to have fewer cellsin the smallto medium sizk bins. animals (F, = 9.8; p < 0.05). In contrast, the 2.2% difference two major findings. First, there was a substantial, regionally in ChAT-positive cell size between the young and aged-unim- selective loss of ChAT-immunoreactive neurons in the aged paired subjectsdid not approach statistical significance. brains. Second, the mean cross-sectionalarea of ChAT-positive A different pattern of resultsemerged at caudal levels of the neurons was larger in the aged monkeys. These findings repre- media1septal nucleus (Fig. 4C). Here, both subgroupsof aged sent the first demonstration ofage-related changesin cholinergic subjectsexhibited approximately a 9% increase in cholincrgic cell number and size in the nonhuman primate. The following cell size relative to the young group [means, in pm* (*SE): sectionsdiscuss the resultsin relation to relevant data from aged young, 209.4 (0.7), aged-unimpaired, 228.5 (3.2), aged-im- rodents and humans. paired, 229.6 (2.1); 1;12,5]= 56.7, p < 0.00 I). Statistical analysis confirmed that the average cross-sectionalarea of labeled cells Ceil loss in the medial septal nucleus was significantly greater in both the aged-unimpaired(F, = 35.6; Overall, there was a 19% loss of ChAT-immunoreactive cells p < 0.05) and aged-impaired animals (F, = 39.9; p < 0.05) in the medial septal nucleus of the aged monkeys. This effect relative to the young group. waslargely restricted to caudal portions of the nucleus,however, The relationship between age-relatedchanges in cell size and where the agedbrains had 4 1% fewer labeledcells than in young cell loss was further analyzed by plotting the frequency distri- subjects. Rostrally, cell number differed by only 6%, and this bution of labeledneurons in bin sizesof 40 pm* (Fig. 5A,B). At erect failed to reach statistical significance. Thus, the loss of rostra1 levels of the medial septal nucleus, where there was no media1septal cholinergic cells in the aged monkey appearsto appreciable cell loss,there was an apparent shift in the distri- be regionally selective. bution amongaged-impaired subjects toward an increasednum- The present resultsare consistent with morphometric studies ber of cells in the larger size bins, and a decreasedincidence of demonstrating a substantialloss ofcholinergiccells in the media1 small cells (Fig. 5A). The distribution of cell sizes for the aged- septal nucleus of the aged rat. Investigations using AChE to unimpaired monkeys, in contrast, more closely approximated visualize media1septal cholinergic neuronshave reported vary- the distribution observed in young subjects.Thus, ChAT-pos- ing degreesof age-related cell loss ranging from 25% in 24- itive cells at rostra1 levels of the medial septal nucleus appear month-old rats of the Brown-Norway strain, to 45O/bin 22-24- to undergo significant hypertrophy in at least a subpopulation month-old Wista-Kyoto, Sprague-Dawley, and 30-month-old ofaged monkeys. At caudal levels of the nucleus,however, there Brown-Norway rats (Gilad et al., 1987; Fischer et al., 1989). was no indication of an increasein the number of large cells in Similar results have been reported in immunohistochemical the agedmonkeys (Fig. 5B). In addition, the substantialcell loss studiesusing antibodies directed againstNGF receptor (NGFr) observed caudally was largely restricted to neuronsin the small (Koh et al., 1989; Markram and Segal, 1990). The vast majority to medium size bins. These findings suggestthat the age-related of NGFr-positive neuronsare also immunoreactive for ChAT increasein the mean cross-sectionalarea of ChAT-positive cells (Kordower et al., 1988; Batchelor et al., 1989; Mufson et al., at caudal levels of the medial septal nucleus may in part be 1989), and results from these morphometric studies therefore attributable to the preferential lossofsmall to medium size cells. provide additional support for the view that there is a substantial degreeof cholinergic cell loss in the medial septal nucleus of Discussion the aged rat. Morphometric analysis of ChAT-immunoreactive cells in the Although relatively few investigations have specifically ana- media1septal nucleus of young and agedrhesus monkeys yielded lyzed the medial septal nucleus in the , there is 1942 Stroessner-Johnson et al. * Morphological Alterations in Aged Monkey Medial Septal Nucleus considerable evidence that cell number is prominently affected years of age to a maximum of 17% by age 60. At more advanced in other components of the basal forebrain cholinergic system ages, cross-sectional area decreased, but remained marginally as a consequence of normal aging. Earlier reports noted varying greater (5%) at 100 years of age relative to the youngest brains degrees of age-dependent cell loss in the basal nucleus of Mey- analyzed. Moreover, cells located at rostra1 levels of the basal nert, ranging from approximately 30% to 50% (Mann et al., nucleus, where no age-related cell loss occurred, were approx- 1984a,b; McGeer et al., 1984). In a recent large-scale investi- imately 30% larger at 60 years of age relative to young subjects. gation, de Lacalle et al. (199 1) analyzed Nissl-stained prepa- These findings in the human are therefore consistent with our rations through the basal nucleus of Meynert in 39 neurologi- observations in the monkey indicating that cholinergic cells in tally intact individuals ranging from 16 to 110 years of age. the primate brain undergo significant hypertrophy as a conse- Total cell counts across all levels of the nucleus revealed a 50% quence of normal aging. decrease by age 90. Particularly intriguing, given our results in the monkey, was the finding that cell loss was most pronounced Interpretations of observedchanges in cell number and size at caudal levels of the basal nucleus, where a decrease of ap- Two plausible explanations can be put forward to account for proximately 65% was observed. Intermediate rostrocaudal lev- the decreasein cholinergic cell number we have observed in the els of the nucleus exhibited a less marked, but significant, 42% medial septal nucleus of the aged monkey. First, this change loss of cells. In contrast, there was no age-related difference in may reflect frank cell death in a proportion of medial septal the number of cells counted in the most rostra1 levels of the neurons.Alternatively, thesecells may survive during aging,but nucleus. Thus, as we have observed in the medial septal nucleus ceaseproducing sufficient levels of ChAT to be detected by the of the aged monkey, cell loss during normal aging in humans immunohistochemical proceduresused here (Lams et al., 1988). appears to be more prominent at caudal levels of the basal Regardlessof which hypothesis proves to be correct, the present forebrain. Indeed, this regional selectivity may be one factor resultsclearly suggestthat the functional integrity of the medial contributing to the lack of age-related cell loss observed in stud- septal nucleus is compromised as a consequenceof agingin the ies that have examined only a limited rostrocaudal extent of the monkey. basal forebrain choline& system (Whitehouse et al., 1983; There are a number of plausibleexplanations for the cellular Chui et al., 1984). hypertrophy that we have observed in the medial septalnucleus. One possibility is that cell hypertrophy in the aged monkey may Cell hypertrophy in the medial septal nucleus reflect regenerative or compensatory processes.Studies of neural Our analysis of the cross-sectional area of medial septal cholin- plasticity in young adult animals provide direct evidence that ergic cells revealed that the mean size of ChAT-labeled neurons the size of basalforebrain cholinergic neuronscan increasesub- in the aged brains was significantly larger than in young animals. stantially in responseto experimental brain damage.Pearson et Neuronal hypertrophy was most evident at rostra1 levels of the al. (1987) for example, found that medial septal cholinergic medial septal nucleus where the number of ChAT-positive cells cells located contralateral to a unilateral hippocampectomy or was comparable across age groups. Interestingly, the increase in fimbrial transection show a significant 24% increase in cross- cell size was almost entirely attributable to cell hypertrophy in sectional area relative to intact rats. This change in cell size brains from aged subjects that were behaviorally impaired. These persisted for at least 3 12 d after the lesion. The proposed ex- monkeys exhibited a significant 13% increase in the mean cross- planation for this hypertrophy is that cholinergic terminals in sectional area of labeled cells relative to the young subjects. Cell the hippocampusmay undergo reactive synaptogenesisin order size was similar at rostra1 levels in brains from the young and to occupy synaptic sitesvacated as a consequenceof the lesion. unimpaired aged animals. It is important to note that a larger The cholinergic cells of origin, which now must support an number of aged animals will need to be analyzed in order to increasedaxonal plexus, expand to accommodate this greater establish a firm relationship between cholinergic cell hypertro- metabolic demand. Studies reporting similar findings in other phy and age-related memory impairment. brain regions provide additional support for the view that cell At more caudal levels, both unimpaired and impaired aged hypertrophy is a frequent correlate of the synaptic reorganiza- monkeys showed a mean cell size increase of approximately 9% tion that occursas a result of experimental brain damage(Gold- relative to young animals. The interpretation of this finding, Schmidt and Steward, 1980; Hendrickson and Dineen, 1982; however, is complicated by the prominent loss of small to me- Krishnan, 1983; Pearsonand Powell, 1986; Ruigrok et al., 1990). dium size neurons that occurred caudally. Thus, the shift in the These findings raise the possibility that, in the aged brain, nat- distribution of cell size for the aged brains may reflect both urally occurring degeneration of noncholinergic afferents to the hypertrophy and cell death, or simply the loss of small to me- hippocampal formation (e.g., the perforant path input from the dium size ChAT-positive neurons. entorhinal cortex) may induce sprouting in the septohippocam- In contrast to the results of the present study, the majority of pal projection and hypertrophy of at least some of the septal investigations in aged rodents have reported that cholinergic cholinergic neurons. neurons undergo significant atrophy as a consequence of aging (Hornberger et al., 1985; Mesulam et al., 1987; Fischer et al., Relationship between morphological alterations and behavior 1989; Markram and Segal, 1990). Indeed, only one investigation Although relatively few memory-impaired aged subjectswere has found a significant hypertrophy of medial septal cholinergic analyzed in the present experiments, several interesting rela- cells in aged rats (Gilad et al., 1987). Although cell size has been tionships emergedbetween the morphometric data and the be- a less frequently used measure in morphometric studies of the havioral status of the aged monkeys. First, changesin the num- aged human brain, de Lacalle et al. (199 1) have recently reported ber ofChAT-positive cellsfailed to distinguish between memory findings on cell size in normal aged humans that closely parallel impaired and unimpaired aged subjects;both subgroupsexhib- our observations in the monkey. Mean cell size in the basal ited a comparable4 1% lossof cells in caudal levels of the medial nucleus of Meynert was found to increase gradually from 16 septal nucleus. 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