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Neurobiology of Aging 21 (2000) 125–134

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Cholinergic medial septum neurons do not degenerate in aged 129/Sv control or p75NGFR⫺/⫺mice夞 Nicole L. Warda, Lianne E. Stanfordb, Richard E. Brownb, Theo Hagga,* a

Department of Anatomy and Neurobiology, Tupper Building, Dalhousie University, Halifax, N.S., B3H 4H7, Canada b Department of Psychology, Dalhousie University, Halifax, N.S., B3H 4H7, Canada Received 29 June 1999; received in revised form 9 November 1999; accepted 15 December 1999

Abstract Cholinergic medial septum neurons express TrkA and p75 nerve growth factor receptor (p75NGFR) and interactions between TrkA and p75NGFR are necessary for high-affinity binding and signaling of nerve growth factor (NGF) through TrkA. In adult p75NGFR-deficient (⫺ /⫺) mice, retrograde transport of NGF and other neurotrophins by these neurons is greatly reduced, however, these neurons maintain their cholinergic phenotype and size. Reduced transport of NGF has been proposed to play a role in Alzheimer’s disease. Here, we investigated whether chronic and long-term absence of p75NGFR (and possibly reduced NGF transport and TrkA binding) would affect the cholinergic septohippocampal system during aging in mice. In young (6 – 8 months), middle aged (12–18 months), and aged (19 –23 months) 129/Sv control mice the total number of choline acetyltransferase-positive medial septum neurons and the mean diameter and cross sectional area of the cholinergic cell bodies were similar. The cholinergic hippocampal innervation, as measured by the density of acetylcholinesterasepositive fibers in the outer molecular layer of the dentate gyrus was also similar across all ages. These parameters also did not change during aging in p75NGFR ⫺/⫺ mice and the number and size of the choline acetyltransferase-positive neurons and the cholinergic innervation density were largely similar as in control mice at all ages. These results suggest that p75NGFR does not play a major role in the maintenance of the number or morphology of the cholinergic basal forebrain neurons during aging of these mice. Alternatively, p75NGFR ⫺/⫺ mice may have developed compensatory mechanisms in response to the absence of p75NGFR. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Aging; Basal forebrain; Cell body size; Central nervous system; Cholinergic; Dentate gyrus; Hippocampal formation; Medial septum; Mouse; Nerve growth factor; Neurons; Neurotrophin; Receptor; Transgenic

1. Introduction The neurotrophins are a family of growth factors that promote cell survival, neurite outgrowth, phenotypic maturation, and synaptic functioning [31,37]. Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4) are all members of the neurotrophin family. NGF mediates its survival-promoting effects via the transmembrane tyrosine

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This work was supported by a scholarship from the Izaak Walton Killam Foundation (NLW), studentships (NLW, LES) and a scholarship (TH) from the Medical Research Council of Canada, a grant from the Alzheimer Society of Canada (TH), and a grant from Natural Science and Engineering Council of Canada (REB). * Corresponding author. Tel.: ⫹1-902-494-6622; fax: ⫹1-902-4942670. E-mail address: [email protected] (T. Hagg).

kinase receptor TrkA, BDNF and NT-4 via TrkB, and NT-3 via TrkC [10]. All neurotrophins also can bind to the p75 NGF receptor (p75NGFR), whose role includes assisting in neurotrophin transport, enhancing ligand binding specificity, and increasing Trk functioning [7,10,13,16] and under certain conditions, induction of apoptosis [11,12]. Basal forebrain cholinergic neurons provide a model system for studying the in vivo role of neurotrophins and their receptors. In the medial septum of the basal forebrain of adult mammals, cholinergic neurons express TrkA and p75NGFR [33,38,48]. In the medial septum, NGF maintains cell body size and choline acetyltransferase (ChAT) levels [41,53], and can prevent or reverse degenerative changes caused by axotomy or aging [22,27,29,39,55]. These effects seem to be regulated through NGF:TrkA signaling since transgenic mice with a genetic deletion of NGF (⫹/⫺) or TrkA (⫺/⫺) have fewer and smaller ChAT-positive medial septum neurons [14,19]. The medial septum of adult

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p75NGFR⫺/⫺ mice has similar numbers of ChAT-positive neurons as control mice ([54], but see [56]). These p75NGFR⫺/⫺ mice have larger cell bodies, and possibly increased levels of ChAT [54,56], which is unexpected since their transport of NGF is greatly reduced [34]. Also, p75NGFR seems to be necessary for TrkA functioning [7,10, 13]. It is possible that detrimental effects of the absence of p75NGFR are only revealed during aging. During aging, the number of basal forebrain cholinergic neurons in rodents has been reported to decrease [2,3,21, 23]. Others have only found that these neurons atrophy [5,15,17,20,22,26,39,40], a process that can be reversed with NGF treatment [5,22,26,39]. Aged animals reportedly also have reduced levels of ChAT and acetylcholinesterase (AChE) activities [6,50], and reduced expression of TrkA mRNA [15]. Moreover, NGF transport and responsiveness to NGF by the basal forebrain cholinergic neurons is reduced in aged rats [15,32], and possibly in Alzheimer’s disease [43,47] where these neurons die. Basal forebrain cholinergic neurons project their axons throughout the hippocampal formation and neocortex and are important for learning and memory [8,18]. Decreased learning- and memory-related performance has been reported in aged rodents [4,5,22,35,39,52]. In an attempt to determine whether chronic and longterm absence of p75NGFR (and possibly reduced NGF transport and TrkA binding) would alter the normal aging process, we investigated whether the cholinergic septohippocampal system would be differentially affected in young, middle-aged, and aged p75NGFR⫺/⫺ mice compared to 129/Sv control mice.

2. Methods 2.1. Animals Breeding pairs of mice homozygous (⫺/⫺) for a deletion of the p75NGFR gene ([36]; Cat. JR2124) and breeding pairs of one of their DNA controls (129/Sv; Cat. JR2448) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA; for further information regarding background and breeding see: www.jax.org). There is no physiological control strain available for the p75NGFR ⫺/⫺ mice, which are on a mixed 129/Sv-Balb/c background. We have previously shown that in young adult mice, the 129/Sv strain more closely resembles the p75NGFR⫺/⫺ line in terms of the numbers of cholinergic neurons and density of cholinergic hippocampal innervation than the Balb/c DNA control strain [54]. Sixty-four male and female offspring from these breeding pairs were sacrificed at either young (6 – 8 months of age, average 7 months), middle aged (12–18 months of age, average 14 months), or aged (19 –23 months of age, average 20 months) time points. All animal protocols were approved by the Dalhousie University Animal Care Committee and conformed to Canadian Council on Animal Care

guidelines. Anesthesia was achieved by intraperitoneal (i.p.) injection of 6.5 mg/kg sodium pentobarbitol. 2.2. Tissue processing, immunohistochemistry, and histochemistry At “young,” “middle aged,” or “aged” time points, anesthetized mice were perfused transcardially with cold (4°C; 15 ml) phosphate-buffered saline (PBS), followed by cold 4% paraformaldehyde in 0.1 M phosphate buffer (4°C; 30 ml). After 24 h post-fixation in 4% paraformaldehyde, the brains were cryoprotected in a 30% sucrose/phosphate buffer solution for 24 h in preparation for sectioning. Brains were marked on the left hemisphere and 30 ␮m thick coronal sections through the septum and hippocampal formation were cut with a freezing microtome. Immunohistochemistry was performed to visualize the cholinergic neuronal cell bodies. Every third section through the entire septal nucleus (total number of sections ⫽ 14) was processed for immunohistochemistry by using an affinity-purified polyclonal goat antibody against ChAT (Ab144P, Chemicon, Temecula, CA, USA). Free-floating tissue sections were incubated sequentially with 3% rabbit serum in Tris buffered saline (TBS) containing 0.3% Triton X-100 for thirty minutes, primary antibody Ab144P at a 1 : 4000 dilution in 1% rabbit serum TBS-triton for 16 h at 4°C, biotinylated rabbit anti-goat IgG (1:300; Vector laboratories; Burlingame, CA, USA) in TBS for 90 min, and avidin-biotin-peroxidase complex (1 : 600; ABC Elite kit, Vector Laboratories) in TBS for 1 h. Immunoreactive products were revealed with a diaminobenzidine reaction in the presence of 0.67% ammonium nickel sulfate for intensification. In between steps, the sections were washed 3 ⫻ 10 min in TBS. All stained sections were mounted on gelatin-coated glass slides, dehydrated, and coverslipped in Permount. Cholinergic axons in the hippocampal formation were visualized by AChE staining in a series of four sections spanning ⬃700 ␮m in the dorso-rostral part of the hippocampal formation. Hippocampal tissues from all mice were mounted on gelatin-coated slides and all slides were processed with random strain and age assignment to one of three batches for AChE histochemistry [28]. Adaptations to the original protocol were made such that tissue was incubated twice for thirty minutes in freshly prepared enzyme solution and Promethazine (0.2 mM, Sigma, St. Louis, MO, USA) was used as an inhibitor of non-specific esterases. As an added control for technical variability, all perfusions, sectioning, and staining were performed at various times on mice of different ages and strains simultaneously. This was possible because of sequential starts of breeding different age groups. 2.3. Quantification In each mouse, every third section through the entire rostro-caudal extent of the medial septum (total ⫽ 14 sec-

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tions) was used to determine the number of ChAT-positive neurons. To ensure consistency between animals, section number 0 (zero) was identified as the most rostral section through the decussation of the anterior commissure. These sections spanned the region between the rostro-caudal midpoint of the decussation (which is 180 ␮m caudal to section 0) and 990 ␮m rostral to the decussation. Neuronal cell body profiles larger than 9 ␮m in longest diameter were counted on the left side of the medial septum, with the ventral border defined by an imaginary line through the arms of the anterior commissure. The total number of cholinergic neurons in each region was estimated according to the formula: Total number ⫽ 3 ⫻ (counted profiles ⫻ section thickness/(mean cell body diameter ⫹ section thickness)) [1]. This value was then used to estimate the total number in both hemispheres. The mean diameter was determined by measuring the longest diameter of 25 cholinergic medial septum neurons in one section (570 ␮m rostral to section 0) per animal. These neurons were randomly chosen from either side of the medial septum without bias to size, in a dorsal-ventral manner. Digitized images of the sections that were used to determine the cell body diameter were used to determine the cross sectional area of ChATpositive neuronal cell bodies larger than 9 ␮m in diameter (NIH Image, Dr. Wayne Rasband, NIH, USA; http://rsb. info.nih.gov/nih-image/). For each of the AChE-stained sections through the hippocampal formation (four per animal), three lines (120 ␮m each) were placed at 60 ␮m intervals over the left dorsal blade of the outer molecular layer of the dentate gyrus. The number of stained fibers intersecting each of these three distinct lines were counted in each of the sections and represented as a density measure, i.e., total number of counted fibers per section/total length of the three lines (0.36 mm). The width of the entire molecular layer was measured in the same sections. All tissue processing and data collection was completed by an experimenter who was blind to the animal age and genotype. Between group analyses were performed with ANOVA and post-hoc Student Newman–Keuls analysis using Statistica software (Statsoft; Tulsa, OK, USA) with an alpha of 0.05. For young, middle aged, and aged 129/Sv control mice: n ⫽ 11 (0 females), 8 (2 females), 15 (7 females), respectively and for young, middle aged, and aged p75NGFR⫺/⫺ mice: n ⫽ 9 (0 females), 9 (5 females), 12 (4 females), respectively.

3. Results 3.1. Young, middle aged, and aged p75NGFR ⫺/⫺ and control mice do not differ in the number of medial septum cholinergic neurons The appearance of the cholinergic neurons in the medial septum did not differ between control and p75NGFR⫺/⫺

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mice at young, middle aged, and aged time-points (Fig. 1), and no obviously distorted neurons were observed at any age in either strain. ChAT-positive neurons were readily detectable at all three time-points, and the intensity of ChAT staining or the number of ChAT-positive neurons did not seem to change with age. The ANOVA for overall effects revealed that the number of ChAT-positive neurons in the medial septum differed significantly between mouse strains (P ⬍ 0.02, ANOVA, F(1, 58) ⫽ 6.72) but not with age. Post-hoc analyses of the main effect of strain (i.e., no age group separation) indicated that 129/Sv mice had more ChAT-positive medial septum neurons than p75NGFR ⫺/⫺ mice (P ⬍ 0.011). In contrast to the overall effects, which indicated a difference between control and p75NGFR ⫺/⫺ mice, analyses of the simple effects of strain failed to reveal any differences. Control and p75NGFR ⫺/⫺ mice did not differ in the number of cholinergic neurons in the young (P ⫽ 0.37; 129/Sv 1357 ⫾ 49; p75NGFR ⫺/⫺ 1277 ⫾ 70), middle aged (P ⫽ 0.06; 129/Sv 1327 ⫾ 100; p75NGFR ⫺/⫺ 1141 ⫾ 71), or aged groups (P ⫽ 0.15; 129/Sv 1345 ⫾ 54; p75NGFR ⫺/⫺1180 ⫾ 92) (Fig. 2A). Analyses of the simple effects of age were consistent with the overall effects, i.e., no differences in the number of cholinergic neurons were observed in control or p75NGFR ⫺/⫺ mice between young, middle aged, and aged groups (Figs. 1 and 2). However, the variability in the number of ChAT-positive neurons seemed to increase with age in p75NGFR ⫺/⫺ mice. In the middle-aged and aged groups, a sub-population of 4 and 5 animals, respectively, had fewer ChAT-positive neurons than any of the control mice at any age (Fig. 2A; P ⬍ 0.01). For all groups at all ages containing female mice, the number of ChAT-positive neurons did not differ between males and females. 3.2. Cholinergic cell body size does not change with age in either p75NGFR⫺/⫺ or control mice Analyses of the overall effects revealed that the diameter of the cholinergic cell bodies differed significantly between mouse strains (P ⬍ 0.003, ANOVA, F(1, 58) ⫽ 9.66) but not with age. Post-hoc analyses of the strain difference suggested that overall, p75NGFR ⫺/⫺ mice have larger cholinergic cell bodies than 129/Sv mice (P ⬍ 0.0026). Analyses of the simple effects of strain revealed differences only at the aged time-point, such that the cholinergic neurons of p75NGFR⫺/⫺ mice had an ⬃12% greater diameter (19.7 ⫾ 0.4 ␮m) than those of 129/Sv mice (17.6 ⫾ 0.3 ␮m) (Fig. 2B). No differences were observed between the two strains of mice at the young (129/Sv 18.1 ⫾ 0.5 ␮m; p75NGFR ⫺/⫺ 19.0 ⫾ 0.6 ␮m) or middle aged (129/Sv 17.9 ⫾ 0.5 ␮m; p75NGFR ⫺/⫺ 18.4 ⫾ 0.6 ␮m) time-points. Consistent with the overall effects, the cell body diameter of cholinergic neurons in 129/Sv and p75NGFR ⫺/⫺ mice did not change between young, middle aged, or aged timepoints (Fig. 2B).

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Fig. 1. Cholinergic medial septum neurons appear similar in young and aged 129/Sv and p75NGFR⫺/⫺ mice. ChAT-immunostained coronal sections through the medial septum of 129/Sv mice (A and B) do not seem different from p75NGFR⫺/⫺ mice (C and D) at young (A and C) and aged (B and D) time-points. Arrowheads indicate the midline of the medial septum. Arrows indicate the position of the anterior commissure (not shown) used to define the ventral boundary of the medial septum. Magnification bar ⫽ 200 ␮m.

Measurements of cross-sectional area (Fig. 2C) confirmed the cell body diameter results. Across all ages, p75NGFR⫺/⫺ mice had larger cholinergic cross-sectional areas than 129/Sv mice (P ⬍ 0.0029; F(1, 58) ⫽ 9.71). Again, no changes in size were found with age.

Analyses of the simple effects of strain revealed that the cholinergic neuronal cell bodies of aged p75NGFR ⫺/⫺ mice had an ⬃14% larger cross-sectional area (147 ⫾ 7 ␮m2 than those of aged 129/Sv mice (P ⬍ 0.0026; 129 ⫾ 4 ␮m2). At the middle-aged time-point, p75NGFR ⫺/⫺ mice (138 ⫾ 4

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Fig. 2. The number and size of cholinergic medial septum neurons remain constant during aging in 129/Sv and p75NGFR⫺/⫺ mice. Presented is the total number of ChAT-positive neurons in both hemispheres of the medial septum area (A), the average cholinergic cell body diameter (B) and cross-sectional area (C) for each p75NGFR ⫺/⫺ (open triangles) and 129/Sv (closed triangles) mouse at young, middle aged, and aged time-points. Note that a sub-group of middle-aged and aged p75NGFR⫺/⫺ mice has fewer neurons than any of the 129/Sv mice. 4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™

served at the young time-point (129/Sv 135 ⫾ 3 ␮m2; p75NGFR ⫺/⫺ 137 ⫾ 4 ␮m2). No sub-groupings of mice were evident for both cell body diameter and cross-sectional area. No significant differences were apparent between males and females in any of the groups. 3.3. Cholinergic innervation of the outer molecular layer of the dentate gyrus does not differ in young, middle aged, and aged p75NGFR ⫺/⫺ and control mice The density of cholinergic fibers in the molecular layer of the denate gyrus, and the overall size of the hippocampal formation (not shown) seemed similar for control and p75NGFR⫺/⫺ mice at young, middle aged, and aged timepoints (Fig. 3). In some mice, AChE staining seemed denser in the inner molecular layer as compared to the outer molecular layer of the dentate gyrus, but this was not limited to a specific age or strain. The density of AChE-positive fibers in the outer molecular layer of the dentate gyrus did not differ between 129/Sv and p75NGFR⫺/⫺ mice at any age. In addition, no differences were found between young (129/Sv 347 ⫾ 8 fibers per mm; p75NGFR ⫺/⫺ 334 ⫾ 8), middle aged (129/Sv 339 ⫾ 10; p75NGFR ⫺/⫺ 354 ⫾ 13), and aged mice (129/Sv 338 ⫾ 2; p75NGFR ⫺/⫺ 350 ⫾ 8) (Fig. 4A). To account for possible changes in the width of the entire molecular layer of the dentate gyrus associated with aging or absence of p75NGFR, we measured its width in the same sections as were used to determine the fiber density. No differences were observed between 129/Sv and p75NGFR⫺/⫺ mice at any age or between young (129/Sv 160 ⫾ 2 ␮m; p75NGFR ⫺/⫺ 156 ⫾ 3 ␮m), middle aged (129/Sv 157 ⫾ 3 ␮m; p75NGFR ⫺/⫺ 160 ⫾ 2 ␮m), or aged (129/Sv 157 ⫾ 1 ␮m; p75NGFR ⫺/⫺ 158 ⫾ 3 ␮m) mice (Fig. 4B). No significant differences were observed between male and female mice for the innervation density or width of the molecular layer of the dentate gyrus.

4. Discussion

␮m seemed to have larger cross-sectional areas than 129/Sv mice (123 ⫾ 6 ␮m2, however the p-value did not reach significance (P ⬍ 0.065). No differences were ob2

The present studies provides evidence that the absence of p75NGFR does not obviously affect the normal aging process

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Fig. 3. The density of cholinergic innervation of the dentate gyrus appears similar in young and aged p75NGFR ⫺/⫺ and 129/Sv mice. AChE-stained coronal sections through the dentate gyrus of the hippocampal formation of 129/Sv (A and B) mice do not seem different than those of p75NGFR ⫺/⫺ mice at young (A and C) and aged (B and D) time-points. The asterisk indicates the granule cell layer of the dentate gyrus. Magnification bar ⫽ 50 ␮m.

of the cholinergic septohippocampal system. In both 129/Sv control and p75NGFR ⫺/⫺ mice the: 1) number of ChATpositive neurons remains constant; 2) cholinergic neuronal cell bodies do not atrophy; and 3) cholinergic innervation of the outer molecular layer of the dentate gyrus remains unchanged from 6 to 23 months of age. 4.1. Potential methodological limitations and caveats Our conclusions must be considered in context of the following potential caveats. We did not use an unbiased stereological optical dissector approach. However, we used a well-established direct method of counting corrected for caps and section thickness, combined with a systematic sampling of sections throughout the entire medial septum nucleus. The current number of cholinergic neurons in the medial septum of 129/Sv control mice (⬃1,350) is in the range of that reported by others (⬃1,700 [57]; ⬃1,200 [20]; and Dr. J. Long, preliminary data suggests ⬃1,600, personal communication), who used unbiased stereological quantification methods, and consistent with what others have reported for aged CD1 and C57Bl/6 mice [20,30,40]. In addition, only recently has a stereological technique been designed for measuring fiber length in coronal sections and it is still in the development stage [42]. Therefore, we report our fiber innervation results as a density measure in the

coronal plane instead of a total length value. Lastly, we have not accounted for potential differential shrinkage of the whole brain tissue due to age, although all ChAT-positive cells larger than 9 ␮m were counted in each section, and the same number of sections were collected throughout the entire nucleus and analyzed for each mouse. We cannot exclude the possibility that neuronal cell bodies would shrink differentially in response to the fixation and processing depending on the age of the mice. 4.2. Medial septum cholinergic neuron numbers do not change in aged control or p75NGFR⫺/⫺ mice The number of ChAT-positive neurons in the medial septum of young, middle aged, and aged 129/Sv mice did not differ across age. This suggests that cholinergic neuron loss or a reduction of ChAT below detectable levels does not occur during the normal aging process in these mice. This lack of age-related cell loss is in contrast to what has been reported to be a natural component of the aging process in rats [2,3,21,23], but confirms what others have described in rats [5,15,17,22,26,35] and mice [20,30,40]. It is possible that mice age differently than rats, i.e., all mouse strains maintaining sufficient ChAT-staining to recognize the cholinergic neurons. On the other hand, rats have been studied more extensively and it is possible that cholinergic

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Fig. 4. The density of AChE-positive fibers in the outer molecular layer of the dentate gyrus remains constant during aging of 129/Sv and p75NGFR ⫺/⫺ mice. A) The density of AChE-positive cholinergic fibers intersecting three perpendicularly placed lines over the outer molecular layer of the dorsal blade of the left dentate gyrus did not differ between 129/Sv (closed triangles) and p75NGFR⫺/⫺ mice (open triangles) at young, middle aged, and aged time-points. Values represent the mean of four sections through the dorsal hippocampal formation of each animal. B) No differences were observed for the width of the entire molecular layer of the dentate gyrus between 129/Sv (closed triangles) and p75NGFR ⫺/⫺ mice (open triangles) in the same tissue sections. Note that similar values may overlap and appear as a single point.

neurons disappear in some mouse strains that have not yet been analyzed. The apparent discrepancy between the results for rats most likely reflects differences in quantification techniques and not strain differences, as cell loss or absence of cell loss has been described in the same strains, e.g. Sprague–Dawley ([21] vs. [15,17,35]) and Fischer 344 rats ([3] vs. [5,26]). In the young mouse group, the number of ChAT-positive

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neurons was the same in p75NGFR ⫺/⫺ mice as in 129/Sv controls, confirming a recent report by our laboratory [54]. This is in contrast to reports by others suggesting that p75NGFR ⫺/⫺ mice have more [56], or fewer [45] ChATpositive neurons. These discrepancies are likely due to differences in breeding strategy that results in a different background strain of the controls and potentially the formation of sub-strains of the p75NGFR ⫺/⫺ mice, as discussed elsewhere [54]. In the middle-aged and aged p75NGFR ⫺/⫺ mouse groups, at least one third of the mice had fewer ChATpositive neurons. This might reflect a decrease in ChAT below detectability, as the density of cholinergic innervation (detected by AChE) was not reduced, which would have been expected if cholinergic neurons had been lost. This suggests the possibility that chronic reduction in p75NGFR leads to reductions in ChAT enzyme in some mice. Endogenous NGF is important for the expression of ChAT [41,53]. P75NGFR is necessary for TrkA functioning and NGF signaling [13], and for efficient neurotrophin transport from the hippocampal formation to the cholinergic medial septum neurons [34]. Moreover, NGF and general axonal transport is reduced during aging [15,17]. Thus, it is somewhat unexpected that in p75NGFR⫺/⫺ mice, on average the cholinergic neurons of the medial septum maintained their ChAT expression with age. On the other hand, both NGF and TrkA-deficient mice also have ChAT-positive basal forebrain neurons [14,19]. Thus, it is possible that an alternative means of trophic support developed in these transgenic mice. The cholinergic neurons of p75NGFR⫺/⫺ mice apparently do not compensate by utilizing other neurotrophins from the hippocampal formation, as their transport is also reduced ([34] and see [16]). Moreover, these neurons most likely do not utilize NGF from another source, as their immunoreactivity for NGF is reduced, even in the presence of increased amounts of NGF (Dr. M. Kawaja, personal communication). It is possible that the cholinergic neurons in the p75NGFR⫺/⫺ mice utilize alternative neurotrophic factors. 4.3. Cholinergic neurons do not atrophy with age in control or p75NGFR ⫺/⫺ mice In 129/Sv mice the cell body size did not change with age. This is in contrast to previous reports, which suggested that cell body atrophy occurs with age in C57Bl/6 [20] and CD1 mice [40]. The apparent discrepancy is probably not due to differences in the age of the “aged” mice, as previous reports analyzed similarly aged mice (⬃24 –25 months of age; [20,40]). Moreover, Hornberger and colleagues [30] analyzed both cell body diameter and surface area in C57Bl/ 6NNIA mice at 53 months of age, and did not find cell atrophy in the medial septum. It is possible that in the 129/Sv mouse strain, cell body atrophy occurs only in very aged animals. We did not analyze mice older than ⬃23 months of age, as they become very frail. The current cell

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area measures (⬃130 ␮m2) are in the range that others report for young control mice (⬃130 ␮m2, [40]; ⬃125 ␮m2, [57]; ⬃150 ␮m2, [19]), thus it is unlikely that differences in technique account for the apparent discrepancy. More likely, the differences between various studies are due to the use of different mouse strains. In aged rats, cell body atrophy is reportedly correlated with the extent of behavioral impairment [21,22]. Here, the lack of age-related morphological changes in the 129/Sv control mice is consistent with the lack of deficits in learning and memory-related behaviors, as will be extensively presented elsewhere (but see [51]). Despite the apparently reduced utilization of NGF by the cholinergic neurons in p75NGFR⫺/⫺ mice [34], and the evidence that hippocampal-derived NGF is important for the regulation of the cholinergic cell body size [17,49], these neurons did not atrophy during aging, i.e., even after chronic long-term absence of p75NGFR. As discussed before, the neurons in p75NGFR⫺/⫺ mice may have developed compensatory mechanisms. Future experiments with pharmacological manipulations with p75NGFR -blocking agents or with an inducible null-mutant animal may resolve this question. In Alzheimer’s disease, the cholinergic basal forebrain neurons have reduced levels of ChAT, reduced NGF transport, atrophy and degenerate. Moreover, TrkA levels [9,44,46] and NGF transport [43,47] are reduced in Alzheimer’s disease, most likely leading to a reduction in NGF signaling. Our current results in mice suggest that long-term reduction in NGF transport and possibly NGF signaling, do not lead to degenerative changes. Given the possibility of developmental compensation in p75NGFR⫺/⫺ mice, these findings are not inconsistent with the previous observations in Alzheimer’s disease. Previously we [54] and others [56] have reported that young adult (3–5 months old) p75NGFR⫺/⫺ mice have larger cholinergic medial septum neurons than control mice. Here, the cell body size in p75NGFR⫺/⫺ mice was similar as in controls at 6 – 8 months (“young”) and at the middle-aged time point. During normal development, a transient increase in cell body size is observed between P6 and P15 followed by a decrease to adult levels [25]. In p75NGFR⫺/⫺ mice, this decrease to adult levels at ⬃3 to 5 months is less than in control mice [54]. Therefore, it is conceivable that p75NGFR⫺/⫺ mice may mature more slowly, reaching their “adult” sizes at 6 to 8 months. 4.4. Cholinergic hippocampal innervation remains constant during aging and in the absence of p75NGFR Axons from cholinergic medial septum neurons project to the hippocampal formation. Here, the density of AChEpositive fibers in the outer molecular layer of the dentate gyrus and the width of the molecular layer remained unchanged between young, middle aged, and aged timepoints. This is consistent with the observed maintenance of

cell size of the cholinergic medial septum neurons during aging and the idea that cell body size may relate to the total volume of their processes [33]. Others [20] have reported an increase in cholinergic fiber density in normal aging mice, despite atrophy of the medial septum cholinergic neurons. A potential explanation for the apparent discrepancy between our studies may be a technical one: Fagan and colleagues [20], used a cycloid probe designed for use in “vertical” sections (sections in which the sectioning plane has been randomized; [24]) in coronal sections, and no corrections for tissue shrinkage seem to have been used. It should be noted that currently available techniques for quantification of a structure in a single (e.g. coronal) plane, including the use of density measures, do not consider the three dimensional nature of such structures. The necessity of analyzing neural structures, in particular fibers, in a variety of randomly sectioned planes is impractical, as this makes identification of anatomical structures very difficult. A new method that is able to measure the total length of fibers in standard “anatomical” sections is currently under development [42]. The AChE-positive cholinergic fiber density of p75NGFR⫺/⫺ mice did not differ from that seen in control mice. Yeo et al. [56], have provided qualitative evidence that young adult p75NGFR ⫺/⫺ mice have an increase in ChAT-immunostaining in the molecular layer of the dentate gyrus and quantitative evidence for an increase in the number of ChAT-immunoreactive fibers in the CA1 region of the hippocampus. However, these authors also report an increase in the number of ChAT-positive medial septum neurons. These differences between our studies, as discussed more extensively elsewhere [54], may reflect differences in maintenance of mouse colonies and choice of control strains. The density of cholinergic fibers in the dentate gyrus did not change during aging in 129/Sv or p75NGFR⫺/⫺ mice. Thus, the lack of p75NGFR does not seem to make the cholinergic neurons and their axons more vulnerable to the aging process in these mice. As discussed above, alternative strategies for trophic support may have developed in these p75NGFR ⫺/⫺ mice to compensate for the reduced efficiency of neurotrophin binding and transport. On the other hand, neurotrophins may not be involved in maintenance of axonal projections during adulthood and aging. In summary, these results suggest that the number and size of cholinergic neurons in the medial septum and their innervation of the hippocampal formation does not change during aging, and that the absence of p75NGFR does not have an obvious effect on these morphological parameters. Acknowledgments We would like to thank Julie Bunker and Jennifer Stapleton for their excellent technical assistance.

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References [1] Abercrombie U. Estimation of nuclear populations from microtome sections. Anat Rec 1946;94:239 – 47. [2] Altavista MC, Rossi P, Bentivoglio AR, Crociani P, Albanese A. Aging is associated with a diffuse impairment of forebrain cholinergic neurons. Brain Res 1990;508:51–9. [3] Armstrong DM, Sheffield R, Buzsaki G, Chen KS, Hersh LB, Nearing B, Gage FH. Morphologic alterations of choline acetyltransferasepositive neurons in the basal forebrain of aged behaviorally characterized Fisher 344 rats. Neurobiol Aging 1993;14:457–70. [4] Aubert I, Rowe W, Meaney MJ, Gauthier S, Quirion R. Cholinergic markers in aged cognitively impaired Long-Evans rats. Neuroscience 1995;67:277–92. [5] Backman C, Rose GM, Hoffer BJ, Henry MA, Bartus RT, Friden P, Granholm A. Systemic administration of a nerve growth factor conjugate reverses age-related cognitive dysfunction and prevents cholinergic atrophy. J Neurosci 1996;16:5437– 42. [6] Baxter MG, Buccie DJ, Sobel TJ, Williams MJ, Gorman LK, Gallagher M. Intact spatial learning following lesions of the basal forebrain cholinergic neurons. Neuroreport 1996;7:1417–20. [7] Bibel M, Hoppe E, Barde YA. Biochemical and functional interactions between the neurotrophin receptors Trk and p75NTR. EMBO J 1999;18:616 –22. [8] Bigl V, Woolf NJ, Butcher LL. Cholinergic projections from the basal forebrain to frontal, parietal, temporal, occipital, and cingulate cortices: a combined fluorescent tracer and acetylcholinesterase analysis. Brain Res Bull 1982;8:727– 49. [9] Boissiere F, Faucheux B, Ruberg M, Agid Y, Hirsch EC. Decreased TrkA gene expression in cholinergic neurons of the striatum and basal forebrain of patients with Alzheimer’s disease. Exp Neurol 1997;145: 245–52. [10] Bothwell M. Functional interactions of neurotrophins and neurotrophin receptors. Annu Rev Neurosci 1995;18:223–53. [11] Carter BD, Lewin GR. Neurotrophins live or let die: does p75NTR decide? Neuron 1997;18:187–90. [12] Chao M, Casaccia Bonnefil P, Carter B, Chittka A, Kong H, Yoon SO. Neurotrophin receptors: mediators of life and death. Brain Res Brain Res Rev 1998;26:295–301. [13] Chao MV, Hempstead BL. p75, and Trk. a two-receptor system. Trends Neurosci 1995;18:321– 6. [14] Chen KS, Nishimura MC, Armanini MP, Crowley C, Spencer SD, Phillips HS. Disruption of a single allele of the nerve growth factor gene results in atrophy of basal forebrain cholinergic neurons and memory deficits. J Neurosci 1997;17:7288 –96. [15] Cooper JD, Lindholm D, Sofroniew MV. Reduced transport of [125I]nerve growth factor by cholinergic neurons and down-regulated TrkA expression in the medial septum of aged rats. Neuroscience 1994;62:625–9. [16] Curtis R, Adryan KM, Stark JL, Park JS, Compton DL, Weskamp G, Huber LJ, Chao MV, Jaenisch R, Lee KF, Lindsay RM, DiStefano PS. Differential role of the low affinity neurotrophin receptor (p75) in retrograde axonal transport of the neurotrophins. Neuron 1995;14: 1201–11. [17] De Lacalle S, Cooper JD, Svendsen CN, Dunnett SB, Sofroniew MV. Reduced retrograde labeling with fluorescent tracer accompanies neuronal atrophy of basal forebrain cholinergic neurons in aged rats. Neuroscience 1996;75:19 –27. [18] Everitt BJ, Robbins TW. Central cholinergic systems and cognition. Annu Rev Psychol 1997;48:649 – 84. [19] Fagan AM, Garber M, Barbacid M, Silos Santiago I, Holtzman DM. A role for TrkA during maturation of striatal and basal forebrain cholinergic neurons in vivo. J Neurosci 1997;17:7644 –54. [20] Fagan AM, Murphy BA, Patel SN, Kilbridge JF, Mobley WC, Bu G, Holtzman DM. Evidence for normal aging of the septo-hippocampal cholinergic system in apoE (⫺/⫺) mice but impaired clearance of

[21]

[22]

[23]

[24] [25] [26]

[27]

[28]

[29] [30]

[31] [32] [33]

[34]

[35]

[36]

[37] [38]

[39]

[40]

133

axonal degeneration products following injury. Exp Neurol 1998;151: 314 –25. Fischer W, Bjo¨rklund A, Chen K, Gage FH. NGF improves spatial memory in aged rodents as a function of age. J Neurosci 1991;11: 1889 –906. Fischer W, Wictorin K, Bjo¨rklund A, Williams LR, Varon S, Gage FH. Amelioration of cholinergic neuron atrophy and spatial memory impairment in aged rats by nerve growth factor. Nature 1987;329: 65– 8. Gilad GM, Rabey JM, Tizabi Y, Gilad VH. Age-dependent loss and compensatory changes of septohippocampal cholinergic neurons in two rat strains differing in longevity and response to stress. Brain Res 1987;436:311–22. Gokhale AM. Unbiased estimation of curve length in 3-D using vertical slices. J Microsci 1990;159:133– 41. Gould E, Woolf NJ, Butcher LL. Postnatal development of cholinergic neurons in the rat. I. Forebrain. Brain Res Bull 1991;27:767– 89. Gustilo MC, Markowski AJ, Breckler SJ, Fleischman CA, Price DL, Koliatsos VE. Evidence that nerve growth factor influences recent memory through structural changes in septohippocampal cholinergic neurons. J Comp Neurol 1999;405:491–507. Hagg T, Fass Holmes B, Vahlsing HL, Manthorpe M, Conner JM, Varon S. Nerve growth factor (NGF) reverses axotomy-induced decreases in choline acetyltransferase, NGF receptor and size of medial septum cholinergic neurons. Brain Res 1989;505:29 –38. Hedreen JC, Bacon SJ, Price DL. A modified histochemical technique to visualize acetylcholinesterase-containing axons. J Histochem Cytochem 1985;33:134 – 40. Hefti F. Nerve growth factor promotes survival of septal cholinergic neurons after fimbrial transections. J Neurosci 1986;6:2155– 62. Hornberger JC, Buell SJ, Flood DG, McNeill TH, Coleman PD. Stability of numbers but not size of mouse forebrain cholinergic neurons to 53 months. Neurobiol Aging 1985;6:269 –75. Ip NY, Yancopoulos GD. Neurotrophic factors and their receptors. Prog Brain Res 1995;105:189 –95. Koh S, Loy R. Age-related loss of nerve growth factor sensitivity in rat basal forebrain neurons. Brain Res 1988;440:396 – 401. Koh S, Loy R. Localization and development of nerve growth factorsensitive rat basal forebrain neurons and their afferent projections to hippocampus and neocortex. J Neurosci 1989;9:2999 –3318. Kramer BMR, Van der Zee CEEM, Hagg T. p75 nerve growth factor receptor is important for retrograde transport of neurotrophins in adult chinergic based forebrain neurons. Neuroscience 1999;94:1163–73. Lee JM, Ross ER, Gower A, Paris M, Martensson R, Lorens SA. Spatial learning deficits in the aged rat: neuroanatomical and neurochemical correlates. Brain Res Bull 1994;33:489 –500. Lee KF, Li E, Huber LJ, Landis SC, Sharpe AH, Chao MV, Jaenisch R. Targeted mutation of the gene encoding the low affinity NGF receptor p75 leads to deficits in the peripheral sensory nervous system. Cell 1992;69:737– 49. Lewin GR, Barde YA. Physiology of the neurotrophins. Annu Rev Neurosci 1996;19:289 –317. Li Y, Holtzman DM, Kromer LF, Kaplan DR, Chua Couzens J, Clary DO, Knu¨sel B, Mobley WC. Regulation of TrkA and ChAT expression in developing rat basal forebrain: evidence that both exogenous and endogenous NGF regulate differentiation of cholinergic neurons. J Neurosci 1995;15:2888 –905. Martinez Serrano A, Fischer W, Bjo¨rklund A. Reversal of agedependent cognitive impairments and cholinergic neuron atrophy by NGF-secreting neural progenitors grafted to the basal forebrain. Neuron 1995;15:473– 84. Mesulam MM, Mufson EJ, Rogers J. Age-related shrinkage of cortically projecting cholinergic neurons: a selective effect. Ann Neurol 1987;22:31– 6.

134

N.L. Ward et al. / Neurobiology of Aging 21 (2000) 125–134

[41] Mobley WC, Rutkowski JL, Tennekoon GI, Gemski J, Buchanan K, Johnston MV. Nerve growth factor increases choline acetyltransferase activity in developing basal forebrain neurons. Brain Res 1986;387:53– 62. [42] Mouton PR, Ward NL, West MJ, Gokhale AM. Unbiased estimation of length density for linear objects on arbitrary sections using isotropic probes (“Space Balls”). Conference on Stochastic Geometry Abstracts, August, Banff, Alberta, 1999. [43] Mufson EJ, Conner JM, Kordower JH. Nerve growth factor in Alzheimer’s disease: defective retrograde transport to nucleus basalis. Neuroreport 1995;6:1063– 6. [44] Mufson EJ, Lavine N, Jaffar S, Kordower JH, Quirion R, Saragovi HU. Reduction in p140-TrkA receptor protein within the nucleus basalis and cortex in Alzheimer’s disease. Exp Neurol 1997;146:91–103. [45] Peterson DA, Dickinson–Anson HA, Leppert JT, Lee KF, Gage FH. Central neuronal loss and behavioral impairment in mice lacking neurotrophin receptor p75. J Comp Neurol 1999;404:1–20. [46] Salehi A, Verhaagen J, Dijkhuizen PA, Swaab DF. Co-localization of high-affinity neurotrophin receptors in nucleus basalis of Meynert neurons and their differential reduction in Alzheimer’s disease. Neuroscience 1996;75:373– 87. [47] Scott SA, Mufson EJ, Weingartner JA, Skau KA, Crutcher KA. Nerve growth factor in Alzheimer’s disease: increased levels throughout the brain coupled with declines in nucleus basalis. J Neurosci 1995;15: 6213–21. [48] Sobreviela T, Clary DO, Reichardt LF, Brandabur MM, Kordower JH, Mufson EJ. TrkA-immunoreactive profiles in the central nervous system: colocalization with neurons containing p75 nerve growth factor receptor, choline acetyltransferase, and serotonin. J Comp Neurol 1994;350:587– 611.

[49] Sofroniew MV, Cooper JD, Svendsen CN, Crossman P, Ip NY, Lindsay RM, Zafra F, Lindholm D. Atrophy but not death of adult septal cholinergic neurons after ablation of target capacity to produce mRNAs for NGF, BDNF, and NT3. J Neurosci 1993;13:5263–76. [50] Springer JE, Koh S, Tayrien MW, Loy R. Basal forebrain magnocellular neurons stain for nerve growth factor receptor: correlation with cholinergic cell bodies and effects of axotomy. J Neurosci Res 1987; 17:111– 8. [51] Stanford LE, Ward NL, Hagg T, Brown RE. p75NGFR-deficient mice show faster learning but decreased working memory in Hebb–Williams maze. Soc Neurosci Abstr 1998;24:179. [52] Sugaya K, Greene R, Personett D, Robbins M, Kent C, Bryan D, Skiba E, Gallagher M, McKinney M. Septo-hippocampal cholinergic and neurotrophin markers in age-induced cognitive decline. Neurobiol Aging. 1998;19:351– 61. [53] Vantini G, Schiavo N, Di Martino A, Polato P, Triban C, Callegaro L, Toffano G, Leon A. Evidence for a physiological role of nerve growth factor in the central nervous system of neonatal rats. Neuron. 1989;3:267–73. [54] Ward NL, Hagg T. p75NGFR and cholinergic neurons in the developing forebrain: a re-examination. Dev Brain Res 1999;118:79 –91. [55] Williams LR, Varon S, Peterson GM, Wictorin K, Fischer W, Bjo¨rklund A, Gage FH. Continuous infusion of nerve growth factor prevents basal forebrain neuronal death after fimbria fornix transection. Proc Natl Acad Sci USA 1986;83:9231–5. [56] Yeo TT, Chua Couzens J, Butcher LL, Bredesen DE, Cooper JD, Valletta JS, Mobley WC, Longo FM. Absence of p75NTR causes increased basal forebrain cholinergic neuron size, choline acetyltransferase activity, and target innervation. J Neurosci 1997;17:7594 – 605.