Immunohistochemical and neurochemical correlates of learning deficits in aged rats

Learning deficits in aged rats

Pergamon PII: S0306-4522(99)00561-8

Neuroscience Vol. 96, No. 2, pp. 275–289, 2000 275 Copyright q 2000 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/00 $20.00+0.00

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IMMUNOHISTOCHEMICAL AND NEUROCHEMICAL CORRELATES OF LEARNING DEFICITS IN AGED RATS J. STEMMELIN, C. LAZARUS, S. CASSEL, C. KELCHE and J.-C. CASSEL* Laboratoire de Neurosciences Comportementales et Cognitives, UMR 7521, CNRS, Universite´ Louis Pasteur, 67000 Strasbourg, France

Abstract—This study examined whether cholinergic and monoaminergic dysfunctions in the brain could be related to spatial learning capabilities in 26-month-old, as compared to three-month-old, Long–Evans female rats. Performances were evaluated in the water maze task and used to constitute subgroups with a cluster analysis statistical procedure. In the first experiment (histological approach), the first cluster contained young rats and aged unimpaired rats, the second one aged rats with moderate impairment and the third one aged rats with severe impairment. Aged rats showed a reduced number of choline acetyltransferaseand p75 NTR-positive neurons in the nucleus basalis magnocellularis, and choline acetyltransferase-positive neurons in the striatum. In the second experiment (neurochemical approach), the three clusters comprised young rats, aged rats with moderate impairment and aged rats with severe impairment. Alterations related to aging consisted of reduced concentration of acetylcholine, norepinephrine and serotonin in the striatum, serotonin in the occipital cortex, dopamine and norepinephrine in the dorsal hippocampus, and norepinephrine in the ventral hippocampus. In the first experiment, there were significant correlations between water maze performance and the number of; (i) choline acetyltransferase- and p75 NTR-positive neurons in the nucleus basalis magnocellularis; (ii) choline acetyltransferase-positive neurons in the striatum and; (iii) p75 NTR-positive neurons in the medial septum. In the second experiment, water maze performance was correlated with the concentration of; (i) acetylcholine and serotonin in the striatum; (ii) serotonin and norepinephrine in the dorsal hippocampus; (iii) norepinephrine in the frontoparietal cortex and; (iv) with other functional markers such as the 5-hydroxyindoleacetic acid/serotonin ratio in the striatum, 3,4-dihydroxyphenylacetic acid/dopamine ratio in the dorsal hippocampus, 5-hydroxyindoleacetic acid/serotonin and homovanillic acid/dopamine ratios in the frontoparietal cortex, and 3,4-dihydroxyphenylacetic acid/dopamine ratio in the occipital cortex. The results indicate that cognitive deficits related to aging might involve concomitant alterations of various neurochemical systems in several brain regions such as the striatum, the hippocampus or the cortex. It also seems that these alterations occur in a complex way which, in addition to the loss of cholinergic neurons in the basal forebrain, affects dopaminergic, noradrenergic and serotonergic processes. q 2000 IBRO. Published by Elsevier Science Ltd. Key words: acetylcholine, basal forebrain, cortex, hippocampus, monoamines, striatum.

The question of a potential involvement of cholinergic dysfunctions in age-related degradation of learning and memory has emerged with the demonstration that the muscarinic antagonist scopolamine disrupted learning in young adults, 39 and the finding that cholinergic neurons degenerate in the forebrain of Alzheimer’s disease (AD) patients. 19,31,109 Since that time, the cholinergic hypothesis of cognitive deficiencies associated with aging 8 has undergone intense experimental exploration using, amongst other approaches, cholinergic drugs, or lesions of defined pathways or structures in the brain. 22,26,42,43 Altogether, the results confirmed a crucial role for cholinergic mechanisms in learning and memory processes. However, besides other arguments, 23,33,127 even experiments using the highly specific cholinergic immunotoxin 192 immunoglobulin G–saporin have suggested that learning and memory are not an exclusive

matter of central cholinergic function, particularly, but not only, when spatial memory is concerned. 10,16,25,135 Extensive and highly specific damage to basalocortical or septohippocampal cholinergic neurons produces weaker cognitive effects than less specific electrolytic, aspiration, N-methyld-aspartate, ibotenate, etc. lesions encroaching on to cholinergic nuclei or pathways. In aged rodents, cognitive impairments were found in an important variety of learning tasks. 68 When the distribution of performances in spatial tasks, such as the water maze or the radial maze, is considered, there appears to be an important interindividual variability: some aged rats perform as accurately as their young counterparts, whereas others are extremely bad performers. 36,49,51 In a previous experiment, 128 we proposed that a possible explanation for the heterogeneity of performance in aged rats could be that the cognitive decline does not occur with a comparable speed and/or extent in all subjects. It has been shown in several reports that the consideration of individual differences was crucial for interpreting the neural determinants of cognitive decline. Most experiments carried out so far were first interested in the cholinergic basis of age-related memory decline. It was demonstrated not only that markers of cholinergic functions were altered, but also that the severity of these alterations could be linked to the decline of cognitive faculties. 4,5,47,67,72,130 In other reports, non-cholinergic markers were also considered, but seldom was attention paid to a possible link with individual cognitive capabilities. 52,75,81 The possibility that alterations in such systems might participate

*To whom correspondence should be addressed. Tel.: 1 33-3-88-35-8435; fax: 1 33-3-88-35-84-42. E-mail address: [email protected] (J.-C. Cassel). Abbreviations: ACh, acetylcholine; AD, Alzheimer’s disease; AMI, aged moderately impaired; ASI, aged severely impaired; AU, aged unimpaired; ChAT, choline acetyltransferase; DA, dopamine; DOPAC, 3,4dihydroxyphenylacetic acid; EDTA, ethylenediaminetetra-acetate; 5HIAA, 5-hydroxyindoleacetic acid; HPLC, high-performance liquid chromatography; 5-HT, serotonin (5-hydroxytryptamine); HVA, homovanillic acid; IR, immunoreactive; MS, medial septum; NBM, nucleus basalis magnocellularis; NE, norepinephrine; p75 NTR, low-affinity neurotrophin receptor; PBS, phosphate-buffered saline; STR, striatum; vDBB, vertical limb of the diagonal band of Broca. 275

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in the gravity of cognitive impairments, whether interacting or not with cholinergic dysfunctions, is compatible with the outcome of experimental work on the implication of interactions between various neurotransmitter systems in cognitive processes. 23,33,127 Furthermore, it is also a fact that neurotransmitter systems other than the cholinergic one are altered in AD patients, 85,104 or even during normal aging. 105 The aim of the present experiments was to further explore which type of age-related alterations in the brain might account for cognitive disabilities. In a first step, using a water maze, we measured spatial reference memory performance in young and aged (26 months) female rats in order to define their cognitive status (i.e. young, aged unimpaired, aged moderately impaired, aged severely impaired). In a second step, the rats were killed and their brains prepared for immunohistochemistry or high-performance liquid chromatography (HPLC) measurements of cholinergic, indolaminergic and catecholaminergic markers. Immunohistochemistry used antibodies raised against choline acetyltransferase (ChAT) or the low-affinity neurotrophin receptor (p75 NTR). The immunoreactive (IR) neurons were counted in the medial septum (MS), the vertical limb of the diagonal band of Broca (vDBB), the nucleus basalis magnocellularis (NBM) and the striatum (STR). All these anatomical entities are cholinergic nuclei or contain cholinergic neurons. Neurochemical markers were measured in the frontoparietal, entorhinal and occipital cortices, the hippocampus and the STR. Immunohistochemistry and neurochemical determinations were made in separate experiments. EXPERIMENTAL PROCEDURES

Experiment 1 Subjects. The first experiment used 12 three-month-old and 32 26month-old Long–Evans female rats (R. Janvier, France). The aged rats were obtained at the age of three months and housed in our laboratory in Makrolon cages (59 cm × 38 cm × 20 cm) in groups of six until the age of 20 months. Subsequently, they were housed in groups of 10 in larger Makrolon cages (100 cm × 60 cm × 25 cm). Before water maze testing, they were placed three per cage (42 cm × 26 cm × 15 cm; one adult rat with two aged rats or three aged rats). Food and water were available ad libitum. The colony and the testing rooms were maintained on a 12-h light/12-h dark cycle (lights on at 07.00) under controlled temperature (228C). All procedures involving animals and their care were conducted in conformity with the institutional guidelines that are in compliance with national (council directive no. 87848, 19 October 1987, Ministe`re de l’Agriculture et de la Foreˆt, Service Ve´te´rinaire de la Sante´ et de la Protection Animales; permission no. 6212 to J.-C.C., no. 2108 to C.K. and J.S. under the responsibility of the former) and international (NIH publication no. 86-23, revised 1985) laws and policies. All efforts were made to keep the number of subjects used to a minimum regarding statistical constraints, and to minimize suffering throughout the experiments. Behavioral testing: water maze. The water maze consisted of a circular pool (diameter 160 cm, height 60 cm) filled to half its height with water. The water (208C) was made opaque with addition of powdered milk. The pool was divided into four virtual quadrants of equal surface. The experimental room contained different extra-maze cues and the illumination was provided by a neon light placed 1.80 m above the center of the pool. A transparent circular platform (diameter 11 cm) was submerged 1 cm underneath the water surface at a constant position in the middle of one quadrant (Q3). Each rat was tested for five days with four consecutive trials per day. For each trial, the rat was placed in the pool, facing the wall at a randomly designed starting point from where it was released. A maximum of 60 s was allowed to reach the submerged platform. When the rat had climbed on to the platform, it remained there for 10 s before being removed and placed on the next starting point. If the rat did not find the platform in time, it was placed

on the platform for 10 s by the experimenter. Such a testing procedure places emphasis on spatial reference memory. Swim paths were recorded using a video camera connected to a tracking software (Ethovision, Noldus, The Netherlands). The time taken by the rat to reach the platform (latency), as well as the distance swum during a given trial, were taken into account for statistical analysis. Immediately after the third trial of the fifth testing day, the platform was removed and each rat was given a probe trial in which it had to swim for 60 s. The time spent in the quadrant where the platform was previously located and the crossings of the former position of the platform were recorded. Histological verifications. Five young and 15 aged rats were used for immunostaining. The remaining rats were used for neuroanatomical determinations in another study. These 20 rats were submitted to a slow (5 ml/min) intracardiac perfusion of 300 ml of a 2% paraformaldehyde solution with 250 mg heparin added. Brains were postfixed in 2% paraformaldehyde for 2 h and transferred into 0.1 M phosphatebuffered saccharose (20%) for 48 h. Subsequently, they were quickly frozen and cut into 10-mm-thick coronal sections using a cryostat (2238C). Sections passing through the MS, STR, vDBB and NBM were collected on to gelatin-coated slices and processed for ChAT or p75 NTR immunohistochemistry. Anti-choline acetyltransferase immunohistochemistry. Sections collected on slides were dried at room temperature and washed in 0.1 M phosphate-buffered saline (PBS). After 20-min incubation in a “blocker” (10% methanol/3% H2O2/PBS), followed by a 30-min incubation in 2% bovine serum albumin/PBS/0.4% Triton X-100, sections were placed for 72 h into the monoclonal anti-ChAT antisera (1:20 dilution, 48C; Boehringer, Germany), which was diluted in PBS/ 0.4% Triton X-100/2% blocker. After washing in PBS, the sections were placed for 30 min in secondary biotinylated antibody (Vector Labs, Burlingame, CA, U.S.A.), followed by the avidin–biotin complex (Elite, ABC kit, Vector Labs). The sections were reacted in 0.1% diaminobenzidine/0.2% H2O2 in Tris-buffered saline. After dehydration through alcohol/xylene baths, slides were mounted with Eukitt for microscope observation. Control sections were prepared as above except that normal serum was substituted for anti-ChAT antiserum. Anti-p75 NTR immunohistochemistry. Adjacent sections to the ones stained for anti-ChAT were used for anti-p75 NTR. The procedure described by Kawaja and Gage, 70 which is almost the same as the one used for ChAT immunostaining, was used for the incubation in the presence of the anti-p75 NTR antiserum (1:10 dilution, 48C; Boehringer, Germany) for 24 h. Nickel chloride was also added to the final diaminobenzidine reaction solution to obtain a dark staining of the cells. ChAT- and p75 NTR-IR cell counts were assessed under a light microscope (Olympus). For each rat, counts were made on about five sections in the regions shown in Fig. 1. These included the STR, the NBM, the MS and the vDBB. In the NBM region, cell counts were performed on a restricted area of each section. A correction index was applied according to the variable spreading of the sections on the slides and analyses were made on corrected data. Counts were made bilaterally by an experimenter who was unaware of the performances of the rats. Experiment 2 Subjects. Ten three-month-old and 26 26-month-old Long–Evans female rats were used in the second experiment. Rats were bred in the same conditions as in Experiment 1. Their spatial reference memory capabilities were also assessed in the water maze prior to killing for neurochemical determination. The testing procedure used was the same as in Experiment 1. Neurochemical analyses. Ten to 15 days after water maze testing, the rats were killed by focused microwave irradiation (Sairem, Villeurbanne, France) in order to induce a rapid inactivation of brain enzymes such as, for instance, acetylcholinesterase. The waves (1.4 s, 4.5 kW) were focused on the head of the rat. Brains were removed and placed on a cold plate, and the frontoparietal cortex, the STR, the dorsal and ventral halves of the hippocampus, the occipital cortex and the entorhinal cortex were dissected out separately. Tissue samples were

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determined using data analysis software (Baseline 810, Waters) and are expressed as ng/mg irradiated tissue. Data analysis Behavioral performances were first compared among groups of young and aged rats using an ANOVA. The corresponding effect is described as the Age effect hereafter. The distances swum to reach the hidden platform on each trial were corrected according to the method described by Lindner. 78 To further analyse the distances, a statistical distinction between different subpopulations of rats was made according to the cluster analysis procedure described by Ghirardi et al. 56 Thereby, the entire population of rats (young 1 aged) was subdivided into three subpopulations. The corresponding effects are described as Subgroup effects hereafter. In Experiment 1, cluster one included all young rats and nine aged rats which showed the best performances (aged unimpaired, AU). Cluster two (n ˆ 9) included aged rats with moderate impairment (AMI). Cluster three (n ˆ 14) included aged rats with severe impairment (ASI). In Experiment 2, only one aged rat showed no impairment; 13 aged rats were moderately impaired and 12 rats showed severe deficits. Based on this distinction, a second ANOVA was run and, where pertinent, two-by-two comparisons were made using a Newman–Keuls test. 141 Immunohistochemical and neurochemical data were also analysed with ANOVA, and twoby-two comparisons used the Newman–Keuls test. In aged rats, correlations between neurochemical data or cell counts and cognitive performances were assessed using Pearson’s parametric regression method (r). An effect, a difference or a correlation was considered significant when the value of P was less than 0.05. RESULTS

Experiment 1

Fig. 1. Schematic cross-sections through the basal forebrain showing the localization of the regions in which cell counting was performed. Adapted from Paxinos and Watson. 107 Top: 10.7 from bregma; bottom: 21.4 from bregma.

weighed, immediately frozen and stored at 2808C until neurochemical determinations. Concentrations of acetylcholine (ACh), norepinephrine (NE), 3,4dihydroxyphenylacetic acid (DOPAC), dopamine (DA), 5-hydroxyindoleacetic acid (5-HIAA), homovanillic acid (HVA) and serotonin (5-hydroxytryptamine, 5-HT) were measured using HPLC with electrochemical detection. 13 Tissue samples (left and right pooled) were prepared for HPLC by homogenization in 1 N formic acid/ acetone (15:85, v/v) and the formic extracts were used for determination of the concentration of ACh, monoamines and related metabolites. The monoamines and metabolites were measured without further purification. The HPLC system consisted of an ESA liquid chromatography pump (ESA Bedford) and an ESA Coulochem II detector (Eurosep Instruments) equipped with a 5014 high-performance analytical cell (ESA Bedford). Detector potential at the analytical cell was set at 10.4 V. HPLC analysis was performed on a C18 Spherisorb ODS2 reverse phase column (5 mm pore size, 4.6 mm i.d., 25 cm long). The mobile phase consisted of 0.1 M NaH2PO4 (pH 3) containing 0.1 mM/l EDTA, 1.7 mM/l 1-octanesulfonic acid sodium salt and 10% acetonitrile. The flow rate was 1 ml/min. ACh was measured following an additional purification of the formic extracts, consisting of tetraphenylboron exchange of the amines in 3-heptanone, followed by 0.1 N HCl extraction. 13 HPLC analysis was performed on a C18 Spherisorb ODS2 reverse phase column (3 mm pore size, 7 mm i.d., 10 cm long). The mobile phase consisted of 0.05 M KH2PO4 (pH 7) containing 600 mg/l tetramethylammonium chloride and 25 mg/l sodium octane sulfate. The flow rate was 0.8 ml/ min. ACh was converted into betaine in a post-column reactor with covalently bounded acetylcholinesterase (EC 3.1.1.7). The resulting H2O2 was detected electrochemically using a 5040 ESA-cell working electrode at 10.3 V. Concentrations of ACh, monoamines and metabolites were

Water maze testing. Acquisition. Data are shown in Table 1. When all aged rats were considered as a single population, ANOVA of distances swum showed significant Age (F1,42 ˆ 11.8, P , 0.01) and Day (F4,168 ˆ 29.7, P , 0.001) effects, but no significant interaction between both factors. The Age effect was due to distances which were significantly longer in aged as compared to young rats. The Day effect reflected a significant decrease of the path length, particularly from day 1 to days 2, 3 and 4 (respectively, P , 0.001), but no difference was observed between days 4 and 5. When the analysis considered the young rats and the three subpopulations of aged rats separately, it showed significant Subgroup (F3,40 ˆ 24.1, P , 0.001) and Day (F4,160 ˆ 46.4, P , 0.001) effects, as well as a significant interaction between both factors (F12,160 ˆ 4.83, P , 0.001). The Subgroup effect was due to a significantly longer distance in AMI and ASI rats as compared to young adult or AU rats (P , 0.001 in each case), but also to a significantly longer distance in ASI rats as compared to AMI rats (P , 0.05). The Day effect was due to overall distances which decreased significantly from day 1 to days 2, 3 and 4 (respectively, P , 0.001), but no difference was observed between days 4 and 5. Finally, the interaction can be interpreted as reflecting a between-day shortening of the path length, which was rapid in young adult and AU rats, slower in AMI rats, and almost absent in ASI rats. Probe trial. Data are shown in Table 1. When all aged rats were considered together, ANOVAs of the time spent in the quadrant where the platform was previously located (time in Q3) showed a significant Age effect (F1,42 ˆ 21.95, P , 0.001). There was also a significant Subgroup effect (F3,40 ˆ 14.1, P , 0.001). Newman–Keuls comparisons showed that AMI and ASI rats spent significantly less time in the correct quadrant than young adult and AU rats (P , 0.001). There was no difference between the scores of

Distances (cm; mean ^ S.E.M.) in daily blocks of four-trial sessions recorded during the acquisition phase, average time (^S.E.M.) spent in quadrant Q3 and average number (^S.E.M.) of platform crossings during the probe trial, in young adult and aged rats (all rats), are included. Aged rats are subdivided into aged unimpaired (AU), aged moderately impaired (AMI) and aged severely impaired (ASI) rats. Statistics: *P , 0.05, **P , 0.01 and ***P , 0.001 compared to young adults.

594.0 ^ 64.5 1049.8 ^ 52.5*** 988.3 ^ 82.5*** 1167.4 ^ 32.9*** 1122.9 ^ 39.4 1107.9 ^ 31.0 1075.2 ^ 43.1 1132.1 ^ 47.5 Experiment 2 Young Aged AMI ASI

730.9 ^ 74.9 1163.1 ^ 24.1*** 1169.1 ^ 32.7*** 1160.7 ^ 39.9***

500.9 ^ 42.1 978.1 ^ 58.1*** 791.6 ^ 63.8** 1229.4 ^ 27.8***

371.7 ^ 50.1 910.3 ^ 55.7*** 736.1 ^ 56.7*** 1141.2 ^ 41.2***

25.8 ^ 1.7 18.2 ^ 1.1** 19.5 ^ 1.6* 16.4 ^ 1.5**

5.6 ^ 0.4 2.7 ^ 0.3*** 3.6 ^ 0.5** 3.2 ^ 0.5** 1.7 ^ 0.3*** 24.8 ^ 1.5 16.2 ^ 0.9*** 21.2 ^ 1.7 15.1 ^ 1.3*** 13.7 ^ 1.2*** 413.0 ^ 69.3 572.1 ^ 41.5* 403.5 ^ 59.8 439.4 ^ 50.2 765.7 ^ 42.9*** 429.9 ^ 57.4 603.1 ^ 44.5* 429.3 ^ 83.3 470.6 ^ 51.9 799.9 ^ 40.6*** 518.0 ^ 57.2 668.9 ^ 37.2 425.5 ^ 32.2 773.9 ^ 47.7** 757.8 ^ 46.4** 612.6 ^ 49.0 813.3 ^ 27.7** 683.4 ^ 53.8 913.8 ^ 31.7*** 832.1 ^ 35.8* 867.9 ^ 35.6 896.7 ^ 20.8 849.6 ^ 54.6 958.1 ^ 11.4 887.5 ^ 28.7 Experiment 1 Young Aged AU AMI ASI

Acquisition day 1 (distances) Acquisition day 2 (distances) Acquisition day 3 (distances) Acquisition day 4 (distances) Acquisition day 5 (distances) Probe trial (time in Q3) Probe trial (crossings)

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Table 1. Water maze data (Experiments 1 and 2)

278

young adult and AU rats, or those of AMI and ASI rats. When compared to young rats, aged rats also showed a significant reduction in the number of crossings over the platform area (crossings; F1,42 ˆ 26.7, P , 0.001). When young rats and the three subpopulations of aged rats were considered separately, ANOVA showed a significant Subgroup effect (F3,40 ˆ 13.5, P , 0.001). Post hoc tests indicated that AU rats made fewer crossings over the platform area than young rats (P , 0.01), as was the case for AMI (P , 0.01) and ASI rats (P , 0.001). The number of crossings did not differ significantly amongst AU and AMI rats. ASI rats crossed the platform area significantly less often than AU (P , 0.05) or AMI rats (P , 0.05). Anti-choline acetyltransferase immunohistochemistry. Typical examples of IR neurons are shown in Fig. 2 and data are shown in Table 2. ANOVA of the number of ChAT-IR neurons showed significant Age effects in the NBM (F1,24 ˆ 8.7, P , 0.01) and the STR (F1,18 ˆ 13.2, P , 0.01), but not in the MS or the vDBB. Significant Subgroup effects were also found in the NBM (F3,22 ˆ 5.6, P , 0.01) and the STR (F3,16 ˆ 19.5, P , 0.001), but not in the MS or the vDBB, when the three subpopulations of aged rats were considered separately. Newman–Keuls comparisons of IR cell counts in the NBM showed significant reductions in AMI and ASI rats as compared to young rats (P , 0.05), but also in ASI rats as compared to AU rats (P , 0.05). The differences between young adult and AU rats, AU and AMI rats, or AMI and ASI rats were not significant. In the STR, no difference was found between young and AU rats, but AMI and ASI rats showed a significant decrease of the number of IR cells when compared to young adult rats (P , 0.01 and P , 0.001, respectively) or AU rats (P , 0.05 and P , 0.001, respectively). In ASI rats, this number was also significantly lower than in AMI rats (P , 0.05). In aged rats, significant correlations were found between the number of ChAT-IR cells in the NBM and acquisition performance (mean distances and latencies) in the water maze (r ˆ 20.65, P , 0.01 and r ˆ 20.61, P , 0.01, respectively), or the number of platform crossings during the probe trial (r ˆ 0.47, P , 0.05). Other significant correlations were found between the number of striatal ChAT-IR cells and the time spent in the training quadrant during the probe trial (r ˆ 0.53, P , 0.05), the number of platform crossings (r ˆ 0.57, P , 0.05) and the acquisition performances (distances: r ˆ 20.65, P , 0.01; latencies: r ˆ 20.64, P , 0.01). Anti-p75 NTR immunohistochemistry. Typical examples of IR neurons are shown in Fig. 3 and data are shown in Table 2. ANOVA of the number of p75 NTR-IR cells showed a significant Age effect in the NBM (F1,22 ˆ 10.13, P , 0.01), but not in the MS or vDBB. When the three subpopulations of aged rats were considered separately, significant Subgroup effects were found in the NBM (F3,20 ˆ 3.73, P , 0.05) and the MS (F3,18 ˆ 6.6, P , 0.01), but not in the vDBB. Two-by-two comparisons of the number of IR cells in the NBM showed a significant reduction in AU, AMI and ASI rats as compared to young adult rats (P , 0.05); no difference was found amongst the three subpopulations of aged rats. In the MS, a significant reduction of the number of IR cells was observed in ASI rats as compared to young adult and AU rats (P , 0.05 and P , 0.01, respectively), and in AMI rats as compared to AU rats (P , 0.05). In aged rats, significant correlations were

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Fig. 2. Photomicrograph of ChAT-positive neurons in the NBM of young (A), AU (B), AMI (C) and ASI (D) rats. Scale bar ˆ 100 mm.

found between the number of p75 NTR-IR cells in the MS and the acquisition performances (distances: r ˆ 20.77, P , 0.001; latencies: r ˆ 20.74, P , 0.01). In the NBM, cell counts were correlated with the number of platform crossings during the probe trial (r ˆ 0.59, P , 0.01).

Experiment 2 Water maze data. Acquisition data are shown in Table 1. As the cluster analysis distinguished only one AU rat, this rat was not taken into account for further analysis. Briefly,

Table 2. Number of choline acetyltransferase- and p75 NTR-positive cells per section (mean ^ S.E.M.) in young adult and aged rats (all rats) Young

Aged

ChAT NBM MS vDBB STR

27.17 ^ 1.83 55.62 ^ 1.46 69.20 ^ 4.10 81.38 ^ 4.80

18.42 ^ 1.20** 55.83 ^ 2.85 59.28 ^ 2.59 56.41 ^ 3.18**

p75 NTR NBM MS vDBB

28.87 ^ 3.23 57.26 ^ 4.16 54.20 ^ 4.49

19.71 ^ 1.12** 53.05 ^ 2.43 51.96 ^ 2.73

AU

AMI

ASI

21.31 ^ 2.13 58.98 ^ 5.91 57.37 ^ 4.41 72.00 ^ 3.89

19.12 ^ 2.37* 57.76 ^ 5.15 61.38 ^ 3.54 58.04 ^ 2.33**

15.00 ^ 1.25* 51.23 ^ 3.66 59.62 ^ 5.30 46.34 ^ 3.24***

18.22 ^ 2.60* 62.83 ^ 3.48 57.20 ^ 5.74

21.72 ^ 2.15* 51.20 ^ 0.49 50.80 ^ 2.87

19.30 ^ 1.34* 44.83 ^ 2.83* 48.58 ^ 5.03

Aged rats are subdivided into aged unimpaired (AU), aged moderately impaired (AMI) and aged severely impaired (ASI) rats. Statistics: *P , 0.05, **P , 0.01 and ***P , 0.001 compared to young adults.

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Fig. 3. Photomicrograph of p75 NTR-positive neurons in the NBM of young (A), AU (B), AMI (C) and ASI rats (D). Scale bar ˆ 100 mm.

ANOVA of the acquisition data showed significant Age (F1,34 ˆ 61.1, P , 0.001) and Day (F4,136 ˆ 24.0, P , 0.001) effects, and a significant interaction between both factors (F4,136 ˆ 8.7, P , 0.001). When the two subpopulations of aged rats were taken into account, ANOVA also showed significant Subgroup (F2,32 ˆ 124.3, P , 0.001) and Day (F4,128 ˆ 22.1, P , 0.001) effects, and a significant interaction between both factors (F8,128 ˆ 10.1, P , 0.001). The Subgroup effect was due to path lengths which were significantly longer in AMI and ASI rats as compared to young adult rats (P , 0.001 in each case), but also in ASI rats as compared to AMI rats (P , 0.001). During the probe trial (Table 1), ANOVA of the time spent in the training quadrant showed significant Age (F1,34 ˆ 12.9, P , 0.001) and Subgroup (F2,32 ˆ 8.0, P , 0.01) effects. Two-by-two comparisons showed significant differences between young adult rats and either AMI or ASI rats (P , 0.05 and 0.01, respectively). High-performance liquid chromatography data. Acetylcholine content. Data are shown in Table 3. ANOVA of the striatal ACh content showed significant Age (F1,24 ˆ 13.6,

P , 0.01; young versus aged rats) and Subgroup (F2,23 ˆ 6.5, P , 0.01; young and two subpopulations of aged rats) effects. The ACh content was significantly reduced in AMI and ASI rats as compared to young rats (P , 0.01 and 0.05, respectively). There was no significant Age or Subgroup effect in the frontoparietal, occipital or entorhinal cortices, or in the hippocampus (ventral or dorsal half). In aged rats, a significant correlation was found between the striatal ACh content and the probe trial scores (r ˆ 0.45, P , 0.05). Monoamine content. Data are shown in Table 3. ANOVA showed a significant Age effect on the 5-HT content in the STR and the occipital cortex (F1,24 ˆ 7.14, P , 0.05 and F1,21 ˆ 4.43, P , 0.05, respectively). Such an effect was also found on the 5-HIAA/5-HT ratio in the frontoparietal (F1,22 ˆ 6.0, P , 0.05) and the entorhinal (F1,20 ˆ 4.78, P , 0.05) cortices, as well as in the hippocampus (dorsal half: F1,22 ˆ 5.76, P , 0.05; ventral half: F1,20 ˆ 6.23, P , 0.05). When the two subpopulations of aged rats were considered separately, a significant Subgroup effect was found on striatal 5-HT content (F2,23 ˆ 4.55, P , 0.05); this

Table 3. Neurochemical measures (mean ^ S.E.M.) in brain homogenates of young adult and aged rats (all rats) Young

Aged

AMI

ASI

ACh Frontoparietal cortex Striatum Dorsal hippocampus Ventral hippocampus Occipital cortex Entorhinal cortex

0.38 ^ 0.04 2.78 ^ 0.40 0.91 ^ 0.17 0.98 ^ 0.11 0.39 ^ 0.06 1.02 ^ 0.21

0.40 ^ 0.02 1.72 ^ 0.07** 0.82 ^ 0.02 1.07 ^ 0.04 0.42 ^ 0.02 0.96 ^ 0.07

0.40 ^ 0.02 1.73 ^ 0.11** 0.79 ^ 0.03 1.11 ^ 0.04 0.42 ^ 0.02 0.94 ^ 0.11

0.39 ^ 0.02 1.71 ^ 0.09* 0.85 ^ 0.03 1.02 ^ 0.06 0.42 ^ 0.03 0.98 ^ 0.09

5-HT Frontoparietal cortex Striatum Dorsal hippocampus Ventral hippocampus Occipital cortex Entorhinal cortex

2.43 ^ 0.25 3.88 ^ 0.35 3.37 ^ 0.22 3.38 ^ 0.13 2.04 ^ 0.21 5.09 ^ 0.49

2.65 ^ 0.18 3.10 ^ 0.12* 2.87 ^ 0.13 2.95 ^ 0.24 1.64 ^ 0.08* 5.01 ^ 0.19

2.66 ^ 0.30 3.29 ^ 0.19 2.73 ^ 0.41 3.01 ^ 0.17 1.56 ^ 0.14 5.02 ^ 0.21

2.50 ^ 0.22 2.86 ^ 0.14* 2.77 ^ 0.25 2.91 ^ 0.20 1.70 ^ 0.09 5.00 ^ 0.34

5-HIAA Frontoparietal cortex Striatum Dorsal hippocampus Ventral hippocampus Occipital cortex Entorhinal cortex

1.67 ^ 0.16 5.19 ^ 0.59 3.49 ^ 0.17 3.14 ^ 0.09 1.34 ^ 0.13 2.39 ^ 0.32

2.68 ^ 0.33 6.06 ^ 0.71 4.97 ^ 0.60 4.41 ^ 0.55 1.74 ^ 0.31 3.45 ^ 0.37

2.65 ^ 0.59 6.75 ^ 1.14 4.84 ^ 0.78 4.33 ^ 0.68 1.63 ^ 0.25 3.22 ^ 0.49

2.58 ^ 0.43 5.13 ^ 0.92 4.76 ^ 0.96 4.49 ^ 0.92 1.85 ^ 0.36 3.72 ^ 0.59

5-HIAA/5-HT Frontoparietal cortex Striatum Dorsal hippocampus Ventral hippocampus Occipital cortex Entorhinal cortex

0.68 ^ 0.02 1.44 ^ 0.17 1.03 ^ 0.05 0.92 ^ 0.03 0.66 ^ 0.04 0.45 ^ 0.05

1.03 ^ 0.09* 1.89 ^ 0.20 1.65 ^ 0.17* 1.51 ^ 0.16* 1.04 ^ 0.14 0.73 ^ 0.08*

1.06 ^ 0.17 1.99 ^ 0.27 1.68 ^ 0.32 1.54 ^ 0.23 1.04 ^ 0.24 0.69 ^ 0.12

1.01 ^ 0.12 1.78 ^ 0.33 1.62 ^ 0.21 1.49 ^ 0.23 1.05 ^ 0.16 0.76 ^ 0.13

DA Frontoparietal cortex Striatum Dorsal hippocampus Ventral hippocampus Occipital cortex Entorhinal cortex

0.75 ^ 0.11 4.67 ^ 0.36 0.72 ^ 0.09 1.00 ^ 0.08 0.17 ^ 0.01 1.03 ^ 0.19

1.09 ^ 0.11 4.11 ^ 0.16 0.39 ^ 0.04** 0.84 ^ 0.10 0.24 ^ 0.03 0.94 ^ 0.09

1.04 ^ 0.16 4.36 ^ 0.31 0.39 ^ 0.05** 0.89 ^ 0.16 0.24 ^ 0.06 0.96 ^ 0.13

1.17 ^ 0.17 3.89 ^ 0.16 0.39 ^ 0.06* 0.80 ^ 0.14 0.23 ^ 0.05 0.93 ^ 0.12

DOPAC Frontoparietal cortex Striatum Dorsal hippocampus Ventral hippocampus Occipital cortex Entorhinal cortex

0.26 ^ 0.04 2.64 ^ 0.23 0.22 ^ 0.02 0.23 ^ 0.03 0.08 ^ 0.01 0.31 ^ 0.03

0.28 ^ 0.02 2.23 ^ 0.10 0.19 ^ 0.02 0.18 ^ 0.02 0.07 ^ 0.006 0.24 ^ 0.02

0.28 ^ 0.03 2.39 ^ 0.11 0.21 ^ 0.03 0.19 ^ 0.03 0.07 ^ 0.01 0.23 ^ 0.03

0.29 ^ 0.04 2.07 ^ 0.16 0.18 ^ 0.03 0.17 ^ 0.03 0.07 ^ 0.009 0.26 ^ 0.02

HVA Frontoparietal cortex Striatum

0.25 ^ 0.06 3.37 ^ 0.16

0.29 ^ 0.04 2.61 ^ 0.14**

0.33 ^ 0.06 2.73 ^ 0.21*

0.29 ^ 0.06 2.58 ^ 0.20*

DOPAC/DA Frontoparietal cortex Striatum Dorsal hippocampus Ventral hippocampus Occipital cortex Entorhinal cortex

0.35 ^ 0.03 0.56 ^ 0.02 0.33 ^ 0.04 0.24 ^ 0.03 0.47 ^ 0.06 0.37 ^ 0.07

0.25 ^ 0.01* 0.54 ^ 0.02 0.47 ^ 0.04* 0.23 ^ 0.01 0.38 ^ 0.05 0.28 ^ 0.02

0.22 ^ 0.01* 0.54 ^ 0.02 0.55 ^ 0.07* 0.23 ^ 0.01 0.36 ^ 0.08 0.25 ^ 0.02

0.26 ^ 0.03* 0.53 ^ 0.04 0.40 ^ 0.02 0.23 ^ 0.03 0.39 ^ 0.06 0.30 ^ 0.04

HVA/DA Frontoparietal cortex Striatum

0.39 ^ 0.05 0.74 ^ 0.04

0.29 ^ 0.02 0.63 ^ 0.03

0.32 ^ 0.04 0.61 ^ 0.04

0.27 ^ 0.04 0.67 ^ 0.05

NE Frontoparietal cortex Striatum Dorsal hippocampus Ventral hippocampus Occipital cortex Entorhinal cortex

1.46 ^ 0.09 1.15 ^ 0.12 1.80 ^ 0.12 1.82 ^ 0.09 1.16 ^ 0.10 2.64 ^ 0.16

1.37 ^ 0.08 0.84 ^ 0.04** 1.40 ^ 0.08* 1.43 ^ 0.07** 1.03 ^ 0.05 2.47 ^ 0.21

1.45 ^ 0.11 0.87 ^ 0.07* 1.53 ^ 0.09 1.51 ^ 0.07* 1.10 ^ 0.05 2.49 ^ 0.34

1.25 ^ 0.12 0.78 ^ 0.05* 1.28 ^ 0.13* 1.35 ^ 0.11** 0.95 ^ 0.09 2.45 ^ 0.24

Aged rats are subdivided into aged moderately impaired (AMI) and aged severely impaired rats (ASI). Values are expressed as ng/mg irradiated tissue. Statistics: *P , 0.05 and **P , 0.01 compared to young adults.

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was due to a significant reduction of the 5-HT concentration in ASI rats (P , 0.05) as compared to young rats. In aged rats, the probe trial scores were correlated with the 5-HT and 5HIAA concentrations in the dorsal hippocampus (r ˆ 0.53, P , 0.05 and r ˆ 0.52, P , 0.05, respectively), 5-HIAA in the striatum or the frontoparietal cortex (r ˆ 0.47, P , 0.05 and r ˆ 0.49, P , 0.05, respectively), and 5-HIAA/5-HT ratio in the striatum or the frontoparietal cortex (r ˆ 0.46, P , 0.05 and r ˆ 0.51, P , 0.05, respectively). A correlation was also found between the striatal 5-HT concentration and acquisition data (r ˆ 20.56, P , 0.05). ANOVA of the DA content in the dorsal hippocampus showed significant Age (F1,23 ˆ 12.9, P , 0.01) and Subgroup (F2,22 ˆ 6.17, P , 0.01) effects. The Subgroup effect was due to a significant reduction of DA concentration in AMI and ASI rats, as compared to young rats (P , 0.01 and P , 0.05, respectively). Significant Age and Subgroup effects were also detected for striatal HVA (F1,25 ˆ 9.13, P , 0.01 and F2,24 ˆ 4.58, P , 0.05, respectively); this was due to a significant reduction in AMI and ASI rats, as compared to young rats (P , 0.05 in each case). In aged rats, no correlation was found between the water maze scores and the DA, DOPAC or HVA content. ANOVA of the DOPAC/DA ratios in the frontoparietal cortex and the dorsal hippocampus showed significant Age (F1,23 ˆ 7.67, P , 0.05 and F1,23 ˆ 4.73, P , 0.05, respectively) and Subgroup effects (F2,22 ˆ 4.18, P , 0.05 and F2,22 ˆ 4.91, P , 0.05, respectively). The Subgroup effect was due to a significant decrease of the ratio in the frontoparietal cortex of AMI and ASI rats as compared to young adult rats (P , 0.05, in each case), and to a significant increase in the dorsal hippocampus of AMI rats as compared to young adult (P , 0.05) and ASI (P , 0.05) rats. In aged rats, we found correlations between the probe trial scores and the DOPAC/DA ratios in the occipital cortex (r ˆ 0.52, P , 0.05) and the HVA/DA ratios in the frontoparietal cortex (r ˆ 0.61, P , 0.05). A correlation was also found between the acquisition data and the DOPAC/DA ratios in the dorsal hippocampus (r ˆ 20.62, P , 0.01). ANOVA of the striatal and hippocampal NE contents showed significant Age (STR: F1,24 ˆ 8.94, P , 0.01; dorsal hippocampus: F1,23 ˆ 7.02, P , 0.05; ventral hippocampus: F1,22 ˆ 11.3, P , 0.01) and Subgroup effects (STR: F2,23 ˆ 4.63, P , 0.05; dorsal hippocampus: F2,22 ˆ 4.86, P , 0.05; ventral hippocampus: F2,21 ˆ 6.38, P , 0.01). Post hoc tests indicated that NE content was reduced in the STR of AMI and ASI rats as compared to young rats (P , 0.05, in each case), as well as in the ventral hippocampus (P , 0.05 and P , 0.01, respectively). A significant decrease of the NE content in the dorsal hippocampus was only found in ASI rats as compared to young rats (P , 0.05). In aged rats, we found significant correlations between the probe trial scores and the NE content in the dorsal hippocampus (r ˆ 0.57, P , 0.05), and between the acquisition data and the NE content in the frontoparietal cortex (r ˆ 20.47, P , 0.05). DISCUSSION

The present study investigated whether putative alterations of cholinergic and monoaminergic functions in the basal forebrain and target structures of aged Long–Evans female rats might account for learning and memory deficits in a water maze task. Globally, aged rats showed a reduced number of ChAT- and p75 NTR-positive neurons in the NBM and

ChAT-positive neurons in the STR. Other age-related reductions concerned the concentration of ACh, NE and 5-HT in the STR, 5-HT in the occipital cortex, DA and NE in the dorsal hippocampus, and NE in the ventral hippocampus. In the first experiment, we found a significant correlation between water maze performance and the number of; (i) ChAT- and p75 NTR-positive neurons in the NBM; (ii) ChAT-positive neurons in the STR and; (iii) p75 NTR-positive neurons in the MS. In the second experiment, water maze performance correlated with the concentrations of; (i) ACh and 5-HT in the STR; (ii) 5-HT and NE in the dorsal hippocampus; (iii) NE in the frontoparietal cortex, and (iv) with other functional markers such as 5-HIAA/5-HT ratio in the STR, DOPAC/DA ratio in the dorsal hippocampus, 5-HIAA/ 5-HT and HVA/DA ratios in the frontoparietal cortex, and DOPAC/DA ratio in the occipital cortex. Aging and water maze performances Our behavioral data are in line with previous findings suggesting that aging is not necessarily accompanied by alterations of learning and memory faculties. 51,113 From Experiment 1, it is clear that a subpopulation of aged rats showed water maze performances comparable to those found in their young adult counterparts. It is also noteworthy that, amongst aged rats with altered cognitive capabilities, individual differences exist as to the severity of the impairment: some rats show a moderate learning impairment whereas others show almost no learning. As stipulated previously, 47,128 such individual differences might be linked to an age-related cognitive decline that does not necessarily occur with a comparable speed or extent in all subjects. Due to that, the cognitive status of an aged rat might have only a temporary validity. This is the reason for which the delay between testing and immunohistochemical or neurochemical assessments in the present study was as short as technically possible. Since aging also results in motor and sensorimotor impairment, 68 it can be questioned whether the age-related impairment in the water maze task does not reflect motor or sensorimotor dysfunctions rather than cognitive disabilities. Although this question has been of major concern in a recent critical review article, 78 several authors have argued in favor of a dissociation between motor, sensorimotor and cognitive dysfunctions assessed in a water maze test. 49–51,86 Also, in a previous study, we failed to find any statistical relationship between motor or sensorimotor dysfunctions and water maze performances in aged rats. 128 Furthermore, in the present study, only the distances from the starting point to the platform were considered for the cluster analysis, and this variable is generally regarded as poorly sensitive to sensorimotor biases. The aforementioned individual differences might thus reflect differential alterations of cognitive functions, and it seems sound that at least part of these alterations parallel different degrees of dysfunction of neurotransmitter systems in the brain. In this study, we paid attention to the STR, the basalocortical and the septohippocampal systems. Alterations in the striatum The STR is a target structure of glutamatergic afferents from the cortex 95 and thalamic nuclei, 14 dopaminergic afferents

Learning deficits in aged rats

from the substantia nigra pars compacta, 100 noradrenergic afferents from the locus coeruleus, 89 and serotonergic afferents from the raphe. 136 It also contains GABAergic neurons and cholinergic interneurons. Glutamatergic and GABAergic markers were not measured in our present series of bioassays and immunostainings. Age-related alterations of cholinergic function in the STR are evidenced by a reduction of the number of ChAT-positive neurons, as well as a marked decrease of ACh concentration. These observations are in line with previous reports showing aged rodents to exhibit altered levels of ChAT activity, 57,82,96 reduced amounts of ChAT-positive neurons 27,46 or depleted ACh concentrations. 67,91 They are, however, at variance with a few other studies in which ChAT activity 5,47 and striatal ACh content 120 were not affected in aged rats. Part of this variability might be due to differences in the strain, gender, age or cognitive status (which was not assessed systematically) of the experimental animals. As described by other authors, 62,81,97,132 we also found evidence for decreased 5-HT function, a finding which is at variance with studies showing that serotonergic markers are not altered in the STR of aged rats. 52,58,75,133 Luine et al. 81 also found the striatal 5-HT content to be correlated with place learning in aged rats. Our results clearly show that a decrease of striatal 5-HT concentration may be linked, in one way or another, to the severity of the age-related cognitive impairments: only rats with the most severe impairment exhibited a significant decrease of 5-HT content. In AMI rats (moderate impairment), the 5-HIAA content and the 5-HIAA/ 5-HT ratio were increased, though not significantly, an observation suggesting possible up-regulating adaptative mechanisms which, in some rats, might have compensated for possible dysfunctions in striatal serotonergic neurotransmission. Although it is based on a statistical tendency in the present study, this interpretation might be consistent with other reports 139 and with clinical observations in AD patients. 106 As to markers of dopaminergic function, only the HVA content was reduced significantly. This finding contrasts with reports showing an age-related decrease in dopaminergic function, 59,88,97,99,121 but is in line with other studies. 58,79,81 As proposed by Tanila et al., 132 and as already mentioned for cholinergic markers, such a discrepancy could be due to strain or gender differences. Interestingly, a sparing of dopaminergic markers in the STR and a reduced level of HVA have also been described in AD patients. 108 Finally, as concerns NE, its level in the STR was most often described as unchanged in aged rats, 99,121,139 although Tanila et al. 132 reported a reduction. In aged humans and AD patients, early studies showed degeneration of neurons from the locus coeruleus, 85 an observation also reported in aged mice 76 in which the alteration of noradrenergic function was correlated with memory deficits. Thus, in the present study, it is possible that the reduced content of noradrenaline reflects degeneration of locus coeruleus neurons. Regarding our correlation data, it seems that the alteration of both cholinergic and serotonergic striatal functions may have played some role in the age-related mnesic deficits found. The STR is involved in motor function, 63 but may also play some role in cognition. 101 From pharmacological studies, it is known that striatal cholinergic neurotransmission is important for acquisition and retention in a passive avoidance task 112 or in positively reinforced learning tasks. 17 To our knowledge, the effects of modulating striatal cholinergic

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or serotonergic neurotransmission have not been assessed in a radial maze or water maze task. Nevertheless, lesion studies have shown that systems such as the hippocampus and the STR may operate simultaneously and in parallel in processing different types of spatial information. 92,93 Also, in rats with lesions of the caudate–putamen, place learning in a water maze is impaired 37,140 (but see Refs. 93 and 103). The dorsal striatum was shown to play some role in procedural, 111 stimulus–response 92 or rule-based 138 learning. Hence, it seems plausible that extra-hippocampal systems contribute to place learning, perhaps via interaction with the hippocampal formation. 94 In any case, these results suggest that the alteration of cholinergic and serotonergic functions in the STR of our aged rats might have contributed to the water maze deficits observed. Also, the degree to which these functions or their interaction is affected might have determined the severity of the impairment. Interactions between cholinergic and serotonergic mechanisms do play some role in cognition, 23,127 particularly in relation to hippocampal and cortical function, and it cannot be excluded that such interactive operations may be important for cognitive functions involving striatal mechanisms. Such an issue would require future experiments to investigate the effects of intrastriatal injections of cholinergic and serotonergic drugs on water maze performances. Alterations in the basalocortical system Following injection of retrograde tracers into the neocortex, about 80% of the labeled neurons in the NBM are cholinergic. 116 The entire cortical mantle shelters serotonergic terminals from the dorsal and median raphe nuclei, 114 a diffuse noradrenergic innervation from the locus coeruleus, 77 and a dopaminergic innervation restricted to the deep layers of the frontal and occipital areas. 15 Cortical nerve growth factor provides a trophic supply for basal forebrain cholinergic neurons via its binding to the TrkA/p75 receptor complex located on cholinergic terminals, internalization and retrograde axonal transport. 119 The colocalization of ChAT and p75 NTR has been shown immunohistochemically on NBM cholinergic neurons projecting to cortical regions. 71 With aging, this cholinergic system undergoes alterations such as a reduction in the number of ChATand p75 NTR-positive neurons. In rodents, these age-related changes have been described often and are in line with our present results. 35,46,60,72,73,115 However, as the activity of enzymes may also be altered with aging, it is possible that the reduced number of IR neurons reflects weakened cholinergic function rather than loss of neurons per se. This possibility is supported by the observations of Koh and Loy, 73 who found p75 NTR-IR neurons in the NBM of aged rats, but they were vacuolated and shrunken. An age-related alteration of retrograde transport mechanisms in the cholinergic cells might account for this reduced p75 NTR immunoreactivity. 35 In the literature, there are discrepancies as to the consequences of these alterations on cholinergic markers in cortical regions. In some reports, levels of cortical ACh were not affected, 120,134 whereas in others they were. 67,91 Such discrepancies were also found with other markers such as, for example, ChAT activity, which was either not different from normal 5,18,28,47,69,82,99,126 or reduced. 1,3,96,129 Again, such contradictory issues might be due to differences in strain, gender, age or methodological aspects. Age-related individual differences might also be a possible factor to account for these

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discrepancies, and this is particularly evident in our present study. Actually, we found the number of ChAT- and p75-IR cells in the NBM to correlate with the rats’ cognitive capabilities assessed in a water maze task, an observation also made in other studies, 46,72 and which could indicate a direct relationship between cholinergic dysfunction in the NBM and cognitive capabilities. Such a consideration would fit with studies using excitotoxic lesions of the NBM and which, despite poor selectivity, found damage of cholinergic neurons projecting to the cortex to disrupt various learning and memory capabilities, including those required for achieving correct performance in a water maze or a radial maze. 34,42 It was also demonstrated that cholinomimetic drugs reversed lesioninduced memory impairments found after infusion of ibotenic acid into the NBM. 41,66,84 However, with the use of highly selective toxins such as 192 immunoglobulin G–saporin, it was shown that specific damage to cholinergic neurons of the NBM induced only weak spatial memory impairments in the water maze, 16 or even no impairment. 9,38,135 Thus, it could be assumed that the responsibility of the basalocortical cholinergic axis in learning and memory processes is not as important as suspected so far. It seems that this axis has a more important part in attentional processes, as argued in recent reviews. 45,53,117 Therefore, part of the spatial memory impairments found in aged rats could reflect attentional deficits related to degeneration or dysfunction of cholinergic neurons in the NBM. Further experiments using appropriate behavioral tasks to dissociate between these components of cognitive processes are required to clarify this important issue. An alternative, but not necessarily exclusive, possibility would consider systems of neurotransmission other than the basalocortical cholinergic system. At the cortical level, and particularly in the frontoparietal area, correlations were found between the level of some monoamines, metabolites or turnover rates and water maze performance in aged rats. The frontoparietal cortex plays a role in cognition, and a modification of the tonus exerted by various afferences on pyramidal cortical cells may produce an imbalance involved in agerelated memory decline. 48 It has also been suggested that the interactions between monoaminergic and cholinergic systems may be of importance in the expression of cognitive function. 23,64,127 Thus, it is possible that our results suggest that the degradation of multiple neurotransmitter systems accounts for the age-related cognitive decline. In our present study, the cortical 5-HT content was reduced, but only in the occipital cortex, while the 5-HIAA/ 5-HT ratio was increased in the frontoparietal cortex. These observations might indicate that, in some cortical territories, serotonergic alterations may occur with aging. However, a possible increase in serotonergic activity as a consequence of neuron loss was evoked by other authors in aged rats, 52,59,62,110,137,139 aged non-demented humans and patients with AD. 61,105 There are also reports showing serotonergic functions to be preserved with aging. 28,58,79,99,125,132 Although our present findings are closer to the latter series of reports, the overall picture seems to be rather complicated. Furthermore, if serotonergic functions are altered, it could be that this alteration is only a general feature of aging, with no particular direct involvement in cognitive dysfunction. Such a statement would be in line with our present results and with others 52,81 showing an absence of correlation between cortical 5-HT contents and memory performance in aged rats. Concerning the cortical DA content, the evidence of no

major alteration related to aging is not surprising as the cortical DA innervation is quite weak, and others have also reported close to normal levels of DA and its metabolites in the cortex of aged rats. 52,132,137,139 In patients with AD, few changes were found in cortical levels of DA; while no cell loss could be evidenced, an increase of DA metabolites was observed on some occasions. 61,105 In line with the reduced DOPAC/DA ratio in the frontoparietal cortex of our aged rats, a decrease in DA or metabolite contents, or even in the turnover of DA, was described in some experiments. 28,62,81,99 In addition, part of these changes were correlated with the degree of cognitive impairments, 75,81 an observation in line with the correlation found in the present study. Similarly, cortical levels of NE were unchanged with age, a result already described in the literature. 28,75,79,97 At variance with such observations, there are studies showing that, in the cortex of aged rats, the level of NE could be increased 52,99,132,139 or decreased. 81,110 The latter assertion is also true with respect to AD patients. 105 As already mentioned for cholinergic and other markers, such contradictory issues might be explained by differences in strain, gender, age or methods used. Our correlation data, however, showed that aged rats with the most severe cognitive impairments were also those with the lowest NE level in the frontoparietal cortex. Alterations in the septohippocampal system Cholinergic projections from the MS–vDBB complex innervate the hippocampus, the entorhinal and cingulate cortices. 2 The hippocampus is also innervated by serotonergic fibers from the medial raphe nuclei, 6 noradrenergic fibers from the dorsal part of the locus coeruleus, 80 and, in a minor proportion, dopaminergic fibers from the ventral tegmental area and the substantia nigra. 55 The entorhinal cortex shelters serotonergic, noradrenergic and dopaminergic terminals arising repectively from the central and dorsal raphe nuclei, 74 the locus coeruleus 80 and the ventral tegmental area. 131 Only weak changes of cholinergic markers were found in the septohippocampal system of aged rats. In the MS, the immunoreactivity for p75 NTR was reduced in the rats showing cognitive impairments, but ACh levels were unaltered in the hippocampus and the entorhinal cortex. A decrease of the number and/or the size of cholinergic neurons of the MS and the vDBB is a common observation in aged rats, 29,35,60,73,87,124 and is often correlated with cognitive impairments. 4,46,72 However, these modifications do not seem to be reflected by a dramatic alteration of cholinergic markers in the hippocampus, 91,120,134 although, in one study on aged mice, decreased levels of ACh could be statistically linked to spatial memory deficits. 67 Preserved hippocampal and entorhinal ChAT activity was also observed in aged rats 120,126,129 and the ChAT activity failed to be correlated with the memory impairments. 5,47,52,82 However, in a few studies, high levels of presynaptic cholinergic markers were associated with better performances in aged rats. 11,40,69 The hippocampal formation plays a crucial role in spatial cognition. 44 Place learning is impaired by hippocampal 98 but also entorhinal cortex lesions. 118 Lesions of the fimbria– fornix pathways deprive the hippocampus of a significant part of its afferents (including the cholinergic ones) and produce lasting spatial learning deficits (for review, see

Learning deficits in aged rats

Ref. 22). That the damage to the cholinergic component of these pathways can, at least partly, account for the observed behavioral deficits is in line with studies using muscarinic blockade, 20,21,24 but also with experiments in which cholinomimetic drugs were found to attenuate the cognitive deficits resulting from MS or fimbria–fornix lesions. 90 Based on results obtained after highly specific lesions of cholinergic neurons in the basal forebrain, the role of the cholinergic septohippocampal fibers in spatial cognition has been somewhat qualified more recently. After intraseptal injections of the cholinergic immunotoxin 192 immunoglobulin G– saporin, impairments weaker than those described with other types of lesions were reported. 16 In some experiments, even no impairment was noticed in young rats 135 or in aged lesioned rats previously identified as good performers. 12 The latter result fits with our present data and suggests that a cholinergic deprivation of the hippocampus or the entorhinal cortex cannot alone explain the magnitude of the spatial impairments associated with aging. The interactions that the cholinergic system may exert with other neurotransmitter systems may be one of the keys towards a better understanding of age-related memory dysfunctions, and this issue clearly deserves further investigation. In both parts of the hippocampus of aged rats, 5-HT and metabolite levels were unchanged, but there was an increased 5-HIAA/5-HT ratio. It is likely that this increase reflected an enhanced neuronal activity in serotonergic afferents, as argued in previous reports. 58,59,75,81,132,139 While this enhancement was found in both hippocampal regions, the dorsal and ventral hippocampus might be dissociated on the basis of our behavioral/neurochemical correlation analysis. The 5-HT and 5-HIAA levels in the dorsal, but not those in the ventral, hippocampus were significantly correlated with the behavioral performances of the rats. Therefore, one is tempted to hypothesize that 5-HT and metabolite levels in the dorsal hippocampus may play some role in age-related spatial memory performance, as already suggested by Luine et al. 81 This role, however, is most probably indirect, as massive serotonergic depletions with 5,7-dihydroxytryptamine are usually without effect on learning and memory abilities. None the less, such lesions were shown to potentiate the cognitive alterations induced by cholinergic lesions or muscarinic blockade (for review, see Refs. 23 and 127). Therefore, it is possible that the serotonergic deficit in the hippocampus contributed to the age-related cognitive deficits by potentiating the effects of other neurotransmitter dysfunctions. A decrease in DA levels and an increase in DA turnover were found in the dorsal (but not the ventral) part of the hippocampus of aged rats, suggesting a region-specific dopaminergic dysfunction. If one excludes the report of Godefroy et al., 58 it seems that hippocampal DA levels are usually unchanged in aged rodents 75,99,132,137,139 and humans. 105 The idea that the mesohippocampal DA system could play some role in cognitive processes was evaluated with selective DA lesions in adult rats, and it was found that place learning in a water maze was dependent upon the integrity of these projections. 54 In our aged rats, we found spatial memory performance to be correlated with the DOPAC/DA ratio, an index of DA turnover. Luine et al. 81 also noticed a link between behavioral performances and DA content in the dorsal hippocampus. These observations suggest that the alterations of dopaminergic functions in the dorsal hippocampus could also contribute to the behavioral deficits related to aging.

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This is compatible with some recent observations showing that treatment with a D1/D5 agonist is able to attenuate the defect in the late phase of hippocampal long-term potentiation in aged mice, 7 and the spatial memory deficits in aged mice 7 and rats. 65 As to the aged rats, Hersi et al. 65 have discussed this drug-induced improvement of spatial memory in terms of interactions between dopaminergic and cholinergic functions in the hippocampus. Although a global decrease in NE content was observed in both hippocampal regions of aged rats, there were individual differences which correlated with the cognitive status of these rats. Aged rats with the most severe memory impairments were also those with the lowest NE content in the dorsal hippocampus. In the ventral hippocampus, the depletion was massive but failed to correlate with the behavioral performances. That the magnitude of cognitive impairment could be related to hippocampal NE levels in aged rats has been suggested by Sirvio¨ et al. 123 However, in their study and in line with other reports, hippocampal NE levels were increased with age. 122,137,139 There are also reports showing NE concentrations to be unchanged. 75,81,99,132 Whereas an explanation for the apparent discrepancies between our present and other studies could again be that of gender and strain differences, we would propose another possibility. Cholinergic denervation of the hippocampus induces growth of noradrenergic peripheral fibers into the hippocampal parenchyma, a phenomenon called sympathetic sprouting 30 and which increases the noradrenergic markers above normal levels after several weeks. 83 As cholinergic changes were found to be relatively modest and might have been insufficient to elicit sympathetic sprouting in our aged rats, it could be that the decreased NE level was the consequence of a combination of the loss of central noradrenergic neurons in the locus coeruleus and poor or virtually no compensatory sympathetic ingrowth. One may suppose that both phenomena may lead to an imbalance of hippocampal neurotransmitters and may, thereby, be detrimental to cognitive processes. That subtle changes in the interaction mechanisms between cholinergic and noradrenergic mechanisms (for review, see Ref. 32), together with the other aforementioned alterations, might account for the memory impairments in aged rats is another working hypothesis deserving particular attention in future research. CONCLUSIONS

Altogether, our present findings suggest that the cognitive deficits related to aging involve more or less simultaneous alterations in several regions of the brain (STR, septohippocampal and basalocortical systems), and not necessarily of the same neurochemical population of neurons in each of them. They also indicate that, besides cholinergic dysfunctions, alterations in various other transmitter systems can be related, at least statistically, to the level of cognitive impairment. Thus, it seems that age-related deficits can be considered the result of multitransmitter alterations in which the severity of the degeneration in the different transmitter systems and the possibility for compensatory mechanisms to slow down the emergence of functional deficits both account for the interindividual variability of cognitive function. From the present study, it also appears that these alterations occur in a rather complex way which, besides the loss of cholinergic neurons, also seems to concern changes in dopaminergic, noradrenergic

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and serotonergic mechanisms. Whether the latter are participating in the cognitive deficits directly or indirectly, or whether these systems are directly affected by aging or only undergo functional modifications in response to damage to other neurotransmitter systems, is a question that remains largely open. Accordingly, one should also mention that age-related alterations in glutamate and GABA neurotransmissions, two aspects not investigated in the present study, may be of importance in the understanding of the cognitive dysfunctions associated with aging. Finally, we would like to emphasize that the present experiment was performed on aged female rats, contrary to most earlier studies, which used male rats. This is an important issue that might account for at least

part of the discrepancies between our present findings and those on male rats. Indeed, factors such as the hormonal status (e.g., estrogens) of the animal might also account for some of the observed alterations. For instance, steroid hormones were shown to interact with several neurotransmitter systems, particularly the cholinergic ones, in memory modulation. 102 Acknowledgements—We wish to thank Mr O. Bildstein and Mr R. Paul for their technical assistance on animal care. We would like to thank Lactina S.A. (Strasbourg, France) for generous supply of powdered milk. This work was supported by a grant to J.S. from the Fondation pour la Recherche Me´dicale (no. FC000494-01), and to C.K. and J.-C.C. from the CNRS (appel d’offres Sante´ et Socie´te´; no. 98N72/ 0047).

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