Abstract

Aging is known to markedly affect the number and structural characteristics of both pre- and post-synaptic sites in the cerebral cortex. There is evidence that lamina V pyramidal neurons, and their basilar dendrites in particular, are affected by age-related decline. Furthermore, layer V is the area where the greatest overall age- related losses in the total population of synaptic boutons and of cholinergic boutons are observed. Since both pyramidal neurons and cortical cholinergic input are characteristically compromised in aging, we investigated whether aging altered the pattern of cholinergic boutons in apposition to the soma, proximal and distal basal dendrites of intracellularly labeled lamina V large pyramidal neurons in the parietal cortex of young and aged rats. We observed a significant age-related decrease in the population of both total and cholinergic boutons apposed to proximal and distal dendrites of layer V large pyramidal neurons. However, the age-related decreases of cholinergic presynaptic boutons were higher than those in the total bouton population apposed to the pyramidal neurons. The average decrease in cholinergic boutons in aged rats was 3.7-fold more pronounced than the diminution in the overall number of presynaptic boutons. Our results add important new evidence in support of the concept that the age-related learning and memory deficits are attributable, at least partially, to a decline in the functional integrity of the forebrain cholinergic systems.

Introduction

The cholinergic innervation of the cerebral cortex has been extensively investigated because of its role in arousal, learning and memory (Bartus et al., 1985; McCormick, 1989, 1992; Olton et al., 1991; Metherate et al., 1992; Voytko et al., 1994). Cholinergic neurons in the nucleus basalis magnocellularis (NBM) and associated forebrain nuclei are the major sources of the extrinsic cholinergic innervation of the cortex (Fibiger, 1982; Mesulam et al., 1983). In rodents, a small portion of the cortical cholinergic innervation is also derived from intrinsic neurons (Eckenstein and Baughman, 1983). The density of cholinergic terminals is particularly high in cortical layer V (Houser et al., 1985). The role of acetylcholine (ACh) in memory has become of interest since the learning and memory deficits of aging and Alzheimer's disease have been attributed, at least in part, to a decline in the functional integrity of the forebrain cholinergic systems (Bowen and Smith, 1976; Davies and Maloney, 1976; Whitehouse et al., 1981, 1982; Bartus et al., 1982; Coyle et al., 1983). Stronger support for a critical role of cortical ACh in age- and dementia-associated cognitive decline has been provided by studies demonstrating a correlation between decreases in markers of cortical ACh and the severity of dementia (McGeer et al., 1984; Etienne et al., 1986; Bigl et al., 1987; Sparks et al., 1992). Several studies support the hypothesis that the integrity of the basal forebrain cholinergic neurons that project to cortical areas is impaired during normal aging and in dementia. With aging, neurons in the basal forebrain undergo a process of atrophy, as judged from morphological and bio-chemical parameters (Fischer et al., 1991; Rylett and Williams, 1994). In addition, preliminary data from our laboratory showed that there is an age-related loss of cholinergic boutons in the parietal cortex with a more pronounced decline in layers V and VI (Marchese et al., 1998). Interestingly, layer V is the area where the highest age-related loss in the total population of synaptic boutons is observed (Wong et al., 1998). Since pyramidal neurons are both a prominent cell type of layer V and constitute the main cortical output (DeFelipe and Farinas, 1992), we investigated whether aging altered the pattern of cholinergic terminals apposed to the soma, proximal and distal basal dendrites of layer V large pyramidal neurons in parietal cortex I of young adult and aged Brown Norway × Fisher 344 F1 (BN×F344 F1) hybrid rats.

For these investigations, individual layer V large pyramidal neurons were intracellularly labeled with biocytin during whole cell patch-clamp recording in brain slices. Slices with only one labeled neuron were immunostained for the vesicular acetyl-choline transporter (VAChT), as a marker for cholinergic boutons. The relative number of cholinergic boutons, both synaptic and non-synaptic, apposed to the intracellularly labeled cell, per unit of length of membrane, was quantified at the electron microscopic level.

We report here that aging causes a significant decrease of both the cholinergic and total (cholinergic and non-cholinergic) bouton population apposed to the pyramidal neurons, with a marked preferential loss of cholinergic terminals. Some of the data have been presented in a preliminary communication (Casu et al., 1999).

Materials and Methods

The sequence in which procedures were applied in this study is represented in a diagrammatic fashion in Figure 1.

Animals

Twenty-two young adult (2–4 months old) and 22 aged (29–37 months old) BN×F344 F1 hybrid rats obtained from the National Institute for Aging (NIH) were used in this study. This strain has the advantage of enhanced resistance to tumors and other genetic diseases compared to its parental inbred strains (Hazzard et al., 1992; Spangler et al., 1994). Efforts were made to minimize the number of animals used and their suffering. All procedures were approved beforehand by the Animal Care Committee of McGill University and followed guidelines set down by the Canadian Institutes of Health Research.

Slice Preparation

Rats were anesthetized with pentobarbital (2.5 ml/kg) and perfused through the left ventricle with ice-cold sucrose artificial cerebrospinal fluid (S-ACSF) containing (in mM): 252 sucrose, 2.5 KCl, 2 CaCl2, 2 MgCl2, 10 glucose, 26 NaHCO3, 1.25 NaH2PO4 (Fisher, Montreal, Quebec, Canada), 5 kynurenic acid, and 1 pyruvic acid (Sigma, Oakville, Ontario, Canada; pH 7.35; 340–350 mOsm). Coronal sections (400 μm thick) from the brains were cut with a Vibratome (Lancer 1000) between coordinates bregma 0.5 mm and –3.0 mm, which comprise parietal regions I and II (Paxinos and Watson, 1986). The slices were incubated in S-ACSF for 30 min at room temperature, and subsequently transferred to a storage chamber filled with oxygenated normal ACSF at room temperature (126 mM NaCl instead of sucrose, 300–310 mOsm). After a minimum incubation of 1 h, slices were transferred to a recording chamber and perfused at ~1 ml/min with oxygenated ACSF containing 5 mM KCl at 33°C. All these steps were performed within a period of less than 3 h so as to reduce tissue damage.

Patch pipettes were pulled from borosilicate glass capillaries. Pipettes were filled with an intracellular solution composed of (in mM): 110 cesium gluconate, 5 CsCl, 10 HEPES, 2 MgCl2, 1 CaCl2, 11 BAPTA, 4 ATP, 0.4 GTP, 0.5% Lucifer Yellow, and 0.2% biocytin.

Electron Microscopic Analysis

After 10 min of recording under a whole-cell configuration to allow biocytin to diffuse inside the cell, slices were immersed in fixative containing 4% paraformaldehyde (PF) and 0.5% glutaraldehyde (GA) in 0.1 M phosphate buffer (PB) for 2 h at room temperature and then post-fixed overnight in 4% PF in PB at 4°C. The tissue was then embedded in 10% gelatin (Han et al., 1993; Buhl et al., 1994; Cobb et al., 1997), post-fixed for 1 h with 4% PF, 0.1% GA and 15% picric acid in PB, and re-sectioned into 50 μm thick sections with a Vibratome and infiltrated for 2 h in 30% sucrose and 10% glycerol. The tissue was quickly frozen in liquid nitrogen-cooled isopentane and thawed at room temperature. Subsequently, biocytin-labeled large pyramidal neurons were revealed using an avidin–biotin complex (1:1000, Vector, Burlington, Ontario, Canada). Following two washes in 0.01 M phosphate-buffered saline (PBS), pH 7.4, the tissue was incubated in 3,3′-diaminobenzidine (DAB, Sigma) with cobalt chloride and nickel ammonium sulfate, followed by the same solution with H2O2 added as described in detail elsewhere (Ribeiro-da-Silva et al., 1993). The sections containing the labeled cells were further processed for VAChT-immunoreactive (IR). An anti-VAChT serum generated in rabbit (Gilmor et al., 1996) (a gift from Dr R. H. Edwards, University of California at San Francisco) was applied. PBS was used for washing and to dilute the immunoreagents, and two PBS washes were performed between incubations. Following a long incubation in the primary antibody for 48 h at 4°C, the tissue was incubated for 2 h in a biotinylated goat anti-rabbit antibody (Vector), followed by a 2 h incubation in an ABC complex (Vector). The DAB reaction was carried out without intensification. Subsequently, the tissue was osmicated, dehydrated in ascending alcohols and propylene oxide, and finally flat-embedded in Epon. The different parts of the labeled neuron, as observed in flat-embedded slices, were photographed and drawn with a camera lucida. This procedure allowed a reconstruction of the whole morphology of each labeled neuron. We applied to each drawing of the neuronal ‘tree’ one circle centered on the cell body and with a radius of 100 μm. The dendrites located inside the circle were considered as proximal, whereas those located outside the circle represented distal dendrites. Samples of the different portions of the labeled neuron (cell body, proximal and distal dendrites) were selected and re-embedded in Epon blocks. Subsequently, 4 μm thick plastic sections were cut serially with an ultramicrotome, photographed and compared to the original drawings for identification of the parts of the labeled neuron present in each section. The selected 4 μm thick sections were then re-embedded in Epon and ultrathin sections were cut, collected onto one-slot formvar-coated grids, counterstained with uranyl acetate and lead citrate and, finally, observed with a Philips 410 electron microscope. This technique has been described in detail elsewhere (De Koninck et al., 1993; Ma et al., 1996).

The quantitative analysis was performed on five large pyramidal neurons from each age group, each originating from a different animal. The only criterion for selecting cells for detailed quantitative analysis was the quality of the ultrastructural preservation. For each cell, the number of VAChT-IR axonal varicosities and the total number of boutons apposed to the proximal and distal dendrites and to the cell body were counted on the electron microscope screen at high magnification. Subsequently, the entire electron microscopic field was photographed at low magnification (×4400) to measure the entire length of cell membrane of identified pyramidal neurons present in the field. At least five random-selected fields, each corresponding to an ultrathin section cut from a re-embedded semithin section, for each of the three parts of the cell (cell body, proximal dendritic tree, distal dendritic tree) were counted. Within each ultrathin section, all boutons – independent of their being synaptic or nonsynaptic – apposed to profiles of the cell were counted. To measure the length of the pyramidal neuron profiles present in each low-magnification electron micrograph, the negative plates were placed on a light box and the images captured into an image analysis system (MCID-M4 system; Imaging Research Inc., St Catharines, Ontario, Canada) using a black and white CCD camera. In each dendritic profile, the length of membrane corresponding to the dendritic spines was measured as part of the total profile perimeter. The densities of VAChT-IR boutons (number of VAChT-IR boutons per 100 μm of cell membrane length) and of total boutons (number of VAChT-IR and non-IR boutons per 100 μm of cell membrane length) were obtained for each labeled neuron. Although synaptic contacts were often observed, they were not always easy to recognize due to the DAB reaction product used to reveal the intracellularly labeled neurons; a side effect of this process is that the postsynaptic thickenings are often obscured. Therefore, we were not able to quantify the total number of profiles that established synapses, much less distinguish symmetric from asymmetric contacts. In consequence, quantitative results presented in this manuscript refer to number of boutons apposed to the cell profiles, independent of their being synaptic or nonsynaptic.

Statistical Analysis

Non-parametric Mann–Whitney U tests were used to compare the morphological parameters between aged and young rats. Statistical significance was set at P < 0.05. All data are expressed as mean±SEM.

Results

Neocortical neurons were obtained from a total of 22 young and 22 aged BN×F344 F1 hybrid rats. The cells were identified as large pyramidal neurons based on several morphological parameters, including a triangular-shaped cell body with dendritic structures composed of basal dendrites, oblique dendrites, and an apical dendrite (Feldman, 1984; DeFelipe and Farinas, 1992). From these neurons, five large pyramidal cells from young and five from aged rats were selected for detailed electron microscopical analysis. Each neuron was obtained from a different animal. The morphological and immunocytochemical properties of a biocytin-labeled lamina V pyramidal neuron from young and aged rats are shown, respectively, in Figures 2 and 3. As previously described, the neurons possessed a pyramidal or cone-shaped cell body, from the base of which emerged a system of large basal dendrites directed laterally and downward; and a large apical dendrite which emerged from the upper extremity of the cell body and reached layer I where it ended in a tuft of branches (Figs 2A and 3A). Laterally orientated dendrites were seen emerging from the apical dendrite (oblique dendrites). In this study, we focused on the appositions of cholinergic boutons on the cell body and basal dendrites. At the ultrastructural level, we detected an absence of appositions of VAChT-IR boutons on the cell body of both young and aged animals (Figs 2D and 3D). Appositions of VAChT-IR boutons were observed on the shaft of proximal and distal basal dendrites (Figs 2E,F and 3E,F), but not on dendritic spines. Their incidence was apparently lower in the aged than in young animals.

Quantitative Analysis

In these investigations, the density of axonal varicosities (boutons) represents the number of boutons observed in direct contact (apposition) to the cell membrane per 100 μm of membrane length (including dendritic spine membrane length). The values of the densities of total boutons (number of VAChT-positive and negative boutons per 100 μm membrane length) apposed to the pyramidal neurons from young rats were higher in the proximal and distal dendrites than in the cell body. Substantially significant age-related losses in the density of total boutons apposed to cell body, proximal and distal dendrites of the pyramidal neurons were found (Fig. 4). In young rats, the densities of VAChT-IR boutons contacting the neuron were not significantly different when comparing proximal and distal dendrites, but no cholinergic appositions on the cell body were observed. In the pyramidal neurons of aged rats, a marked loss of cholinergic varicosities contacting the proximal and distal dendrites was detected; however, this decrease was more noticeable in distal dendrites (Fig. 5). We further calculated the relative number of cholinergic boutons apposed to pyramidal neurons with respect to the total bouton population contacting the same cells. This analysis revealed a selective decrease of the cholinergic population (Fig. 6). In aged rats, the decrease of cholinergic boutons apposed to proximal and distal dendrites of pyramidal neurons was, respectively, 2.21 and 5.17 folds higher than the diminution in the overall number (independently of their neurotransmitter nature) of boutons apposed to the same regions of the cells (Table 1). The above values corresponded to an average decrease of cholinergic boutons that was 3.7-fold higher than the overall decrease of bouton appositions on the dendritic tree of pyramidal neurons.

Discussion

The present results revealed an age-related decrease in the population of both cholinergic and total (cholinergic + non-cholinergic) boutons in apposition to the soma, proximal and distal basal dendrites of large layer V pyramidal neurons. The detected overall decrease in boutons apposed to the labeled neurons supports several previous studies showing an age-related decline in the total number of cortical pre-synaptic boutons (Adams and Jones, 1982; Markus and Petit, 1987; Wong et al., 1998). Previous studies from our laboratory have provided evidence that this synaptic attrition is accompanied by an overall behavioral impairment in the BN×F344 F1 hybrid rat strain (Wong et al., 1998). It should be stressed that, although we observed an age-related decrease in the total number of boutons apposed to layer V pyramidal neurons, the loss of the cholinergic subpopulation was notably higher. The average decrease in cholinergic boutons in aged rats was several-fold more pronounced than the diminution in the overall bouton numbers, thus providing additional evidence that aging preferentially affects the functional integrity of forebrain cholinergic systems.

In these investigations we used an intracellular labeling approach in which only one neuron was stained per brain slice so as to avoid the overlap of dendrites from other labeled neurons that occurs when multiple neurons are stained in, for example, the Golgi method. The present technique gives a better estimate of dendritic parameters than is possible to obtain with these other approaches and allows the study of the transmitter-specific innervation patterns of individual neurons. Thus, we investigated the precise patterns of cholinergic innervation by terminals contacting the soma, proximal and distal basal dendrites of lamina V pyramidal neurons of the neocortex (parietal). This has been possible by carefully optimizing the experimental conditions for the brain slice preparations, including perfusing the animals with an ice-cold solution as described in Materials and Methods. Furthermore, a pre-incubation medium known to reduce NMDA receptor activation, free radical formation and cell swelling was applied (Aitken et al., 1995). This approach has proven to be very satisfactory, as the inter-animal variation in quantitative analysis is very small, as shown by the small size of the error bars in the graphs (Figs 4–6).

Our study was focused on layer V because this is where the large pyramidal neurons are located and because it possesses a high density of cholinergic terminals (Houser et al., 1985). Furthermore, changes in deeper cortical layers appear prominent in the aging process (Wong et al., 1998). Thus, it has been shown that there is an age-related decline in the total number of cortical pre-synaptic boutons in the cerebral cortex (Adams and Jones, 1982; Markus and Petit, 1987) and that the superficial cortical laminae (lamina I to III) are relatively resistant to the age-related changes in presynaptic varicosity numbers when compared with cortical laminae IV to VI (Wong et al., 1998). In addition, preliminary data from our laboratory provided evidence that the cholinergic innervation of the cerebral cortex was considerably more sensitive to the aging process than the overall cortical innervation. Thus, at the light microscopical level, a profound depletion of cholinergic boutons was detected in relation to overall innervation which was more pronounced in deeper cortical laminae (Marchese et al., 1998). Similarly, studies have shown changes with aging in postsynaptic structures. In fact, studies in primates, including human, have shown that layer V pyramidal neurons exhibit an age-related atrophy of the dendritic structures (Cupp and Uemura, 1980; Nakamura et al., 1985; Jacobs et al., 1997; de Brabander et al., 1998). Data from our laboratory obtained in rat have revealed that changes were particularly marked in the dendrites located in layers V and VI (Wong et al., 2000). We have found that in the deeper layers there was a decrease in the number of basal dendritic branches, shortening of the total length of basal dendrites, decrease in cell body size, and loss of dendritic spines whereas there was no significant decrease in dendritic length and branching in the oblique and tuft regions of the same pyramidal neuron that reached layers I and II.

Interestingly, no cholinergic appositions on the cell body were found in either young or aged rats. Previous immuno-cytochemical studies indicate that the vast majority of synapses on pyramidal cell somata (90–95%) are formed by GABAergic terminals (Hendry et al., 1983; Farinas and DeFelipe, 1991), some of them co-localizing neuropeptides. In fact, CCK (Freund et al., 1986), somatostatin (de Lima and Morrison, 1989) and tachykinins (DeFelipe et al., 1990) were identified in boutons apposed to the cell body of pyramidal cells. Most axosomatic synapses are assumed to be inhibitory, diminishing the depolarizing inputs arriving from the cell dendrites (Jack et al., 1975). It should be stressed that ACh has been shown to have an excitatory effect on cortical neurons in layers V and VI (Krnjevic and Phillis, 1963; Crawford, 1970) and, in consequence, it is not surprising that we found in this study a lack of cholinergic axosomatic appositions on pyramidal neurons. Our observations are compatible with previous reports demonstrating a very low frequency of cholinergic appositions on cell bodies in the cerebral cortex of rats (Beaulieu and Somogyi, 1991) and primates (Mrzljak et al., 1995). As the cell type in these studies have not been characterized, it is possible that the cell bodies reputed as receiving cholinergic terminals were non-pyramidal neurons.

The profound age-related decreases in the number of cholinergic appositions on pyramidal neurons detected in this study (see Figs 5 and 6) are in line with previous studies (Landry et al., 1984; Biegon et al., 1986; Altavista et al., 1990; Fischer et al., 1991) showing that cell bodies from the cholinergic basal forebrain neurons projecting to the cortex are particularly vulnerable to aging. Since cell bodies of NBM neurons experience an age-related atrophy, it is likely that a retraction of the cholinergic axonal processes in the cerebral cortex also takes place. As the dendrites of pyramidal neurons also undergo atrophy (Leuba, 1983; de Brabander et al., 1998; Wong et al., 2000), there will be a concomitant reduction in available pyramidal dendrite membrane for the cholinergic boutons to establish synapses. The combined reduction of the pre- and post-synaptic structures would result in a significant reduction in cholinergic transmission. In line with this, studies using microdialysis have shown that the output of endogenous ACh from the cerebral cortex is significantly reduced in aged rats (Wu et al., 1988). It could be argued that the decreased ACh release might simply be the consequence of a reduction in ACh synthesis. However, estimations on the enzymatic activity of choline acetyltransferase (ChAT) in aged rats are somewhat ambiguous. Thus little (McGeer et al., 1971; Lai et al., 1981) or no changes in ChAT activity were found in cortical areas (Bartus et al., 1982; Luine and Hearns, 1990). Therefore, the reduction in the number of cortical cholinergic boutons in aged animals, as observed in this study, is the most likely substrate for the diminished release of endogenous ACh reported in aging.

Regarding the correlative expression of cholinergic receptors, it is well known that layer V pyramidal neurons are immuno-reactive for both muscarinic (Van der Zee and Luiten, 1999) and nicotinic receptors (Bravo and Karten, 1992). In aging, a loss of nicotinic receptor immunoreactivity has been observed in the human neocortex (Schröder et al., 1991) and a decrease in the mRNA of nicotinic receptor sub-units α4-1 and α5 has been detected in the cerebral cortex of rats (Birtsch et al., 1997). Although there is strong evidence for an age-related nicotinic receptor loss, the data for muscarinic receptors in the rat neocortex is still controversial. Some studies report no changes in the number of muscarinic binding sites and others a decrease or even an increase [for review see (Decker, 1987; Van der Zee and Luiten, 1999)]. These findings suggest that aging could preferentially affect the cortical cholinergic innervation rather than the cholinergic receptor expression.

There is evidence that synaptic density is related to cognitive function (Eastwood et al., 1994). For instance, acquisition of cognitive tasks corresponds to an increase in the number of synapses in the motor cortex (Kleim et al., 1996) and, conversely, a reduction in synaptic density in the frontal cortex in Alzheimer's disease is correlated with cognitive decline (DeKosky and Scheff, 1990; Terry et al., 1991). Therefore, it is reasonable to assume that the decline in cognitive function in aging is related to the diminution in cortical synaptic number in general, and in particular to the decline in cholinergic synapses. In this regard, it is interesting to note that in rats bearing lesions of the NBM, the cortical implantation of genetically modified cells that produce ACh improves performances in the Morris water maze task (Winkler et al., 1995). Furthermore, in aged animals, the application of nerve growth factor (NGF) can revert the loss of cholinergic markers and lead to behavioral impairments (Fischer et al., 1987). Moreover, in rats bearing cortical stroke-type lesions, the infusion of NGF results in de novo cortical cholinergic synaptogenesis concomitant with the retention of acquired behaviors (Garofalo et al., 1992; Garofalo and Cuello, 1994). The above experimental data supports a crucial role for cortical ACh in cognitive functions and its involvement in age-related cognitive decline. Furthermore, age-impaired rats display atrophy in forebrain cholinergic bodies, and both the behavioral impairment and the cholinergic atrophy are reduced following NGF treatment (Fischer et al., 1987).

In conclusion, our results show an age-related preferential loss of cholinergic boutons in apposition to neocortical large pyramidal neurons. This new evidence supports the concept that the diminution in the learning and memory capabilities in aging and dementia could be attributed, at least partially, to a decline in the integrity of the forebrain cholinergic innervation.

Notes

This study was supported by grants from the Canadian Institutes of Health Research to A.C.C. and National Institute of Neurological Disorders and Stroke to Y. De K. The authors would also like to acknowledge a grant on ‘Structural/Functional Modeling and Imaging’ received from SmithKline Beecham (Canada). The authors thank Dr R. H. Edwards (UCSF) for the generous gift of anti-VAChT antibodies, and Drs P. Somogyi and P. Bolam (Oxford) for valuable technical suggestions. In addition, they would like to thank Marie Ballak for expert technical assistance in the EM studies, Alan Foster for photographic expertise, and Sid Parkinson for editorial assistance. Y. De K. is a Scholar of the Canadian Institutes of Health Research. T.P.W. was a recipient of a Doctoral Award from the Alzheimer Society of Canada.

Table 1

Age-related decline of the densities of total boutons (cholinergic + non-cholinergic) and cholinergic boutons apposed to the different parts of pyramidal neurons (in percentages)

 Decline of density of total boutons (%) Decline of density of VAChT-IR boutons (%) Ratio of the decrease (VAChT-IR/total boutons) 
Note the marked decrease of both total boutons and cholinergic boutons. The percentage decrease in the density of cholinergic boutons was higher in distal dendrites of characterized pyramidal neurons. 
Cell body 38 N/A N/A 
Proximal dendrites 28 62 2.21 
Distal dendrites 18 93 5.17 
 Decline of density of total boutons (%) Decline of density of VAChT-IR boutons (%) Ratio of the decrease (VAChT-IR/total boutons) 
Note the marked decrease of both total boutons and cholinergic boutons. The percentage decrease in the density of cholinergic boutons was higher in distal dendrites of characterized pyramidal neurons. 
Cell body 38 N/A N/A 
Proximal dendrites 28 62 2.21 
Distal dendrites 18 93 5.17 
Figure 1.

Diagrammatic representation of the protocol for ultrastructural study of intracellularly labeled layer V pyramidal neurons. See Materials and Methods for details.

Figure 1.

Diagrammatic representation of the protocol for ultrastructural study of intracellularly labeled layer V pyramidal neurons. See Materials and Methods for details.

Figure 2.

Morphological and immunocytochemical properties of a biocytin-labeled lamina V pyramidal neuron from a young rat.(A) Camera lucida reconstruction of the cell. (B) Micrograph of part of the cell, obtained from a 50 μm thick plastic section. (C) Micrograph from a 4 μm thick plastic section obtained after Epon re-embedding of the 50 μm thick section from (B). (D) Electron micrograph of the cell body area obtained after further re-embedding of the 4 μm thick section shown in (C). Note the absence of VAChT-IR boutons apposed to the cell body and elsewhere in the surrounding neuropile. (E, F) Electron micrographs of distal and proximal dendrites, respectively; note the VAChT-IR boutons apposed to the dendrites (arrows). Scale bars in (A), (B) and (C) = 50 μm; (D) = 5 μm; (E) and (F) = 1 μm.

Figure 2.

Morphological and immunocytochemical properties of a biocytin-labeled lamina V pyramidal neuron from a young rat.(A) Camera lucida reconstruction of the cell. (B) Micrograph of part of the cell, obtained from a 50 μm thick plastic section. (C) Micrograph from a 4 μm thick plastic section obtained after Epon re-embedding of the 50 μm thick section from (B). (D) Electron micrograph of the cell body area obtained after further re-embedding of the 4 μm thick section shown in (C). Note the absence of VAChT-IR boutons apposed to the cell body and elsewhere in the surrounding neuropile. (E, F) Electron micrographs of distal and proximal dendrites, respectively; note the VAChT-IR boutons apposed to the dendrites (arrows). Scale bars in (A), (B) and (C) = 50 μm; (D) = 5 μm; (E) and (F) = 1 μm.

Figure 3.

Morphological and immunocytochemical properties of a biocytin-labeled lamina V pyramidal neuron from an aged rat.(A) Camera lucida reconstruction of the cell. (B) Micrograph of part of the cell, obtained from a 50 μm thick plastic section. (C) Micrograph from a 4 μm thick plastic section obtained after Epon re-embedding of the 50 μm thick section from B. (D) Electron micrograph of the cell body area obtained after further re-embedding of the 4 μm thick section shown in (C). Note the absence of VAChT-IR boutons apposed to the cell body and elsewhere in the surrounding neuropile. (E, F) Electron micrographs of proximal and distal dendrites, respectively; note the VAChT-IR boutons apposed to the dendrites (arrows). Scale bars in (A), (B) and (C) = 50 μm; (D) = 5 μm; (E) and (F) = 1 μm.

Figure 3.

Morphological and immunocytochemical properties of a biocytin-labeled lamina V pyramidal neuron from an aged rat.(A) Camera lucida reconstruction of the cell. (B) Micrograph of part of the cell, obtained from a 50 μm thick plastic section. (C) Micrograph from a 4 μm thick plastic section obtained after Epon re-embedding of the 50 μm thick section from B. (D) Electron micrograph of the cell body area obtained after further re-embedding of the 4 μm thick section shown in (C). Note the absence of VAChT-IR boutons apposed to the cell body and elsewhere in the surrounding neuropile. (E, F) Electron micrographs of proximal and distal dendrites, respectively; note the VAChT-IR boutons apposed to the dendrites (arrows). Scale bars in (A), (B) and (C) = 50 μm; (D) = 5 μm; (E) and (F) = 1 μm.

Figure 4.

Densities of total boutons (number of VAChT-positive and negative boutons/100 μm membrane length) apposed to the pyramidal neurons from young and aged rats in the proximal and distal dendrites, and in the cell body. A significant age-related loss in the densities of total boutons apposed to the pyramidal neurons was found. *P < 0.05, **P < 0.01.

Figure 4.

Densities of total boutons (number of VAChT-positive and negative boutons/100 μm membrane length) apposed to the pyramidal neurons from young and aged rats in the proximal and distal dendrites, and in the cell body. A significant age-related loss in the densities of total boutons apposed to the pyramidal neurons was found. *P < 0.05, **P < 0.01.

Figure 5.

Densities of VAChT-IR boutons apposed to the pyramidal neurons from young and aged rats. The densities of VAChT-IR boutons were not significantly different between proximal and distal dendrites, but no cholinergic appositions on the cell body were found in young rats. Note the marked loss of cholinergic varicosities contacting the proximal and distal dendrites of pyramidal neurons of aged rats. **P < 0.01.

Figure 5.

Densities of VAChT-IR boutons apposed to the pyramidal neurons from young and aged rats. The densities of VAChT-IR boutons were not significantly different between proximal and distal dendrites, but no cholinergic appositions on the cell body were found in young rats. Note the marked loss of cholinergic varicosities contacting the proximal and distal dendrites of pyramidal neurons of aged rats. **P < 0.01.

Figure 6.

Percentage of the total number of boutons apposed to pyramidal neurons expressing VAChT immunoreactivity. In neurons from young rats, 7% of the total number of bouton appositions were from cholinergic boutons. Note that in the aged rats the proportional loss of cholinergic appositions was very pronounced (down to less than 3% of the total number of appositions). **P < 0.01.

Figure 6.

Percentage of the total number of boutons apposed to pyramidal neurons expressing VAChT immunoreactivity. In neurons from young rats, 7% of the total number of bouton appositions were from cholinergic boutons. Note that in the aged rats the proportional loss of cholinergic appositions was very pronounced (down to less than 3% of the total number of appositions). **P < 0.01.

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