1 Departments of Pharmacology & Therapeutics and , 2 Anatomy & Cell Biology, McGill University, Montreal, Quebec, Canada, H3G 1Y6 and , 3 Neurobiologie Cellulaire Centre de recherche, Université LavalRobert Giffard, Quebec, Quebec, Canada, G1J 2G3
Dr A. Claudio Cuello, Departments of Pharmacology & Therapeutics, McGill University, 3655 Promenade Sir William Osler, Montreal, Quebec H3G 1Y6, Canada. Email: accuello{at}pharma.mcgill.ca.
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Abstract |
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Introduction |
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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).
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Materials and Methods |
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Twenty-two young adult (24 months old) and 22 aged (2937 months old) BNxF344 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; 340350 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, 300310 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 avidinbiotin 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 (x4400) 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 MannWhitney 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.
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Results |
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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.
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Discussion |
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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 46
).
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 (9095%) 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
4-1 and
5 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.
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Notes |
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References |
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