Elsevier

Neurobiology of Aging

Volume 33, Issue 7, July 2012, Pages 1466-1480
Neurobiology of Aging

Regular paper
Correlation of cognitive performance and morphological changes in neocortical pyramidal neurons in aging

https://doi.org/10.1016/j.neurobiolaging.2010.10.011Get rights and content

Abstract

It is well established that the cerebral cortex undergoes extensive remodeling in aging. In this study, we used behaviorally characterized rats to correlate age-related morphological changes with cognitive impairment. For this, young and aged animals were tested in the Morris water maze to evaluate their cognitive performance. Following behavioral characterization, the animals were perfused and a combination of intracellular labeling and immunohistochemistry was applied. Using this approach, we characterized the dendritic morphology of cortical pyramidal neurons as well as the pattern of glutamatergic and GABAergic appositions on their cell bodies and dendrites. We focused on the association region of the parietal cortex (LtPA) and the medial prefrontal cortex (mPFC) for their involvement in the Morris water maze task. We found an age-related atrophy of distal basal dendrites that did not differ between aged cognitively unimpaired (AU) and aged cognitively impaired animals (AI). Dendritic spines and glutamatergic appositions generally decreased from young to AU and from AU to AI rats. On the other hand, GABAergic appositions only showed a trend towards a decrease in AU rats. Collectively, the data show that the ratio of excitatory/inhibitory inputs was only altered in AI animals. When cortical cholinergic varicosities were labeled on alternate sections, we found that AI animals also had a significant reduction of cortical cholinergic boutons compared with AU or young animals. In aged animals, the density of cortical cholinergic varicosities correlated with the excitatory/inhibitory ratio. Our data suggest that both cholinergic atrophy and an imbalance towards inhibition may contribute to the observed age-associated behavioral impairment.

Introduction

The cerebral cortex has long been known to be involved in cognitive processing. There is extensive evidence showing that a fine balance between excitatory and inhibitory currents is crucial for information processing in the cerebral cortex (Hausser and Clark, 1997, Miura et al., 2007, Le et al., 2008). This excitatory and inhibitory synaptic transmission is altered by numerous neuromodulators (e.g., acetylcholine) that have been shown to be important for proper cognitive function (Sarter and Bruno, 1997, Gu, 2002). Normal aging has detrimental effects on cognition and is associated with cortical remodeling (for review see Morrison and Hof, 2002, Dickstein et al., 2007). Contrary to what was believed previously, normal aging is not associated with extensive cell loss in the rat (Pugnaloni et al., 1998, Merrill et al., 2001) or human (Terry et al., 1987, Haug and Eggers, 1991) cortex. However there is abundant evidence in the literature showing neuronal atrophy and synaptic loss in humans, primates and rodents (Wong et al., 2000, Jacobs et al., 2001; Uylings and de Brabander, 2002, Duan et al., 2003). In experimental animals, a widely used and accepted paradigm to evaluate cognitive function is the Morris water maze (MWM) which assesses spatial learning and memory (Morris, 1984, Brandeis et al., 1989, McNamara and Skelton, 1993). Lesion studies have demonstrated that the parietal and prefrontal cortices play a crucial role in spatial memory formation (Sutherland et al., 1988, Kolb et al., 1997, Ragozzino et al., 1998, Gottlieb, 2002). As the water maze task depends on the function of both the hippocampus and the cerebral cortex, the systems consolidation model of memory formation states that memories are first encoded by the hippocampus and then consolidated in the cerebral cortex (Maviel et al., 2004). Consistently, lesions or inactivation of the medial prefrontal cortex (mPFC) impair memory consolidation in the hours following a single day of water maze training (Kraemer et al., 1996, Leon et al., 2010). As well, lesions affecting the parietal cortex significantly impair the retention of the platform location following training (Elliott et al., 1989). Aged rats show altered performances in the MWM, accompanied by an age-related atrophy of pyramidal neurons as well as synaptic loss in the above-mentioned cortical regions (Wong et al., 1998a, Wong et al., 1998b, Markham and Juraska, 2002, Majdi et al., 2007). By means of whole cell patch clamp recordings on acute cortical slices from behaviorally characterized young and aged animals, it has recently been shown that pyramidal neurons from lamina V of the parietal cortex of aged cognitively impaired animals had an altered ratio of mEPSC and mIPSC (Wong et al., 2006a). More precisely, aged cognitively unimpaired rats had a decrease in the frequency of both excitatory and inhibitory mPSC. On the other hand, aged cognitively impaired rats had decreased mEPSCs while the frequency of mIPSCs was comparable to that of young animals. According to quantal analysis, since the frequencies were altered but the amplitudes of the currents were unchanged, the data suggest alterations in synaptic numbers as opposed to changes in receptor levels. Alternatively, differences in the average release probability of apposing presynaptic boutons could also lead to the observed physiological changes. A detailed morphological analysis of excitatory and inhibitory appositions on pyramidal neurons can provide valuable information regarding the mechanisms driving the imbalance towards inhibition. In our laboratory, we have been using an approach that allows the simultaneous visualization of dendrites from intracellularly-labeled cortical pyramidal neurons and the appositions on them from presynaptic boutons of specific neurotransmitter systems. By exploiting this combination of immunocytochemistry and intracellular fillings on fixed tissue, we were able to acquire morphological data from a higher number of animals in many cortical regions. Our objective was to quantify the age-related dendritic atrophy and to verify if it correlated with cognitive loss. Moreover, we investigated whether there was a change in the excitatory and inhibitory appositions on morphologically characterized cortical pyramidal neurons that would explain the previously described imbalance towards inhibition. The advantage to our approach is that we can sacrifice all the animals at the same time following behavioral characterization and, as the tissue is fixed, we have sufficient time to analyze morphological parameters from many neurons across cortical regions. We were mainly interested in the association region of the parietal cortex (LtPA) and the mPFC as both structures have been shown to be involved in the cognitive processing associated with performing the MWM (Sutherland et al., 1982, Kolb et al., 1997) or during memory consolidation and recall (Leon et al., 2010). We were specifically interested in the morphological and synaptic analysis of cortical pyramidal neurons since they are the main output system of the cortex (Ramón y Cajal, 1909, Rockland and Pandya, 1979) and their age-related morphological and physiological changes are better documented than interneurons. We also wanted to discriminate between pyramidal neurons from lamina V and lamina III as their circuitry and connectivity are different (Callaway, 2002, Bannister, 2005). Furthermore, since different dendritic compartments have a different physiology and receive different inputs (Thomson and Deuchars, 1997, Berghuis et al., 2004a, Bannister, 2005), we compared synaptic densities on proximal and distal basal dendrites as well as on proximal and distal apical dendrites. For example, a combination of physiological recordings, intracellular labeling and immunocytochemistry has demonstrated that the perisomatic region of pyramidal neurons has the highest density of inhibitory appositions and that most of those synapses are made by fast spiking, paravalbumin positive interneurons. Conversely, inhibitory synapses made on more distal dendrites generally come from adapting, cholecystokinin positive interneurons (Berghuis et al., 2004a).

Section snippets

Methods

Twelve young (6 months old) and 93 aged (24 months old) animals were used for the behavioral characterization. All animals were male Fischer 344 rats obtained from the National Institute for Aging (NIH). For this study, brains from 6 young and 12 aged animals were used. All procedures were approved by the McGill University Animal Care Committee and followed the guidelines of the Canadian Council on Animal Care and of the NIH.

Pyramidal cell morphology

The quantification of total dendritic lengths for each branching order revealed that aging is accompanied by a preferential reduction of the most distal dendritic compartments (Fig. 1, Fig. 2). However, of the cortical regions analyzed, these changes reached statistical significance only in lamina V of the LtPA (Fig. 2A) and lamina III of the mPFC (Fig. 2B). More precisely, there was a statistically significant reduction in the total lengths of quaternary order dendrites in both AU and AI rats

Discussion

The present study investigated for the first time the changes in immunocytochemically characterized input to intracellularly labeled neocortical pyramidal neurons in aged-impaired compared with aged-unimpaired and young rats. Our findings show a change in the ratio of inputs favoring inhibition in specific layers of 2 cortical areas.

Conclusions

Using a combination of cognitive screening, intracellular filling and immunocytochemistry, we were able to measure a cholinergic loss accompanied by a decrease in the ratio of glutamatergic/GABAergic appositions on distal basal dendrites from cortical pyramidal neurons. Both of these changes correlated with poorer cognitive performance in aged rats. Regression analysis revealed a positive correlation between the density of cortical cholinergic varicosities and the ratio of E/I appositions in

Disclosure statement

The authors do not have financial interests or other situations which would incur a conflict of interest with this study.

Acknowledgments

This work was supported by the National Institutes of Health grant R01AG020529 (ARS and ACC) and the Canadian Institutes of Health Research grants MOP-79411 to ARS and MOP-62735 to ACC. ACC is the holder of the McGill University Charles E. Frosst Merck Chair of Pharmacology. ACC is grateful for the support from Alan Frosst and the Frosst family. We would also like to thank Javier DeFelipe for sharing some of his expertise regarding intracellular injections. We are grateful to Manon St-Louis,

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