Frontiers in Systems Neuroscience (Feb 2014)
Premature aging phenotype in mice lacking high affinity nicotinic receptors: region specific changes in layer V pyramidal cell morphology
Abstract
The mechanisms by which aging leads to alterations in brain structure and cognitive deficits are unclear. A central yet presently unresolved issue in aging research concerns the distinction between normal/successful aging, consisting of a moderate decline in cognitive performance, and pathological aging, manifested as mild cognitive impairment or full-blown neurodegeneration and dementia. In particular, it has been proposed that the age-related decline in cognitive abilities may be an age-related escalation of early-life cognitive limitations, rather than an abruptly emerging neuropathological process that occurs in old age (Elias et al., 2000; Small et al., 2000; Sarter and Bruno, 2004; Amieva et al., 2005; Tyas et al., 2007). In this scenario, early abnormalities or incompletely matured neural systems would interact with age-related processes to explain the cognitive decline in later ages. However this proposal remains controversial (Nilsson et al., 2009; Salthouse, 2009) and, to our knowledge, has not been explored at the morphological/structural level. Hence it is important to identify factors that may confer a predisposition to pathological aging and examine how they interact with the process of aging per se. One such factor is the integrity of the cholinergic system: cholinergic basal forebrain neurons and their projections to the cortex show increased vulnerability to aging (Fischer et al., 1987; Altavista et al., 1990; Casu et al., 2002) and cognitive decline is associated with selective loss of neuronal nicotinic acetylcholine receptor (nAChR) function (Hellstrom-Lindahl and Court, 2000; Schliebs and Arendt, 2011). In this respect, animals with specific cholinergic deficits are important tools for understanding the neurobiology of successful aging. One such animal model is the β2-/- mouse, in which the gene encoding the β2 subunit of the nAChR is genetically deleted (Picciotto et al., 1995). Aged β2-/- mice have been proposed as a model of accelerated cognitive aging, based on structural alterations and spatial learning deficits only evident in old animals (Zoli et al., 1999; Picciotto and Zoli, 2002). However a systematic comparison of neuronal microanatomy in adult and aged animals has not been done to date. In the present study adult (4-6months) and old (22-24months) WT and β2-/- animals were used to examine the respective contributions of age and genotype on neuronal structure. We focus on layer V pyramidal cells because: (i) they constitute the main cortical output (DeFelipe and Farinas, 1992; Romand et al., 2011)) (ii) they are often reported to exhibit increased sensitivity to aging (Nakamura et al., 1985; Baskys et al., 1990; De Brabander et al., 1998; Turner et al., 2005); (iii) they possess a high density of cholinergic terminals (Houser et al., 1985) and, in contrast to layer III cells, they exhibit strong presynaptic modulation by β2 containing nAChRs and are activated by nAChR stimulation (Poorthuis et al., 2013); hence they would be a sensitive readout for the lack of high affinity nicotinic receptors. Furthermore, to examine the degree of age-related vulnerability across distinct cortical areas we used YFP-H mice that express yellow fluorescent protein (YFP) in specific populations of thick-tufted layer V pyramidal neurons across the cortical mantle (Feng et al., 2000; Sugino et al., 2006). We used mutants crossed with YFP+ mice in order to have the same labeled populations in both genotypes, and we examined cells in primary visual cortex (V1) and anterior cingulate cortex (ACC), two cortical regions that receive similar cholinergic inputs (McKinney et al., 1983; Jacobowitz and Creed, 1983; Everitt and Robbins, 1997; Laplante et al., 2005) but have distinct cytoarchitecture and functional role (Elston et al., 2005). We ask whether neurons in old β2-/- mice exhibit greater structural deficits than aged-matched controls and whether deficits appear in old age or are already present earlier. Brains from 21 adult (4-6 months) and old (22-24 months) C57Bl6J wildtype and β2-/- mice were used in this study (WT adult n=5; WT old n=5; β2-/- adult n=5; β2-/- old n=6). Each brain was fixed overnight in 4% paraformaldehyde and subsequently transferred to saline solution until ready for use. The two hemispheres were separated and embedded in 3% agarose. Sections (100µm) were cut using a vibratome from V1 (both sagittal and coronal sections) and ACC (coronal sections). Complete neurons were defined as YFP+ pyramidal cells whose soma, basal dendrites, apical dendrite with its branches and apical tuft could be clearly distinguished under the fluorescence microscope. Argon laser (488 nm) was used for YFP excitation, and Z-series of images were captured by Leica Confocal Software (LAS AF). Measurements were restricted to parameters that could be fully visualized and quantified within the section thickness in order to avoid errors associated with distal dendrites that would be absent due to the cutting procedure, thereby biasing our sample in favour of smaller dendritic trees. Hence, measurements were taken for the following 12 parameters, with respect to the three neuronal compartments: (i) soma: volume (in 3 dimensions, for optimal estimation of absolute size; horizontal and vertical Feret diameter which gives additional information on the shape of the cell body); (ii) apical dendrite: length of dendrite from soma to the apical tuft bifurcation, number of apical branches, diameter (measured at a distance of 60µm from the soma), vertical and horizontal coverage of apical tuft; (iii) basal dendrites: number of primary dendrites, diameter of all primary dendrites (measurements taken at 2 µm from the soma), length of all primary dendrites (up to first bifurcation point), number of all secondary dendrites. More distal dendritic segments could not be measured reliably in all cells and were excluded from analysis. Several additional parameters were calculated from these measurements: “tuft area” was estimated by multiplying the vertical and the horizontal dimensions of the apical tuft to indicate the extent of its areal coverage and “cell body elongation” was calculated as the horizontal divided by the vertical diameter of the cell body. Cortical thickness was measured as the distance between the pia and ventral border of layer 6 in all sections from which cells were imaged. Our data revealed substantial morphological differences between YFP+ cells of the ACC and V1, in both genotypes, implying different synaptic integration properties and functional role for cells in the two cortical areas. We found an increased susceptibility to aging in cells located in ACC, a region associated with higher cognitive functions. In addition, we found that the lack of the β2 subunit is associated with an appearance of premature aging in layer V pyramidal cells, which is preferentially expressed in ACC. In morphological terms, ACC neurons already look ‘old’ at 4-6 months, whereas V1 cells are minimally affected. Interestingly, the same parameters affected by the mutation are also the ones most prominently affected by aging in normal animals, suggesting possible common underlying mechanisms. In contrast, V1 cells are less affected by aging in WT animals and the impact of the mutation is only apparent in aged individuals. To our knowledge this is the first study that examines the combined effects of aging and genetic predisposition on neuronal subpopulations with distinct areal identities and connectivity patterns but same layer identity and comparable intrinsic properties, thereby allowing an examination of their respective contributions to the aging process. We have shown that high-affinity nicotinic signaling plays a region-specific role both on morphogenesis and/or maintenance of identified layer V pyramidal neurons, as well as on the process of aging per se, by promoting or enhancing the age-related decline in neuronal structure. Hence, the phenotype of YFP+ cells in aged β2-/- mice is the result of the interaction of aging with the absence of high affinity nAChRs over a relatively prolonged period of life span. This, in turn, may contribute to structural degeneration especially of the circuits that participate in high order functional connectivity of the cerebral cortex. Thus we propose that β2-/- mice can serve as an appropriate animal model with which to study the factors that confer cell-type specific vulnerability to aging and provide a useful tool with which to examine the possible interventions that could restore successful cognitive aging.
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