- Email: [email protected]
Morphological alterations in neurons forming corticocortical projections in the neocortex of aged Patas monkeys
Neuroscience Letters 317 (2002) 37–41 www.elsevier.com/locate/neulet
Morphological alterations in neurons forming corticocortical projections in the neocortex of aged Patas monkeys Tanya L. Page a, Michael Einstein a,b, Huiling Duan a,b,c, Yong He a,c, Tony Flores a, Daniil Rolshud a,b, Joseph M. Erwin d,e, Susan L. Wearne b,c,f, John H. Morrison a,b,c, Patrick R. Hof a,b,c,e,* a
Kastor Neurobiology of Aging Laboratories, Box 1639, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA b Computational Neurobiology and Imaging Center, Mount Sinai School of Medicine, New York, NY, USA c Fishberg Research Center for Neurobiology, Mount Sinai School of Medicine, New York, NY, USA d Division of Neurobiology, Behavior and Genetics, Bioqual Inc., Rockville, MD, USA e Foundation for Comparative and Conservation Biology, Rockville, MD, USA f Department of Biomathematical Sciences, Mount Sinai School of Medicine, New York, NY, USA Received 30 July 2001; received in revised form 17 September 2001; accepted 26 September 2001
Abstract Recent studies indicate that the cognitive processes mediated by the prefrontal cortex, such as working memory, are impaired during normal aging. These disturbances in cortical function may be a consequence of abnormalities in neocortical circuits, even though the numbers of cortical neurons are preserved in normal aging. We performed retrograde tract-tracing of cortical projections connecting the temporal cortex to the prefrontal cortex in combination with dye-filling and three-dimensional neuronal reconstructions in aged patas monkeys. Age-related changes affected the apparent complexity of the apical dendrites of projection neurons and caused a significant loss of dendritic spines at all levels of their dendritic trees. These results indicate that normal aging is accompanied by neuronal changes that are quite subtle, and possibly involves discrete cellular components of certain cortical neurons selectively rather than inducing major alterations such as cell death. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Brain aging; Corticocortical projections; Patas monkeys; Prefrontal cortex; Primates; Pyramidal neurons
A distinct subpopulation of neurons forming long corticocortical projections in the association neocortex is highly vulnerable to the degenerative process in Alzheimer’s disease [7,11]. However, the degree to which age-related molecular and morphologic alterations of identifiable neuronal populations reflect early cellular degeneration leading to functional deficits has not yet been fully investigated in the aging brain. No neuronal loss is observed in the course of normal aging in non-human primates, although significant cognitive changes can be observed in animals older than 19 years of age [3,5,8,15–17]. It is likely that such changes are related to pervasive pathologic processes that occur in select classes of cortical neurons. In this context, it is worth noting * Corresponding author. Kastor Neurobiology of Aging Laboratories, Box 1639, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA. Tel.: 11-212659-5904; fax: 11-212-849-2510. E-mail address: [email protected] (P.R. Hof).
that recent studies of the prefrontal cortex and primary visual cortex in behaviorally tested old macaque monkeys have generally failed to reveal any objective age-related alteration at the light microscopy level. However, electron microscopic investigations have demonstrated consistent pathological changes in oligodendrocyte and axonal myelin sheath morphology in aged non-human primates [15–17], pointing to the possible involvement of certain cortical projection systems in aging. Several studies indicate that the cognitive processes mediated by the prefrontal cortex are impaired during the normal aging processes [3,14–16]. In particular, old macaque monkeys show consistently lower performance in delayed response and delayed nonmatching to sample tasks that test prefrontal cortex function, when compared to young animals [3,14,15]. These and other data indicate that age-related cognitive deficits in nonhuman primates may be a consequence of abnormalities in cortical circuits that clearly do not include loss of neurons
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 02 42 8- 4
38
T.L. Page et al. / Neuroscience Letters 317 (2002) 37–41
but rather involve subcellular compartments of the neurons at risk. The goals of the present study were to develop methodologies to quantify age-related morphological changes at the single cell level in identified populations of corticocortically-projecting neurons in the Patas monkey, a cercopithecine species closely related to the macaques. Six Patas monkeys (Erythrocebus patas; 8–14.5 kg, of either sex) were used in the present study. There was one young male and two young females (10–12 years old), and one old female and two old males (21–25 years old). These age groups were defined based on evidence from macaque monkey aging [3–5,8,14,16]. These animals were never involved in any pharmacological or other invasive studies, and had been subjected to regular serological tests and annual physical examinations. Necropsy analyses did not reveal any factor that may have influenced the findings obtained in this study. Importantly, potential effects of estrogen levels could be ruled out, since animals of either sex were included in the young and old groups. All experimental protocols were conducted within NIH guidelines for animal research and were approved by the Institutional Animal Care and Use Committee at Mount Sinai School of Medicine. For surgery, animals were tranquilized with ketamine hydrochloride (35 mg/kg i.m.), intubated, and maintained under isoflurane general anesthesia (0.5–1.5% as necessary), and strict sterile surgical conditions [13]. Following craniotomy, aqueous solutions of Fast Blue (FB, 4%; Sigma, St. Louis, MO) or Diamidino Yellow (DY, 4%; Sigma) were injected into the left prefrontal cortex in the lower bank of the principal sulcus corresponding to ventral area 46 (500 nl at each site). After a survival time of 3 weeks to permit optimal retrograde transport, the animals were deeply anesthetized with ketamine hydrochloride (40 mg/kg) and pentobarbital sodium (20–35 mg/kg i.v.), and perfused transcardially with cold 1% paraformaldehyde in phosphate-buffered saline (PBS) and then for 14 min with cold 4% paraformaldehyde/0.125% glutaraldehyde in PBS. Following perfusion, the brain was removed from the skull and 4–5 mm-thick blocks were obtained from the ipsilateral cortex located in the fundus of the superior temporal sulcus corresponding to areas IPa and TEa. These blocks were postfixed for 2 h in the same fixative and cut at 400 mm on a Vibratome. FB or DY retrogradely labeled pyramidal cells in layers III and V of sections from the temporal neocortex were identified under epifluorescence with a UV filter, impaled with sharp micropipettes, and loaded with 5% Lucifer Yellow (LY; Molecular Probes, Eugene, OR) under a DC current of 3–8 nA for 10–12 min [13]. Filled neurons were reconstructed using a computer-assisted morphometry system consisting of a Zeiss Axiophot photomicroscope equipped with a Zeiss MSP65 computercontrolled motorized stage, a Zeiss ZVS-47E video camera system, a Macintosh G3 computer, and custom designed morphometry software NeuroZoom [8,13]. Neurons were drawn using a Zeiss Apochromat 100 £ objective with a
numerical aperture of 1.4 and mapping was performed by moving the stage in 1 mm steps through the z-axis along the length of each dendrite. Spines were plotted at the same time as they appeared in sharp focus so that the x-y-z coordinates of each dendritic segment or spine were recorded to enable later three-dimensional representation and rotation of the reconstructed neuron [6,13]. The NeuroZoom datasets were then exported to generate three-dimensional renderings of the traced neurons using NeuroGL, a self-contained, compact, and fast application to visualize structural details of neurons, including branching patterns, spine location and spine density, at a range of scales and in an interactive threedimensional environment [6]. Total dendritic length, dendritic segment counts, spine density and total spine numbers were calculated in the apical and basal dendritic arbors. Neurons that were improperly filled, had cut dendrites or had light fluorescence were not reconstructed. Tissue blocks containing the injection sites were sectioned at 40 mm and
Fig. 1. Examples of retrogradely traced neurons filled with LY and reconstructed in three dimensions using NeuroZoom and NeuroGL software applications. Neuron A is from layer III of the temporal cortex of a 24-year-old animal and neuron B is from the same region of a 12-year-old animal. Note that some differences in the complexity of the apical dendrites are visible. Higher magnification views centered on the soma of the same neurons as in A and B are shown in C and D. Age-related differences in spine numbers are apparent at this magnification (yellow dots; compare A–C with B–D). Higher zoom factors are shown on isolated dendritic fragments in E (third order apical dendritic branch, in an old animal), and F (matching level in a young animal). Scale bar, 150 mm (A,B), 60 mm (C,D) and 20 mm (E,F).
T.L. Page et al. / Neuroscience Letters 317 (2002) 37–41
stained with Cresyl Violet, and the extent of the injection sites was verified and reconstructed using NeuroZoom [13]. Statistical testing was performed using analysis of variance to assess possible differences in the various morphometric parameters among cases within each group. As the variance between was smaller than that within animals and the variances of each subject did not significantly differ for each of the measured parameters, data were expressed as means from the three animals in each group. Age-related differences in these variables across the total, apical, and basal dendritic domains were then assessed by comparing the means of the young and old groups using Tukey tests for groups with unequal numbers. The injections of FB or DY in area 46 resulted in comparable numbers of retrogradely labeled neurons in the superior temporal cortex in both age groups. All of the filled neurons exhibited a typical pyramidal morphology with extensive dendritic arborization and large numbers of spines (Fig. 1). Similar data were obtained from neurons located in layers III and V, and these results were therefore pooled in the final analysis. As no significant differences in the measured parameters were observed among individuals in either the young or old animal groups, data from the three animals in each age group were pooled in Table 1. Based on the three-dimensional reconstructions of these neurons, few differences in dendritic morphology could be observed between the age groups (Fig. 1 and Table 1). The total length of the dendrites in the basal and apical domains was not different in old animals compared to the young ones, and the number of dendritic branches did not change with aging in either type of projection, even though qualitatively some neurons appeared less complex in old than in young animals (Fig. 1A,B and Table 1). A minor effect of
39
age was visible on the length of dendritic segments in old animals, which did not reach statistical significance (Table 1). The most consistent finding in aged animals was a reduction in dendritic spine numbers and densities along the dendritic branches (Fig. 1C–F and Table 1). Depending on the neuron analyzed, the total number of spines decreased by 28–37% in the basal and apical dendrites of aged animals compared to young ones (P , 0:001). The spine densities per mm of dendritic length decreased by about 23% overall (P , 0:05) and were consistent across the entire dendritic arborization after the second order of dendritic branching. Cognitive functions are impaired during normal aging, yet the neurobiological factors underlying the age-related cognitive decline remain largely unknown. A loss of cortical neurons has long been considered a probable cause of cognitive deficits in normal aging. However, stereologic methods have failed to reveal a decrease in cerebral cortical neuron numbers or cortical volume in aged monkeys and humans [5,8,11,14–16]. Therefore, it is likely that more subtle cellular changes occur during aging and account for the observed functional deficits. The present data suggest that a significant decrease in the density of dendritic spines occurs during aging in neurons forming long corticocortical projections from the temporal to the prefrontal cortex in a nonhuman primate. A certain impoverishment of the complexity of dendritic arborizations may also occur in old animals, although additional morphometric tools will be necessary to quantify these differences appropriately [6]. The observed change in spine numbers may lead to a potential deficit in the excitatory drive on the neurons that receive these inputs. In a related fashion, it is interesting to note that levels of Nmethyl-d-aspartate receptor protein subunit 1 decrease specifically and consistently in the dentate gyrus in aged
Table 1 Summary of quantitative analyses of reconstructed neurons a Parameter Length of segments Apical Basal Total Number of segments Apical Basal Total Number of spines Apical Basal Total Spine density Apical Basal Total a
Young (n ¼ 3)
Old (n ¼ 3)
% Difference
2056.5 ^ 200.0 3036.8 ^ 176.2 5093.3 ^ 533.5
1757.4 ^ 210.1 2448.6 ^ 320.0 4206.0 ^ 480.8
214.5 219.4 217.4
27.7 ^ 5.2 43.7 ^ 4.5 71.4 ^ 4.6
28.3 ^ 6.0 48.8 ^ 2.7 77.1 ^ 3.0
12.1 111.7 18.0
1089.9 ^ 90.0 1488.0 ^ 125.0 2597.6 ^ 580.0
738.1 ^ 62.8 955.0 ^ 80.0 1682.4 ^ 130.3
232.3 235.8 235.2
0.53 ^ 0.02 0.49 ^ 0.02 0.51 ^ 0.02
0.42 ^ 0.01 0.39 ^ 0.03 0.40 ^ 0.03
224.5 220.4 223.5
Data represent the mean ^ SD from the three subjects in each group. Segment lengths are in mm and spine densities are expressed per mm of dendrite. The differences between young and old animals are indicated and the statistically significant results (P , 0:001, spine numbers; P , 0:05, spine densities) are emphasized in bold. Note the consistent loss of spines in old monkeys. The trend of a reduced length of segments in old animals did not reach statistical significance.
40
T.L. Page et al. / Neuroscience Letters 317 (2002) 37–41
macaque monkeys in the absence of neuronal loss [4,5,11], suggesting that the intradendritic parcellation of a neurotransmitter receptor is modifiable in an age-related and circuit-specific manner and that such changes may provide a substrate for age-related memory impairment. Our observations support data from the brains of Alzheimer’s disease patients, where large layer III pyramidal neurons are consistently affected [7,11], as well as several morphometric findings on the cerebral cortex in aged human and non-human primates. Scheibel et al. [18] first reported regressive dendritic changes of cortical pyramidal neurons during aging in layer III pyramidal cells of the prefrontal and superior temporal cortex. Nakamura et al. [12] showed that the number of dendrites of pyramidal cells in the human motor cortex decreased with advancing age. Jacobs et al. [9,10] examined the total dendritic length, mean segment length, dendritic segment number, dendritic spine number, and dendritic spine density of basal dendrites of supragranular pyramidal cells in the human prefrontal and occipital cortex. They reported a 9–11% decrease in total dendritic length and about a 50% decrease in spine count estimates in the older group in both areas. Dendritic regression of layer V cortical pyramidal neurons with aging was also observed in the prefrontal cortex by de Brabander et al. [1]. Since the dendritic surface receives most of the synapses on a given neuron, alterations in dendritic morphology, and particularly in spine numbers, may lead to concomitant changes in synaptic density. Interestingly, such a parallel decrease in spine numbers and synaptic density during aging has been observed in quantitative electron microscopy studies [17,20]. A recent study reported a loss of apical dendritic tufts of pyramidal cells in layer I of area 46 of old compared to young monkeys [17]. These authors estimated that 50% of spines were lost in layer I of area 46 of the old monkeys. Direct comparison between these and the present studies is, however, subject to caution due to methodological differences, namely a focus on layer I [17], or the use of the Golgi stain [20]. Also, other studies using stereology have failed to show an age-related decline in synapses and spines in the dentate gyrus of aged monkeys [19], possibly reflecting projection-specific differences during aging, and it should be kept in mind that recent studies have shown a considerable variability in the morphology of pyramidal neurons among neocortical regions and among cortical layers [1,2,6,13]. In spite of these discrepancies, age-related changes in dendrites and dendritic spines of cortical pyramidal cells, and an accompanying decrease in synaptic density in aged individuals may lead to a disruption of cortical circuits during normal aging. Although future studies will need to extend the present data to additional cortical circuits, and address the specificity of these morphological alterations in behaviorally tested aged animals [14], our findings support the notion that memory impairment in aging is unlikely to be due to major morphological alterations in cortical circuits, and link age-related changes in dendritic morphology and spine densities of
pyramidal neurons to identified, specific neocortical circuits. We thank B. Wicinski, B. Seabrook, W.G.M. Janssen, and J. and S. Harbaugh for help with surgery and animal care, A. Rodriguez, K.T. Kelliher, R.A. Shah, D. Ehlenberger, and Dr W.G. Young for software development, and A.P. Leonard for expert technical assistance. Drs G.N. Elston, E.A. Nimchinsky, B.I. Henry, and P. Rothnie provided valuable discussion. This study was supported by NIH grants AG05138, AG06647, AG14308, MH58911, and DC04632, the Howard Hughes Medical Institute, and Bioqual, Inc. T.L.P. and M.E. were recipients of Hartford/AFAR fellowships. T.F. and D.R. received student fellowships from the Mount Sinai School of Medicine. [1] de Brabander, J.M., Kramers, R.J. and Uylings, H.B., Layerspecific dendritic regression of pyramidal cells with ageing in the human prefrontal cortex, Eur. J. Neurosci., 10 (1998) 1261–1269. [2] Elston, G.N., Interlaminar differences in the pyramidal cell phenotype in cortical areas 7m and STP (the superior temporal polysensory area) of the macaque monkey, Exp. Brain Res., 138 (2001) 141–152. [3] Gallagher, M. and Rapp, P.R., The use of animal models to study the effects of aging on cognition, Annu. Rev. Psychol., 48 (1997) 339–370. [4] Gazzaley, A.H., Siegel, S.J., Kordower, J.H., Mufson, E.J. and Morrison, J.H., Circuit-specific alterations of Nmethyl-d-aspartate receptor subunit 1 in the dentate gyrus of aged monkeys, Proc. Natl. Acad. Sci. USA, 93 (1996) 3121–3125. [5] Gazzaley, A.H., Thakker, M.M., Hof, P.R. and Morrison, J.H., Preserved number of entorhinal cortex layer II neurons in aged macaque monkeys, Neurobiol. Aging, 18 (1997) 549– 553. [6] Henry, B.I., Hof, P.R., Rothnie, P. and Wearne, S.L., Fractal analysis of aggregates of non-uniformly sized particles: an application to macaque monkey cortical pyramidal neurons, Fractals, (2001) in press. [7] Hof, P.R., Cox, K. and Morrison, J.H., Quantitative analysis of a vulnerable subset of pyramidal neurons in Alzheimer’s disease: I. Superior frontal and inferior temporal cortex, J. Comp. Neurol., 301 (1990) 44–54. [8] Hof, P.R., Nimchinsky, E.A., Young, W.G. and Morrison, J.H., Numbers of Meynert and layer IVB cells in area V1: a stereologic analysis in young and aged macaque monkeys, J. Comp. Neurol., 420 (2000) 113–126. [9] Jacobs, B., Driscoll, L. and Schall, M., Life-span dendritic and spine changes in areas 10 and 18 of human cortex: a quantitative Golgi study, J. Comp. Neurol., 386 (1997) 661– 680. [10] Jacobs, B., Schall, M., Prather, M., Kapler, E., Driscoll, L., Baca, S., Jacobs, J., Ford, K., Wainwright, M. and Treml, M., Regional dendritic and spine variations in human cerebral cortex: a quantitative Golgi study, Cereb. Cortex, 11 (2001) 558–571. [11] Morrison, J.H. and Hof, P.R., Life and death of neurons in the aging brain, Science, 278 (1997) 412–419. [12] Nakamura, S., Akiguchi, I., Kameyama, M. and Mizuno, N., Age-related changes of pyramidal cell basal dendrites in layers III and V of human motor cortex: a quantitative Golgi study, Acta Neuropathol., 65 (1985) 281–284. [13] Nimchinsky, E.A., Hof, P.R., Young, W.G. and Morrison,
T.L. Page et al. / Neuroscience Letters 317 (2002) 37–41 J.H., Neurochemical, morphologic, and laminar characterization of cortical projection neurons in the cingulate motor areas of the macaque monkey, J. Comp. Neurol., 374 (1996) 136–160. [14] O’Donnell, K.A., Rapp, P.R. and Hof, P.R., Preservation of prefrontal cortical volume in behaviorally characterized aged macaque monkeys, Exp. Neurol., 160 (1999) 300–310. [15] Peters, A., Morrison, J.H., Rosene, D.L. and Hyman, B.T., Feature article: are neurons lost from the primate cerebral cortex during normal aging? Cereb. Cortex, 8 (1998) 295– 300. [16] Peters, A., Rosene, D.L., Moss, M.B., Kemper, T.L., Abraham, C.R., Tigges, J. and Albert, M.S., Neurobiological bases of age-related cognitive decline in the rhesus
[17]
[18]
[19]
[20]
41
monkey, J. Neuropathol. Exp. Neurol., 55 (1996) 861– 874. Peters, A., Sethares, C. and Moss, M.B., The effects of aging on layer 1 in area 46 of prefrontal cortex in the rhesus monkey, Cereb. Cortex, 8 (1998) 671–684. Scheibel, M.E., Lindsay, R.D., Tomiyasu, U. and Scheibel, A.B., Progressive dendritic changes in aging human cortex, Exp. Neurol., 47 (1975) 392–403. Tigges, J., Herndon, J.G. and Rosene, D.L., Mild age-related changes in the dentate gyrus of adult rhesus monkeys, Acta Anat., 153 (1995) 39–48. Uemura, E., Age-related changes in prefrontal cortex of Macaca mulatta: synaptic density, Exp. Neurol., 69 (1980) 164–172.
Morphological alterations in neurons forming corticocortical projections in the neocortex of aged Patas monkeys Tanya L. Page a, Michael Einstein a,b, Huiling Duan a,b,c, Yong He a,c, Tony Flores a, Daniil Rolshud a,b, Joseph M. Erwin d,e, Susan L. Wearne b,c,f, John H. Morrison a,b,c, Patrick R. Hof a,b,c,e,* a
Kastor Neurobiology of Aging Laboratories, Box 1639, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA b Computational Neurobiology and Imaging Center, Mount Sinai School of Medicine, New York, NY, USA c Fishberg Research Center for Neurobiology, Mount Sinai School of Medicine, New York, NY, USA d Division of Neurobiology, Behavior and Genetics, Bioqual Inc., Rockville, MD, USA e Foundation for Comparative and Conservation Biology, Rockville, MD, USA f Department of Biomathematical Sciences, Mount Sinai School of Medicine, New York, NY, USA Received 30 July 2001; received in revised form 17 September 2001; accepted 26 September 2001
Abstract Recent studies indicate that the cognitive processes mediated by the prefrontal cortex, such as working memory, are impaired during normal aging. These disturbances in cortical function may be a consequence of abnormalities in neocortical circuits, even though the numbers of cortical neurons are preserved in normal aging. We performed retrograde tract-tracing of cortical projections connecting the temporal cortex to the prefrontal cortex in combination with dye-filling and three-dimensional neuronal reconstructions in aged patas monkeys. Age-related changes affected the apparent complexity of the apical dendrites of projection neurons and caused a significant loss of dendritic spines at all levels of their dendritic trees. These results indicate that normal aging is accompanied by neuronal changes that are quite subtle, and possibly involves discrete cellular components of certain cortical neurons selectively rather than inducing major alterations such as cell death. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Brain aging; Corticocortical projections; Patas monkeys; Prefrontal cortex; Primates; Pyramidal neurons
A distinct subpopulation of neurons forming long corticocortical projections in the association neocortex is highly vulnerable to the degenerative process in Alzheimer’s disease [7,11]. However, the degree to which age-related molecular and morphologic alterations of identifiable neuronal populations reflect early cellular degeneration leading to functional deficits has not yet been fully investigated in the aging brain. No neuronal loss is observed in the course of normal aging in non-human primates, although significant cognitive changes can be observed in animals older than 19 years of age [3,5,8,15–17]. It is likely that such changes are related to pervasive pathologic processes that occur in select classes of cortical neurons. In this context, it is worth noting * Corresponding author. Kastor Neurobiology of Aging Laboratories, Box 1639, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA. Tel.: 11-212659-5904; fax: 11-212-849-2510. E-mail address: [email protected] (P.R. Hof).
that recent studies of the prefrontal cortex and primary visual cortex in behaviorally tested old macaque monkeys have generally failed to reveal any objective age-related alteration at the light microscopy level. However, electron microscopic investigations have demonstrated consistent pathological changes in oligodendrocyte and axonal myelin sheath morphology in aged non-human primates [15–17], pointing to the possible involvement of certain cortical projection systems in aging. Several studies indicate that the cognitive processes mediated by the prefrontal cortex are impaired during the normal aging processes [3,14–16]. In particular, old macaque monkeys show consistently lower performance in delayed response and delayed nonmatching to sample tasks that test prefrontal cortex function, when compared to young animals [3,14,15]. These and other data indicate that age-related cognitive deficits in nonhuman primates may be a consequence of abnormalities in cortical circuits that clearly do not include loss of neurons
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 02 42 8- 4
38
T.L. Page et al. / Neuroscience Letters 317 (2002) 37–41
but rather involve subcellular compartments of the neurons at risk. The goals of the present study were to develop methodologies to quantify age-related morphological changes at the single cell level in identified populations of corticocortically-projecting neurons in the Patas monkey, a cercopithecine species closely related to the macaques. Six Patas monkeys (Erythrocebus patas; 8–14.5 kg, of either sex) were used in the present study. There was one young male and two young females (10–12 years old), and one old female and two old males (21–25 years old). These age groups were defined based on evidence from macaque monkey aging [3–5,8,14,16]. These animals were never involved in any pharmacological or other invasive studies, and had been subjected to regular serological tests and annual physical examinations. Necropsy analyses did not reveal any factor that may have influenced the findings obtained in this study. Importantly, potential effects of estrogen levels could be ruled out, since animals of either sex were included in the young and old groups. All experimental protocols were conducted within NIH guidelines for animal research and were approved by the Institutional Animal Care and Use Committee at Mount Sinai School of Medicine. For surgery, animals were tranquilized with ketamine hydrochloride (35 mg/kg i.m.), intubated, and maintained under isoflurane general anesthesia (0.5–1.5% as necessary), and strict sterile surgical conditions [13]. Following craniotomy, aqueous solutions of Fast Blue (FB, 4%; Sigma, St. Louis, MO) or Diamidino Yellow (DY, 4%; Sigma) were injected into the left prefrontal cortex in the lower bank of the principal sulcus corresponding to ventral area 46 (500 nl at each site). After a survival time of 3 weeks to permit optimal retrograde transport, the animals were deeply anesthetized with ketamine hydrochloride (40 mg/kg) and pentobarbital sodium (20–35 mg/kg i.v.), and perfused transcardially with cold 1% paraformaldehyde in phosphate-buffered saline (PBS) and then for 14 min with cold 4% paraformaldehyde/0.125% glutaraldehyde in PBS. Following perfusion, the brain was removed from the skull and 4–5 mm-thick blocks were obtained from the ipsilateral cortex located in the fundus of the superior temporal sulcus corresponding to areas IPa and TEa. These blocks were postfixed for 2 h in the same fixative and cut at 400 mm on a Vibratome. FB or DY retrogradely labeled pyramidal cells in layers III and V of sections from the temporal neocortex were identified under epifluorescence with a UV filter, impaled with sharp micropipettes, and loaded with 5% Lucifer Yellow (LY; Molecular Probes, Eugene, OR) under a DC current of 3–8 nA for 10–12 min [13]. Filled neurons were reconstructed using a computer-assisted morphometry system consisting of a Zeiss Axiophot photomicroscope equipped with a Zeiss MSP65 computercontrolled motorized stage, a Zeiss ZVS-47E video camera system, a Macintosh G3 computer, and custom designed morphometry software NeuroZoom [8,13]. Neurons were drawn using a Zeiss Apochromat 100 £ objective with a
numerical aperture of 1.4 and mapping was performed by moving the stage in 1 mm steps through the z-axis along the length of each dendrite. Spines were plotted at the same time as they appeared in sharp focus so that the x-y-z coordinates of each dendritic segment or spine were recorded to enable later three-dimensional representation and rotation of the reconstructed neuron [6,13]. The NeuroZoom datasets were then exported to generate three-dimensional renderings of the traced neurons using NeuroGL, a self-contained, compact, and fast application to visualize structural details of neurons, including branching patterns, spine location and spine density, at a range of scales and in an interactive threedimensional environment [6]. Total dendritic length, dendritic segment counts, spine density and total spine numbers were calculated in the apical and basal dendritic arbors. Neurons that were improperly filled, had cut dendrites or had light fluorescence were not reconstructed. Tissue blocks containing the injection sites were sectioned at 40 mm and
Fig. 1. Examples of retrogradely traced neurons filled with LY and reconstructed in three dimensions using NeuroZoom and NeuroGL software applications. Neuron A is from layer III of the temporal cortex of a 24-year-old animal and neuron B is from the same region of a 12-year-old animal. Note that some differences in the complexity of the apical dendrites are visible. Higher magnification views centered on the soma of the same neurons as in A and B are shown in C and D. Age-related differences in spine numbers are apparent at this magnification (yellow dots; compare A–C with B–D). Higher zoom factors are shown on isolated dendritic fragments in E (third order apical dendritic branch, in an old animal), and F (matching level in a young animal). Scale bar, 150 mm (A,B), 60 mm (C,D) and 20 mm (E,F).
T.L. Page et al. / Neuroscience Letters 317 (2002) 37–41
stained with Cresyl Violet, and the extent of the injection sites was verified and reconstructed using NeuroZoom [13]. Statistical testing was performed using analysis of variance to assess possible differences in the various morphometric parameters among cases within each group. As the variance between was smaller than that within animals and the variances of each subject did not significantly differ for each of the measured parameters, data were expressed as means from the three animals in each group. Age-related differences in these variables across the total, apical, and basal dendritic domains were then assessed by comparing the means of the young and old groups using Tukey tests for groups with unequal numbers. The injections of FB or DY in area 46 resulted in comparable numbers of retrogradely labeled neurons in the superior temporal cortex in both age groups. All of the filled neurons exhibited a typical pyramidal morphology with extensive dendritic arborization and large numbers of spines (Fig. 1). Similar data were obtained from neurons located in layers III and V, and these results were therefore pooled in the final analysis. As no significant differences in the measured parameters were observed among individuals in either the young or old animal groups, data from the three animals in each age group were pooled in Table 1. Based on the three-dimensional reconstructions of these neurons, few differences in dendritic morphology could be observed between the age groups (Fig. 1 and Table 1). The total length of the dendrites in the basal and apical domains was not different in old animals compared to the young ones, and the number of dendritic branches did not change with aging in either type of projection, even though qualitatively some neurons appeared less complex in old than in young animals (Fig. 1A,B and Table 1). A minor effect of
39
age was visible on the length of dendritic segments in old animals, which did not reach statistical significance (Table 1). The most consistent finding in aged animals was a reduction in dendritic spine numbers and densities along the dendritic branches (Fig. 1C–F and Table 1). Depending on the neuron analyzed, the total number of spines decreased by 28–37% in the basal and apical dendrites of aged animals compared to young ones (P , 0:001). The spine densities per mm of dendritic length decreased by about 23% overall (P , 0:05) and were consistent across the entire dendritic arborization after the second order of dendritic branching. Cognitive functions are impaired during normal aging, yet the neurobiological factors underlying the age-related cognitive decline remain largely unknown. A loss of cortical neurons has long been considered a probable cause of cognitive deficits in normal aging. However, stereologic methods have failed to reveal a decrease in cerebral cortical neuron numbers or cortical volume in aged monkeys and humans [5,8,11,14–16]. Therefore, it is likely that more subtle cellular changes occur during aging and account for the observed functional deficits. The present data suggest that a significant decrease in the density of dendritic spines occurs during aging in neurons forming long corticocortical projections from the temporal to the prefrontal cortex in a nonhuman primate. A certain impoverishment of the complexity of dendritic arborizations may also occur in old animals, although additional morphometric tools will be necessary to quantify these differences appropriately [6]. The observed change in spine numbers may lead to a potential deficit in the excitatory drive on the neurons that receive these inputs. In a related fashion, it is interesting to note that levels of Nmethyl-d-aspartate receptor protein subunit 1 decrease specifically and consistently in the dentate gyrus in aged
Table 1 Summary of quantitative analyses of reconstructed neurons a Parameter Length of segments Apical Basal Total Number of segments Apical Basal Total Number of spines Apical Basal Total Spine density Apical Basal Total a
Young (n ¼ 3)
Old (n ¼ 3)
% Difference
2056.5 ^ 200.0 3036.8 ^ 176.2 5093.3 ^ 533.5
1757.4 ^ 210.1 2448.6 ^ 320.0 4206.0 ^ 480.8
214.5 219.4 217.4
27.7 ^ 5.2 43.7 ^ 4.5 71.4 ^ 4.6
28.3 ^ 6.0 48.8 ^ 2.7 77.1 ^ 3.0
12.1 111.7 18.0
1089.9 ^ 90.0 1488.0 ^ 125.0 2597.6 ^ 580.0
738.1 ^ 62.8 955.0 ^ 80.0 1682.4 ^ 130.3
232.3 235.8 235.2
0.53 ^ 0.02 0.49 ^ 0.02 0.51 ^ 0.02
0.42 ^ 0.01 0.39 ^ 0.03 0.40 ^ 0.03
224.5 220.4 223.5
Data represent the mean ^ SD from the three subjects in each group. Segment lengths are in mm and spine densities are expressed per mm of dendrite. The differences between young and old animals are indicated and the statistically significant results (P , 0:001, spine numbers; P , 0:05, spine densities) are emphasized in bold. Note the consistent loss of spines in old monkeys. The trend of a reduced length of segments in old animals did not reach statistical significance.
40
T.L. Page et al. / Neuroscience Letters 317 (2002) 37–41
macaque monkeys in the absence of neuronal loss [4,5,11], suggesting that the intradendritic parcellation of a neurotransmitter receptor is modifiable in an age-related and circuit-specific manner and that such changes may provide a substrate for age-related memory impairment. Our observations support data from the brains of Alzheimer’s disease patients, where large layer III pyramidal neurons are consistently affected [7,11], as well as several morphometric findings on the cerebral cortex in aged human and non-human primates. Scheibel et al. [18] first reported regressive dendritic changes of cortical pyramidal neurons during aging in layer III pyramidal cells of the prefrontal and superior temporal cortex. Nakamura et al. [12] showed that the number of dendrites of pyramidal cells in the human motor cortex decreased with advancing age. Jacobs et al. [9,10] examined the total dendritic length, mean segment length, dendritic segment number, dendritic spine number, and dendritic spine density of basal dendrites of supragranular pyramidal cells in the human prefrontal and occipital cortex. They reported a 9–11% decrease in total dendritic length and about a 50% decrease in spine count estimates in the older group in both areas. Dendritic regression of layer V cortical pyramidal neurons with aging was also observed in the prefrontal cortex by de Brabander et al. [1]. Since the dendritic surface receives most of the synapses on a given neuron, alterations in dendritic morphology, and particularly in spine numbers, may lead to concomitant changes in synaptic density. Interestingly, such a parallel decrease in spine numbers and synaptic density during aging has been observed in quantitative electron microscopy studies [17,20]. A recent study reported a loss of apical dendritic tufts of pyramidal cells in layer I of area 46 of old compared to young monkeys [17]. These authors estimated that 50% of spines were lost in layer I of area 46 of the old monkeys. Direct comparison between these and the present studies is, however, subject to caution due to methodological differences, namely a focus on layer I [17], or the use of the Golgi stain [20]. Also, other studies using stereology have failed to show an age-related decline in synapses and spines in the dentate gyrus of aged monkeys [19], possibly reflecting projection-specific differences during aging, and it should be kept in mind that recent studies have shown a considerable variability in the morphology of pyramidal neurons among neocortical regions and among cortical layers [1,2,6,13]. In spite of these discrepancies, age-related changes in dendrites and dendritic spines of cortical pyramidal cells, and an accompanying decrease in synaptic density in aged individuals may lead to a disruption of cortical circuits during normal aging. Although future studies will need to extend the present data to additional cortical circuits, and address the specificity of these morphological alterations in behaviorally tested aged animals [14], our findings support the notion that memory impairment in aging is unlikely to be due to major morphological alterations in cortical circuits, and link age-related changes in dendritic morphology and spine densities of
pyramidal neurons to identified, specific neocortical circuits. We thank B. Wicinski, B. Seabrook, W.G.M. Janssen, and J. and S. Harbaugh for help with surgery and animal care, A. Rodriguez, K.T. Kelliher, R.A. Shah, D. Ehlenberger, and Dr W.G. Young for software development, and A.P. Leonard for expert technical assistance. Drs G.N. Elston, E.A. Nimchinsky, B.I. Henry, and P. Rothnie provided valuable discussion. This study was supported by NIH grants AG05138, AG06647, AG14308, MH58911, and DC04632, the Howard Hughes Medical Institute, and Bioqual, Inc. T.L.P. and M.E. were recipients of Hartford/AFAR fellowships. T.F. and D.R. received student fellowships from the Mount Sinai School of Medicine. [1] de Brabander, J.M., Kramers, R.J. and Uylings, H.B., Layerspecific dendritic regression of pyramidal cells with ageing in the human prefrontal cortex, Eur. J. Neurosci., 10 (1998) 1261–1269. [2] Elston, G.N., Interlaminar differences in the pyramidal cell phenotype in cortical areas 7m and STP (the superior temporal polysensory area) of the macaque monkey, Exp. Brain Res., 138 (2001) 141–152. [3] Gallagher, M. and Rapp, P.R., The use of animal models to study the effects of aging on cognition, Annu. Rev. Psychol., 48 (1997) 339–370. [4] Gazzaley, A.H., Siegel, S.J., Kordower, J.H., Mufson, E.J. and Morrison, J.H., Circuit-specific alterations of Nmethyl-d-aspartate receptor subunit 1 in the dentate gyrus of aged monkeys, Proc. Natl. Acad. Sci. USA, 93 (1996) 3121–3125. [5] Gazzaley, A.H., Thakker, M.M., Hof, P.R. and Morrison, J.H., Preserved number of entorhinal cortex layer II neurons in aged macaque monkeys, Neurobiol. Aging, 18 (1997) 549– 553. [6] Henry, B.I., Hof, P.R., Rothnie, P. and Wearne, S.L., Fractal analysis of aggregates of non-uniformly sized particles: an application to macaque monkey cortical pyramidal neurons, Fractals, (2001) in press. [7] Hof, P.R., Cox, K. and Morrison, J.H., Quantitative analysis of a vulnerable subset of pyramidal neurons in Alzheimer’s disease: I. Superior frontal and inferior temporal cortex, J. Comp. Neurol., 301 (1990) 44–54. [8] Hof, P.R., Nimchinsky, E.A., Young, W.G. and Morrison, J.H., Numbers of Meynert and layer IVB cells in area V1: a stereologic analysis in young and aged macaque monkeys, J. Comp. Neurol., 420 (2000) 113–126. [9] Jacobs, B., Driscoll, L. and Schall, M., Life-span dendritic and spine changes in areas 10 and 18 of human cortex: a quantitative Golgi study, J. Comp. Neurol., 386 (1997) 661– 680. [10] Jacobs, B., Schall, M., Prather, M., Kapler, E., Driscoll, L., Baca, S., Jacobs, J., Ford, K., Wainwright, M. and Treml, M., Regional dendritic and spine variations in human cerebral cortex: a quantitative Golgi study, Cereb. Cortex, 11 (2001) 558–571. [11] Morrison, J.H. and Hof, P.R., Life and death of neurons in the aging brain, Science, 278 (1997) 412–419. [12] Nakamura, S., Akiguchi, I., Kameyama, M. and Mizuno, N., Age-related changes of pyramidal cell basal dendrites in layers III and V of human motor cortex: a quantitative Golgi study, Acta Neuropathol., 65 (1985) 281–284. [13] Nimchinsky, E.A., Hof, P.R., Young, W.G. and Morrison,
T.L. Page et al. / Neuroscience Letters 317 (2002) 37–41 J.H., Neurochemical, morphologic, and laminar characterization of cortical projection neurons in the cingulate motor areas of the macaque monkey, J. Comp. Neurol., 374 (1996) 136–160. [14] O’Donnell, K.A., Rapp, P.R. and Hof, P.R., Preservation of prefrontal cortical volume in behaviorally characterized aged macaque monkeys, Exp. Neurol., 160 (1999) 300–310. [15] Peters, A., Morrison, J.H., Rosene, D.L. and Hyman, B.T., Feature article: are neurons lost from the primate cerebral cortex during normal aging? Cereb. Cortex, 8 (1998) 295– 300. [16] Peters, A., Rosene, D.L., Moss, M.B., Kemper, T.L., Abraham, C.R., Tigges, J. and Albert, M.S., Neurobiological bases of age-related cognitive decline in the rhesus
[17]
[18]
[19]
[20]
41
monkey, J. Neuropathol. Exp. Neurol., 55 (1996) 861– 874. Peters, A., Sethares, C. and Moss, M.B., The effects of aging on layer 1 in area 46 of prefrontal cortex in the rhesus monkey, Cereb. Cortex, 8 (1998) 671–684. Scheibel, M.E., Lindsay, R.D., Tomiyasu, U. and Scheibel, A.B., Progressive dendritic changes in aging human cortex, Exp. Neurol., 47 (1975) 392–403. Tigges, J., Herndon, J.G. and Rosene, D.L., Mild age-related changes in the dentate gyrus of adult rhesus monkeys, Acta Anat., 153 (1995) 39–48. Uemura, E., Age-related changes in prefrontal cortex of Macaca mulatta: synaptic density, Exp. Neurol., 69 (1980) 164–172.