Download Quantitative morphological changes in neurons from the dorsal

THE ANATOMICAL RECORD 248:137–141 (1997)
Quantitative Morphological Changes in Neurons From the Dorsal
Lateral Geniculate Nucleus of Young and Old Rats
ALICIA VILLENA,1* FLORENTINA DÍAZ,1 VIRGINIA REQUENA,1
ISABEL CHAVARRÍA,1 FRANCISCA RIUS,2 AND IGNACIO PÉREZ DE VARGAS1
1Department of Normal and Pathological Morphology, Faculty of Medicine,
University of Málaga, Málaga, Spain
2Department of Public Health, Faculty of Medicine, University of Málaga, Málaga, Spain
ABSTRACT
Background: We studied the morphological changes occurring in neurons from the dorsal lateral geniculate nucleus (dLGN) during
aging by analysing the size and shape of cell bodies and nuclei.
Methods: Male albino Wistar rats, aged 3, 18, 24, and 30 months, were
used. After appropriate tissue preparation and following the usual histological procedure, the profiles of 1,920 neuronal bodies and nuclei were
drawn using a camera lucida. Data was later recorded and processed with
a semiautomatic image analyser.
Results and conclusions: We observed that dLGN neurons do not change
in size from the age of 3–24 months. Between 24 and 30 months, the soma
and nucleus of the cell undergo hypertrophy, 32.8% and 35.6%, respectively, when compared to those from 3-month-old animals (P F 0.01).
Furthermore, we found a high correlation between cell body size/nucleus
size, which does not disappear with age. The r values (correlation coefficient) were 0.7998, 0.8662, 0.8433 and 0.7304, and R2 (determination
coefficient) was equal to 0.6397, 0.7504, 0.7112, and 0.5335. These latter
values show that in 63.97%, 75.04%, 71.12%, and 53.35% of cases, respectively, modifications in somata size were accompanied by similar changes
in nucleus size, and vice-versa. The study of the shape of the soma and
nucleus of the cell revealed that both structures have a rounded-oval
configuration that does not change in a significant way from adulthood to
old age. Anat. Rec. 248:137–141, 1997. r 1997 Wiley-Liss, Inc.
Key words: dLGN; aging; quantitative; size; shape
The morphological, neurochemical, and physiological
characteristics of the dLGN have been exhaustively
studied from postnatal development (Guillery, 1966;
Parnavelas et al., 1977; Heumann and Rabinowicz,
1980; Gottlieb et al., 1985; Aggelopoulos et al., 1989;
Villena et al., 1989, 1991; Dı́az et al., 1994) to adulthood
(Casagrande and Norton, 1991; Grossman et al., 1973;
Montero and Zempel, 1986; Pasik et al., 1990). However, data are scarce regarding changes occurring in
this and other brain regions during aging. Such data
should help to identify which brain areas are more
susceptible to aging and also what are the specific
changes taking place in each region during senescence.
In general, the basic morphological modifications
undergone by neurons during aging are known, but the
findings are controversial. Thus there is no agreement
regarding the different parameters currently under
study, e.g., neuronal size, extension of the dendritic
tree, or the number of synapses per cell (Curcio and
Hinds, 1983; Coleman and Flood, 1986; Markus et al.,
1987; Flood and Coleman, 1988; de Lacalle et al., 1991;
Crespo et al., 1992; Stroessner-Johnson et al., 1992;
r 1997 WILEY-LISS, INC.
Flood and Coleman, 1993; Peinado et al., 1993; Rance et
al., 1993). Similarly, it is argued whether or not neuronal loss occurs during old age (Diamond et al., 1977;
Curcio and Coleman, 1982; Satorre et al., 1985; Sturrock, 1989). All these data are fundamental for understanding the aging process in the brain and needed for
interpreting results coming from the application of
physiological, biochemical, and pharmacological methods.
In this study we have analysed the morphological
modifications occurring in the dLGN neurons from
adulthood to senescence. We used quantitative methods
that enabled us to assess changes in the size and shape
of the soma and nucleus of the cell. In this sense, the
quantification of somatic and nuclear size provides us
with very useful information to evaluate the integrity of
the neurons left in the senescent brain.
Received 23 July 1996; accepted 23 December 1996.
*Correspondence to: Department of Normal and Pathological Morphology, Faculty of Medicine, University of Málaga, 29080-Málaga,
Spain.
138
A. VILLENA ET AL.
MATERIALS AND METHODS
Animals
Sixteen male albino rats (Wistar strain) were used in
this study. They were subdivided into four groups of 3,
18, 24, and 30 months old (n 5 4 per age group). The
mean lifespan of this strain of rats is 27 months
(Janicke et al., 1985). Animals were provided with food
and water ad libitum and maintained in a temperaturecontrolled room with a 12-hour dark/light cycle. Their
weight was between 275 6 21 gm and 410 6 30.8 gm
(Table 1).
Tissue Preparation
Animals were anaesthetised with 8% chloral hydrate
(0.1 ml/30 gm weight). Fixation was achieved by intracardiac perfusion with buffered 10% formaldehyde (after a saline wash). The entire brains were then removed
and a block containing the dLGN dissected out and
placed in the same fixative for 48 hours. Thereafter,
they were processed in the standard way for embedding
in paraffin. Serial 8 µm coronal sections containing the
dLGN were made through the block’s entire length and
then stained with gallocyanine (Einarson, 1951).
Morphometric Study
A morphometric analysis of the size and shape of the
neuronal soma and nuclei was carried out using these
sections. The outlines of these cells were drawn with a
camera lucida attached to a Leitz Orthoplan microscope
using a 3100 oil objective. A total of 1,920 neuronal
bodies and nuclei profiles were measured (480 per
group, i.e., 120 per animal). The data was recorded and
processed with a semiautomatic image analyser (Kontron MOP-AM2). We chose only those neuron cell bodies
whose nucleoli were clearly visible (Konigsmark, 1970).
Neuron cell bodies were distinguished from glia on the
TABLE 1. Mean weight (mean 6 SEM) of rats from each
age group
Age (months)
Mean weight (g)
275 6 21
375 6 38.9
410 6 30.8
370 6 10
3
18
24
30
basis of size (neurons are much larger than glia in the
dLGN), the presence of a large and relatively pale
nucleus, and well-defined Nissl material in the cytoplasm of neurons. The following soma and nuclear
parameters were calculated: to evaluate the size (area,
perimeter, and maximum diameter) and to evaluate the
shape (form factor).
Statistical Analysis
The data are presented as mean 6 SEM (standard
error mean). Statistical analysis was performed first by
a computerised Kolmogorov-Smirnov test to evaluate
whether or not the data followed a normal distribution.
Because this test refuted the hypothesis of a normal
distribution in most parameters, the results obtained
were compared using the Kruskall-Wallis test (P , 0.05
and P , 0.01).
Finally, bivariate linear models were used to describe
the statistical relationship between soma area/nuclear
area.
RESULTS
Size Parameters
Area, perimeter, and maximum diameter of neuronal
cell bodies and nuclei followed similar modifications
during aging (Tables 2 and 3). Thus we observed that no
significant changes occurred in any parameter from the
3rd to the 24th month except for a slight increase (7.2%,
P , 0.05) in the maximum somatic diameter in the
24-month-old rats when compared to the 3-month-olds.
However, from the 24th month onward a significant
increase (P , 0.01) is observed in each parameter reaching their peak value in the 30th month (Tables 2 and 3).
In order to compare the behaviour of the area parameter among the different age groups, we analysed the
frequency distribution in the cell body and in the
nucleus (Fig. 1). At the age of 3 months, it was observed
that in the neuronal soma (Fig. 1A), the main percentage (26.66%) had an average size of between 110–130
µm2; at the age of 18 months, 25% of the soma were
between 130–150 µm2; at the 24th month 26.66% were
between 150–170 µm2; and finally at the age of 30 months,
27.5% had a size of 170–190 µm2. So, as the animal
advanced in age a deviation toward the right occurred in
the frequency distribution. In the nuclear area we observed
a similar pattern: 35% and 22.5% of the 3- and 18-month-
TABLE 2. Size and shape somatic parameters (mean 6 SEM) between 3 and 30
months of age1
Age (months)
3
Area (µm2)
Perimeter (µm)
Maximum diameter (µm)
Form factor
18
24
30
138.06 6 2.45
141.98 6 2.88
150.23 6 2.83* 183.47 6 2.88**,***,****
(100)
(102.8)
(108.8)
(132.8)
44.54 6 0.42
44.90 6 0.45
45.42 6 0.54
50.96 6 0.41**,***,****
(100)
(100.8)
(101.9)
(114.4)
13.73 6 0.19
14.66 6 0.20
14.73 6 0.38
15.73 6 0.21**,***,****
(100)
(106.7)
(107.2)
(114.5)
0.874 6 0.006
0.879 6 0.006
0.892 6 0.006
0.884 6 0.005
(100)
(100.5)
(102)
(101.1)
1Percent of values with respect to 3 months old in parens.
*P , 0.05 as compared to 3–24 months old.
**P , 0.01 as compared to 3–30 months old.
***P , 0.01 as compared to 18–30 months old.
****P , 0.01 as compared to 24–30 months old.
139
CHANGES IN NEURONS FROM dLGN DURING AGING
TABLE 3. Size and shape nuclear parameters (mean 6 SEM) between 3 and 30 months of age1
Age (months)
Area (µm2)
Perimeter
Maximum diameter (µm)
Form factor
3
18
24
30
78.61 6 1.79
(100)
33.10 6 0.34
(100)
10.29 6 0.15
(100)
0.922 6 0.005
(100)
80.87 6 2.19
(102.8)
33.58 6 0.42
(101.4)
10.83 6 0.18
(105.2)
0.906 6 0.006
(98.2)
85.03 6 1.73
(108.1)
34.84 6 0.34*
(105.2)
10.92 6 0.15
(106.1)
0.900 6 0.006
(97.6)
106.60 6 1.96**,***,****
(135.6)
38.46 6 0.32**,***,****
(116.1)
12.02 6 0.15**,***,****
(116.8)
0.925 6 0.005
(100.3)
1Percent of values with respect to 3 months old in parens.
*P , 0.05 as compared to 3–24 months old.
**P , 0.01 as compared to 3–30 months old.
***P , 0.01 as compared to 18–30 months old.
****P , 0.01 as compared to 24–30 months old.
Fig. 1. Frequency distribution for somatic (A) and nuclear (B) cross-sectional areas. Comparison
between the different ages shows a tendency to increasing sizes with advancing age.
old rats, respectively, had an average nucleus size of 70–85
µm2; 32.5% of the 24-month-old group had an average
between 85–100 µm2; and at the age of 30 months, 36.6%
showed a nucleus size of between 100–115 µm2 (Fig. 1B).
To determine the degree of dependence between
nuclear and somatic size and possible changes as age
advances, we calculated the correlation coefficient (r)
and the determination coefficient (R2 ) as well as their
corresponding regression lines in each age group (Fig.
2A–D). These revealed a high correlation at all ages
studied with values of 0.7998, 0.8662, 0.8433, and
0.7304 for r, and 0.6397, 0.7504, 0.7112, and 0.5335 for
R2. Therefore, changes in somatic size are accompanied
by similar changes in nuclear size, and vice-versa.
Shape Parameter
We calculated the form factor to detect possible
modifications in neuron cell bodies and nuclei shape
during the process of aging. We observed that in both
cases values were close to 1, but more so in the nucleus
(Tables 2 and 3). This shows that both the neuronal cell
body and the nucleus have a rounded-oval shape. Aging
does not seem to affect this parameter in a significant
way (Tables 2 and 3).
DISCUSSION
The growing interest in the process of aging has
encouraged great advances and diversification in neurobiological methodologies, which are applicable not only
to humans but to other animal species. Given the
difficulty of carrying out this type of research on humans,
laboratory animals are used. Among these, rodents present
many advantages (Masoro, 1990). Changes found in rodents can lead to an understanding of the processes
taking place during human aging (John, 1972).
Our investigation was carried out on dLGN neurons
from albino rats. The aim was to explore some basic
neurobiological questions concerning cell aging and
death, in line with earlier studies in which we had
already analysed some other aspects of this nucleus
during postnatal development and adulthood (Villena
et al., 1989, 1991; Dı́az et al., 1994).
We performed a neuronal morphological analysis
following morphometric methodology. Size parameters
such as area, perimeter, and maximum diameter were
evaluated. Area was given more importance as it is
considered the most useful parameter regarding neuronal size evaluation (Ewart et al., 1989; de Lacalle et al.,
140
A. VILLENA ET AL.
Fig. 2. Regression lines illustrating the relationship between somatic area/nuclear area at 3 (A), 18 (B),
24 (C), and 30 (D) months old. r 5 correlation coefficient. R2 5 determination coefficient.
1991; Peinado et al., 1993; Rance et al., 1993). We also
analysed shape parameters such as form factor with
values ranging from 0 to 1, the latter being the definition of a sphere.
Our results show that there are no significant changes
in somata and nuclear size between the ages of 3 and 24
months. However, between the 24th and the 30th
months, an important increase in all these parameters
is observed (P , 0.01). Thus the area of the cell body
increases from 150.23 6 2.83% µm2 to 183.47 6 2.88
µm2 and that of the nucleus increases from 85.03 6 1.73
µm2 to 106.60 6 1.96 µm2. The total area increase, as
compared with the young adult (3 months), is 32.8%
and 35.6% for the somata and the nucleus, respectively.
This coincides with observations of Ahmad and Spear
(1993) in monkey dLGN, who found an increase during
aging of 31% and 36% in the neuronal bodies of the
magno and parvicellular layers, respectively.
The increase observed in the mean somata size could
be due to a decrease in the soma of small neurons and/or
an increase in larger neurons’ soma as the animal’s age
advances (Sturrock, 1989; Stroessner-Johnson et al.,
1992). In this regard, by analysing the frequency distribution, we demonstrated that both processes are present. Thus at the 3rd month 14.99% of neurons are small
(,110 µm2 ), but at the 30th month this falls to 5%.
Furthermore, we observed that at the age of 3 months
most of the neuronal population (73.32%) have somata
ranging in size from 110–170 µm2, but in the 30th
month, 56.66% of them have somata ranging in size from
170–210 µm2. In the 3-month-old animals neuron cell
bodies larger than 210 µm2 are not found, but in the
30-month-old rats these neurons represent 19.16%. The
analysis of nuclear size revealed the self-same facts.
It can be inferred from our study that dLGN neurons
undergo a process of hypertrophy during senescence,
i.e., from the 24th month onward in the Wistar rat. This
phenomenon has been observed in different centres of
the central nervous system by other researchers (Flood
and Coleman, 1988; de Lacalle et al., 1991; Ahmad and
Spear, 1993; Stroessner-Johnson et al., 1992; Rance et
al., 1993). Nevertheless, other studies have found just
the opposite, i.e., stability and a shrinkage in size
(Luine et al., 1986; Peinado et al., 1993; Willot et al.,
1994). In some cases, the discrepancies might be due to
the varying susceptibility of the different centres of the
nerve system to aging, and in others to the species and
breeds employed as well as the different methodologies.
From previous results (submitted), we also know that
during aging there is an increase in volume of dLGN
and a decrease in neuronal density and number/mm3.
Thus the somatic hypertrophy observed in this study
CHANGES IN NEURONS FROM dLGN DURING AGING
from the 24th month onward could be explained as a
compensatory mechanism to maintain the same total
somatic volume in spite of the increase in the total
volume in the dLGN. This, according to Hinds and
McNelly (1977), Flood et al., (1985), and Coleman and
Flood (1986), could be related to an increase in the
dendritic tree in order to maintain the existing synaptic
connections, despite the increase in the dLGN total
volume, or perhaps, to compensate for the decrease in
the number of synapses and/or in their efficiency during
aging (Ahmad and Spear, 1993).
The data concerning the degree of correlation or
dependency between the size of the soma and nucleus of
the cell and their possible modifications during aging
have proved of interest. By analysing the correlation
and determination coefficients (r and R2, respectively)
we found that there was a high reciprocal dependence
between both parameters, which does not disappear in
the older animals. In 63.97%, 75.04%, 71.12%, and
53.35% of cases, modifications in somata size were
accompanied by a similar change in nuclear size, and
vice-versa. These data corroborate that found by de
Lacalle et al. (1991) during the same period in the
nucleus basalis of Meynert.
Finally, when the cell body and the nucleus shape
were studied, it was confirmed that dLGN neurons and
their nuclei have a rounded-oval shape with a value
close to 0.90 for the cell body and slightly higher for the
nucleus. It is important to note that this parameter
does not change from adulthood to senescence.
In summary, our findings reflect that during aging
the dLGN neurons undergo somatic hypertrophy with
an increase in the nucleus size and that a high correlation between the somatic and nuclear size is maintained throughout adulthood to aging. No modifications
in the shape of somata and nucleus were found during
senescence.
LITERATURE CITED
Aggelopoulos, N., J.G. Parnavelas, and S. Edmunds 1989 Synaptogenesis in the dorsal lateral geniculate nucleus of the rat. Anat.
Embryol., 180:243–257.
Ahmad, A., and P.D. Spear 1993 Effects of aging on the size, density
and number of rhesus monkey lateral geniculate nucleus. J.
Comp. Neurol., 334:631–643.
Casagrande, V.A., and T.T. Norton 1991 Lateral geniculate nucleus: A
review of its physiology and function. In: The Neuronal Basis of Visual
Function. A.G. Leventhal, ed. CRCP, Boca Ratón, pp. 41–48.
Coleman, P.D., and D.G. Flood 1986 Dendritic proliferation in the
aging brain as a compensatory repair mechanism. Prog. Brain
Res., 70:227–236.
Crespo, D., C. Fernández-Viadero, and C. González 1992 The influence
of age on supraoptic nucleus neurons of the rat: Morphometric and
morphologic changes. Mech. Ageing Develop., 62:223–228.
Curcio, C.A., and P.D. Coleman 1982 Stability of neurons number in
cortical bands of aging mice. J. Comp. Neurol., 212:158–172.
Curcio, C.A., and J.W. Hinds 1983 Stability of synaptic density and
spine volume in dentate gyrus of aged rats. Neurobiol. Aging,
4:77–87.
de Lacalle, S., I. Iraizoz, and M.A. Gonzalo 1991 Differential changes
in cell size and number in topographical subdivisions of human
basal nucleus in normal aging. Neuroscience, 43:445–456.
Diamond, M.C., R.E. Johnson, and M.W. Gold 1977 Changes in neuron
number and size and glia number in the young, adult and aging
rat medial occipital cortex. Behav. Biol., 20:409–418.
Dı́az, F., A. Villena, V. Requena, and I. Pérez de Vargas 1994
Cytochrome oxidase activity in the lateral geniculate nucleus of
postnatal rats. Brain Res. Bull., 35:269–271.
Einarson, L. 1951 On the theory of the gallocyanin-chromalum
staining and its applications for quantitative estimation of basophilia. Acta Path. Microbiol. Scand., 28:82.
Ewart, D.P., W.M. Ruzon, S.S. Fish, and N.H. McNee 1989 Nerve fibre
141
morphometry: A comparison of techniques. J. Neurosci. Meth.,
29:143–150.
Flood, D.G., and P.D. Coleman 1988 Neuron numbers and sizes in
aging brain: Comparisons of human, monkey, and rodent data.
Neurobiol. Aging, 9:453–463.
Flood, D.G., and P.D. Coleman 1993 Dendritic regression dissociated
from neuronal death but associated with partial deafferentation
in aging rat supraoptic nucleus. Neurobiol. Aging, 14:575–587.
Flood, D.G., S.J. Buell, C.H. DiFiore, G.J. Horwitz, and P.D. Coleman
1985 Age-related dendritic growth in dentate gyrus of human
brain is followed by regression in the ‘‘oldest old.’’ Brain Res.,
345:366–368.
Gottlieb, M.D., P. Pasik, and T. Pasik 1985 Early postnatal development of the monkey visual system. I. Growth of the lateral
geniculate nucleus and striate cortex. Dev. Brain Res., 17:53–62.
Grossman, A., A.R. Lieberman, and K.E. Webster 1973 A Golgi study of
the rat dorsal lateral geniculate nucleus. J. Comp. Neurol.,
150:441–466.
Guillery, R.W. 1966 A study of Golgi preparations from the dLGN of the
adult cat. J. Comp. Neurol., 128:21–50.
Heumann, D., and Th. Rabinowicz 1980 Postnatal development of the
dorsal lateral geniculate nucleus in the normal and enucleated
albino mouse. Exp. Brain Res., 38:75–85.
Hinds, J.W., and N.A. McNelly 1977 Aging of the rat olfactory bulb: An
atrophy of constituent layers and changes in size and number of
mitral cells. J. Comp. Neurol., 171:345–366.
Janicke, B., D. Wrobel, and G. Schulze 1985 The effects of various
drugs on performance capacity of old rats. Pharmacopsychiatric,
18:136–137.
John, H.A. 1972 The relevance of the rodent as a model system of aging
in man. In: Development of the rodent as a model system of aging,
D.C. Gibson, ed. DHEW Publ. No. (NIH) 72-121, pp. 3–6.
Konigsmark, B.W. 1970 Methods for the counting of neurons. In:
Contemporary Research Methods in Neuroanatomy. W.J.H. Nauta
and S.O.E. Ebbesson, eds. Springer-Verlag, New York, pp. 315–340.
Luine, V.N., K.J. Renner, S. Heady, and K.J. Jones 1986 Age and
sex-dependent decreases in ChAT in basal forebrain nuclei.
Neurobiol. Aging 7:193–198.
Markus, E.J., T.L. Petit, and J.C. LeBoutillier 1987 Synaptic structural changes during development and aging. Dev. Brain Res.,
35:239–248.
Masoro, E. 1990 Animal models in aging research. In: Handbook of the
Biology on Aging, E.L. Schneider and J.W. Rowe, eds. Academic
Press, New York, pp. 71–94.
Montero, V.M., and J. Zempel 1986 The proportion and size of
GABA-immunoreactive neurons in the magnocellular and parvocellular layers of the lateral geniculate nucleus of the rhesus
monkey. Exp. Brain Res., 62:215–223.
Parnavelas, J.G., R. Bradford, E.J. Mounty, and A.R. Lieberman 1977
Postnatal growth of neuronal perikarya in the dorsal lateral
geniculate nucleus of the rat. Neurosci. Lett., 5:33–37.
Pasik, P., R. Molinar-Rode, and T. Pasik 1990 Chemically specified
systems in the dorsal lateral geniculate nucleus of mammals. In:
Vision and the Brain: The Organization of the Central Visual
System. B. Cohen and I. Bodis-Wollner, eds. Raven Press, New
York, pp. 43–83.
Peinado, M.A., M. Martı́nez, J.A. Pedrosa, A. Quesada, and J.M.
Peinado 1993 Quantitative morphological changes in neurons and
glia in the frontal lobe of the aging rat. Anat. Rec., 237:104–108.
Rance, N.E., S.V. Uswandi, and N.T. McMullen 1993 Neuronal hypertrophy in the hypothalamus of older men. Neurobiol. Aging,
14:337–342.
Satorre, J., J. Cano, and F. Reinoso-Suarez 1985 Stability of the
neuronal population of the dorsal lateral geniculate nucleus
(LGNd) of aged rats. Brain Res., 339:375–377.
Stroessner-Johnson, H.M., P.R. Rapp, and D.G. Amaral 1992 Cholinergic cell loss and hypertrophy in the medial septal nucleus of the
behaviorally characterized aged rhesus monkey. J. Neurosci.,
12:1936–1944.
Sturrock, R.R. 1989 A quantitative histological study of the anterodorsal thalamic nucleus and the lateral mammilary nucleus of aging
mice. J. Hirnforsch., 30:191–195.
Villena, A., F. Dı́az, V. Requena, L. Vidal, and I. Pérez de Vargas 1989
Quantitative study of the dorsal lateral geniculate nucleus of the
rat during the postnatal development. J. Hirnforsch., 30:185–190.
Villena, A., V. Requena, F. Dı́az, and I. Pérez de Vargas 1991
Histochemical study of RNA content of neurones in the dorsal
lateral geniculate nucleus during postnatal development. Mech.
Ageing Develop., 57:275–282.
Willot, J.F., L.S. Bross, and S.L. McFadden 1994 Morphology of the
inferior colliculus in C57BL/6J and CBA/J mice across the life
span. Neurobiol. Aging, 15:175–183.