Download Age-related changes in the hippocampal subdivisions of the rat

Age-related changes in the
hippocampal subdivisions of the rat
Mohammad Hosseini-sharifabad, PhD
Department of Anatomy
Yazd University of Medical sciences
E-mail:[email protected]
Background

Normal aging is commonly linked to a decline in learning
and memory.
•
Understanding the mechanisms responsible for age-related
cognitive changes remains a critical challenge in
neurobiology.

Many studies examining cellular substrates of this agerelated cognitive decline have focused on the hippocampal
formation because its structural integrity is crucial for
normal learning and memory and because it is especially
vulnerable to the process of aging.
Background

The hippocampal formation shows early signs of agerelated changes in the brains of normal humans. Age
differences in total volume of the hippocampus have
been observed in healthy humans with the use of both
brain–imaging methods and stereological techniques.

Studies using modern stereological methods of
quantification have established that, in rats, mice,
monkeys and humans, the total number of granule cells
in the dentate gyrus, and pyramidal neurons in CA3 and
CA1 fields, preserved over the life span.
Background

These findings support the hypothesis that an agerelated decline in hippocampal-dependent learning and
memory may result from changes in other morphometric
parameters, rather than a loss of hippocampal neurons.

age-related changes in dendrites are of particular
interest since dendrites are the targets of the majority of
synapses and since dendrites remain subject to
structural changes even into adulthood. Recent
experimental studies and models of dendritic processing
suggest that both the extent and pattern of the dendritic
arbor could influence how synaptic inputs are integrated.
Background

In human studies, the influence of age on the
hippocampus is confounded with other variables, such
as inadequate nutrition, psychological and physical stress.

Therefore, this study aimed to determine the effects of
normal advanced aging on the hippocampal subdivisions
using animal model of rat.
Background

The present study investigated the effects of aging on
the volumes of the layers in hippocampal subregions,
where the respective cell bodies or its processes were
located.

we also performed a quantitative morphological analysis
of
dendritic
architecture
of
Golgi-impregnated
hippocampal neurons from young and aged rats.
Animals and Housing

Male wistar rats were housed in a temperature- controlled
(22± 2 ºC) animal room and on a 12 hr light/ dark cycle
(light on at 07.00–19.00 hours) and provided food and
water until sacrifice at 6(young) and 24 (old) months of
age.
Methods

Rats were deeply anesthetized with urethan and
transcardially perfused with a phosphate-buffered
solution of 4% formaldehyde and 1% glutaraldehyde.
Each brain was numbered and cerebellum and olfactory
bulb were removed. The brains divided into
hemispheres.
One hemisphere was selected at random for estimating the
volumes of layers, and the other for morphometric analysis
of neuronal dendrites. Posterior portion of each
hemisphere, which contained hippocampus, was taken.
Coronal sections of 100µm thickness were cut
serially with a calibrated vibratome into a bath of
3% potassium dichromate in distilled water.
Delineation of the hippocampal regions

Discrimination between the different
subdivisions of the hippocampal
formation was made according to
cell morphology.

CA1 and CA3 are fields of Cornu
Ammonis; DG, dentate gyrus; M;
Molecular layer; G, Granular layer;
H, Hilus, O, Oriens layer; P,
Pyramidal layer; R, Radiatum layer.
Hematoxilin stain.
Volume estimation

The Cavalieri principle used to
estimate the reference volume of
the constituent layers of the
hippocampal formation. A grid with
a tessellation of points, randomly
positioned on each section, and the
points hitting each layer of
hippocampal layer were counted.
Volume estimation

The number of points, P, multiplied
with the area associated with each
point, a (P), to obtain an unbiased
estimate of sectional area of each
profile. The sum of sectional areas
of the layers was used to estimate
reference volume, V (ref), from the
following relationship, where t
represents the distance between
sections.
V (ref) = t.  P. a (p) = t.  A
Staining

Incubate in 3% potassium dichromate in distilled water overnight

Rinse in distilled water

Mount on slides and glue coverslip over the sections at four corners

Incubate in 1.5% silver nitrate in distilled water overnight in the dark

The following day, dismantle the slide assemblies

Rinse the tissue sections

Rinse in distilled water

Dehydrate in 95%, followed by absolute ethanol.

Clear the sections in xylene

Mount onto gelatinized slides

Coverslip
Cell selection

The criteria employed for selecting
the neurons to be measured were the
following:
(1) Dark and consistent
impregnation throughout the extent
of dendrites
(2) Cell bodies located in the middle
part of the section thickness to
minimize branch segments cut off at
the plan of the section
(3) Relative isolation from
neighbouring impregnated cells in
order not to irresolvable overlap the
dendrites of adjacent cells.
Number of dendritic segments

A Camera lucida drawing of the
granule cell in which the
classification of the dendritic
segments is shown. The numbers
represent the degree of dendrites:
1: terminal segments ; 2:
intermediate segments originating
two terminals ; 3, 4: intermediate
segments divided in a terminal
and an intermediate segment.

The centrifugal ordering of
dendritic trees was used to
estimate the number of dendritic
segments per cell.
Dendritic branching density

The branching density of dendritic trees
was evaluated by applying the method
of concentric rings. The number of
dendritic intersections crossing each
concentric ring centered in the cell body
was counted. The concentric rings were
calculated at interval of 20 µm for
granule cells and 25 µm for CA3 and
CA1 pyramidal cells. Whenever the
dendrites extended beyond 375 µm
(circle 15), they were included in circle
15.
Results

Statistical analysis revealed there is only a
significant influence of age in the stratum
radiatum and lacunosum-molecular of the
CA1 pyramidal field, in where the volume
was lower in aged than young rats
(P<0.05). Results also showed that there
is no significant difference in total volume
of hippocampus between aged and young
rats.
Results

T-test revealed a significant effect of normal aging on
the total number of dendritic segments per cell in the
CA1 pyramidal cells ( P=0.01) but not in the dentate
granule cells and CA3 pyramidal cells ( table 1).

The number of dendritic intersections showed the effect
of aging was significant for circles 11, 12, 13, 14 and 15
in CA1 pyramidal cells (P<0.05). In these circles aged
had a lower number of intersections than young rats. No
significant age-related difference was detected in the
dendritic branching density of granule cells and CA3
pyramidal cells.
The total number of dendritic segments in granule cells and CA3 and CA1
pyramidal cells of rat hippocampus in young and aged rats.
Animals
Hippocampal Region
Granule cells
CA3
CA1
Young
20.9 ±2.0
41.0±3.6
41.0±3.6
Aged
18.5 ±2.0
39.1 ±3.6
36.5±1.8
P=0.210
P=0.314
P=0.01
Graphic representation of the dendritic branching density of hippocampal
granule cells in the aged and young rats. Vertical bars represent SD.
Aged (24 months)
Young (6 months)
Number of intersections
Granule cells
8
7
6
5
4
3
2
1
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Graphic representation of dendritic branching density of CA1 pyramidal cells in
the aged and young rats. Vertical bars represent SD. Circles 11, 12, 13, 14 and
.
15, P<0.05
Number of Intersections
CA1 pyramidal cells
8
7
6
5
*
4
*
3
*
*
2
*
1
0
1
B
2
3
4
5
6
7
8
Circles
9
10 11 12 13 14 15
Graphic representation of dendritic branching density of CA3 pyramidal cells in
the aged and young rats. Vertical bars represent SD.
Number of intersections
CA3 pyramidal cells
9
8
7
6
5
4
3
2
1
0
1
C
2
3
4
5
6
7
8
Circles
9
10 11 12 13 14 15
Discussion

Our present findings revealed the alterations in the
terminal segments of the apical arbors of the CA1
hippocampal cells due to normal aging.

Reports on age-related alterations in morphology of the
rodent CA fields have been inconsistent, however. Some
indicate a reduction in synaptic contacts and dendrites
while others suggest preservation of connectivity. It is
likely that methodological differences, such as rodent
strain, age of subjects, and precise hippocampal region
examined, contribute to the disparity of findings.
Discussion

Although there are several descriptions of age-related
dendritic changes, the mechanisms controlling dendritic
morphology in the adult and aging brain have not been
elucidated.

Since individual trophic factors promote dendritic growth
of specific populations of neurons and can act within
restricted dendritic domains, it is reasonable to propose
that differential trophic support continues across the
lifespan and that age-related declines in trophic support
lead to dendritic regression in some neural regions.
Discussion

Throughout development and adulthood the availability
of growth factors is likely to differ among cortical regions
and layers due to differences in capillary density and
blood flow.

As levels of many blood-borne trophic factors decline
with age, the focal differences in blood flow that exist
throughout the lifespan, coupled with age-related
vascular changes, may result in local deficiencies in one
or more critical trophic factors that lead to dendritic
regression among some neurons.
Discussion

The CA1 region is the hippocampal subdivision in which
the cytoarchitectural organization and size have changed
most during mammalian and its structural integrity has
been reported to be particularly susceptible to ischemia.

It is also reported that Microtubule –associated 2 protein
(MAP2) has been decreased in the dendrites and axons
of hippocampal CA1 neurons in aged mice. These
findings demonstrate that dendrites and axons in the
hippocampal CA1 neurons are particularly susceptible to
aging processes.
Conclusion

This and similar descriptive studies demonstrate that
throughout adulthood and senescence dendritic extent is
regulated locally and that changes affect specific
populations of neurons and restricted regions within the
dendritic arbors. Recognition of which neurons are
subject to dendritic regression and of how dendritic
geometry is altered will facilitate experimental studies to
assess the significance of dendritic change for neural
function, as well as the mechanisms by which dendritic
extent is regulated throughout the lifespan.
Thank you for your attention