Age-related changes in the hippocampal subdivisions of the rat

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Age-related changes in the hippocampal
subdivisions of the rat

• Mohammad Hosseini-sharifabad, PhD
• Department of Anatomy
• Yazd University of Medical sciences
• E-mailmhosseini81_at_yahoo.com

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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 age-related 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.

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Background

• The hippocampal formation shows early signs of
  age- related 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 brainimaging 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.

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Background

• These findings support the hypothesis that an
  age-related 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.

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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.

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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.

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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 07.0019.00 hours)
  and provided food and water until sacrifice at
  6(young) and 24 (old) months of age.

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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.

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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.
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Coronal sections of 100µm thickness were cut
serially with a calibrated vibratome into a bath
of 3 potassium dichromate in distilled water.
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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.

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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.

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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

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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

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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.

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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.

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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.

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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 (Plt0.05). Results also
  showed that there is no significant difference in
  total volume of hippocampus between aged and
  young rats.

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Results

• T-test revealed a significant effect of normal
  aging on the total number of dendritic segments
  per cell in the CA1 pyramidal cells ( P0.01) 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
  (Plt0.05). 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.

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The total number of dendritic segments in granule
cells and CA3 and CA1 pyramidal cells of rat
hippocampus in young and aged rats.
Hippocampal Region Granule cells CA3 CA1 Hippocampal Region Granule cells CA3 CA1 Hippocampal Region Granule cells CA3 CA1 Animals
41.03.6 41.03.6 20.9 2.0 Young
36.51.8 39.1 3.6 18.5 2.0 Aged
P0.01 P0.314 P0.210
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Graphic representation of the dendritic branching
density of hippocampal granule cells in the aged
and young rats. Vertical bars represent SD.
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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, Plt0.05.
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Graphic representation of dendritic branching
density of CA3 pyramidal cells in the aged and
young rats. Vertical bars represent SD.
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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.

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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.

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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.

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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.

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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.

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Thank you for your attention