Age-related changes in the Age-related changes in the

hippocampal subdivisions of the rathippocampal subdivisions of the rat

Mohammad Hosseini-sharifabad, PhDMohammad Hosseini-sharifabad, PhDDepartment of AnatomyDepartment of Anatomy

Yazd University of Medical sciencesYazd University of Medical sciencesE-mail:mhosseini81@yahoo.comE-mail:mhosseini81@yahoo.com

BackgroundBackground Normal aging is commonly linked to a decline in Normal aging is commonly linked to a decline in

learning and memory.learning and memory.

• Understanding the mechanisms responsible for age-Understanding the mechanisms responsible for age-related cognitive changes remains a critical related cognitive changes remains a critical challenge in neurobiology.challenge in neurobiology.

Many studies examining cellular substrates of this Many studies examining cellular substrates of this age-related cognitive decline have focused on the age-related cognitive decline have focused on the

hippocampal formation because its structural hippocampal formation because its structural integrity is crucial for normal learning and memory integrity is crucial for normal learning and memory

and because it is especially vulnerable to the and because it is especially vulnerable to the process of agingprocess of aging.

BackgroundBackground

The hippocampal formation shows early signs of age- The hippocampal formation shows early signs of age- related changes in the brains of normal humans. Age related changes in the brains of normal humans. Age differences in total volume of the hippocampus have differences in total volume of the hippocampus have been observed in healthy humans with the use of both been observed in healthy humans with the use of both brain–imaging methods and stereological techniques.brain–imaging methods and stereological techniques.

Studies using modern stereological methods of Studies using modern stereological methods of quantification have established that, in rats, mice, quantification have established that, in rats, mice, monkeys and humans, the total number of granule monkeys and humans, the total number of granule cells in the dentate gyrus, and pyramidal neurons in cells in the dentate gyrus, and pyramidal neurons in CA3 and CA1 fields, preserved over the life span.CA3 and CA1 fields, preserved over the life span.

BackgroundBackground

These findings support the hypothesis that an age-These findings support the hypothesis that an age-related decline in hippocampal-dependent learning and related decline in hippocampal-dependent learning and memory may result from changes in other morphometric memory may result from changes in other morphometric parameters, rather than a loss of hippocampal neurons.parameters, rather than a loss of hippocampal neurons.

age-related changes in dendrites are of particular age-related changes in dendrites are of particular

interest since dendrites are the targets of the majority of interest since dendrites are the targets of the majority of synapses and since dendrites remain subject to synapses and since dendrites remain subject to structural changes even into adulthood. Recent structural changes even into adulthood. Recent experimental studies and models of dendritic processing experimental studies and models of dendritic processing suggest that both the extent and pattern of the dendritic suggest that both the extent and pattern of the dendritic arbor could influence how synaptic inputs are integrated.arbor could influence how synaptic inputs are integrated.

BackgroundBackground

In human studies, the influence of age on the In human studies, the influence of age on the hippocampus is confounded with other variables, hippocampus is confounded with other variables, such as inadequate nutrition, psychological and such as inadequate nutrition, psychological and physical stress.physical stress.

Therefore, this study aimed to determine the Therefore, this study aimed to determine the effects of normal advanced aging on the effects of normal advanced aging on the hippocampal subdivisions using animal model of hippocampal subdivisions using animal model of rat.rat.

BackgroundBackground

The present study investigated the effects of The present study investigated the effects of aging on the volumes of the layers in aging on the volumes of the layers in hippocampal subregions, where the respective hippocampal subregions, where the respective cell bodies or its processes were located.cell bodies or its processes were located.

we also performed a quantitative morphological we also performed a quantitative morphological analysis of dendritic architecture of Golgi-analysis of dendritic architecture of Golgi-impregnated hippocampal neurons from young impregnated hippocampal neurons from young and aged rats. and aged rats.

Animals and HousingAnimals and Housing

Male wistar rats were housed in a temperature- Male wistar rats were housed in a temperature-

controlled (22± 2 ºC) animal room and on a 12 hr controlled (22± 2 ºC) animal room and on a 12 hr

light/ dark cycle (light on at 07.00–19.00 hours) light/ dark cycle (light on at 07.00–19.00 hours)

and provided foodand provided food and water and water until sacrifice at until sacrifice at

6(young) and 24 (old) months of age6(young) and 24 (old) months of age..

MethodsMethods

RatsRats were deeply anesthetized with urethan and were deeply anesthetized with urethan and transcardially perfused with a phosphate-buffered transcardially perfused with a phosphate-buffered solution of 4% formaldehyde and 1% solution of 4% formaldehyde and 1% glutaraldehyde.glutaraldehyde. Each brain was numbered and Each brain was numbered and cerebellum and olfactory bulb were removed. The cerebellum and olfactory bulb were removed. The brains divided into hemispheres.brains divided into hemispheres.

One hemisphere was selected at random for One hemisphere was selected at random for estimating the volumes of layers, and the other for estimating the volumes of layers, and the other for morphometric analysis of neuronal dendrites. morphometric analysis of neuronal dendrites. Posterior portion of each hemisphere, Posterior portion of each hemisphere, which which contained hippocampus, was takencontained hippocampus, was taken..

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

Delineation of the hippocampal Delineation of the hippocampal regionsregions

Discrimination between the Discrimination between the

different subdivisions of the different subdivisions of the

hippocampal formation hippocampal formation was was

made according to cellmade according to cell

morphology.morphology.

CA1 and CA3 are fields of Cornu CA1 and CA3 are fields of Cornu

Ammonis; DG, dentate gyrus; M; Ammonis; DG, dentate gyrus; M;

Molecular layer; G, Granular Molecular layer; G, Granular

layer; H, Hilus, O, Oriens layer; layer; H, Hilus, O, Oriens layer;

P, Pyramidal layer; R, Radiatum P, Pyramidal layer; R, Radiatum

layer. Hematoxilin stain.layer. Hematoxilin stain.

Volume estimationVolume estimation

The Cavalieri principle used to The Cavalieri principle used to

estimate the reference volume estimate the reference volume

of of the constituent layers of the the constituent layers of the

hippocampal formationhippocampal formation. A grid . A grid

with a tessellation of points, with a tessellation of points,

randomly positioned on each randomly positioned on each

section, and the points hitting section, and the points hitting

each layer of hippocampal layer each layer of hippocampal layer

were counted. were counted.

Volume estimationVolume estimation

The number of points, The number of points, P, P, multiplied with the area multiplied with the area associated with each point, a associated with each point, a (P), to obtain an unbiased (P), to obtain an unbiased estimate of sectional area of estimate of sectional area of each profile. The sum of each profile. The sum of sectional areas of the layers sectional areas of the layers was used to estimate reference was used to estimate reference volume, V (ref), from the volume, V (ref), from the following relationship, where t following relationship, where t represents the distance represents the distance between sections. between sections.

V (ref) = t. V (ref) = t. P. a (p) = t. P. a (p) = t. A A

StainingStaining Incubate in 3% potassium dichromate in distilled water overnightIncubate in 3% potassium dichromate in distilled water overnight

Rinse in distilled waterRinse in distilled water

Mount on slides and glue coverslip over the sections at four cornersMount on slides and glue coverslip over the sections at four corners

Incubate in 1.5% silver nitrate in distilled water overnight in the darkIncubate in 1.5% silver nitrate in distilled water overnight in the dark

The following day, dismantle the slide assembliesThe following day, dismantle the slide assemblies

Rinse the tissue sectionsRinse the tissue sections

Rinse in distilled waterRinse in distilled water

Dehydrate in 95%, followed by absolute ethanol.Dehydrate in 95%, followed by absolute ethanol.

Clear the sections in xyleneClear the sections in xylene

Mount onto gelatinized slidesMount onto gelatinized slides

CoverslipCoverslip

CellCell selectionselection

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.

NumberNumber ofof dendriticdendritic segmentssegments 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 densityDendritic branching density

The branching density of dendritic The branching density of dendritic trees was evaluated by applying trees was evaluated by applying the method of concentric rings. the method of concentric rings. The number of dendritic The number of dendritic intersections crossing each intersections crossing each concentric ring centered in the cell concentric ring centered in the cell body was counted. The concentric body was counted. The concentric rings were calculated at interval of rings were calculated at interval of 20 µm for granule cells and 25 µm 20 µm for granule cells and 25 µm for CA3 and CA1 pyramidal cells. for CA3 and CA1 pyramidal cells. Whenever the dendrites extended Whenever the dendrites extended beyond 375 µm (circle 15), they beyond 375 µm (circle 15), they were included in circle 15.were included in circle 15.

ResultsResults

Statistical analysis revealed there is Statistical analysis revealed there is only a significant influence of age in the only a significant influence of age in the stratum radiatum and lacunosum-stratum radiatum and lacunosum-molecular of the CA1 pyramidal field, in molecular of the CA1 pyramidal field, in where the volume was lower in aged where the volume was lower in aged than young rats (P<0.05). Results also than young rats (P<0.05). Results also showed that there is no significant showed that there is no significant difference in total volume of difference in total volume of hippocampus between aged and young hippocampus between aged and young rats.rats.

ResultsResults T-test revealed a significant effect of normal aging T-test revealed a significant effect of normal aging

on the total number of dendritic segments per cell on the total number of dendritic segments per cell in the CA1 pyramidal cells ( P=0.01) but not in the in the CA1 pyramidal cells ( P=0.01) but not in the dentate granule cells and CA3 pyramidal cells dentate granule cells and CA3 pyramidal cells ( table 1).( table 1).

The number of dendritic intersections showed the The number of dendritic intersections showed the

effect of aging was significant for circles 11, 12, 13, effect of aging was significant for circles 11, 12, 13, 14 and 15 in CA1 pyramidal cells (P<0.05). In these 14 and 15 in CA1 pyramidal cells (P<0.05). In these circles aged had a lower number of intersections circles aged had a lower number of intersections than young rats. No significant age-related than young rats. No significant age-related difference was detected in the dendritic branching difference was detected in the dendritic branching density of granule cells and CA3 pyramidal cells. density of granule cells and CA3 pyramidal cells.

The total number of dendritic segmentsThe total number of dendritic segments in granule cells and CA3 and in granule cells and CA3 and CA1 pyramidal cells of rat hippocampus in young and aged rats.CA1 pyramidal cells of rat hippocampus in young and aged rats.

AnimalsAnimalsHippocampal RegionHippocampal Region

Granule cells CA3Granule cells CA3 CA1CA1

YoungYoung20.9 ±2.020.9 ±2.041.0±3.641.0±3.641.0±3.641.0±3.6

AgedAged18.5 ±2.018.5 ±2.039.1 ±3.639.1 ±3.636.5±1.836.5±1.8

P=P=0.2100.210P=P=0.3140.314P=P=0.010.01

Graphic representation of the dendritic branching density of Graphic representation of the dendritic branching density of hippocampal granule cells in the aged and young rats. Vertical bars hippocampal granule cells in the aged and young rats. Vertical bars represent SD.represent SD.

Granule cells

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Aged (24 months) Young (6 months)

Graphic representation of dendritic branching density of CA1 Graphic representation of dendritic branching density of CA1 pyramidal cells in the aged and young rats. Vertical bars represent pyramidal cells in the aged and young rats. Vertical bars represent

SD. Circles 11, 12, 13, 14 and 15, P<0.05SD. Circles 11, 12, 13, 14 and 15, P<0.05..

CA1 pyramidal cells

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Graphic representation of dendritic branching density of CA3 Graphic representation of dendritic branching density of CA3 pyramidal cells in the aged and young rats. Vertical bars represent pyramidal cells in the aged and young rats. Vertical bars represent SD.SD.

CA3 pyramidal cells

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DiscussionDiscussion

Our present findings revealed the alterations in the Our present findings revealed the alterations in the terminal segments of the apical arbors of the CA1 terminal segments of the apical arbors of the CA1 hippocampal cells due to normal aging.hippocampal cells due to normal aging.

Reports on age-related alterations in morphology Reports on age-related alterations in morphology of the rodent CA fields have been inconsistent, of the rodent CA fields have been inconsistent, however. Some indicate a reduction in synaptic however. Some indicate a reduction in synaptic contacts and dendrites while others suggest contacts and dendrites while others suggest preservation of connectivity. It is likely that preservation of connectivity. It is likely that methodological differences, such as rodent strain, methodological differences, such as rodent strain, age of subjects, and precise hippocampal region age of subjects, and precise hippocampal region examined, contribute to the disparity of findings.examined, contribute to the disparity of findings.

DiscussionDiscussion

Although there are several descriptions of age-Although there are several descriptions of age-

related dendritic changes, the mechanisms related dendritic changes, the mechanisms

controlling dendritic morphology in the adult and controlling dendritic morphology in the adult and

aging brain have not been elucidated.aging brain have not been elucidated.

Since individual trophic factors promote dendritic Since individual trophic factors promote dendritic

growth of specific populations of neurons and can growth of specific populations of neurons and can

act within restricted dendritic domains, it is act within restricted dendritic domains, it is

reasonable to propose that differential trophic reasonable to propose that differential trophic

support continues across the lifespan and that age-support continues across the lifespan and that age-

related declines in trophic support lead to dendritic related declines in trophic support lead to dendritic

regression in some neural regions.regression in some neural regions.

DiscussionDiscussion

Throughout development and adulthood the availability Throughout development and adulthood the availability of growth factors is likely to differ among cortical of growth factors is likely to differ among cortical regions and layers due to differences in capillary density regions and layers due to differences in capillary density and blood flow. and blood flow.

As levels of many blood-borne trophic factors decline As levels of many blood-borne trophic factors decline with age, the focal differences in blood flow that exist with age, the focal differences in blood flow that exist throughout the lifespan, coupled with age-related throughout the lifespan, coupled with age-related vascular changes, may result in local deficiencies in one vascular changes, may result in local deficiencies in one or more critical trophic factors that lead to dendritic or more critical trophic factors that lead to dendritic regression among some neurons.regression among some neurons.

DiscussionDiscussion

The CA1 region is the hippocampal subdivision in The CA1 region is the hippocampal subdivision in

which the cytoarchitectural organization and size have which the cytoarchitectural organization and size have

changed most during mammalian and its structural changed most during mammalian and its structural

integrity has been reported to be particularly integrity has been reported to be particularly

susceptible to ischemia.susceptible to ischemia.

It is also reported that Microtubule –associated 2 It is also reported that Microtubule –associated 2

protein (MAP2) has been decreased in the dendrites protein (MAP2) has been decreased in the dendrites

and axons of hippocampal CA1 neurons in aged mice. and axons of hippocampal CA1 neurons in aged mice.

These findings demonstrate that dendrites and axons These findings demonstrate that dendrites and axons

in the hippocampal CA1 neurons are particularly in the hippocampal CA1 neurons are particularly

susceptible to aging processes.susceptible to aging processes.

ConclusionConclusion

This and similar descriptive studies demonstrate This and similar descriptive studies demonstrate

that throughout adulthood and senescence dendritic that throughout adulthood and senescence dendritic

extent is regulated locally and that changes affect extent is regulated locally and that changes affect

specific populations of neurons and restricted specific populations of neurons and restricted

regions within the dendritic arbors. Recognition of regions within the dendritic arbors. Recognition of

which neurons are subject to dendritic regression which neurons are subject to dendritic regression

and of how dendritic geometry is altered will and of how dendritic geometry is altered will

facilitate experimental studies to assess the facilitate experimental studies to assess the

significance of dendritic change for neural function, significance of dendritic change for neural function,

as well as the mechanisms by which dendritic as well as the mechanisms by which dendritic

extent is regulated throughout the lifespan.extent is regulated throughout the lifespan.

Thank you for your attention