Transcript Altman presentation - Neuron Development

1. EARLY WORK WITH
3H-LEUCINE
Unstained autoradiogram
of the spinal cord from a
rat that was injected with
2 µc of 3H-leucine 2
hours before it was
killed. The accumulated
grains indicates the level
of protein metabolism.
Abbreviations:
cc – central canal
DH – Dorsal horn
VH – ventral horn
2. EARLY WORK
WITH 3H-LEUCINE
Examples of the differences in
3H-leucine uptake by motor
neurons in the cervical spinal
cord in resting (left panel) and
exercised (right panel) rats.
RESTING
EXERCISED
3. EARLY WORK WITH 3H-LEUCINE
Examples of the differences
in 3H-leucine uptake by
neurons in the reticular
formation in resting (left
panel) and exercised (right
panel) rats.
RESTING
EXERCISED
4. EARLY WORK WITH 3H-LEUCINE
The results of uptake studies
showed great variability
between animals during and
after exercise.
I began to doubt the
repeatability of this work, and
I turned to 3H-thymidine to
standardize the autoradiographic technique.
• 3H-thymidine autoradiography is the
ideal technique to study cell proliferation.
• The nervous system contains many
proliferating glial cells in mature animals.
• To our surprise, we found evidence that
the small granule cells (NEURONS) in the
dentate gyrus of the hippocampus are
generated in the ADULT rat brain.
The Problem of Adult Neurogenesis
The great Spanish neuroscientist,
Santiago Ramón y Cajal, had an
interest in the regeneration of the
injured brain.
To his disappointment, he could
find no evidence of neuron
multiplication in adults.
That gave rise to the Dogma:
NO ADULT NEUROGENESIS
THE RAT HIPPOCAMPUS
This is a specialized area of the cerebral cortex in mammalian brains.
Ammon’s horn (AH) contains a thin layer of pyramidal cells (pl) that
form a large C-shaped curve. The dentate gyrus (DG) has a thick
layer of small granule cells (gl).
Examples of
granule cells
(red arrows)
that were
generated in
an adult rat
brain.
From: Altman, J., 1963
Autoradiographic
investigation of cell
proliferation in the brains
of rats and cats. The
Anatomical Record, 145
(4):573-591.
Adult Neurogenesis:
A Systematic Research Program
• To link adult hippocampal neurogenesis with
embryonic, fetal, infantile, and juvenile
neurogenesis, we did a systematic study of
neuron development in rats.
• With 3H-thymidine autoradiography Shirley
Bayer established that 85% of granule cells in
the dentate gyrus are generated postnatally.
• Later, Bayer found that the total number of
dentate granule cells increases during
adulthood.
The dentate granular layer
in a rat that was injected
with 3H-thymidine on
postnatal day (P) 2 and
killed on P60. Note only
the most superficial
granule cells are
unlabeled. All the labeled
cells were formed after
P2.
The same in a rat that was
injected with 3H-thymidine
on P13 and survived to
P60. Note the deep
placement of the labeled
granule cells.
From Altman, J. 1966.
Autoradiographic investigations … II.
Journal of Comparative Neurology
128:431-474.
In the right dentate gyrus, the
number of granule cells
continually increases from
around 900,000 (30 days) to
over 1,275,000 (365 days).
THE ADULT-GENERATED
NEURONS SUBSTANTIALLY
ADD TO THE POPULATION
OF GRANULE CELLS IN THE
HIPPOCAMPUS.
Over 1,100 neurons are added
to the population EVERY DAY
between 30 and 365 days
Bayer, SA et al., 1982 Science 216:890892.
Bayer, SA 1982 Experimental Brain
• While Ramón y Cajal was correct
that neurons do not multiply, we
showed that precursor cells of
neurons, now called STEM
CELLS, do multiply.
• We called the layer of precursor
cells in the dentate gyrus, the
SUBGRANULAR ZONE.
The subgranular zone
in a rat injected with
3H-thymidine on P5-P8
and killed on P9.
The dentate gyrus in a
rat injected with 3Hthymidine on P10 and
killed 2 months later.
Note the predominantly
deep placement of the
labeled cells.
We subsequently showed
that the subgranular zone
is present in the dentate
gyrus of other mammals,
such as the guinea pig
and the cat.
GUINEA PIG SUBGRANULAR ZONE
6 DAYS
18 DAYS
CAT SUBGRANULAR ZONE
30 DAYS
60 DAYS
The complete story of
hippocampal development
in diagrammatic form.
On Embryonic day (E)18, the
neuroepithelium (NEP) is
differentiated and is beginning to
generate neurons that just start
to migrate (arrows show
trajectories).
Most granule cells come from
a dispersed germinal matrix,
the SUBGRANULAR ZONE,
not directly from the
neuroepithelium.
On P30, only the subgranular
zone is still present AND
REMAINS to generate granule
cells in juvenile and adult
brains.
•
•
Postnatally-forming
precursors of neurons in the
hippocampus are highly
vulnerable and are killed
with low-level X-irradiation.
This permanently interferes
with adult neurogenesis.
Normal and
X-irradiated
Hippocampus
Normal rat hippocampus
at 3 months, showing the
dentate gyrus (DG) with
the thick granular layer
(gl) and Ammon’s horn
(AH) with a thin pyramidal
cell layer (pl).
The SELECTIVELYreduced granular layer
(arrows) in a 3 month-old
rat that was given 8 doses
of X-rays between P2 and
P15.
We put the X-irradiated rats
through a series of tests to
study consequent
behavioral abnormalities.
Hippocampal X-irradiation: Behavioral Studies
OPEN FIELD
TEST
A rat is placed in
the central
square, and the
number of
squares entered
during a
specified time is
recorded.
Hippocampal X-irradiation: Behavioral Studies
The hippocampal
X-irradiated animals
are HYPERACTIVE
in an open field.
Hippocampal X-irradiation: Behavioral
Studies
SPONTANEOUS
ALTERNATION
TEST.
Will a rat re-enter an
arm of a T-maze that it
has already explored?
Normal rats
spontaneously
alternate above chance
level on the second
trial.
Hippocampal X-irradiation: Behavioral
Studies
The X-irradiated animals
have SHORT-TERM
MEMORY DEFICITS since
they readily re-enter the
same arm of a T-maze in
successive trials.
Hippocampal Xirradiation:
Behavioral Studies
The PASSIVE AVOIDANCE
apparatus first shapes hungry rats to
eat from a food cup when the door is
opened.
A day later the rats are shocked when
they eat from the food cup.
On the next testing day, the hungry
rats are again placed in the
apparatus and the door is opened.
The time it takes for the rat to
approach the food cup is recorded.
Normal rats stay in the start box for a
long time to passively avoid being
Hippocampal X-irradiation: Behavioral
Studies
The X-irradiated
animals have
LEARNING DEFICITS
to avoid shock
because they
approach the food cup
after a shorter delay
than normal rats.
CONCLUSION: The “Good”
News
• Our research, and those of many other
developmental neurobiologists,
established that the regenerative capacity
of the nervous system is FAR GREATER
than it was believed.
• Is it possible that adult-generated neurons
can be coaxed into therapies to effectively
remedy developmental disorders like
autism, or degenerative disorders like
Alzheimer’s disease?
CONCLUSION: The “Bad” News
• The stem cells that give rise to neurons in
adult brains are highly vulnerable and can
be killed by exposure to radiation, alcohol,
drugs, etc.
• The absence of postnatal and adultgenerated neurons affects the function of
specific brain structures and leads to
learning disabilities, abnormal behavior,
and other disorders.
Please check our two websites for
our latest research.
neurondevelopment.org
Here you can find pdf files of our past publications and our
current work on MENTAL EVOLUTION.
braindevelopmentmaps.org
This is a new website that archives our histological collection of
normal developing rat brains with high resolution images that
can be downloaded and used for your own research.