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Running head: KITTENS EXPERIMENT
Proposal to Extend the Classic Hubel and Wiesel Kittens Experiment
Nathan Sommer
Creighton University
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Abstract
This paper proposes an extension for the Nobel Prize (1981) winning work of Hubel and Wiesel.
Mimicking the classic experiment, kittens will be deprived of visual sensory stimulation from
birth until the end of the critical period of brain development. Neural precursor cells will then be
injected into the C-4 Layer of the visual cortex to restore a totally functioning visual processing
center. The procedure for the experiment is outlined and expected results are discussed in light
of relevant scientific studies.
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Proposal to Extend the Classic Hubel and Wiesel Kittens Experiment
In a famous Nobel Prize winning experiment which was recognized in 1981, Hubel and
Weisel took kittens at birth and deprived them of visual sensory stimulation during the critical
period of brain development. After the critical period had passed, the kittens were allowed to
fully experience visual sensory stimulation. However the brains of those kittens were unable to
process visual information and the kittens were in effect, blind (Purves et al., 2001). Decades
after that experiment, I am proposing that there be an extension of Hubel and Wiesel’s work.
My proposal is aimed at investigating the establishment of a new critical period of brain
development with respect to the visual system. Specifically I will examine whether injecting
neural precursor cells into the 4-C layer of the visual cortex of kittens raised in the manner done
by Hubel and Wiesel, can enable normal development of the ocular dominance columns of 4-C
and visual sensory neural pathways outside of the critical period of brain development. The
therapeutic ramifications and the knowledge that can be gained by such an experiment are vast.
Consider Mike May. Blind most of his life, he is now a married adult with children.
Recently doctors were able to repair his dysfunctional photoreceptors using stem cells. His eyes
have been repaired to such a degree that there is no physical reason as to why his eyes do not
allow him to recognize his wife or kids (Abrams, 2002). The real problem lies within the fact
that his brain developed as a young child without receiving visual sensory stimulation from the
eyes which would enable the proper formation of synaptic connections within the visual cortex.
Even though Mike’s eyes are now capable of relaying visual sensory information to his brain, the
brain is incapable of interpreting this information and he remains blind.
A similar scenario can be seen in individuals with severe hearing loss or deafness who do
not receive hearing aids or cochlear implants until later on in life. These individuals have been
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given the ability to send the brain audio sensory information through technology. However the
brain remains incapable of completely interpreting the sensory information it is receiving even
after months of adjustment and intense language therapy (Hensh et al., 1998, p. 1504). Hensh
summarizes this reality by stating “sensory experiences in early life shapes the mammalian brain.
The process of growth is one of activity dependant refinement of functional connections within
the cerebral cortex” (Hensh et al., 1998, p. 1504). Therefore the key to recuperation in
individuals suffering from the early onset of hearing loss or blindness lies within extending or reestablishing the critical period of development of the sensory neural pathways or creating a new
period of development altogether. I seek to do the latter.
The initial stages of the proposed experiment will mimic that of Hubel and Wiesel’s
classic experiment. Immediately after birth, the eyes of a dozen kittens will be sewn shut to
prevent visual stimulation. After the critical period of brain development has passed, which is
twelve weeks after birth in kittens (Ferster, 2004), the stitches will be removed. The kittens will
then have neural precursor cells injected into the visual cortex in hopes of establishing new
visual sensory pathways. The injections will be followed by a lengthy period of two years of
normal exposure to day/night cycles and light intensities. Whenever possible, the environment
will be held constant among test subjects. This includes but is not limited to, light exposure,
diet, and exercise. During and after that period, the development of neural activity will be
closely monitored.
The neural precursor cells must be injected into layer 4-C of the visual cortex. All neural
layers pertinent to the study up to layer 4-C of the visual cortex form independently of 4-C and
do not require sensory stimulation to form synaptic connections. However layer 4-C requires
sensory stimulation for neurons to survive and form healthy synaptic connections in accordance
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with the Hebbian rules (Dr. Shibata, personal communication, Spring 2004). The Hebbian rules
follow the use it or lose it principle and simply state that neurons that fire together, wire together.
Synaptic connections are formed between neurons that fire together and these neurons grow
stronger at the expense of weaker ones because neurotrophic factors are released by target cells
which keep active neurons alive (Dr. Shibata, personal communication, Spring 2004). The
release of neurotrophic factors is stimulated by active pre-synaptic neurons (Dr. Shibata,
personal communication, Spring 2004). Essentially survival and growth of neurons depends on
an exchange between pre-synaptic neurons and target neurons in which activation is exchanged
for neurotrophic factors.
As noted before, the layers up to 4-C will have developed with a good degree of
normalcy. However the synaptic connections between these layers and 4-C will not have
developed. Thus the ocular dominance columns will not be formed (Dr. Shibata, personal
communication, Spring 2004). The axons immediately preceding layer 4-C are the genicoulo
cortical axons (Dr. Shibata, personal communication, Spring 2004). To increase neural activity
in the targeted region and thus facilitate integration of the newly developing 4-C layer, NMDA
will be added to increase activity among the genicoulo cortical axons (Dr. Shibata, personal
communication, Spring 2004). The NMDA will stimulate growth by mimicking the effects of
the neurotrophic factors known as glutamate which are normally released by endogenous cells
(Dr. Shibata, personal communication, Spring 2004). Thus the NMDA will increase the rate of
maturation of cortical neurons (Dr. Shibata, personal communication, Spring 2004).
Park et al. (2003) found that “transplantation of fetal dopaminergic neurons in patients
suffering from Parkinson’s disease, yielded dramatic relief from symptoms” (Park et al., 2003, p.
91). From these results it is expected that the integration of cloned neural precursor cells should
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go smoothly and begin to assume normal neural connections and the functions with which such
connections are associated. However, to provide a sufficient quantity of cells to be transplanted
as well as avoiding rejection by the immune system, it will be necessary to culture a large
population of clone cells.
For those reasons and to eliminate genetic variation as a variable in the experiment, I
recommend using only cloned kittens if possible. Obviously though, this will entail a significant
financial cost. The use of cloned stem cells is a necessity, however much can still be learned
through a study of non-cloned kittens.
In one pertinent experiment, students with normal vision were taught to read Braille
while their eyes were closed (Dr. Snipp, personal communication, Spring 2004). After many
weeks of study they developed the ability to read Braille. However, when the students were
allowed to see while attempting to read Braille, they were unable to do so (Dr. Snipp, personal
communication, Spring 2004). The Braille experiment goes to show that the brain is capable of
reorganizing how it processes sensory information.
Those findings were confirmed in a University of California-Berkley study. DeAngelis,
Anzai, Ohzaawa, and Freeman (1995) report that “sensory areas of the adult cerebral cortex can
reorganize in response to long-term alterations in patterns of afferent signals” (DeAngelis, Anzai,
Ohzaawa, & Freeman, 1995, p. 9682). In fact, “topographic reorganization is likely based on
plasticity at the cortical level” (Calford et al., 2000, p. 587). The sum of the Braille and Berkley
experiments suggests that a brain which has undergone some degree of development, even if
certain sensory areas of the cerebral cortex do not develop, is still capable of reorganizing to
such a degree that there should be successful implementation of neural sensory pathways which
did not exist during the normal course of development during the critical period.
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At the University of Michigan, it was shown that “primitive neural cells respond to brain
injuries by migrating to the injured area and attempting to form new neurons” (Pobojewski,
2002, p. 1). The primitive cells used were neuroblasts, “cells midway between a stem cell and a
fully developed neuron” (Pobojewski, 2002, p. 1). Once neuroblasts migrated to the injured
area, they began to multiply and form neural connections (Pobojewski, 2002).
The knowledge obtained by the University of Michigan study is useful in the proposed
experiment in several ways. First, it reveals that neural precursor cells injected into the 4-C layer
of the primary visual cortex, by the occipital lobe, may not stay in one place. Rather, they will
migrate to different areas where the precursor cells have the ability to develop into fully
functioning neurons, potentially capable of restoring a totally functioning visual processing
center in kittens initially deprived of developing such a center. This migration phenomena is
beneficial to the experiment in that the experimenters will hopefully not have to rely on hit or
miss trials to find where neural precursor cells need to be injected to achieve the desired results.
Instead, the injections can occur sporadically throughout the 4-C layer of the cerebral cortex and
the body’s natural migration effect will take over in directing the cells to the necessary locations.
If in the initial trials the cells are labeled with some type of radioactive dye and tracked to their
final destinations, subsequent trial injections can be made with greater accuracy. The
recommended tracking method would be to infect the cloned neural precursor cells with a virus
genetically modified not to kill the cell and to produce a green fluorescent protein (Dr. Shibata,
personal communication, Spring 2004). The results of such monitoring would likely be an
increased understanding in how the primitive sensory processing centers develop as well as
increased knowledge of the location of various neurons in the brain and their function. The
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fluorescent marker would also enable researchers to differentiate between injected and
endogenous cells.
The University of Michigan study also provides some information about the maturity
stage required of the injected neural precursor cells required to develop a functioning visual
system in the kittens. It is very likely that the most effective precursor cells in this experiment
will not be the embryonic stem cells with the most plasticity, but rather the stem cells which have
achieved some level of maturation towards the adult neuron stage. Determining what degree of
maturation is optimal will enable the scientific community to develop a better understanding of
neural differentiation, patterns of migration and plasticity and how it relates to plasticity in the
brain. If the genetic component is analyzed in great detail, it will also provide information
pertaining to how various genetic sequences are turned on and off in the process of neural
development. If such knowledge is obtained, it may soon be possible to artificially induce that
state by undoing some of the on and off switching which has occurred in the genome of neurons.
Rolling back the clock of development of neural synaptic connections of neurons may enable the
nervous system to repair itself. That ability would have tremendous therapeutic possibilities.
Even if such repair in the visual system is not possible, the knowledge will be useful in finding
new potential therapies.
Finally, the University of Michigan study seems to suggest that in rats with injured
brains, the body seemed to recognize what should exist but does not. Evidence for such a
statement lies in the fact that the migration of neural precursor cells in rats was specifically
towards the injured area. If such an outlook is true, then it can be expected that the precursor
cells injected into the 4-C layer of the visually deprived kittens will somehow know where they
need to migrate to. That knowledge may be a recognition that visual sensory neural pathways do
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not exist where they should, or on a more basic level, the individual survival drive of the
precursor cells may drive them to migrate and develop connections in areas in which they have
the greatest potential to survive.
The Hebbian rules mentioned previously, seem to support the latter theory in that the
rules recognize that neurons are competing for resources. Those that are able to gain access to
the survival resources grow stronger at the expense of the other neurons, which grow weaker
(Dr. Shibata, personal communication, Spring 2004). The Michigan study proposed a similar
theory in stating that there seems to be some “growth factors or neurotrophic factors that
stimulate the proliferation and migration of precursor cells” (Pobojewski, 2002, p. 1). It can be
hoped that the proposed experiment may offer some insight into what such factors those may be.
Further evidence of growth and neurotrophic factors can be seen in the loss of
responsiveness to an eye initiated by enhancing GABA levels (Fagionlini et al., 2004). GABA is
an inhibitory neurotransmitter which down regulates electrical messages between neurons. The
presence of GABA hyper polarizes neurons and thus decreases the likelihood of a neuron
reaching its threshold and firing an action potential (Dr. Shibata, personal communication,
Spring 2004). It was shown that disrupting production of GABA on the genetic level “delayed
the onset of the critical period indefinitely” (Fagionlini et al., 2004, p. 1681). Thus it will likely
be important to monitor and limit GABA levels in the visual cortex while it is being attempted to
restore visual sensory pathways.
Also of great relevance to the experiment is the issue of premature visual stimulation. In
mammals there is usually a great deal of development in the brain prior to exposure to sources of
sensory stimulation such as visual stimulation. Therefore one might question as to what the
effects will be of early exposure to visual stimulation by immature neurons and visual neural
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pathways. An experiment done by Bourgeois, Pawel, Jasterboff, and Rakic (1989) suggests that
there will be no effect. In their experiment rhesus monkeys were delivered by caesarean section
three weeks prior to term. They were then exposed to normal light through day and night cycles.
Their results state that they “found that premature visual stimulation does not affect the rate of
synaptic accretion and overproduction” (Bourgeois, Pawel, Jasterboff, & Rakic, 1989, p. 4297).
Thus, it should be expected that the unusual time in the visual cortex’s development, due to the
injection of immature neural precursor cells and immediate exposure to light versus a several
month incubation period, in which the kittens will be exposed to visual stimulation should not
result in abnormal effects that would hinder normal neural development.
At the end of the experiment observations of the synaptic connections and formation of
ocular columns will provide insight into the plasticity of the visual sensory pathways with
respect to the rest of the nervous system. Specifically, the experiment will give insight into the
possibility of developing or repairing sensory pathways apart from the normal development of
the brain. If the experiment succeeds and it is possible to impart normal visual function outside
of the critical period of development, it will mark the beginnings of a path towards therapeutic
possibilities of restoring other normal human functions lost in individuals due to the limited time
frame of the critical period in brain development. A short list of possibilities includes restoring
language, hearing, and vision capabilities in those who were not able to develop those functions
in the normal course of early development. Therapeutic benefits may also exist for those
suffering from strokes, neurodegenerative diseases, spinal cord injuries, or other severe brain
injuries.
The likely method of testing which synaptic connections and columns were formed
would involve radioactive dyes, fluorescent markers, or strategic placement of electrodes in the
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brain to trace electrical activity in the kittens and compare to a standard. These tests would be of
known pathways including, but not limited to, color, motion, and horizontal and vertical light
(Dr. Shibata, personal communication, Spring 2004).
Alternative or follow up research possibilities should include other forms of sensory
deprivation and restoration such as hearing. The basic experimental procedure would remain in
tact. It would follow the course of blocking of sensory stimulation with ear plugs. After the
critical period has passed, neural precursor cells would be injected into targeted cerebral cortex
layers of the desired region in the brain. In the case of hearing, the region would be in the
parietal lobes. Sensory stimulation would then assume normal levels. An extended period of
development would be allowed during which there would be frequent monitoring of neural
activity in the same manner as described in how to trace electrical activity along the visual
sensory pathways in kittens. It is expected that the results would be similar for the vast majority
of sensory pathways in mammals.
I do not wish to make light of animal treatment concerns and recognize the necessity of
addressing such concerns. During the course of the experiment every effort will be made to
minimize the pain felt by the kittens and ensure their overall health through the use of anesthetics
and proper animal care techniques. I believe the potential benefits for millions of people in
America and worldwide outweigh the unusual course of development that the kittens will
experience. Furthermore it is not possible for this experiment to be done on humans until, and if
it can be refined to a therapeutic stage.
The pertinence of this proposed experiment to genetics and behavior is far reaching. The
use of cloned stem cells is deeply intertwined with the frontiers of genetics. It has been
recognized here and elsewhere that genetic and environmental variation can influence behavior
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and thus the experiment seeks to minimize the variation in both. Furthermore, the classic work
by Hubel and Wiesel is deeply imbedded in psychology and neurobiology, demonstrating that
the environment affects behavior. It is my hope that an extension of their work will yield new
information in the fields and lead to innovative therapeutic options for individuals in need.
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References
Abrams, M. (2002). Sight unseen. Discover, 58-63.
Bourgeois, J., Jastreboff, P., & Rakic, P. (1989). Synaptogenesis in visual cortex of normal and
preterm monkeys: evidence for intrinsic regulation of synaptic overproduction.
Neurobiology, 86, 4297.
Calford, M., Wang, C., Taglianetti, V., Waleszyczyk, W., Burke, W., & Dreher, B. (2000).
plasticity in adult cat visual cortex (area 17) following circumscribed monocular
lesions of all retinal layers. Journal of Physiology, 587-588.
DeAngelis, G., Anzai, A., Ohzaawa, I., & Freeman, R. (1995). Receptive field structure in the
visual cortex: does selective stimulation induce plasticity? Neurobiology, 92, 9682.
Fagiolini, M., Fritschy, J., Low, K., Mohler, H., Rudoph, U., Hensch, T. (2004). Specific GABA
circuits for visual cortical plasticity. Science, 303, 1681.
Ferster, H. (2004). Blocking plasticity in the visual cortex. Science, 303, 1619-1620.
Hensch, T., Fagiolini, M., Mataga, N., Stryker, M., Baekkeskov, S., & Kash, S. (1998). Local
GABA circuit control of experience-dependent plasticity in developing visual cortex.
Science, 282, 1504.
Park, S., Kim, E., Ghil, G., Joo, W., Wang, K., Kim, S., Lee., & Lim, J. (2003). Genetically
modified human embryonic stem cells relieve symptomatic motor behavior in a rat
model of parkinson’s disease. Neuroscience Letters, 353, 91.
Pobojewski, S. (2002). Neural stem cells move to damaged areas of brain after injury.
University of Michigan Medical School Press Release, 1.
Purves, D., Fitzpatrick, D., Williams, S., McNamara, J., Augustine, G., Katz, L., & LaMantia, A.
(2001). Critical periods, cortical plasticity, and ambyopia in humans. Neuroscience.