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Literature Review
Introduction
Planarians are primitive, aquatic flatworms that have a sophisticated nervous
system and that demonstrate remarkable regenerative capabilities. Though they have
rudimentary responses to external stimuli, their memory retention is similar to that of
humans. Planarians negatively respond to light stimuli through their ocelli, eyespots
located on the dorsal area of their body, and they express their affinity and
amalgamation towards chemicals through their auricles. Though most testing of
memory retention in planarians has been practiced through the conditioning of their
negative photo-tactic behaviors, there is little evidence of training using chemo-tactic
behaviors or a combination of both. Excising, or removing, ocelli will likely induce
brain damage, so the planarians will be in the process of regeneration (which may
affect their behavior) (Received via electronic messaging). By combining both the
habituation of photo tactic and chemotactic training, the planarians with excised
ocelli may offset the impairments caused by neurological damage. Furthermore, this
new area of research may provide results that are applicable to the fields of behavior
and functional recovery and therapy.
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Planarians: Anatomy and Physiology and Central Nervous System
Planarians, a group of flatworms classified under the phylum Platyhelminthes,
are one of the most primitive organisms to have a nervous system and to exhibit
bilateral symmetry (Tyler 2015 Turbellaria). Planarians ingest through an orifice
located at the ventral side of their bodies. They ingest and excrete food through the
same orifice Planarians do not have blood within their vascular system; rather, their
tissue is composed of unspecialized and differentiated cells (Tyler, 2015).
The planarian nervous system branches out from the dorsal area to the body.
The structure of the planarian brain resembles an inverted U-shape with nine
branches coming out from it. Two ocelli are located at the end of dorsal side of the
planarian, where visual axons cross at the dorsal-inner region of the brain. The
auricles of the planarian closely correspond to the 6th to 9th branches in the brain.
Auricles serve as chemoreceptive organs. Though both auricles and ocelli are
structurally and functionally diverse, the genes that are expressed
(DjotxA, DjotxB and Djotp) are located near each other, and they are called
homeobox genes. DjotxA and Djotp genes determine chemosensory and photo-sensory
traits, whereas the DjotxB gene determines sensory to mechanical pressures (Agata
and Yoshihiko 2008).
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The planarian nervous system is found on the dorsal side of the body. The anatomy of the nervous
system is displayed in the figure. (Agata et al, 2008)
Planarians: Regeneration
Though planarians are composed of a myriad of differentiated cells, a majority
of the cells are neoblasts. Neoblasts are non-differentiated, pluripotent cells, that are
roundish cells with a 5-10μm diameter, discernible organelles, free ribosomes, a large
nucleus, and little heterochromatin. Neoblasts have been linked as a key role in
planarian regeneration when researchers noticed a heavy congregation of neoblasts
upon an inflicted site followed by a sudden loss of neoblasts. Following this
observation, it appears that neoblasts are the only cells in planarians with the
capability to divide (Rink 2013).
There is a proper balance that exists between neoblasts and specialized cells.
For example, adult vertebrates contain tissue specific stem cells, and are therefore
limited in terms of differentiation. However, the neoblasts in planarians are unique in
that they are pluripotent; they can diverge into any non-reproductive cell (Rink
2013).
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Regarding the nervous system, after truncation of a planarian, it takes about
five days for the regeneration of the brain to be complete. The process of
regenerating the brain can be distinguished into five processes: anterior blastemal
formation, brain rudiment formation, pattern formation, neural network formation,
and functional recovery. After the neoblasts close up the wound, the anterior
blastema begins development. The brain rudiments are developed inside the anterior
blastema. These processes encompass a combined total of approximately 24 hours,
but it is hard to distinguish when each individual step initiates. This rudiment then
further develops into the inverted U-shaped form, with branches coming out.
Following the development, structural recovery, such as the branches formed from
the Djotp, begin to develop and connect with the brain. Fully developed structures
appear four days into the regeneration process, and phototactic behaviors appear on
the fifth day (Agata and Yoshihiko 2008).
The five stages of neural regeneration are: anterior blastemal formation, brain rudiment
formation, pattern formation, neural network formation, and functional recovery. These steps
take a total of approximately 120 hours. (Agata et al, 2008)
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Planarians: Ocelli and Visual Regeneration
The ocelli are composed of pigmented cells and neurons and are located on the
dorsal (back) side of the planarians. The ocelli are arranged into a semilunar eyecup,
and the dendrites from these neurons are organized inside the pigmented cells to
form rhabdomeres (a structure containing light-sensitive cells, lenses, pigments, etc.)
At the optic chiasma located around the center of the dorsal region in planarians,
both inputs from the left ocellus and right ocellus are incorporated as inputs for a
phototactic responses (Inoue et al, 2004).
Unlike most animals, planarians are considered different because they exhibit a
behavior referred to as negative phototaxis. Essentially, planarians exhibit a
photophobic reaction when put under white light. The presence and application of
phototaxis on planarian yield a myriad of effects regarding memory acquisition and
retention and visual organ regrowth. However, most of these results are present only
when planarians are subjected to visual light, which is synthesized from a plethora of
various wavelengths (infrared, UV). Planarians appear to display an inverse
relationship when exposed to light over a period of time. Longer wavelengths such as
IR waves yield a smaller photophobic response in planarians than UV waves, shorter
waves which yield the highest photophobic response in planarians. However,
planarians yield both specific and non-specific responses to wavelengths. Non-specific
responses occur when planarians, originally kept in a dim lighting, were suddenly
exposed to a different light, which induced a photophobic response. However, in
relation to red light, planarians became more accustomed to light as time went on,
which is an example of a specific behavioral response (Paskin Jellies and Beane 2014).
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The planarian can act on a multitude of ways depending on the wavelength of light received by the
photoreceptor cells. (Paskin T. et al, 2014)
Planarians: Chemotaxis and Auricle Anatomy
Many organisms have demonstrated that ciliated organs have the capability to
exhibit a myriad of responses towards different stimuli. For instance, the vertebrates’
and invertebrates’ auditory organs as well as the chemo-sensing organs in
invertebrates contain ciliated ends. These processes demonstrated by the sensory
receiving organs suggest that the cilia are the predominant factors behind successful
expression. Planarians, for instance, exhibit elongation on the anterior sides of their
bodies when placed in a solution saturated with chemo-attractants. This is primarily
due to the location of the auricles, one of such ciliated organs that are capable of
exhibiting an affinity towards chemo-attractants. When initially placed into a
solution, the planarians hazily translate themselves across the aqueous solution;
within a few centimeters; however, the auricles are able to accurately direct the
planarians into a straight path. The excision of a single auricle results in the repeated
rotation of the planarian to the opposite side. Interpreting these results leads to the
idea that planarians contain two primary chemo-receptors on the anterior sections of
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their cerebral ganglia, and that these chemo-receptors are symmetrical to each
other. By coupling these receptors with an idea about a planarian coordinate system
(planarians have the capability to map out their surrounding), the planarians are able
to recognize and respond accurately to chemical stimulation The morphology behind
the sensory organs in invertebrates are similar to that of vertebrates, but the
abundance of minor variations in organ structure imply that the processes are
insufficient grounds to assume the presence of similar processes. Furthermore, though
the morphological structures and variations among the organs have been identified,
the actual mechanical and chemical processes behind the stimulation of the ciliated
sensory organs remain obscure (MacRae 492-493).
The presence of rhodopsin-like proteins was detected in both the ocelli and
auricles of the planarian species Dugesia Japonica. Ocelli and head excised specimens
displayed no signs of negative photo-taxis when subjected to light, but auricleexcised planarians showed photo tactic behaviors. The regeneration of this rhodopsin
protein is associated to the planarian’s ability to recover its photo-tactic behavior.
Only Auricular excisions served to increase asexual fission when subjected under light.
This information suggests that planarians use the rhodopsin in the auricles for
regulating asexual fission and circadian rhythms and planarians utilize rhodopsin in
the ocelli to establish negative photo-taxis (Fujita J. et. al).
Planarians of length 8 mm has approximately 2.0-3.0*104 neurons. To observe
and quantify planarian chemical sensing behavior, liver extract would be added into
the solution. Chemo-taxis would be described as the planarians in an assay that would
recognize and head to the chemo-attractant. When planarians were placed into a
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chemo-attractant assay without the chemoattractant, observations suggest that the
planarians initially spent a majority of their time on the surface of the assay.
However, intact planarians demonstrated chemotaxis when the liver extract was
diffused throughout the solution. In contrast, planarians that had their heads
truncated failed to recognize the presence of the chemoattractant, and thus
wandered throughout the maze. Observations also suggest that when exposed to
chemo-attractants, intact planarians have a tendency to move up a chemical
gradient.
Chemical and visual sensing and the ability to respond to stimuli involving these
senses are essential for locating food, communicating, and escaping predators. Intact
planarians spend an abnormally large amount of time in areas with a high
concentration of chemo-attractants, thus they demonstrate the ability to recognize
chemical gradients. Planarians that have lost their entire head are completely
degenerate of photo-taxis, chemotaxis, and thigmo-taxis (aggregation by touch).
Housing and Caring for Planarians
Planarians generally exist in freshwater and pond water environments; it is very
important to regulate the quality of water that the planarians live in. Minute changes
in the quality of water can result in the extermination of the entire test group. Tap
water contains lethal amounts of chlorine to planarians, but it can still be used if the
water was aerated for approximately 24-48 hours. An alternative method is to
artificially create a medium for planarians grown in. (McConnel 1965). Poland Spring
and alternative vendors that produce spring water are also suitable for the housing
and caring of planarians.
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Maze Training for Planarians
Planarians are not only capable of regenerating their entire system post truncation,
but research shows that Planarians have some aspects of long-term memory.
Therefore, there must be some way to study brain regeneration and memory in the
same animal. There are currently two known ways to process information in
organisms: spatial information and temporal information. Spatial information is
necessary to maintain the structural and functional integrity of anatomical structures
during embryogenesis, the process of the embryonal growth and development.
Temporal information is a post- developmental method of neurological processing;
external stimuli are abstracted and stored in the central nervous system of the
organism over time. (Shomrat, T., Levin, M. 2013)
Free- living planarians are classified as one of the first types of organism to
harbor a centralized brain with adequate synaptic transmission. The centralized
brains in planarians have many similarities to the centralized brains in vertebrate
animals. The planarians can utilize their rudimentary sensory organisms to recognize
differences in chemical gradients, vibration, electric fields, and magnetic fields.
These sensory capabilities are integrated into the planarians nervous system which is
the primary reason for their comprehensive behavior given basic sensory organs
(Shomrat, T., Levin, M. 2013).
Planarians were first demonstrated to learn how to overcome a training
challenge and still retain the memory of the habituation involved in the training after
they were bisected in 1965. Additionally, the tail segment, which eventually grows
into another planarian, retains the memory of the training sessions. This evidence
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suggests that planarians are capable of storing their memory elsewhere in the nervous
system; they do not limit their storage in the brain. The process of training planarians
to follow and overcome a challenge is referred to as classical conditioning. The most
popular way to use classical conditioning methodology on planarians was based on
their well-known photosensitivity. Temporal processing and storage was proven to
exist outside of the brain after a series of tests showed both the posterior and
anterior fragments of the planarians displayed a faster learning rate against the same
training paradigm assigned to the previously intact planarian. (Shomrat, T., Levin, M.
2013)
Learning has been proven to be the origin for behavioral changes given
different stimuli because quantitative analyses indicate distinct behavioral motions
(wayward movement given chemoattractant or directional preference in a maze)
(Shomrat, T., Levin, M. 2013).
The planarian starts at one end of the maze. Then, the planarian comes across a point of
intersection. The incorrect turn (0) results in the LED laser turning on. This is an example of
positive classical conditioning (action followed by stigma).
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Literature Cited
Tyler, Seth. (2015). Tricladida. In AccessScience. McGraw-Hill
Education. http://dx.doi.org/10.1036/1097-8542.709300
Paskin, T., Jellies, J., Bacher, J., & Beane, W. (2014). Planarian Phototactic Assay Reveals
Differential Behavioral Responses Based on Wavelength. PLoS ONE.
Rink, J. C. (2013). Stem cell systems and regeneration in planaria. Development Genes and
Evolution, 223(1-2), 67–84. http://doi.org/10.1007/s00427-012-0426-4
McConnel, J. (1965). On the Procuring and Care of Planarians. In A Manual of Psychological
Experiments on Planarians (pp. 10-21). Ann Arbor, Michigan: The Worm Runner's
Digest.
McConnel, J. (1965). Apparatus and Procedure. In A Manual of Psychological
Experiments on Planarians (pp. 21-58). Ann Arbor, Michigan: The Worm Runner's
Digest.
Curtin, Charles B. (2014). Sense organ. InAccessScience. McGraw-Hill
Education. http://dx.doi.org/10.1036/1097-8542.614700
Agata, Kiyokazu, and Yoshihiko Umesono. “Brain Regeneration from Pluripotent Stem Cells in
Planarian.” Philosophical Transactions of the Royal Society B: Biological
Sciences 363.1500 (2008): 2071–2078. PMC. Web. 1 Nov. 2015.
Inoue, T., Kumamoto, H., Okamoto, K., Umesono, Y., Sakai, M., Alvarado, A. S., & Agata, K.
(2004). Morphological and functional recovery of the planarian photosensing system
during head regeneration. Zoological Science, 21(3), 275-283. doi:10.2108/zsj.21.275
Asano, T., Nakamura, S., Ishida, S, Azuma, K.,Shinozawa, T.” RHODOPSIN-LIKE PROTEINS
IN PLANARIAN EYE AND AURICLE: DETECTION AND FUNCTIONAL ANALYSIS”
Asano, Y., Nakamura, S., Sachiko, I., Azuma, K., & Shinozawa, T. (1998). RHODOPSIN-LIKE
PROTEINS IN PLANARIAN EYE AND AURICLE: DETECTION AND FUNCTIONAL ANALYSIS.
Retrieved November 9, 2015
Shomrat, T., & Levin, M. (2013). An automated training paradigm reveals long-term memory
in planarians and its persistence through head regeneration. Journal of Experimental Biology,
3799-3810.
Wisenden, B., & Millard, M. (n.d.). Aquatic flatworms use chemical cues from injured
conspecifics to assess predation risk and to associate risk with novel cues. Animal
Behaviour, 761-766.
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MacRae, E K (01/01/1967). "The fine structure of sensory receptor processes in the
auricular epithelium of the planarian, Dugesia tigrina". Zeitschrift für Zellforschung
und mikroskopische Anatomie (1948) (0340-0336), 82 (4), p. 479.