Monday, January 16, 2012

Thesis Part 1 of 6 Introduction and Background Information






A Study of Gamma Lactones and Their Ability to Antagonize Cocaine’s Effects in Planarians and the Consequential Change in the Expression of Dopaminergic Membrane Proteins

Debra Baker

Department of Biology

West Chester University

M.S. Thesis























Table of Contents






Introduction …………………………………………………………………………. ….. 6

            Information about Planarians ……………………………………………………………..6

            Dopamine ………………………………………………………………………………...7

            Cocaine’s Effect on the CNS ……………………………………………………………..8

            Information about Cocaine ……………………………………………………………….9

                        Cocaine as a Local Anesthetic …………………………………………………..10

            The Evolution of the CNS……………………………………………………………….13

            Parthenolide and Gamma Lactones……………………………………………………..16

            Planarians as a Model Organism…………………………………………………………17

            Previous Research using Planarians ……………………………………………………..19

                        The Use of DMSO as a Solvent………………………………………………… 20

            Testing Sesquiterpenes ………………………………………………………………….21



Part One …………………………………………………………………………………………24


            Methods and Materials………………………………………………………………….. 26

            General Procedure ………………………………………………………………………26

            Surface Area to Volume Ratio ………………………………………………………….30

            Results……………………………………………………………………………………31

                        The Effect of γ-Lactones on Planarian Motility…………………………………31

            Discussion ………………………………………………………………………………34

            Similar Results in Mammals……………………………………………………………. 35

Part Two …………………………………………………………………………………………36

            Introduction …….………………………………………………………………………. 37

            Methods ………………………………………………………………………………….38

            Procedure………………………………………………………………………………...39

            Analysis of Results………………………………………………………………………42

            Expected Outcomes ……………………………………………………………………..43

            Alternative Approaches …………………………………………………………………46

Part Three ……………………………………………………………………………………… 47

            Introduction ……………………………………………………………………………...48

            Methods and Materials …………………………………………………………………..48

            Substances Tested ……………………………………………………………………….50

Expected Outcomes/Alternative Approaches ………………………………………….. 51

Room for Improvement …………………………………………………………………51

Part Four………………………………………………………………………………………XX

            Introduction……………………………………………………………………………XX

            Materials………………………………………………………………………………XX

            Experimental Design……………………………………………………………………XX

                        Experimental Design for NMDA………………………………………………XX

                        Experimental Design for Cytisine………………………………………………XX

            Results………………………………………………………………………………….XX

            Discussion………………………………………………………………………………XX

            Room for Improvement…………………………………………………………………XX

            Future Direction………………………………………………………………………XXx

Bibliography ………………………………………………………….…………………………52

Appendix…………………………………………………………………………………………57

Published Papers Based Upon This Work……………………………………………………XX

































“Science and research, and technology are like treasure.” Hannah started warming to her favorite subject and glad to have a receptive audience, “Its lying there, hidden in plain sight, ready to be discovered, has *always* existed but not yet known. We are fortunate to live in the right time and place and given the opportunity to discover it” She continued, “The heart of a person guides actions with intention. Radiation has been used to kill people but radiation has also been used to heal; without love we are noise but with love…”



“Love is the currency of Heaven,” continued Anna thinking of Miss Viola.























Acknowledgements

I thank God for the gift of life and for my unique temperament

I thank my parents, Joan Sander and James Sander, for doing the best they could with what they were given.

I want to thank my husband, Pat, for supporting me

Also my children, Jessica, Jennifer, Joanna, Jonathan, Jeanette, Joshua, Joseph, and Julianna, who inspire me with their own uniqueness

And my granddaughter, Fiona, who was born as this was being written and all of her future siblings and cousins

Dr Gestl, who put up with endless questions

Dr. Mbuy, who encouraged creative thought and teaches by provoking learning

Dr.Pagan, who told me to believe in myself.

Sean Deats, who met me in the middle of the Biology-Psychology bridge; thank you for brainstorming with me.

All my lab partners, Sean, Peter, Daniel, Matthew, Erica, Clinita, Dharini, and Galina; Whenever I remember you it will include laughter.












Introduction and
Background Information



























Introduction



Planarian worms are being used as model organisms in research related to Neuroscience because they occupy a unique niche in the evolution of the Central Nervous System. They are believed to be the extant species that is the closest, morphologically, to the common ancestor shared by all subsequent species that possess a centralized nervous system.  The planarian nervous system is relatively simple but, paradoxically, it possesses the same neuronal elements that are found in more evolved species.  Genes of the central nervous system are very highly conserved.  Consequentially, there is reason to believe results obtained by observing planarians have the potential to contribute to a better understanding of similar processes in mammalian species including humans. Because the planarian genome has been sequenced and resulting data are being made available, observing and analyzing behavior in planaria has become an attractive subject for investigation.

Another quality that is expressed intensely in planarians is an extraordinary ability to regenerate. Planarians have the highest concentration of Pluripotent stem cells found in adult organisms.  The potential applications using stem cells is just beginning to be explored.  Because planarians are both able to respond to psychoactive substances in a quantifiable and predictable manner and they are able to regenerate any organ in their system including the cerebral ganglion, also known as a brain, we have been able to determine the onset of function in the planarian brain that is necessary to evoke a response upon exposure to cocaine and other substances that act upon the nervous system.


 Planaria is the common name for several genera of free-living non-parasitic flatworms. Taxonomically, planarians belong to the phylum Platyhelminthes, class Turbellaria, order Tricladida (Carranza, et al. 1998).  Planarians make up various family, genera, and species.  Although they possess three germ layers, planarians are acoelomates.

Planarian Biologists in Asian labs tend to work with Dugesia japonica.  As its name implies, it is a common species of planaria in Japan.  Dugesia tigrina is the most common species in the United States but it is the genome of Schmidtea mediterranea has been sequenced by Dr. Alejandro Sánchez Alvarado and his colleagues at the University of Utah. The accomplishment of the Sanchez lab opens up access to genetic assays that will enable biologists to determine transcription and expressing rate while siRNA will be used to silence genes to determine function (Gentile, et al., 2011; Sofia, 2007).   It is not unreasonable to predict S. mediterranea will replace both D. tigrina and D. japonica in the next five years (Gentile, et al., 2011).

Planarians occupy an auspicious position in the evolution of the central nervous system the details of which can be found on page cite.  Although their system is simple, they possess features that suggest a pivotal place in evolution They are closely related to the first organism to evolve a brain and share pivotal central nervous system genes with almost every contemporary organism that possess a CNS (cite source.)  Planarians process and dispose of nitrogenous waste using flame cells.  Recently, the physiology of flame cells has been studied and these cells form a prototype of a nephron (Need citation.)  

Planarians are triclad but do not possess a coelem(get spelling) and do not have an alimentary canal.  They access food using an appendage called a proboscis. Solid waste exits the body via the proboscis (cite.)

Most species of planarians (including the ones used in this project) are hermaphroditic possessing both male and female genitalia (cite.) 







Information about Dopamine

Figure 1: Dopamine Molecule
Dopamine is a neurotransmitter that can be found in the brain of planarians.  It is synthesized from the amino acid Tyrosine.  A hydroxyl group is added by Tyrosine Hydroxylase and the intermediary, 3, 4-Dihydroxyphenylalanine is formed after which the carboxyl group is removed by DOPA decarboxylase to form Dopamine. Dopamine is stored in vesicles in the end of an axon. With the appropriate stimulation, dopamine is secreted into the synaptic cleft.  Dopamine travels across the synaptic cleft to the receptor on the neuron on the other side of the cleft.


There are two major types of dopamine receptors and planarians have both types. They are D1 (including D1 like) and D2 (including D2 like.) The major difference between these two receptors is that, upon stimulation, D1 receptors are coupled with the G-protein that stimulates the release of adenylate cyclase (cAMP.)  Conversely, when D2 receptors are stimulated, the activity level of cAMP decreases or is unaffected (Venturini, et al., 1989).

The dopamine transporter (DAT) is a transmembrane protein that pumps dopamine out of the synapse and into the neuron from which it originated (Venturin, et al., 1989).  Dopamine is repackaged into vesicles to be used again.

Dopamine can be modified by Beta-hydroxylase and processed into norepinephrine. Because it was regarded (correctly) as a precursor of norepinephrine and epinephrine, Dopamine was not recognized as a neurotransmitter in its own right until the 1950’s (get source for this factoid.)

Cocaine is an addictive drug as a result of the way that it exploits the dopaminergic neural pathways, experienced as a sense of euphoria in human subjects.  Dopamine is prevented from being cleared from the synapse when cocaine acts as a competitive reuptake inhibitor. Eventually, more cocaine is needed for the addict to experience the same intensity of euphoria.  Recently, knockout mice without dopamine transporters have demonstrated addictive “seeking” behaviors which led to the discovery that cocaine affected serotonin transporters as well as dopamine transporters (Ribeiro, et al., 2005;  Iversen, 2009; and NIH.1998).

Because dopamine and serotonin are both associated with motivation, pleasure, and reward, cocaine is an extremely addictive substance. The dopaminergic system is actually altered in chronic addicts (Volkow, et al., 1993).  Because cocaine blocks the dopamine transporter, and excessive dopamine accumulates in the synapse, the number of postsynaptic receptors is actually decreased in long-term addicts (NIH, 1998). 

Additionally, some physiological effects of cocaine exposure include tachycardia, elevated body temperature, decreased appetite, and an increase in available energy; the result of cocaine’s effect on epinephrine. (Koehntop, et al., 1977)



Information about Cocaine

Cocaine is a derivative of the coca plant, Erythroxylum coca, which thrives in its woodland environment of South America. There are actually several species of shrub that are in the Erythroxylum Genus but the species most used and most associated with cocaine is Erythroxylum coca. 

 Indigenous peoples of that region have taken advantage of the medicinal properties coca leaf and have also incorporated it into their religious rituals. Generally, indigenous peoples struck a balance with their natural environment and used the coca leaves with respect and moderation (Personal conversation with Sue Mustalisch, Professor of Health Science at West Chester University). There have been no documented cases of addiction as the result of using coca leaves (find source.)

According to anthropologists, natives of Peru were aware of the anesthetic properties of the coca plant. They would chew the leaves and drool on the part of the body that needed to be cut. Herbal treatments derived from coca leaves were applied to the wounds associated with trepanning procedures that were performed into the early part of the twentieth century (Musto, 1991).

There are some accounts of the indigenous use of white powder and herbs that would increase energy and take away painful sensation.   These accounts were documented by the priests and scribes that accompanied the Spanish in the fifteenth and sixteenth century (Calatayud, and  Gonzalez 2005).

Cocaine was initially transported to Europe and studied by Friedrich Gaedcke.  Starting in 1855, he extracted an “amorphous substance” from the coca plant and identified it as an alkaloid naming it Erythroxyline.  In 1860, another chemist, Dr. Carl Sherzer, transported cocaine to Europe and, along with his colleague, Albert Niemann, extracted cocaine from Erythoxyline.  Niemann was the first European known to observe the anesthetic effect of cocaine when it numbed his tongue (Koller, 1941).

Figure 2: Cocaine Molecule
In 1868, Thomas Morenoy Maiz, a surgeon in the Peruvian army, wrote a book based on records he kept while experimenting on animals in his care (Moreno y Maiz, T., 1868).  In 1879, Vassili von Anrep noticed that applying cocaine in solution would numb the skin on the exposed region. He also dropped the same solution onto the eye of an animal which would numb the eyeball and dilate the pupil (Vandam, 1987).  For some reason, it took another five years before cocaine was recognized as a local anesthetic.


Cocaine was the first clinically used local anesthetic (Sholz, 2002).  Albert Niemann isolated cocaine from the coca leaves (Koller, 1941) and Sigmund Freud was the first one to treat a patient with it. Ironically, Freud used cocaine to wean his patients from morphine. He and colleague, Karl Koller, were also the first to demonstrate its numbing effect when applied occularly.  A conundrum exists in this narrative that may lead the reader to believe that two men were given credit for discovering cocaine as a local anesthetic.   Although Koller cited Anrep’s previous work that was published, Koller was credited with the discovery because his work that confirmed Anrep’s original findings was published in a professional journal that was prominent to the surgeons of his era (Yentis and Vlassakov, 1999) Dr. William Stewart Halsted is credited to be the first to describe the effect when cocaine is injected into the sensory nerve trunk (Osborne, 2007).  Unfortunately, no one knew about the addictive qualities of cocaine. Freud, as well as many other prominent people of the late Victorian era, became cocaine addicts before laws could be passed to regulate the substance (Koller, 1941).

 The mechanism of action that interferes with the transmission of sensation is a disturbance in the resting potential in the sodium gated ion channels.  Most theories involve open/closed, active/inactive channels. Other theories propose a disturbance in the phospholipid bilayer of the cell membrane.  Local anesthetics are generally divided into amides and esters (Vandam, 1987).

Amylocaine was the first synthetic local anesthetic. It was synthesized in 1903 and was quickly overshadowed by the formulation of procaine in 1904. Both Amylocaine and Procaine (Novacaine) are esters and analogs of cocaine (McLure and Rubin, 2005).   The slight change in molecular conformation was sufficient to lose the addictive properties associated with cocaine while preserving its effectiveness as a local anesthetic. Soon after Procaine was first synthesized in 1904, it became widely available in dental offices and emergency rooms.  During the second half of the twentieth century, Amides took the place of esters and local anesthetics took effect more rapidly and had a lower incidence of allergic reactions (Calatayud and Gonzalez, 2005).

From the plant’s perspective, cocaine evolved as a potent weapon in E.coca’s botanical arsenal. Cocaine affords effective protection against predation. It is worth mentioning the likelihood that many potential future pharmaceuticals may be found in the arsenal of hundreds of thousands of known and yet-to-be discovered plants, animals, and microscopic life forms that are players in the evolutionary arms race that share habitat with Erythroxylum coca (Perfecto and Vandermeer, 2008). The cocaine molecule has four chiral centers which allow it to form 16 isomers (McLure and Rubin, 2005). 





Parthenolide and Gamma Lactones



Figure 3: Parthenolide
Parthenolide, a Sesquiterpene lactone, has been established to antagonize behavior that is associated with cocaine in planarians.  Parthenolide is a relatively complex ringed structure. Because of this, determining the simplest structure that has the capacity to antagonize cocaine in planarians has the potential to contribute to a better understanding of the mechanisms that work to antagonize the behavioral effects cocaine has on planarians.  Recent findings have identified γ-Nonalactone as the simplest structure needed to effectively antagonize cocaine toxicity in planarians (Baker, et al., 2011).  Established procedures will be used to determine the genetic and biochemical properties that cause these lactones to be effective cocaine antagonists in planarians. (Raffa, et al., 2005).


Determining any changes in the transcription rate of key components in the cocaine-sensitive dopaminergic pathway will contribute to a more comprehensive understanding of the mechanism of action that lactones employ as cocaine antagonists.

In one sense, parthenolide was an obvious choice A detailed explanation of the reasoning behind choosing parthenolide can be found on page (fill in.) A few roadblocks exist chief among them is the very low solubility of parthenolide. There is some effort on the part of chemists to synthesize an analog of parthenolide with high solubility



Gamma Lactones were not selected randomly for examination.   When several compounds were introduced to the aqueous environment in which planarians were contained, the compounds with the five-carbon lactone ring structure were found to alleviate the withdrawal symptoms in planarians with Parthenolide demonstrating the greatest effect (Pagán, et al., 2008).  Parthenolide is similar in many ways to compounds such as β-eudesmol, which shares similarities in molecular structure that had no significant effect on planarians that were exposed to cocaine. The structures that were active often had the lactone structure (a“lactone” is a ringed ester.)  These ringed structures can also be found in many traditional herbal medicines that have been used by people for thousands of years. Feverfew (Tanacetum  parthenium) is a good example. Parthenolide is one of the identified molecules in this herbal remedy (Erlich, 2008).







The Evolution of the CNS



Development of bilateral symmetry and cephalization were important precursors to the evolution of the Central Nervous System (CNS).  Bilaterians most likely descended from a common flatworm-like ancestor during the Cambrian geological period approximately 550-600 million years ago (Jacobs, et al., 2005).                         

 Until a few years ago, the dominant theory was that vertebrates, insects, and flatworms diverged from a common bilaterians ancestor, appropriately named Urbilateria, and went on to evolve giving rise to three distinct nervous systems; the ganglion and ladder-like nerve cord of planarians, the segmented ganglia of arthropods, and the brain and spinal cord and inverted dorsoventral axis of vertebrate systems, (Sarnat and Netsky, 1985).  New evidence suggests the Urbilateria had not only developed bilateralism but also acquired the genes that form the foundation of a CNS (Jacobs, et al. 2005). All organisms with a defined CNS have origins in this common ancestor.  This is reflected in the evidence that many associated genes are extremely highly conserved across all species that possess a CNS (Mineta, et al., 2003).  Although significant differences exist in the morphology of individual species, each possesses corresponding regions that, even when their neurological systems are morphologically dissimilar, posses, “similar molecular fingerprints,” that reflects a high degree of conservation (Sarnat and Netsky, 2002).


Figure 5:Map of Planarian Brain Regions. Picture Courtesy of K. Agata.
Planarians possess a CNS with simple primitive morphology. The, “brain,” is a centralized U-shaped ganglion with ventral nerve cords. The ganglion has nine branches that form pairs of connections to symmetrical sensory organs. The cephalic ganglion is similar to more complex vertebrate CNS.  Some examples of features both share include multipolarized neurons and homologs of the otx (orthodenticle) genes that are expressed in the anterior region of the planarian cephalic ganglion (Ribiero, 2005).  


A large commissure crosses medially uniting planarian hemispheres reminiscent of a mammalian corpus callosum (Sarnat and Netsky, 1985).  The planarian ganglion is, indeed, considered a primitive brain.  The simple representation in Figure 5 shows bilateralism with the yellow regions being devoted to mechano-sensory activities, the green are chemosensory, blue represents light-sensory, and red represents inter-neuron activity (Agata, 1998).  Planarians also possess a peripheral nervous system that is distinct from the central nervous system. The planarian cephalic ganglion exhibits many morphological features similar to the vertebrate CNS, such as multipolarized neurons.  The ratio of brain size to body mass in planarians is similar to rats and young planarians have a higher ratio compared to adults. This is similar to mammalian species but is not a feature in other simple animal examples (cite source.)

A consequence of evolving a central nervous system is the emergence of special sense organs and these are represented in simple presumably newly evolved form in the planarian brain. The eyespots are not eyes as mammals experience eyes but are organs able to sense light from dark. The auricles are associated with hearing but in planarians, they are not quite hearing organs. They have ciliated neurons that resemble olfactory bulbs and are actually chemo receptors.  Some species of planarians have a statocyst which is a simple vestibular organ (Sarnat and Netsky, 1985).

Taxonomically, planarians belong to the phylum Platyhelminthes. The taxonomic niches are being adapted by molecular analyses of the 18s rRNA sequences and, using this measure, bilateral animals are being classified into three groups: Deuterostomia, Ecdysozoa, and Lophotrochozoa (Huson and Scornavacca, 2011).   Platyhelminthes are found near the root of Lophotrochozoa.

 Because planarians are so close to the morphology of the early bilateral ancestor and because planarians occupy a unique place wherein they possess qualities shared by vertebrates, they are an ideal species to use in the study of the evolution of the CNS (Agata, et al., 1998)











Key Genes for the CNS and Their Conservation Across Species


Figure 6: Source K. Agata

Figure 6: Key Genes for the CNS

When key genes in the central nervous system were compared across species, 110 of 116 head genes found in planarians were shared by all of the bilateral animals that have been examined (C. elegans, D. melanogaster, and H. sapiens.) The absent genes , the 6 out of 116 genes that were analyzed, were absent in C. elegans and/or in D. melanogaster. These three species belong to the three different groups of bilaterians that have a CNS (Agata, et al., 1998).  These genes include FGF signaling molecules, noggin, and frizzled, among many others.  Because so many functional genes are so highly conserved, one leading theory suggests that all bilaterians with a CNS share a common ancestor (Sarnat and Netsky, 1985).






Figure 7: Bilateral Symmetry in Planarians



Because the CNS seems to be so highly conserved and because planarians appear to be the closest extant organism to a shared common ancestor, several research laboratories have been busy observing and documenting normal behavioral patterns in planarians that have been exposed to various chemicals that act upon various neurotransmission pathways.  Responses to compounds such as cocaine (Raffa, et al., 2003), caffeine (Pagan, et al., 2009), nicotine, (Pagan, et al., 2009), aspartame (unpublished data, Baker, 2010), and NMDA (Rawls, et al., 2007), have been observed in planarians.  These observations have established a variety of behaviors that are considered normal which has, in turn, made it possible to identify behavior that departs from the standard (Raffa, et al., 2001).

With respect to cocaine, planarians exhibit predictable behavior patterns upon exposure to cocaine at sufficient concentrations.  Their movement is retarded significantly. They also go through seizure-like movements during which they frequently curl up into a, “C-like position.”  This is a spasm-like behavior that is distinguishable from the more smooth movements that are within the planarians repertoire.  Another behavior that is associated with cocaine toxicity is the, “head bop,” in which they appear to have the tail pinned to the bottom of the dish while they waggle back and forth (Raffa, et al., 2001; Pagán, et al., 2008).



























Planarians as a Model Organism

By the turn of the twentieth century, Biologists were aware of the regenerative abilities of planarians.  The earliest known reference was found in a Japanese encyclopedia dating to the seventeenth century (Sanchez, 2010).   Over 600 scholarly papers about planarians were published around the turn of the twentieth century.   Research varied from light-dark studies to regeneration after exposure to radiation.  

The reason planarians stopped being used in advanced research was a direct result of the intellectual rivalry between two distinguished developmental and genetic biologists, namely Charles Manning Child and Thomas Hunt Morgan (Clark, 1992). 

Thomas Hunt Morgan embraced Mendelian Inheritance theories and believed there was a distinction between somatic and germ line genetics. Since Drosophila melanogaster expresses its germ line earlier in development, D. melanogaster became a model organism for use in developmental and genetics research (Nobel Prize Biography).

Child used planarians in his research and was much more interested in environmental influences and phenotypes.   Child was not quite convinced that genes were stored on chromosomes.  He also rejected the notion that two discrete sets of molecular instructions exist; the somatic and germ line (Clark, A., 1992).

Child and Morgan chose to ignore one another’s existence and certainly did not acknowledge the other’s contribution to genetics and developmental biology.

Eventually, Morgan’s ideas were supported by subsequent discoveries and his work with D. melanogaster became associated with the emerging discipline while planarian research fell out of favor (Clark, 1992). Ironically, some trends have come full circle and the work Morgan pioneered in the field of genetics has resulted in the mapping of the human genome as well as the genomes of many other species.  As a result, the focus of research has shifted to epigenetics; the study of how environment interacts with the basic instructions contained within the cell, and stem cell technology that holds the hope of innovative therapies.  Because they have adult pluripotent stem cells distributed throughout their body, planarians are able to allow regeneration of an entire individual from a tiny fragment of tissue. As a consequence of their remarkable ability to regenerate and because of their status as the least evolved organism with a central nervous system, planarians are being rediscovered as model organisms in labs all around the world (Gentile, 2011).



Advancements in sequencing DNA have contributed to resurgence in planarian Biology.  Dr. Alejandro Sánchez Alvarado was among the first to recognize the potential benefits of sequencing the genome of at least one species of planarians. It was necessary for him to harvest planarians from a fountain in Spain. The genus and species of his planarians just happened to be, “Schmidtea mediterranea,” which is why this particular genus and species will most likely emerge as the model species for planarian research into regeneration (Newmark and Sánchez-Alvarado, 2003).

Planarians are emerging as model organisms in the areas of regeneration research, particularly using adult stem cells and in the area of neurological and neuropharmacological research (Gentile, et al., 2011).  Along with the ability to regenerate and their position on the evolutionary clade that places them as the most anciently evolved species with a centralized nervous system and rudimentary special sensory organs, planarians are able to reproduce sexually and asexually. S. mediterranea houses its genome on four stable diploid chromosomes (Lau, et al., 2007).

The lack of a stable genome within the Dugesia genus will adversely affect the ability to conduct the tests necessary to discern what is happening to the transcription rate of the associated RNA and subsequent changes to the expressing level of key protein products.





























The Use of DMSO as a Solvent



Figure 8: Diethyl Sulfoxide (DMSO)
Some of the substances that are evaluated in this work are poorly soluble in an aqueous environment (cite.) Getting things such as Parthenolide into solution is aided by Dimethyl Sulfoxide (cite).  Dimethyl Sulfoxide (DMSO) is a polar aprotic organic molecule. Because of these qualities, DMSO serves as an excellent solvent.  Unfortunately, DMSO is also controversial since it has been associated with at least one human death and has been rumored to be teratogenic to the developing brain. The Material Safety Data Sheet does not consider DMSO to be teratogenic or dangerous unless the concentration was high (MSDS).  A solvent was needed to put some substances that were being tested into solution and DMSO was an attractive option but it was found to be toxic in planarians at a high concentration. Tests have been conducted in order to determine the level of DMSO necessary for effectiveness but in a concentration that will not affect the behavioral responses in planarians.  For normal, 1-2cm long planarians, this ideal concentration is about 0.1% (Pagán, et al., 2006).






Testing Sesquiterpenes



Various organic compounds have been tested to determine if any of them was able to antagonize cocaine’s effect in planarians.  Eventually, a group of ringed organic Sesquiterpenes were suspected to be good candidates for investigation. After careful observation, a Sesquiterpene with the five sided gamma lactone, Parthenolide, was found to antagonize the symptoms of cocaine’s activity in planarians (Pagan, et al., 2008).

 The idea of testing Parthenolide against cocaine in planarians originated in Dr. One Pagan’s dissertation. The following narrative is a direct quote taken from Dr. Pagan’s dissertation defense.

Why parthenolide?  The idea of using parthenolide as a cocaine-displacing ligand came from its close structural similarity to another class of natural products, the cembranolides.  Cembranolides are cyclic diterpenoids widely distributed in nature.  More than 300 examples have been described (Wahlberg & Eklund 1992). 



The cembranolides are established ligands of the nicotinic acetylcholine receptor (nAChR).  The nAChR is the best-known member of the ligand-gated ion channel superfamily.  This receptor will not be discussed here, and the reader is referred elsewhere (Karlin, 2002).  Cembranolides bind in a mutually-exclusive manner with the local anesthetic procaine, on the nAChR (Pagán, 1998; Pagán et al., 2001).     

The “line of thought” that led me to consider parthenolide as a cocaine antagonist was:

1.  Cembranolides and an example of a local anesthetic bind to nicotinic acetylcholine receptors (nAChRs) in a mutually-exclusive manner (Pagán, 1998, Pagán et al., 2001).

2.  Cocaine is the prototype of the local anesthetic class of molecules, with neuronal sodium channels as the target (Ruetsch et al., 2001; Scholz, 2002).  However, its behavioral effects are due to cocaine’s interaction with neurotransmitter transporters, mainly the dopamine transporter (Uhl et al., 2002).

3.  Parthenolide shows close structural similarity with cembranolides.

4.  Parthenolide interacts with nAChRs and is capable of alleviating cocaine inhibition of this receptor in electrophysiological studies (Pagán and Hess, unpublished results).

Based on the points above, I thought that parthenolide and cocaine had the potential to interact with the dopamine transporter” (Pagan, 2005).



Dr. Pagan’s educated hunch seems to have served him well as yet-to-be published data that is the Master’s thesis of one of his graduate students strongly supports the hypothesis articulated in Dr. Pagan’s dissertation.  Thomas M. Graczyk, MS, has transfected dopamine transporter genes (DAT) into embryonic kidney cells that normally do not possess dopamine transporters.  He then exposed these cell lines to procaine (an analog of cocaine) in the presence and absence of parthenolide and the parthenolide reverses the effect procaine has upon the DAT. His experimental design included negative and positive controls one of which allowed for repeated tests in cloned cell lines that did not possess DAT. These non-DAT lines did not see the same effects (cite thesis as source.) One of the more intriguing results of this endeavor includes the discovery that the binding site of parthenolide to the dopamine transporter is allosteric and non-competative with dopamine or cocaine.  It apparently increases the affinity for dopamine. Up until this point, the model for the binding site cocaine occupied to antagonize movement and increase seizure-like movements was thought to be both orthosteric and competitive with dopamine at the transporter site (cite.)

Cocaine ester acts as an anesthetic as well as a dopamine reuptake antagonist (Koehntop, 1977). Cocaine amide acts as a local anesthetic but does not produce the behavioral effects that are associated with excessive dopamine levels (Iversen, 2009).


Figure 9; Parthenolide (left) and Euniolide (right) Shown together to illustrate structural similarity.

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