Formatting the Bibliography

Bibliography

Debra:  Please use a consistent format for the references.  We have talked about it….  Use the following format:

Mohr JT, Gribble GW, Lin SS, Eckenhoff RG and Robert S. Cantor RS (2005) Anesthetic potency of two novel synthetic polyhydric alkanols longer than the n-alkanol cutoff: evidence for a bilayer-mediated mechanism of anesthesia? J Med Chem. 48:4172-4176.

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Agata, K., Soejima, Y., Kato, K., Kobayashi, C., Umasono, Y., Watanabe, K. (1998) Structure of the planarian central nervous system (CNS) revealed by neuronal cell markers.  Zoolog Sci. Jun 1;15(3):433-40.

Baker, D., Deats, S., Boor, P., Pruitt, J., Pagán, O.R. (2011) Minimal structural requirements of alkyl γ-lactones capable of antagonizing the cocaine-induced motility decrease in planarians. Pharmacology Biochemistry and Behavior Volume 100, Issue 1, November 2011, Pages 174-179

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Benya, E., Leal-Zanchet, A., Santos, W., Hauser, J., Erdtmann, B. (2007)  Chromosome polymorphism and complements in populations of Girardia species (Platyhelminthes, Tricladia, Paludicola) from southern Brazil. Brazilian Journal of Braz. J. Biol. Vol.67 no.4 supp1.0 Sao Carlos Dec. 2007.

Carranza,S., Littlewood, D T., Clough, K A., Ruiz-Trillo, I., Baguñà, J., and Riutort, M.(1998)  A robust molecular phylogeny of the Tricladida (Platyhelminthes: Seriata) with a discussion on morphological synapomorphies.  Proc Biol Sci. April 7; 265(1396): 631=640.

Calatayud, J. Gonzalez, A. (2005) History of the Development and Evolution of Local Anesthesia Since the Coca Leaf. Anesthesiology. V 98, No 6, jun 2005.

Callier, S., Snapyan, M., Le Crom, S., Prou, D., Vincent, J.D., Vernier, P. (2003) Evolution and cell biology of dopamine receptors in vertebrates. Biol Cell. 95(7):489-502.

Carlsson, A. et al. (1957) 3,4-dihydroxyphenylalanine and 5-hydroxytryptophan as reserpine antagonists. Nature 180, 1200

Chong, T., Stary, J., Want, Y., Newmark, P. (2011)  Molecular markers to characterize the hermaphroditic reproductive system of the planarian Schmidtea mediterranea.  BMC Developmental Biology 2011, 11:69 doi:10.1186/1471-213X-11-69.

Eckenhoff RG, Tanner JW, Johansson JS. (1999) Steric hindrance is not required for n-alkanol cutoff in soluble proteins. Mol Pharmacol 56:414-418.

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Gentile, L., Cebria, F., Bartscherer, K., (2011) The planarian flatworm: an in vivo model for stem cell biology and nervous system regeneration . Published by The Company of Biologists Ltd PMCID: PMC3014342

Graczyk, Thomas (2011) An Investigation of the Effects of the Local Anesthetic Procainamide and the Sesquiterpene Lactones Parthenolide, Santonin, and Costunolide at the Human Dopamine Transporter Using a Fluorescence-Based Uptake Assay. T. Graczyk’s Master’s Thesis Copyright 2011 Unpublished at this writing.

Guzman, M., Rossi, R., Neelakantan, S., Li, X., Corbett, C., Hassane, D., Becker, M., Bennett, J., Sullivan, E., Lachowicz, J., Vaughan, A., Sweeney, C., Matthews, W., Carroll, M., Liesveld, J., Crooks, P., and Jordan, C. (2007) An orally bioavailable parthenolide analog selectively eradicates acute myelogenous leukemia stem and progenitor cells.  Blood. Published  online September 5, 2007.  Blood December 15, 2007 vol. 110 no. 13 442 7-4435.

Hall, F.S., Sora I, D.R., Gonova, J., Li, X.F., Goeb, M., Uhl, G.R. (2004). Molecular mechanisms underlying the rewarding effects of cocaine. Ann NY Acad Sci. 1025:47-56.

Hertting, G., Axelrod, J. (1961) Fate of tritiated noradrenaline at the sympathetic nerve-endings. Nature. 192:172-3.

Hertting, G., Axelrod, J., Patrick, R.W. (1961) Actions of cocaine and tyramine on the uptake and release of H3-norepinephrine in the heart. Biochem Pharmacol. 8:246-8.

Huson, D., Scornavacca, C., (2011) A Survey of Combinatorial Methods for Phylogenetic Networks. , Genome Biol Evol  3 23-35. doi: 10.1093/gbe/evq077

Hyman, S. E. (2005) Neurotransmitters. Cur Bio 15:R 154-R 158.

Iversen, Leslie.(2009) Introduction to neuropsychopharmacology.  Oxford University Press.

Jacobs, D., Hughes, N., Fitzgibbon, S., Winchell, C. (2005) Terminal addition, the Cambrian radiation and the Phanerozoic evolution of bilaterian form. EVOLUTION & DEVELOPMENT 7:6, 498–514

Jayanthi, L., Apparsundaram ,  S., Malone, M., Ward, E., Miller, D., Eppler, M. Blakely, R. (1998) The Caenorhabditis elegans GeneT23G5.5 Encodes an Antidepressant- and Cocaine-Sensitive Dopamine Transporter.  Molecular Pharmacology October 1, 1998 vol. 54 no. 4 601-609.

Koehntop, D., Chia , L, Van Bergen, F. (1977) Effects of Pharmacologic Alterations of Adrenergic Mechanisms by Cocaine, Tropolone, Aminophylline, and Ketamine on Epinephrine-induced Arrhythmias during Halothane-Nitrous Oxide Anesthesia. Anesthesiology Volume 46 - Issue 2 pp: 83-158

Koller,C. (1941) History of cocaine as a local anesthetic, JAMA 117: 1284

Kurian, M., Zhen, J., Cheng, S., Santosh S., Mordekar, R., Jardine, P., Morgan, N., Meyer, E., Tee, L., Pasha, D., Wassmer, E., Heales, S., Paul Gissen, P., Reith, M., Eamonn R. Maher, E. (2009)Homozygous loss-of-function mutations in the gene encoding the dopamine transporter are associated with infantile parkinsonism-dystonia Published in Volume 119, Issue 6  J Clin Invest. 2009; 119(6):1595–1603 doi:10.1172/JCI39060

Lau, A. H., Knakievicz, T., Prá, D. and Erdtmann, B. (2007) Freshwater planarians as novel organisms for genotoxicity testing: Analysis of chromosome aberrations. Environmental and Molecular Mutagenesis, 48: 475–482. doi: 10.1002/em.20307

Margotta V, Caronti B, Meco G, Merante A, Ruggieri S, Venturini G and Palladini G.(1997) Effects of cocaine treatment on the nervous system of planaria (Dugesia gonocephala s.l.). Histochemical and ultrastructural observations. Eur J Histochem 41:223-30.

Mateo, Y. Budygin, E., Johansen, C. (2004)  Role of Serotonin in Cocaine Effects in Mice with Reduced Dopamine Transporter Function. National Academy of Sciences

 Material Safety Data Sheet MSDS #Gcc1-7  Dimethyl Sulfoxide (DMSO) August 20, 2004

McLure, H.and Rubin, A. (2005) Review of Local Anesthetic Agents.  Anestesia 2005 ; 73: 59-74.

Mineta, K., Masumi, N., Cebria, F., Ikeo, K., Agata, K., Gojobori, T. (2003) Origin and evolutionary process of the CNS elucidated by comparative genomics analysis of planarian ESTs. PNAS June 24, 2003 vol. 100 no. 13 7666-7671

book Mitman G and Fausto-Sterling A (1992) "Whatever happened to Planaria? C.M. Child

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Mohr JT, Gribble GW, Lin SS, Eckenhoff RG and Robert S. Cantor RS. (2005) Anesthetic potency of two novel synthetic polyhydric alkanols longer than the n-alkanol cutoff: evidence for a bilayer-mediated mechanism of anesthesia? J Med Chem. 48:4172-4176.

Moreno y Maiz, T. (1868) Recherche cliniques et physiologiques sur l'érythoxylon coca du Perou et la cocaine. Thèse, 1868, Paris.

Musto, D. (1991) Opium, cocaine and marijuana in American history. SciAm. 1991                                                                                                   

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Nathanson, J., Hunnicutt, E., Lakshmi, K., Scavone, C. (1993) Cocaine as a Naturally Occurring Insecticide. PNAS Page 9645-9658.

Newmark, P. A. and A. Sánchez Alvarado. (2002)  Not your father’s planarian: a classic model enters the era of functional genomics. Nature Reviews Genetics 3: 210-219.

O’Donnell, M. J., (1997) Mechanisms of Excretion and Ion Transport in Invertebrates. Supplement 30: Handbook of Physiology, Comparative Physiology. 10.1002/cphy.cp130217

Osborne, M., (2007) William Stewart Halsted: his life and contributions to surgery. The Lancet Oncology, Volume 8, Issue 3, Pages 256 - 265.

Pagán OR (2005) Synthetic local anesthetics as alleviators of cocaine inhibition of the human dopamine transporter.  Ph.D. Dissertation, Field of Pharmacology and Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY

Pagán OR, Rowlands AL, Fattore AL, Coudron T, Urban KR, Bidja AH, Eterovic VA. (2009) A cembranoid  from tobacco prevents the expressionof nicotine-induced withdrawal behavior in planarian worms. Eur J Pharmacol 615:118–124.

Pagán OR, Rowlands AL and Urban KR. (2006)Toxicity and behavioral effects of dimethylsulfoxide in planaria. Neurosci Lett 2006; 407:274-8.

Pagán OR, Rowlands AL, Azam M, Urban KR, Bidja AH, Roy DM, Feeney RB, Afshari LK. Reversal of cocaine-induced planarian behavior by parthenolide and related sesquiterpene lactones. Pharmacol Biochem Behav 2008; 89:160-170.

Palladini G, Ruggeri S, Stocchi F, De Pandis MF, Venturini G, Margotta V. (1996) A pharmacological study of cocaine activity in planaria. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol 1996; 115:41-5.

Perfecto, I. and Vandermeer, J. (2008) Biodiversity Conservation in Tropical Agroecosystems. Annals of the New York Academy of Sciences, 1134: 173–200. doi: 10.1196/annals.1439.011

Personal Correspondence with Dr. Chen May 4, 2011.

Raffa RB and Desai P. (2005) Description and quantification of cocaine withdrawal signs in Planaria. Brain Res 2005; 1032:200-2.

Raffa RB, Holland LJ and Schulingkamp RJ.(2001) Quantitative assessment of dopamine D2 antagonist activity using invertebrate (Planaria) locomotion as a functional endpoint. J Pharmacol Toxicol Methods 2001; 45:223-6.

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Two Peer-Reviewed Papers That are Products of This Effort

Baker, D., Deats, S., Boor, P., Pruitt, J., Pagán, O. (2011) Minimal structural requirements of alkyl γ-lactones capable of antagonizing the cocaine-induced motility decrease in planarians. Accepted for Publication 12 August, 2011, published online version 22 August, 2011, Published Volume 100, Issue 1, pages 174-179  Pharmacology, Biochemistry, and Behavior

 Oné R. Pagán, Debra Baker, Sean Deats, Erica Montgomery, Matthew Tenaglia, Clinita Randolph, Dharini Kotturu, Christopher Tallarida, Daniel Bach, Galia Wilk, Scott Rawls And Robert B. Raffa (2012) Planarians in pharmacology: Parthenolide is a specific behavioral antagonist of cocaine in the planarian Girardia tigrina. (Accepted, December 22, 2011, Made available online and in print March 15, 2012, published March 22, 2012) International Journal of Developmental Biology (2012) 56: 193-196

A word about Formatting

The thesis is too large for one post and the formatting is wonky but if you want to take a chunk and help with flow and spelling/grammar, go for it.

Almost parts 3 and 4

In light of this information, results showing that parthenolide prevented the behavioral effects of cocaine but not amphetamine was unexpected, since cocaine and amphetamines both interact with neurotransmitter transporters, albeit through different mechanisms. Cocaine is a reuptake blocker, preventing the transport of the neurotransmitter back to the presynaptic site; amphetamine is considered a “releaser”, which acts as a false substrate of the transporter (Riddle et al., 2005). In both cases, the net result is the abnormal increase of neurotransmitter molecules in the synaptic cleft, which accounts for the drug’s psychoactive properties (Iversen, 2006; Sager and Torres, 2011).

Using vertebrate pharmacology as a point of reference, our results suggests that parthenolide prevents the pSLM induced by cocaine by inhibiting an interaction with dopaminergic systems. However, this is not consistent with the amphetamine results (Figure 1E) or by the fact that no significant pSLMs are detected when exposing the worms to a dopamine concentration of 1 mM (data not shown). Interestingly, preliminary results with another planarian species, Dugesia dorotocephala, seem to indicate that parthenolide alleviates amphetamine-induced pSLM (Rawls et al., unpublished data). A possible interpretation is that in planarians, any catecholamine responses are modulated by compounds such as norepinephrine, octopamine or tyramine, as opposed to dopamine. Another implication of our results is that the pSLM are induced by cocaine/amphetamines and by cholinergic/glutamatergic compounds by interacting with different protein targets. This is amenable to pharmacological dissection of these distinct mechanisms.

In future experiments, we will study the effect of parthenolide against cocaine and amphetamine-like compounds in planarian behavior using paradigms such as conditioned place preference (Rawls et al., 2011), cross-sensitization (Rawls et al., 2010) and withdrawal-like behavior (Sacavage et al., 2008).

In a broader context, this work highlights the usefulness of planarians as an important animal model in pharmacology. In addition to the multiple advantages of using planarians described above, this model has been demonstrated to be relevant to mammalian pharmacology (Schwarz, 2011). Furthermore, since they can be studied all the way from molecular biology to behavior, these organisms will likely be developed as important tools in drug discovery research.

 

 

 

 

 

 

 

 

 

 

Part Four, Using and Seizure-like Responses to Determine Gain of Function in the Planarian Regenerating Brain

 

 

 

 

 

 

 

 

 

 

 

Introduction

When planarian regeneration is taken into consideration along with the lack of responsiveness in decapitated planarians that are exposed to cocaine, it is logical to consider utilizing this information in order to help determine the onset of function in the regenerating brain in planarians.  Planarians have the highest concentration of adult Pluripotent stem cells found in any animal species (cite.) This affords a unique opportunity to be able to observe the onset of function in regenerating cells in a centralized nervous system.  Although there is relatively little plasticity in mammalian CNS, enough homology exists between genes that govern the formation and expression of the CNS (between species) some of the results gained from such observations have the potential to contribute to the understanding of when these systems cross a threshold needed to go from not functional to functional.

 

Experimental Design

Planarians that were selected to be of similar size were selected and placed in a vial of APW and a similar cohort of planarians were decapitated and placed in an identical vial of APW. Both groups were segregated on Day 0.  The first of a set of observations was conducted on Day 0. Each planarian was placed in an observation well and immersed in a solution of APW or 1mM solution of cocaine and observed for seizure-like positions for ten minutes. Four observations were made for each of the following: Control Intact, Control Decapitated (on Day Zero) or Regenerating (From Day One through Day Seven), Cocaine Intact, Cocaine Decapitated (on day Zero) or Regenerating (From Day One through Day Seven.) This set of observations was repeated on Day 2, 4, 5, 6, and 7.

When Seizure-like positions are observed in regenerating planarians, there is a strong likelihood that the brain has regenerated to the degree that function has been sufficiently restored such that the effect of cocaine upon the nervous system of the regenerating planarians is able to respond to the cocaine in a quantifiable and replicable way.

 

 

 

 

 

 

 

 

 

 

 

 

 

Graph 2

A Comparison of Seizure-like Responses in Intact Vs. Decapitated Planarians

 

This graph demonstrates results in intact and decapitated planarians in the presence and absence of cocaine.  All of the decapitated planarians (control, cocaine, and the combination of cocaine and Parthenolide) display no significant response. The intact planarians’ control elicits no response while the intact planarian in the solution of cocaine demonstrates seizure-like positions at a rate that is consistent with previous observations.  Similarly, when the solution of cocaine is augmented with Parthenolide at a concentration of 50µM, the seizure-like behavior is reduced by approximately 50%.

 

Figure 1: Comparing Planarian Seizure-Like Movements

Figure 1 comparing the frequency of seizure-like movements in L-glutamic acid (p=0.491), NMDA (p=0.0007), Nicotine (p=0.592), and Cocaine (p=0.00002). A two-tailed paired t-test was conducted on each data set (intact verses decapitated.)

Figure 2

Difference Between Groups on the Same Day

Day	P-value	Significant?
0	0.005008	Yes
2	0.011431	Yes
4	0.009077	Yes
5	0.230201	No
6	0.134488	No
7	0.099728	No

This figure is the result of a Two-tailed T-test comparing the number of seizure-like positions of intact planarians with regenerating planarians on a given day. When the p-value is not significant (significance is 0.05%).  When the transition from significance to that of no significance difference is realized, there is reason to believe these data support the presence of a brain that is necessary to elicit the response that is being observed.

Graph 3

Control, Seizure Like Positions (or the lack thereof) in Intact and Decapitated Planarians

This graph establishes controls elicit no significant seizure-like behaviors in planarians.

Graph Four

Seizure-like Positioning in Intact and Decapitated Planarians Upon Exposure to Cocaine at 1mM Concentration

Figure X shows seizure like positions in intact and regenerating planarians over a period of seven days.  The sample size for days 0-6 was n=4.  On Day 7, the sample size was n=3.  Data were evaluated using a 2-way ANOVA with a p-value of 0.02.

Discussion

This series of observations afforded an opportunity to unite the disciplines of neuropharmocology with regenerative biology by employing the unique properties that planarians enjoy as a result of their regenerative abilities.  Naturally, the results, while substantive, have raised more questions about the nature of the structures in the planarian brain that are recruited when exposed to cocaine.  The partial reduction of seizure-like behavior when planarians are exposed to NMDA is also sparking questions. What is causing this partial antagonism of the normal neuronal response upon exposure to NMDA? Are two different receptors affected by NMDA? Is there a common pathway shared by whatever is being used upon exposure to cocaine? 

While cocaine works on the dopaminergic and serotonergic pathways, Cytisine affects nicotinic acetylcholine receptors.  In the meantime, NMDA is known to affect the Glutamate/GABA pathways (cite sources.)

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Additional Information About the Experimental Design for NMDA

Seizure-like behavior for NMDA was reduced to 50% of its normal levels when planarians were decapitated (unpublished data.) The objective of this experiment is to determine when the planarian brain has recovered the ability to express seizure like positions at normal levels when the planarian has regenerated its brain. NMDA levels are at 1.0 mM concentration.

Approximately 50 planarians (D. tigrina) will serve as the intact controls while 50 were decapitated on Day Zero. 

Starting on Day Zero, observations were conducted in order to quantify the number of seizure-like positions an individual planarian makes in ten minutes when immersed in a solution of NMDA at a concentration of 1.0mM.  Observations were made between 8 and 11 AM in order to provide consistency and rule out differences that were a result of circadian rhythms.

Sample size is n=4 for each of the following; Control Intact, Control Decapitated/Regenerating, NMDA Intact, NMDA Decapitated/Regenerating. Observations will be made on Day Zero, 2, 4, 5, 6, and 7.

Additional Information About the Experimental Design of Cytisine

Figure 16: Cytisine Molecule (Public Domain)

Cytisine (not to be mistaken for the pyrimidine, Cytosine,) is derived from laburnum seeds and has been used to help people stop smoking (cite source.) Cytisine was one of the neuronally active compounds that were being observed in the Pagan Laboratory during the Fall 2011 semester.  When one of the planarians did not respond to being in a solution of 1mM Cytisine, it was observed under the dissecting microscope and was found to be regenerating a head.  This observation resulted in Cytisine being used in a seven day regeneration observation that was similar to the previously reported observations using cocaine and NMDA.  

One of the more fascinating elements of this study is the notion that these three compounds tend to work within three separate neurotransmission pathways. Cocaine works within the dopaminergic and serotonergic pathways while Cytisine works with the Acyetlcholine pathways and NMDA uses the GABA/Glutamic system. 

Future Direction

It is the nature of Hypothesis Testing to finish with more questions to explore than those that were answered or even initially asked.  There are several avenues of exploration that have presented themselves among them include but are not limited to establishing any pattern of change in the transcription rate of structures such as the Dopamine Transporter, Dopamine and Serotonin receptor sites. Conducting assays to detect metabolites of suspected ligands or neurotransmitters would also provide clues to piece together a larger neuropharmacological picture. 

Another direction would be to continue work with the human embryonic kidney cells that were transfected with the dopamine transporter.  Receptors and serotonergic receptors and transporters could also be transfected into this cell line. 

With respect to the regeneration studies, it would be helpful to track the regeneration of ganglion as well as the regeneration of receptor sites in order to correlate their appearance with gain of function.  This could be augmented by a microarray, perhaps a microarray could be conducted at the same time the p-slp observations are made.

Room for Improvement

Schmidtea mediterranea is presently not available for purchase in the United States and, as this is being penned, efforts are being made to secure a colony of S. mediterranea from Dr. Nester Oviedo’s lab.   It will become necessary to achieve proficiency at propagating colonies of planarians.  Maintaining a colony of S. mediterranea would increase the efficiency of genetic analysis that is important when investigating the molecular mechanism(s) of action at the transcriptional level.  Getting primers from Dugesia japonica to work with Dugesia tigrina with genetic sequences of Schmidtea mediterranea is precarious at best and a waste of time and reagents at worst.

Additionally, planarians seem to have some observable irregularities in their behavior that has yet to be explained. The surface area to volume ratio helps to account for individual differences but there seems to be more than one factor that creates an irregular pattern in planarians.  There is some speculation that the planarians may be affected by some type of circadian-type rhythm that has not been identified.                            

Finally, blindness should be incorporated into the observation process in order to reduce or eliminate the influence of test-giver bias.  It is possible, for example, to decant the various solutions into individual 5.0 mL. containers and label the container with a code.  The vials would be given to others to test afterwards; the code would be matched to its true identity.