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species, Dugesia dorotocephala, seem to indicate that parthenolide alleviates amphetamine-induced pSLM (Rawls et al., unpublished data).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.