SLC6A4 Gene and Functional Response to MDMA Application in Octopus Serotonergic System
|✅ Paper Type: Free Essay||✅ Subject: Biology|
|✅ Wordcount: 4009 words||✅ Published: 23rd Sep 2019|
Conservation of SLC6A4 Gene and Functional Response to MDMA Application in Octopus Serotonergic System
1) Specific Aims:
a) Aim 1: To investigate the conservation of transmembrane domain and amino acid regions necessary for MDMA binding between octopus and humans. Prior phylogenetic analysis has shown orthologs of the human SLC6A4 gene, which encodes the serotonin transporter SERT or 5-HTT, to be present in octopus. This study will examine the degree of functional conservation of MDMA effects in octopus and human with regard to social behavior, due to the commonalities in their serotonergic systems.
b) Aim 2: To determine whether the observed changes in social behavior are related to the serotonergic system, the serotonin transporter will be blocked prior to the experiment in one test group. It can be reasoned that the MDMA-induced changes are not due to serotonin if the results are consistent between SERT-inactive and SERT-active groups.
c) Aim 3: To characterize the social behavior caused by MDMA application in octopus. Prior studies have shown that movements post-MDMA were indelicate as compared to the control. It is possible that the observed social behavior could be explained by past experimental design that has provoked aggression between octopuses, which are generally asocial species. Another hypothesis is that hunting behavior could have been increased by drug application and, as there is a cannibalistic tendency among octopuses, post-MDMA movement is one of foraging aggression.
Serotonin is considered to be ancient on the evolutionary scale as a neurotransmitter and has been linked to social behaviors in both vertebrates and invertebrates (Dehal and Boore, 2005). In the past decade, the degree to which mechanisms within the serotonergic system are conserved between invertebrates and vertebrates has been further elucidated (Kristensen et al, 2011). For instance, studies have shown that the serotonin transporter SERT, which is encoded by the SLC6A4 gene, is conserved in the Octopus bimaculoides genome (Kristensen et al, 2011). However, research has not yet uncovered the functional conservation of serotonin signaling and whether social behaviors linked to the serotonergic system may be traced back to these genetic commonalities between octopus and humans. A recent experiment performed by Edsinger and Dölen (2018) attempted to begin testing this relationship based on the fact that the serotonin transporter encoded by the SLC6A4 gene is a binding site for MDMA, also known as 3,4-Methylenedioxymethamphetamine and Ecstasy, a man-made drug that is commonly known for its illicit recreational use.
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MDMA is a substance that has received attention for being a “happy” drug, which is due to its tendency to increase levels of three neurotransmitters: serotonin, norepinephrine, and dopamine (Hellerman, 2016). When the drug is administered, greater serotonin levels result in symptoms associated with elevated mood. Common side effects of MDMA include increased desire for or feelings of social connection, euphoric state of mind, and distortion of spatial-temporal perceptions. Its stimulation of wellbeing is what has brought MDMA into the research spotlight in recent years as a possible therapeutic for treatment-resistant conditions such as PTSD. Within the veteran population specifically, individuals with high anxiety and PTSD symptoms are routinely being prescribed SSRIs, or selective serotonin reuptake inhibitors, a class of antidepressants that also target the serotonergic system. However, only one in three veterans has found effective treatment for their PTSD after being prescribed recommended drugs such as SSRIs (Phillips, 2018). Using MDMA as a PTSD therapeutic, although less common, has thus far produced more positive states in the veteran population, providing a path to recovery in many cases.
This is why understanding the functional effects of MDMA’s interaction with the serotonergic system in vertebrates has received great attention in recent years. Invertebrate models, specifically octopuses, have been used to understand the drug-behavior relationship because the MDMA binding site in the ion transporter shows one hundred percent conservation between the octopus and human (Edsinger and Dölen, 2018). Due to this great degree of conservation, researchers can hypothesize that MDMA-application may result in comparable behavioral side effects in both octopus and humans. Especially as Octopus bimaculoides is typically an asocial organism that engages in minimal intraspecies contact, the increased social connection and physical touch created by MDMA should be a highly observable phenotypic difference.
Understanding the degree of functional conservation of SLC6A4 gene in both octopuses and humans may prove to be highly advantageous in the realm of contemporary medicine, but it also poses dilemmas in terms of the practical use of research results. The adverse side effects of MDMA cannot be ignored when considering its potential as a therapeutic—after a serotonin supply is released into the synaptic cleft due to administration, subjects experience withdrawal-like symptoms after a period due to depression of the neurotransmitter in the brain. Interestingly, effects observed post-MDMA use include depression, irritability and aggression. Side effects may continue in many cases unless doses of MDMA administered are gradually increased, which can result in dependency. Currently, the trials in which MDMA has been administered to patients have been highly controlled, under which a designated dose of MDMA is given within a clinical setting by a physician (Phillips, 2018). Patients are not permitted to self-administer doses of MDMA, in order to prevent adverse side effects. Regardless, if a strong functional conservation of the SERT system is elucidated in octopuses and humans, then studies may be constructed in order to examine the time-dependent effects of MDMA in order to understand factors such as tolerance and significant changes in social behavior.
This study, unlike prior research, will consider multiple factors that have proven to be limitations in the past. Edsinger and Dölen, who initiated many aspects of the discussion on cephalopod serotonergic system conservation, failed to conclusively demonstrate functional conservation by way of increased asociality in Octopus bimaculoides as a result of MDMA application (2018). The current study will control for factors such as upbringing by observing laboratory-hatched and wild-caught octopuses in parallel, assuring that the sample size is sufficient for statistically significant results. Another arm will vary the tank environments and diets of cephalopods in order to determine effects upon aggression-like symptoms due to foraging behavior stimulated by MDMA. Furthermore, boredom-initiated aggression will also be watched for by varying tank size. Most importantly, our study will observe the effects of blocking the serotonin transporter and observing whether social behavior changes post-MDMA application to determine if changes in behavior are related to the serotonergic system.
3) Experimental Procedures
Research Strategies for Aim 1: Continuation of Phylogenetic and Functional Analysis of SLC6 and SLC6A4
Comparative Sequence Analysis: The reference gene set that will be utilized in this experiment is the reference gene set made in prior studies using nineteen human SLC6 proteins along with the orthologs identified in fly and worm using Ensembl. The nineteen gene set was used by Edsinger and Dölen (2018) as well as Kristensen (2011) and the sequences may be accessed from UniProt v. 2018. Pairwise protein alignments will be determined of both the human SLC6A4 genes and their corresponding octopus SCL6A4 genes. As performed in previous genomic studies, the domain alignment and percent identity per protein and per domain will be established (Edsinger and Dölen, 2018).
Phylogenetic Analysis: For this experiment, we are going to generate a consensus tree in order to find the tree that represents the greatest likelihood of the SLC6A gene family for use in this experiment. The clades of the phylogenetic gene family will include the SLC6A4 family, the monoamine transporters, and the outgroup monoamine transporters. Formatting the tree in order to show the composition of species in each clade is preferred by our research protocol. In this part of the experiment, it can be seen that the monoamine transporters (such as SERT, our transporter under study) have been through a range of evolutionary patterns. There are two orthologs found in the octopus and mollusc genomes.
Behavior and Sociality Analysis: This experiment will utilize an experimental design slightly distinct from prior studies in order to test sociality pre- and post-MDMA application. Edsinger and Dölen (2018) utilized a tank that was segmented into three regions, a central region with a region on either side. One side included an object with which the octopuses could interact and the other side included another octopus kept beneath a barred area, which the octopus under study could access but not fully interact with. Tanks with a similar layout will be utilized in this experiment but there will be different arms – the factors that will be compared are serotonin transporter blockage, wild or laboratory upbringing, and the influence of hunger on aggression after MDMA application. The octopuses will be placed in the tank in 30-minute intervals as done previously – once prior to MDMA application and once after. The degree and type of social interaction will be closely monitored in both cases, noting how octopuses behave in relation to factors such as serotonin transporter inactivation, upbringing, and hunger levels. In order to test hunger as a possible stimulator of foraging-related aggression, octopuses will either be placed in the tanks after being washed in MDMA and being fed a healthy diet of crustaceans and fish or without any nourishment. This will allow for testing of aggression-like symptoms, determining to which extent, if any, they are hunger-related. Varying tank sizes will allow for further determination of aggressive behavior causation – when wild animals are kept in confinement, boredom may manifest itself in the animal through destructive behavior that may be directed towards the tank itself, the materials within the tank, or the mates that it is sharing the tank with (Mather and Anderson, 2007). The effect of tank size, confinement, and boredom on aggression must be evaluated in this experiment (Mather and Anderson, 2007). Wild-caught octopuses will also be used in addition to laboratory-hatched in order to understand the effects of upbringing and predation, as well as the ability for these results to be generalized as responses common to O. bimaculoides, an oceanic species.
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The ultimate factor that will be tested is the degree to which serotonin transporter blockage will produce changes in social behavior post-MDMA application. Since MDMA is a man-made drug that increases the amount of serotonin in the synaptic cleft, increased “sociality” or aggression-like behaviors are hypothesized to be prevented under blockage of the serotonin transporter. A large increase in the typical symptoms of high serotonin levels, such as elevated mood and increased physical touch should generally not be observed. If the symptoms persist, then the functional conservation of SLC6A4 must be investigated further and under different conditions, as this experiment may not prove the link between the functional similarities due to evolutionary conservation of the serotonin system in O. bimaculoides and humans.
Experimental Design to Optimize Social Behavior Accuracy
Keeping size constant is an important factor of this experiment, whether of the live objects or the inanimate objects that the subject is interacting with. Edsinger and Dölen (2018) showed that both male and female octopuses interacted more with female octopuses, but this could be a matter of size. If foraging behavior and aggression are present instead of increased sociality, then test subjects are likely to show less inhibition towards smaller targets. Size of both the inanimate and animate objects will be normalized in this experiment.
Pharmacology and Drug Information
When administering a drug, it is important to test the subjects in the window of the drug’s maximum efficacy. MDMA effects are greatest within a half hour to hour after being administered or ingested. As in Edsinger and Dölen’s study (2018), MDMA will be administered in the form of a bath, entering the test animal through its gills (Edsinger and Dölen, 2018). The recommended dose from prior studies will be used so as to prevent extreme phenotypes in response to high doses of the drug.
Two-factor ANOVA will be used in this study, followed by the Shapiro-Wilk Test and Tukey’s multiple comparison test, or the two-tailed unpaired Student’s t-tests.
Availability of Data
Tree text data and sequences will be available for public access.
Research Strategies for Aim 2: Specimen Collection and Characterization
Specimen Collection: The model organism to be used is O. bimaculoides, the California Two-Spot Octopus. Edsinger and Dölen (2018) gathered a wild female octopus in the brooding process and delivered the organism to the laboratory, where the octopuses for the study were to be hatched. It is important to note that, in their study, animals were kept together in tanks with hundreds of others for a period of nearly a month, followed by a 7-month social isolation period. Their experiment ultimately only used seven octopuses as its sample size, which does not provide conclusive, statistically significant evidence (Edsinger and Dölen, 2018). This experiment will improve on prior research in terms of the conditions in which the octopuses are raised. Although isolated from the wild in this experiment, they are generally an asocial species and do not fare well in restricted space with other organisms of their species present. The initial stage of Edsinger and Dölen’s experiment could have imposed stress upon the organisms, as they were housed with many other animals prior to their period of social isolation (2018). Also, when observing social behavior and extrapolating laboratory data to correspond to an oceanic environment, it is significant that the octopuses hatched in the laboratory were not exposed to the same environment in their study and matters of upbringing must be considered before generalizing behavioral observations (Edsinger and Dölen, 2018). The one-month period prior to isolation is not sufficient in terms of its social mimicking of an oceanic environment as the conditions were made due to housing restrictions and not actively chosen on part of the researchers in order to recreate the species or environmental stimuli that the octopuses might encounter in the wild. In this study, specimen collection will thus include laboratory hatched octopuses as done previously and those born in and collected from the wild, observed in parallel, in order to control for upbringing and environmental differences. Observing octopuses over a wider age range, as opposed to siblings born from the same mother, would be beneficial for our study and for the characterization of octopuses’ social behavior.
Research Strategies for Aim 3: Construction of Laboratory Tanks to Reduce Impact of Confounding Variables
Ethical Considerations: It is important to note that the previous study (Edsinger and Dölen, 2018) in which cephalopods were used as model organisms to understand the serotonergic system, a specific committee was not available for overseeing the researcher’s protocols and use of invertebrate species. Instead, a general adherence to the Animal Welfare Act was followed (Edsinger and Dölen, 2018). This experiment will continue with the prior’s abidance to animal welfare laws but also incorporate suggestions made specifically by animal ethicists, as invertebrate use in research is growing and the necessary conditions for octopus housing have been specified.
Housing Conditions: Tanks should include seawater of temperature between 20-23 degrees C, with seawater recirculating each hour. Tank sides must be covered if there are adjacent tanks in order for sufficient isolation for animals from stimuli outside the study. Appropriate seasonal light cycles should be used in the room. In prior studies, 12:12 or 14:10 light: dark circles were used (Edsinger and Dölen, 2018).
Tank Construction: In terms of an efficient and humane approach to our experimental design, it is significant that the welfare of invertebrates implicated in scientific research has received increasing attention in recent years and scientists have been held more accountable for their laboratory protocols. Creating a suitable laboratory environment for the use of cephalopods, specifically octopuses, as model organisms depends upon certain crucial factors. First, tank size should allow for the organisms’ fluid movement, providing ample swimming space (Mather and Anderson, 2007). The tank environment should also include a variety of objects for the octopuses to interact with and grasp, as well as materials with which they can construct dens for sleeping (Mather and Anderson, 2007). There should be food available within the tank for the organisms to freely choose – the use of live food specimens may allow the organisms’ predatory behavior to emerge and be observed. Even though this food must be administered in a non-oceanic environment, Mather and Anderson (2007) assert that the organisms can receive appropriate nutrition if the live food consists of freshwater fish and crustaceans. The safety of the tank environment must be considered in terms of the tank materials. The authors of this paper assert that only tanks with smooth sides should be used so that octopuses cannot damage themselves by swimming into tank corners. Such damage, if left untreated, can result in infection and fatality. Another consideration to be made is the inherent nature of the organisms and their interactions with each other – when designing the experimental environment, it should be noted that octopuses are solitary beings and should not be kept together in tanks with restrictions on space. Post-MDMA application, octopuses must be placed in tanks with each other in order to interact and for changes in social behavior to be monitored. Social behavior will also be monitored under identical conditions without MDMA application, but octopuses should be kept in separate adequately sized tanks in order to prevent any aggression due to space restriction at all other times during the experiment.
Tank Enhancement: This experiment will use rock, clay, sand, and other oceanic materials common to the octopuses’ environment in the tank in order for sleeping dens to be constructed by the octopuses. Prior studies have used live crabs and snails as nourishment, but freshwater fish will be included to meet diet requirements (Mather and Anderson, 2007). In this experiment, it is crucial that animals should be inspected for signs of emotional stress or tension at certain checkpoints throughout the study, as this can have significant effects on the observed social behavior recorded by researchers. Specifically, as many factors as possible are being controlled to prevent the onset of aggression in O. bimaculoides. After and before drug administration are the bare minimum number of checkpoints at which stress levels should be recorded.
Conclusion: If this study provides statistically significant results, it will build upon existing literature determining the functional conservation of SLC6A4 gene action and its relationship with MDMA via effects on social behavior. Observed behavioral reactions should be consistent with the properties of MDMA drug action and, if successful, the research will be instructive in terms of therapeutic approaches to MDMA and its incorporation into a clinical setting due to incredibly high conservation of the SERT transporter between humans and octopuses.
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