NHRC _22_P4
Derron R. Taite, Mohammad Kutub Ali
Introduction
N-Methyl-D-Aspartate receptors (NMDArs) are a of
family membrane bound glutamic receptors/ ion channels
found mostly in neurons. These receptors are sensitive to
the concentration of extracellular glutamate, which can
bind to NMDArs allowing the ion channels to open. This
then triggers an influx of extracellular calcium cations
(Ca2+) into the neuron, which ultimately leads to an
excitatory response. Noxious stimuli (stimuli which are
associated with pain) triggers the release of glutamate,
therefore, NMDArs can be considered as a type of pain
receptor. Monosodium glutamate (MSG), a salt of
glutamate, is a common food additive used particularly in
Asian cuisine. MSG consumption is associated with some
unwanted reactions, including headaches, facial pain and
mechanical sensitivity in pericranial muscles. MSG has a
similar structure to glutamate, which gives it the ability to
bind to NMDArs in-vitro and initiate a pain response.
2-Phenoxyethanol ethanol (POE), a commonly used fish
anaesthetic in aquaculture. Its mechanism of anaesthesia is
not fully understood; however, previous studies have
indicated that POE reduces NMDAr generated membrane
potentials considerably in a reversible and concentration
dependent manner. Nicotine and alcohol also affect
NMDArs in different ways, where, nicotine changes the
structures of the subunits of NMDArs making substrates
unable to bind to the receptor to trigger pain responses.
Alcohol reduces the protein synthesis of NMDArs and
should lead to an increase in pain response, however, it
can also negatively affect the integration of these receptors
into cell membrane.
Objective
• To observe whether POE sensitivity in zebrafish differs
between individuals and if there are sensitivity changes
in the presence of alcohol, nicotine and monosodium
glutamate (MSG).
Methods
Materials
Adult zebrafish, Wray and Nephew white overproof rum
(65% vol/vol), 500 mL containers, 1.8% e-liquid nicotine,
distilled water, 2-phenoxyethanol 99% vol/vol (Sigma-
Aldrich), Logitech 5 MP camera, pins, Island Spice MSG,
laptop running iSpy recording software.
RESEARCH POSTER PRESENTATION DESIGN © 2015
www.PosterPresentations.com
Nicotine alters susceptibility to anesthesia and pain response in zebrafish via NDMA receptors
Department of Basic Medical Sciences, Faculty of Medical Sciences, University of the West Indies Mona campus, Kingston Jamaica
Methods
2-Phenoxyethanol treatment solutions preparation
•A 5% POE stock solution was generated by pipetting the appropriate
volume of 99% POE into 500 mL of distilled water. From this
solution all further treatment solutions were generated.
•Subsequent treatment solutions of 0.0450, 0.0375, 0.0370, 0.0360,
0.350, 0.340, 0.0330, 0.0325 and 0.0300% POE were made by
pipetting appropriate volumes of the 5% stock POE solution into 400
mL of distilled water.
Phenoxyethanol Assay
•Different groups of six (6) adult healthy zebrafish were exposed to
400 mL solutions of POE ranging from 0.0450-0.0300% for 45
minutes.
•Videos of the fish behaviour during this 45 min treatment period
were recorded using iSpy software.
•During the treatment the tail response reflex was assessed by gently
pricking the tails of the treated fish with a pin at regular intervals.
The loss of this reflex signifies that the fish is fully anesthetized.
•These experiments were repeated and a graph was generated to find
the optimal concentrations of POE to be used in the further
experiments.
Drug Assays
•Different groups of six (6) adult healthy zebrafish were subjected to
400 mL solutions of 0.0350% POE containing either 50 mg/L MSG,
0.5% alcohol, 0.5 mg/L nicotine and 1 mg/L nicotine over a 20-
minute treatment period.
•Videos of the behavioural changes of the fish over this treatment
period were generated using iSpy software. The loss of the tail
response reflex was assessed for the treated fish.
•The susceptibility for anaesthesia was then generated and compared
under the different treatment conditions
Statistical Analysis
•All graphs and the appropriate statistical tests such as; one way
ANOVA and t-tests, were done using Graphpad prism version 6
statistical software.
•P values </= 0.05 were considered to be statistically significant
Results
Graph #1:
Graph #2:
Graph #3:
* of receptors to fully stop POE from showing its influence.
* Indicates degree of statistical significance. A slightly significant decrease
(p=0.0384) in susceptibility was seen in the 1 mg/mL nicotine treatment
when compared to the 0.035% phenoxyethanol control.
Conclusions
POE Assay
• With increases in POE concentrations, a general decrease in sedation times
can be seen in the fish. POE is a competitive inhibitor of NMDArs, therefore,
with increases of POE concentrations there would be an increase in POE
binding to these receptors causing more inhibition, ultimately reducing pain
perception.
• 0.035% POE was selected because its average anaesthesia time fell in middle
of all the concentrations assayed. This gives ample time to detect anaesthesia
without risking fish death as seen in higher concentrations of POE. The fish
were also more less immobile when placed in this solution making it easier to
detect the tail reflex. In lower concentrations the fish were disorientated and
very much mobile, this makes it harder to detect anaesthesia, as well as, their
increased mobility posed a higher risk of injuring the fish during testing.
Drug Assays
• Alcohol cannot directly bind to pain receptors, and can only affect their
activities in indirect ways. Alcohol can affect the activity of NMDArs
indirectly in two ways; increasing the expression of NDMArs and/or
affecting the integration of these receptors into the cell membrane,
therefore, alcohol can be a pain reducer or agitator or both. Based on
results alcohol played no direct part in the binding of POE to already
membrane bound receptors, therefore, having no perceived effect. Even if
alcohol caused the production of more receptors, if the membranes of the
cells are already saturated with receptors newly formed ones are going to
find it hard to integrate into the cell.
• MSG is unable to cross the blood brain barrier and it effects are mostly
because of the interaction with the peripheral nervous system (PNS). The
effect of anaesthesia starts at the central nervous system (CNS) and
reduces/blocks the perception of pain stimuli. POE was already bound to
the receptors in CNS and can competitively bind to those in PNS, thus,
making either MSG unable to bind to receptors or blocking pathways to
the CNS which pain is perceived, thus, explaining the result observed.
• Nicotine can directly affect NMDArs or indirectly through the activity
Nicotinic acetyl choline receptors (NaChRs) and can easily pass through
the blood brain barrier to affect the CNS. In both instances nicotine
increases the activities of NMDArs by binding directly to them and changing
the structures of the subunits to make certain substances unable to bind
to them to close the receptors or either through NaChRs triggering the
release of extracellular glutamate which binds to receptors. In this case
what was observed seemed to be explained via NaChRs route, this is
because nicotine is very fast acting and if it was bound directly to the
NMDArs, the lower concentration of nicotine would have prevented the
binding POE, thus significantly reducing the anaesthesia susceptibility. This
was not seen, however, when the concentration of nicotine increased
there was a significant reduction in susceptibility indicating somewhat of a
competitive relationship between nicotine and POE activity. This suggests
that it may have released extra glutamate, which bind to the receptors
before the arrival of POE to central nervous system. This does not entirely
rule out the directly influence theory as the low concentration of nicotine
may have not been enough to trigger significant changes in large number
• Overall Conclusion
Zebrafish pain responses differ between individual fish. Nicotine can directly
act pain receptors and limit the effect/binding of different substrates to
these receptors, which may explain the reduction in susceptibility observed.
Alcohol and MSG does not directly affect the activity of POE, thus indicating
that they may act on different sites in the nervous system.
References
Mußhoff, U., Madeja, M., Binding, N., Witting, U., & Speckmann, E. J. (1999). Effects of 2-phenoxyethanol
on N-methyl- d -aspartate (NMDA) receptor-mediated ion currents. Archives of Toxicology, 73(1), 55–
59. https://doi.org/10.1007/s002040050586
Petrenko, A. B., Yamakura, T., Baba, H., & Shimoji, K. (2003). The Role of N-Methyl-d-Aspartate (NMDA)
Receptors in Pain: A Review. Anesthesia &Amp; Analgesia, 1108–1116.
https://doi.org/10.1213/01.ane.0000081061.12235.55
Woodside, B., Borroni, A., Hammonds, M., & Teyler, T. (2004). NMDA receptors and voltage-dependent
calcium channels mediate different aspects of acquisition and retention of a spatial memory task.
Neurobiology of Learning and Memory, 81(2), 105–114. https://doi.org/10.1016/j.nlm.2003.10.003
Zanfirescu, A., Cristea, A., Nitulescu, G., Velescu, B., & Gradinaru, D. (2017). Chronic Monosodium
Glutamate Administration Induced Hyperalgesia in Mice. Nutrients, 10(1), 1.
https://doi.org/10.3390/nu10010001
Zappettini, S., Grilli, M., Olivero, G., Chen, J., Padolecchia, C., Pittaluga, A., Tomé, A. R., Cunha, R. A., &
Marchi, M. (2014). Nicotinic α7 receptor activation selectively potentiates the function of NMDA
receptors in glutamatergic terminals of the nucleus accumbens. Frontiers in Cellular Neuroscience, 8.
https://doi.org/10.3389/fncel.2014.00332