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    • 2013 Publication

Response to acoustic stimuli in the plainfin midshipman
Jack Howard ('19)

Abstract:    While the function of sensory systems within the brain are well documented, the inside-out modulation, or efferent modulation, of the brain is fairly unknown. This efferent system was explored using the model species Porichthys notatus, a species of toadfish called the plainfin midshipman, which has been documented to exhibit sexual dimorphism and seasonal reproductive behavior. Catecholamines, which are neurotransmitters, are proven modulators of vocal, auditory, and reproductive behavior across all vertebrates, and are the concentration of many studies focusing on hearing loss. Tyrosine hydroxylase, a rate-limiting enzyme of all catecholamines, is activated when phosphorylated by protein kinase enzymes. Phosphorylated tyrosine hydroxylase was observed within efferent regions of the brain within the midshipman to determine why tyrosine hydroxylase is activated and what function it serves within these regions. Female midshipman were exposed to three different noises: mating “advertisement” calls, ambient background noise mimicking their habitat, and noise whose wavelength is identical to the mating calls. Tyrosine hydroxylase was anticipated to activate at different levels in response to the advertisement calls and the background noise recordings. Our results show a higher density of phosphorylated tyrosine hydroxylase within midshipman exposed to mating calls than in background noise groups within two regions of the brain projecting catecholamines to the ear.
Introduction:         
​Catecholamines are amines naturally produced in the brain that function similarly to neurotransmitters or other naturally-occurring hormones (Forlano
et al., 2015). Made up of both catechol and amine groups, catecholamines consist of dopamine, adrenaline, and noradrenaline and are known to trigger emotions in humans such as but not limited to: motivation, drive, sexual interest, aggression, overall cognitive function, impulse, irritability, and anxiety (Forlano et al., 2015).

Catecholamines serve as modulators of vocal, auditory, and sexual behavior in all vertebrates (Ghahramani et al., 2015) and have been observed at elevated levels in the human bloodstream as the amount of noise in a workplace increases (Sudo et al., 1996). These studies reveal that increased catecholamine production is a result of auditory or vocal behavior in vertebrates. Catecholamines are important factors in hearing loss. However, the role of dopamine and its presence within efferent brain regions is unknown in a greater context and is not funded by those invested in understanding hearing loss.
Picture
Figure 1: The synthesis of all catecholamines begins when TH is added to tyrosine to produce dopamine, the building block for all other catecholamines. (Sharaff and Freestone, 2011)

Tyrosine hydroxylase (TH) is a rate-limiting enzyme for all catecholamines. TH is crucial for the synthesis of all catecholamines (see figure 1); once added to tyrosine, it becomes dopamine, which is the first synthesized catecholamine (Fernstrom, 2007). Dopamine beta-hydroxylase (DBH) is a necessary ingredient in the synthesis of noradrenaline or adrenaline from dopamine, meaning that DBH indicates the presence of adrenaline and/or noradrenaline (Fernstrom, 2007). When TH is present in the absence of DBH, it can be assumed that the only catecholaminergic activity is dopaminergic, as opposed to adrenaline or noradrenaline.
Phosphorylated TH (pTH) is a form of TH that has only recently been a focus of study within the Forlano Lab. When protein kinase enzymes phosphorylate TH, there is usually an increase in movement, indicating the release and activation of TH (Joh et al., 1978), thus showing the synthesis of catecholamines. TH, which has been a longtime focus in the Forlano Lab, marks all TH activity within the brain—active and inactive. However, pTH only marks “active” TH neurons, meaning that it depicts the regions in which TH is being produced and synthesized.
The plainfin midshipman (Porichthys notatus) is a species of toadfish native to the Pacific West coast of the United States, and has three different morphs: females, Type I males, and Type II males (Sisneros, Forlano, Knapp, & Bass, 2004). Type I males and Type II males can be distinguished in three main ways, two of which are easily observed, the third being more ambiguous, and thus the focus of the Forlano lab. Both behaviorally and physically, the Type I and Type II male midshipmen exhibit vastly different traits and qualities. The Type I midshipman has a much larger body mass than the Type II male (Ghahramani, et al., 2015). However, the sexual organs of Type I males are much smaller relative to their body size than Type II males (Ghahramani, et al., 2015). The internal differences are not well-documented, and could very well shine light upon previously unknown similarities or differences between the two male morphs.
In a previous study focusing on catecholamine levels in the midshipman brain, it was found that during the reproductive season, female midshipman fish had significantly greater TH innervation (concentration) within regions of the brain near the saccule (the primary end organ for hearing—the plainfin midshipman ear) during their reproductive season than during the winter (Forlano et al., 2015). Other regions, such as octavolateralis efferent nucleus (OE) and the periventricular posterior tuberculum (TPp), yielded results showing an increase of TH during the reproductive season, whereas other regions further from the saccule yielded less TH activity during the reproductive season than the winter season (Forlano et al., 2015).
The TPp is an efferent region of the brain that is dopaminergic (Tay et al., 2011), which sends dopamine to regions of the brain that project to the saccule. The OE, which is within the hindbrain, projects catecholamines to the saccule as well as the lateral line system (Bass et al., 2000), a group of organs found in aquatic vertebrates that detect movement and differences in pressure. It has been identified to serve as a suppressor of noise to the saccule, insulating it when loud noises are being absorbed by the ear, but making it more sensitive to noise when less noise is present.
The goal of this work is to observe the OE and TPp’s dopaminergic response to different acoustic stimuli.  These two efferent regions project dopamine to the saccule, which is the main hearing organ of the midshipman. Much is unknown about dopamine’s role in sensory processing. In past studies, TH has been observed to fluctuate as a result of different stimuli; however pTH’s role remains as an  unknown at this point. It is hypothesized that female midshipman when exposed to acoustic stimuli mimicking male vocal calls will contain differing amounts of pTH in the TPp and OE regions of the brain than when not exposed to acoustic stimuli.
Materials and Methods:Female plainfin midshipman were harvested between June and July of 2018 from intertidal breeding sites in Friday Harbor, Washington, and brought to flow-through tanks at Friday Harbor Labs (University of Washington). Due to the time and location of their capture, all female fish were in their reproductive state. The fish were then placed in one of three different test groups: hum, noise, or control.
The hum group was exposed to multiple Type I male midshipman calls looped together continuously. The noise group served as a secondary control group that was exposed to a type of noise called “brown noise” that mimicked the wavelengths of the hums, while not having the exact makeup of a hum call. The control group was a recording of the intertidal breeding zone of the midshipman without any calls from fish.  In both the hum and noise groups, sounds were played from 100-500 Hz. However, the hum group’s audio contained some harmonic noises at different frequencies that were not present in the noise group’s audio. If the hum and noise groups were to yield similar results, this would indicate that the female midshipman ear cannot distinguish between reproductive and nonreproductive calls by wavelength. This would mean that the brain regions react to qualitative differences between auditory stimuli, rather than quantitative differences—wavelength. However, if noise and hum groups were to yield different results, this may indicate that female midshipman can perceive reproductive calls. The fish within each group were exposed to their respective noise condition for 15 minutes continuously in a tank, followed by a 30 minute period of no noise being played. The fish were able to move freely in their tanks, and the majority of fish remained on the outside of the tank, meaning most fish received playback of similar volume.
    After the 30 minute period of no noise being played, the fish were euthanized in glass jars of seawater mixed with anesthesia. Once euthanized, fish were dissected, and their brains were removed and preserved in 4% paraformaldehyde in the refrigerator. The brain was treated using immunohistochemistry (a process in which brain slices are stained with antibodies in order to excite neurotransmitters different colors for quantification) before being put in cryogel and sliced using a cryostat at around -20°C. Antibodies were used to stain and identify TH as green, pTH as red, and the natural anatomy of the fish as blue. During slicing, every second slice was archived on a slide in order to have backups of each region if others were to get destroyed. All usable slices that contained TPp or OE anatomy were imaged by a microscope and run through a macro system using the open-source ImageJ application.  Then, the regions were traced and the signal of TH and pTH were thresholded and quantified. Within the ImageJ protocol, the average intensity of each channel was found. Average intensity is the average amount of a channel (in this case pTH) that was quantified in each image.

​Results:

Brains were sectioned and stained using immunohistochemical techniques. The tissue with stained with markers for phosphorylated TH and TH. Images were derived from fluorescent microscopy and analyzed on image J. Average intensity were measured using Image J.

Picture
Figure 3: The average intensity of pTH within the OE while being exposed to the 3 different noise types. Somata and Dendrite indicatethe location of the pTH within cell bodies in the OE.
There were 22 animals tested for the TPp region of the brain, and 43 images per animal. There were 7 animals each for the noise and hum groups, and 8 animals tested for the control. Within the TPp animals, a significantly lower average intensity of pTH was found in the hum group in relation to the ambient noise and control groups. There was not a significant difference of pTH intensity between the ambient noise and control groups.
    Seventeen animals were used for the OE analysis; Five animals were discarded from analysis because of damage during the sectioning process. The results demonstrate a decrease in pTH intensity within the hum group in comparison to the background noise group. There was an insignificant difference between the hum group and the control, and an insignificant difference between the control and the hum groups.
Discussion and Conclusion:Within the OE and TPp, the noise and control groups had nearly identical pTH levels, confirming the hypothesis that they would vary from the group exposed to advertisement hums. Further evidence must be found in order to determine whether pTH within the OE indicates dopaminergic suppression of hearing or if dopamine enhances the hearing mechanisms. These preliminary results suggest that a decrease in pTH while exposed to advertisement hums means that dopaminergic activity within the OE increases sensitivity in the saccule. Thus, an increase in calls significant to reproduction, or noises significant to hearing organs, would decrease the amount of noise entering the ear. This supports the established function of the OE, which is to filter noise through the saccule and serve as an insulation of sorts. However, this interpretation could be revised in order to account for the duration of the presence of pTH, which remains to be unknown.
From previous research, it is known that the TPp and OE send dopaminergic projections to the ear of the midshipman. Additionally, the TPp sends projects to the OE. It is interesting that the TPp would make direct projections to the ear and then also send inadvertent signals to the OE, which also projects to the ear. The Forlano Lab hypothesizes that the dopaminergic projections from the TPp to the OE assist and increase signals from the OE to the ear, aiding hearing.
Whether dopamine is amplifying or attenuating signal, we are unsure—as we know that when acoustic stimuli is decreased, so is dopamine production. This could indicate that dopamine allows for the midshipman reduce the total amount of noise entering the ear, in order to parse through background noise better. However, it is also possible that dopamine amplifies noise in the ear in order to better hear noises crucial to their reproduction.
Observing the pTH levels within the TPp and OE show that the plainfin midshipman uses more than just wavelengths of noise to discern what stimuli is connected to reproductive and social behavior crucial for the passing down of its genes.
    Future work will include the study of regions not efferent to the saccule, the results of which will be compared to the OE and TPp regions. These results indicate a function of the OE that very closely monitors the saccule’s input—a function that is likely only found in very few brain regions. Similar regions will be explored through the lens of pTH and results will be compared and contrasted to those of this study.


Acknowledgements:
Thank you to Kelsey Hom and Dr. Paul Forlano for their continued support and facilitation of this work at Brooklyn College and beyond. Additionally, thank you to Ms. Schmitz and Ms. Machac for their coordination of my research within the Science Research Program. All of these individuals are integral to this research, and their assistance is greatly appreciated.
References:Bass, A. H., Bodnar, D. A., & Marchaterre, M. A. (2000). Midbrain acoustic circuitry in a
vocalizing fish. Journal of Comparative Neurology, 419(4), 505-531.
Bass, A. H., & Mckibben, J. R. (2003). Neural mechanisms and behaviors for acoustic
communication in teleost fish. Progress in Neurobiology,69(1), 1-26.
doi:10.1016/s0301-0082(03)00004-2
Fernstrom, J. D., & Fernstrom, M. H. (2007). Tyrosine, phenylalanine, and catecholamine
synthesis and function in the brain. The Journal of nutrition, 137(6), 1539S-1547S.
Forlano, P. M., Ghahramani, Z. N., Monestime, C. M., Kurochkin, P., Chernenko, A., &
Milkis, D. (2015). Catecholaminergic Innervation of Central and Peripheral
Auditory Circuitry Varies with Reproductive State in Female Midshipman Fish,
Porichthys notatus. Plos One,10(4). doi:10.1371/journal.pone.0121914.
Forlano, P. M., Sisneros, J. A., Rohmann, K. N., & Bass, A. H. (2015). Neuroendocrine
control of seasonal plasticity in the auditory and vocal systems of fish. Frontiers
in Neuroendocrinology,37, 129-145. doi:10.1016/j.yfrne.2014.08.002
Ghahramani, Z. N., Timothy, M., Kaur, G., Gorbonosov, M., Chernenko, A., & Forlano, P.
M. (2015). Catecholaminergic Fiber Innervation of the Vocal Motor System Is
Intrasexually Dimorphic in a Teleost with Alternative Reproductive Tactics. Brain,
Behavior and Evolution,86(2), 131-144. doi:10.1159/000438720
Optimizing Efficacy and Tolerability of Antidepressant Therapy: Does Selectivity of
Action Matter?: The Alphabet Soup of Antidepressant Pharmacology: From TCAs
and MAOIs to SSRIs, SNRIs, and Beyond. (n.d.). Retrieved December 4, 2017,
from https://www.medscape.org/viewarticle/458647_2.
Sharaff, F., & Freestone, P. (2011). Microbial endocrinology. Central European Journal of
Biology, 6(5), 685.
Sisneros, J. A., Forlano, P. M., Knapp, R., & Bass, A. H. (2004). Seasonal variation of
steroid hormone levels in an intertidal-nesting fish, the vocal plainfin midshipman.
General and Comparative Endocrinology,136(1), 101-116.
doi:10.1016/j.ygcen.2003.12.007
Sudo, A., Luong, N. A., Jonai, H., Matsuda, S., Villanueva, M. B. G.,
Sotoyama, M., ... & SY, N. (1996). Effects of earplugs on catecholamine and
cortisol excretion in noise-exposed textile workers. Industrial health, 34(3),
279-286.
Tay, T. L., Ronneberger, O., Ryu, S., Nitschke, R., & Driever, W. (2011). Comprehensive
catecholaminergic projectome analysis reveals single-neuron integration of zebrafish
ascending and descending dopaminergic systems. Nature communications, 2, 171.
The Editors of Encyclopædia Britannica. (2017, November 24). Catecholamine.
Retrieved February 28, 2018, from https://www.britannica.com/science/catecholamine.
Zug, G. R. (2018, May 03). Lateral line system. Retrieved October 8, 2018, from
https://www.britannica.com/science/lateral-line-system.

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    • 2017 Publication >
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      • Mathematic and Scientific Exploration
      • Reactions and Responses
      • Scientific Research
    • 2016 Publication >
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      • Mathematic and Scientific Explorations
      • Scientific Research
      • Reactions and Responses
      • Artistic Creations
    • 2015 Publication >
      • Historical and Current Explanations >
        • Bell Curves
        • Birds Vs. Turbines
        • Energy in the Obama Era
        • The Future of Neuroscience
        • Gender Gap in Math
        • GMOs--Yes or No?
        • The History of Minecraft: How a Swedish Indie Game Came to Dominate the World
        • The Effect of Prozac on the Brain
        • Philae Lander's Discovery of Organic Molecules
        • Advantages and Disadvantages of Wind Turbines
        • Your Own Worst Enemy: An Overview of Lupus
        • The Methylhex Ban
        • The Effect of Lyme Disease on the Immune system
        • Infectious Mononucleosis
        • Replacing CFCs
        • The Switch
      • Mathematic and Scientific Explorations >
        • The 43rd Figure
        • The Clock
        • The Collatz Conjecture
        • Constructing a Soccer Ball
        • Determining how Ballparks Affect Batter's Ability to Create Hits
        • The Rotating Conundrum
        • Pythagorean Puzzle
        • Mathematic and Scientific Explorations
        • Kinetics Lab
        • Math in the Restaurant Business
        • Math as a Vessel for Social Change
        • Sustainability of Bottled Vs. Tap Water
        • Thoughts on the Lottery
        • Understanding Player Efficiency Rating
      • Scientific Research >
        • Communicating With Computers
        • The Mystery of Asthma
        • The Nanoscopic War Against Cancer
        • Phytochemistry
        • Solving the energy crisis with Intermediate Band Solar Cells
        • A Pain That Never Ends
        • Rapamycin Resistance
        • Ampacity of a Single Core Horizontal Cable
        • Morphological Properties of Texting Acronym Formation
        • cGAS and STING Expression
      • Reactions and Responses >
        • Can Humans Survive the Climate Crisis?
        • My Experience as a Teacher's Assistant
        • Ted Talk Responses
        • Teens For Food Justice
      • Artistic Creations >
        • Chandelier
        • Deltoidal Hexacontrahedon
        • Dodecahedron Card Trick
        • Eye of the Triangle
        • Free Radric Delantic Davis
        • The Grid
        • What Does A Randomly Composed Song Sound Like?
        • Science Wing Mural
    • 2014 Publication >
      • Cover Photo
      • Artistic Creations >
        • Art Using the Fibonacci Sequence
        • Computer Generated Architecture and Designs
        • Mathematical Landscape
        • Math Art
        • Math in Music
      • Historical and Current Explanations >
        • Algae Bio-Fuel
        • An Energy Alternative
        • Clean Energy In Transportation
        • Calorie Restriction
        • Creating Energy in the Modern World
        • Dietary Intervention Impact on Gut Microbial Gene Richness
        • Earthly Applications for NASA Technology
        • Explaining Relative Motion
        • Exploring Artificial Inteligence
        • Gamma Function
        • How Leaves Work
        • Hydrogen Fuel Cells
        • Music and Brain Development
        • Programming Calculators
        • The Science of Microsatellites
        • Sci-Fi Taser
        • Sloane's Gap
        • Sustainable Energy: Why Some Ideas Shine Brighter than Others
        • Understanding The Galvanic Cell
        • The Virus: Our Unforeseen Philosopher's Stone
        • What Are Fuel Cells and How Do They Work?
      • Mathematic and Scientific Explorations >
        • Astrocytes Expressing ALS-Linked Mutated SOD1 Release Factors Selectively Toxic to Motor Neurons
        • Big Bang
        • Dictyostelium Discoideum
        • The Future of Solar Cell Technology
        • And Many More...
      • Reactions and Responses >
        • Alternative Energy Sources, New but Unused
        • An Insight Into the Curious World of Ethnobotany
        • Challenging What We Think We Know
        • The Current State of American Education
        • Discovering New Numbers
        • Interview With an Architect
        • Life of Pi Response
        • Mathematical Art Video Commentary
        • Missing from Science Class
        • The Museum of Math
        • The Inside Scoop on a Real Mathematician
    • 2013 Publication