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5-3-2021
Function of spiny dorsal fin erector muscles in the bluegill, Lepomis macrochirus
Zakiyat Djabakatie
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Function of spiny dorsal fin erector muscles
in the bluegill,
Lepomis macrochirus
By:
Zakiyat Djabakatie
An Honors Project in Fulfillment
of the Requirement for Honors in
The Department of Biology
The School of Arts and Sciences
Rhode Island College
April, 2021
Acknowledgements
“Research means you do not know, but you are willing to find out” Dr. Charles F. Kettering.
This thesis ties together my research experience. A journey that started with the McNair Program, for which I am thankful for introducing me to research for the first time. I would like to extend my gratitude to Dr. Deborah E. Britt who offered me her guidance by teaching me how to productively read literature reviews via concise note takings to avoid plagiarism.
I would also like to express my gratitude to Dr. Anika Toorie for giving me the chance to work in her lab and for always including her research students in new research techniques and encouraging us to perform such techniques independently. I am also thankful for Dr. Toorie’s detailed lecture in Cell
Molecular lab on how to dissect research articles section by section. From this lecture, I have learned how to extract crucial information from each section first and piece it all together at the end for a complete yet summarized comprehension of the study at hand.
I am thankful to the Biology Department for choosing me as a 2020-2021 recipient of the Theodore
Lemeshka award and an honorable mention for the Joseph and Norma DiLibero scholarship. I am also extending my gratitude to the Biology Honors committee for their time and consideration for reading this thesis and for their insightful comments.
I am extending my appreciation to all my independent research peers in Dr. Maia’s lab. I am thankful for Amina K. Chamanlal for teaching me how to make and clamp electrodes, test water pH in the aquaria and perform water changes, surgical procedures on bluegill, and the use of LabScribe. I thank Nathan
Pimental and Meghan E. Hatcher for taking their personal time to review data analysis on LabScribe over zoom.
Lastly, I am exceedingly indebted to Dr. Anabela Maia for her infinite guidance at every step of this process, this project would not have been possible without her support. It has been such an honor to be part of her lab and learn from her. No part of this thesis would have been possible without her mentorship.
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Research to me is an eye-opening experience, one that would be worthless without a dedicated and passionate PI such as her. I am thankful for always being available, consistent, and flexible with our scheduled and unscheduled meetings regarding data analysis. Thank you for taking me under your wings.
Funding for this honors thesis was supported primarily by the Rhode Island Institutional
Development Award (IDeA) Network of Biomedical Research Excellence from the National Institute of
General Medical Sciences of the National Institutes of Health under grant number P20GM103430.
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Abstract
Local motor control directly contributes to stability, which can be compromised by injury or multiple neuromuscular disorders. In addition, lack of sensory perception as experienced by decreased limb sensation can further deteriorate one’s quality of life. The goal of this study is to use bluegill (Lepomis macrochirus) fins as model systems to study and gain insights on local motor control and sensory perception to improve stability and locomotion in humans, especially in a rehabilitative state. We hypothesize that 1) when exposed to turbulence (T), bluegill will use the spiny dorsal fin to recover stability and muscle intensity and duration will increase in the spiny dorsal fin erector muscles; 2) we expect the removal of afferent information, by injection of lidocaine, to decrease muscle intensity and duration compared to saline (control); and 3) the removal of muscle control by injection of flaxedil, to dramatically decrease muscle intensity and duration compared to saline. Upon sedation of the bluegill, the epaxial and spine erector muscles were implanted bilaterally with double insulated electrodes via hypodermic needles, followed by injection of 0.6 mL (0.1 mL per erector muscle) with one of the three treatments: flaxedil (0.04mg/mL), lidocaine (1.25mg/mL), or buffered saline. Muscle activity was recorded with an iWire-BIO8 biopotential recorder module and two high-speed cameras for the top and side views of the tank. The findings showed that turbulence within each treatment did not affect magnitude, relative intensity, burst duration, cycle duration, or duty factor of erector or epaxial muscle bursts. However, within no turbulence conditions, lidocaine treated fish had higher muscle activity in the spiny erector muscle magnitude than the flaxedil treatment. Activity bursts of spiny erector muscle were shorter under flaxedil than control under turbulent conditions. While under no turbulence relative intensity also decreased under flaxedil conditions. We found that in lidocaine treated fish, the erector muscles were unable to correctly modulate muscle activity and that in flaxedil fish, erector muscle function was compromised. Findings on impaired motor control and blocked sensory perception may be applied to enhance human prosthetic control and thus achieve stability.
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Introduction
In Biology, the function of a particular structure can dictate its role within an organism. This direct connection became very relevant to me in a comparative vertebrate anatomy class, where the morphology of a structure also represents its visual physiology. The specific arrangement, orientation, and composition of a structure enable and dictate this structure’s function. For example, fishes have multiple fins, all of which play distinct roles, due to the difference in form and placement. It is, however, the summation of each distinct contribution that results in the smooth act of swimming. Derived bony fishes, like bluegill, have a total of five types of fins - the dorsal, pectoral, pelvic, anal, and caudal fins (Standen
& Lauder, 2005). The anal, caudal, and dorsal fins are known as the median fins (Standen & Lauder,
2005). Fins may also be classified in another two categories: unpaired and paired. The unpaired fins are the dorsal, caudal, and anal; while the paired fins are the pectorals and pelvics, or ventrals (Larouche et al., 2017). The dorsal fin, located at the superior surface of the fish, is used for swimming and stabilization by increasing the lateral surface of the body, during swimming, especially during a C-start movement, i.e., an escape reflex (Bruce et al, 1996). In derived bony fish, the dorsal fin is made up of spinous (9-12 sharp spines) and soft-rayed (no spines) portions (Lauder 2011). Though both portions appear as a single continuous fin in bluegills, the spiny anterior and soft posterior dorsal fins actually contribute differently to locomotion (Chadwell & Ashley-Ross, 2011). The soft posterior dorsal fin may either contribute to thrusting or stability depending on the species (Drucker and Lauder 2001). In teleosts, it generates propulsive force by a similar hydrodynamic mechanism during forward locomotion and turning (Chadwell & Ashley-Ross, 2011). In some teleost species, the dorsal fin extends along most of the body, with the soft rays creating undulating waves that transmit many small amplitude waves along the entire dorsal fin surface, allowing the fish to steadily swim (Shupliakov, et al., 1992). Unlike the posterior soft dorsal fin, the anterior spiny dorsal fin in bluegill does not oscillate laterally along the entire dorsal fin body during locomotion, instead it depresses during higher speed swimming (Standen & Lauder,
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2005). Drucker and Lauder (2001) also concluded that soft ray dorsal fin withstands hydrodynamic forces.
The pectoral fins, located near the operculum, are a pair of fins that help in maneuvering and braking (Higham, 2007). The pelvic fins, also paired fins, are located on the ventral surface of the fish and help in braking, maneuvering, and steering in an up and down direction (Higham 2007). The anal fin, a median fin also located on the ventral region posteriorly to the anus, can contribute to stability or thrust depending on the species (Standen &Lauder, 2007). The caudal fin or tail is the main propulsor in fish that use body caudal swimming modes (Korsmeyer et al. 2002) and can contribute to steering with its rounded lobes that allow for sudden bursts at short distances (Flammang & Lauder, 2009).
Paired fins evolutionarily gave rise to tetrapod limbs, a process known as the fin-to-limb transition (Yano and Tamura 2013). This was characterized by the loss of dermal fin rays (dermal skeletal rods with a web-like structure) and the evolutionary origin of digits (Yano and Tamura 2013). Stewart et al. (2020) focused on the evolution of the endoskeleton, as well as the structure and function of the dermal rays of the pectoral fins in three tetrapodomorph taxa, including Sauripterus taylori (Rhizodontida),
Eusthenopteron foordi (Tristichopteridae), and Tiktaalik roseae (Elpistostegalia) to reveal the fin ray pattern in the fin-to-limb transition. The tetrapod class is a crown group of Tetrapodomorph, whose paired fins were plesiomorphically adapted to support hydrodynamic forces (Stewart et al., 2020). Prior to the fin-to-limb transition, fin rays had evolved morphologies convergent to benthic actinopterygians, which had consolidated fin rays and reduced fin web (Shubin et al., 2006). The transition to benthic lifestyle was adapted by the increased dorsoventral asymmetry of the fins to maintain an elevated and fin-supported posture in Tiktaalik (Stewart et al., 2020). This transitory step was followed by the origin of digits due to the consolidation of fin rays via reduced segmentation and branching, reduction of the fin web, and the evolution of dorsoventral asymmetry of fin rays. Such trends led to transitioning of tetrapods to terrestrial biomes (Stewart et al., 2020).
As we can see, each fin has a distinctive role based on its form and composition. However, the summation of each individual role results in swimming. This could not be accomplished without nervous
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system control, specifically from the motor cortex in the cerebrum, the cerebellum as it controls movement, posture, balance, and coordination and the spinal cord (Uematsu, 2008). The mechanism of swimming is characterized by forces generated by undulatory movements of the trunk muscles, which are composed of myomeres that are innervated by different spinal segments (Uematsu, 2008). Those spinal segments include interconnected neural circuits of different interneurons and motor neurons, which together form a central pattern generator (CPG), the main driver behind swimming rhythms (Uematsu,
2008). However, this ability is regulated by the excitatory and inhibitory stimulations of the interneurons and brain (Liu &Westerfield, 1988). Fin movement is achieved via muscle contraction after firing of motor neurons. Fine motor control and response to perturbations at the level of the fins is known to be possible experimentally (Grillner and El Manira 2020) but local neural networks are still unknown.
In aquatic locomotion, stability and maneuverability requirements define fish swimming and establish tradeoffs. Stability is defined as the resistance to, and recovery from, disturbances or perturbations to an intended trajectory (Sefati et al., 2013). It is also characterized by the relative positions of the center of mass and center of buoyancy, with the notion that a resting position is stable when the center of buoyancy is above the center of mass (Weihs, 2002). Stability may be achieved actively or passively (Webb & Weiss, 2015). Neurological control is required for active stabilization as it activates musculoskeletal components to compensate for the effects of external perturbations (Jing & Kanso,
2013). Passive stability of the locomotion, defined by body shape, fin position and swim bladder position, however, does not require energy input by the fish, reducing the energetic cost of locomotion (Webb &
Weiss, 2015, Figure 1). Thus, from an evolutionary point of view, body shapes that promote stability are under selective pressure (Jing & Kanso, 2013).
Stability, or - on the reverse side of the coin – maneuverability, are achieved via a dynamic interaction between linear velocity and angular velocity of the fins relative to the water flow (Webb &
Weiss, 2015, Figure 1). Such forces balance out in a neutrally buoyant fish in comparison to water, i.e., where fish density equals medium density, if the animal is moving at a constant speed (Webb & Weiss,
2015, Figure 1). However, in a non-neutral buoyancy state due to turbulent conditions or perturbations,
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the fish is expected to experience a linear or rotational acceleration due to Newton’s second law of motion, causing an imbalance or instability (Weihs, 2002). Stability is disrupted by external forces such as water current and drag forces during locomotion (Standen & Lauder, 2005). The fish motion is unstable in the presence of perturbations that results in forces and moments that lead to loss of stability, and a stable state can only be achieved when forces originating on the fish – such as the deployment of the spiny dorsal fin - counteract the perturbations (Tritico & Cotel, 2010). Turbulence conditions that are specifically oriented to produce horizontal vortices near the fish are expected to produce higher vorticity and angular momentum, therefore creating greater stability challenges for a free-swimming fish (Tritico
& Cotel, 2010). However, the size and position of the fin in relation to the fish’s center of mass (COM) can help overcome static instability (Standen & Lauder, 2005). Movement coordination is achieved by the use of multiple fins, essential to control body posture. The recruitment of fins in response to perturbations allows fish to quickly correct for the impact of external forces and torques that result from swimming in a turbulent flow (Standen & Lauder, 2005, Figure 1).
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Figure 1. Classification of the types of stability in fish along with corresponding control systems (adapted from Webb & Weihs, 2015).
Stability is not always negative, as procured loss of stability is required for maneuverability
(Webb, 2002). Maneuverability is the fish’s capacity to change direction, and is characterized by the turning radius, relative body size, and flexibility (Webb, 2002). For instance, a larger and rigid bodied fish, such as a tuna, placed in a small tank will have more difficulty in maneuvering, whereas a smaller and flexible with a deeper body and fins arranged around the COM fish, such as a blue tang, in the same tank will maneuver the same environment with little to no challenges. Given that maneuverability is employed due to instability, it is understandable that body designs and fin motions adapted for stability are not suitable for maneuverability (Jing & Kanso, 2013). In a three-dimensional environment, there are six degrees of motion or moments which can be categorized in three rotational axis -roll, pitch and yaw-
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and three translational or linear motions of -surge, sway, and heave (Jing & Kanso, 2013). Roll, pitch, and yaw are the motions of rotating along the longitudinal horizontal axis, vertical axis, and lateral horizontal axis, respectively (Webb, 2005). Surge, sway, and heave are the motions of forwards and backwards movement along the longitudinal horizontal axis, left and right along the lateral horizontal axis, and up and down along the vertical axis, respectively (Webb, 2005). Changes in the flapping motion (a transition from stable to maneuverable) suggest that fish can change stability behavior according to the environmental stimulus (Jing & Kanso, 2013). This also suggests that live organisms that can control their flapping motion can switch between maneuverability and stability depending on the locomotion task. For example, a fish may transition from stable periodic or steady body caudal swimming to an unstable maneuver when evading a predator (Jing & Kanso, 2013).
Fish fins have been used as a proxy for human limbs and to study limb evolution, development and possible regeneration (Coates and Ruta 2008). Fish fins are also a natural system to study how local motor control is used to improve stability in humans. Injuries to the nervous system or recovery from stroke can lead to difficulty in smooth motor control (Travlos et al., 2017).
Other pathological conditions such as Multiple Sclerosis, and Parkinson’s disease are also known to impair motor control (Travlos et al., 2017). The absence of motor control, as characterized by the inability or difficulty in regulating one’s voluntary muscle contraction, combined with the lack of sensory perception is one of the main causes of decreased quality of life in many of these pathologies (Travlos et al., 2017). Such conditions result in decreased sensation in the limb extremities, which dramatically affects their quality of life and may cause further health deterioration. Prosthetic devices may just be the solution; however, it is crucial to note the lack of stability and control as potential problems with current available prosthetics (Barone et al. 2017).
If prosthetic devices are to fully replace the functionality of the limb extremities and therefore provide better rehabilitation, we need to work on finer motor control and response times. Motor control and sensory perception, as key features of locomotion, can be studied in simpler systems such as the spiny dorsal fin of fishes.
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The present study focuses on the function of spiny dorsal fin erector muscles in bluegill when subjected to perturbations during locomotion. The musculature of the spiny (anterior) portion of the dorsal fin consists of sets of muscles attached to the bases of dorsal fin spines (Chadwell et al. 2012).
Each of the sets, equal in number to the number of spines (variable 9-10 among individual specimens) consists of one pair of erector, depressor, and inclinator muscles that originate from the fascia between the skin and axial musculature (Schneider & Sulner, 2006). The erector and depressor originate from the pterygiophore, an endoskeletal structure that supports the spiny rays’ lateral surface (Chadwell
& Ashley-Ross, 2012). The erector muscle inserts onto the anterolateral processes of the head and erects the spiny ray, while the depressor muscle inserts in the posterolateral processes to depress the spiny ray
(Chadwell & Ashley-Ross, 2012). The contraction of inclinator muscles controls the lateral movement and curvature of the soft dorsal fin rays, therefore is primarily associated with lateral flexion of the fish fin (Jayne et al., 1996). There is a webbing connection between the most posterior spine and the most anterior soft ray of the dorsal fin (Chadwell & Ashley-Ross, 2012). Due to the continuous fin web, activity of a dorsal fin inclinator, erector, or depressor can affect both the spiny dorsal fin (anterior, made of fins spines) portion and the soft dorsal fin (posterior, made of segmented and branched lepidotrichia), even at distant locations from the corresponding muscles (Chadwell & Ashley-Ross, 2012).
The overall objectives of this study are to measure the muscle activity and assess the fin response in vivo in the presence of turbulence (T) or absence of turbulence (NT) during locomotion. By removing afferent and efferent control with the use of specific pharmaceuticals (flaxedil and lidocaine), we will test the role of neuromuscular innervation and sensory integration. This will provide insights on the local feedback loops involved. Flaxedil is a neuromuscular blocker that works through competitive binding to the acetylcholine receptors at the neuromuscular junction (Riker and Wescoe 1951).
Lidocaine is a local anesthetic that works by blocking the voltage gated sodium channels present in afferent neurons (Estebe, 2017). We hypothesize that: 1) under turbulence (T) condition, muscle intensity and duration will increase when the spiny dorsal fin erector muscles are injected with saline
(control), flaxedil, and lidocaine and we expect the muscle intensity and duration to decrease with the
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same treatments under no turbulence (NT) condition, 2) we expect the removal of afferent information to decrease muscle intensity and duration compared to saline, and 3) the removal of muscle control to dramatically decrease muscle intensity and duration compared to saline (Table 1).
Table 1. Summary of the hypothesis for this study.
Materials and Methods
Fish
This experiment used 10 Lepomis macrochirus (bluegill), with a mean total length of 14.2 cm
(range 11.4-17 cm). The individuals were maintained in pairs in separate 20-gallon aquaria (63.5 cm x43.82 cm x33.66 cm LxHxW) in a recirculating system equipped with physical, chemical and UV filters and aeration systems. Water was maintained at an ambient temperature of 21-24°C and animals were monitored daily, with weekly water changes and siphoning to maintain chemical parameters (pH, nitrates, nitrites and ammonia). Each individual was fed frozen dried shrimp or brine shrimp ad libitum daily.
Surgical Procedures
Fish were sedated in a 2L bath of non-lethal dose of Tricaine methanesulfonate (MS 222) at
0.15g/L in aerated conditioned water for 10 minutes prior to surgery. Electrodes, made of 1.5m of double stranded insulated copper wire (diameter 0.11mm, California Fine Wire) were thread through 27-gauge injection needles (outer diameter of 0.42mm). Insulation was removed under a scope by scrapping the last
1mm of the wires with a scalpel and a 0.5mm long anchor-like hook was created that then sat exposed
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forming positive and negative poles on the tip of the needle (Maia and Wilga 2013). Epaxial (axial musculature) and spine erector muscles were implanted bilaterally (left and right sides) using hypodermic needles. The left and right epaxial muscle electrodes were located underneath the second fin ray and used as a reference. The other three muscles were all fin erectors, located anteriorly at the base of the third, fifth and seventh fin rays (Figures 2 and 3B). The needle was then carefully removed through the length of the electrode and the ends farther away from the fish were stripped of insulation using a lighter and sequentially crimped onto connector pins and attached to an iWire-BIO8 biopotential recorder module
(iWorx Systems Inc.). After electrode placement and still under sedation, the fish was also injected with a total 0.6 mL, 0.1 mL per erector muscle and side with one of the three treatments: Flaxedil (0.04 mg/mL), lidocaine (1.25 mg/mL), or buffered saline. The fish was then transferred to a 60-gallon experimental tank
(4121.92 cm x33.02 cm x60.96 cm, LxHxW) and allowed to recover before recording started.
Figure 2. Experimental location of electrodes for muscle activity. The spiny dorsal fin rays are numbered
1-9 and the soft dorsal fin is located. Electrodes shown as blue and black lines, were inserted in the epaxial and spiny erector muscles on both sides R (right) and L (left), shown only on the left side.
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Kinematics and Electromyography Recordings
Muscle activity was recorded with two high-speed cameras for the top and side views of the tank
(GoPro Hero Black 7 and GoPro Hero 3) at 125 frames per second and synchronized with the electromyographical recordings by pressing an event trigger in front of the cameras. Electromyographic signals were acquired at 10 kHz via an iWorx RA-834 data acquisition system (iWorx Systems Inc.) with an eight channel biopotential recorder module (iWire-BIO8, iWorx Systems Inc.) connected to a dedicated laptop.
Experimental Setup
After the fish fully recovered and has resumed normal swimming and correct posture, video and electromyographic recordings are obtained for 10 different trials with and without turbulence. Horizontal turbulence was generated by a submerged Fluval Sea CP1 Circulation Pump (Fluval) controlled from outside the tank (Figure 3A). After the experiments are concluded, the fish was lightly sedated to removed, the electrodes and monitored in its own holding tank.
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Figure 3 – Experimental setup showing fish in tank in front of turbine that can generate turbulence (A), detailed view of the fish with the implanted electrodes (B) and rectified electromyographic sample trace showing the variables analyzed (C).
Electromyographic analysis
Electromyographic data was analyzed for the temporal and intensity muscle mechanics variables using LabScribe 4.0 software (iWorx Systems Inc.) following best practices (Roberts & Gabaldón, 2008
Maia & Wilga, 2013; 2016). Three independent trials per experiment and turbulence conditions were analyzed. First, all channels are rectified using the mathematical function, absolute value. Muscle activity baseline, i.e., when the muscle is not active, was identified using the mean function and timing of onset and offset of muscle activity was determined when the baseline means was exceeded by 15%. Onset and offset allow to determine muscle burst duration by simple subtraction, while cycle duration was determined by the time elapsed between one onset and the next onset of muscle activity. Muscle frequency can be determined based on cycle duration as 1/ (cycle duration). Duty cycle was calculated by dividing burst duration by cycle duration. Muscle magnitude (in mV) can be determined using the maximum minus minimum function in LabScribe 4.0. Maximal intensity of all bursts for each channel was also calculated for each experiment to serve as a correction factor to calculate relative intensity
(Figure 3C).
Statistical analysis
Electromyographic data from L and R were pooled for the epaxial muscles, while data from L and R as well as third, fifth and seventh erector muscles were pooled for spiny erector activity.
Electromyographic variables defined above – burst duration, cycle duration, frequency, duty factor, magnitude and relative intensity - were analyzed between turbulence and no turbulence conditions for epaxial and spiny erector muscles using T-tests with equal variance or unequal variance depending on the data distribution. The same variables were analyzed for the effects of treatment – control (saline), flaxedil
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or lidocaine – using a one-way ANOVA. The combined effects of turbulence and treatment were investigated using a two-way ANOVA. Data Analysis VBA Toolpak in Excel was used. 2-way ANOVAs were run using Sigmaplot 11.0 (Systat Software Inc.). Statistical significance was set a priori at alpha level of 0.05.
Results
Overall, our findings partially supported our hypothesis that muscle activity (magnitude, intensity and cycle duration, and duty factor) was affected by the different treatments, albeit not exactly as expected. We observed that muscle activity was affected by both treatment and turbulence. However, contrary to our expectations, turbulence and no turbulence for the same treatment did not differ as much as expected.
Muscle activity under control treatment
Despite apparent changes in activity patterns between turbulent and no turbulent conditions
(Figure 4), the variability in the activity between different fish and turbulence conditions was high. Under control conditions, magnitude (t=1.21, d.f.=112, p=0.30) was found to be the same in turbulence (T) and no turbulence (NT) in the spiny erector muscles (Figure 5A). A similar trend was found with the cycle duration (Figure 5B). Relative intensity and duty factor were found to be not significantly higher in no turbulence compared to turbulence conditions (Figure 5C and D, respectively). Our findings showed that turbulence did not affect magnitude (t=1.21, d.f.=112, p=0.60), relative intensity (t=0.91, d.f=391, p=0.72), burst duration (t=-2.69, d.f=367, p=0.74), or cycle duration (t=-2.57, d.f=347, p=0.54) in erector muscles under the control treatment (Figure 5, Panels A-E). Similar patterns were observed in epaxial muscles under control conditions (Figure 6, Panels A-E), where presence or absence of turbulence did not affect magnitude (t=-0.25, d.f=119, p=0.84), relative intensity (t=0.28 d.f=111, p=0.98), burst duration
(t=-2.43, d.f=138, p=0.65), or cycle duration (t=-1.78, d.f=11, p=0.78). These results indicate that under control condition, the activities of both the erector and epaxial muscles are not affected by the presence of
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turbulence. These findings do not support the first part of our initial hypothesis (Table 1), which predicted that an increase in muscle activity (magnitude, relative intensity and burst duration) under turbulence (T) and a decrease in muscle activity under no turbulence (NT).
Figure 4. Representative raw electromyographic recordings on the same fish and experiment from no turbulence (left) and turbulence (right) under control conditions.
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A A 4.5 B 1.4 1) - 1.2 3.5 1 2.5 0.8 0.6 1.5 0.4 Magnitude (mV)
0.5 Relative Intensity (0 0.2 0 -0.5 NT T NT T
6 C 1.2 D 1 5 0.8 4 0.6 3 0.4 2 Cycle Duration (s) Burst Duration (s) 0.2 1 0 0 NT T NT T
E 1 0.8 1) - 0.6
0.4
Duty Factor (0 0.2
0 NT T Figure 5. Magnitude, relative intensity, cycle duration, burst duration, and duty factor of the erector muscles under control conditions in turbulence and no turbulence. Turbulence did not affect magnitude
(p=0.43), cycle duration (p=0.52), relative intensity (p=0.18), burst duration (p=0.37), or duty factor
(p=0.41) under control conditions.
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Figure 6. Magnitude, relative intensity, cycle duration, burst duration, and duty factor of the epaxial muscles under control conditions in turbulence and no turbulence. Turbulence did not affect magnitude
(p=0.42), cycle duration (p=0.39), relative intensity (p=0.49), burst duration (p=0.83), or duty factor
(p=0.25) under control conditions.
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Muscle activity under impaired muscle control
The removal of muscle control dramatically decreased muscle intensity and duration compared to control conditions (ANOVA, F= 5.70, d.f=2, 243, p=0.004). Our main findings only partially support our hypothesis as treatment with flaxedil decreases cycle duration and burst duration in the erector muscles under turbulence, contrary to what happens under control conditions. However, there was no change in relative intensity (t=0.91, d.f=391, p=0.36), magnitude (t=0.17, d.f=353, p=0.86), or duty factor (t=-0.23, d.f=336, p=0.82) in the erector muscle (Figure 7, Panels A-E) under turbulence. In the epaxial muscles
(Figure 8, Panels A-E), our findings showed no differences in cycle duration (t=-1.78, d.f=11, p=0.39), duty factor (t=0.68, d.f=105, p=0.50), magnitude (t=-0.25, d.f=119, p=0.84), relative intensity (t=0.28, d.f=111, p=0.98), or burst duration (t=-2.42, d.f=138, p=0.65) in turbulence.
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A B 1 4.5
4 1) - 0.8 3.5 3 0.6 2.5 2 0.4 1.5
Magnitude (mV) 1 0.2 0.5 Relative Intensity (0 0 0 NT T NT T
C D
1.2 6 1 5 0.8 4 0.6 3 0.4 2 Burst Duration (s)
0.2 Cycle Duration (s) 1 0 0 NT T NT T E 1
0.8 1) - 0.6
0.4
Duty Factor (0 0.2
0 NT T
Figure 7. Magnitude, relative intensity, cycle duration, burst duration, and duty factor of the erector muscles under removal of motor control – flaxedil - in turbulence and no turbulence. Turbulence did not affect cycle duration (p=0.52), relative intensity (p=0.18), burst duration (p=0.37), or duty factor (p=0.41) under control conditions.
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Figure 8. Magnitude, cycle duration, burst duration, relative intensity, and duty factor of the epaxial muscles under control conditions in turbulence and no turbulence. Turbulence did not affect magnitude
(p=0.42), cycle duration (p=0.39), relative intensity (p=0.49), burst duration (p=0.83), or duty factor (p =
0.25) under control conditions.
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Muscle activity under impaired sensation
Under lidocaine treatment, turbulence did not affect magnitude (t=0.17, d.f=353, p=0.86 ), relative intensity (t=0.91, d.f=391, p=0.36), duty factor (t=-0.23, d.f=336, p=0.82), or burst duration (t=-
2.69, d.f=367, p=0.74) in the erector muscles (Figure 9, Panels A-E). Under the same treatment in epaxial muscles (Figure 10, Panels A-E), our findings showed no differences in relative intensity (t=0.28, d.f=111, p=0.49), cycle duration (t=-1.78, d.f=11, p=0.39), or burst duration (t=-2.43, d.f=138, p=0.74) under turbulence condition. Duty factor and magnitude also remained unchanged between turbulence and no turbulence (magnitude: t=-0.25, d.f=119, p=0.84. and duty factor: t=0.68, d.f=105, p=0.50). The second part of our initial hypothesis (Table 1) predicted that muscle activity would be greater under turbulence even with removal of afferent information (via the administration of a numbing agent such as lidocaine). These findings do not support our hypothesis as muscle mechanics variables remained unchanged in turbulence respectively in the erector muscles.
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Figure 9. Magnitude, relative intensity, cycle duration, burst duration, and duty factor of the erector muscles with impaired sensation under turbulence and no turbulence. Turbulence did not affect magnitude
(p=0.43), cycle duration (p=0.52), relative intensity (p=0.18), burst duration (p=0.37), or duty factor
(p=0.41) under lidocaine conditions.
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Figure 10. Magnitude, relative intensity, cycle duration, burst duration, and duty factor of the epaxial muscles under impaired sensation under turbulence and no turbulence. Turbulence decreased cycle duration of muscle contraction under lidocaine conditions. Turbulence did not affect magnitude (p=0.43), cycle duration (p=0.52), relative intensity (p=0.18), burst duration (p=0.37), or duty factor (p=0.41) under lidocaine conditions.
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Comparison between treatments
Within turbulence, treatment did not affect the magnitude of spiny erector muscles (ANOVA, F=0.24, df=2, 202, p=0.79, Figure 11). However, within no turbulence conditions, lidocaine treated fish had higher magnitude of muscle contraction of the spiny erector than the flaxedil treatment (ANOVA, F=4.43, df=2, 185, p=0.013, Figure 11). Activity bursts of spiny erector muscle were shorter under flaxedil than control under turbulent conditions (Figure 13, ANOVA, F=5.703, d.f.=2, 59, p=0.004). While under no turbulence relative intensity also decreased under flaxedil conditions (ANOVA, F=4.426, d.f.=2, 56, p=0.013).
4
3.5
3
2.5
2
1.5 Magnitude (mV) 1
0.5
0 Control erector Flaxedil erector Lidocaine erector
No Turb Turb
Figure 11. Erector muscles have higher contraction magnitude under control and lidocaine and lower when muscle control is impaired.
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6
5
4
3
2 Magnitude (mV)
1
0 Control epaxial Flaxedil epaxial Lidocaine epaxial
No Turb Turb
Figure 12. Epaxial muscles contraction magnitude is not significantly affected by treatment or turbulence.
100 90 80 70 60 50 40 30 Burst duration (ms) 20 10 0 Control erector Flaxedil erector Lidocaine erector
No Turb Turb
Figure 13. Erector muscles have longer burst duration under control conditions and lidocaine under no turbulence and lower burst durations when muscle control is impaired.
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100
80 Control≠Flaxedil=Lidocaine
60
40
20 Burst duration (ms)
0 Control epaxial Flaxedil epaxial Lidocaine epaxial -20 No Turb Turb
Figure 14. Epaxial muscles have longer muscle contraction under control conditions independently of turbulence.
Discussion
The goal of the present study was to assess the activity of the erector and epaxial muscles focusing on magnitude, relative intensity, and burst duration, when fish are perturbed by the presence of turbulence and when the spinal dorsal fin erector muscles undergo three different treatments
(saline/control, flaxedil, and lidocaine). We also explored additional muscle activity parameters such as cycle duration and duty factor, which can provide information on how muscle frequency and power changes under different conditions.
Our findings showed that turbulence did not affect magnitude, relative intensity, burst duration, cycle duration, or duty factor, important variables to characterize individual burst dynamics in erector muscles under control treatment (Figure 5), despite the fact that whole trial traces show differences in how often muscles are activated (Figure 4). This could be explained due to analyzing only bursts that were present, thus disregarding the times when the muscle was not contracting. Since the goal of this study was to determine the effects of loss of sensation and motor control on stability erector and epaxial muscles, this will not affect the conclusions we are able to draw. However, in the future we plan to
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conduct a different analysis that will focus not only on the characteristics of the bursts present but also to quantify the burst activity between turbulence and no turbulence trials. In epaxial muscles, as expected, since these muscles are responsible for regular fish swimming (Jayne &Lauder, 1994) and not for increased stability like the dorsal fin erectors (Chadwell & Ashley-Ross, 2013), no differences were observed between turbulence and no turbulence for the muscle activity variables analyzed: magnitude, relative intensity, duty factor, burst duration, and cycle duration (Figure 6). These results indicate that under control condition, the activities of both the erector and epaxial muscles remained unchanged. These findings do not support the first part of our initial hypothesis (Table 1), which predicted higher muscle magnitude/relative intensity and longer bursts in turbulence under control treatment.
Our findings showed that under lidocaine treatment, turbulence did not affect relative intensity, magnitude, duty factor, cycle duration, or burst duration in the erector muscles (Figure 9). This pattern was also observed for the epaxial muscles (Figure 10). Similarities between turbulence and no turbulence when sensation is removed are to be expected as the fish is not able to sense the turbulence and thus not expected to adapt muscle mechanics. These findings support the second part of our hypothesis, which predicted that muscle activity in both muscle types to be the same in turbulence and no turbulence under loss of sensation. This means that the absence of afferent information via the administration of numbing pharmaceutical (lidocaine) showed no change in muscle mechanics under turbulence and no turbulence in the epaxial muscle.
Fin ray afferents have been shown to provide proprioceptive feedback, which allows for the movement of rays (Hardy & Hale, 2020). In bluegill, there are mechanoreceptor structures, connected to the nerves at base and distal tip of the fin rays, that provide sensory functions (Hardy et al., 2016).
Mechanoreceptors play an additional role in relaying key sensory information to facilitate navigation and orientation around the surrounding environment (Flammang & Lauder, 2013). In pictus catfish
(Pimelodus pictus), afferents innervating the pectoral fin are capable of responding to surface pressure and touch (Hardy et al., 2016). Histology work from our lab has also shown profuse nerve endings in the fin web of the spiny dorsal fin (unpubl. data, Cindy Rodriguez and Nick Saygeh). It is thus not surprising
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that in the lidocaine treatment, input on the turbulence hitting the fish from the turbine was missing. This further supports the important role that the spiny dorsal fin plays, not only in correcting perturbations like roll induced by horizontal vortices, but also as a sensory apparatus that might send information to the brain and affect the motor activity of other fins. Kinematic analysis (not shown here) corroborates these findings, as fish treated with lidocaine have a hard time maintaining position in turbulence.
Our findings showed that under flaxedil treatment, turbulence did not affect relative intensity, magnitude, duty factor, cycle duration, or burst duration in erector muscles (Figure 7). Under the same treatment in the epaxial muscles, turbulence also did not change the following muscle mechanics: duty factor, cycle duration, burst duration, magnitude, and relative intensity (Figure 8). As we had predicted muscle activity was still present under turbulence. However, contrary to what we predicted, muscle activity was also present in the absence of turbulence. This suggests that a higher concentration of flaxedil would have been required to completely block the neuromuscular junction.
In largemouth bass, another Centrarchidae like the bluegill, the unilateral activation of epaxial muscles causes a lateral bending of the body for undulatory locomotion, which requires the activation of the epaxial muscles (Jimenez & Brainerd, 2020). Epaxial musculature has primarily been linked to locomotion, but is now known to also generate power for suction feeding via the bilateral activation of epaxial muscle (Jimenez & Brainerd, 2020). In our experiments we saw that epaxial magnitude was always higher than the spiny erector muscle, most likely related to the large powerful contractions that these fast glycolytic locomotor muscles are known for (Ellerby & Altringham, 2001). In general, fishes activate all epaxial regions (ventral and dorsal) when performing fast-starts or predatory escape (Jayne &
Lauder, 1995). Though the activation of both regions generates the force necessary for rapid escape, each activated region serves a specific role. For instance, trout only activate the ventral region for sprinting
(Ellerby & Altringham, 2001). Often, when the turbine was on, fish lost their position in the water column and performed fast-starts, leading to very high epaxial contractions. We know more about the epaxial muscles, than we know about the spiny erector muscles. Thus, using the epaxial musculature as a reference enables us to understand how local motor control and sensory integration. The epaxial muscle’s
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dual function was the result of dorsoventral regionalization (Jimenez & Brainerd, 2020). Primary and secondary motoneurons innervate the axial muscle fibers (Westerfield et al.,1986). Complete activation is achieved by primary motoneurons by receiving inputs from Mauthner neurons, innervating larger portion of the axial musculature to attain high intensities for rapid escapes (Ampatzis et al., 2013). Secondary motoneurons, however, achieve graded activation, which innervates a smaller portion of the axial musculature to attain lower intensities for non-rapid escapes (Ampatzis et al., 2013). Further analysis of the number of motoneurons innervating the spiny erector muscles is needed to be able to understand how these parameters are modulated. However, the fact that bluegill muscle mechanics are not as different between turbulence and no turbulence conditions within each treatment seems to suggest temporal modulation and absence of activity as a way to overcome turbulence. When sensory information is not present, then timing of muscle activity (not shown here) is affected, and the fish is unable to recover position.
While we know more about how the spiny erector muscle contracts and what information it needs to respond to turbulence, more research is needed to understand the temporal patterns and how impaired sensation affects stability. We plan to also analyze the onset and offset of muscle activity collected during these experiments. Lastly, during these same experiments we collected video and analyzing the body and fin motion in relation to the muscle activity can provide further insight into how the fine motor differences result in relevant behaviors for the animal.
Conclusion
This study focused on the aspects of local motor control and sensory perception and its effects on the maintenance of stability in response to perturbation conditions. The innervation of the spiny dorsal fin of bluegill and the fin circuit’s reaction to perturbations applies to human prosthetic control. We found that as expected impaired motor control results in abnormal muscle mechanics. More strikingly, the absence of sensation resulted in overcompensation of muscle activity. The results of this study are important as they provide additional insights that can be applicable in the enhancement of prosthetic
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devices in response pathological conditions such as Multiple Sclerosis, and Parkinson’s disease driven by impaired motor control, disrupted voluntary muscle contraction, and lack of sensory perception. Such specific parameters required to achieve stability and resume correct control can help solve existing problems with current available prosthetic devices.
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