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Introduction
Introduction
Psychiatric disorder prevalence is increasing throughout the world, with depression one of the most diagnosed mental illnesses (Robert et al., 2019). Thus, medicinal drugs such as antidepressants are more frequently consumed and found within aquatic environments (Fong & Ford, 2014). For example, antidepressants that are not taken up within the body are excreted directly, with no change to the compound (Rodriguez Gil, 2021). Once they are excreted from the body, they travel to sewage water. Treated sewage water helps filter out compounds, but antidepressants such as fluoxetine remain unchanged and find their way into water systems (Prozac’s in the Water, 2004). Depending on location, fluoxetine can be found in treated waterways at concentrations between 0.122 μg /L and 0.540 μg /L (Mesquita et al., 2011).
Known by the trade name Prozac, fluoxetine is one of the most commonly used antidepressants. As a selective serotonin reuptake inhibitor (SSRI), fluoxetine increases synaptic serotonin (5-HT) levels and effects on cognition and behavior (Bacque-Cazenave et al., 2020). SSRIs are generally used to treat disorders such as depression, generalized anxiety disorder, and bulimia eating disorder (Edinoff et al., 2021). In the body, fluoxetine has one of the longest half-lives of SSRIs: four to six days with an additional seven to fifteen days for norfluoxetine, an active metabolite (Edinoff et al., 2021). The resistance to breakdown in ways such as these is additionally significant in terms of its increasing prevalence in the environment. In multiple crab species, exposure to fluoxetine increases high-risk behaviors such as foraging, mobility, and species interaction (Peters et al., 2017; Chabenat et al., 2021; Mesquita et al., 2011).
5-HT is implicated in different physiological processes. 5-HT influences the behavior of multiple aquatic species including crayfish and the blue swimming crab, in ways such as regulating neuronal activity, changing color, and neurogenesis (Nakeim et al., 2020). 5-HT also affects behaviors such as anxiety, depression, high-risk behaviors, fight initiation, and feeding behavior (Bacque-Cazenave et al., 2020; Nakeim et al., 2020). In crustaceans, 5-HT facilitates synaptic transmission at neuromuscular junctions within the food chewing system and decreases the frequency of chewing network activity in vitro (DeLong, 2009). This suggests that fluoxetine exposure could impact crustacean feeding at the level of the nervous and muscular system.
Although food intake is impacted by increased 5-HT (Tierney, 2022), there is no knowledge of whether antidepressants in the water acting through serotonergic signaling affect feeding behaviors upon food ingestion. Furthermore, feeding increases C. borealis heart rate (Smith, Johnson, Burke, Blitz, unpublished) and injection of fluoxetine raises crustacean heart rate (Listerman et al., 2000; Robert et al., 2019), meaning there may be interactions between the effects of feeding and fluoxetine exposure on heart rate. Therefore, we aim to explore whether fluoxetine within the water influences and interacts with feeding-related changes in heart rate. The gastric mill (chewing) rhythm is a slow (~10 sec period), periodically active rhythm that breaks down food within the foregut of the animal (Marder & Bucher, 2007). The release of 5-HT causes an increased duration of one phase of the rhythm, thereby slowing the overall frequency and can also increase the contraction strength (amplitude) of muscles involved in chewing (DeLong, Beenhakker, & Nusbaum, 2009; Jorge-Rivera et al., 1998). One of these striated gastric mill chewing muscles is gm1, located immediately under the crab shell; this makes it accessible to non-invasive recordings in a behaving animal (Smith, Johnson, Burke, Blitz, unpublished).
Research Question:
Research Question:
How does the exposure of SSRI fluoxetine (Prozac) affect chewing muscle contractions and heart rate during feeding?
Methods
Methods
Figure 1. PPG sensors placed on carapace above heart and GM1 muscles and the resulting recordings (3 hour windows). Pop outs depict the general oscillation pattern and define the interspike intervals.
Figure 2. Dissection of the Gm1 muscle (top) and heart (bottom).
In-vivo Recordings of Heart and Stomach Muscle Contractions
In-vivo Recordings of Heart and Stomach Muscle Contractions
In vivo recording procedures:
Photoplethysmography (PPG) is a noninvasive technique that emits light through the animal’s carapace which then reflects back to the photosensor to visualize muscle movements (Depledge, 1983; Kushinsky, 2019; Smith, Johnson, Burke, Blitz, unpublished). We used PPG recordings of heart and chewing muscle (gm1) activity (Figure 1). This technique puts minimal stress on the animals and allows for stable recordings over days-weeks during repeated feedings for the same animals. We used both squid and sardine since these different food types result in different muscle activity (Smith, Johnson, Burke, Blitz, unpublished).
Crabs were placed on ice for 45 minutes to anesthetize them for sensor placement. PPG sensors (Newshift) were then glued to the carapace above the heart and Gm1 muscle (Figure 2) with cyanoacrylate glue. Layers of dental wax and clay over the sensors were used to create a stable base and a secure attachment to the crab such that it could last weeks of recording. Marine Adhesive sealant was then used to waterproof the structure. The crabs were then introduced to a tank containing 16 L of saltwater in an incubator at 11°C on a 12-hour light/dark cycle.
Heart rate and gastric mill muscle activity was recorded and analyzed using Spike2 software (Newshift AMP03-U amplifier with MK1401 hardware).
Sardine (2.4 ±0.36 kg/cm2) and squid (8.57 ± 0.163kg/cm2) were alternatively fed to the crab 2X per week on Mondays and Thursdays, consistent with their feeding schedule. Squid is significantly harder (t-test; p<0.0001) than sardine (n=3). The initial order of food type was randomized. The data selected for analysis was at least two days post introduction of the crab to the tank, to allow time for acclimatization.
Water changes occurred on days after feeding, and water quality tests for nitrate, nitrite, ammonia, oxygen, and pH were performed daily.
After adding fluoxetine (16 mL of 0.12 mg/mL stock solution) for a target tank concentration of 0.12 mg/L, the crab acclimated for at least two days before feeding under fluoxetine conditions. Both food types were fed once again.
Data reported as mean ± SD
Fluoxetine exposure working concentration: 0.120 mg/L
Data Analysis:
Interspike intervals (ISI) were identified for both heart and chewing muscle contractions within a 25 minute timespan. These ISIs measure the time between each each spike in the recording, which reflect muscular contractions. The distribution of ISIs between the saltwater and the fluoxetine conditions was compared using cumulative histograms. This was done for both food types as well as during time periods where the crab was not feeding.
ISIs were also determined for 3-hour windows of cardiac recordings at noon and midnight. This allowed the number of acardia events during times of nonfeeding to be counted and compared for both saltwater and fluoxetine conditions.
Results
Results
Fluoxetine Exposure Slows Heart Rate
Fluoxetine Exposure Slows Heart Rate
During periods of nonfeeding, the distribution of Interspike intervals in a 30 minute window showed that there are shorter ISIs in saltwater compared to fluoxetine exposure (Fig. 3a & 3b). The cumulative percentage helps visualize this distribution. This translates to a slower heart rate. When the cumulative histograms for saltwater and fluoxetine conditions are overlaid during feeding times, this decrease in heart rate is more apparent. This is true for both sardine (Fig. 3c) and squid (Fig. 2d) food types.
Note that the cumulative percentage during nonfeeding times does not reach 100% (Fig. 3a & 3b). This is due to events of acardia, which are present during rest and are outside the range of typical cardiac ISIs.
Figure 3. Cumulative histograms of cardiac ISI (s) for both environmental conditions when nonfeeding (top graphs) and during sardine (bottom left) and squid (bottom right) feedings.
Figure 4. Bar graph showing the number of acardia events within a three hour window starting at noon and midnight for both environmental conditions.
Acardia Occurs Less Frequently with Fluoxetine
Acardia Occurs Less Frequently with Fluoxetine
Acardia is defined as a period of ≥5 seconds between heart muscle contractions (McGaw, I. J. & Nancollas, S. J., 2021). Our cardiac recording data shows that during nonfeeding times, a cyclic burst pattern with intervals of acardia is observed with a cycle period of approximately 20 minutes. To account for this, we analyzed 3-hour intervals to investigate the effect of fluoxetine on acardia (traces below). We looked at noon and midnight as they are times where the crab has not been disturbed. Our analysis shows that there are less acardia events in the fluoxetine condition at both times compared to the saltwater condition (Fig. 4).
Fluoxetine Increases Chewing Muscle Contraction Rate
Fluoxetine Increases Chewing Muscle Contraction Rate
In our analysis of of gastric interspike intervals (ISI), the ISI lengths are more consistent in squid feeding compared to sardine feeding. ISIs in the sardine data for the fluoxetine condition ranges from 5 seconds to 11 seconds, whereas the saltwater condition ISI ranges from 6 seconds 14 seconds between muscle contractions (Fig. 5). The squid ISI data shows that the fluoxetine condition has more consistency of ISIs across the 30 minute recording, staying around the 6 second range, where the saltwater condition is a little more distributed, spanning from 6 seconds to 8 seconds between contractions (Fig. 6). Overall, shorter ISIs occur during fluoxetine exposure for both food types, which reflects a faster gm1 contraction rate.
Figure 5. Cumulative histogram of gastric ISI (s) for sardine feeding in both environmental conditions.
Figure 6. Cumulative histogram of gastric ISI (s) for squid feedings during both environmental conditions
Conclusions
Conclusions
Future Directions
Future Directions
Although these are preliminary findings with a n=1, we see that fluoxetine affects physiological processes, such as heart rate and contraction patterns as well as feeding behaviors.
Fluoxetine decreases the heart rate during feeding.
Fluoxetine decreases the amount of acardia events.
Fluoxetine increases chewing muscle contractions during feeding.
With increasing prevalence of antidepressant prescriptions and a lack of filtration from waterways, the environmental impact of these compounds should be considered. These findings are important as they give us insight on the effects of antidepressants on marine life.
Quantifying the recordings.
We have considered looking at our data thus far and analyzing the amplitude of both the gm1 and heart recordings to see if there is a difference in the force of the muscle contractions between the environmental conditions.
Changing the dose of SSRIs.
The SSRI concentration (0.120 mg/L), although higher than environmental concentrations, is fairly low compared to a typical human dosage. Determining the differences in muscle contraction during feeding depending on a range of concentration could have interesting implications on determining environmentally unsafe fluoxetine concentrations.
Other application methods of SSRIs.
We have considered injection of fluoxetine to determine if this direct method of administration would have a similar or more pronounced effect on muscular contraction. We have found so far that this may affect appetite and further testing and development of experimental design is required.
Increase the sample size to see if the effect is the same.
Perhaps most importantly, further trials are required to determine the true effect of fluoxetine on C. borealis during feeding. Since this project has only just begun, replicates have not yet been performed, although this initial data has promising implications.
References
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