Using Drosophila melanogaster to Elucidate Sleep and Circadian Rhythm Disruptions after Traumatic Brain Injury

Shan Lateef
Thomas Jefferson High School for Science and Technology

Abstract

Traumatic brain injury (TBI) is a leading international cause of morbidity and mortality. Regardless of severity, TBI can significantly disrupt sleep-wake physiology by mechanisms that are not fully defined. The goals of this project were to utilize Drosophila melanogaster as an animal model and determine whether therapeutic hypothermia could minimize disruptions in circadian rhythms and sleep caused by TBI. We built a high-impact trauma (HIT) device that uses a spring-based mechanism to propel flies against the wall of a vial, causing mechanical damage to the brain. Sleep and circadian data was obtained by monitoring fly locomotor activity with the Drosophila Activity Monitoring (DAM) system, which uses infrared beams to detect and quantify animal movement over time. Male and female data were independently analyzed due to known sexual dimorphism of sleep architecture in D. melanogaster. We discovered that after both single and recurrent TBI (rTBI), female flies demonstrated increased daytime somnolence while males subjected to rTBI had decreased night sleep amounts. Therapeutic hypothermia did not mitigate the symptoms associated with sleep-wake cycle disturbances in females after TBI; however, cooling did increase the amount of night sleep in male flies subsequent to rTBI. The circadian rhythm appeared more resilient to disruption after TBI, but we did observe increased numbers of arrhythmic flies, particularly among the female cohort, after TBI. Recurrent TBI in flies shows altered sleep patterns and disrupted circadian rhythms, with distinct differences among male and female D. melanogaster. Thus, male and female brains may respond differently to TBI, and this must be considered while addressing their disrupted sleep and circadian physiology.

Introduction

Traumatic brain injury (TBI) is a major public health concern and a leading international cause of morbidity and mortality [11]. In the United States, approximately 1.6-3.8 million sports-related TBIs occur annually [13], and the prevalence of TBI among returning service members in the military ranges from 15.2% to 22.8%, affecting as many as 320,000 troops [20]. Regardless of severity, TBI can commonly cause sleep-wake disturbances that can significantly impede neurologic recovery [23]. Between 30-70% of individuals experience sleep disturbances after TBI, and in rehabilitation units, up to 68% of patients experience trouble sleeping [18]. Besides sleep quality, TBI can also have a deleterious impact on circadian function. Circadian rhythms are daily rhythms in behavior and biologic systems that recur approximately every 24 hours, and when those rhythms are disrupted, it can affect everything from learning and memory to metabolism and cardiovascular health [26]. In addition to neurologic and metabolic alterations, sleep-wake disturbances also decrease cognitive function and lead to an increase in pain and psychological distress [23]. Despite the recognition that sleep-wake disturbances are prominent after TBI, specific disruptions in sleep architecture are yet to be clearly established, and there is a need for effective interventions.

Only recently have studies started utilizing animal models, which can better probe the underlying pathophysiology of sleep and circadian disorders that occur after TBI [23]. Systematically assessing the impact of altered physiology in mammalian models is difficult due to high costs and their relatively long life spans. Drosophila melanogaster have been used successfully to model human neurodegenerative disorders, and it was found that fundamental characteristics of human TBI also occur in flies [1, 12]. The fly and human brain have similar structural features, and the fly cuticle, like the human cranium, is relatively inflexible and protects the fly brain from environmental insults. D. melanogaster also have the potential to reveal the complex neuroanatomy and neurophysiology involved in sleep and circadian cycles. Due to evolutionary conservation, the neurochemistry of D. melanogaster sleep-wake systems parallels those of mammals [8]; they share many of the sleep and wake promoting neurotransmitters and neuropeptides, including dopamine and adenosine (Sehgal and Mignot, 2011). In fact, the master gene that controls circadian rhythms in all mammals, called “period”, was first isolated in fruit flies [9].

Phase 1 of this study created a successful animal model of single and rTBI using Drosophila melanogaster and a “high-impact trauma” (HIT) device [15]. Recurrent TBI had a measurable and significant negative impact on lifespan, time to sedate, and behaviors such as phototaxis. The most intriguing findings were that therapeutic hypothermia could significantly attenuate the deleterious effects of rTBI on lifespan, of single TBI and rTBI on time to complete sedation, and of rTBI on phototaxis [15]. The main goal of this project was to examine potential disruptions in sleep and circadian physiology in Drosophila melanogaster after TBI and also to determine whether therapeutic hypothermia can mitigate these adverse health effects.

Circadian rhythms are daily rhythms in behavior or physiology that reoccur approximately every 24 hours. Circadian rhythms can be entrained by external environmental cues, such as light, but persist in the absence of these cues with periods that deviate slightly from the expected 24 hours in stable environmental conditions. The assay most reliably used to study circadian rhythms in D. melanogaster includes an assessment of their locomotor activity. This activity in D. melanogaster is organized such that in a 12:12 hour light:dark (LD) cycle, flies exhibit peaks of activity during dawn and dusk (morning and evening peaks), with increases in activity typically occurring slightly before (anticipating) the lights-on and lights-off transitions [8]. Additionally, sleep in flies closely resembles sleep in larger organisms and is marked by measurable periods of quiescence. Based on studies of arousal threshold, sleep in D. melanogaster is commonly defined as a period of inactivity lasting five minutes or longer [25]. Thus, by studying and quantifying locomotor activity in flies, we are able to reliably derive their sleep and circadian profiles.

One factor that has been shown to worsen the effects of TBI in humans is hyperthermia, or increased body temperature [4]. Theoretically, hypothermia should mitigate the toxic effects of trauma, which include excitotoxicity, free radical-induced alterations, inflammatory events, and disruption of the blood-brain barrier [1]. Studies have been conducted to determine the utility of hypothermia in severe TBI patients, but with conflicting results [16]. If temperature control could improve TBI outcomes, then it is crucial that we study this intervention systematically. Using an animal model would be a first step in this major initiative. D. melanogaster, being ectotherms, cannot endogenously regulate body temperature and essentially take on the temperature of their environments as long as this is physiologically permissible [7]. Prior research has shown that hypothermia, or cooling D. melanogaster to 170 C, has a protective effect on brain potassium homeostasis during repetitive anoxia [22]. It has also been reported that hypothermic treatment improved the D. melanogaster’s longevity and response to sedation, phototaxis, and geotaxis following rTBI [14]. Thus, the purpose of this study was to use a validated model of TBI in Drosophila melanogaster and observe the effects of TBI on the sleep-wake patterns and circadian rhythms of flies as well as analyze whether hypothermic treatment immediately following TBI and rTBI could attenuate these deleterious effects.

The main objectives of this study were to determine whether total amount and distribution of sleep are significantly altered after single and recurrent TBI in Drosophila melanogaster, to assess disruptions in circadian rhythm after single and recurrent TBI in D. melanogaster, and to study whether therapeutic hypothermia applied after each bout of TBI can minimize disruption to circadian rhythms in D. melanogaster.

Methods and Material

Fly stocks (wild-type, Oregon R strain D. melanogaster) were obtained from Bloomington Stock Center (Bloomington, Indiana) and raised at 23o C on a standard cornmeal-molasses medium. D. melanogaster sleep consists of two temporally distinct components: daytime sleep and nighttime sleep, which differ by sex. Male flies spend considerable time sleeping during both the day and night, with the amount of sleep during the day often comparable to that at night. Although mature mated females sleep as much as their male counterparts during the night, they exhibit sustained activity during the day and sleep much less during this period [10]. Thus, male and female flies were studied independently in this project.

As previously developed by Katzenberger et al. [12], a fly model of TBI was created by inflicting mechanical injury on flies using rapid acceleration and deceleration forces. This produces outcomes similar to those of closed head TBI in humans. A HIT device was constructed, consisting of a metal spring clamped at one end to a wooden board with the free end positioned over a polyurethane pad (Figure 1). When the spring is deflected and released, the vial contacts the pad, and the flies contact the vial and rebound, providing a model of TBI. The degree of injury can be adjusted by changing the extent of the deflection or by varying the number of strikes.

Figure 1a. Inflicting TBI on Flies Katzenberger, 2013 — Figure 1a. Inflicting TBI on Flies
Katzenberger, 2013

Figure 1b. Lateef HIT device Lateef et al. 2019 — Figure 1b. Lateef HIT device
Lateef et al. 2019

Katzenberger et al. showed that deflection of the spring to 90° resulted in an impact velocity of ∼3.0 m/s (∼6.7 miles/h) and an average force of 2.5 N [12]. Those flies subjected to a single strike with the spring deflected to 90° became temporarily incapacitated and fell to the bottom of the vial; however, there was no obvious external damage to the head, body, or appendages. During the first minute after a strike, 8.8 ± 3.8% of flies were incapacitated, but most of these flies recovered locomotor activity within five minutes, as measured by climbing ability. Although mobility was reduced, it gradually returned over a two day period. The immediate loss of motor ability followed by ataxia and gradual recovery of mobility were reminiscent of concussion in humans and consistent with the idea that the HIT device inflicts brain injury in flies [12]. We observed a very similar response to single TBI in our experiment.

To test the effect of hypothermia on traumatic brain injury, a deflection angle of 90° was used, and a total of five hits were applied, with one hit every two minutes. A one day recovery period was allowed after the recurrent TBI prior to starting the molecular assays. Moderate hypothermia in human trials was considered to be between 32-34.5 °C, which is approximately 1-5 degrees lower than normal human body temperature. Therefore, our goal was to change the fly’s environmental temperature to 5-6 degrees below its baseline. Hypothermia was induced for 3 minutes in a refrigerator at 16oC after all hits were performed, similar to prior experiments. A one day recovery period was allowed after concussion or rTBI prior to starting the circadian and sleep assays. Katzenberger’s model of TBI showed that primary injuries cause death within 24 hours only if the injuries exceed a specific threshold. In addition, death from primary injuries was complete after 24 hours because the percent survival at 24 hours was not substantially different from the percent survival at 48 hours.

Figure 2. Drosophila Activity Monitor System

In order to analyze sleep and circadian rhythms, flies were allowed to recover for one day after TBI and hypothermia (for applicable groups). For sleep analysis, individual flies were transferred to 5 × 65 mm glass tubes containing fly media using CO2 anesthesia and placed into a DAM5 System (Trikinetics Inc.,Waltham, MA, USA) (Figure 2). Flies were allowed to recover and acclimate for an additional two days under 12 hour:12 hour LD cycle conditions before being assayed in constant dark (DD) conditions. Movement or locomotor activity was recorded as the number of infrared beam breaks collected in one minute bins for six days using DAM system v3.8 software. As described previously, sleep was measured using the definition of locomotor inactivity lasting a minimum of five minutes [25] and analyzed using custom-designed MATLAB (Mathworks, Natick, MA) based software. Flies that died during the sleep study periods were removed from the analysis. The waking activity during the day, daily sleep bouts, and total sleep time during the day and night sleep bouts were calculated using the Sleep-Lab software. Means were compared using the t-test, where a p-value less than 0.05 was considered significant. Circadian rhythm disturbance was recorded by observing the percentage of flies that retained rhythmic activity patterns after being placed in constant dark conditions. Outcomes measured included period (which defines the length of circadian rhythms), rhythmicity index (which defines the strength and regularity of the rhythmic process), and percent rhythmic flies.

Results

Female flies subjected to both single and recurrent TBI demonstrated an increased number of daytime sleep bouts as shown in Figure 3. In addition, rTBI female flies also experienced significantly higher day sleep amounts (Figure 3b) and decreased daytime waking activity (Figure 3c) compared with female flies not subjected to TBI. Male flies did not demonstrate such daytime sleepiness and diminished motor activity during the day. Hypothermia did not alleviate these symptoms among the female TBI flies. In contrast to females, the male TBI flies showed decreased night sleep amounts after being subjected to recurrent TBI when compared to male flies who were not injured (Figure 3d). Female flies did not have any significant changes in night sleep amounts.

Figure 3. Key – TBI (1) is 1 hit, TBI (1)+C is 1 hit with cooling, TBI (4) is 4 hits, and TBI (4)+C is 4 hits with cooling

Representative actograms (30-minute bins) for male flies from each of the five different treatment conditions are illustrated in Figure 7 (a, b, c, d). Double plotted actograms show the activity of flies entrained to a 12/12-hour LD cycle and then released in constant darkness to determine their period. Each day is plotted twice, first on the right and then duplicated on the left half of the next line. Non-injured control flies generally maintained a rhythmic pattern of activity, moving throughout the subjective day and sleeping throughout the subjective night (Figure 7a) with minimal disruption after a single TBI (Figure 7b). In contrast, many of the rTBI-treated flies did not exhibit a rhythmic pattern of activity (Figure 7c). Importantly, cooling appeared to restore some rhythmicity to the actogram (Figure 7d).

Figure 7. Drosophila actograms displayed of a) control flies b) single TBI. c) recurrent TBI and d) recurrent TBI + cooling. No disruption of circadian rhythms is observed after a single TBI, whereas recurrent TBI is associated with disrupted rhythms. Hypothermia appears to attenuate the effect on rTBI on circadian rhythms.

Overall period and rhythmicity index did not differ among the TBI and non-TBI flies for both males and females (Table 1). We also examined the proportion of arrhythmic flies in each group, compared to the controls for both females and males, and these results are shown in Figure 8a and 8b respectively. Proportion of female arrhythmic flies after single TBI was 58.6% (95% CI: 49.4%-67.7%) compared with controls, where arrhythmic flies were 34% (95% CI: 25.6 % - 43%). After rTBI, the proportion of arrhythmic flies rose to 70.0% (95% CI: 60.4% – 77.6%). Cooling diminished the proportion of arrhythmic flies in both single and rTBI groups but not to a significant degree.

Table 1. Period and average rhythmicity index after TBI

Male flies across all groups tended to have less arrhythmic individuals, with the control group having 17.2% (95% CI: 10.2% – 24.3%) arrhythmic flies. After rTBI, the arrhythmic proportion increased to 45.2% (95% CI: 36.2% - 54.1%). rTBI male flies which were cooled had a proportion of arrhythmic flies that was 26.7% (95% CI: 18.6% - 34.7%).

Figure 8a. Proportion of arrhythmicity in female fly sample

Figure 8b. Proportion of arrhythmicity in male fly sample

Discussions and Conclusions

The purpose of this experiment was to assess the effect of single and recurrent TBI on the sleep-wake cycle of Drosophila melanogaster, specifically the day sleep bouts, waking activity, and total day and night sleep amounts, as well as to evaluate if the administration of therapeutic hypothermia would mitigate these adverse effects. We found that after both single and rTBI, female flies demonstrated increased daytime somnolence while males subjected to rTBI had decreased night sleep amounts. Therapeutic hypothermia did not prove to mitigate the symptoms associated with sleep-wake cycle disturbances in females after TBI; however, cooling did increase the amount of night sleep in male flies subsequent to rTBI. The circadian rhythm appeared more resilient to disruption after TBI, but we did observe increased numbers of arrhythmic flies, particularly females, after TBI.

Observed alterations in sleep patterns after TBI could have occurred due to disturbed arousal systems, which involve the neurotransmitter glutamate in the brain stem, hypothalamus, and basal forebrain [23]. Interestingly, our results showed a distinct difference in how TBI affects the male and female D. melanogaster brain, with females becoming more somnolent during the day and males unable to attain restful sleep at night. Post-menopausal human females have significant alterations in their sleep-wake cycles due to the drastic decline in their reproductive hormones, estrogen and progesterone. Likewise, the disrupted daytime sleep in female flies in our experiments could be explained by altered signaling of the steroid hormone, ecdysone, which plays a significant reproductive role in female flies [10]. TBI likely affects the neurologically controlled endocrine system, which is responsible for the production and transport of hormones such as ecdysone, thereby disrupting the female flies’ ability to remain wakeful during the day. Furthermore, with increasing age, female flies exhibit increased daytime sleep and decreased nighttime sleep [19]. Thus, the increasing daytime somnolence in female TBI flies may be a manifestation of neurodegenerative processes that mimic aging occurring in the fly brain. Male flies showed hypervigilance and decreased nighttime sleep, very reminiscent of the insomnia that occurs after human TBI.

The infliction of rTBI produced arrhythmic patterns of activity in both sexes. It increased the number of arrhythmic female flies by 36% and the number of arrhythmic male flies by 28%. These unfavorable alterations to the circadian rhythm brought about by rTBI can most likely be attributed to TBI’s capability to deregulate the expression of key circadian clock genes, such as Bmal1 and Cry1, suggesting perturbation of transcriptional-translational feedback loops that are central to circadian timing [3]. Another potential contributing factor may be the aberrant production or release of the neuropeptide PDF, which is normally released by clock neurons to promote arousal and signal time of day to other brain regions [1, 21]. Also, several studies have demonstrated that sleep deprivation results in displayed pro-inflammatory cytokine production. Thus, the disturbed sleep-wake cycles could contribute to the systemic inflammation observed in rTBI flies, as shown in prior work [14].

Interestingly, TBI affected the arrhythmic females more severely than the arrhythmic males. This observation can be explained by sexual dimorphism, where the male flies demonstrate increased amounts of day time sleep and exhibit more consolidated sleep than individual female flies [17]. Since female flies have a more defined diurnal sleep-wake cycle, they are likely more susceptible to fragmentation of circadian rhythms and disruption of sleep-wake cycles compared to male flies.

In males subjected to rTBI, hypothermia mitigated the effect of decreased nighttime sleep, possibly due to its ability to reduce concentrations of excitatory and inflammatory neurotransmitters [24]. Therapeutic hypothermia failed to ameliorate any of the sleep disturbances evoked by TBI in females, possibly because hypothermia has minimal influence over neuroendocrine signaling, which may be the primary mechanism of disturbed daytime sleep in female flies. To our knowledge, this differential impact of TBI on male and female circadian physiology has not been previously reported. It is also possible that hypothermia was not beneficial in this experiment as it was applied after all the bouts of TBI as opposed to being applied after each bout of TBI.

Many treatments, both pharmacological and non-pharmacological, exist to treat sleep-wake disorders in both non-injured and TBI patients. The most notable non-medicinal agents for the treatment of sleep disturbances include cognitive behavioral therapy, acupuncture, and blue light therapy. Medicinal aids for sleep-wake disorders include ORX receptor antagonists such as Suvorexant and melatonin, which provide relief from insomnia, and stimulants such as Modafinil, which decrease the effects of hypersomnia [23]. Though these treatments can provide patients with some relief of sleep-wake disorder symptoms, their effects on patients with sleep disturbances post-TBI has been inconclusive [2]. Additionally, the aforementioned treatments are often used only after sleep disturbance symptoms have developed in the patient. Thus, potential interventions such as hypothermia, which may be applied prior to symptom evolution, must be explored.

Recurrent TBI in flies causes altered sleep patterns and increased circadian arrhythmicity with remarkably distinct differences among male and female D. melanogaster. Thus, male and female brains may respond differently to TBI, which must be considered while addressing their disrupted sleep and circadian physiology. More research to investigate the remedial effects of therapeutic hypothermia on recurrent TBI is essential since it may prevent life-altering outcomes related to neurologic injury.

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