Circadian Clock Behavior Analysis
The Circadian clock generates physiological and behavioral rhythms with a period roughly 24 hours. It is thought that Circadian dysfunctions underlie the pathogenesis of many psychiatric disorders such as mania-like behavior and bipolar disorder (McClung, 2013). Attention Deficit Hyperactivity Disorder (ADHD) is one of the most widespread psychiatric disorders in humans. Somewhere between 3 and 5% of the population is affected by ADHD (Geissler and Lesch, 2011). The ADHD phenotype is characterized by increases in hyperactivity, inattentiveness and impulsivity (Kendal et al., 2008). Hyperactivity that results in sleep deprivation is one of the key behavioral manifestations of ADHD (Philipsen et al., 2006). Mice with the dominant negative Clock
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mutation have have increased hyperactivity, decreased sleep, lowered depression-like behavior, reduced anxiety and an increased reward value association with increased dopaminergic activities (McClung et al., 2005; Roybal et al., 2007). However, the mechanisms by which the circadian clock impacts the pathogenisis of ADHD, are uncertain.
A lack of several circadian clock genes have been shown to elicit ADHD-like symptoms.
Adult humans with dysrhythmias of melatonin production and altered expression of the circadian clock genes BMAL1 and PER2 appear to have a higher incidence of adult ADHD (Baird et al., 2012). Circadian nuclear receptor Rev-erbα (Nr1d1) knock-out mice display mania-like behavior such as hyperactivity and a central hyperdopaminergic state (Chung et al. 2014). Per1 knock-out mice also display ADHD-like symptoms and reduced levels of dopamine. This indicates that a disruption of a circadian clock gene a elicits ADHD-like syndrome more generally. Zebrafish with circadian gene period1b (per1b) mutations display hyperactive, impulsive-like and ADHD-like behaviors as well as low levels of dopamine. This trend across three different species indicates a strong correlation between Circadian clock, dopamine and ADHD like behaviors. The circadian model for understanding ADHD sheds light on the pathogenesis and could potentially open avenues for exploring novel targets for diagnosis and therapy for this common psychiatric disorder. Huang et al. show that zebrafish mutants with per1b mutation display hyperactivity, impulsivity-like and inattentive-like behaviors as well as low levels of dopamine (DA), reminiscent of human ADHD …show more content…
patients.
Zebrafish (Danio rerio) have proven to be a useful model for studying the genetic and developmental basis of behaviors more generally (Lieschke and Currie, 2007). Period1b (per1b an ortholog of human PER1) acts via enzymes critical for DA metabolism to directly regulate the endogenous DA levels, and also through genes critical for development of dopaminergic neurons to possibly regulate their number and spatial organization.
Huang et. al. generated Per1 mutant zebrafish using a retroviral insertion approach. The experimenters raised zebrafish in normal day-night lighting (LD) and compared them to fish raised in night-night (DD) lighting. Results showed that wild types expressed per1b rhythmically under both LD and DD conditions, but homozygous per1b retroviral insertion mutant zebrafish showed only ~10% of the wild-type per1b transcript levels. This was achieved using western blotting and immunofluorescence with per1b monoclonal antibody. Western blotting showed barely any detectible signal in the per1b mutant population and substantial signal in the wild type population. Immunofluorescence indicated that the per1b protein was expressed primarily in the eye in wild types, primarily in the inner nuclear layer of the retina, while per1b mutants showed little per1b concentration. This successfully demonstrated that the generation of per1b mutant population was successful.
The behavior of these per1b mutant fish was measured using a variety of methods to study whether they demonstrated ADHD like symptoms. In DD conditions, the per1b mutant population demonstrated a shorter circadian period than the wild type population by approximately 1.2 hours. Zebrafish hyperactivity was measured using a variety of methods.. The mutant population demonstrated a significantly higher amplitude of locomotor activity in the larvae stage than the wild type control population. The experimenters also saw that under LD conditions, per1b mutant zebrafish were approximately 3 times more active than the control group during both daytime and nighttime. It was also measured that the average swimming speed of the mutant fish population was approximately twice the average speed of the control group during the daytime and one and a half times during the nighttime. It was also shown that the per1b mutant fish were less averse to swimming through the center of their tank than the wild type which seemed to preferred to swim around the extremes of the tank. This indicates lower levels of anxiety and increased exploratory behavior. The mutant fish also demonstrated higher levels of aggression when posed with a perceived threat. To show that this hyperactive behavior was caused by the per1b mutation rather than some closely linked third genetic aberration, the experimenters microinjected per1b capped mRNA into one-cell zebrafish embryos. They observed that hyperactivity of mutant larvae injected with per1b mRNA was returned to normal levels. This is really exciting as it shows that both gain of function and loss of function are possible by manipulating per1b.
The ADHD phenotype is characterized by an increased difficulty learning and an impaired memory. To investigate whether the per1b zebrafish demonstrate these characteristics, Huang et al employed the AAC paradigm which measures a fish’s ability to learn to avoid electric shocks. They found that the per1b mutant fish were less adept at learning to avoid the electric shocks. The experimenters also estimated the memory of fish by measuring the ratio between training and testing scores on the AAC test. They found that the wild type fish performed better on this test than the mutant fish. It was also observed that no significant difference was observed between the control group and the per1b mRNA injected mutant population. Given the more explorative and aggressive nature of the per1b mutant fish as was demonstrated earlier, this does not seem to be the best mechanism for quantifying memory or learning aptitude. A better experiment might involve testing a the zebrafish’s ability to learn to traverse a maze to reach a reward.
Impulsivity is one of the most characteristic behavioral characteristics of ADHD in humans. Huang et al found that the per1b mutant population demonstrate higher levels of impulsivity-like behavior than the wild type population. This was accomplished by training the fish to swim over to a green light by giving it food every time the green light was turned on. After a week of training, the experimenters began withholding the reward for 4 minutes after the green light was switched on. It was observed that the wild type fish were more likely to wait by the green light than the per1b mutant population. This shows that the mutant zebrafish had less patience than the wild-type fish.
Abnormal dopamine (DA) levels have been implicated in the etiology and pathogenesis of ADHD.
Studying the physiological basis for ADHD like behaviors in per1b mutant zebrafish showed that per1b mutant zebrafish had significantly lower levels of DA than wild types further strengthening the link between per1b and ADHD-like behavior. Human ADHD is characterized by similarly low levels of DA. The circadian rhythm has been implicated in regulating DA synthesis, metabolism and signaling. The experimenters found that mao, a gene which encodes mooamineoxidases for decomposing DA, was upregulated in per1b mutant zebrafish. The mutant population was shown to additionally have excess β hydroxylase (dbh) which converts DA into NE. It was also observed that there was a significant phase difference (about 8 hrs) between per1b and wild type population rev-erbα expression which inhibits mechanisms associated with the production of mao and dbh. This effect is also found in mice and is likely responsible for the irregularities in DA levels in the mutant
population.
The study shows a clear link between per1b and ADHD like symptoms such as altered circadian rhythms, hyperactive behavior, defective learning and memory, and impulsivity-like symptoms. Based on their findings, the correlation is strong however, it is still entirely possible that ADHD is polygenic and there might be many factors that cause human ADHD-like behaviors. Ultimately, the study of developmental abnormalities is only useful insofar as it is able to lead to treatment. The experiment carried out by Huang et al where the experimenters microinjected per1b capped mRNA into one-cell zebrafish embryos showed that ADHD can be eliminated in zygotes. If this procedure could be safely applied to in-vitro fertilized human zygotes, then it could have enormous implications for society. Future studies could explore the safety and reliability of this method.
mutation have have increased hyperactivity, decreased sleep, lowered depression-like behavior, reduced anxiety and an increased reward value association with increased dopaminergic activities (McClung et al., 2005; Roybal et al., 2007). However, the mechanisms by which the circadian clock impacts the pathogenisis of ADHD, are uncertain.
A lack of several circadian clock genes have been shown to elicit ADHD-like symptoms.
Adult humans with dysrhythmias of melatonin production and altered expression of the circadian clock genes BMAL1 and PER2 appear to have a higher incidence of adult ADHD (Baird et al., 2012). Circadian nuclear receptor Rev-erbα (Nr1d1) knock-out mice display mania-like behavior such as hyperactivity and a central hyperdopaminergic state (Chung et al. 2014). Per1 knock-out mice also display ADHD-like symptoms and reduced levels of dopamine. This indicates that a disruption of a circadian clock gene a elicits ADHD-like syndrome more generally. Zebrafish with circadian gene period1b (per1b) mutations display hyperactive, impulsive-like and ADHD-like behaviors as well as low levels of dopamine. This trend across three different species indicates a strong correlation between Circadian clock, dopamine and ADHD like behaviors. The circadian model for understanding ADHD sheds light on the pathogenesis and could potentially open avenues for exploring novel targets for diagnosis and therapy for this common psychiatric disorder. Huang et al. show that zebrafish mutants with per1b mutation display hyperactivity, impulsivity-like and inattentive-like behaviors as well as low levels of dopamine (DA), reminiscent of human ADHD …show more content…
patients.
Zebrafish (Danio rerio) have proven to be a useful model for studying the genetic and developmental basis of behaviors more generally (Lieschke and Currie, 2007). Period1b (per1b an ortholog of human PER1) acts via enzymes critical for DA metabolism to directly regulate the endogenous DA levels, and also through genes critical for development of dopaminergic neurons to possibly regulate their number and spatial organization.
Huang et. al. generated Per1 mutant zebrafish using a retroviral insertion approach. The experimenters raised zebrafish in normal day-night lighting (LD) and compared them to fish raised in night-night (DD) lighting. Results showed that wild types expressed per1b rhythmically under both LD and DD conditions, but homozygous per1b retroviral insertion mutant zebrafish showed only ~10% of the wild-type per1b transcript levels. This was achieved using western blotting and immunofluorescence with per1b monoclonal antibody. Western blotting showed barely any detectible signal in the per1b mutant population and substantial signal in the wild type population. Immunofluorescence indicated that the per1b protein was expressed primarily in the eye in wild types, primarily in the inner nuclear layer of the retina, while per1b mutants showed little per1b concentration. This successfully demonstrated that the generation of per1b mutant population was successful.
The behavior of these per1b mutant fish was measured using a variety of methods to study whether they demonstrated ADHD like symptoms. In DD conditions, the per1b mutant population demonstrated a shorter circadian period than the wild type population by approximately 1.2 hours. Zebrafish hyperactivity was measured using a variety of methods.. The mutant population demonstrated a significantly higher amplitude of locomotor activity in the larvae stage than the wild type control population. The experimenters also saw that under LD conditions, per1b mutant zebrafish were approximately 3 times more active than the control group during both daytime and nighttime. It was also measured that the average swimming speed of the mutant fish population was approximately twice the average speed of the control group during the daytime and one and a half times during the nighttime. It was also shown that the per1b mutant fish were less averse to swimming through the center of their tank than the wild type which seemed to preferred to swim around the extremes of the tank. This indicates lower levels of anxiety and increased exploratory behavior. The mutant fish also demonstrated higher levels of aggression when posed with a perceived threat. To show that this hyperactive behavior was caused by the per1b mutation rather than some closely linked third genetic aberration, the experimenters microinjected per1b capped mRNA into one-cell zebrafish embryos. They observed that hyperactivity of mutant larvae injected with per1b mRNA was returned to normal levels. This is really exciting as it shows that both gain of function and loss of function are possible by manipulating per1b.
The ADHD phenotype is characterized by an increased difficulty learning and an impaired memory. To investigate whether the per1b zebrafish demonstrate these characteristics, Huang et al employed the AAC paradigm which measures a fish’s ability to learn to avoid electric shocks. They found that the per1b mutant fish were less adept at learning to avoid the electric shocks. The experimenters also estimated the memory of fish by measuring the ratio between training and testing scores on the AAC test. They found that the wild type fish performed better on this test than the mutant fish. It was also observed that no significant difference was observed between the control group and the per1b mRNA injected mutant population. Given the more explorative and aggressive nature of the per1b mutant fish as was demonstrated earlier, this does not seem to be the best mechanism for quantifying memory or learning aptitude. A better experiment might involve testing a the zebrafish’s ability to learn to traverse a maze to reach a reward.
Impulsivity is one of the most characteristic behavioral characteristics of ADHD in humans. Huang et al found that the per1b mutant population demonstrate higher levels of impulsivity-like behavior than the wild type population. This was accomplished by training the fish to swim over to a green light by giving it food every time the green light was turned on. After a week of training, the experimenters began withholding the reward for 4 minutes after the green light was switched on. It was observed that the wild type fish were more likely to wait by the green light than the per1b mutant population. This shows that the mutant zebrafish had less patience than the wild-type fish.
Abnormal dopamine (DA) levels have been implicated in the etiology and pathogenesis of ADHD.
Studying the physiological basis for ADHD like behaviors in per1b mutant zebrafish showed that per1b mutant zebrafish had significantly lower levels of DA than wild types further strengthening the link between per1b and ADHD-like behavior. Human ADHD is characterized by similarly low levels of DA. The circadian rhythm has been implicated in regulating DA synthesis, metabolism and signaling. The experimenters found that mao, a gene which encodes mooamineoxidases for decomposing DA, was upregulated in per1b mutant zebrafish. The mutant population was shown to additionally have excess β hydroxylase (dbh) which converts DA into NE. It was also observed that there was a significant phase difference (about 8 hrs) between per1b and wild type population rev-erbα expression which inhibits mechanisms associated with the production of mao and dbh. This effect is also found in mice and is likely responsible for the irregularities in DA levels in the mutant
population.
The study shows a clear link between per1b and ADHD like symptoms such as altered circadian rhythms, hyperactive behavior, defective learning and memory, and impulsivity-like symptoms. Based on their findings, the correlation is strong however, it is still entirely possible that ADHD is polygenic and there might be many factors that cause human ADHD-like behaviors. Ultimately, the study of developmental abnormalities is only useful insofar as it is able to lead to treatment. The experiment carried out by Huang et al where the experimenters microinjected per1b capped mRNA into one-cell zebrafish embryos showed that ADHD can be eliminated in zygotes. If this procedure could be safely applied to in-vitro fertilized human zygotes, then it could have enormous implications for society. Future studies could explore the safety and reliability of this method.