Bipolar Disorder (BD) is a psychiatric disorder that is characterized by mood episodes: mania, depression, and mixed episodes that cycle with vary bouts of euthymia between them (Gold and Einat, 2007). Bipolar I disorder (BD1) is characterized by the onset of manic episodes whereas bipolar II disorder (BD2) is characterized with depressive and hypomania episodes. Symptoms that are exhibited through mood disorders can cause substantial impairments in an individual’s life. Many researchers have sought to find the specific cause of this disorder but little is known about the mechanisms of the disorder. There has been an established link between sleep problems and BD and treatment of CRDs have been shown to improve individual functionality in BD individuals (Bradley et al., 2017; Sylvia et al., 2012). Disturbances to sleep patterns through experimental manipulations in patients with BD1 can cause an individual to exhibit manic episodes with insomnia and in BD2 sleep deprivation had similar effects to antidepressants (Wu et al, 1990). Other data has suggested abnormal sleep/CRDs were an influential trait in expression of manic episodes in BD1 individuals and that sleep deprivation helped remedy the depressive episode in BD2 patients (Bradley et al., 2017). CLOCK gene mutations resulted in manic episodes but didn’t show cyclic mood activity to depressive episodes (Roybal et al., 2007). Further research is need to create a model that can exhibit manic and depressive episodes in an organism. For instance, low serotonin (5HT) levels has been implicated in many mood and anxiety disorders, and involved in increased susceptibility to depression (Richardson-Jones et al., 2010). A study showed that 5-HT1A receptor knockout mice represent a model for anxiety and depression (Toth, 2003). The goal of my project is to create a double transgenic mouse model that exhibits the clock gene mutation and a knockout 5-HT1A to better capitulate BD psychopathology and use multiple behavioral assays to measure their behavior.
Specific Aim 1: I will create a double transgenic mouse model with both a clock gene mutation and knockout 5-HT1A genotype and use behavioral testing to ascertain if this mouse model will recapitulate BD psychopathology.
I will procure C.B6-Clockm1Jt/J (Clock gene Mutation) from Jackson Laboratory and B6.129(Cg)-Slc6a4tm1Kpl/J 5-HT1A knockout mice and breed them together. I will breed them and genotype them to make sure the mice exhibit both genetic manipulations. I will have a transgenic clock mutation group, 5-HT1A KO group, double transgenic clock/5-HT1A KO mice, and wildtype mice. I will then use a circadian rhythm assay to develop a baseline of circadian rhythm and the force swim test to measure hyperactivity and depressive-like behavioral baselines. Due to the implications of these genes in Bipolar Disorder, I hypothesize that the double transgenic mice will exhibit variability in the behavioral tests, the clock mutation mice will have longer circadian rhythms and spend less time immobile in the FST, and the 5-HT1A mice will have smaller circadian rhythms and exhibit more time immobile in the FST. The wildtype mice, clock mutation mice, and 5-HT1A mice will be used to establish a baseline to compare behaviors in the double transgenic mice.
Specific Aim 2: I will investigate how environmental factors affect the manifestation of different mood episodes. I will expose mice to different environmental stressors or enrichment, such as social housing or isolation and environmental enrichment/no environmental enrichment then use behavioral tests to assess the effect of environmental manipulation on manic-like and depressive-like behavior.
It has been shown that the Clock gene mutation mice exhibited behavior consistent with human manic episodes and that the 5-HT1A KO mice exhibit increased anxiety and depressive-like behaviors (Roybal et al., 2007; Toth, 2003). Due to the nature of my genetic manipulations on mice I believe these tests will examine cyclic manic and depressive behavior dependent on environmental stressors or enrichment. I hypothesize that by giving or taking away enrichment, the double transgenic mice will exhibit cyclic behavioral mood episode. Using social housing, isolation, and environmental enrichment I hypothesize that these factors can influence the presentation of mood episodes in mice.
Background Significance: Bipolar Disorder is a poorly understood neuropsychiatric illness making it difficult to recapitulate the symptoms of the disorder in animal models. Due to ethical laws regarding scientific research in humans, researchers must rely on animal models that can exhibit behavior associated with Bipolar Disorder. Trying to make a valid model of human behavior in animals is difficult so researchers must meet three validity criteria: construct, face, and predictive validity Construct validity refers to how closely the human disease is replicated in animals, face validity refers to if the tests measure what it intends to measure, and predictive validity refers to if the treatment used in humans has the same effect in animals and vice versa. Many proposed animal models of the BD are limited in their validity, usually only satisfying one of three validity criteria. Many of the symptoms of BD rely on subjective experiences of the individual making it difficult to assess these behaviors in animals, for example, depressed mood, feelings of worthlessness, and suicide. Researchers must rely on biochemical/genetic manipulations in animals so they exhibit behaviors like those seen in human BD psychopathology and reliable behavioral test to measure these behaviors accurately to increase the validity of animal model. Bipolar Disorder consists of cyclic bouts of depressive and/or (hypo)manic episodes. There are currently no known animal models of BD that exhibit spontaneous recurrent mood episodes. Most models that test for bipolar usually focus on one aspect, either depressive or manic episodes. Past research has shown genetic manipulations that are implicated in depression and mania and I have inferred that both these manipulations may be able to be represented in one organism which will exhibit cyclic mood episodes of depression and mania.
The Clock gene which regulates circadian rhythm has been implicated as a genetic issue highly correlated with BD. One reliable animal model of BD used a Clock mutant mice model. These mutant mice exhibited manic like symptoms of hyperactivity, reduced behavioral despair, reduced anxiety, sleep disturbances and increases in reward seeking behavior (Roybal et al., 2007). Lithium, a widely-used drug used to treat mania in BD, was shown to normalize the behavior of the Clock mutant mice providing this model with face and predictive validity. Another aspect of BD that is studied is the role of serotonin and serotonin receptors. One study showed that 5HT1A KO mice were more anxious, less reactive, and show behavior that is like depression in humans (Zhuang et al., 1999; Holmes et al., 2003, Lira et al., 2003). These KO mice have also been documented as showing reduced exploratory behavior and sensitization to fear cues (Parks et al., 1998; Gross et al., 2000; Toth, 2003). There are other studies that implicate corticosteroids, stress hormone in desensitizing or downregulate postsynaptic 5-HT1a receptors in various brain regions after exposure to chronic stress or corticosteroid administration (Flugg, 1995; Flugg et al., 1998; Chalmers et al., 1994; Watanabe et al., 1993; Vicentic et al., 2006). The proposed BD animal model that exhibits a Clock mutation and 5-HT receptor KO, mentioned in greater detail below, is expected to recapitulate BD pathophysiology and that this model will meet the three criteria of validity.
As mentioned above, mutations in the Clock gene and alterations in 5-HT1A receptor expression plays a role in the pathophysiology of mania and depression in BD, respectively. The significance of this study is to combine these two different phenotypes into one organism to meet the three-validity criterion. The proposed BD animal model, mentioned in greater detail below, is expected to meet these criteria of validity using genetic manipulations of depression and mania and use environmental stressors or enrichment to instigate cyclic moods in rodents. If the validity of this model is represented in the data, which it is expected to, then future studies can be utilized to better understand the pathophysiology of these genetic manipulations together to better treat or cure this disorder.
Approach
Specific Aim 1: I will create a double transgenic mouse model with both a clock gene mutation and knockout 5-HT1A genotype and use behavioral testing to ascertain a baseline prior to manipulating the mice with environment enrichment or stressors.
Rational: Clock mutant mice show similar symptoms of human BD such as disrupted circadian rhythms, decreased need for sleep and reduced anxiety (Roybal et al., 2007). Other studies have shown that genetic manipulations in mice that play a role in circadian rhythm spent less time immobile in the FST and more time in the center of an open field and had significantly longer circadian period (Roybal et al., 2007; Landgraf at al., 2016). These studies have reliably ascertained the implications of Clock gene manipulation in rodent manic mood-episode to correlate to human symptoms of BD mania. Other studies have shown mice can exhibit depressive like symptoms, such as anxiety and lower activity levels, through genetic manipulations of the serotonergic systems, using the FST and Circadian Rhythm/Free Running Assays (Smith et al., 2015; Soiza-Reilly, Goodfellow, Lambe, and Commons, 2015). 5-HT KO mice also showed increased sensitivity to stress and it is suggested that 5-HT1A receptors can be influence by gene and environmental stressors. (Smith et al., 2015; Toth 2003; Soiza-Reilly, Goodfellow, Lambe, and Commons, 2015). Though these are stand-alone genetic manipulations, the data collected has shown results that are reliable and consistent across multiple experiments. The goal is to combine these two genetic mutations that are related to bipolar disorder and it is expected that these double transgenic mice will exhibit cyclic mood episodes. The purpose of this initial procedure is to examine fertility and mortality in these newly created mice as well as establish a baseline for their exhibited behavior.
Transgenic Mouse Model Creation: Mice carrying the delta 19 Clock mutation strain, C.B6-Clockm1Jt/J, on a BALB/cJ genetics background will be obtained from The Jackson Laboratory (Bar Harbor, ME). Serotonin Transporter knock-out mice (5-HTT-/-) strain B6.129(Cg)-Slc6a4tm1Kpl/J, will also be obtained from The Jackson Laboratory (Bar Harbor, ME) and these mice are homozygous for the 5-HTT mutation. C.B6-Clockm1Jt/J male mice will be bred with B6.129(Cg)-Slc6a4tm1Kpl/J mice and progeny will be genotyped using reverse transcription polymerase chain reaction (RT-PCR) procedure to assess phenotypes. The hope is that cross breeding will produce a double transgenic Clock-Slc6a4 mouse that exhibits the KO 5-HT gene and the mutated Clock gene. The Clock mutated mice and 5-HT KO mice were created through backcrossing mice on a BALB/cJ and C57BL/6J backgrounds, respectively, and serve as controls. Also Clock mutant mice and the 5-HT KO mice will also serve as a baseline of their respective phenotypes.
Behavioral Approach
Open Field Test: Mice will be placed in the periphery of a novel open field environment in a dimly lit room. The field will be marked with a grid and square crossings with the center of the field marked with a different color to differentiate from other squares. Mice will be allowed to explore for 5 minutes and be monitored using an overhead video camera connected to a tracking software to measure time, distance moved, line crossing, center square entries, and center square durations.
Circadian Rhythm/Free Running Test: This test will be utilized to measure locomotor activity with mice being individually housed in a running-wheel equipped cages with access to food and water for 1 week. Equipment will monitor and record wheel running activity to measure hyperactivity as well as the length of the individuals CRs.
Forced Swim Test: Mice will be tested at 8-weeks and be performed as described by Porsolt et al. (1977). Two Plexiglas tubes (47 x 18 cm) will be filled with water (25° C). Water will be changed after each testing interval. Two tubes will be used at the same time and separated by an opaque divider. The testing area will be concealed completely by an opaque shower curtain. All tests will be recorded using a video recording equipment placed to look at the test horizontally. All mice will be tested for one trial in a random order randomized by an online generator. Animals will be placed in transport cages individually and transported to testing room. They will be allowed to habituate to the room for 5 minutes prior to testing. Each animal will be given a 6-minute trial. At the end of the trial the animals will be dried with a towel under a heat lamp and returned to their transport cage. When testing is finished, the recordings will be analyzed and hand scored by two research assistants blind to experimental groups to measure time spent immobile. Immobility is defined as remaining motionless and only making movements to keep head above the water (Porsolt et al., 1977).
Expected Outcomes: Behavioral testing will consist of first using the OFT then the CR/Free running test and then the FST. The order in which these behavioral tests will be performed is to limit stress caused by the test which could impact the mice’s behavior. Using the open field test, CR/Free running assay and the forced swim test, I hypothesize that the Clock mutant mice will exhibit hyperactivity (measured in locomotor activity) and stay active for longer periods of time in the CR/Free running test, spend more time in the center of the open field test and less time immobile in the FST. I also hypothesize that the 5-HT KO mice will exhibit the opposite results compared to the Clock Mutant mice.. These non-mutant mice will be breed together to create a C57-Balb mixed mouse to serve as a control. Due to the uncertainty of the double transgenic manipulation these tests will just be used to assess and create a baseline prior to environmental manipulations that will be performed in the second part of the experimental procedure.
Potential Complications: The behavioral testing for the Clock and 5-HTKO mice are documented and consistent but combining these two genetic manipulations has not been performed. It is unknown if the double transgenic mouse will be fertile or have a high mortality rate. If these mice have high mortality rates then brain extractions and necropsies of the mice will be performed and different immunohistochemistry procedures will be utilized to examine cause of brain abnormalities as a potential cause of death. If the mortality is related to the 5-HT KO then I believe instead of knocking out 5-HT receptors I could limit the expression of these receptors. It is also uncertain how the double transgenic mice will perform in behavioral testing, which will be resolved during this initial phase to ascertain behavioral phenotypes.
Specific Aim 2: I will investigate how environmental factors affect the manifestation of different mood episodes. I will use circadian rhythm test, sucrose preference, forced swim test, and open-field test to assess manic-like and depressive-like behavior.
Rational: It has been shown that the Clock gene mutation mice exhibited behavior consistent with human manic episodes and that the 5-HT1A KO mice exhibit increased anxiety and depressive-like behaviors (Roybal et al., 2007; Toth, 2003). These are genetic factors that play a role in the pathophysiology of mood disorders but little is known how environmental factors influence expression of mood episodes in BD. Environmental enrichment is defined as “a combination of complex inanimate and social stimulation” (Rosenzweig et al., 1978). It has also been suggested that depression pathophysiology is a combination of genotype and the occurrence of at least three adverse life events (Caspi et al., 2003). Environmental enrichment has also been shown to have protective physiological effects on the brain and that environmental enrichment can counteract pathological deficits (Rosenzweig and Bennett, 1996; Nithianantharajah and Hannan, 2009). Housing multiple animals in a large enough cage can limit stressful conditions and social isolation in small cages can induce chronic stress (Stairs and Bardo, 2009; Laviola et al., 2008; Nithianantharajah and Hannan, 2009; Hall, 1998). Due to the nature of my genetic manipulations on mice I believe these tests will examine cyclic manic and depressive behavior dependent on environmental stress or enrichment provided. After the initial testing phase mentioned above, mice will be separated into groups [Table 1] for two weeks before behavioral testing. I hypothesize that through environmental enrichment or environmental stressors, the double transgenic mice will exhibit cyclic behavioral mood episode. Using social housing, isolation, and environmental enrichment I hypothesize that these factors can influence the presentation of mood episodes in mice.
Environmental Manipulations
Housing: Isolation/EE groups will be housed individually in a standard size cage with a toy and running wheel. Isolation/No EE groups will be housed individually with no toys or running wheel. Social Housed/EE groups will be housed in standard cages of with three mice in each and provided toys and a running wheel. Socially Housed/No EE groups will be housed 3 to a cage with no toys or running wheel. Mice will be randomly assigned conditions.
Behavioral Approach
Open Field Test: Mice will be placed in the periphery of a novel open field environment in a dimly lit room. The field will be marked with a grid and square crossings with the center of the field marked with a different color to differentiate from other squares. Mice will be allowed to explore for 5 minutes and be monitored using an overhead video camera connected to a tracking software to measure time, distance moved, line crossing, center square entries, and center square durations.
Circadian Rhythm/Free Running Test: This test will be utilized to measure locomotor activity with mice being individually housed in a running-wheel equipped cages with access to food and water for 1 week. Equipment will monitor and record wheel running activity to measure hyperactivity as well as the length of the individuals CRs.
Forced Swim Test: Mice will be tested after 2 weeks after environmental manipulation and be performed as described by Porsolt et al. (1977). Two Plexiglas tubes (47 x 18 cm) will be filled with water (25° C). Water will be changed after each testing interval. Two tubes will be used at the same time and separated by an opaque divider. The testing area will be concealed completely by an opaque shower curtain. All tests will be recorded using a video recording equipment placed to look at the test horizontally. All mice will be tested for one trial in a random order randomized by an online generator. Animals will be placed in transport cages individually and transported to testing room. They will be allowed to habituate to the room for 5 minutes prior to testing. Each animal will be given a 6-minute trial. At the end of the trial the animals will be dried with a towel under a heat lamp and returned to their transport cage. When testing is finished, the recordings will be analyzed and hand scored by two research assistants blind to experimental groups to measure time spent immobile. Immobility is defined as remaining motionless and only making movements to keep head above the water (Porsolt et al., 1977).
Expected Outcomes:
Supplementary Enrichment versus No Enrichment: I expect that through isolation and No EE double transgenic mice will show depressive-like behavior such as decreased locomotor activity and shorter CRs, spend more time spent around the walls of the open field, and longer time spent immobile in the FST due to the chronic stress on no social or inanimate object enrichment. I also believe that enrichment through social housing and inanimate object enrichment the double transgenic mice will show more manic-like behavior defined as increased locomotor activity and longer CRs, more time spent in the center of the open field, and less time immobile in the FST. It is expected that the 5-HT mutated mice that are socially housed and provided EE will exhibit elevated locomotor activity and increased time spent in the center of the open field, increased locomotion and normal CRs, and spend lower time immobile in the FST. The 5-HT KO mice who are isolated and provided no enrichment will show significantly less time spent in the center of the open field, decreased locomotion and lowered CRs, and spend more time immobile in the FST. It is expected that Clock mutant mice who are socially housed and provided EE will exhibit more time spent in the center of the open field, increased locomotion and longer CRs, and spend less time immobile during the FST. Clock mutant mice who are isolated without EE should exhibit the same results as the Clock+[Social Housed/EE] mice. I expect that the WT mice will exhibit a similar but less pronounced effects of the 5-HT KO and CLOCK mutant mice with scores falling in-between these two genotypes.
Partial Enrichment: For the groups, Isolation/EE and Socially Housed/No EE, I expect that enrichment through social housing will have a more profound effect on than through inanimate environmental enrichment in the all the genotypes. It is unclear how much of an effect just social housing or inanimate object enrichment can affects physiology as it has been reported that social grouping alone cannot account for cerebral effects due to enriched environments and that inanimate stimulus enrichment must also be considered (Rosenzweig et al., 1978).
Potential Complications: Potential pitfalls of this experiment is that these tests have never been performed on this double transgenic mouse model. The WT, 5-HT KO and CLOCK mutant mice will have to serve as controls and baselines to properly assess the effects of social enrichment and inanimate object enrichment or both are needed to profoundly affect pathophysiology in these genotypes. This part of the experiment will have to serve as an initial study to examine the behavior observed and in the future the effects of antidepressants and mood stabilizers can be used to assess pharmacological interventions on behavior.