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Collaborative Cross recombinant inbred inter-crosses (RIX) for the study of the behavioral and structural consequences of chronic antipsychotic treatment Daniela DeCristo Abstract Schizophrenia is an idiopathic disorder that affects approximately 1% of the global population, and presents with persistent delusions, hallucinations, and disorganized behaviors. Antipsychotics are the standard treatment of schizophrenia, but are frequently discontinued by patients due to inefficacy and/or intolerable side effects. Chronic treatment with the antipsychotic haloperidol causes tardive dyskinesia in about 30% of patients, manifested in humans as involuntary and often permanent orofacial movements. Tardive dyskinesia can be effectively modeled in rodents by means of vacuous chewing movements. Recombinant inbred inter-crossed (RIX) mice, derived from the genetically diverse Collaborative Cross murine population, were treated with haloperidol and subjected to a panel of behavioral assessments to monitor the development of side effects. Furthermore, transition electron microscopy (TEM) was used to assess the impact of chronic haloperidol treatment on synaptic structures in wildtype C57BL/6 mice. While there were similar neuronal synaptic architectures in the corpus striata across treatment groups, distinctions were found in phenotypic behavior between strain genotypes. These findings add to our understanding of the genetic basis of the side effects of haloperidol with the aim of higher efficacy and lower burden of haloperidol treatment. Introduction Schizophrenia is a highly complex and heritable psychiatric disorder with many possible genetic and environmental determinants. Affecting over 51 million people 1 worldwide, schizophrenia is among the top ten leading causes of disability and is accompanied with high costs for patients, their caretakers, and society as a whole1-4. Although schizophrenia was first described more than 100 years ago, an underlying cause remains unknown. While genome-wide association studies (GWAS) and structural variation studies have led to many advancements in our understanding of the disorder in the past 5-10 years, its pathophysiology is still not well-defined. Additionally, symptoms are often difficult to identify and can manifest in varying degrees depending on the patient. As a result, doctors are tasked with correctly diagnosing patients with schizophrenia using a vague clinical and research definition and an imprecise therapeutic approach. Recent innovations in genomics have shed light on the genetic basis of schizophrenia. The Psychiatric Genomics Consortium, led by Dr. Patrick Sullivan, applied GWAS using a large sample set of 36,989 schizophrenia patients and 113,075 unaffected individuals in order to determine possible candidate genes. The Consortium found 108 loci in the human genome that confer an increased risk for the disorder. Within this set of loci, there is an overrepresentation of genes with neuronal and brain expression, including DRD25,6. DRD2 codes for the production of brain receptors for dopamine, a neurotransmitter highly involved in complex thought, movement, reward and other behaviors. The gene is also the target of all clinically-effective antipsychotic medications currently in use; these drugs antagonistically interact with dopamine receptors to reduce hallucinations and delusions7. Unfortunately, antipsychotic drugs frequently lead to serious adverse drug reactions (ADRs). Treatment of schizophrenia with the antipsychotic drug, haloperidol, is 2 no exception. Haloperidol is a prototypical antipsychotic and potent antagonist of the dopamine receptor D2 in the striatum, a brain region that plans and moderates movement. In addition to other side effects, a subset of patients will experience severe and often irreversible motor ADRs including uncontrolled and purposeless jaw movements, termed tardive dyskinesia (TD)8. Prevalent in about 30% of patients and permanent in half of those affected, the reason why haloperidol causes this ADR has not been discovered9-11. Currently, there is no compelling way to predict adverse drug reactions (ADRs) or drug efficacy using the genetic makeup of a patient, an ability that would make drug treatment of schizophrenia using antipsychotics safer and more effective. The laboratory mouse can be used as a proxy to study certain human pharmacogenetic phenotypes. Collaborative Cross RIX mice recapitulate many of the features of TD with haloperidol treatment including jaw tremors, tongue protrusions, and vacuous chewing movements (VCMs), making them an ideal model system. A major goal of the Center for Integrated Systems Genomics at the University of North Carolina at Chapel Hill is to harness the genetic diversity of RIX mice to explore the genetic basis of antipsychotic side effects12. This study is a further exploration of the preliminary data published by Crowley et al. that determined the validity of murine VCMs as a model of TD induced by haloperidol and the heritability of risk of adverse drug effects10. Previous studies have explored morphological and synaptic changes that are thought to be associated with haloperidol treatment and TD in the brain using rodents and transition electron microscopy. Results from these studies are inconclusive and contradictory at times. Kerns et al. suggested that alterations in synaptic architecture follow a sequence (enlargement, perforation, and double synapse formation) after 3 observing an enhanced number of synaptic boutons and an increase in perforated and double synapses due to haloperidol treatment13. In contrast, Benes et al. found that changes in synapse numbers were region-specific, and that striatal neuronal density and relative number of nerve terminals were unchanged between treatment groups14. Additional studies have similarly reported an increase in perforated synapses in the caudate nucleus and striatum13-17, yet no change was found in the nucleus accumbens or the medial prefrontal cortex in a separate study17. Other studies examining asymmetric synapses, or synapses that exhibit larger postsynaptic densities than presynaptic densities and are typical of excitatory inputs, found decreases in asymmetric synaptic densities16,18,19. Roberts and Lapidus suggested that this decrease in excitatory inputs may protect against VCMs in their examination of the relationship between haloperidol, VCMs, and asymmetric synapses as a result of finding differentially affected asymmetric synaptic densities in the low and high VCM groups, with a high amount of asymmetric synaptic densities only in the high VCM group19. The current problem at hand is that there is no compelling way to predict drug efficacy or adverse drug reactions such as TD. The goal of this study is to elucidate the genetic basis of TD by determining sensitivity to haloperidol-induced adverse drug effects in the genetically diverse RIX mouse population. Synaptic morphology of striatal tissue was also analyzed using electron microscopy due to the critical role of the striatum in modulating motor function19. Behavioral phenotypes and synaptic ultrastructure were examined between mice treated with haloperidol and those treated with placebo. This paper discusses findings from the behavioral characterization and synaptic architecture analysis of RIX mice to explore the influence of genetic variation on haloperidol-induced 4 TD susceptibility and contributes to the growing predictive power of pharmacogenomics for improved personalized treatment of patients impacted by schizophrenia. Methods Animals During the course of this study 846 male and female mice from 73 different RIX lines derived from the Collaborative Cross (The Jackson Laboratory, Bar Harbor, ME) were phenotyped. Mice were housed on a cycle of 12 hours of light and 12 hours of dark with lights on at 0700 hours in a room held at a consistent temperature range of 20-24 °C. Two mice of the same sex were housed per standard ventilated cage with water and Purina ProLab IsoPro 3000 food available constantly and positioned on the roof of the cage. For each strain, mice were grouped in three batches so that three replicate mice per sex and treatment combination were tested by the end of the study. The study strictly followed the ‘Guide for the Care and Use of Laboratory Animals’ with approval by the Institutional Animal Care and Use Committee of the University of North Carolina20. Haloperidol treatment and activity testing Methods for drug administration were adopted from pilot studies indicating that the optimal human-like steady-state concentration of haloperidol could be obtained using a 30-day release tablet that delivered 3.0 mg kg-1 per day to the mouse10,21. Mice were anesthetized for 2 minutes with isoflurane, and haloperidol pellets were implanted into the mice using a trocar22. The drug was administered after mice were 8 weeks old. The activity of the mice was monitored using open field testing in activity chambers with infrared beams to measure locomotor and exploratory behavior of mice before treatment and 28 days post-treatment (Fig. 1). 5 Scoring orofacial movements Video recording of vacuous chewing movements (VCMs) was carried out after 28 days post-treatment. To this end, mice were briefly anesthetized with isoflurane and restrained for 25 minutes using a plastic collar. Collars were made from two plastic semicircular pieces that were adjustable based on neck size and to achieve the most comfortable position for the mouse. The collar partially immobilized the mice at the neck but still permitted head movement to allow for video recording of jaw movements by JVC Everio digital camcorders. Digital videotapes were made using the protocol developed by Tomiyama et al.23. The first 10 minutes of video were not analyzed in order to allow the mice to adjust to the collar and to relax. The last 15 minutes of the video were scored for orofacial movement. Videos were randomized and scored by a singleblinded rater to increase consistency and to reduce any deviation or bias between raters. The rater was trained by an expert and a set of standard training videos used in the study by Crowley et al. to align the rater with correct identification of VCMs according to the scoring from the previous study10. Drift was monitored by re-scoring random videos throughout the course of the study. The movements that were specifically analyzed were tongue protrusions, jaw tremors, overt chewing movements, and subtle chewing movements. Individual events of each movement with the exception of tremors were counted; tremors were measured by duration in seconds. Subtle chewing movements were defined as instances of vertical jaw movement in which the inside cavity of the mouth could not be seen and the jaw was not open for a long period of time. Overt chewing movements occurred when a larger vertical movement was observed in which the cavity could be seen and the jaw was open for an 6 extended length of time. The videos were scored using The Observer XT (Noldus Inc., Wageningen, Netherlands) observational data analysis program. Behavioral analysis Overall analysis of activity was performed using R (The R Foundation, Vienna, Austria). Statistical analyses of scored observations were carried out using JMP software (version 12.0.1, SAS Institute Inc., Cary, NC). Data from 846 RIX mice were collected from 73 strains in 51 batches. Due to the very minor distinctions between subtle and overt VCMs, the counts of these two phenotypes were combined for analysis. The data for VCMs between treatment groups was fit following Two-Way ANOVA with interaction: Yijk = μ+αi+βj+γij+εijk εijk ~ N(0,σ2) i = 1,…,ni j = 1,…,nj Where the α are strain effects, β are the treatment effects, and γ are the interactions thereof. After dropping strain 5080x6750, as there was only a single mouse of this strain, ni = 72, nj = 2, the model was fit in SAS PROC MIXED version 9.4. The following hypothesis was then tested: H0: γij = 0 ∀ i,j In words, the significance of the interactions - that is, whether the treatment effect differs by strain – was tested. For this, the F-test was employed. Estimates of the treatment effect for each strain were calculated with combined subtle and overt VCM totals as the dependent variable and treatment by strain as the parameter, along with the associated p- 7 values, which have been adjusted to control False Discovery Rate at 0.05. Tissue preparation Using the same 30-day treatment protocol previously described, ten C57BL/6 mice were perfused with a solution of 2% paraformaldehyde/2.5% glutaraldehyde in 0.15 M sodium phosphate buffer, pH 7.4. A 2 mm thick coronal section (Bregma coordinates from +1.0 to -1.0) was dissected and the ventral striatum (~1 mm thickness) was manually isolated from each mouse. Samples were post-fixed in 1% osmium tetroxide in 0.15 M sodium phosphate buffer, pH 7.4, for one hour. Following gradual dehydration with ethanol and propylene oxide, samples were infiltrated and embedded in PolyBed 812 epoxy resin (Polysciences, Inc., Warrington, PA). Light microscopy sections (1 µm) were cut, mounted on slides, and stained with 1% toluidine blue O in 1% sodium borate. After selecting the region of interest, 70 nm ultrathin sections were cut using a Leica Ultracut UCT microtome (Leica Microsystems, Inc., Bannockburn, IL) and a diamond knife. The sections were mounted on 200 mesh copper grids and contrasted with 4% uranyl acetate and Reynolds' lead citrate stains. Transition electron microscopy Samples were observed using a LEO EM 910 transmission electron microscope at 80 kV (Carl Zeiss SMT, Inc., Thornwood, NY) and digital images were acquired with a Gatan Orius SC1000 CCD camera and Digital Micrograph Software (version 2.3.1, Gatan, Inc., Pleasanton, CA). With striatal samples from 10 different mice, 10 locations per section were mapped to separate grids. Images were taken on a single plane to eliminate any possibility of counting a synapse twice. For each location, one image was taken at 10,000 × magnification and four images were taken from that image at 25,000 × 8 without overlap. Four images per mouse were also acquired at 50,000 × for very close examination of synapses. Image analysis Using Fiji, an open-source ImageJ software, synapses and mitochondria were counted in each of the 100 electron microscopy images taken at 10,000 × using the cell counter tool24. General observations of the amount of symmetric versus asymmetric synapses were made. Perforated synapses were defined as having breaks in synaptic continuity greater than 0.05 μm and double synapses were those that exhibited active zones with two different postsynaptic structures in accordance with similar previous studies (Fig. 2)13,25. Perforated and double synapses were also counted in the 100 images at 10,000 ×. The scaling and measurement tools were used in Fiji to validate distances between synaptic densities for perforated synapses. Counts of the four measures of number of synapses, mitochondria, perforated synapses and double synapses were totaled for each subject from their respective 10 images and evaluated using a Student’s t-test with an alpha of 0.0125 after Bonferroni correction in JMP (version 12.0.1, SAS Institute Inc., Cary, NC). Results Behavior analysis Overall open field activity analysis between treatment groups indicates reduced activity in the mice treated with haloperidol in contrast to mice treated with placebo. Haloperidol-treated mice spent less time exploring the open field both horizontally and vertically. Levels of anxiety (as measured using time in center) and repetitive behaviors (measured via stereotypy) were shown to be the same for both groups (Fig. 3). 9 VCMs analysis Haloperidol-treated mice showed greater susceptibility to subtle and overt VCMs than placebo-treated mice with no significant differences between groups in number of tremors and tongue movements (Fig. 4). Strain-by-strain analysis of the recorded number of subtle, overt, tremors, and tongue protrusion VCMs indicate trends similar to the overall trend observed between treatment groups (Fig. 5-8). From the Two-Way ANOVA of 72 strains, the null hypothesis that there was no difference in treatment effect between strains was rejected at the α = 0.05 level of significance with a calculated p-value of 0.0224. Thus, the effect of treatment differs by strain (Table 1). Individual statistical analysis of each strain allowed for clear identification of strains significantly affected by haloperidol. In fourteen strains (8016x8034, 559x8031, 3140x3015, 6513x6188, 8049x8046, 5306x5612, 3252x3154, 3032x6188, 8031x3609, 8008x8016, 5156x1566, 6188x3252, 8005x8002, 867x3252), the number of combined subtle and overt VCMs in mice treated with haloperidol were significantly different from the number of VCMs recorded in mice administered placebo after controlling FDR at 0.05 (Table 2). Striatal ultrastructure analysis Most of the synapses observed in every image were asymmetric and had larger post-synaptic densities than pre-synaptic densities. No differences were found between 30-day C57BL/6 treatment groups for the four measured ultrastructural components: number of synapses, mitochondria, perforated synapses, and double synapses per sample. Calculated p-values do not pass the Bonferroni-corrected threshold of significance of 0.0125 (Fig. 9, Table 3). 10 Discussion The Collaborative Cross-derived RIX lines have proven to be a vital means for exploring heterogeneity. The results of this study validate the use of RIX mice for the study of VCMs due to the differential haloperidol sensitivities observed in the strains tested. The discovery of fourteen RIX strains that are highly susceptible to haloperidolinduced TD contributes to the aim to identify genes regulating this susceptibility. Using these lines diminishes the need for human studies with very large sample sizes required to effectively parallel the diversity found in the entire population. Genetic mapping of candidate regions identified in mice will allow precious human samples to be used only for highly probable regions of interest as part of future research. While significant differences were not found in the synaptic architecture between drug and placebo treatment groups, increased power with a larger sample size or more electron microscopy imaging may be necessary to reveal any differences or confirm results. Additionally, images of another location in the striatum or at a different timepoint may also illustrate a more significant impact of the drug than what was captured in this study. The results of this study suggest that the underlying mechanism of TD by chronic haloperidol treatment does not involve alterations in striatal ultrastructure in congruence with findings by Kessas et al.26. Further investigations of neuroplasticity, mitochondrial amounts, and synaptic counts will shed more light on this area of research. The genetic variation found in the human population is difficult to model using animals that have been bred to be genetically similar for many generations. These difficulties arise when trying to solve problems and answer questions related to human diseases and disorders. Though much of the genome is conserved between individuals, 11 some of the smallest variances like single nucleotide polymorphisms and variable number tandem repeats have been associated with significant phenotypic differences between individuals. The Collaborative Cross murine population may be a valuable tool to assess these genetic features. Due to the genetic diversity and intricate breeding system of these Collaborative Cross RIX lines, complex modeling of sex effects, effect of parental lines, within group variability and between group variability is being carried out to examine the results in a larger context and elucidate relationships between strains for genetic candidate mapping. These efforts aim to better understand the relationship between gene expression and chronic haloperidol treatment and to correlate phenotypes with strain genotypes and RNA-sequencing data. Genome analysis will be done to try and identify regions of the genome that correlate with those mice that reacted most strongly to the drug. The recent popularity and intrigue of personalized medicine has generated much discussion and research in health care. Evaluation of an individual patient’s genome, familial genetic history, and pertinent environmental factors all to arrive at a precise approach to treatment may lead to greater therapeutic success as this approach is refined. The identification of genes involved in higher risk of TD development due to chronic haloperidol treatment in RIX lines allows for focused application in human treatment to increase physicians’ predictive power to prevent harmful side effects and maximize benefit in the treatment of patients with schizophrenia. Conclusions RIX strains had wide-ranging sensitivities to haloperidol-induced VCMs, with fourteen strains being particularly susceptible. Striatal imaging results suggest that 12 haloperidol does not influence synaptic amount or morphology. Further explorations of changes in striatal neuroplasticity will clarify these results. This study will aid in the discovery of the genetic determinants of susceptibility to tardive dyskinesia with more indepth analysis underway. References 1 World Health Organization. The Global Burden of Disease: 2004 Update. Geneva: WHO Press, 2008. 2 Murray CJL and Lopez AD. Alternative projections of mortality and disability by cause 1990-2020: Global Burden of Disease Study. Lancet. 1997;349(9064):1498-504. 3 Knapp M, Mangalore R, Simon J. The global costs of schizophrenia. Schizophrenia bulletin 2004; 30: 279-293. 4 Saha S, Chant D, McGrath J. A systematic review of mortality in schizophrenia: is the differential mortality gap worsening over time? Archives Gen Psych 2007; 64: 1123-31. 5 Sullivan P, Daly M, O’Donovan M. Genetic architectures of psychiatric disorders: the emerging picture and its implications. Nat Reviews 2012; 13: 537-551. 6 Ripke, S. et al. Genome-wide association analysis identifies 13 new risk loci for schizophrenia. Nature Genetics 2013; 45:1150-9. 7 Roth B, Sheffler D, Kroeze W. Magic shotguns versus magic bullets: selectively nonselective for mood disorders and schizophrenia. Nature Reviews 2004; 3: 353-359. 8 Kinon B, Lieberman J. Mechanisms of action of atypical antipsychotic drugs: a critical analysis. Psychopharmacology 1996; 124: 2-34. 9 Dayalu P, Chou KL. Antipsychotic-induced extrapyramidal symptoms and their management. Expert Opin Pharmacother 2008; 9: 1451–1462. 13 10 Crowley J, Adkins D, Pratt A, Quackenbush C, van den Oord E, Moy S, Wilhelmsen K, Cooper T, Bogue M, McLeod H, Sullivan P. Antipsychotic-induced vacuous chewing movements and extrapyramidal side effects are highly heritable in mice. Pharmacogen J 2010; 12: 147-155. 11 Soares-Weiser K, Fernandez H. Tardive dyskinesia. Semin Neurol 2007; 27: 159– 169. 12 Threadgill D, Miller D, Churchill G, Pardo-Manuel de Villena F. The Collaborative Cross: a recombinant inbred mouse population for the systems genetics era. ILAR Journal 2001; 52: 24-31. 13 Kerns JM, Sierens DK, Koa LC, Klawans HL, Carvey PM. Synaptic plasticity in the rat striatum following chronic haloperidol treatment. Clin Neuropharm. 1992; 15(6):488500. 14 Benes FM, Paskevich PA, Davidson J, Domesick VB. The effects of haloperidol on synaptic pattern in the rat striatum. Brain Res. 1985; 329:265-174. 15 Meshul CK, Stallbaumer RK, Taylor B, Janowsky A. Haloperidol-induced morphological changes in striatum are associated with glutamate synapses. Brain Res. 1994; 648(2):181-95. 16 Andreassen O, Meshul CK, Moore C, and Jorgensen HA. Oral dyskinesias and morphological changes in rat striatum during long-term haloperidol administration. Psychopharm. 2001; 157: 11-19. 17 Meshul CK, Janowsky A, Casey DE, Stallbaumer RK, Taylor B. Effect of haloperidol and clozapine on the density of "perforated" synapses in caudate, nucleus accumbens, and medial prefrontal cortex. Psychopharm. 1992; 106: 45-52. 14 18 Benes FM, Paskevich PA, Davidson J, Domesick VB. Synaptic rearrangements in medial prefrontal cortex of haloperidol-treated rats. Brain Res. 1985; 348: 15-20. 19 Roberts RC and Lapidus B. Ultrastructural correlates of haloperidol-induced oral dyskinesias in rats: a study of unlabeled and enkephalin-labeled striatal terminals. J Neural Transm. 2003; 110: 961–975. 20 National Research Council (US) Committee for the Update of the Guide for the Care and Use of Laboratory Animals. Guide for the Care and Use of Laboratory Animals. 8th edition. Washington (DC): National Academies Press (US); 2011. 21 Fleischmann N, Christ G, Sclafani T, Melman A. The effect of ovariectomy and longterm estrogen replacement on bladder structure and function in the rat. J Urol 2002; 168: 1265–1268. 22 Hsin-Tung E, Simpson G. Medication-induced movement disorders. In: Kaplan HI, Sadock BJ (eds). Comprehensive Textbook of Psychiatry. Lippincott Williams and Wilkins: Philadephia, PA, 2000; 2265–2270. 23 Tomiyama K, McNamara FN, Clifford JJ, Kinsella A, Koshikawa N, Waddington JL. Topographical assessment and pharmacological char- acterization of orofacial movements in mice: dopamine D(1)-like vs. D(2)-like receptor regulation. Eur J Pharmacol 2001; 418: 47–54. 24 Schindelin J, Arganda-Carreras I, Frise E et al. Fiji: an open-source platform for biological-image analysis. Nature methods 2012; 9(7): 676-682. 25 Meshul CK, Casey DE. Regional, reversible ultrastructural changes in rat brain with chronic neuroleptic treatment. Brain Research. 1989; 489 (2): 338-346. 15 26 Kessas M, Creed M, Nobrega JN. An examination of synaptic proteins following chronic haloperidol in a rat model of tardive dyskinesia. Psychology & Neuroscience 2010; 3(2): 229-237. 16 Supplementary Tables and Figures aged7% 7weeks% weeks !!!!aged% Figure 1. RIX phenotyping pipeline. 8-week old mice (846 male and female mice) from 73 RIX strains were phenotyped in 51 batches using this phenotyping pipeline to identify strain differences in response to haloperidol treatment. Open field activity (Activity) was assessed before and after treatment. Fecal boli will be stored at -80 °C for potential microbiomics work (*). Extrapyramidal symptoms (EPS), or acute side effects, were tested soon after treatment. Vacuous chewing movements (VCMs; i.e. Orofacial) were recorded after 28 days of drug treatment. 17 * Figure 2. Transmission electron microscopy (TEM) was used to assess changes in synaptic density after chronic haloperidol treatment. Representative electron microscopy image of striatal tissue with a perforated synapse (single arrow) defined by a break between synaptic densities of greater than 0.05 μm, a double synapse (double arrow) where active zones are entering two different synaptic structures and a mitochondria (*). Many mitochondria and synapses are clearly seen in this image. 18 Figure 3. Haloperidol-treated mice exhibit reduced horizontal and vertical activity. Open field activity was used to measure changes in activity upon chronic haloperidol treatment. Open field activity testing showed that haloperidol treatment reduces horizontal exploratory distance and vertical rearing activities with no significant differences in anxiety levels (time in centroid) or repetitive behaviors (stereotypy) between treatment groups. 19 Figure 4. Haloperidol-treated mice exhibit larger amounts of subtle and overt VCMs. Scoring of video recordings of vacuous chewing movements (VCMs) after 28 days of haloperidol treatment indicates that haloperidol-treated mice exhibit VCMs at a higher frequency than placebo-treated mice with no significant differences between groups in number of tremors and tongue movements. 20 Figure 5. Differential susceptibility to haloperidol-induced subtle VCMs between RIX strains. Strain-by-strain analysis of the recorded number of subtle VCMs organized by increasing mean of subtle VCMs counts for placebo groups (black squares). Means of counts for haloperidol groups are overlaid (red squares), and individual samples are also indicated (black dots for placebo-treated, red dots for drug-treated). 21 Figure 6. Differential susceptibility to haloperidol-induced overt VCMs between RIX strains. Strain-by-strain analysis of the recorded number of overt VCMs organized by increasing mean of overt VCMs counts for placebo groups (black squares). Means of counts for haloperidol groups are overlaid (red squares), and individual samples are also indicated (black dots for placebo-treated, red dots for drug-treated). 22 Figure 7. Differential susceptibility to tremors between RIX strains. Strain-by-strain analysis of the recorded number of tremors VCMs organized by increasing mean of tremors VCMs counts for placebo groups (black squares). Means of counts for haloperidol groups are overlaid (red squares), and individual samples are also indicated (black dots for placebo-treated, red dots for drug-treated). 23 Figure 8. RIX strains do not exhibit differential susceptibility to tongue movements. Strain-by-strain analysis of the recorded number of tongue VCMs organized by increasing mean of tongue VCMs counts for placebo groups (black squares). Means of counts for haloperidol groups are overlaid (red squares), and individual samples are also indicated (black dots for placebo-treated, red dots for drug-treated). 24 Table 1. Treatment effect on subtle and overt VCMs significantly differs between strains. We fit the data to a Two-Way ANOVA with interaction (after dropping strain 5080x6750 as there only a single mouse of this strain). The null hypothesis that there was no difference in treatment effect between strains was rejected at alpha=0.05 with a calculated p-value of 0.0224 for the combined effect of strain and treatment. Thus, the effect of haloperidol and placebo on combined subtle and overt VCM amount differs between strains. Source DF SS F-value P-value 71 1041180 7.3 0.0001 Strain 1 199349 89.9 0.0001 Treatment 71 199838 1.4 0.0224 Strain*Treatment 591 1191456 Error 25 Table 2. Haloperidol-treated and placebo-treated groups within 14 strains are significantly different. Significant differences in the combined subtle and overt VCMs phenotype were found between groups within fourteen strains using a two-way ANOVA and controlling False Discovery Rate at alpha=0.05. Strain Estimate StdErr tValue Probt False Discovery Rate 28.3971875 3.97 <.0001 0.0072 8016x8034 112.8 95.833333 25.922967 3.7 0.0002 0.0086 559x8031 25.922967 3.58 0.0004 0.0089 3140x3015 92.833333 28.1424991 3.39 0.0007 0.011 6513x6188 95.5 0.001 0.011 8049x8046 105.333333 31.7490208 3.32 31.7490208 3.31 0.001 0.011 5306x5612 105 25.922967 3.24 0.0013 0.011 3252x3154 84 25.922967 3.21 0.0014 0.011 3032x6188 83.333333 25.922967 3.21 0.0014 0.011 8031x3609 83.333333 27.1882371 2.91 0.0037 0.027 8008x8016 79.133333 25.922967 2.85 0.0045 0.0292 5156x1566 74 27.1882371 2.78 0.0056 0.0333 6188x3252 75.666667 28.3971875 2.73 0.0065 0.0358 8005x8002 77.6 71.5 27.1882371 2.63 0.0088 0.0451 867x3252 26 Figure 9. Chronic haloperidol treatment does not appear to impact synaptic structures. Box plots with whiskers extending to the most extreme sample values of the four ultrastructural components of the sum of the number of synapses, mitochondria, perforated synapses, and double synapses per sample (indicated by black dots) measured in striatal tissue and compared between haloperidol and placebo groups. 27 Table 3. No significant differences in synaptic architecture and mitochondria amount in 30-day treated C57BL/6 mice. Values after t-test analyses of the sum of synapses, mitochondria, perforated synapses, and double synapses per sample indicate that there is no difference between haloperidol-treated and placebo-treated groups because p-values do not pass the set threshold of 0.0125 after Bonferroni correction. Observations DF t Ratio P-value (prob > |t|, α = 0.0125) 10 8 0.911597 0.3886 Sum(synapses) 10 8 -0.91259 0.3881 Sum(mitochondria) 10 8 0.452438 0.663 Sum(perforated synapses) 10 8 0.286446 0.7818 Sum(double synapses) 28