The purpose of this report was to initiate investigation of murine VCMs with the eventual goal of improving understanding of the genomics of human TD. If VCMs are a reasonable analogue of TD, then it might be possible to accelerate discovery by using MTH (mouse-then-human) designs whereby mouse genetic mapping resources are used to screen the genomic search space to derive high-probability targets whose orthologs can be studied in human samples. In this way, the multiple testing burden is paid in a relatively inexpensive and experimentally tractable system and precious human samples are used only for the most crucial targets.
To achieve this end, we pursued a number of systematic goals. The initial consideration was the ability to deliver human-like concentrations of haloperidol in a sustained, consistent, and reliable manner as haloperidol exposure is a critical risk factor for TD 14
. A literature review 27
and pilot experiments (Table S2
) demonstrated that implantable pellets yielded lower coefficients of variation for haloperidol levels in plasma (20.6%) and brain (11.6%) than other methods of drug delivery (>34%). Additional dose-ranging experiments showed that dosing mice with implantable pellets at 3.0 mg/kg/day (60 day sustained release) reliably yielded human-like plasma concentrations. The data that form the backbone of this report ( and Table S3
) indicate that implantable pellets yield low coefficients of variation in plasma haloperidol concentrations (median 0.19) and nearly always achieved human-like plasma concentrations (>10 nM in 98.1% of mice). Note that two months of haloperidol exposure is an appreciable fraction of the ~1.5–2 year lifespan of a laboratory mouse and thus constitutes chronic exposure.
Second, we demonstrated that haloperidol concentrations are highly variable between inbred strains. The observed variability was not influenced by potential confounders such as the dose implanted or body mass. Moreover, within- was considerably less than between-strain variation leading to heritability estimates for haloperidol concentrations of ~0.7 during the times when the drug was being actively released. Future work will attempt to identify the genetic determinants of haloperidol concentrations in a genome-wide and unbiased manner.
Third, we developed a battery of tests to assess the observable effects of haloperidol. Following careful rater training and calibration, assessment of VCMs in randomly assigned tapes by raters blinded to experimental date was reproducible. An immediate observation was that exposure to haloperidol yielded marked average changes across multiple domains. Four measures of activity in the open field, one measure of EPS, and four measures of orofacial movement all exhibited, on average, marked changes following haloperidol exposure (). These changes did not return to baseline values by the end of the study (120 days). Crucially, these measures were independent of haloperidol plasma level and other covariates. Strain was again the major predictor of phenotypic variation.
Fourth, we observed that the three domains we assessed – orofacial movements, activity in the open field, and EPS – were not discrete constructs but rather loaded onto two factors. Analysis of human data from a large randomized clinical trial (CATIE) 28
,show a similar pattern: subjects with TD by consensus criteria 29
had greater EPS and akathisia (P
values < 0.0001) 30
. To investigate this with an approach closer to that in , we analyzed the CATIE baseline data for total Abnormal Involuntary Movement scale (AIMS) score 31
, EPS measured by the Abbreviated Simpson–Angus Side Effect Scale 32
, and the Barnes Akathisia Scale 33
. Similar to the analyses in , two principal components accounted for 78% of the variance (see Table S5
), and the first component had similar loadings: AIMS total (0.61), EPS (0.56), and akathisia (0.56). Therefore, the human and mouse data converge and suggest that each of these scales is an imprecise but informative assessment of a more fundamental latent construct.
Fifth, we estimated heritability for haloperidol-induced effects on VCMs, activity in the open field, and EPS. provides the results at successive levels of processing. Given that our design was inherently longitudinal, analysis of the response trajectories of individual mice is superior to individual time points and, given the inter-relatedness of the phenotype domains, analysis of factors from factor analysis of response trajectories is superior to trajectory analysis. Thus, we believe heritability estimates in provide the best representation of our study and of the phenotypes themselves. The heritability of factor 1 was 0.86 and 0.96 for factor 2. To our knowledge, these are the first estimates of heritability for VCMs. Variation in behavioral responses to chronic haloperidol exposure is predominantly determined by genetic influences. These data strongly support efforts to identify the specific genetic basis of variation in these traits and augment the case for the study of genetic influences on human TD.
A major assumption of the mouse-then-human approach is that murine VCMs are an adequate model for the human adverse drug reaction TD. Three criteria are generally applied to establish the validity of a mouse model of a human phenotype 34
. The pun is unavoidable: VCMs have face validity for human TD. Key behavioral markers that define the predominant type of TD (repetitive and purposeless movements of the mouth and jaw) are highly similar to VCMs (purposeless jaw movements in the vertical plane). A board-certified neurologist (KW) with considerable experience in the diagnosis and treatment of TD reviewed tapes of mice with VCMs and also noted the face validity of VCMs for TD. The face validity of VCMs is augmented by multiple additional lines of argument (summarized in Table S1
), and their persistence over large fractions of the mouse lifespan is compelling (Figure S1
Evaluation of the remaining two criteria for VCMs as a valid model of human TD are for future studies. Construct validity requires a conceptual analogy to the cause of human TD. This criterion is partially fulfilled given the crucial role of typical antipsychotics in the etiology of both VCMs and TD. However, fully evaluating this criterion is a fundamental goal of the MTH design – if susceptibility loci can be identified in mouse, do the orthologous regions in human contain susceptibility loci for TD? The final criterion – predictive validity – assesses (in part) specificity of treatment response. Although this could be studied in the absence of precise pathophysiology, it is far more interesting to conduct such studies with the greater insight afforded by a clear understanding of how genes and haloperidol exposure interact to produce VCMs.