Traumatic brain injury (TBI) has been frequently linked to affective disorders such as anxiety and depression. However, much remains to be understood about the underlying molecular and signaling mechanisms that mediate affective dysfunctions following injury. A lack of consensus in animal studies regarding what the affective sequelae of TBI are has been a major hurdle that has slowed progress, with studies reporting the full range of effects: increase, decrease, and no change in anxiety following injury. Here, we addressed this issue directly by investigating long-term anxiety outcomes in mice following a moderate to severe controlled cortical impact (CCI) injury using a battery of standard behavioral tests—the open field (OF), elevated zero maze (EZM), and elevated plus maze (EPM). Mice were tested on weeks 1, 3, 5 and 7 post-injury. Our results show that the effect of injury is time- and task-dependent. Early on—up to 3 weeks post-injury, there is an increase in anxiety-like behaviors in the elevated plus and zero mazes. However, after 5 weeks post-injury, anxiety-like behavior decreases, as measured in the OF and EZM. Immunostaining in the basolateral amygdala (BLA) for GAD, a marker for GABA, at the end of the behavioral testing showed the late decrease in anxiety behavior was correlated with upregulation of inhibition. The approach adopted in this study reveals a complex trajectory of affective outcomes following injury, and highlights the importance of comparing outcomes in different assays and time-points, to ensure that the affective consequences of injury are adequately assessed.

Anxiety outcomes following traumatic brain injury (TBI) are complex, and the underlying neural mechanisms are poorly understood. Here, we developed a multidimensional behavioral profiling approach to investigate anxiety-like outcomes in mice that takes into account individual variability.Departing from the tradition of comparing outcomes in TBI versus sham groups, we identified animals within the TBI group that are vulnerable to anxiety dysfunction by applying dimensionality reduction, clustering and post-hoc validation to behavioral data obtained from multiple assays for anxiety at several post-injury timepoints. These vulnerable animals expressed distinct molecular profiles in the corticolimbic network, with downregulation in GABA and glutamate, and upregulation in NPY markers. Indeed, among vulnerable animals, not resilient or sham controls, severity of anxiety outcomes correlated strongly with expression of molecular markers. Our results establish a foundational approach, with predictive power, for reliably identifying maladaptive anxiety outcomes following TBI and uncovering neural signatures of vulnerability to anxiety..

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11Anxiety outcomes following traumatic brain injury (TBI) are complex, and the underlying neural 12 mechanisms are poorly understood. Here, we developed a multidimensional behavioral profiling 13 approach to investigate anxiety-like outcomes in mice that takes into account individual variability. 14 Departing from the tradition of comparing outcomes in TBI versus sham groups, we identified animals 15 within the TBI group that are vulnerable to anxiety dysfunction by applying dimensionality reduction, 16 clustering and post-hoc validation to behavioral data obtained from multiple assays for anxiety at 17 several post-injury timepoints. These vulnerable animals expressed distinct molecular profiles in the 18 corticolimbic network, with downregulation in GABA and glutamate, and upregulation in NPY 19 markers. Indeed, among vulnerable animals, not resilient or sham controls, severity of anxiety 20 outcomes correlated strongly with expression of molecular markers. Our results establish a 21 foundational approach, with predictive power, for reliably identifying maladaptive anxiety outcomes 22 following TBI and uncovering neural signatures of vulnerability to anxiety.23 24 101 sections (Methods).102 103 Figure 1. Multidimensional behavioral clustering with validation (MBCV) for identifying animals 104 vulnerable to anxiety after TBI 105 (A) Experimental timeline and protocol for measuring anxiety-like behaviors before and after injury. EZM 106 -elevated zero maze; OFT -open field test; EPM -elevated zero maze; all standard assays for 107 5 measuring anxiety-like behaviors in rodents. CCI -Controlled cortical impact injury model. IHC -108 immunohistochemistry. 109 (B) Anxiety behavioral 'profile' of a mouse: A 11-dimensional vector which combines behavioral data on 110 the EZM, OFT and EPM across time points. Each element in the vector is the proportion of time spent by 111 the mouse in the anxiogenic zones of one of the assays (ex. EZM) at one of the post-injury time points 112 (ex. Week 7), normalized to the animal's pre-injury baseline value in that assay (Methods). 113(C) Multidimensional behavioral clustering with validation approach: Behavioral profiles of mice exposed 114 TBI are first subject to principal components analysis (PCA), and then to k-means clustering (with k=2) to 115 identify two clusters in principal component (PC) space. The final validation step determines (i) whether 116 animals in the two clusters exhibit distinct anxiety phenotypes after TBI, and if so, (ii) whether (and 117 which) cluster consists of animals vulnerable to anxiety outcomes after TBI. This involves plotting and 118 comparing between the clusters, anxiety metrics from each assay. 120We asked if, based on their multidimensional anxiety behavioral profiles, mice that underwent TBI could 121 be separated into two distinct groups such that one exhibited severe affective behavioral consequences 122 of injury, and the other was largely resistant to effects of injury. To address this question, we developed 123 a data-driven approach. W...