Long-Term Effects of Traumatic Brain Injury on Anxiety-Like Behaviors in Mice: Behavioral and Neural Correlates

Abstract

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.

Neural markers of vulnerability to anxiety outcomes following traumatic brain injury

“…In the mPFC, whereas E-I signaling changes linked to cognitive and fear dysfunction following injury have been explored (Kobori and Dash, 2006, Schneider et al, 2016), those associated with anxiety behaviors are less clear. In the BLA, results regarding GABAergic and glutamatergic signaling are contradictory, with some studies reporting increases and other reporting decreases following injury (Ajao et al, 2012, Reger et al, 2012, Malkesman et al, 2013, Almeida-Suhett et al, 2014, Palmer et al, 2016, Popovitz et al, 2019, Beitchman et al, 2020). In the hippocampus, dysfunction of inhibitory synapses and reduction in glutamate precursors have been reported following injury (Witgen et al, 2005, Cole et al, 2010).…”

“…We found substantial overlap in the distributions of anxiety outcomes between TBI versus sham injured animals, supporting this hypothesis, and also demonstrating that standard approach of comparing outcomes between TBI and sham groups may be flawed. Moreover, a multidimensional behavior profile has been found to be important in characterizing affective behavioral responses following injury (Popovitz et al, 2019), which makes the application of simple criteria-based thresholds to separate behavioral subsets difficult. (Cohen et al, 2004, Elliott et al, 2010).…”

“…These studies report a wide range of (conflicting) effects: from (i) a decrease in anxiety-like behaviors following a controlled cortical impact injury (Washington et al, 2012) and weight-drop injury (Pandey et al, 2009) at 20 to 30 days post-TBI, to (ii) an increase in anxiety-like behaviors following weight-drop (Meyer et al, 2012), lateral fluid percussion (Johnstone et al, 2015), and blast overpressure (Awwad et al, 2015), up to 3 months after later, as well as (iii) no change in anxiety-like behaviors in animals exposed to controlled cortical impact injury (CCI) (Sierra-Mercado et al, 2015), repeated weight-drop (Fidan et al, 2016) and lateral fluid percussion injury (Ferreira et al, 2014), up to 3 months after injury. Indeed, even when the injury model is fixed, the specific behavioral assay used to measure anxiety, the metric used to quantify it, as well as the time points of measurement, can all play a role in the results regarding anxiety outcomes following injury (Popovitz et al, 2019). This has made it difficult to identify reliably, the nature of anxiety dysfunction following TBI in pre-clinical models, as well as to probe the underlying neural mechanisms.…”

“…Here, we set out to leverage the potential heterogeneity among individuals in anxiety responses to a CCI model of injury, in order to develop a pre-clinical approach analogous to the use of inclusion criteria in clinical TBI studies. Because of the inherent complexity of measuring anxiety in animal models, and the impact of different assays and timepoints (Popovitz et al, 2019), we reasoned that the approach in the stress literature of defining thresholds on pre-selected, unidimensional severity measures (‘cut-off behavioral criteria’ or CBC (Cohen and Zohar, 2004), would be ineffective for distinguishing vulnerability to anxiety responses following injury. In response, we developed an objective, multidimensional behavioral profiling, clustering and validation approach to identify vulnerable and resilient sub-groups of injured animals with distinct anxiety phenotypes.…”

Genetic Background Influences Acute Response to TBI in Kindling-Susceptible, Kindling-Resistant, and Outbred Rats

“…As the time course relevant for the development of TBI-related sequela such as PTE and cognitive deficits is unknown, we examined at the earliest time points for divergent responses to TBI in the inbred strains and outbred rats. In addition to the divergent responses to seizureinduction in the kindling model, these strains also demonstrate differences in behavior and learning paradigms which are known to change in brain injured animals (26)(27)(28)(29)(30). Therefore we examined acute electrophysiological alterations and BDNF expression after TBI in these unique, complementary strains as well as outbred SD rats.…”