The dynamic balance between neuronal volume and chloride handling in network excitability after traumatic brain injury

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Project Summary/Abstract Cerebral edema is the most significant cause of morbidity and mortality following traumatic brain injury (TBI). Though a major target for intervention, current treatments have limited success, perhaps due to the incomplete understanding of edema mechanisms. The task of this proposal is to dissect potential mechanisms underlying TBI-associated neuronal edema, with a particular focus on how edema affects neuronal and network excitability after mild and severe injury. We recently found that neuronal swelling is associated with reduced expression of the cation chloride transporter KCC2 48 hours after controlled cortical impact (CCI) TBI. To our initial surprise, we discovered that neuronal swelling coincided with reduced cellular and network excitability 48 hours post- CCI. And furthermore, when we treated neuronal swelling we observed increased excitability - suggesting that neuronal volume and excitability changes are intimately linked. These results were unexpected, as we predicted that lower levels of KCC2 expression would increase intraneuronal chloride and reduce the efficacy of inhibition – an effect that would increase excitability. However, the excitability of neurons is also affected by their shape: an edematous neuron is less excitable, which may overpower effects of KCC2 on chloride homeostasis. Moreover, despite continued reductions in KCC2 expression, neuronal volume is normalized by one-week post-CCI. This implies that mechanisms beyond KCC2 expression are involved in neuronal volume regulation. This proposal focuses on mechanisms of neuronal edema and its resolution after TBI. In addition, these studies will examine the contributions of neuronal and astrocytic mechanisms on excitability following injury and during recovery. We will test our hypotheses using genetic manipulations, simultaneous in vivo whole cell recording and two- photon microscopy, and histological techniques. In Aim 1, we examine the mechanisms of neuronal edema at 48 hours and at 1 week after CCI and mild TBI, time points when edema is maximal and resolved, respectively. We hypothesize that extracellular impermeant anions are the missing element in the edema response. In Aims 2 & 3, we examine how neuronal volume (and associated chloride shifts) affect excitability at 48 hours and one week after TBI. In Aim 2, we examine to what extent: 1. GABAergic activity is rendered excitatory after TBI, and 2. neuronal volume impacts mechanisms of chloride and water movement across neuronal membranes to influence inhibition. In Aim 3, we assess the effects of neurons (i.e., neuronal volume and chloride transport) and astrocytes (potassium and glutamate clearance) on excitability. In particular, we will examine susceptibility to seizures and spreading depolarizations, which are two key consequences of the hyperexcitable network after TBI. We hypothesize that failure to reuptake potassium and glutamate by astrocytes may contribute - in conjunction with neuronal effects - to enhanced excitability. Our findings will result in a mechanistic understanding of neuronal edema and excitability, including potential drug targets, which in turn could prompt a reassessment of how and why we treat edema clinically.