The Importance of Mouse Medial Premotor Cortex, Early Re-Training, and Fluoxetine In The Recovery From a Focal Stroke Induced Motor Deficit. Steven R. Zeiler, M.D., Ph.D., Ellen M. Gibson, B.A., Robert E. Hoesch, M.D., Ph.D., Ming Y Li, B.A., Paul F Worley, M.D., Richard J. O’Brien, M.D., Ph.D., John W. Krakauer, M.D. Functional recovery after stroke, which we define as improved success at a task mediated either through reduced impairment or through compensatory behavior, occurs and is enhanced by rehabilitative training. In rodent models, cortical reorganization, i.e. a change in motor output pre- compared to post-stroke measured by intra-cortical stimulation or as measured by behavior, seems to be involved in both reduction of impairment as well as compensatory behavior1, 2. For example, cortex adjacent to infarcted cortex (peri-infarct cortex)2 as well as more distant premotor areas3, 4 have been shown to reorganize so as to reestablish function of stroke-damaged areas. Recent data has suggested that such reorganization is likely dependent on a change in the excitation and inhibition balance of surviving cortex,5 as well as the timing of therapeutic interventions6. Therefore, we hypothesized that mouse neo-cortical reorganization after stroke is related to recovery of success at a motor task and is (1) detectable in part through changes in the inhibitory interneuron markers parvalbumin (PV), calretinin (CR), and calbindin (CB), (2) that there is critical window of time during which rehabilitative training can affect recovery, and, (3) based upon data suggesting that fluoxetine can enhance recovery of motor deficits after stroke,7 one can pharmacologically modify recovery during this window via the use of fluoxetine. We trained wild type mice to perform a skilled reaching task whereupon they reached a maximum accuracy after approximately 9 training days (Figure 1C). Photocoagulation induced contralateral forelimb area (CFA) infarction led to a significant decrement in skilled motor reaching accuracy which recovered after 6-7 training days (Figure 1C). We saw no decrement in skilled motor reaching accuracy following sham procedures (Figure 1D) or with isolated focal strokes in AGm (Figure 3B). In pilot data, we noted a reduction in inhibitory interneuron marker expression in what appeared to be medial pre-motor cortex. To define a counting area anatomically (and not based upon visually perceived differences of marker expression) we identified AGm on coronal sections and generated a 1.8X107 µm3 volume of brain tissue from the indicated conditions (Figure 2). Blinded quantification of cells expressing PV, CR, and CB showed a statistically significant decrease in ipsilesional versus contralesional AGm but only if the animals experienced a CFA stroke followed by post-stroke training (Figure 2). Since AGm is known to make cortical-spinal projections and displayed a focal reduction in inhibitory interneuron markers, we hypothesized that AGm played a role in the recovery of skilled motor reaching after CFA stroke. To test this, we trained mice to perform the skilled motor reaching task, induced a focal motor stroke in CFA, and observed their return to baseline reaching by 6 days; these mice then underwent a second focal stroke in AGm which led to a decline in skilled motor reaching accuracy only if the infarct was ipsilesional to the initial CFA stroke (Figure 3C) and not contralesional (data not shown). To test if there is a window of time during which rehabilitative training leads to return of success at a reaching task, we trained wild type mice to perform a skilled reaching task followed by CFA infarction with no training for 7 days following the infarct. On the 8th day, the mice were retrained. CFA stroke led to a significant decrement in skilled motor reaching accuracy and without the robust recovery seen if training was initiated 24 hours after the infarction (Figure 4A). However, providing once daily fluoxetine 10mg/kg IP beginning 24 hours post-stroke and during the training days lead to a significant increase in reaching accuracy even if the training was delayed for 7 days. We conclude that with training, AGm can reorganize after a focal motor stroke and serve as a new control area for prehension. Reduced inhibition may represent a marker for reorganization or it is necessary but not sufficient for reorganization. Second, we conclude that training is most effective during a window of time after stroke. Finally, we conclude that fluoxetine modifies this window in such a way to allow delayed training to remain effective. Our mouse model, with all of the attendant genetic benefits, may allow us to determine at the cellular and molecular level how behavioral training and endogenous plasticity interact to mediate recovery. References: 1. Murphy TH, Corbett D. Plasticity during stroke recovery: From synapse to behaviour. Nat Rev Neurosci. 2009;10:861-872 2. Winship IR, Murphy TH. Remapping the somatosensory cortex after stroke: Insight from imaging the synapse to network. Neuroscientist. 2009;15:507-524 3. Conner JM, Chiba AA, Tuszynski MH. The basal forebrain cholinergic system is essential for cortical plasticity and functional recovery following brain injury. Neuron. 2005;46:173-179 4. Krakauer JW, Carmichael ST, Corbett D, Wittenberg GF. Getting neurorehabilitation right: What can be learned from animal models? Neurorehabil Neural Repair. 2012 5. Carmichael ST. Brain excitability in stroke: The yin and yang of stroke progression. Arch Neurol. 2011 6. Maurer D, Hensch TK. Amblyopia: Background to the special issue on stroke recovery. Developmental psychobiology. 2012;54:224-238 7. Chollet F, DiPiero V, Wise RJ, Brooks DJ, Dolan RJ, Frackowiak RS. The functional anatomy of motor recovery after stroke in humans: A study with positron emission tomography. Ann Neurol. 1991;29:63-71