Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive loss of motor neurons in both the motor cortex and the spinal cord. Currently, there is no curative therapy. The objective of this project is to characterize the mechanisms of ALS pathogenesis, with the long-term goal of identifying new therapeutic targets. Glutamate-induced excitotoxicity of motor neurons is considered a primary mechanism of ALS pathogenesis. While most previous studies have focused on the lack of glutamate transporter as the primary mediator of excitotoxicity, little attention has been paid to a potential alternative cause of excitotoxicity: dysfunctional inhibitor mechanisms. The cortical hyperexcitability hypothesis states that degeneration of cortical inhibitory interneurons (CIIN) causes excessive excitability of cortical motor neurons (CMN) and their ultimate degeneration. Despite the existence of this hypothesis for decades, it remains unclear whether CIIN indeed undergo degeneration in ALS, whether loss of CIIN precedes loss of CMN, and whether loss of CIIN results in CMN death. Using an innovative two-photon microscopy system that we designed for in vivo imaging of neuronal morphology in mice, we aim to test the cortical hyperexcitability hypothesis using two mouse models of ALS. Our preliminary studies in ALS mice demonstrated significant loss of dendritic branches in CIIN and marked dendritic blebbing in CMN in the motor cortex, both occurring prior to disease onset. These findings suggest that both CIIN and CMN undergo degeneration in ALS, consistent with the hyperexcitability hypothesis. We propose to test this hypothesis using in vivo microscopy imaging and in vivo functional tests to elucidate the morphological changes of CIIN and the functional consequences of their degeneration in ALS mice. We will: Aim 1) Determine if CIIN degeneration is an initiating factor in ALS pathogenesis, through in vivo imaging in ALS mouse models; and Aim 2) Determine if ablation of CIIN in the motor cortex causes CMN death and accelerates ALS disease progression. Successful completion of this project will establish a foundation for understanding the role of CIIN in ALS pathogenesis, paving the way for future research targeting CIIN as a novel therapeutic for ALS.