Abstract/Summary 3D Immunohistochemistry (IHC) analysis to enhance the rigor of motoneuronal somatic reconstruction and potassium channel characterization. Clinical Relevance: Amyotrophic lateral sclerosis (ALS) has few therapies with only modest benefits despite decades of research. Barriers arise from complex, multifarious pathologies and conflicting reports in the ALS literature, often due to inconsistent methods and unaccounted for biological variables. Parent grant data implicates Kv2.1 and SK potassium ion channels in the excitability dysregulation of motoneurons (MNs) in ALS, specifically in SOD1-G93A and rNLS8 ALS mouse lines. The objective of this research is to develop novel 3D IHC analysis methods to rigorously examine somatic morphology and potassium channel structure in normal vs. diseased states to elucidate the roles of Kv2.1 and SK in ALS. Rationale: Current 2D IHC methods do not provide accurate measures of soma size or ion channel structure. Also, standard manual tracing to assess 2D area involves human bias. Notably, MN soma volume indicates vulnerability to ALS (largest cells die first). Also, the soma surface area impacts membrane capacity for ion channels. Thus, 3D somatic measurements are vital to the parent grant. Also, the multiple, complex roles of potassium channels in regulating MN excitability, and the fact that these channels are understudied in spinal MNs, makes rigorous characterization of Kv2.1 and SK critical to the parent grant’s work. Thus, our Specific Aims are: SA 1) Develop algorithms for accurate 3D reconstruction and measurement of MN somas in IHC: 2D soma measurements require subjective and tedious manual cell body tracing, which is sensitive to human error and biased by image orientation. Also, current 3D software- renderings are low-resolution and insufficient for 3D ion channel quantitation. Thus, the candidate will develop unbiased algorithms to automatically trace the 3D somatic membrane and quantitate soma volume and surface area. SA 2) Rigorously characterize somatic potassium channel structure and calibrate analysis to known channel phosphorylation states in healthy spinal MNs: Kv2.1 and SK structure each exhibit a structural range from tightly clustered to loosely dephosphorylated depending on their functional state. These graded clustering changes are not thoroughly characterized by visual inspection, as proposed in the parent grant. Thus, the candidate will develop quantitation algorithms for 3D intensity maps to characterize Kv2.1 and SK structure. SA 3) Characterize Kv2.1 and SK structure in ALS spinal MNs: While Kv2.1 and SK qualitative structural changes have been observed across healthy cell function, both channels have been understudied in ALS and never characterized in rNLS8 specifically. Thus, we will rigorously characterize Kv2.1 and SK clustering changes in rNLS8 MNs and compare/contrast these changes to those measured in SOD1 MNs. Completion of these studies will link potassium chann...