ABSTRACT Methamphetamine (meth) is an addictive psychostimulant. Our pilot studies show that chronic meth resulted in degeneration of substantia nigra pars compacta (SNc) dopamine (DA) and locus coeruleus (LC) norepinephrine (NE) but not ventral tegmental area (VTA) DA or dorsal raphe (DR) serotonin (5-HT) neurons in mice. DA, NE, and 5-HT are monoamine neurotransmitters synthesized and packaged into vesicles in axonal compartments. Meth increases cytosolic monoamines and MAO-dependent mitochondrial stress in SNc axons but not the soma. Pilot studies show that MAO inhibition prevented meth-induced SNc degeneration. These data suggest that degeneration is driven by MAO-dependent axonal mitochondrial stress. However, pilot data show meth also increased LC, VTA, and DR axonal mitochondrial stress yet chronic meth induced SNc and LC but not VTA or DR degeneration. We found L-type Ca2+ channel (LCC)-dependent mitochondrial stress in SNc but not VTA axons. We propose a `two-hit' hypothesis wherein both MAO- and LCC-dependent axonal mitochondrial stress are required for degeneration. The degeneration `trigger' is meth-induced MAO-dependent axonal mitochondrial stress and vulnerability is a consequence of axonal LCC-dependent mitochondrial stress. Since MAO- and LCC- dependent stress converge in axons, we propose that axonal loss precedes somatic loss in a `dying-back' pattern of degeneration. Pilot data indicate that SNc axonal mitochondrial stress was increased during abstinence. We hypothesize this stress is MAO-dependent and contributes to degeneration during abstinence. Lastly, meth- induced degeneration is expected to impair novel object recognition and fear conditioning which are dependent on DA and NE, respectively. Hypotheses will be tested using two-photon laser scanning microscopy in brain slices, genetically encoded biosensors, pharmacological and genetic manipulation of MAO/LCCs, and immunohistochemistry. Aim 1: Determine mechanisms of mitochondrial stress in monoaminergic axons, consequences of chronic meth on mitochondria, and whether mitochondrial stress continues during abstinence. We hypothesize that SNc, VTA, LC, and DR axons all have meth-induced MAO-dependent mitochondrial stress whereas SNc and LC but not VTA or DR axons will have LCC-induced mitochondrial stress. We also hypothesize that axonal stress will remain elevated throughout abstinence from chronic meth and will be MAO-dependent. Aim 2: Determine the roles of MAO and LCCs in chronic meth-induced degeneration and behavioral consequences. We hypothesize that pharmacologically inhibiting or genetically deleting MAO or LCCs will prevent chronic meth-induced degeneration of vulnerable (SNc and LC) neurons and deleting the Ca2+ buffering protein calbindin from resistant (VTA and DR) neurons will render them vulnerable to degeneration. We further hypothesize that degeneration will be progressive throughout abstinence and MAO- dependent. Lastly, we hypothesize that SNc and LC degen...