We previously found that human heroin addicts and mice chronically exposed to morphine exhibit a significant increase in the number of detected hypocretin/orexin (Hcrt) producing neurons. However it remains unknown how this increase affects target areas of the hypocretin system involved in opioid withdrawal, including the locus coeruleus (LC) and the A1/A2 medullary regions. Using a combination of immunohistochemical, biochemical, imaging, and behavioral techniques we now show that the increase in detected hypocretin cell number translates into a significant increase in hypocretin innervation and tyrosine hydroxylase (TH) levels in the LC without affecting norepinephrine (NE) containing neuronal cell number. We show that the increase in TH is completely dependent in Hcrt innervation. The A1/A2 regions were unaffected by morphine treatment. Manipulation of the Hcrt system may affect opioid addiction and withdrawal. SIGNIFICANT STATEMENT Previously we have shown that the hypothalamic hypocretin system und...