Identification of Opioid Receptors in Mixed Cortical Cultures

Introduction: The six layers of the rat cerebral cortex consists of around twenty-one million cells which can be sub-divided into distinct neuronal and glial populations (e.g. GABAnergic, Glutaminergic, Astrocytic). These neuronal populations are differentially targeted by both licit and illicit drugs to alter cellular states, excitability, and/or behavioral outcome. Opioids alter cortical excitability through inhibition of GABAnergic inhibitory interneurons leading to disinhibition of glutaminergic neurons and downstream excitation of dopaminergic neurons. Resulting in altered reward evaluation and opioid induce analgesia. Objective: To use Immunocytochemistry - Immunofluorescence to identify the key cellular targets of opioids in the cortex. Methods: Cortical neurons were isolated from (P0) rat pups, cultured on glass coverslips and allowed to form mature synapses over 12 days. On days 13-20 cultures were Immuno-labeled with neuronal (Anti-NeuN), Astrocytic (Anti-GFAP), GABAnergic (Anti-GAD67) and Mu1 Opioid receptor (Anti-OPRM1) conjugated antibodies and imaged at 405nm, 488nm, 550nm, and 633nm respectively on an inverted confocal microscope. Results: Opioid receptors were found to be co-localized with GAD67 and NeuN labeling with little to no detectable expression on astrocytes or other neuronal subpopulations. Conclusion: These data support previous evidence that opioids selectively act on specific subpopulations of cortical neurons that suppress and limit cortical excitability