Neurochemical assessment of identified phrenic motor neurons via flow cytometry

Abstract

Flow cytometry (FCM) utilizes high powered, laser‐based tools to perform multi‐parametric analyses on cell populations. Although FCM is routinely utilized in immunology and cancer biology fields, it is rarely used in the study of neurobiology. Here, we show a novel use of FCM to identify and isolate phrenic motor neurons, a small, but critical pool of respiratory motor neurons innervating the diaphragm. Rat phrenic motor neurons were retrogradely labeled via bilateral intrapleural injections of fluorescently‐conjugated cholera toxin fragment B (CtB‐AlexaFluor 647). After 72 hours, rats were perfused and the cervical spinal cords dissected, dissociated, fixed in a modified zinc‐based fixative, and stained with DAPI to discriminate intact cells from debris. Using FCM, a clearly defined group of intact cells were identified as CtB‐AlexaFluor 647 positive, corresponding closely with available estimates of phrenic motor neuron numbers in rats. These cells were confirmed to be neurons by co‐staining with the neuronal marker NeuN. Our results demonstrate the feasibility of using FCM to identify, characterize, and sort phrenic motor neurons. Coupling FCM with neurophysiology provides an innovative approach to the study of phrenic motor neuron biology, while lending new insights into mechanisms of spinal respiratory neuroplasticity in models of traumatic or neurodegenerative injury.