Protocol for live neuron imaging analysis of basal surface fraction and dynamic availability of the dopamine transporter using DAT-pHluorin.

Surface availability of the dopamine (DA) transporter (DAT) critically influences DA transmission. Here, we present a protocol that describes the preparation of mouse ventral midbrain neurons, the expression of a new optical sensor, DAT-pHluorin, and the utilization of this sensor to analyze the surface availability of DAT in live neurons via fluorescent microscopy. This approach allows quantitative measures of basal surface DAT fraction under genetic backgrounds of interest and live trafficking of DAT in response to psychoactive substances.

Synaptojanin1 Modifies Endolysosomal Parameters in Cultured Ventral Midbrain Neurons.

The accumulation of α-synuclein (α-syn)-enriched protein aggregates is thought to arise from dysfunction in degradation systems within the brain. Recently, missense mutations of encoding the SAC1 and 5'-phosphatase domains have been found in families with hereditary early-onset Parkinsonism. Previous studies showed that haploinsufficiency (+/-) leads to accumulation of the autophagy substrate p62 and pathologic α-syn proteins in the midbrain (MB) and striatum of aged mice.