SUMMARY Parkinson disease is characterized by loss of dopamine neurons in the substantia nigra 1 . Similar to other major neurodegenerative disorders, no disease-modifying treatment exists. While most treatment strategies aim to prevent neuronal loss or protect vulnerable neuronal circuits, a potential alternative is to replace lost neurons to reconstruct disrupted circuits 2 . Herein we report an efficient single-step conversion of isolated mouse and human astrocytes into functional neurons by depleting the RNA binding protein PTB. Applying this approach to the mouse brain, we demonstrate progressive conversion of astrocytes into new neurons that can innervate into endogenous neural circuits. Astrocytes in different brain regions are found to convert into different neuronal subtypes. Using a chemically induced model of Parkinson’s disease, we show conversion of midbrain astrocytes into dopaminergic neurons whose axons reconstruct the nigro-striatal circuit. Significantly, re-innervation of striatum is accompanied by restoration of dopamine levels and rescue of motor deficits. Similar disease phenotype reversal is also accomplished by converting astrocytes to neurons using antisense oligonucleotides to transiently suppress PTB. These findings identify a potentially powerful and clinically feasible new approach to treating neurodegeneration by replacing lost neurons.

SUMMARY1. The calcium binding capacity (Ks) of bovine chromaffin cells preloaded with fura-2 was measured during nystatin-perforated-patch recordings.2. Subsequently, the perforated patch was ruptured to obtain a whole-cell recording situation, and the time course of KS was monitored during periods of up to one hour.

1. Simultaneous fluorescence and whole-cell current measurements using the calcium indicator dye fura-2 were made in HEK 293 cells expressing recombinant glutamate receptor (GluR) channels, and fractional Ca2+ currents (the proportion of whole-cell current carried by CP2+ 4. In cells expressing a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPAR) subunits which were unedited at the Q/R site of the putative transmembrane segment TM2 (Q-form), or in cells coexpressing unedited and edited subunits (R-form), the glutamateevoked Ca2+ inflow increased from 20 to -80 mV in an almost linear way. 5. Fractional Ca2+ currents through AMPAR channels depended on subunit composition.Pf values of Q-form homomeric channels at -60 mV and 1X8 mm [Ca2+]o were between 3-2 and 3 9%. They were slightly voltage dependent and increased with [Ca2+]o in the range 1 8-10 mm. Pf values in cells co-expressing Q-and R-form subunits were almost one order of magnitude smaller (0 54 %). 6. Relative concentrations of Q-form and R-form GluR-B subunit-specific cDNAs used for cell transfection determined the expression of functionally different heteromeric AMPARS.Pf decreased with increasing relative concentration of R-form encoding cDNAs from 3-4 to 1-4 %, demonstrating that editing of the Q/R site of GluR-B subunits decreases Ca2P inflow through heteromeric AMPARs. 7. Cells expressing the GluR-6 subunit of the kainate receptor (KAR) family were characterized by Pf values which depended on the editing in the TM1 and TM2 segments.Pf values were largest for the Q-form (1 -55-2 0 %) and lowest for R-form channels (< 0 2 %), suggesting that Q/R site editing also decreases Ca2P inflow through KAR channels. Cells coexpressing both subunit forms showed an intermediate value (0 58 %).

Developments in miniaturized microscopes have enabled visualization of brain activities and structural dynamics in animals engaging in self-determined behaviors. However, it remains a challenge to resolve activity at single dendritic spines in freely behaving animals. Here, we report the design and application of a fast high-resolution, miniaturized two-photon microscope (FHIRM-TPM) that accomplishes this goal. With a headpiece weighing 2.15 g and a hollow-core photonic crystal fiber delivering 920-nm femtosecond laser pulses, the FHIRM-TPM is capable of imaging commonly used biosensors (GFP and GCaMP6) at high spatiotemporal resolution (0.64 μm laterally and 3.35 μm axially, 40 Hz at 256 × 256 pixels for raster scanning and 10,000 Hz for free-line scanning). We demonstrate the microscope's robustness with hour-long recordings of neuronal activities at the level of spines in mice experiencing vigorous body movements.