The Journal of Neuroscience, August 8, 2007, ():
Functional Properties of Neurons Derived from In Vitro Reprogrammed Postnatal Astroglia
J. Neurosci. Berninger et al.
27: 8654
Supplemental Data
Files in this Data Supplement:
- supplemental material -
Supplemental Movie Legend
- supplemental material
-
Supplemental Figure 1. Characterisation of cell types constituting the astroglia enriched culture. a. Glial fibrillary acidic protein (GFAP) immunoreactivity demonstrates astroglial identity of the vast majority of cells in cultures prepared from P7 cortex and grown as described in Materials and Methods. b. Quantification of the number of cells expressing different cell type specific markers. c. GFAP positive cells incorporate preferentially BrdU during the first 24 h indicating that astroglial cells are highly proliferative in these cultures. d. Frequency of cells incorporating BrdU expressing GFAP, NG2 or TuJ1, normalized to the total number of cells as determined by DAPI staining. e. Cells targeted by the retrovirus co-express GFAP indicating their astroglial identity.
- supplemental material
-
Supplemental Figure 2. Example of spike adaptation and accomodation in a neuron derived from a Ngn2 transduced astroglial precursor. Left panel: fluorescence micrograph. Middle panel: Spike pattern in response to current injection. Right panel: First two action potentials at higher temporal resolution. Note the fast and the slow phase of afterhyperpolarization marked with * and **, respectively.
- supplemental material
-
Supplemental Figure 3. Neurons derived from reprogrammed astroglial precursors do not receive spontaneous synaptic input in the absence of co-cultured neurons. a. Fluorescence micrograph of a neuron derived from an astroglial precursor transduced with Pax6-IRES-GFP. Current injection elicited APs in this cell (right). Current trace in voltage clamp (below) is devoid of any spontaneous synaptic event b. Fluorescence micrograph of a neuron derived from an astroglial precursor transduced with Mash1-IRES-GFP. Current injection elicited a series of APs in this cell (right). Current trace in voltage clamp (below) shows rare recording of a spontaneous synaptic event (see enlarged trace).
- supplemental material
-
Supplemental Figure 4. Neurons derived from reprogrammed astroglial precursors fail to form synapses onto neighbouring neurons. a. Representative example of dual recording of a GFP positive neuron derived from an Ngn2 reprogrammed astroglial precursor and a co-cultured E16 cortical neuron. The GFP positive cell (marked by asterisk) was step-depolarised in current clamp until it elicited trains of firing action potentials (middle traces), yet no postsynaptic response was evoked in the cortical neuron (kept in voltage clamp, upper traces). The traces shown in red depict a single discharge of multiple APs in the astroglia-derived neuron (middle traces) and the absence of a response in the embryonic cortical neuron (upper traces). The deflection in the red trace before onset of stimulation depicts a spontaneous synaptic event in the cortical neuron. b. Pair of cortical neurons present in the same culture. Stimulating the neuron (marked with the asterisks) by depolarizing steps from -70 to +30 mV elicited a response consisting of mono- and multiple polysynaptic events (lower traces). The response was blocked by CNQX indicating the glutamatergic identity of the presynaptic neuron.
- supplemental material
-
Supplemental Figure 5. Dual recording of a neuron derived from a Pax6 reprogrammed astroglial precursor and a co-cultured neuron obtained from E16 cerebral cortex, 8 days after plating. a. Left panel shows bright field image. Middle panel shows GFP fluorescence. Right panel shows GFP fluorescence in the tip of the pipette after recording. b. Spiking properties of the two neurons. c. Only the neuron of embryonic origin received spontaneous synaptic input.
- supplemental material
-
Supplemental Movie 1
- supplemental material
-
Supplemental Movie 2
- supplemental material
-
Supplemental Movie 3
