Abstract
We describe here methods for dissociating retinal ganglion cells from adult goldfish and rat without proteolytic enzymes, and show responses of ganglion cells isolated this way to step-wise voltage changes and fluctuating current injections. Taking advantage of the laminar organization of vertebrate retinas, photoreceptors and other cells were lifted away from the distal side of freshly isolated goldfish retinas, after contact with pieces of membrane filter. Likewise, cells were sliced away from the distal side of freshly isolated rat retinas, after these adhered to a membrane filter. The remaining portions of retina were incubated in an enzyme-free, low Ca2+ solution, and triturated. After aliquots of the resulting cell suspension were plated, ganglion cells could be identified by dye retrogradely transported via the optic nerve. These cells showed no obvious morphological degeneration for several days of culture. Perforated-patch whole-cell recordings showed that the goldfish ganglion cells spike tonically in response to depolarizing constant current injections, that these spikes are temporally precise in response to fluctuating current injections, and that the largest voltage-gated Na+ currents of these cells were larger than those of ganglion cells isolated with a neutral protease.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 25-35 |
| Number of pages | 11 |
| Journal | Journal of Neuroscience Methods |
| Volume | 137 |
| Issue number | 1 |
| DOIs | |
| State | Published - Aug 15 2004 |
Fingerprint
Keywords
- Enzyme-free dissociation
- Goldfish
- Na current
- Rat
- Retinal ganglion cell
- Spikes
ASJC Scopus subject areas
- Neuroscience(all)
Cite this
Dissociation of retinal ganglion cells without enzymes. / Hayashida, Yuki; Partida, Gloria J.; Ishida, Andrew.
In: Journal of Neuroscience Methods, Vol. 137, No. 1, 15.08.2004, p. 25-35.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Dissociation of retinal ganglion cells without enzymes
AU - Hayashida, Yuki
AU - Partida, Gloria J.
AU - Ishida, Andrew
PY - 2004/8/15
Y1 - 2004/8/15
N2 - We describe here methods for dissociating retinal ganglion cells from adult goldfish and rat without proteolytic enzymes, and show responses of ganglion cells isolated this way to step-wise voltage changes and fluctuating current injections. Taking advantage of the laminar organization of vertebrate retinas, photoreceptors and other cells were lifted away from the distal side of freshly isolated goldfish retinas, after contact with pieces of membrane filter. Likewise, cells were sliced away from the distal side of freshly isolated rat retinas, after these adhered to a membrane filter. The remaining portions of retina were incubated in an enzyme-free, low Ca2+ solution, and triturated. After aliquots of the resulting cell suspension were plated, ganglion cells could be identified by dye retrogradely transported via the optic nerve. These cells showed no obvious morphological degeneration for several days of culture. Perforated-patch whole-cell recordings showed that the goldfish ganglion cells spike tonically in response to depolarizing constant current injections, that these spikes are temporally precise in response to fluctuating current injections, and that the largest voltage-gated Na+ currents of these cells were larger than those of ganglion cells isolated with a neutral protease.
AB - We describe here methods for dissociating retinal ganglion cells from adult goldfish and rat without proteolytic enzymes, and show responses of ganglion cells isolated this way to step-wise voltage changes and fluctuating current injections. Taking advantage of the laminar organization of vertebrate retinas, photoreceptors and other cells were lifted away from the distal side of freshly isolated goldfish retinas, after contact with pieces of membrane filter. Likewise, cells were sliced away from the distal side of freshly isolated rat retinas, after these adhered to a membrane filter. The remaining portions of retina were incubated in an enzyme-free, low Ca2+ solution, and triturated. After aliquots of the resulting cell suspension were plated, ganglion cells could be identified by dye retrogradely transported via the optic nerve. These cells showed no obvious morphological degeneration for several days of culture. Perforated-patch whole-cell recordings showed that the goldfish ganglion cells spike tonically in response to depolarizing constant current injections, that these spikes are temporally precise in response to fluctuating current injections, and that the largest voltage-gated Na+ currents of these cells were larger than those of ganglion cells isolated with a neutral protease.
KW - Enzyme-free dissociation
KW - Goldfish
KW - Na current
KW - Rat
KW - Retinal ganglion cell
KW - Spikes
UR - http://www.scopus.com/inward/record.url?scp=2942685418&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=2942685418&partnerID=8YFLogxK
U2 - 10.1016/j.jneumeth.2004.02.008
DO - 10.1016/j.jneumeth.2004.02.008
M3 - Article
C2 - 15196824
AN - SCOPUS:2942685418
VL - 137
SP - 25
EP - 35
JO - Journal of Neuroscience Methods
JF - Journal of Neuroscience Methods
SN - 0165-0270
IS - 1
ER -