Biomedical Engineering Reference
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type, since modulations of spike frequency are used to code information about
stimulus contrast. In addition, several studies now indicate that spike timing
between multiple retinal neurons is often very precise [14, 15], suggesting that
a prosthetic device may need to generate spikes with high temporal precision.
Here, we have developed a paradigm to generate precise temporal patterns of
spiking activity in retinal ganglion cells.
Methods
Animal Preparation
The care and use of animals followed all federal and institutional guidelines
and all protocols were approved by the Animal Care and Use Committee,
UC Berkeley. New Zealand white rabbits,
25 kg, were anesthetized with
injections of xylazine/ketamine and subsequently euthanized with an intracardial
injection of sodium pentobarbital. Immediately after death, the right eye was
removed. All subsequent procedures were performed under dim red illumination.
The front of the eye was removed, the vitreous eliminated and the eyecup
dissected so that the visual streak and regions ventral were left intact - all other
areas were discarded. Three rectangular pieces of eyecup each approximately
5
∼
7mm were extracted and stored in oxygenated Ames medium (see below)
prior to use. Storage times ranged from 15 minutes to 8 hours. Just prior to use,
the retina was dissected from the eyecup and mounted, photoreceptor side down,
to a 10mm square piece of Millipore paper which was mounted with vacuum
grease to the recording chamber (
×
10ml volume). The Millipore paper had a
4mm square hole in its centre which allowed light from below to be projected
on to the photoreceptors. Retinas were superfused continuously at 7-10ml/min
with Ames medium (Sigma; pH 7.4, 36
C), equilibrated with 95% O
2
and 5%
CO
2
. Kanamycin sulphate antimicrobial was added to the Ames medium.
∼
Electrophysiology
Patch pipettes were used to make small holes in the inner limiting membrane and
ganglion cells were targeted under visual control. Spiking was recorded with a
cell-attached patch electrode 5-6M, filled with superfusate. Light responses
were used to confirm the viability of the recording setup as well as the general
health of the cell.
Pharmacological agents were applied locally by micro-injection through a
glass pipette using a PicoPump (World Precision Instruments, PV830). This
method generated a quick wash in and out of the drug and our results
were consistent with bath-applied methods. Spiking was blocked with 25M
Tetrodotoxin (TTX, Sigma). For the synaptic blocker experiments we used a
combination of 5mM Curare, 1mM CNQX and 10mM AP-7 (Sigma). Data was
analyzed in Matlab (MathWorks).
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