Human In Vitro Retina, number 2

Summary

1. On November 9, 1999 a series of in vitro measurements were made on a sample of living human retina, taken from the eye of a patient with cancer of the eye socket. The patient's eye was removed as part of the cancer treatment.
2. Excitation thresholds were measured for several cells. Monopolar thresholds ranged from roughly 0.1-0.4 microamps for a 400 microsecond per phasic anodic first balanced biphasic pulse pair with 400 microseconds intraphase delay, consistent with previous experiments in rabbit.
3. Partial maps were made of 2 cells (no complete maps from which a reliable estimate of the fiber location could be made).
4. A few bipolar threshold measurements were made on 1 cell. Thresholds were somewhat higher for stimulation across the fiber than for stimulation along the fiber, as in rabbits.
5. Exposure to 5min bright illumination did not cause any change in threshold.
6. Addition of 200uM cadmium largely eliminated the spontaneous activity, but caused no change in threshold. The retina did not respond at all to light pre-cadmium, probably due to bleaching during the dissection which was performed under very bright illumination. Since there was no light response there was no independent means of judging cadmium efficacy, but the abrupt disappearance of spontaneous activity clearly indicates that the cadmium did something.
7. I did not find any single unit responses upon switching the positions of stimulating and recording electrodes. Pre-cadmium, did observe highly variable "late" activity for the highest currents tested (2-2.4uA) in the 3-20ms interval following the stimulus (Quantifying this activity may be challenging). Post-cadmium, found late activity only in the 3-8ms interval under the same conditions.

Timeline

• 3:25pm:Eye is removed from the body at the Mass. Eye and Ear Infirmary.
• 3:45pm:Eye arrives at MIT. Dissection is performed under bright illumination.
• 4:35pm:A patch of retina, taken from the same area where in vivo stimulation took place, is mounted on the array and recording commences. The unused portions of retina are stored in Ames' medium and placed in a cold water bath.
• 4:45-4:55pm:Spontaneous activity is evident. Recorded spikes resemble those seen in rabbit, though are a bit small (about 50microvolts) in amplitude.
• 5-6pm:Trying to electrically stimulate the retina without success. A possible cause is that the retina patch is not aligned correctly on the array. Ideally the axons should run vertically. It is possible that we lost track of the axon direction during disssection.
• 6-6:30pm:The retina patch is removed from the array and set aside in a petri dish with Ames' medium.
• 6:30pm:A new patch of retina, from which the axon direction could be more readily deduced but from an area that was not used for in vivo stimulation, is mounted on the array. Vigorous spontaneous activity is observered immediately with large spike amplitudes.(hundreds of microvolts)
• 6:55pm-12:15am:Thresholds for electric stimulation are measured.
• 12:15am-1:30am:Retina samples are placed in paraformaldehyde, with the first patch (from the region where in vivo stimulation was applied) in a separate container. Cleanup.

Monopolar Thresholds

Partial monopolar threshold maps were made for two cells as shown in the tables below. Thresholds are listed in microamperes. Note that two values are listed in boxes corresponding to stimulating electrode positions where a second threshold measurement was made as a control. -- = threshold was not determined.

Recording Electrode: A2	XX -- -- -- -- XX .21 .21 >.40 -- -- -- -- .19 .27 .30 .32 -- -- -- .29 .22 -- -- -- -- .28 .19 -- -- -- -- XX .21 -- -- -- XX
Recording Electrode: F5	XX -- -- -- -- XX -- -- -- -- -- -- -- .19 .29 -- -- -- -- .29 .18 -- -- -- -- -- .16 -- -- -- XX -- .18 -- -- XX

A strength-duration curve was measured for the cell picked up by recording electrode A2. Stimuli were symmetric, charge-balanced, anodic-first stimuli with phase durations listed and an intra-phase delay of 400 microseconds.

The effects of bright light and cadmium on threshold were measured for the cell picked up by recording electrode F5. In all these measurements the stimulus was monopolar, applied between stimulating electrode H1 (see below) and a distant return.

• Threshold prior to 5 minutes illumination with the room lights on (they were normally dim) and a flashlight pointed directly at the preparation: .156 microamps
• Threshold after 5 minutes illumination: .154 microamps

• Threshold prior to application of 200 micromolar cadmium chloride (CdCl₂): .154 microamps
• Threshold after 10 minutes in cadmium, during which spontaneous activity largely disappeared: .156 microamps

Bipolar Thresholds

Here are the thresholds, measured for the cell picked up by recording electrode A2 (see partial monopolar threshold map above):