Four male rhesus monkeys (Macaca mulatta) were used in this study. Prior to enucleation, animals were deeply and terminally anesthetized by other experimenters, in accordance with institutional guidelines for the care and use of animals. Immediately after enucleation, the anterior portion of the eye and vitreous were removed in room light and the eye cup was placed in a bicarbonate-buffered Ames' solution (Sigma, St. Louis, MO). The tissue was stored in darkness at 32–34°C (pH 7.4) before dissection.
Under infrared illumination, pieces of peripheral retina 1–2 mm in diameter (eccentricity 8-11 mm) were isolated from the retinal pigment epithelium and placed flat against a planar array of 61 microelectrodes, with the ganglion cell layer facing the array. A transparent membrane was positioned over the tissue to exert gentle pressure on the preparation. The assembly was then mounted on a circuit board attached to an inverted microscope and continuously superfused with Ames' solution bubbled with 95% oxygen and 5% carbon dioxide at a flow rate of 2-4 ml/min (chamber volume 0.4 ml) and maintained at 30-33°C.
The array has been described in detail elsewhere (Litke, 1998
; Litke et al., 2003
; Sekirnjak et al., 2006
). Briefly, it consisted of a hexagonal arrangement of 61 extracellular electrodes, used both to record action potentials from ganglion cells and to inject current into the tissue. Each electrode was formed by microwells which were electroplated with platinum prior to an experiment. Electrode diameter varied between 9 and 15 μm, with a fixed inter-electrode spacing of 60 μm. The planar electrode area (πr2
) was used to calculate charge densities. All stimulations were performed using a monopolar configuration (current flow from electrode to distant ground wire).
Electrical stimulation and recording
The stimulation pulse consisted of a cathodic (negative) current pulse of amplitude A and duration d, followed immediately by an anodic (positive) pulse of amplitude A/2 and duration 2d. All pulses were calibrated to produce stimuli with zero net charge to minimize electrode corrosion and tissue injury. Pulse durations in this study refer to the duration d of the cathodic phase and current values refer to the cathodic phase amplitude A. In most cases, pulse duration was 0.05 ms, and in several cases 0.1 ms. Stimulation frequency was typically 5-10 Hz. All electrical pulses were delivered in darkness.
For each cell, the electrode that recorded the largest spikes was designated as the “primary electrode”. Most stimulation pulses were delivered through a neighboring electrode on the array, 60 μm distant to the primary electrode. This approach significantly reduced the stimulus artifact and avoided amplifier saturation (Sekirnjak et al., 2006
). In a few cells, stimulation at the primary electrode was attempted. This was limited to cases in which exceptionally large spikes could be detected on the stimulating electrode during the artifact, or in which a clearly isolated signal from the same cell could be recorded at a neighboring electrode.
Selection of the stimulation site was aided by a map of spike amplitudes surrounding each primary electrode. Since large signals presumably indicate closer proximity to the soma, stimulation was usually attempted using an adjacent electrode with a large spike amplitude.
In the four retinas studied, a total of 123 ON cells and 75 OFF cells were identified. Several cells of the ON and OFF parasol type were targeted for electrical stimulation in each retina, with a success rate of 46 ± 7 %. A cell was abandoned when stimulation attempts at several neighboring electrodes failed to reliably yield an unambiguous evoked spike. In most cases, unsuccessful stimulation could be attributed to a large stimulus artifact which precluded the identification of the evoked response in the sub-millisecond range. Cells with small spike amplitudes were particularly difficult to detect in the nearly saturated amplifier signal. Furthermore, cells located near the edge of the array often lacked a neighboring electrode with a large spike amplitude which could have served as a low-threshold stimulation site. Stimulation success rate improved with practice from 29% in the first primate retina to 57% in the final retina.
Stimulation was typically commenced by using the lowest available current setting and was then increased systematically if no response was seen. The increase in stimulus amplitude was halted when amplifier saturation and the shape of the stimulus artifact prevented the unambiguous detection of evoked responses, typically above 0.07 – 0.15 mC/cm2. Threshold was defined as the lowest current which produced a spike on 50% of stimulus pulses while stimulating at 5-10 Hz. The exact threshold value was interpolated from several pulse strengths near threshold using the pooled data shown in . Assuming random fluctuations of the stimulation threshold, the threshold data in were fit with a cumulative Gaussian (error function):
Spike responses to electrical stimulation
The error function G(x) ranges from -1 to +1; the free parameter a represents the half-maximum response rate, b is the stimulus strength scaling, c is the offset of the midpoint on the response rate axis, and d is the offset of the midpoint on the stimulus strength axis.
Spontaneous spikes were readily distinguished from evoked spikes since they bore no temporal relationship to the stimulus pulse, while evoked spikes were locked to the stimulus onset.
All sub-millisecond spike responses were partially obscured by the stimulus artifact. To remove the artifact, several hundred pulses were applied around spike threshold. About half of the pulses evoked spikes while the remainder did not. Successes and failures were averaged and subtracted to cleanly reveal the evoked spike (). This subtraction method was also used to analyze responses below and above threshold as long as a few traces without evoked spikes were available. A detailed description of this technique can be found elsewhere (Sekirnjak et al., 2006
Latency was defined as the time between the onset of a 50 μs pulse and the first unambiguous downward deflection of the voltage signal indicating the evoked spike (). In cases where artifact subtraction yielded distorted or truncated spikes (indicating amplifier saturation), signals from 4-8 more distant electrodes (60-180 μm away) were used to align the spike waveforms and accurately determine spike latency at the primary electrode.
Precision of sub-millisecond latency responses to electrical stimulation
To quantify spike precision, histograms of spike latencies (number of spikes per histogram: 199 ± 91) were fitted with a function representing the impulse response of a sequence of low-pass filters, specifically:
The free parameters t0 and α respectively represent the latency and amplitude of the response, τ represents the time constant of the individual filters, and n represents the number of filters. The full width at half maximum of the best fit curve was measured and reported.
Receptive field analysis
An optically reduced stimulus from a cathode ray tube computer display refreshing at 120 Hz was focused on the photoreceptor outer segments. The photopic intensity was controlled by neutral density filters in the light path. Spatiotemporal receptive fields were measured using a dynamic checkerboard (white noise) stimulus in which the intensity of each display phosphor was selected randomly and independently over space and time from a binary distribution. The pixel size was selected to accurately capture the spatial structure of macaque parasol cell receptive fields (Chichilnisky, 2001
The voltage signal on each electrode during the white noise presentation was digitized at 20 kHz and stored for off-line analysis. Details of the recording and spike sorting methods are given elsewhere (Litke et al., 2003
; Litke et al., 2004
). To describe how the cell integrates visual inputs over space and time, the spike-triggered average (STA) stimulus was computed for each ganglion cell () (Chichilnisky, 2001
). An elliptical two-dimensional Gaussian function was fit to the spatial profile. Receptive field diameter was defined as the diameter of a circle with the same area as the 1 standard deviation boundary of the Gaussian center profile (Chichilnisky and Kalmar, 2002
). White noise data were collected continuously during several 20-30 minute data runs before and after electrical stimulation attempts.
White noise visual stimulation
Receptive field location did not always match the location of the array electrode recording the largest spikes. The variable pressure exerted by the transparent membrane likely caused horizontal displacements of the photoreceptor layer; this was typically observed as an expansion of the entire visual field. To compare results across retinas, a linear scaling factor (1.03 – 1.41) was applied to bring the centers of parasol cell receptive fields in register with the underlying electrodes on which they were recorded. All receptive field diameters reported here have been adjusted in this fashion.
Care was taken to match electrically stimulated cells with cells identified from visual stimulation. Average waveforms of electrically and visually evoked spikes were compared on the primary electrode as well as on several neighboring electrodes to verify cell identity.
Data from visual stimulation runs were also used for the calculation of electrical images (Litke et al., 2004
). The electrical image was calculated separately for each recorded neuron and provides an illustration of the spatial extent of electrical signals produced by the cell. An automated spike sorting and neuron identification procedure (Litke et al., 2004
) was used to detect lower amplitude signals on nearby electrodes arising from the same cell as recorded on the primary electrode. To calculate the image, the average spike amplitude on each electrode on the array was determined and normalized to the amplitude of the electrode recording the largest spike (by definition, the primary electrode). The shape of each voltage signal allowed for the distinction of axonal (triphasic signals), somatic (biphasic signals with large negative-first deflections), dendritic (smaller signals with positive-first deflections), as well as somatodendritic spikes (for waveform examples, see Litke et al., 2004
). Electrodes with axonal spikes along the expected axon path (see below) were excluded, leaving only somatic and dendritic spikes. The electrical image was generated by interpolating (non-linear Delaunay triangulation) between electrodes and producing an iso-amplitude plot, which typically showed the primary electrode in the center (). An elliptical two-dimensional Gaussian function was fit to this plot and a boundary at 1 SD was drawn. Electrical image diameter was defined as the diameter of a circle with the same area as this boundary. Cells located at the edge of the array (with primary electrodes on the array perimeter) provided insufficient amplitude data to accurately determine electrical image size and were excluded from the electrical image analysis.
Distance from the soma
The distance between the stimulating electrode and the soma was measured for each cell from the same set of visual stimulation data as above. The approximate soma position was triangulated by calculating a center-of-mass location from the spike amplitudes on each electrode: after excluding electrodes with spike amplitudes of less than 10% of the primary electrode signal, the center-of-mass coordinates were calculated as:
with Ai being the spike amplitude on electrode i and xi and yi its coordinates on the array. The distance between this center-of-mass and the stimulating electrode was used as a soma proximity measure (). Included in the analysis were several cells stimulated at the primary electrode, which had some of the smallest distances in the dataset. Note that these calculations assume a uniformly conductive medium between the electrodes and the soma, as well as equal sensitivity of all electrodes.
Sensitivity to spatial location of stimulation
Axon path analysis and threshold map
The path of the axon was estimated by lining up all electrodes showing small triphasic signals (Litke et al., 2004
). Within the same piece of retina, axons tended to run parallel to one another as expected based on their trajectory toward the optic disk. This fact was used to establish an average axon direction for each preparation by calculating the mean angle of several unambiguous axons (n=4-9) in a given retina. The angle between the average axon direction and a line connecting the triangulated soma center and the stimulating electrode was then measured for each cell. To overlay stimulation sites from all cells in one plot, the position coordinates for each cell were rotated about the estimated soma center location so that each axon path pointed to the right ().
An elliptical two-dimensional Gaussian function was fit to the stimulation position coordinates using 1/threshold as a measure of sensitivity at each location. The center of this Gaussian fit indicates the estimated location of maximal sensitivity to electrical stimulation and was located to the right of the origin (soma center). An estimate of the error in the location of this point was obtained by resampling from the collection of recorded cells with replacement, and calculating the Gaussian center for each resampled data set. The resampled centers were located close to the original center of sensitivity, indicating that the observed right-shift of the location of maximal sensitivity was statistically significant.
The position of maximum sensitivity was further verified by calculating a center-of-mass for a subset of stimulation positions with coordinate average in the origin (x=0, y=0). The resulting center of mass was located to the right of the origin, similarly to the location obtained from the Gaussian fit.
Spatial specificity analysis
To analyze the spatial spread of a single stimulus pulse, artifact subtraction was performed on every parasol cell in both the ON- and OFF-cell mosaics using data from the same stimulation run. All cells (n=6) were directly stimulated at the primary electrode and the pulse duration in all stimulations was 0.05 ms. The number of applied pulses in this analysis was 130 ± 8. Response rates were calculated by counting all spikes from a given cell in a 2 ms window after stimulation onset, divided by the total number of stimulation pulses. Several cells were spontaneously active but fired no spikes in the 2 ms counting window. The response rate of these cells was <1% and they were labeled with “<1” in to convey that the cell was capable of spiking at the time the stimulation was performed. The stimulation charge in the six cells analyzed was 44 ± 3 pC and the stimulation charge density 0.037 ± 0.007 mC/cm2, which was at or above threshold (118 ± 8 % of threshold).
Data processing and reporting
Multi-electrode data was analyzed offline using Labview and Igor Pro. Means and standard errors were calculated in Excel; the statistical tests were performed in Igor Pro. Images were processed in Photoshop and Intaglio. Statistical significance was calculated by performing a two-tailed two-sample Wilcoxon-Mann-Whitney test with a significance limit of p < 0.05. All reported error values are standard errors of the mean (SE).