Effects of intravitreal furosemide injections on the responses of NOT-DTN neurons to moving light (ON) and dark (OFF) edges.

<p>Comparison of the responses during ON (A) and OFF (B) moving edge stimulation in preferred (PD, grey bars) and non-preferred (NPD, white bars) directions prior to (control) and after intravitreous injection of the drug furosemide. Horizontal lines indicate the median, boxes the 25–75%, whiskers the 10–90%, and black dots the 5–95 percentiles of the non-parametric statistical comparison. Furosemide reduces responses in preferred and in non preferred direction.</p

Table 1 summarizes the solvents used for the stem solutions, the concentration of the stem solutions, the concentration of the working solutions, the volume injected, the concentration in the vitreous body based on an estimated volume of 50 µl [50] up to 100 µl (own measurements in older animals (>400 g), and the number of tests performed.

<p>Numbers in parentheses give the number of tests where drug administration resulted in changes of the neuronal activity. APB: 2-amino-4-phosphonobutyrat; PTX: picrotoxin; PBS: phosphate buffered saline.</p

Effects of 2-amino-4-phosphonobutyrat (40–80 µM APB) on neuronal activity of single units.

<p>Responses of 3 units (mean and standard deviation of 10 measurements) to flashed and moving ON (A) and OFF (B) visual stimuli before (control) and after intravitreous drug injection (APB; intravitreous concentration 40–80 µM). Ordinate: neuronal activity in spikes per second, abscissa: experimental conditions. APB blocks responses to all ON stimuli but spares the OFF flash response and the responses to the moving dark edge, albeit moderately decreased. Grey boxes: flash responses; black boxes: responses to moving edges in preferred direction; white boxes: responses in non-preferred direction.</p

NOT-DTN neurons respond to flashed as well as moving ON- and OFF visual stimuli.

<p>A: Response to flash stimulation, light onset at 500 ms, light offset at 1500 ms. This neuron exhibits clear phasic responses to both ON and OFF stimulation, the tonic response is low during ON stimulation. Spontaneous activity is represented by the first 500 ms of the PSTH. Ordinate: activity in spikes per second (spikes/s), abscissa: time in milliseconds (ms). B: direction selective response to a moving light edge (ON). Movement onset at 1000 ms. PD: preferred direction, NPD: non-preferred direction (opposite to PD). C: direction selective response to a moving dark edge (OFF).</p

Effects of intravitreal bumetanide injections on the responses of NOT-DTN neurons to moving light (ON) and dark (OFF) edges.

<p>Comparison of the activity during ON (A) and OFF (B) moving edge stimulation in preferred (PD, grey bars) and non-preferred (NPD, white bars) directions prior to (control) and after intravitreous injection of the drug (bumetanide). Horizontal lines indicate the median, boxes the 25–75%, whiskers the 10–90%, and black dots the 5–95 percentiles of the non-parametric statistical comparison. Bumetanide reduces responses in preferred direction and enhances responses in the non preferred direction, especially in the responses to the OFF stimulus.</p

Schematic demonstration of the visual stimuli used in the present study.

<p>A: stationary flash stimulus, duration of the presentation of the bright (ON) and the dark (OFF) monitor lasted for 1s each. B: moving ON stimulus. A light edge moved across the dark monitor until it was fully bright in the four cardinal directions. C: moving OFF stimulus. A dark edge moved across the bright monitor until it was fully dark in the four cardinal directions (for further description see methods).</p

Effects of 2-amino-4-phosphonobutyrat (APB) and picrotoxin (PTX) on the directional tuning of the light edge and dark edge responses in an example NOT-DTN unit.

<p>Polar plot of the responses during stimulation with ON (A) and OFF (B) edges moving in the four cardinal directions prior to drug application (black curves), after intravitreous injection of APB (red curves), and after additional intravitreous injection of PTX (green curves). Distance from the center of the polar plots indicates neuronal activity in spikes per second given by the numbers on the circles. PD: preferred horizontal direction, NPD: non-preferred horizontal direction. Note that after APB followed by a PTX injection an increased but direction unselective response to the moving dark edge (OFF) is uncovered.</p

Effects of 2-amino-4-phosphonobutyrat (APB) and picrotoxin (PTX) on neuronal activity in all units tested.

<p>Neuronal responses in all 25 single- and multi-unit recordings during ON A) and OFF (B) stimulation in preferred (PD, grey boxes) and non-preferred (NPD, white boxes) direction before (control; left 2 boxes), after intravitreous application of the drug APB (middle 2 boxes) and after subsequent intravitreous application of PTX (right 2 boxes). Horizontal lines indicate the median, boxes the 25–75%, and whiskers the 10–90% percentiles of non-parametric statistical comparison. Ordinate: neuronal activity in spikes per second, abscissa: experimental conditions. At approximately 100–200 µM intravitreal concentration, APB completely blocks the response driven by the moving ON stimulus (p<0.005) but also decreases the response to the moving OFF stimulus (p<0.05). The activity in the non-preferred direction is unaltered as is spontaneous activity. PTX increases the responses in PD and in NPD to about the same values during ON- as well as during OFF stimulation.</p

Effects of bumetanide on direction selectivity.

<p>Comparison of the direction selectivity index DSI (ordinate) during ON and OFF stimulation before (grey columns) and after (white columns) intravitreous application of bumetanide averaged (mean and standard deviation) over all units recorded. Bumetanide abolishes direction selectivity in the retina and subsequently in NOT-DTN retinal slip cells.</p

Effects of furosemide on direction selectivity.

<p>Comparison of the direction selectivity index DSI (ordinate) during ON and OFF stimulation before (grey columns) and after (white columns) intravitreous application of furosemide. averaged (mean and standard deviation) over all units recorded. Furosemide abolishes direction selectivity in the retina and subsequently in the ON and to a lesser degree in the OFF responses in NOT-DTN retinal slip cells.</p