David K. Merwine

Necessity of acetylcholine for retinal directionally selective responses to drifting gratings in rabbit

1 A model for retinal directional selectivity postulates that GABAergic inhibition of responses to motions in the null (anti‐preferred) direction underlies this selectivity. An alternative model postulates that besides this inhibition, there exists an asymmetric, nicotinic acetylcholine (ACh) input from starburst amacrine cells. It is possible for the latter but not the former model that stimuli could exist such that nicotinic blockade eliminates directional selectivity. Such stimuli would drive the cholinergic but not the GABAergic system well. 2 So far, attempts to eliminate directional selectivity with nicotinic blockade have failed, but they always used isolated, moving bars as the stimulus. We confirmed this failure for On‐Off directionally selective (DS) ganglion cells in our preparation of the rabbits retina. 3 However, while recording from these cells, we discovered that nicotinic blockade eliminated directional selectivity to drifting, low spatial frequency sine‐ and square‐wave gratings. 4 This effect was not just due to the smallness of the responses under nicotinic blockade. NMDA blockade caused even smaller responses, but no loss of directional selectivity. 5 This result is consistent with a two‐asymmetric‐pathways model of directional selectivity, but inconsistent with an asymmetric‐GABA‐only model. 6 We conclude that asymmetric nicotinic inputs extend the range of stimuli that can elicit directional selectivity to include moving textures, that is, those with multiple peaks in their spatial luminance profile.

Complementary roles of two excitatory pathways in retinal directional selectivity.

The two major excitatory synapses onto ON-OFF directionally selective (DS) ganglion cells of the rabbit retina appear to be nicotinic cholinergic and NMDA glutamatergic. Blockade of either of these synapses with antagonists does not eliminate directional selectivity. This suggests that these synapses may have complementary roles in the computation of the direction of motion. To test this hypothesis, quantitative features of the DS cell excitatory pathways were determined by collecting responses, under nicotinic and/or NMDA blockade, to a sweeping bar, hyperacute apparent motions, or a drifting sinusoidal grating. Sweeping bar responses were reduced, but directional selectivity not eliminated, by blockade of either excitatory path, as previously shown (Cohen & Miller, 1995; Kittila & Massey, 1997). However, residual responses under combined blockades were not statistically significantly DS. NMDA blockade reduced responses more than nicotinic blockade for each protocol, and shifted hyperacute motion thresholds to higher values. This supported the notion that glutamate provides the main excitatory drive to DS cells, that is, the one responsible for contrast sensitivity. In turn, nicotinic, but not NMDA blockade eliminated directional selectivity to a drifting low spatial-frequency sinusoidal grating in these cells. This suggested that acetylcholine (ACh) is the main excitatory input with regards to directional selectivity for some textured stimuli, that is, those with multiple peaks in their spatial luminance profile. Moreover, nicotinic blockade raised the low temporal-frequency cutoff of the grating responses, consistent with the proposal that preferred-direction facilitation, which is temporally sustained, is dependent on the cholinergic input. These different properties of the NMDA and nicotinic pathways are consistent with a recently proposed two-asymmetric-pathways model of directional selectivity.

Extra-receptive-field motion facilitation in on-off directionally selective ganglion cells of the rabbit retina

The excitatory receptive-field centers of On-Off directionally selective (DS) ganglion cells of the rabbit retina correspond closely to the lateral extent of their dendritic arborizations. Some investigators have hypothesized from this that theories for directional selectivity that entail a lateral spread of excitation from outside the ganglion cell dendritic tree, such as from starburst amacrine cells, are therefore untenable. We show here that significant motion facilitation is conducted from well outside the classical excitatory receptive-field center (and, therefore, dendritic arborization) of On-Off DS ganglion cells for preferred-direction, but not null-direction moving stimuli. These results are consistent with a role in directional selectivity for cells with processes lying beyond the On-Off ganglion cells excitatory receptive-field center. These results also highlight the fundamental distinction in retinal ganglion cell receptive-field organization between classical excitatory mechanisms and those that facilitate other excitation without producing directly observable excitation by themselves.

Non-monotonic contrast behavior in directionally selective ganglion cells and evidence for its dependence on their GABAergic input

We serendipitously discovered that the preferred-direction responses of ON-OFF directionally selective (DS) ganglion cells in the rabbit retina fall as a function of contrast when the contrast of a moving bar exceeds about 100%. Null-direction responses did not fall for contrasts up to 400%. Because the non-monotonic (rise-then-fall) behavior as a function of contrast occurred only for preferred-direction responses, it must depend on the mechanism of directional selectivity. It became thus of interest to investigate how this non-monotonicity depends on the major synapses involved in directional selectivity. Blockades of nicotinic acetylcholine (ACh) and NMDA glutamate receptors reduced responses without eliminating preferred-response non-monotonicity. Blocking GABAergic inhibition, however, did eliminate non-monotonicity. These results pose a difficult puzzle, since in the accompanying paper (Grzywacz et al., 1998), we showed that residual responses under combined nicotinic and NMDA blockades are not statistically significantly directionally selective. How is it possible that null-direction GABAergic inhibition affects non-nicotinic-non-NMDA residual responses without generating directional selectivity? This may happen if there exists an asymmetric GABAergic input to distal dendrites of the DS cell while the excitatory, non-nicotinic-non-NMDA input is to proximal dendrites. In support of this hypothesis, bath-applied GABA reduces responses to exogenous ACh under synaptic block, providing for the first time in the rabbits retina, direct evidence of GABA receptors on DS cells.

Developmental regulation of the morphology of mouse retinal horizontal cells by visual experience

Visual deprivation during development alters the normal refinement of connections, neurotransmitter expression and physiological function in the retina. We investigated the effects of different forms of visual experience on the anatomy of retinal neurons in the mouse. Although it is generally assumed that outer retinal cells are not affected morphologically by visual experience, we found changes in the outer retinas of animals reared with light but no contrast. In postnatal day 30 animals reared in control, dark and high‐contrast environments, horizontal‐cell processes ramified normally in the outer plexiform layer. However, in postnatal day 30 no‐contrast‐reared retinas, horizontal‐cell processes emerged from the outer plexiform layer and ramified in the inner nuclear layer. Similar sprouting processes of horizontal cells were found in a mouse model of retinitis pigmentosa. In conclusion, our data show that a lack of contrast during development alters the morphology of horizontal cells and may thus affect normal visual processing. This effect may be relevant for young patients with cloudy vision (e.g. cataract).

Choline acetyltransferase-immunoreactive neurons in the retina of normal and dark-reared turtle

Visual deprivation alters retinal‐ganglion‐cell response properties through changes in spontaneous wave‐like activity (Sernagor and Grzywacz [ 1996 ] Curr Biol 6:1503–1508). This activity depends on cholinergic synaptic transmission in the turtle retina (ibid; Sernagor and Mehta [ 2001 ] J Anat 199:375–383). We studied the expression of choline acetyltransferase (ChAT) by immunocytochemistry and Western blot in developing retinas of control and dark‐reared turtles. At postnatal day 0 (P0), right after hatching, ChAT‐immunoreactivity was present in the ganglion cell layer (GCL), in the inner nuclear layer (INL), and in two distinct bands of the inner plexiform layer (IPL). In P14‐ and P28‐control, and P14‐ and P28‐dark‐reared retinas, ChAT‐immunoreactivity showed similar patterns to those in P0. However, in P14‐ and P28‐dark‐reared retinas the density of ChAT‐immunoreactive cells was higher in both the INL and GCL than in P14‐ and P28‐control retinas, respectively. Moreover, Western blotting showed that ChAT protein levels were significantly increased in the dark‐reared retina compared to those of the control. TUNEL studies indicated that the difference between normal and dark‐reared conditions was not due to extra apoptosis in the former. In turn, proliferating‐cell nuclear antigen immunocytochemistry showed no extra proliferating cells in the latter. Finally, nearest‐neighbor analysis revealed that the denser population of cholinergic cells in dark‐reared turtles formed a mosaic as regular as the normal ones in the GCL. Thus, light deprivation increases the expression of ChAT, increasing the apparent density of cholinergic neurons in the developing turtle retina. J. Comp. Neurol. 503:768–778, 2007.

Statistically robust detection of spontaneous, non-stereotypical neural signals

Neural signals of interest are often temporally spontaneous and non-stereotypical in waveform. Detecting such signals is difficult, since one cannot use time-locking or simple template-matching techniques. We have sought a statistical method for automatically estimating the baseline in these conditions, and subsequently detecting the occurrence of neural signals. One could consider the signals as outliers in the distribution of neural activity and thus separate them from the baseline with median-based techniques. However, we found that baseline estimators that rely on the median are problematic. They introduce progressively greater estimation errors as the neural signals duration, amplitude or frequency increases. Therefore, we tested several mode-based algorithms, taking advantage of the most probable state of the neural activity being the baseline. We found that certain mode-based algorithms perform baseline estimation well, with low susceptibility to changes in event duration, amplitude or frequency. Once the baseline is properly established, its median absolute deviation (MAD) can be determined. One can then use it to detect spontaneous signals robustly as outliers from the noise distribution. We also demonstrate how the choice of detection threshold in terms of MADs can be used to bias against false positives, without creating too many false negatives or vice versa.