Franklin R. Amthor

Morphology of on-off direction-selective ganglion cells in the rabbit retina *

On-off direction-selective ganglion cells in rabbit retina have been stained by intracellular injection of horseradish peroxidase (HRP). The dendritic ramification is basically bistratified . Global asymmetries related to the preferred direction are not apparent, but the small diameter dendrites, spines, and complex branching pattern are consistent with models based on local, non-linear mechanisms for direction-selectivity.

Effects of the destruction of starburst-cholinergic amacrine cells by the toxin AF64A on rabbit retinal directional selectivity

The effects of intraocular injections of ethylcholine mustard aziridinium ion (AF64A), an irreversible inhibitor of choline uptake, on the rabbit retina were assessed electrophysiologically, pharmacologically, anatomically, and behaviorally. Survival times from 1 day to 30 days were investigated. After 24 h, the shortest time tested, the directional selectivity of On-Off responding ganglion cells having the characteristic morphology of On-Off directionally selective directionally selective (DS) ganglion cells, as revealed by intracellular dye injection, was significantly reduced, both by an apparent decrease of preferred direction responses and an increase in responses to null-direction movement. No toxin-mediated changes in the dendritic trees of these cells were noted. Cells in AF64A-affected retinas having the DS morphology did not respond significantly to GABAergic or cholinergic agents such as picrotoxin and eserine, but did respond to nicotine. Recordings from a small random sample of other ganglion cell classes in the same retinas yielded no obvious changes in response properties. The direct effects on starburst (cholinergic) amacrine cells, which were identified by intraocular injection of the fluorescent dye DAPI with the AF64A, were investigated by intracellular injections of Lucifer yellow, and by immunohistochemical staining with antibodies to choline acetyltransferase (ChAT). Although starburst amacrine cell somas survived the AF64A treatment for at least several days, the dendrites could not be visualized by fluorescent dye injection in affected retinas due to dye leakage of the injected fluorescent dye from either the soma or proximal dendritic region. ChAT staining revealed a sequence in which ChAT-positive cells were undetectable first in the inner nuclear layer, and then in the ganglion cell layer. Cholinergic amacrine cells in the central retina were also affected before those in the periphery. The electrophysiological changes observed typically preceded the loss of ChAT activity. Behavioral tests for optokinetic nystagmus responses also revealed a lack of such responses in the affected eyes.

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.

Synaptic input to the on-off directionally selective ganglion cell in the rabbit retina

A physiologically identified on–off directionally selective (DS) ganglion cell with its preferred‐null axis defined was stained with horseradish peroxidase (HRP) and prepared for electron microscopy. A continuous series of thin sections were used to examine the cells synaptology. Although the DS cell dendrite received the majority of its synaptic input from a heterogeneous population of amacrine cell processes, a frequently observed synaptic profile consisted of a DS cell dendrite receiving synapses from a cluster of several amacrine cell processes. These clusters of processes were assumed to be from a fascicle of amacrine cells, most of which probably belonged to several different cholinergic starburst amacrine cells. The most frequently observed presynaptic profile within the clusters consisted of a synaptic couplet in which two processes synapsed with each other before one of them finally synapsed with the DS ganglion cell dendrite; occasionally, a chain of three serial synapses was seen. In addition, a specific microcircuit that has the potential to exert lateral feedforward inhibition was also observed. This microcircuit consisted of two cone bipolar cell terminal dyad synapses where one dyad contained an amacrine cell process making a reciprocal synapse and a DS ganglion cell dendrite receiving direct excitation; the other dyad synapse, found lateral to the first dyad, contained two amacrine cell processes that both made reciprocal synapses, but one fed forward to make a putative inhibitory synapse with the DS cell dendrite. J. Comp. Neurol. 456:267–278, 2003.

Quantitative Morphology of Rabbit Retinal Ganglion Cells

Intraretinal (extracellular) injections of horseradish peroxidase were used to stain rabbit retinal ganglion cells. Five basic morphological ganglion cell classes were identified by quantitative analysis of dendritic branching patterns and computer reconstruction of dendritic ramification levels. Type 1 cells are characterized by a unistratified, radial dendritic morphology. The dendritic fields are of medium to large size. Subgroups ramify in either the outer or the inner part of the inner plexiform layer (i. p. l.). Type 2 cells have complex intricately branched dendritic morphologies with wide branch angles. They are comparable with type 1 cells in dendritic field size. Subgroups of this class include unistratified cells ramifying in the outer or inner part of the i. p. l. as well as cells with more complicated i. p. l. ramification schemes. Type 3 cells are somewhate similar to type 1 cells. A particular distinction is that they are much larger than type 1 cells at the same retinal eccentricity. Type 4 cells have a thin elliptical soma and a lobulate dendritic tree structure. Type 5 cells are a somewhat heterogeneous group with very small intricately branched dendritic fields. Since the number of anatomical groups is comparable with the number of physiological classes, it is tenable that the major physiological cell classes are associated with distinct dendritic morphologies.

Parallel optical monitoring of visual signal propagation from the photoreceptors to the inner retina layers

Understanding of visual signal processing can benefit from simultaneous measurement of different types of retinal neurons working together. In this Letter, we demonstrate that intrinsic optical signal (IOS) imaging of frog retina slices allows simultaneous observation of stimulus-evoked responses propagating from the photoreceptors to the inner neurons. High-resolution imaging revealed robust IOSs at the photoreceptor, the inner plexiform, and the ganglion cell layers. While IOSs of the photoreceptor layer were mainly confined to the area directly stimulated by the visible light, IOSs of the inner retinal layers spread from the stimulus site into relatively large areas with a characteristic near-to-far time course.

High spatiotemporal resolution imaging of fast intrinsic optical signals activated by retinal flicker stimulation

High resolution monitoring of stimulus-evoked retinal neural activities is important for understanding retinal neural mechanisms, and can be a powerful tool for retinal disease diagnosis and treatment outcome evaluation. Fast intrinsic optical signals (IOSs), which have the time courses comparable to that of electrophysiological activities in the retina, hold the promise for high resolution imaging of retinal neural activities. However, application of fast IOS imaging has been hindered by the contamination of slow, high magnitude optical responses associated with transient hemodynamic and metabolic changes. In this paper we demonstrate the feasibility of separating fast retinal IOSs from slow optical responses by combining flicker stimulation and dynamic (temporal) differential image processing. A near infrared flood-illumination microscope equipped with a high-speed (1000 Hz) digital camera was used to conduct concurrent optical imaging and ERG measurement of isolated frog retinas. High spatiotemporal resolution imaging revealed that fast IOSs could follow flicker frequency up to at least 6 Hz. Comparable time courses of fast IOSs and ERG kinetics provide evidence that fast IOSs are originated from stimulus activated retinal neurons.

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.

Muscarinic acetylcholine receptor localization and activation effects on ganglion response properties.

PURPOSE The activation and blockade of muscarinic acetylcholine receptors (mAChRs) affects retinal ganglion cell light responses and firing rates. This study was undertaken to identify the full complement of mAChRs expressed in the rabbit retina and to assess mAChR distribution and the functional effects of mAChR activation and blockade on retinal response properties. METHODS RT-PCR, Western blot analysis, and immunohistochemistry were used to identify the complement and distribution of mAChRs in the rabbit retina. Extracellular electrophysiology was used to determine the effects of the activation or blockade of mAChRs on ganglion cell response properties. RESULTS RT-PCR of whole neural retina resulted in the amplification of mRNA transcripts for the m1 to m5 mAChR subtypes. Western blot and immunohistochemical analyses confirmed that all five mAChR subtypes were expressed by subpopulations of bipolar, amacrine, and ganglion cells in the rabbit retina, including subsets of cells in cholinergic and glycinergic circuits. Nonspecific muscarinic activation and blockade resulted in the class-specific modulation of maintained ganglion cell firing rates and light responses. CONCLUSIONS The expression of mAChR subtypes on subsets of bipolar, amacrine, and ganglion cells provides a substrate for both enhancement and suppression of retinal responses via activation by cholinergic agents. Thus, the muscarinic cholinergic system in the retina may contribute to the modulation of complex stimuli. Understanding the distribution and function of mAChRs in the retina has the potential to provide important insights into the visual changes that are caused by decreased ACh in the retinas of Alzheimers patients and the potential visual effects of anticholinergic treatments for ocular diseases.

Rabbit retinal ganglion cell responses mediated by alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors.

The responses of many ganglion cells in the rabbit retina are mediated, at least in part, by acetylcholine (ACh) acting on neuronal nicotinic acetylcholine receptors (nAChRs). nAChRs are comprised of alpha and beta subunits; three beta subunits and nine alpha subunits of nAChRs have been identified and these subunits can combine to form a large number of functionally distinct nAChR subtypes. We examined the effects of cholinergic agents on the light-evoked responses of ganglion cells to determine which nAChR subtypes mediate the effects of ACh. Extracellular recordings of retinal ganglion cells were made in intact everted eyecup preparations and nicotinic agonists and antagonists were added to the superfusate. While several ganglion cell classes exhibited methyllycaconitine (MLA) sensitivity, the directionally selective (DS) ganglion cells were most sensitive; exposure to 30 nanomolar MLA, a concentration reportedly too low to affect alphaBgt-insensitive nAChRs, suppressed the stimulus-evoked responses of DS cells without eliminating directional selectivity. Epibatidine, which at low concentrations is an agonist selective for alphaBgt-insensitive nAChRs, stimulated firing of various cell types including DS ganglion cells at low nanomolar concentrations. The effects of the various agents tested persisted under cobalt-induced synaptic blockade. The low nanomolar MLA and epibatidine sensitivity of DS cells suggests that DS ganglion cells express both alphaBgt-sensitive and alphaBgt-insensitive nAChRs. Other ganglion cell types appear to express only alphaBgt-sensitive nAChRs but not alphaBgt-insensitive nAChRs.