Quantitative light and electron microscopy was used to monitor the extent of retinal degeneration as a function of age and temperature in the white-eyed rdgBKS222 mutant of Drosophila melanogaster. Parallel measurements of the electroretinogram (ERG) of the degenerating retina reveal a new phenomenon – the appearance of spike potentials following illumination with bright light. These spikes, which do not appear in the normal fly retina, have a relatively long duration (20–50 ms), regenerative properties, and a rate of occurrence which increases with increasing light intensity. The spikes differed from the light response in being more susceptible to CO2 and to cuts in the eye. The spikes completely disappeared at low extracellular Ca2+ levels which did not reduce the amplitude of the light response. The spike potentials become triphasic when the recording electrode is advanced to the level of the basement membrane. This suggests that the spike potentials originate from the photoreceptor axons as a result of synchronous opening of voltage-dependent channels in a large number of photoreceptor cells. The occurrence of spike potentials during the process of degeneration was studied. Two phases can be distinguished: (1) Spike potentials appear in retinae of 2–3-day-old flies which display few morphological signs of degeneration. The frequency of appearance of spike potentials decreases in retinae of 14–16-day-old flies which show extensive degeneration of the R1–6 photoreceptor cells but no degeneration of the central R7,8 cells. (2) Spike potentials appear more frequently again in flies of 22–24 d of age. This is probably a consequence of degeneration of the remaining R7,8 photoreceptor cells. Temperature and the light-dark cycle had a critical effect on degeneration. Eight-day-old mutants raised at 19°C in a normal light-dark cycle showed only little degeneration. Eight-day-old mutants raised at 24°C showed only a slight degeneration when raised in the dark. However, the degree of degeneration was greatly enhanced in the mutants raised at 24°C under a light-dark cycle regime.

The combined electrophysiological and morphological study of the degeneration, as a function of age and temperature, revealed that (1) the degeneration process takes place even in darkness, but at a slow rate, while light greatly accelerates the degeneration. (2) The degeneration is negligible at 19°C, even during light, in the first week after eclosion. (3) The appearance of spike potentials at an early stage of the degeneration suggests that changes in the plasma membrane of the photoreceptor cells manifest at an initial stage of the degeneration process.

Chemicals which affect different steps of the phototransduction cascade were used to identify the site of action of the rdgB gene product of Drosophila. In the rdgB mutant, the photoreceptor cells degenerate after several days of exposure to light, whereas raising the flies in the dark largely prevents the degeneration. In the rdgBKS222 mutant, which was used in the present studies, the light-induced degeneration is characterized by (1) selective degeneration of the peripheral but not the central photoreceptor cells; (2) random distribution of degenerated cells among ommatidia; and (3) the degeneration is specific to the rdgB but not the wild-type photoreceptor cells. In the present study, we show that application of specific chemical agents to the eyes of rdgBKS222 flies in the dark mimics the effects of light and causes retinal degeneration indistinguishable from light. The agents used in these studies are the metabolically stable GTP analogs GTPγS and Gpp(NH)p as well as fluoride ions, which are known to activate the transducing guanine nucleotide binding protein (G-protein of fly photoreceptors). It is unlikely that the chemically induced retinal degeneration is mediated by effects on energy metabolism, since application of the metabolic inhibitors CN-and 2-deoxy-D-glucose did not increase the extent of retinal degeneration over that observed in control flies treated with Ringer solution. The GDP analog GDPβS, which inhibits G-protein activity, greatly reduced the extent of retinal degeneration in the dark, over that observed in control flies treated with Ringer solution. These results suggest that activation of the G-protein precedes the step in the transduction cascade that leads to retinal degeneration and provides a powerful tool to investigate the molecular mechanism of light-induced degeneration in the rdgB mutant.

The morphology of the axon which connects the cell body and pedicle of turtle cone photoreceptors was studied by light and electron microscopy. The axon which contains numerous synaptic vesicles, some endoplasmic reticulum, and a few cisternae is basically filled with cytoplasm. The length of the axon is related to the class of cone and varies slightly with retinal location, with axons as short as 3–6 μm found in red cones, and as long as 60 μm in cones containing colorless oil droplets. By simultaneously voltage clamping the cell body and pedicle regions of single isolated cones, we measured the longitudinal axonal resistance and the cell body and pedicle membrane resistances. For each cell studied, the axonal resistance of cones with short axons was lower than the cell and pedicle membrane resistances. Thus, the cell can be considered to be an isopotential structure. However, in some cones with long axons, the axonal, cell body, and pedicle resistances were comparable. The pedicles of these cones, therefore, could act like summing points and may provide a locus for spatial signal integration. Electrical coupling between the principal and accessory members of double cones was also studied. Electron-microscopic observation of the membrane junction between the apposed inner segments of the double cones in the intact retina show narrow segments which resemble gap junctions. However, in every double cone studied in culture, passing currents into one member of the double cone did not result in measurable current flow in the adjacent cell. Thus, the two members of the double cone, isolated from the turtle retina, are not electrically coupled.

A large population of amacrine cells in the retina are thought to use GABA as an inhibitory neurotransmitter in their synaptic interactions within the inner plexiform layer. However, little is known about their synaptic targets; the neurons that express the receptors for GABA have not been clearly identified. Recently, the GABAA receptor has been isolated and antibodies have been raised against it. These antibodies have proven useful for the immunocytochemical localization of the receptor, and two brief reports describing the distribution of GABAA receptor immunoreactivity in the retina have appeared (Richards et al., 1987; Mariani et al., 1987). We used a monoclonal antibody (62–3G1) against the GABAA receptor to study the retina of the New World primate Saimiri sciureus.

Labeled somata were found in the inner nuclear layer (INL) and ganglion cell layer (GCL). The staining was confined to what appeared to be the cell's plasmalemma and small cytoplasmic granules. Most of the labeled neurons in the INL had small somata (5–7 μm in diameter) located at the vitreal edge of the layer. They arborized in two laminae (approximately 2 and 4) of inner plexiform layer (IPL). Ventral to the optic disc (2.5 mm) they comprised 29% of the cells present. A few of the labeled neurons appeared to be interplexiform cells or flat bipolar cells, with labeled processes that extended into both the IPL and the inner half of the outer plexiform layer. In the GCL, the labeled somata were among the largest present (13–20 μm in diameter), and 2.5 mm ventral to the optic disc they made up 15% of the cells present. Experiments in which immunoreactive somata were retrogradely labeled following the injection of fluorescent tracers into the optic tract provided a conclusive demonstration that some of the immunoreactive somata were ganglion cells. The antibody often labeled their axons in the optic fiber layer. This suggests that the GABAA receptors are transported anterogradely to the retinal terminal fields. The dendrites of the immunoreactive ganglion cells extended into the 2 laminae of labeled processes in the IPL, and their primary dendritic arbors were, at any given eccentricity, quite similar in appearance. This homogeneity suggests that they comprise a particular subset of the ganglion cells.

Sections simultaneously labeled with the monoclonal antibody against the GABAA receptor and antisera against either L-glutamic acid decarboxylase (GAD) or GABA revealed that the GAD/GABA was distributed much more widely in the IPL than the GABAA receptor. This variance may reflect either the presence of other, perhaps GABAB, receptors in the IPL or GABA in portions of the IPL where it is ineffective as a neurotransmitter.

We have purified a protein that changes in relative concentration during the development of the kitten visual cortex. It resembles GAP-43 (a neuronal protein that is expressed at elevated levels during periods of development and regenerative axon growth) in the following respects: (1) it is an acidic protein (pI=4.7) whose electrophoretic mobility on SDS-PAGE is similar to, but lower than rat GAP-43, suggesting that the cat protein is larger; (2) its electrophoretic mobility varies with the acrylamide concentration in a manner that is characteristic of GAP-43; (3) its concentration in kitten forebrain is elevated during early postnatal development; (4) the sequence of ten consecutive amino acids from a chemically generated fragment matches the expected sequence from GAP-43; and (5) its amino-acid content also matches GAP-43. We conclude that our purified protein is cat GAP-43. Immunoblots with an antibody prepared against rat GAP-43 suggested that the concentration of GAP-43 in the visual cortex declines with age.

Cat striate cortical neurons were investigated using a new method of studying adaptation aftereffects. Stimuli were sinusoidal gratings of variable contrast, spatial frequency, and drift direction and rate. A series of alternating adapting and test trials was presented while recording from single units. Control trials were completely integrated with the adapted trials in these experiments.

Every cortical cell tested showed selective adaptation aftereffects. Adapting at suprathreshold contrasts invariably reduced contrast sensitivity. Significant aftereffects could be observed even when adapting at low contrasts.

The spatial-frequency tuning of aftereffects varied from cell to cell. Adapting at a given spatial frequency generally resulted in a broad response reduction at test frequencies above and below the adapting frequency. Many cells lost responses predominantly at frequencies lower than the adapting frequency.

The tuning of aftereffects varied with the adapting frequency. In particular, the strongest aftereffects occurred near the adapting frequency. Adapting at frequencies just above the optimum for a cell often altered the spatial-frequency tuning by shifting the peak toward lower frequencies. The fact that the tuning of aftereffects did not simply match the tuning of the cell, but depended on the adapting stimulus, implies that extrinsic mechanisms are involved in adaptation effects.

Adaptation-induced changes in the temporal-frequency tuning and direction selectivity of cat visual cortical cells were studied. Aftereffects were induced largely independent of direction. Adapting in either direction reduced responses in both directions. Aftereffects in the direction opposite that adapted were only slightly weaker than were aftereffects in the adapted direction. No cell showed any enhancement of responses to drifting test stimuli after adapting with moving gratings. Adapting in a cell's null direction usually had no effect. Dramatic differences between the adaptation characteristics of moving and stationary stimuli were observed, however.

Furthermore, aftereffects were temporal frequency specific. Temporal frequency-specific aftereffects were found in both directions: adapting in one direction induced frequency-specific effects in both directions. This bidirectionality of frequency-specific aftereffects applied to the spatial domain as well. Often, aftereffects in the direction opposite that adapted were more narrowly tuned.

In general, adaptation could shift a cell's preferred temporal frequency. Aftereffects were most prominent at high temporal frequencies when testing in the adapted direction. Aftereffects seemed to be more closely linked to temporal frequency than to velocity matching.

These results constrain models of cortical connectivity. In particular, we argue against schemes by which direction selectivity is generated by inhibiting a cell specifically when stimulated in the nonpreferred direction. Instead, we argue that cells receive bidirectional spatially and temporally tuned inputs, which could combine in spatiotemporal quadrature to produce direction selectivity.