Identification of a subtype of cone photoreceptor, likely to be blue sensitive, in the human retina.

A minor population of cone photoreceptors (called B-cones) can be distinguished from the major population (called R-cones) on morphological criteria as seen by light microscopy in foveal and peripheral human retina. The B-cones are characterized by a longer inner segment projecting into subretinal space, a larger-diameter inner segment, an increased staining intensity of the inner segment, and a different distribution relative to the R-cones in the cone mosaic. B-cones occur even in the foveolar center (3-5%) and rise to a maximum (15%) in the foveolar slope. They can also be identified in peripheral retina where they form 7-10% of the total cone population. The B-cone population follows the distribution profile postulated for the blue-sensitive system from histochemical studies on monkeys and from psychophysical studies on humans. The B-cones also share many of the same morphological features of the putative blue cones of the ground squirrel and monkey retinas. For these reasons we suggest that our B-cone group is the blue cone population of the human retina.

ON-OFF amacrine cells in cat retina.

We studied the morphology, photic responses, and synaptic connections of ON-OFF amacrine cells in the cat retina by penetrating them with intracellular electrodes, staining them with horseradish peroxidase, and examining them with the electron microscope. In a sample of seven cells, we found two different morphological types: the A19, which ramifies narrowly in stratum 2 (sublamina a) of the inner plexiform layer, and the A22, which ramifies mostly in stratum 4 (sublamina b) but extends some dendrites to sublamina a. Both of these cell types have axon-like processes that extend > 800 microns from the conventional dendritic arbor. ON-OFF amacrine cells in our sample had receptive fields (1.7 +/- 0.3 mm diameter) that were broader than their dendritic arbors (425 +/- 35 microns diameter) and that extended over the region of axon-like processes. In addition, we found many features in common with ON-OFF amacrine cells in poikilotherm vertebrates: a broad receptive field without surround antagonism, two sizes of spike-like events, narrow dynamic range (1 log unit intensity), and excitatory postsynaptic potentials at light on and light off. Two A19 amacrine cells were examined in the electron microscope: most synaptic inputs (93 and 76%, respectively) to either cell were from amacrine cells, with minor inputs from cone bipolar cells. Synaptic outputs were to bipolar, amacrine, and ganglion cells, including the OFF-alpha cell.

Computer-controlled impalement of cells in retinal wholemounts visualized by infrared CCD imaging on an inverted microscope.

We present a computer-guided microelectrode positioning system that is routinely used in our laboratory for intracellular electrophysiology and functional staining of retinal neurons. Wholemount preparations of isolated retina are kept in a superfusion chamber on the stage of an inverted microscope. Cells and layers of the retina are visualized by Nomarski interference contrast using infrared light in combination with a CCD camera system. After five-point calibration has been performed the electrode can be guided to any point inside the calibrated volume without moving the retina. Electrode deviations from target cells can be corrected by the software further improving the precision of this system. The good visibility of cells avoids prelabeling with fluorescent dyes and makes it possible to work under completely dark adapted conditions.

Optophysiology: depth-resolved probing of retinal physiology with functional ultrahigh-resolution optical coherence tomography.

Noncontact, depth-resolved, optical probing of retinal response to visual stimulation with a <10-microm spatial resolution, achieved by using functional ultrahigh-resolution optical coherence tomography (fUHROCT), is demonstrated in isolated rabbit retinas. The method takes advantage of the fact that physiological changes in dark-adapted retinas caused by light stimulation can result in local variation of the tissue reflectivity. fUHROCT scans were acquired from isolated retinas synchronously with electrical recordings before, during, and after light stimulation. Pronounced stimulus-related changes in the retinal reflectivity profile were observed in the inner/outer segments of the photoreceptor layer and the plexiform layers. Control experiments (e.g., dark adaptation vs. light stimulation), pharmacological inhibition of photoreceptor function, and synaptic transmission to the inner retina confirmed that the origin of the observed optical changes is the altered physiological state of the retina evoked by the light stimulus. We have demonstrated that fUHROCT allows for simultaneous, noninvasive probing of both retinal morphology and function, which could significantly improve the early diagnosis of various ophthalmic pathologies and could lead to better understanding of pathogenesis.

Possible role of amacrine cells in the generation of the mammalian ERG b-wave.

The ERG b-wave is believed to be generated by a change of membrane potential of Müller cells mediated by alteration in extracellular K+ activity. At least two K+ sources have been suggested. From studies with K+-sensitive electrodes there is some evidence that a proximal K+ source is generated by amacrine cell activity. It has been shown autoradiographically that in the rabbit retina gamma aminobutyric acid (GABA) is located in a subpopulation of amacrine cells. Therefore the effect of GABA on the b-wave amplitude of the isolated superfused rabbit retina was investigated by double-flash stimulation. Concentrations below 10(-5) MM GABA did not change the ERG; higher concentrations diminished the b-wave amplitude. With concentrations of 10(-3) MM GABA response to the second flash was clearly less reduced than that to the first. Furthermore the time course of the ERG was altered. Our investigation supports participation of amacrine cells in b-wave generation.

Telodendrial contacts between foveolar cone pedicles in the human retina.

The synaptic pedicles of foveolar cones in the human retina contact each other by means of telodendrial processes. Thus direct lateral coupling of photoreceptor terminals exists even in the area of highest acuity function.

Background-induced flicker enhancement in cat retinal horizontal cells. I. Temporal and spectral properties.

1. Dim backgrounds can enhance small-spot flicker responses of cat retinal horizontal cells by a factor of 2 or more. 2. Intracellular marking with horseradish peroxidase (HRP) reveals that this enhancement effect occurs in--but is not necessarily limited to--the cone-connected, A-type horizontal cell. 3. Flicker amplitudes decrease over a frequency range from 3 to 36 Hz of square-wave photic stimulation. There is little evidence of flicker-response enhancement at 3 Hz. Flicker-response enhancement is typically 2-6 times larger at 35 than at 6 Hz. 4. Inspection of flicker waveforms indicates both a scaling-up of response signals with backgrounds and a distortion composed of 2- to 5-ms-latency decrease, expressed primarily within a quick component of OFF-repolarization. 5. Flicker enhancement first increases as a function of background irradiance and then decreases. The increasing limb has the dynamic range and spectral sensitivity of cat rods (507-nm peak). Enhancement is maintained during rod after-effects. The decreasing limb of the background-versus-intensity function results from light adaptation of cat, long-wavelength (red) cones. 6. The flicker responses themselves peak spectrally at approximately 555 nm and reflect only the activity of cat long-wavelength (red) cones, without evidence of intermixing of other photoreceptor mechanisms. 7. Thus within the first synaptic layer of the cat visual system, rod signals interact with the flicker responses of red cones, both increasing cone-signal amplitudes and modifying cone-signal waveforms. 8. The results are closely analogous to "suppressive rod-cone interaction" (SRCI) as described in human psychophysics. 9. An outer-plexiform-layer circuit involving rods, horizontal cells and cones may mediate rod-induced enhancement of cone flicker. This being the case, notions of horizontal-cell feedback interactions with cones may have to be modified and extended. A specific feedback model is elaborated in the companion paper.

Background-induced flicker enhancement in cat retinal horizontal cells. II. Spatial properties.

1. Intracellular recordings have been made from cat retinal horizontal cells stimulated with flickering test spots. Dim backgrounds increase flicker amplitudes in response to small but not large test stimuli. 2. This background-induced flicker enhancement has been measured for different slit- and square-test stimulus widths and the results compared with two spatial models for the enhancement effect. 3. In the "dark test-region" model it is argued that rods within the test region are unresponsive to background stimuli because of prior saturation by the test stimulus. Background-evoked rod signals decay passively from regions outside the test stimulus through a syncytial network into the recording site, where they act on the cone-to-horizontal-cell synapse, increasing its gain. 4. In the "changing length-constant" model rod signals reduce the length constant of a syncytial network by uncoupling the cells within it. This causes an increased response to small but not large test stimuli. 5. Both models are analytically evaluated with the use of a conductive-sheet approximation to the syncytial network. Expressions are derived for network polarization [(V(0, 0)] as a function of stimulus size. The specific stimulus shapes considered are disks, rectangles, slits, and squares in both bright and dark varieties. From these expressions predictions of response enhancement as a function of stimulus size are made for both models. 6. The dark test-region model provides for an exponential decay of flicker enhancement as a function of slit width but a steeper-than-exponential decay with the width of squares, in close agreement with experimental data. 7. The changing length-constant model makes qualitatively similar predictions. Flicker enhancement declines nearly exponentially with slit width. For square-shaped test stimuli the predicted decline of flicker enhancement with size is somewhat shallower than either the dark test-region-model curve or the experimentally determined curve. 8. As recorded in the same set of cells and under the same set of stimulus conditions (with the use of both slit- and square-test stimuli), the mean length constant of the peak-to-peak flicker component in the horizontal-cell response is 168 +/- 18 (SE) microns with the background and 232 +/- 45 microns in the dark. The mean length constant for the background-induced flicker enhancement, as fit by dark test-region-model curves, is 186 +/- 22 microns (n = 9).(ABSTRACT TRUNCATED AT 250 WORDS)

Functional role of GABA in cat retina: II. Effects of GABAA antagonists.

Putative GABAergic mechanisms were studied in the cat retina by exogenous application of the GABAA antagonists picrotoxin (PTX), native bicuculline (BCC), and bicuculline methyl bromide (BCC MeBr). When recording intracellular responses from horizontal cells (HCs) and amacrine cells as well as electroretinograms (ERGs), drugs were added to the perfusate used to maintain the isolated eyecup; when recording extracellular spikes from ganglion cells of anesthetized cats, drugs were introduced by iontophoretic injection. Both PTX and BCC MeBr had relatively little influence upon the response properties of HCs. In contrast, native BCC tended to decrease the amplitude of and to slow the photic response to light onset and both to quicken and to increase the amplitude of response to light offset; in the presence of native BCC, HC responses were dominated by a prominent spike-like "Off-overshoot." The influence of GABAA agonists upon HC responses was not blocked by GABAA antagonists. ERG b-wave amplitude was reduced both by PTX and by native BCC, but was not influenced by BCC MeBr. Latency (time to half-peak) was increased by low doses of native BCC, and to a lesser extent PTX but not BCC MeBr. Rod-amacrine On-transient responses were increased in amplitude by PTX. Extracellular recordings from On- and Off- X and Y ganglion cell types became considerably more transient with application of either PTX, native BCC, or BCC MeBr; this tendency was greater in Off-type ganglion cells. Collectively, these results strengthen conclusions from the previous paper suggesting that GABA serves to slow onset and offset kinetics of retinal neurons, making them more sustained and less phasic. They also suggest that in mammalian retina heterogeneous types of GABAA receptors exist, segregated into different zones: a distal zone, sensitive only to native BCC, a central zone sensitive to both native BCC and PTX, and a proximal zone sensitive to native BCC, BCC methyl halides (BCC MeH), and PTX. Only the proximal zone obeys conventional GABAA pharmacology.

Experimental vitreous and aqueous replacement with perfluorophenanthrene. Clinical, histologic, and electrophysiologic results.

PURPOSE: Anterior and posterior segment changes of experimental vitreous and aqueous substitution with Perfluorophenanthrene were evaluated. METHODS: In 28 rabbit eyes that underwent vitrectomy, tamponades of 1.2 cc Perfluorophenanthrene remained as long as 8 weeks under clinical and electrophysiologic control. Histologic examinations of the eyes were done 1, 2, 4, and 8 weeks after insertion of the tamponade. In an additional 15 rabbit eyes, Perfluorophenanthrene was injected into the anterior chamber, and in some cases it was removed after 2 and 4 weeks, respectively. Follow-up examinations, which were done no later than 12 weeks after injection, included clinical appearance, endothelial cell counts, corneal pachymetry, tonometry, and histopathology. RESULTS: Histologically we found narrowing of the outer plexiform layer and single macrophages in the inferior retina after 2 weeks. Cell loss in the outer nuclear layer and wrinkling of the outer retinal layers were observed after 4 weeks, which lead to the development of irregularities of all layers inferiorly after 8 weeks. Electroretinograms showed unchanged b-wave amplitudes after maximal light stimulation at each examination, but separate interpretation of low light intensity responses showed a significant decrease in b-wave amplitudes 4 weeks after surgery. Anterior segment intolerance started with stromal edema on the second day after surgery; corneal vascularization and scar formation occurred subsequently. Inferior endothelial cell density decreased to about 50%. Histologically inflammatory reactions, vacuolization of the inferior trabecular meshwork, and closure of the chamber angle between 5 and 7 o'clock were observed. CONCLUSIONS: Perfluorophenanthrene is only suitable for very short-term vitreous replacement unless prolapsing into the anterior chamber. Damages to the retina were observed 2 weeks after surgery.