Existing models of the primate photopic electroretinogram (ERG) attribute the light-adapted b–wave to activity of depolarizing bipolar cells (DBCs), mediated through a release of potassium that is monitored by Müller cells. However, possible ERG contributions from OFF-bipolar cells (HBCs) and horizontal cells (HzCs) have not been explored. We examined the contribution of these hyperpolarizing second-order retinal cells to the photopic ERG of monkey by applying glutamate analogs to suppress photoreceptor transmission selectively to HBC/HzCs vs. DBCs.ERGs of Macaca monkeys were recorded at the cornea before and after intravitreal injection of drugs. Photopic responses were elicited by bright 200–220 ms flashes on a steady background of 3.3 log scotopic troland to suppress rod ERG components.2–amino-4–phosphonobutyric acid (APB), which blocks DBC light responses, abolished the photopic b–wave and indicated that DBC activity is requisite for photopic b–wave production.However, applying cis–2,3–piperidine dicarboxylic acid (PDA) and kynurenic acid (KYN), to suppress HBCs/HzCs and third-order neurons, revealed a novel ERG response that was entirely positive and was sustained for the duration of the flash. The normally phasic b–wave was subsumed into this new response. Applying n–methyl-dl-aspartate (NMA) did not replicate the PDA+KYN effect, indicating that third-order retinal cells are not involved. This suggests that HBC/HzC activity is critical for shaping the phasic b–wave.Components attributable to depolarizing vs. hyperpolarizing cells were separated by subtracting waveforms after each drug from responses immediately before. This analysis indicated that DBCs and HBC/HzCs each can produce large but opposing field potentials that nearly cancel and that normally leave only the residual phasic b–wave response in the photopic ERG.Latency of the DBC component was 5–9 ms slower than the HBC/HzC component. However, once activated, the DBC component had a steeper slope. This resembles properties known for the two types of cone synapses in lower species, in which the sign-preserving HBC/HzC synapse has faster kinetics but probably lower gain than the slower sign-inverting G-protein coupled DBC synapse.A human patient with “unilateral cone dystrophy” was found to have a positive and sustained ERG that mimicked the monkey ERG after PDA+KYN, indicating that these novel positive photopic responses can occur naturally even without drug application.These results demonstrate that hyperpolarizing second-order neurons are important for the primate photopic ERG. A “Push-Pull Model” is proposed in which DBC activity is requisite for b–wave production but in which HBC/HzC activity limits the amplitude and controls the shape of the primate photopic b–wave.

Visual thresholds of mice for the detection of small, brief targets were measured with a novel behavioral methodology in the dark and in the presence of adapting lights spanning ∼8 log10 units of intensity. To help dissect the contributions of rod and cone pathways, both wild-type mice and mice lacking rod (Gnat1−/−) or cone (Gnat2cpfl3) function were studied. Overall, the visual sensitivity of mice was found to be remarkably similar to that of the human peripheral retina. Rod absolute threshold corresponded to 12-15 isomerized pigment molecules (R*) in image fields of 800 to 3000 rods. Rod “dark light” (intrinsic retinal noise in darkness) corresponded to that estimated previously from single-cell recordings, 0.012R*s−1rod−1, indicating that spontaneous thermalisomerizations are responsible. Psychophysical rod saturation was measured for the first time in a nonhman species and found to be very similar to that of the human rod monochromat. Cone threshold corresponded to ∼5 R* cone−1 in an image field of 280 cones. Cone dark light was equivalent to ∼5000 R*s−1 cone−1, consistent with primate single-cell data but 100-fold higher than predicted by recent measurements of the rate of thermal isomerization of mouse cone opsins, indicating that nonopsin sources of noise determine cone threshold. The new, fully automated behavioral method is based on the ability of mice to learn to interrupt spontaneous wheel running on the presentation of a visual cue and provides an efficient and highly reliable means of examining visual function in naturally behaving normal and mutant mice.

MATERIALS AND METHODSElectroretinography. Corneal electroretinograms (ERGs) were elicited by full-field 10-gs xenon photostrobe flashes and recorded at 0.1-1000 Hz by using methods and control values presented previously (6). Molecular Biology. Genomic DNA was isolated from peripheral blood samples. The Gly9OAsp mutation was found by sequencing the entire rhodopsin coding region, with target DNA generated by PCR amplification (7) of genomic DNA during 40 rounds of thermal cycling [94°C for 90 s, 54°C for 60 s, and 72°C for 180 s] followed by 72°C for 10 min. Exon 1 primers were 5'-AGCTCAGGCCTTCGCAGCAT-3' and 5'-GAGGGCTTTGGATAACATTG-3'. The codon 90 region was sequenced by the Sanger dideoxynucleotide chaintermination method (8) with primer 5'-ACGCAGCCCCTTC-GAGTAC-3'. The presence of the Gly9OAsp mutation was assayed in a population of normal subjects by allele-specific oligonucleotide hybridization with primers 5'-TGGT-GAAGCCACCTAGGAC-3' for Gly-90 and 5'-TGGTGA-AGTCACCTAGGAC-3' for Asp-90.Densitometry. The 530-nm test and 880-nm reference beams of a modified Florida retinal densitometer (9) were reflected from the human retina in vivo onto a photomultiplier tube. The 70 test field was centered on the 9.10 bleaching field. Measurements were made at 17.80 in the temporal retina of the left eye of the 38-year-old proband. A bleaching beam was either "white" or "monochromatic" from 10-nm interference filters (Baird-Atomic type B-1). Dark-corrected photon counts of test and reference beams were counted separately in 2-s intervals, and the ratio was averaged to give T, with To at the end of a long full bleach and Td after full dark recovery.(To -Td)/To = (1 -s)[1 -e-23(Am) [1] in which s is the fraction of dark-corrected test counts evoked by photons that have not passed twice through rhodopsin (i.e., stray light) and ,B is rhodopsin density (loge) expressed as a(Am)cl where a(Am) is the molecular extinction coefficient at the test wavelength Am, c is the concentration, and 1 is the optical path length through rhodopsin in the rod outer segment. Since s is not easily measured, it is common to assume that s = 0 and then to solve Eq. 1 for 2,. Converting to loglo gives the "two-way density" A, which is the frequently used parameter of retinal densitometry. At equilibrium, the kinetic equation of rhodopsin bleaching (10) is:Abbreviations: adRP, autosomal dominant retinitis pigmentosa; ERG, electroretinogram; sc-td, scotopic trolands; tvi, threshold versus retinal illuminance curve; Gly9OAsp, Gly-90 -> Asp. tTo whom reprint requests should be addressed at: W. K.

The goal of the present study was to relate the dark and light-adapted flash sensitivity of the scotopic threshold response (STR) and rod b-wave of the electroretinogram (ERG) to behaviorally measured rod increment threshold responses. Small amplitudes of the dark-adapted STR and b-wave, the latter after application of NMDA, were found to increase in proportion to flash intensity. The value obtained for the sensitivity of the b-wave would be expected if signals from rods were summed linearly by the rod bipolar cell. The sensitivity of the STR could not be accounted for in terms of rod signal convergence as the source of this ERG component is still unknown. Increment threshold responses of rats were measured behaviorally in an operant conditioning chamber. At absolute threshold, on average I in 2400 rods were activated by the test flash. Comparison of the adaptive effects of background lights on behaviorally measured scotopic sensitivity and rod ERG sensitivity suggest that the increment threshold sensitivity of rat is regulated at three different sites in the retina.

Corneal electroretinograms (ERGs) were recorded from anesthetized cats under scotopic conditions. We examined whether the scotopic threshold response (STR) of the ERG could be functionally distinguished from scotopic PII and a-wave using intravitreal application of neuroactive agents. We found that neurotransmitters with active sites on third order neurons had several different effects. Results were: (1) glycine and gamma-amino butyric acid (GABA) selectivity suppressed the STR but had relatively small and/or opposite effects on PII; (2) serotonin, acetylcholine and dopamine were nonselective and suppressed both STR and PII; (3) strychnine blocked the suppression of the STR by glycine. GABA-a antagonists alone only partially blocked GABA effects on the STR, and GABA-b antagonists were ineffective; (4) strychnine enhanced the STR. Bicuculline also increased STR amplitudes, but only in the presence of haloperidol. Our results suggest that the retinal pathway that contributes to the rod-driven STR is strongly influenced by cells that release glycine or GABA in the dark. These cells are possibly third order neurons in the retina. Our results also suggest that picrotoxin and bicuculline can facilitate the release of dopamine in the cat retina. Furthermore, the data indicate a light evoked release of dopamine which was first noticeable at about two log units above ERG threshold.