Laura J. Frishman

Response linearity and kinetics of the cat retina: the bipolar cell component of the dark-adapted electroretinogram.

The electroretinogram (ERG) of the dark-adapted cat eye in response to brief ganzfeld flashes of a wide range of intensities was recorded after intravitreal injection of n-methyl DL aspartate (NMDLA, cumulative intravitreal concentration of 1.3-3.9 mM) to suppress inner-retinal components, and after intravitreal DL or L-2-amino-4-phosphonobutyric acid (DL-APB, 1-3 mM; L-APB, 1.2 mM) and 6-cyano-7-nitroquinoxaline-2,3 dione (CNQX, 40-60 microM), to suppress all post-receptoral neuronal responses. Rod PII, the ERG component arising from rod bipolar cells, was derived by subtracting records obtained after APB and CNQX from post-NMDLA records. When we measured the derived response at fixed times after the stimulus, we found that PII initially increased in proportion to stimulus intensity without any sign of a threshold. The leading edge of PII at early times after the stimulus, when the response was still small, was well described by V(t) = kI(t-td)5 where k is a constant, I is the intensity of the stimulus, and td is a brief delay of about 3 ms. Correspondingly, the time for the response to rise to an arbitrary small criterion voltage Vcrit was adequately fitted by tcrit = td + (Vcrit/kI)1/5. The time course of the leading edge of the PII response can be interpreted to indicate that the mechanism generating PII introduces three stages of temporal integration in addition to the three stages that are provided by the mechanism of the rod photoreceptors. This finding is consistent with the operation within the rod bipolar cell of a G-protein cascade similar to that in the rods.

The scotopic threshold response of the dark-adapted electroretinogram of the mouse

The most sensitive response in the dark‐adapted electroretinogram (ERG), the scotopic threshold response (STR) which originates from the proximal retina, has been identified in several mammals including humans, but previously not in the mouse. The current study established the presence and assessed the nature of the mouse STR. ERGs were recorded from adult wild‐type C57/BL6 mice anaesthetized with ketamine (70 mg kg−1) and xylazine (7 mg kg−1). Recordings were between DTL fibres placed under contact lenses on the two eyes. Monocular test stimuli were brief flashes (λmax 462 nm; ‐6.1 to +1.8 log scotopic Troland seconds(sc td s)) under fully dark‐adapted conditions and in the presence of steady adapting backgrounds (‐3.2 to ‐1.7 log sc td). For the weakest test stimuli, ERGs consisted of a slow negative potential maximal ≈200 ms after the flash, with a small positive potential preceding it. The negative wave resembled the STR of other species. As intensity was increased, the negative potential saturated but the positive potential (maximal ≈110 ms) continued to grow as the b‐wave. For stimuli that saturated the b‐wave, the a‐wave emerged. For stimulus strengths up to those at which the a‐wave emerged, ERG amplitudes measured at fixed times after the flash (110 and 200 ms) were fitted with a model assuming an initially linear rise of response amplitude with intensity, followed by saturation of five components of declining sensitivity: a negative STR (nSTR), a positive STR (pSTR), a positive scotopic response (pSR), PII (the bipolar cell component) and PIII (the photoreceptor component). The nSTR and pSTR were approximately 3 times more sensitive than the pSR, which was approximately 7 times more sensitive than PII. The sensitive positive components dominated the b‐wave up to > 5 % of its saturated amplitude. Pharmacological agents that suppress proximal retinal activity (e.g. GABA) minimized the pSTR, nSTR and pSR, essentially isolating PII which rose linearly with intensity before showing hyperbolic saturation. The nSTR, pSTR and pSR were desensitized by weaker backgrounds than those desensitizing PII. In conclusion, ERG components of proximal retinal origin that are more sensitive to test flashes and adapting backgrounds than PII provide the ‘threshold’ negative and positive (b‐wave) responses of the mouse dark‐adapted ERG. These results support the use of the mouse ERG in studies of proximal retinal function.

Dissecting the dark-adapted electroretinogram

Although gross recordings of the ganzfeld flash-evoked electroretinogram (ERG) can potentially provide information about the activity of many, if not all, retinal cell types, it is necessary to dissect the ERG into its components to realize this potential fully. Here we describe various procedures that have been used in intact mammalian eyes to identify and characterize the contributions to the dark-adapted ERG of different cells in the retinal rod pathway. These include (1) examination of the very early part of the response to a flash (believed to reflect directly the photocurrent of rods), (2) application of high-energy probe flashes to provide information about the underlying rod photoreceptor response even when this component is obscured by the responses of other cells, (3) pharmacological suppression of responses of amacrine and ganglion cells to identify the contribution of these cells and to reveal the weaker responses of bipolar cells, (4) use of pharmacological agents that block transmission of signals from rods to more proximal neurons to separate responses of rods from those of later neurons, (5) examination of the ERG changes produced by ganglion-cell degeneration or pharmacological block of nerve-spike generation to identify the contribution of spiking neurons, (6) modeling measured amplitude-energy functions and timecourse of flash responses and (7) using steady backgrounds to obtain differential reductions in sensitivity of different cell types. While some of these procedures can be applied to humans, the results described here have all been obtained in studies of the ERG of anaesthetized cats, or macaque monkeys whose retinas are very similar to those of humans.

Visual field defects and neural losses from experimental glaucoma

Glaucoma is a relatively common disease in which the death of retinal ganglion cells causes a progressive loss of sight, often leading to blindness. Typically, the degree of a patients visual dysfunction is assessed by clinical perimetry, involving subjective measurements of light-sense thresholds across the visual field, but the relationship between visual and neural losses is inexact. Therefore, to better understand of the effects of glaucoma on the visual system, a series of investigations involving psychophysics, electrophysiology, anatomy, and histochemistry were conducted on experimental glaucoma in monkeys. The principal results of the studies showed that, (1) the depth of visual defects with standard clinical perimetry are predicted by a loss of probability summation among retinal detection mechanisms, (2) glaucomatous optic atrophy causes a non-selective reduction of metabolism of neurons in the afferent visual pathway, and (3) objective electrophysiological methods can be as sensitive as standard clinical perimetry in assessing the neural losses from glaucoma. These experimental findings from glaucoma in monkeys provide fundamental data that should be applicable to improving methods for assessing glaucomatous optic neuropathy in patients.

Expression of vesicular glutamate transporter 1 in the mouse retina reveals temporal ordering in development of rod vs. cone and ON vs. OFF circuits

Glutamatergic transmission is crucial to the segregation of ON and OFF pathways in the developing retina. The temporal sequence of maturation of vesicular glutamatergic transmission in rod and cone photoreceptor and ON and OFF bipolar cell terminals is currently unknown. Vesicular glutamate transporters (VGLUTs) that load glutamate into synaptic vesicles are necessary for vesicular glutamatergic transmission. To understand better the formation and maturation of glutamatergic transmission in the rod vs. cone and ON vs. OFF pathways of the retina, we examined the developmental expression of VGLUT1 and VGLUT2 immunocytochemically in the mouse retina. Photoreceptor and bipolar cell terminals showed only VGLUT1‐immunoreactivity (‐IR); no VGLUT2‐IR was present in any synapses of the developing or adult retina. VGLUT1‐IR was first detected in cone photoreceptor terminals at postnatal day 2 (P2), several days before initiation of ribbon synapse formation at P4–P5. Rod terminals showed VGLUT1‐IR by P8, when they invade the outer plexiform layer (OPL) and initiate synaptogenesis. Developing OFF bipolar cell terminals showed VGLUT1‐IR around P8, 2–3 days after bipolar terminals were first identified in the inner plexiform layer (IPL) by labeling for the photoreceptor and bipolar cell terminal marker, synaptic vesicle protein 2B. Although terminals of ON bipolar cells were present in the IPL by P6–P8, most did not show VGLUT1‐IR until P8–P10 and increased dramatically from P12. These data suggest a hierarchical development of glutamatergic transmission in which cone circuits form prior to rod circuits in both the OPL and IPL, and OFF circuits form prior to ON circuits in the IPL. J. Comp. Neurol. 465:480–498, 2003.

Rod and cone contributions to the a‐wave of the electroretinogram of the macaque

The electroretinogram (ERG) of anaesthetised dark‐adapted macaque monkeys was recorded in response to ganzfeld stimulation and rod‐ and cone‐driven receptoral and postreceptoral components were separated and modelled. The test stimuli were brief (< 4.1 ms) flashes. The cone‐driven component was isolated by delivering the stimulus shortly after a rod‐saturating background had been extinguished. The rod‐driven component was derived by subtracting the cone‐driven component from the mixed rod–cone ERG. The initial part of the leading edge of the rod‐driven a‐wave scaled linearly with stimulus energy when energy was sufficiently low and, for times less than about 12 ms after the stimulus, it was well described by a linear model incorporating a distributed delay and three cascaded low‐pass filter elements. Addition of a simple static saturating non‐linearity with a characteristic intermediate between a hyperbolic and an exponential function was sufficient to extend application of the model to most of the leading edge of the saturated responses to high energy stimuli. It was not necessary to assume involvement of any other non‐linearity or that any significant low‐pass filter followed the non‐linear stage of the model. A negative inner‐retinal component contributed to the later part of the rod‐driven a‐wave. After suppressing this component by blocking ionotropic glutamate receptors, the entire a‐wave up to the time of the first zero‐crossing scaled with stimulus energy and was well described by summing the response of the rod model with that of a model describing the leading edge of the rod‐bipolar cell response. The negative inner‐retinal component essentially cancelled the early part of the rod‐bipolar cell component and, for stimuli of moderate energy, made it appear that the photoreceptor current was the only significant component of the leading edge of the a‐wave. The leading edge of the cone‐driven a‐wave included a slow phase that continued up to the peak, and was reduced in amplitude either by a rod‐suppressing background or by the glutamate analogue, cis‐piperidine‐2,3‐dicarboxylic acid (PDA). Thus the slow phase represents a postreceptoral component present in addition to a fast component of the a‐wave generated by the cones themselves. At high stimulus energies, it appeared less than 5 ms after the stimulus. The leading edge of the cone‐driven a‐wave was adequately modelled as the sum of the output of a cone photoreceptor model similar to that for rods and a postreceptoral signal obtained by a single integration of the cone output. In addition, the output of the static non‐linear stage in the cone model was subject to a low‐pass filter with a time constant of no more than 1 ms. In conclusion, postreceptoral components must be taken into account when interpreting the leading edge of the rod‐ and cone‐driven a‐waves of the dark‐adapted ERG.

Identifying Inner Retinal Contributions to the Human Multifocal ERG

Contributions to the multifocal electroretinogram (ERG) from the inner retina (i.e. ganglion and amacrine cells) were identified by recording from monkeys before and after intravitreal injections of n-methyl DL aspartate (NMDLA) and/or tetrodotoxin (TTX). Components similar in waveform to those removed by the drugs were identified in the human multifocal ERG if the stimulus contrast was set at 50% rather than the typically employed 100% contrast. These components were found to be missing or diminished in the records from some patients with glaucoma and diabetes, diseases which affect the inner retina.

Evidence for a ganglion cell contribution to the primate electroretinogram (ERG): effects of TTX on the multifocal ERG in macaque.

To assess the contribution of spiking inner retinal neurons to the multifocal electroretinogram (ERG), recordings were made from four monkeys (Macaca mulatta) before and after intravitreal injections of tetrodotoxin (TTX). TTX blocks all sodium-based action potentials and thus terminates spiking activity of amacrine and ganglion cells. TTX eliminated a large component from the control responses, and this TTX-sensitive component was present as early as 10 ms after the stimulus. Before injection with TTX, the 103 focal ERG responses varied in waveform across the retina. After TTX, the response waveforms were largely independent of retinal position, indicating that it was primarily the TTX-sensitive component of the control response that was dependent upon retinal location. Given that retinal ganglion cells compose a sizable proportion of the retinal elements that produce action potentials, it is likely that part of the TTX-sensitive component is due to the spiking activity of these cells. Further, the systematic change in waveform of the TTX-sensitive component with distance from the optic nerve head suggests that part of the TTX-sensitive component may originate from the activity of the ganglion cell axons. Based on these findings, there is reason to be optimistic that the multifocal technique can be employed to study the effects of glaucoma and other diseases that affect the inner retina.

Photoreceptor and bipolar-cell contributions to the cat electroretinogram: a kinetic model for the early part of the flash response

The time course of the initial negative wave of the flash electroretinogram of the dark-adapted cat has been found to be critically dependent of contributions from cells of the inner retina, not only for very low-intensity flashes for which the negative scotopic threshold response is dominant but also when the stimulus is sufficiently intense for the rods themselves to contribute directly to the electroretinogram. However, if the inner-retinal responses are blocked pharmacologically or are suppressed by a steady adapting background, the initial negative wave of the remaining electroretinogram (the alpha wave) can be explained as the sum of photoreceptor and bipolar-cell components that can be modeled as described by Lamb and Pugh [J. Physiol. (London) 449, 717 (1992)] and Robson and Frishman [Vis. Neurosci. 12, 837 (1995)], respectively.

Rod vision is controlled by dopamine-dependent sensitization of rod bipolar cells by GABA

Dark and light adaptation of retinal neurons allow our vision to operate over an enormous light intensity range. Here we report a mechanism that controls the light sensitivity and operational range of rod-driven bipolar cells that mediate dim-light vision. Our data indicate that the light responses of these cells are enhanced by sustained chloride currents via GABA(C) receptor channels. This sensitizing GABAergic input is controlled by dopamine D1 receptors, with horizontal cells serving as a plausible source of GABA release. Our findings expand the role of dopamine in vision from its well-established function of suppressing rod-driven signals in bright light to enhancing the same signals under dim illumination. They further reveal a role for GABA in sensitizing the circuitry for dim-light vision, thereby complementing GABAs traditional role in providing dynamic feedforward and feedback inhibition in the retina.