Natalia V. Kapousta-Bruneau

A dissection of the electroretinogram from the isolated rat retina with microelectrodes and drugs

The origins of the a- and b-wave of the ERG were studied using simultaneous recordings made across the receptor layer and the full thickness of a piece of isolated albino rat retina. An inwardly directed current flowing across the rod outer segments was eliminated from the recording when postsynaptic activity was blocked with cobalt or when current source density measurements were made along the length of the outer segments. Rod photovoltages were inferred by removing extraneous field potentials from the recordings made across the photoreceptor layer. The spatial properties of the photovoltage indicates the responses came from an area about 100 microm in diameter. The glutamate analog. APB, which blocks depolarizing bipolar cells, eliminated the b-wave but left the a-wave unaffected. The ERG component due to depolarizing bipolar cells was inferred by subtracting recordings obtained before and after APB. After treatment with APB a slow component remained. This component was completely blocked by barium (200 microM), which blocks potassium channels on Müller cells. Barium had virtually no effect on low-intensity photovoltages but did affect the amplitude and shape of the saturated responses. Barium increased the amplitude of the component of the ERG which underlies the b-wave. It was concluded that the depolarizing bipolar cells directly generate the b-wave of the ERG.

Opposite effects of GABAA and GABAC receptor antagonists on the b-wave of ERG recorded from the isolated rat retina

The largest component in the fully dark-adapted ERG is a corneal-positive response, known as the b-wave, and believed to originate from depolarizing (ON-type) bipolar cells. The two types of GABA receptors, GABA(A) and GABA(C) have been reported to exist on bipolar cells in rat retina. The goal of these experiments was to find whether these GABA receptors participate in the generation of the b-wave of electroretinogram (ERG). ERGs were recorded from the isolated rat retinas. The P(2)(t) component, obtained by subtracting the ERGs measured before the application of 50 micrograms APB from those measured after the application of 50 micrograms APB, was used as an indicator of depolarizing bipolar cell activity. Photovoltages, the fast P(3)(t) component of ERG, were registered between the two microelectrodes across the rod outer segments. Bicuculline and 3-aminopropylphosphonic acid (3-APA) were used as selective antagonists of GABA(A) and GABA(C) receptors, respectively. It was found that the GABA(A) and GABA(C) receptors antagonists have opposite effects on the b-wave: bicuculline increased the b-wave amplitude, while 3-APA reduced the amplitude of the b-wave. Neither bicuculline nor 3-APA affect photoreceptors.

Effects of inhibiting glutamine synthetase and blocking glutamate uptake on b-wave generation in the isolated rat retina

The purpose of the present experiments was to evaluate the contribution of the glutamate-glutamine cycle in retinal glial (Müller) cells to photoreceptor cell synaptic transmission. Dark-adapted isolated rat retinas were superfused with oxygenated bicarbonate-buffered media. Recordings were made of the b-wave of the electroretinogram as a measure of light-induced photoreceptor to ON-bipolar neuron transmission. L-methionine sulfoximine (1-10 mM) was added to superfusion media to inhibit glutamine synthetase, a Müller cell specific enzyme, by more than 99% within 5-10 min, thereby disrupting the conversion of glutamate to glutamine in the Müller cells. Threo-hydroxyaspartic acid and D-aspartate were used to block glutamate transporters. The amplitude of the b-wave was well maintained for 1-2 h provided 0.25 mM glutamate or 0.25 mM glutamine was included in the media. Without exogenous glutamate or glutamine the amplitude of the b-wave declined by about 70% within 1 h. Inhibition of glutamate transporters led to a rapid (2-5 min) reversible loss of the b-wave in the presence and absence of the amino acids. In contrast, inhibition of glutamine synthetase did not alter significantly either the amplitude of the b-wave in the presence of glutamate or glutamine or the rate of decline of the b-wave found in the absence of these amino acids. Excellent recovery of the b-wave was found when 0.25 mM glutamate was resupplied to L-methionine sulfoximine-treated retinas. The results suggest that in the isolated rat retina uptake of released glutamate into photoreceptors plays a more important role in transmitter recycling than does uptake of glutamate into Müller cells and its subsequent conversion to glutamine.

Partial rescue of the ocular retardation phenotype by genetic modifiers

The or(J) allele of the murine ocular retardation mutation is caused by a premature stop codon in the homeodomain of the Chx10 gene. When expressed on an inbred 129/Sv strain, the or(J) phenotype is characterized by microphthalmia and a thin, poorly differentiated retina in which the peripheral portion is affected to a greater extent than the central portion. Such mutant retinae lack differentiated bipolar cells and the optic nerve typically fails to form, leading to blindness. Here, we show that progeny from an outcrossed backcross between 129/Sv-or(J) /or(J) and Mus musculus castaneus produce animals that are homozygous for the or(J) mutation and exhibit a much ameliorated eye phenotype. Although not of normal size, such modified or(J) eyes are significantly larger than those in 129/Sv-or(J) /or(J) mice, and contain a better organized retina which includes bipolar cells. Furthermore, optic nerves are frequently present, and the eyes show a degree of function as reflected by electroretinogram and pupillary response. As in 129/Sv-or(J) /or(J) mice, however, modified or(J) eyes show incomplete growth and a lack of cell differentiation in the periphery of the retina. The selective, and apparently nonmodifiable, effect of the ocular retardation phenotype on the periphery of the retina indicates that Chx10 plays an important role in the central-to-peripheral gradient of retinal development. These findings demonstrate that the ocular retardation phenotype can be greatly modified by the genetic background, and help to define a role for Chx10 in ocular development.

Serum-induced changes in the physiology of mammalian retinal glial cells: role of lysophosphatidic acid

1 With a breakdown of the vascular‐CNS barrier, serum enters the nervous system. Although this is a frequent pathophysiological event, knowledge of the effects of serum on the function of the nervous system is limited. In this study, we examined the effects of serum on the activity of ion channels in Müller cells: the principal glia of the retina. 2 Freshly dissociated Müller cells from the bovine and human retina were studied with the perforated‐patch configuration of the patch clamp technique. In other experiments, electroretinograms (ERGs) were recorded from isolated rat retinas. 3 Perforated‐patch recordings revealed that serum induced a calcium‐permeable, non‐specific cation (NSC) current. Approximately 40 s after induction of this current, an outwardly rectifying K+ current was also detected. Sensitivity to charybdotoxin and margatoxin indicated that this K+ current was due to the activation of KV1.3 channels. This increase in the KV1.3 current was dependent on extracellular calcium. 4 The NSC and KV1.3 currents were activated by serum in 100 % and 95 % of the sampled Müller cells, respectively. Also, in a minority (21 %) of the cells, the inwardly rectifying K+ current was inhibited slightly. These changes in ion channel activity were associated with depolarization of the Müller cells. 5 We hypothesized that activation of NSC channels would reduce the siphoning of K+ via the Müller cells. Consistent with this idea, ERGs from isolated retinas showed serum‐induced reductions in the slow PIII component, which is generated by Müller cells responding to light‐evoked changes in the extracellular K+ concentration. 6 Lysophosphatidic acid (LPA), a component of serum, had effects on Müller cells that were qualitatively similar to those induced by serum. 7 Our observations demonstrate that exposure to serum alters the activity of multiple types of ion channels in Müller glial cells of the mammalian retina. When there is a breakdown of the blood‐retina barrier, LPA may be one of the serum‐derived molecules which regulates the physiology of Müller cells.

Electrophysiological properties of a new isolated rat retina preparation.

A piece of rat retina was mounted in an open chamber and perfused with a Ringer solution at 37 degrees C. The electroretinogram (ERG) was recorded between an extracellular microelectrode in contact with the rod outer segments and a reference electrode under the retina. The addition of 250-500 microM of glutamate to the media prevented the b-wave from decaying in amplitude with time. Minor components of the ERG, the scotopic threshold response (STR) and oscillatory potentials (OPs), were well maintained with glutamate in the media. Experiments on the spatial properties of the recordings indicated that a small area immediately around the microelectrode contributes most strongly to the response. The similarity of ERGs recorded in vivo from the cornea to the transretinal ERGs from the isolated retina of the same animal indicated that the functional integrity of the isolated retina was well preserved in the media with glutamate.

Effects of sodium pentobarbital on the components of electroretinogram in the isolated rat retina

Photovoltages, the fast P3(t) component of electroretinogram (ERG), were registered between two microelectrodes across the rod outer segments. The P2(t) component, obtained by subtracting the ERGs measured before the application of 50 microM APB from those measured after the application of 50 microM APB, was used as an indicator of depolarizing bipolar cell activity. Measurements of the scotopic threshold response (STR) and the oscillatory potentials (OPs) were used as indicators of third order neuron activity. The slow P3*(t) component, obtained by subtracting the photovoltages from the transretinal recording in the APB-treated retina was used as an indicator of Müller cell activity. The components of the ERG obtained in normal superfusate medium were compared with those obtained in the presence of 100 microM sodium pentobarbital. We found that sodium pentobarbital slowed the kinetics of the P2(t) component and increased its latency. The fast P3(t) component was not affected by pentobarbital. The slow P3*(t) component was slightly reduced in the presence of pentobarbital. The minor components of the ERG, the STR and the OPs, were strongly suppressed by pentobarbital. These results suggest that in rat retina pentobarbital does not affect photoreceptors, but it does affect bipolar cells and Müller cells, and it suppresses activity of third order neurons.

Plasma-induced changes in the physiology of mammalian retinal glial cells: Role of glutamate

Plasma can leak into the nervous system when the vascular endothelial barrier is compromised. Although this occurs commonly, little is known about the effects of plasma on the function of cells in the central nervous system. In this study, we focused on the responses of glial cells, which, because they ensheathe the blood vessels, are the first cells exposed to leaking plasma. We used the perforated‐patch configuration of the patch‐clamp technique to assess the effects of plasma on freshly dissociated bovine and human Müller cells, the principal glia of the retina. To monitor the function of Müller cells in situ, we recorded electroretinograms from isolated retinas. We found that plasma activates an electrogenic glutamate transporter and inhibits inward‐rectifying K+ channels, as well as a transient outward current. Glutamate, a normal constituent of the blood, mimicked these effects. Unlike our recent findings with serum, which contains molecules generated by the clotting process, plasma neither activated a nonspecific cation conductance nor inhibited the slow PIII component of the electroretinogram, which is generated by Müller cells responding to light‐evoked changes in the extracellular potassium concentration ([K+]O). Taken together, our observations indicate that a leakage of serum into the retina compromises the regulation of [K+]O by Müller cells; however, when plasma enters the retina at sites of a breakdown in the blood‐retinal barrier, these glia can maintain K+ homeostasis while reducing the potentially neurotoxic levels of glutamate. GLIA 25:205–215, 1999.

Evidence that L-AP5 and D,L-AP4 can preferentially block cone signals in the rat retina

Several lines of evidence suggest that, as concentrations of two agonists of group III metabotropic glutamate receptors are increased, cone contributions to the b-wave are blocked before rod contributions. Application of L-AP5 (L-2-amino-5-phosphonobutyric acid) at concentrations of 50 microM and D,L-AP4 (D,L-2-amino-4-phosphonobutyric acid) at concentrations 2 microM had a greater effect in reducing the amplitude of the rat ERG b-wave at high light intensities than at low light intensities. The amplitude reduction occurs at flash intensities that saturate rod photoreceptor responses. When steady backgrounds are used to saturate rod photoreceptors, the b-wave responses show increased long-wavelength sensitivity. Responses on a rod saturating background are blocked by adding L-AP5 or AP4 at the above concentrations to the perfusate. Further evidence for metabotrophic receptors being involved comes from the observation that even when ionotropic glutamate receptors are pharmacologically blocked with MK801 and DNQX, AP4 selectively blocks cone contributions to the b-wave. Thus we suggest that the type III metabotrophic receptors on depolarizing cone bipolar cells or cone synaptic terminals are affected by concentrations of L-AP5 and D,L-AP4 that have minimal effects on rod bipolar cells or rod synaptic terminals.