Normal photoresponses and altered b-wave responses to APB in the mdx(Cv3) mouse isolated retina ERG supports role for dystrophin in synaptic transmission

Abstract

The mdx(Cv3) mouse is a model for Duchenne muscular dystrophy (DMD). DMD is an X-linked disorder with defective expression of the protein dystrophin, and which is associated with a reduced b-wave and has other electro- retinogram (ERG) abnormalities. To assess potential causes for the abnormalities, we recorded ERGs from pieces of isolated C57BL/6J and mdx(Cv3) mouse retinas, including measurements of transretinal and intraretinal potentials. The ERGs from the isolated mdx(Cv3) retina differ from those of control retinas in that they show reduced b-wave amplitudes and increased b-wave implicit times. Photovoltages obtained by recording across the photoreceptor outer segments of the retinas did not differ from normal, suggesting that the likely causes of the reduced b-wave are localized to the photoreceptor to ON-bipolar synapse. At a concentration of 50 microM, the glutamate analog dl-2-amino-4-phosphonobutyric acid (APB) blocks the b-wave component of the ERG, by binding to sites on the postsynaptic membrane. The On-bipolar cell contribution to the ERG was inferred by extracting the component that was blocked by APB. We found that this component was smaller in amplitude and had longer response latencies in the mdx(Cv3) mice, but was of similar overall time course. To assess the sensitivity of sites on the postsynaptic membrane to glutamate, the concentration of APB in the media was systematically varied, and the magnitude of blockage of the light response was quantified. We found that the mdx(Cv3) retina was 5-fold more sensitive to APB than control retinas. The ability of lower concentrations of APB to block the b-wave in mdx(Cv3) suggests that the ERG abnormalities may reflect alterations in either glutamate release, the glutamate postsynaptic binding sites, or in other proteins that modulate glutamate function in ON-bipolar cells.

Fig. 1

(a) Representative ERG responses to bright flashes of light. The tracings are single responses from a mdx^Cv3 and a C57BL/6J mouse (log I = -1.5). This intensity corresponds to a quantal flux of 35,000 quanta/ μm²/s incident on the piece of retina (see Materials and methods for other details). (b) B-wave intensity-response functions for mdx^Cv3 (n = 7) and C57BL/6J (n = 7) mice. The points are the averages ± the standard error of the mean. An intensity of -5.5 corresponds to about 0.1 R*/rod/flash. The b-wave intensity functions in mdx^Cv3 mice were 40 % smaller and shifted to the right on the intensity axis by about 0.5 log units. (c) The time from the start of the flash to the peak of the b-wave (implicit time) as a function of intensity for mdx^Cv3 (n = 7) and C57BL/6J (n = 7) mice. The points are the averages ± the standard error of the mean. There were significant differences between control and mdx^Cv3 mice in b-wave amplitudes and implicit times at all intensities.

Fig. 2

Photovoltages from mdx^Cv3 (n = 4) and C57BL/6J (n = 4) mice. The curves are the averages of a set of responses that were obtained at a range of intensities (log I = -3.5 to -1.5). An intensity of -1.4 log corresponds to a bleaching strength estimated to be about 1000 R*/rod/flash. In both mdx^Cv3 (panel a) and C57BL/6J (panel b), a stimulus intensity of about 25 R*/rod/flash produced a half-saturated response.

Fig. 3

P₂, the component blocked by APB, as a function of nine stimulus intensities (log I = -5.5 to -1.5). Panels a and b show the average P₂'s for mdx^Cv3 mice (n = 5) on two different time scales. Panels c and d show P₂ for C57BL/6J mice (n = 5) . In panels a and c, the P₂(t) responses are shown on a shorter time scale and in b and d on a longer time scale.

Fig. 4

Panels a and b show P₂(t) responses at low intensities (log I = -5.5 to 4.5) for mdx^Cv3 (n = 5) and C57BL/6J (n = 5) mice, respectively. These stimulus intensities correspond to R*/rod/flash of 0.1 to 1.0 . At the lowest intensities the amplitudes of the responses grew linearly with light intensity. Panel c shows the P₂ intensity-response functions for mdx^Cv3 (n = 5) and C57BL/6J (n = 5) mice. The curves fit to the points are of the form V/V_max = I/(I σ) with log sigma = [-4.01, -4.6 ] and V_max = [0.2324, 0.31], respectively. The curved segment to the left of each curve shows a linear growth with flash intensity.

Fig. 5

Representative ERG responses with various concentrations of APB in the perfusate. The tracings in panela are from a mdx^Cv3 mouse. The tracings in panel b are from a C57BL/6J mouse. The stimulus intensity in both was 3 R*/rod/flash.

Fig. 6

APB dose–response functions. The points plot the normalized b-wave amplitudes as a function of APB concentration for a flash of an intensity of 3 R*/rod/flash. The triangles are individual b-wave amplitudes from seven C57BL/6 J mice on which measurements were made at a variety of APB concentrations. A smooth curve is drawn through the averages. The mean and standard deviation of the response amplitudes before APB was administered were 165 ± 78 μV. The circles are individual b-wave amplitudes from seven mdx^Cv3 mice. The mean and standard deviation of the amplitudes before APB were 114 ± 45 μV. The mdx^Cv3 mice are more sensitive to APB.