The ERG is a summed response from a number of different retinal cells. The ERG a-wave is generated by the photoreceptors,
19,20 whereas the b-wave is generated primarily by the bipolar cells.
21,22 The major contribution to the b-wave is likely the ON or depolarizing bipolar cells (DBCs), which produce a large positive-going wave. The positive contribution to the b-wave was demonstrated nicely in pharmacologic experiments using 2-amino-4-phosphoonobutyric acid
30 or by mutant mice such as
nob,
26 in which DBC activity was blocked, leaving only the negative-going a-wave derived from the photoreceptors. However, the HBC also contributed to this response as a negative wave that can be demonstrated by using
cis-2,3-piperidine dicarboxylic acid and kynurenic acid to block their contribution, which results in a larger than normal b-wave.
31 Thus, one possible explanation for the supernormal responses in the
Ant1 −/− mice is that
Ant1 is specifically expressed in the HBCs, such that its loss in the
Ant1 −/− mouse would cause inactivity of these cells, thereby eliminating their negative input to the b-wave, and thus producing a larger b-wave response in
Ant1 −/− mice.
Similar abnormalities in inhibitory pathways have been implicated in clinically recorded supernormal ERG responses in patients with cone dystrophy.
32 –34 Supernormal responses are relatively rare compared with the number of diminished responses seen in retinal disease. A comprehensive retrospective report on supernormal ERG responses by Heckenlively et al.
35 reported that maculopathy could be associated with responses greater than 2 SD above normal for photopic, scotopic, and bright flash stimuli. Because the macula is cone dense, this report could be interpreted as further support for the inhibitory role of the hyperpolarizing cone pathway in the ERG response.
It has been reported that astrocytes of
Ant1 −/− mice have a decrease in glutamate uptake.
36 In the retina, glutamate is released in the darkness because of the flow of calcium through the photoreceptor ion channels. In the light, photoreceptor ion channels close, hyperpolarizing the photoreceptor and causing glutamate release to decrease. Thus, one consequence of the
Ant1 mutation in the retina may be an abundance of glutamate, resulting in more glutamate remaining in the synaptic cleft, which might lead to a more hyperpolarized state of the ON bipolar cells and a smaller b-wave. However, the
Ant1 −/− mice have supernormal b-wave responses. Furthermore, an excess of glutamate could lead to neuronal excitotoxicity and cell death; however, no significant differences were observed in individual layer thicknesses between WT and mutant retinas. Additionally, ATP has been implicated in synaptic transmission and the modulation of neurotransmitter release
37 ; thus, the loss of ATP may produce a change in the kinetics of phototransduction. However, normalizing the leading edge of the a-wave in
Ant1 −/− mice (
Fig. 3C) illustrated that the kinetics of the response were similar in affected and WT mice.
Another consequence of the lack of
Ant1 could be a chronic energy deficiency (as seen in the skeletal and cardiac muscle of Ant1
−/− mice
4 ) that decreases the mitochondrial ATP available for ion transport in retinal neurons. Decreased ATP could open ATP-sensitive potassium channels in retinal neurons,
38 which might simulate the dark-adapted state. However, in this scenario, it might be expected that the
Ant1 −/− retina would have a delay in the implicit time to a single flash or a delay in recovering from a flash of light because of the increased flow of ions. No changes in implicit times or in recovery response (
Fig. 3D) were seen between
Ant1 −/− and age-matched WT mice.
Alternatively, the larger b-wave amplitudes may be caused by an overcompensation of other ANT isoforms in the inner retinal cells to compensate for the loss of the ANT1 isoform. If ANT1 is present only in DBCs, then an abundance of ATP from the additional expression of ANT isoforms could make the DBCs super responders. However, if this hypothesis is correct, an increase in the level of COX and SDH staining might be expected in the plexiform layers, indicating increased OXPHOS activity. In fact, no qualitative or quantitative changes in COX and SDH staining were detected in the
Ant1 −/− retina (
Fig 5;
Table 3). Given that ANT1 is located in a subset of inner retinal cells, it is possible that COX and SDH are not sensitive enough at the light microscopy level to detect any changes. In addition, no upregulation of
Ant2 mRNA was detected in the brains of
Ant1 −/− mice, suggesting no compensation of the loss of the
Ant1 isoform in neural tissue.
4