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Jun Zhang, Nicholas Oesch, Jeffrey Diamond; Abnormalities of retinal synaptic ribbons induced by L-AP4. Invest. Ophthalmol. Vis. Sci. 2013;54(15):6164. doi: https://doi.org/.
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AP4 is a widely used mGluR agonist that hyperpolarizes ON bipolar cells by activating mGluR6, but other potential effects of L-AP4 have not been explored. Here, we have investigated whether L-AP4 causes structural changes that may complicate interpretation of its pharmacological effects.
Acute retinal slices (200 μm thick) were prepared from Sprague Dawley rats (P17-P21) in ACSF media bubbled with 95% O2-5% CO2. After 20-30 minutes in ACSF, retinal slices were treated with (1) L-AP4 (10 μm) for 10 min; (2) L-AP4 (10 μm) for 20 min; (3) no AP4 treatment, and then fixed immediately in 4% paraformaldehyde plus 0.01% glutaraldehyde in 0.1 M PB at pH 7.4 for overnight. All tissues were then processed with routine EM methods. 70 nm thin sections were collected and viewed with transmission EM. Approximately 100 presynaptic RBC profiles and 150 presynaptic rod profiles were collected in each group, after which quantitative analysis was performed.
Prolonged exposure to L-AP4 caused a reduction in the number of synaptic ribbons in both RBC and rod terminals. In 10 minute groups, presynaptic RBC profiles appeared normal when compared with control. However, only 24% (36 of 152) of presynaptic rod profiles had normal synaptic ribbons (Control: 80% [122 of 153]), whereas 47% (72 of 152) of those contained a cluster of broken synaptic ribbons that were truncated or floating in the cytoplasm (Control: 2% [3 of 153]). Following 20 minutes in L-AP4, 43% (47 of 109) of presynaptic RBC profiles had normal synaptic ribbons (Control: 84.5% [87 of 103]), whereas 51% (55 of 109) of those did not contain synaptic ribbons (Control: 15.5% [16 of 103]). Interestingly, rod terminals did not show further damages at this stage when compared with those in 10 minute groups. Additionally, glia processes were often observed in the two synaptic layers in L-AP4 groups.
Our results indicate, for the first time, that prolonged addition of L-AP4 may induce degenerative changes in mammalian retina, particularly at synaptic ribbons. Thus, the appropriate time or/and concentration of L-AP4 should be carefully considered in retinal physiological experiments in which L-AP4 is used as an mGluR6 agonist. Further physiological and pathological experiments to identify the mechanism underlying the L-AP4 effects are under way.
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