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Concerns about toxicity in the very young ferrets made the use of the sodium channel blocker tetrodotoxin impractical as a method for blocking retinal ganglion cell activity. All surgeries were performed according to protocols approved by the University of California at Davis Animal Care and Use Committee, Daily (24 ± 2 hours) eye injections of APB were done, Isofluorane anesthesia was used. Injections were made just posterior to the scierai margin with a 33-gauge needle on a Hamilton syringe. All subsequent injections were made into the same hole. APB dosages were calculated as described elsewhere (9). Injection volumes ranged from 1 to 2.8 μl. After the injections, animals received antibiotic ophthalmic ointment (Chloroptic) and prophylactic broad-spectrum antibiotics intramuscularly (Flocillin). The animals were returned to their mothers and littermates as soon as they awoke from the anesthesia (3 to 5 min).
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10
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0343204550
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note
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In the adult retina, APB is a quite specific blocker of the ON-center pathway (14). However, in very young ferrets (PND 5 to 7) calcium imaging in vitro demonstrates that even very small concentrations of APB (1 μM) block all retinal ganglion cell spontaneous activity (Fig. 2). By PND 21, low concentrations of APB begin to have a more selective effect, but concentrations that fully block ON-center ganglion cells still decrease activity in OFF-center cells (15). The mechanisms by which APB blocks OFF-center activity in the young ferret retina are unknown.
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11
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0342334957
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note
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APB-injected and normal age-matched control animals were euthnanized and perfused with 4% paraformaldehyde. Retinae were dissected out of the eyes, 1-mm "punches" were embedded in 5% agar, and 30-μm cross sections were cut on a vibratome. Sections were mounted on slides, stained for Nissl substance with thionin, cover-slipped, and photographed.
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13
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0343640116
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note
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Five microliters of 5% wheat germ agglutinin-horseradish peroxidase (WGA-HRP) (Sigma) was injected on PND 23 over a period of 5 min, following the same injection protocol used for the APB injections. Animals were euthanized 48 hours after the WGA-HRP injections and perfused with 4% paraformaldehyde. LGNs were embedded in gelatin/albumin and 50-μm vibratome sections were cut. Floating sections were reacted for HRP with a TMB protocol. Sections were mounted on slides, dried overnight, dehydrated, cover-slipped, and photographed. Ipsilateral and contralateral projections were measured with Scion Image software.
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14
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0343204549
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Animals were euthanized and perfused with 4% paraformaldehyde. LGNs were embedded in gelatin/albumin and 50-μm vibratome sections were cut. Sections were mounted on slides, dried overnight, dehydrated, Nissl-stained with cresyl violet, cover-slipped, and photographed.
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0342769864
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note
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Supported by NIH grant EY-11369. E. Anderson, V. Chan, T. Hallam, and S. Shook collected pilot data. C. Wefers gave expert advice on retinal dissections and histology. A. Haines and S. Shah performed LGN measurements. L. Chalupa and L. Stone provided helpful comments on this manuscript.
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