Identification of synaptic acetylcholine receptor sites in retina with peroxidase-labeled α-bungarotoxin

Abstract

An a-bungarotoxin-horseradish peroxidase colijugate, which binds specifically to nicotinic acetylcholine receptors, was synthesized. This conju ate was bound by 5-7% of the synapses in the inner plexiform layer of the chicken retina. Bipolar ribbon synapses as well as amacrine synapses bound the conjugate. The idea that certain neurotransmitter receptors are located predominantly at chemical synapses has been supported by acetylcholine (AcCh) sensitivity mapping of the skeletal muscle membrane (1) and parasympathetic neurons (2) and by electron microscopic identification of nicotinic AcCh receptor sites in the skeletal neuromuscular junction by a-bungarotoxin (aBT) binding (3-5). Radioactively labeled aBT has been used to detect nicotinic AcCh receptors in the central nervous system (6, 7) and to determine their location by light microscopic autoradiography (8)(9)(10)(11)(12). However, there is no evidence, either from high resolution sensitivity mapping or from electron microscopy, for the precise location of the neurotransmitter receptors on neurons in any part of the central nervous system.There is evidence for cholinergic synaptic transmission in the vertebrate retina (for review see ref.13; see also refs. 14-16). The synaptic layers of the retinas of several species, particularly the inner plexiform layer of the chicken retina, contain high concentrations of nicotinic AcCh receptors (9-11). We describe here the use of AI3T crosslinked to horseradish peroxidase (HRP) to identify nicotinic AcCh receptor sites in the chicken retina. We present evidence for synaptic location of nicotinic AcCh receptors and a partial characterization of the synapses that contain the receptors. METHODSSynthesis of aBT-HRP. aBT was purified from Bungarus multicinctus venom (Miami Serpentarium) G-25 fine was prepared with 0.15 M NaCl (saline); 0.2 ml of 1.25% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 6.8, was passed through, and the column was washed thoroughly with saline. The HRP/glutaraldehyde solution was rapidly filtered through this column at 40 with saline. The brown band of "activated" HRP was collected in a tube containing S mg of aBT with 2 X 106 cpm of 125I-labeled aBT in 4 ml of saline. One molar sodium carbonate buffer, pH 9.0, was immediately added to a final concentration of 0.05 M. After 5 hr at 40, the pH of the solution was adjusted to 7.0 with 1 M NaH2PO4. One molar NaCNBH3 (Alpha Chemicals), a specific reagent for the reduction of Schiff bases (20), was freshly prepared in 1 M sodium phosphate buffer, pH 7.0, and was added to the reaction mixture to a final concentration of 10 mM. The solution was kept at 00 for 10-20 hr and then NH4Cl was added to a final concentration of 20 mM. The solution was passed through a column of Sephadex G-100 with 0.15 M NaCl, 0.01 M sodium phosphate buffer, pH 7.4, and the fractions with both high absorbance at 403 nm (indicating HRP) and high cpm (indicating aBT) were pooled and dialyzed against 10 mM sodium acetate, pH 5.0. This mixture, containing aBT-HRP an...

RELEASE OF [3H]-ACETYLCHOLINE FROM THE ISOLATED RETINA OF THE RAT BY POTASSIUM DEPOLARIZATION: DEPENDENCE ON HIGH AFFINITY CHOLINE UPTAKE

“…(Baughman & Bader, 1977). This is consistent with other evidence which suggests that cholinergic synapses in the retina occur predominantly in the inner plexiform layer and that a sub-population of amacrine cells is cholinergic (Nichols & Koelle, 1968;Neal, 1976a;Ross & McDougal, 1976;Masland & Ames, 1976;Vogel, Maloney, Ling & Daniels, 1977;Massey & Neal, 1978). If the sites of [3H]-choline uptake in the rat retina are similar to those in the chicken (but see Neal (1976b) for the importance of species differences in the retina) then the [3H]-ACh released by potassium depolarization presumably originated from amacrine and/or bipolar cells.…”

Expression of Alpha 7 Nicotinic Acetylcholine Receptors by Bipolar, Amacrine, and Ganglion Cells of the Rabbit Retina

“…Specifically, observations in chick (Vogel et al 1977), mouse (Pourcho 1979), goldfish (Zucker and Yazulla 1982), and turtle (James and Klein 1985) all demonstrated aBgt binding associated with bipolar cells or amacrine-bipolar synapses. In addition, previous work from this laboratory (Keyser et al 1993) showed aBgt-sensitive nAChR (a8 subunits) antibody labeling of bipolar cells in the avian retina.…”

Synapse turnover: the formation and termination of transient synapses.

“…All synapses between retina neurons and muscle cells are terminated in 3-10 days depending on the developmental age of the neurons. The half-lives of synapses between muscle cells and retina neurons from 8-, 12-, or (3)(4)(5)(6)(7)(8).…”

“…All synapses between retina neurons and muscle cells are terminated in 3-10 days depending on the developmental age of the neurons. The half-lives of synapses between muscle cells and retina neurons from 8-, 12-, or (3)(4)(5)(6)(7)(8).In this report we examine the specificity of synapse formation by retina neurons. The neurons were cultured with striated muscle cells which also possess abundant nicotinic ACh receptors, to determine whether retina neurons form synapses with muscle cells and whether muscle cells and retina neurons displace one another or are competitive targets for synapse formation by retina neurons that synthesize ACh.…”