Autoradiographic localisation of α-bungarotoxin-binding sites in the central nervous system

[3H]Muscimol was used in the light microscopic autoradiographic localization of 7-amninobutyrip acid (GABA) receptors in the rat cerebellum. The binding ofI3Hl muscimol to the slide-mounted tissue sections had all of the characteristics associated with a GABA receptor. It was saturable with appropriate kinetic constants and was blocked only by other GABAergic drugs. Autoradiographic studies revealed a very high density of receptors over the granule cell layer, a low level over'the molecular layer, and negligible receptor binding over white matter. This suggested a high localization of GABA receptors to granule 'cells, which is supported by other neurochemical studies. The autoradiographic procedure used here should be a useful method in further studies of receptors requiring high sensitivity and increased anatomical resolution.y-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system of mammals and elsewhere. One of the most intensively studied components of the GABAergic system has been the GABA receptor, Recently, conditions have been defined whereby one can utilize [3H]GABA to bind to a GABA receptor in membrane homogenates (1-6). More recently, muscimol (3-hydroxy-5-aminomethylisoxazole), a GABA receptor agonist, has also been used in its radiolabeled form to bind to GABA receptors (7). In binding studies, [3H]muscimol has the advantages that it has only a weak affinity for the GABA uptake system and is not an avid substrate for GABA-2-oxoglutarate aminotransferase (8, 9).Our laboratory has studied the light microscopic, autoradiographic localization of a number of neurotransmitter receptors, including cholinergic muscarinic (10-12), opiate (13-15), and dopaminergic receptors (16,17). We recently developed a method to localize receptors by autoradiographic methods at the light microscopic level that can be used with ligands that do not cross the blood/brain barrier or do not efficiently label receptors by in vivo injection (18). This method involves the in vitro labeling of receptors in slide-mounted tissue sections and the apposing of emulsion-coated cover slips to generate autoradiograms; it gives results identical to those found in earlier studies using in vivo labeling of receptors (18) Receptor Binding. Slides with mounted tissue sections were brought to room temperature and preincubated in 0.31 M Tris-citrate buffer, pH 7.1, at 2-4°C in an ice bath for 20 min. In extensive preliminary experiments, we found that this preincubation resulted in a marked increase in maximal specific binding of [3H]muscimol to the tissues. We suspect that this preincubation resulted in a depletion of endogenous GABA from the tissues, which would inhibit the binding of [3H]muscimol. A similar effect has been reported in published experiments with homogenates in which tissues are commonly washed or treated with Triton X-100 to enhance GABA binding (3,5). After preincubation, the slides were transferred to an incubation medium of 0.31 M Tris citrate (pH 7.1 at 2-40C) buffer contai...

Section: Methodsmentioning

confidence: 99%

Autoradiographic localization of gamma-aminobutyric acid (GABA) receptors in the rat cerebellum.

Palacios¹,

Young²,

Kuhar³

1980

“…Additional support for its identity comes from the specific locations of the a-BTX-binding sites in this tissue (ref. 28; unpublished autoradiographic observations on the chick optic tectum). These sites are seen only in the zones known to contain nicotinic synapses.…”

Section: Affinity Labeling Of the A-btx-binding Components Frommentioning

confidence: 99%

Nicotinic acetylcholine receptor from chick optic lobe.

Norman¹,

Mehraban²,

Barnard³

et al. 1982

An a-bungarotoxin-sensitive nicotinic cholinergic receptor from chick optic lobe has been completely purified. Its standard sedimentation coefficient is 9.1 S. The value near 12 S reported for the related component from other brain regions can be reproduced when the initial extraction is by Triton X-100 (rather than Lubrol PX), but other protein is then complexed with it. A single subunit of apparent molecular weight 54,000 is detected, and this subunit is specifically labeled by bromo-[3H]acetylcholine, but only after disulfide reduction. The same size subunit likewise is labeled in the protein (purified similarly) from the rest ofthe chick brain which can also bind a-bungarotoxin and nicotinic ligands. Immunological crossreactivity is demonstrated between both of these proteins with an antiserum to pure acetylcholine receptor from skeletal muscle. The acetylcholine receptor from chick optic lobe and the a-bungarotoxin-binding protein from the rest of the brain appear similar or identical by a series of criteria and are related to (but with differences from) peripheral acetylcholine receptors.

“…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)(4)(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.…”

mentioning

confidence: 99%

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

Vogel

Maloney

Ling

et al. 1977

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...