Screening for neurotransmitters: A rapid radiochemical procedure

Abstract

SUMMARYAmong the known or suspected neurotransmitter compounds are: acetylcholine, norepinephrine, 7-aminobutyric acid, epinephrine, 5-hydroxytryptamine, dopamine, and octopamine. This communication describes a rapid screening procedure for identifying which of these substances are likely candidates for neurotransmitters in a particular tissue. The method is based on the selective synthesis and localization of transmitters in those cells which are presumed to release them. The procedural steps are: (I) incubation of tissue in a physiological medium containing one or more radiochemical precursors of suspected transmitters; (2) acidic extraction of all or selected parts (e.g., single cells) of the incubated tissue; (3) high-voltage paper electrophoresis of the extract under conditions which separate most of the compounds of interest; and ( 4 ) quantification of isotope in each transmitter candidate. The effectiveness and sensitivity of the method have been shown with several preparations whose transmitters are known. In certain cases the sensitivity can extend to a single cell level. Preliminary results are also reported on the application of the technique to a lobster sensory system of unknown transmitter chemistry, the abdominal muscle receptor organ.

Dopamine elicits feeding motor program in Limax maximus

“…Metabolism of [3HItyrosine. The production of radioactive metabolites from [3H]tyrosine was tested using the methods of Hildebrand et al (1971). Isolated ganglia were incubated at 12 to 15°C for up to 25 hr in the presence of 10 &i/ml of [3H]tyrosine (specific activity 30 Ci/mmol) in Limax saline.…”

Cellular synthesis and axonal transport of gamma‐aminobutyric acid in a photoreceptor cell of the barnacle.

“…Many sensory cells of Crustacea are cholinergic (Florey & Biederman, 1960;Hildebrand, Barker, Herbert & Kravitz, 1971; Barker, Herbert, Hildebrand & Kravitz, 1972). However, barnacle photoreceptor cells did not synthesize acetylcholine from [3H]choline injected into the cells but synthesized gamma-aminobutyric acid (GABA) from labelled glutamate (Koike & Tsuda, 1974, 1979.…”

Postnatal development of dopaminergic neurons in the rabbit retina

“…One milliliter of each eluate was mixed thoroughly with 9 ml of a scintillant mixture (667 ml of toluene, 333 ml of Triton X-100,5.5 gm of PPO (2,5-diphenyloxazole), and 0.1 gm of POPOP (1,4-bis-2-(5-phenyloxazolyl)benzene)), and the radioactivity was measured by a liquid scintillation counter (Beckman, model LS-330). Ten microliters of each eluate was used to determine, by high voltage paper electrophoresis, the percentage of total radioactivity remaining as [3H]dopamine (Hildebrand et al, 1971). The Ca2+ dependence of this release was studied by adding CoC12 to either normal or K+-rich Ringer's solution at a final concentration of 10 mM during the appropriate intervals.…”

“…After each release experiment, the retina was homogenized with a glass homogenizer (from Kontes Glass Co., Vineland, NJ) in 1 ml of 40% trichloroacetic acid. Twenty microliters of the supernatant was used to determine the percentage of the total radioactivity in the retina remaining as [3H]dopamine using high voltage paper electrophoresis (Hildebrand et al, 1971).In order to protect the ["Hldopamine from degradation, sodium ascorbate (from Merck) and pargyline hydrochloride (from Regis Chemical Co.) were added to the Ringer's solution at final concentrations of 1 mM and 15 PM, respectively.Assay of tyrosine hydroxyylase. Tyrosine hydroxylase (EC 1.14.16.2) activities were measured using the methods described by Nagatsu et al (1964) and Coyle (1972) with some modifications.…”