ANNE B. YOUNG,     Department of Neurology, University of Michigan, Ann Arbor, Michigan

Publisher Summary

This chapter reviews the methodologies utilized to investigate [3H]strychnine binding within the central nervous systems of a variety of vertebrate species. [3H]strychnine binding has been associated with central glycine receptors. The site on the receptor complex to which strychnine binds is probably not the glycine recognition site but a site more closely associated with the ionic conductance mechanism. The site to which strychnine binds is the site with which a variety of strychnine analogs, opiate alkaloids, and certain other convulsants interact. It is conceivable that strychnine binding sites may also be associated with inhibitory effects of substances, such as taurine and/or β-alanine in brain. Abnormalities in [3H]strychnine binding have been found in rodent genetic disorders and human motor neuron disease. In the future, it may be possible to develop a suitable ligand for the agonist binding site, independently isolate the various receptor subunits, and develop the type of information about the glycine receptor that has been gathered for the nicotinic cholinergic receptor.

I INTRODUCTION

Glycine has been implicated in the past two decades as a major inhibitory neurotransmitter in the spinal cord and brainstem of mammalian species. Glycine is thought to be the neurotransmitter of the so-called reciprocal Ia interneurons (ten Bruggencate and Engberg, 1968; Curtis et al., 1968; Werman et al., 1968) and Renshaw cells of the spinal cord (Belcher et al., 1976; Curtis et al., 1976) as well as that of interneurons in cuneate nucleus (Galindo et al., 1967), Deiter’s nucleus (ten Bruggencate and Engberg, 1971), hypoglossal nucleus (ten Bruggencate and Sonnhof, 1971), thalamus (Ishida, 1977), substantia nigra (Pycock et al., 1981), ventral tegmental area (Gundlach and Beart, 1981), and retina (Berger et al., 1977; Borbe et al., 1981). In cerebral cortex, glycine is much less active physiologically (Pycock and Kerwin, 1981; Levi et al., 1982). Glycine has also been implicated as a neurotransmitter in other vertebrates such as birds (Reubi and Cuénod, 1976; LeFort et al., 1978) and frogs (Davidoff and Adair, 1976; Müller and Snyder, 1978a).

The synaptic actions of glycine are inhibited specifically by the convulsant alkaloid strychnine (Curtis et al., 1971; Davidoff et al., 1969). Strychnine is known to block glycine receptors at very low concentrations, suggesting high affinity for the receptors (Curtis et al., 1971) and for this reason, it has been used as a ligand for the investigation of synaptic glycine receptors (Young and Snyder, 1973, 1974a). High specific activity [3H]strychnine binding has been studied in a variety of species (Snyder and Young, 1975; Zukin et al., 1975; LeFort et al., 1978; Müller and Snyder, 1978a; de Montis et al., 1982). Strychnine is extremely potent and, as expected, binds with high affinity to the glycine receptor. Strychnine has been used instead of glycine itself to avoid the possible interaction with various membrane-bound enzymes and transport systems that bind glycine directly. Glycine has been shown to bind to synaptic membranes; however, the binding is only weakly inhibited by strychnine and the regional distribution of binding does not parallel that expected of glycine receptors (DeFeudis et al., 1978; Orensanz Muñoz et al., 1977; DeFeudis, 1978). [3H]Glycine also binds to high-affinity transport sites and enzymes (Valdes and Orrego, 1975; DeFeudis, 1978). This chapter will review the methodologies utilized to investigate [3H]strychnine binding within the central nervous systems of a variety of vertebrate species.

II LIGANDS

Ligands available commercially to assay central glycine synaptic receptors are [2,4,11-3H]strychnine sulfate (10–30 Ci/mmol) (Amersham Corporation), [benzene ring-3H]strychnine (15–40 Ci/mmol) (New England Nuclear), and [21,22-3H]dihydrostrychnine (30–60 Ci/mmol) (New England Nuclear). All compounds are quite stable when stored in ethanol–water (4:1) in the dark at −20°C (<1% decomposition per month). Appropriate solvent systems for checking radiochemical purity by thin layer chromatography on silica gel plates are methanol–water (7:3), chloroform–methanol (7:3), n-butanol–n-propanol–20% ammonia (2:2:1), chloroform–ethanol–concentrated ammonia (80:20:0.5), and n-butanol–acetic acid–water (4:1:1). Ultraviolet spectra of labeled and unlabeled strychnine yield absorption maxima at 254, 278, and 288 nm. Most investigators have utilized [3H]strychnine for their studies. [3H]Dihydrostrychnine has a lower affinity for glycine receptors, but the higher specific activity somewhat offsets this disadvantage (see Section V,A).

III TISSUE PREPARATION