4 Neuroscience Letter.s, 161 (1993) 4 8 g~ 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/93/$ 06.00 NSL 09840 Autoradiographical evaluation of [3H]glycine uptake in rat forebrain: cellular localization in the hippocampus Ernesto Fedele 1, David Smith*, Alan C. Foster 2 Merck Sharp and Dohme Research Laboratories Neuroscience Research Centre, Terlings Park Eastwick Road. Harkm; Essex. CM20 2QR ( UK~ (Received 4 February 1993; Revised version received 27 June 1993; Accepted 28 June 1993) Key words: Glycine; Uptake: Hippocampus; Glutamate; Neurotransmission Glia The cellular elements responsible for the uptake of [3H]glycine into rat hippocampal slices were investigated. The diffuse laminar distribution of labelling observed under control conditions was greatly reduced seven days after intrahippocampal injection of a neurotoxic dose of quinolinic acid, suggesting a neuronal localization. Glycine was also taken up into glial cells, since dense clusters of silver grains were present on small sized cells throughout the hippocampus which were apparently increased in number after the lesion. The pattern of [~H]glycine uptake into rat cerebral cortex and cerebellar slices was also consistent with both neuronal and glial localization. These glycine transport sites may be strategically located to control excitatory neurotransmission mediated by the N-methyl-D-aspartate sub-type of glutamate receptors. Interest in the neurotransmitter role of glycine in su- praspinal regions of the mammalian central nervous sys- tem (CNS) has grown over the past few years. Thus, cal- cium dependent release of radiolabelled or endogenous glycine has been demonstrated [9, 13, 15, 18] and strych- nine-sensitive inhibitory responses to glycine have been observed [2, 3, 6, 12, 18, 24]. The realization that glycine is required for activation of the N-methyl-D-aspartate (NMDA) sub-type of glutamate receptors, being a co- agonist with c-glutamate [10, 11], has given a new and important aspect to glycine's influence on neurotrans- mission in higher centres. Consequently, it is of interest to investigate processes which control the intrasynaptic levels of glycine. It has been suggested that the glycine site on the NMDA receptor is fully occupied and not subject to physiological control [11]. However, certain experiments in vitro [25], and in vivo [20, 22], indicate that glycine site agonists can potentiate NMDA receptor mediated re- sponses. These results suggest that local control of syn- aptic glycine levels may indeed occur and play an impor- tant role in regulating excitatory neurotransmission. *Corresponding author. Fax: (1) (714) 855-8210. Wisiting scientist from the Istituto di Farmacologia e Farmacognosia, Universita di Genova, Viale Cembrano 4, Genova 16148, Italy. "-Present address: Gensia, Inc., 11025 Roselle Street, San Diego, CA 92121-1204, USA. This idea has been directly demonstrated in the cerebel- lum, where an NMDA receptor-mediated component of the synaptic response in granule neurons evoked by stim- ulation of the mossy fibres was completely dependent on exogenous glycine [4]. The mossy-fibre granule neurons synapse is in close proximity to the axons of Golgi cells which selectively take up [3H]glycine [28], suggesting a role for this transport system in regulating extracellular glycine levels at this location. Several studies have demonstrated the existence of high and low affinity uptake systems for [3H]glycine in the cerebral cortex [5, 17, 26], the striatum [19] and the cerebellum [27]. In the hippocampus, [3H]glycine is accu- mulated by P2 synaptosomal preparations by both high and low affinity components [8]. Autoradiographical studies of [3H]glycine uptake into hippocampal slices re- vealed a laminar distribution which comprised both dif- fuse labeling and clusters of silver grains suggesting accu- mulation in small, non-neuronal cells [8]. The present study was undertaken to investigate the neuronal or glial localization of [~H]glycine uptake sites. For the autoradiographic studies, sagittal slices of rat hippocampus and coronal slices of cerebellar and cere- bral cortex from male Sprague Dawley rats (200-250 g) were obtained by means of a Lancer 1000 vibratome set at 350 pro, and immediately transferred into ice-cold Krebs medium (composition in mM: NaCI 125, KC1 3, CaCI~ 1.2, MgSO4 1.2, NaHCO~ 22, NaH2PQ4 1, glucose 10, gassed with 95% O~ and 5% CQ2, pH 7.4) until the