In the vascular system, endothelium-derived relaxing factor (EDRF) is the name of the local hormone released from endothelial cells in response to vasodilators such as acetylcholine, bradykinin and histamine. It diffuses into underlying smooth muscle where it causes relaxation by activating guanylate cyclase, so producing a rise in cyclic GMP levels. It has been known for many years that in the central nervous system (CNS) the excitatory neurotransmitter glutamate can elicit large increases in cGMP levels, particularly in the cerebellum where the turnover rate of cGMP is low. Recent evidence indicates that cell-cell interactions are involved in this response. We report here that by acting on NMDA (N-methyl-D-aspartate) receptors on cerebellar cells, glutamate induces the release of a diffusible messenger with strikingly similar properties to EDRF. This messenger is released in a Ca2+-dependent manner and its activity accounts for the cGMP responses that take place following NMDA receptor activation. In the CNS, EDRF may link activation of postsynaptic NMDA receptors to functional modifications in neighbouring presynaptic terminals and glial cells.

Endothelium-derived relaxing factor release on activation of NMDA receptors suggests role as intercellular messenger in the brain.

Glutamate, nitric oxide and cell-cell signalling in the nervous system

Glutamate is ubiquitously distributed in brain tissue, where it is present in a higher concentration than any other amino acid. During the last 50 years glutamate in brain has been the subject of numerous studies, and several different functions have been ascribed to it. Early studies by Krebs (1935) suggested that glutamate played a central metabolic role in brain. The complex compartmentation of glutamate metabolism in brain was first noted by Waelsch and coworkers (Berl et al., 1961). These studies were precipitated by the claim that glutamate improved mental behaviour and was beneficial in several neurological disorders including epilepsy and mental retardation. Other scientists pointed out its function in the detoxification of ammonia in brain (Weil-Malherbe, 1950). Glutamate is also an important building block in the synthesis of proteins and peptides, including glutathione (Meister, 1979). The toxic effect of administered glutamate and its analogues kainic acid, ibotenic acid, and N-methyl aspartic acid on CNS neurones has become a large and independent line of research (Lucas and Newhouse, 1957; Olney et al., 1974; Lund-Karlsen and Fonnum, 1976; Coyle, 1983). Attention has also been focused on the role of glutamate as a precursor for the inhibitory neurotransmitter y-aminobutyric acid (GABA) (Roberts and Frankel, 1950). Electrophysiological studies (Curtis and Watkins, 1961) focused early on the powerful and excitatory action of glutamate on spinal cord neurones. Since the action was widespread and effected by both the Dand Lforms, it was at first difficult to believe that glutamate could be a neurotransmitter. During the last 15 years, however, several studies have provided support for the concept that glutamate is a transmitter in brain (for review see Curtis and Johnston, 1974; Fonnum, 1978; 1981; Roberts et al., 1981; DiChiara and Gessa, 1981). Glutamate satisfies today to a large extent the four main criteria for classification as a neurotransmitter: (1) it is presynaptically localized in specific neurones; (2) it is specifically released by physiological stimuli in concentrations high enough to elicit postsynaptic response; (3) it demonstrates identity of action with the naturally occurring transmitter, including response to antagonists; and (4) mechanisms exist that will terminate transmitter action rapidly. The evidence for glutamate as a transmitter at the locust neuromuscular junction has recently been carefully evaluated by Usherwood (1981). In that case the identity of action of glutamate with the naturally occurring transmitter on the neuromuscular receptor, the release from nerve terminals, and its similarity to acetylcholine at the mammalian neuromuscular junction with regard to presynaptic pharmacology and denervation supersensitivity are compelling evidence for glutamate as a neurotransmitter. The main methods used to identify glutamergic pathways in brain will be critically reviewed and discussed. The effect of lesions on high-affinity uptake and release are particularly important, but immunohistochemical methods to study enzymes and glutamate itself are becoming more interesting. The release of glutamate has been demonstrated by several different procedures using both in vivo and in vitro preparations. The synthesis of large groups of specific agonists and antagonists has been important both for identification and characterization of the glutamate receptor by electrophysiological techniques and for the isolation of glutamate receptors. High and perhaps low-affinity uptake into nerve terminals and glial cells is important for the termination of transmitter action. Particular attention is given in this review to the complex compartmentation of glutamate synthesis and the possibility of identifying the transmitter pool of glutamate.

Glutamate: a neurotransmitter in mammalian brain.

SYNAPTIC transmission of most vertebrate synapses is thought to be terminated by rapid transport of the neurotransmitter into presynaptic nerve terminals or neuroglia1–5. L-Glutamate is the major excitatory transmitter in brain and its transport represents the mechanism by which it is removed from the synaptic cleft and kept below toxic levels5,6. Here we use an antibody against a glial L-glutamate transporter from rat brain7 to isolate a complemen-tary DNA clone encoding this transporter. Expression of this cDNA in transfected HeLa cells indicates that L-glutamate accumulation requires external sodium and internal potassium and transport shows the expected stereospecificity. The cDNA sequence predicts a protein of 573 amino acids with 8–9 putative transmembrane α-helices. Database searches indicate that this protein is not homologous to any identified protein of mammalian origin, including the recently described superfamily of neurotransmitter transporters. This protein therefore seems to be a member of a new family of transport molecules.

Cloning and expression of a rat brain L-glutamate transporter.

While there is an abundance of pharmacological and biochemical evidence to suggest the existence of multiple opioid receptors, their precise localization within the brain is unclear. To help clarify this issue, the present study examined the distributions of the mu, delta, and kappa opioid receptor subtypes in the rat forebrain and midbrain using in vitro autoradiography. Mu and delta receptors were labeled with the selective ligands 3H-DAGO (Tyr- D-Ala-Gly-MePhe-Gly-ol), and 3H-DPDPE (D-Pen2, D-Pen5-enkephalin), respectively, while the kappa receptors were labeled with 3H-(-)bremazocine in the presence of unlabeled DAGO and DPDPE. Based on previous findings in our laboratory, the labeling conditions were such that each ligand selectively occupied approximately 75% of each of the opioid sites. The results demonstrated that all 3 opioid receptor subtypes were differentially distributed in the rat brain. Mu binding was dense in anterior cingulate cortex, neocortex, amygdala, hippocampus, ventral dentate gyrus, presubiculum, nucleus accumbens, caudate putamen, thalamus, habenula, interpeduncular nucleus, pars compacta of the substantia nigra, superior and inferior colliculi, and raphe nuclei. In contrast, delta binding was restricted to only a few brain areas, including anterior cingulate cortex, neocortex, amygdala, olfactory tubercle, nucleus accumbens, and caudate putamen. Kappa binding, while not as widespread as observed with mu binding, was densely distributed in the amygdala, olfactory tubercle, nucleus accumbens, caudate putamen, medial preoptic area, hypothalamus, median eminence, periventricular thalamus, and interpeduncular nucleus. While all 3 opioid receptor subtypes could sometimes be localized within the same brain area, their precise distribution within the region often varied widely. For example, in the caudate putamen, mu binding had a patchy distribution, while delta and kappa sites were diffusely distributed, with delta sites being particularly dense ventrolaterally and kappa sites being concentrated ventromedially. These results support the existence of at least 3 distinct opioid receptors with possibly separate functional roles.

Autoradiographic differentiation of mu, delta, and kappa opioid receptors in the rat forebrain and midbrain

Preparation of cell bodies from the developing cerebellum: Structural and metabolic integrity of the isolated ‘cells’

Evidence for a kappa-opioid receptor on pituitary astrocytes: an autoradiographic study.

Subcellular fractionation of rat cerebellum: an electron microscopic and biochemical investigation. III. Isolation of large fragments of the cerebellar glomeruli.

Part I - CNS Neurons. 1: Cerebellum: granule cells. 2: Cerebellar Purkinje neurons. 3: Nigral & straital neurons. 4: Septal neurons. 5: Proliferation & differentiation in retinal cell cultures. 6: Purified embryonic motoneurones. Part II - CNS glia and non-neuronal cells. 7: Astrocytes. 8: Oligodendrocyte lineage cells from neonatal rat brain. 9: Micrglia: the tissue macrophage of the CNS. 10: Brain endothelium. Part III - PNS neurons and glia. 11: Neural crest cells: strategies to generate lineage diversifaction in vitro. 13: Avian cilliary and lumbar sympathetic ganglion neurons. 14: Enteric ganglion cells. 15: Mouse olfactory epithelium. 16: Schwann cell culture

Graham P. Wilkin

Papers

Preparation of cell bodies from the developing cerebellum: Structural and metabolic integrity of the isolated ‘cells’

Evidence for a kappa-opioid receptor on pituitary astrocytes: an autoradiographic study.

Subcellular fractionation of rat cerebellum: an electron microscopic and biochemical investigation. III. Isolation of large fragments of the cerebellar glomeruli.

Neural cell culture: a practical approach

Guanylate cyclase activities in enriched preparations of neurones, astroglia and a synaptic complex isolated from rat cerebellum