Excitatory amino acid transmitters

Information obtained over the past 25 years indicates that the amino acid glutamate functions as a fast excitatory transmitter in the mammalian brain. Studies completed during the last 15 years have also demonstrated that glutamate is a powerful neurotoxin, capable of killing neurons in the central nervous system when its extracellular concentration is sufficiently high. Recent experiments in a variety of preparations have shown that either blockade of synaptic transmission or the specific antagonism of postsynaptic glutamate receptors greatly diminishes the sensitivity of central neurons to hypoxia and ischemia. These experiments suggest that glutamate plays a key role in ischemic brain damage, and that drugs which decrease the accumulation of glutamate or block its postsynaptic effects may be a rational therapy for stroke.

Glutamate and pathophysiology of hypoxic-ischemic brain damage

Glutamate and the pathophysiology of hypoxic--ischemic brain damage.

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.

Amino acid neurotransmitters and their pathways in the mammalian central nervous system

— Glutamate and aspartate probably serve as transmitters of hippocampal perforant path and commissural afferents, respectively. We therefore used slices of hippocampal regions to evaluate certain biochemical properties as markers for sites of transmission mediated by these amino acids. In these studies content and accumulation of glutamate and aspartate were compared with their Ca2+-dependent effluxes.



Hippocampal regions varied little in their contents of glutamate and aspartate, but slices of regio superior and dentate gyrus accumulated and released more of each than slices of regio inferior. A commissurotomy or bilateral entorhinal lesion altered Ca2+-dependent efflux and accumulation in the same direction, but did not affect the glutamate or aspartate content of any hippocampal region. Elimination of hippocampal mossy fibers reduced the Ca2+-dependent efflux of glutamate and probably aspartate from slices of dentate gyrus, but not of regio inferior, where most mossy fiber synapses are located. The mossy fibers appeared relatively deficient in aspartate in both strains tested, but only in Purdue-Wistar rats were they enriched in glutamate. Removal of the perforant path input to the fascia dentata did not significantly change the activity of any of the enzymes most actively involved in glutamate synthesis.



These results suggest that accumulation or high affinity transport of glutamate or aspartate can be employed to localize afferents which use these amino acids as transmitters, although it is not so reliable or selective a marker as Ca2+-dependent efflux. Enrichment in either glutamate or aspartate content or in the activity of enzymes which synthesize them is not a reliable marker. Neither amino acid is likely to be used as a transmitter by the hippocampal mossy fibers.

Biochemical correlates of transmission mediated by glutamate and aspartate.

— Superfused slices of the rat dentate gyrus were employed to study the release of GABA, glutamate and aspartate, which are considered strong neurotransmitter candidates in this region. The introduction of Ca2+ to a Ca2+-free superfusion medium containing a depolarizing agent augmented the efflux of all three amino acids. The response to application of Ca2+ nearly always occurred within 30 s, the shortest interval tested in these studies. The efflux rate reached a peak within 90 s and then declined to a level slightly greater than the prestimulation baseline. The failure to maintain the maximal rate with continued exposure to Ca2+ and depolarizing influences appeared not to result from a reduction in Ca2+ permeability caused by continuous depolarization. Ca2+ also stimulated the efflux of exogenously loaded radiolabeled GABA, glutamate and aspartate, but not proline. Exogenously loaded GABA was more readily released than endogenous GABA. Otherwise the effects of various treatments on their efflux rates were qualitatively similar.



Mg2+ inhibited Ca2+-dependent efflux. Ba2+, but not Mg2+, stimulated amino acid efflux in the absence of Ca2+. Extracellular Na+ was not required to support Ca2+-dependent efflux. Addition of Ca2+ to a Ca2+-free medium in the absence of a depolarizing agent released GABA from the slices, but not glutamate or aspartate.



K+-enriched medium and the depolarizing alkaloid, veratridine, stimulated both Ca2+-dependent and Ca2+-independent release processes. Na+-free medium enhanced the Ca2+-independent releasing action of elevated K+. Ca2+-independent release was inhibited by raising the Mg2+ concentration by 15 or 30 mM and appeared to be inhibited by Ca2+ as well. Amino acid output in the absence of Ca2+ is probably not directly related to transmission and is considered to result partially from a general increase in membrane permeability induced by depolarization in a Ca2+-free medium and partially from stimulation of carrier-mediated amino acid efflux.



These results support previously suggested transmitter roles for GABA, glutamate and aspartate in the rat dentate gyrus.

K. W. Vaca

Papers

Biochemical correlates of transmission mediated by glutamate and aspartate.

Characterization of putative amino acid transmitter release from slices of rat dentate gyrus.

Calcium-dependent γ-aminobutyrate release by interneurons of rat hippocampal regions: Lesion-induced plasticity