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Many evidence suggest that metabotropic glutamate receptors (mGluRs) may modulate glutamatergic transmission, hence, these receptors are regarded as potential targets for neuroprotective drugs. Since group III mGlu receptor agonists are known to reduce glutamatergic transmission by inhibiting glutamate release, we decided to investigate the neuroprotective potential of the group III mGlu receptor agonist, (1S,3R,4S)-1-aminocyclopentane-1,2,4-tricarboxylic acid (ACPT-I) against kainate (KA)-induced excitotoxicity in vitro and in vivo. In primary neuronal cell cultures ACPT-I (1–200 μM), applied 30 min–3 h after starting the exposure to KA (150 μM), significantly attenuated the KA-induced LDH release, increased cell viability, and inhibited caspase-3 activity both in cortical and hippocampal cell cultures. The effects were dose-, time- and structure-dependent. The neuroprotective effects of ACPT-I were reversed by (RS)-alpha-cyclopropyl-4-phosphonophenyl glycine, a group III mGluR antagonist. In the in vivo studies, KA (2.5 nmol/1 μl) was unilaterally injected into the rat dorsal CA1 hippocampal region and the size of degeneration was examined by stereological counting of surviving neurons in the CA pyramidal layer. It was found that ACPT-I (7.5 or 15 nmol/1 μl), injected into the dorsal hippocampus 30 min, 1 or 3 h after KA in dose-dependent manner prevented the KA-induced neuronal damage. Moreover, in vivo microdialysis studies in the rat hippocampus showed that ACPT-I (200 μM) given simultaneously with KA (50 μM) significantly diminished the KA-induced glutamate release in the hippocampus. This mechanism seems to play a role in mediating the neuroprotective effect of ACPT-I.

Kainate receptors are widely distributed in the CNS, but also in the PNS. Dorsal root ganglia are enriched in this subtype of glutamate ionotropic receptors. In addition to their activity as ligand-gated ion channels, kainate receptors exhibit other properties already characterized in other systems, such as hippocampus, i.e., their ability to induce a metabotropic cascade signalling, through G-protein and PKC activation. With a very similar actuation mechanism as formerly described in the CNS, kainate receptors in the DRG also present other differentiated features, such as the Ca2+ channel blockade and a self-regulation property. The peculiarity of these neurons has served to progress the study of kainate receptors. Nevertheless, many other physiological functions of these receptors remain unclear, as does the related molecular nature of the metabotropic cascade and the involvement of this signalling pathway with sensory transmission of pain.

Kainate receptors (KARs) are members of the family of ionotropic glutamate receptors (iGluRs) which also include NMDA and AMPA receptors. As ionotropic receptors, KARs have been characterized, pre and postsynaptically, in several brain regions. In this chapter we review evidence that suggests that KARs mediate some of their effects without invoking ion-fluxes. Beginning with seminal experiments described some ten years ago, when the notion of a metabotropic action of KAR was first posited in the modulation of GABA release from hippocampal interneurons, increasingly, there have been reports indicating that some KAR functions overtly depend on G-protein activation and involve the participation of intracellular signalling cascades. Thus, KAR activation instigates a cascade involving Gi/o, phospholipase C and protein kinase C to suppress the release of GABA and therefore underpins disinhibition of pyramidal cells in the CA1 region of the hippocampus. This type of metabotropic function of KARs in controlling GABA release represents an additional level of activity-dependent control of synaptic inhibition which is independent of any ionotropic activity of KARs.

Kainate receptors (KARs) mediate postsynaptic responses in CA3 pyramidal cells and CA1 interneurones in the hippocampus. In CA1 pyramidal cells knockout studies have inidcated the presence of functional GluR6-containing KARs, however in this region they made no ionotropic contribution to the synaptic responses. In the meantime, a metabotropic function was reported for presynaptic KARs modulating transmitter release in CA1. We examined the possibility that KARs in CA1 pyramidal cells have a metabotropic function. Kainate is known to inhibit a slow afterhyperpolarization current that regulates excitability in hippocampus and can be modulated by a number of G protein coupled receptors. We showed that KARs activation reduces slow afterhyperpolarization current in CA1 pyramidal cells via metabotropic action and elucidated the transduction mechanism(s) underlying this action.

Most of our knowledge of the synaptic function of kainate receptors stems from a detailed analysis of synaptic transmission between dentate granule cells and CA3 pyramidal neurons, where kainate receptors mediate a slow excitatory current with integrative properties ideally suited for repetitive neuronal firing. Besides this well characterized ionotropic effect of kainate receptors, they can also enhance neuronal excitability by inhibiting the slow Ca2+ activated K+ current IsAHP via a G-protein coupled mechanism. This phenomenon is associated with Ca2+ mobilization and protein-kinase activation and ultimately leads to modulation of ion channels responsible for intrinsic electrical properties such as firing adaptation. The significance for CNS function of these newly emerging metabotropic kainate receptors is poorly understood and as yet proteomic analysis of kainate receptors has yielded little information on signaling molecules associated with the kainate receptor ionophore. This chapter covers the key findings that have led to the proposal that high-affinity postsynaptic kainate receptors trigger a form of metabotropic signaling regulating IsAH P and neuronal firing in CA3 hippocampal neurons.

The kainate-type glutamate receptor displays strong modulation by monovalent anions and cations. This modulation is independent of permeation of the ion channel. Instead, structural, computational and biophysical evidence shows that receptor activity is controlled by binding of sodium and chloride ions at sites that stabilize active dimers of glutamate binding domains. Modulation by monovalent ions is a surprisingly general property across ion channel families. However, evidence of a physiological role for ion-dependent effects on glutamate receptors is lacking, perhaps reflecting the adventitious use of ions as structural components of the kainate receptor. “ergo, Hercules, vita humanior sine sale non quit degree […]” “Heaven known, a civilized life is impossible without salt” —Pliny the Elder, Natural History XXXI 88

Distinct populations of kainate-type ionotropic glutamate receptors (KARs), located at various cell types and subcellular compartments and utilizing diverse downstream signaling mechanisms, represent an intricate system with large capacity for modulatory effects ranging from synapse-specific changes to alterations in the excitability of large neuronal ensembles. However, the way the diverse functions ascribed for KARs are utilized under different physiological and pathological conditions to regulate activity at the level of neuronal networks is still largely unclear. Here, we address the data regarding functions of KARs in the regulation of network activity in the hippocampus, with a main focus on their roles during early postnatal development. We further discuss the evidence suggesting that KAR mediated signaling during the immature type network activity is involved in the formation and maturation of glutamatergic synapses.

Kainate receptors (KAR) form a class of glutamate receptors that have been implicated in epilepsy, stroke, Alzheimer’s and neuropathic pain.1 KAR subtypes are known to be segregated to specific locations within neurons and play significant roles in synaptic transmission and plasticity.2 Increasing evidence suggests a the role for ubiqutination in regulating the number of synaptic neurotransmitter receptors.3, 4, 5 The ubiquitin pathway consists of activation (E1), conjugation (E2) and ligation (E3). Cullins form the largest family of E3 ligase complexes. We have recently shown that the BTB/Kelch domain proteins, actinfilin and mayven, bind both Cul3 and specific KAR subtypes (GluR6 and GluR5-2b) to target these KARs for ubiquitination and degradation.5 In this chapter we will review how these interactions occur, what they mean for the stability of KARs and their associated proteins and how, in turn, they may affect synaptic functions in the central nervous system.

1 Ionotropic glutamate receptors are ligand-gated ion channels that mediate excitatory synaptic transmission in the central nervous system. Based on their structural and pharmacological properties, ionotropic glutamate receptors can be divided into three groups, which include α-amino-3-hydroxy-5-methyl-4-isoxasolepropionic acid (AMPA) receptors, kainate receptors, and N-methyl-D-aspartate (NMDA) receptors. Historically, these groups were named on the basis of the activating agonist. AMPA and kainate receptors are discussed in the chapter by Mayer. NMDA receptors are distinguished from other ionotropic glutamate receptors by their unique features including requirement for simultaneous binding of the co-agonists glycine and glu- tamate, voltage-dependent Mg 2+ block, and high permeability to Ca 2+ . NMDA recep- tors contribute to the slow component of the excitatory postsynaptic current (EPSC) and play key roles in neuronal development, synaptic plasticity, learning, and mem- ory, as well as in a number of pathophysiological conditions including epilepsy, and Alzheimer's disease), and psychiatric disorders (e.g. schizophrenia). Therefore, understanding the relationship between structure and function of the NMDA receptor will provide valuable insights into the mechanisms of synaptic transmission, as well as pathophysiology of a number of disorders in the central nervous system. Under- standing this relationship will also facilitate the development of therapeutically use- ful compounds.

Structural studies on glutamate receptor ion channels (iGluRs) have reached a level of sophistication that was impossible to imagine when the individual receptor genes were cloned in the 1980s. Because these membrane proteins mediate excitatory responses at approximately 60% of synapses in the brain, they have attracted enormous interest due to their role in synaptic plasticity, learning and memory, development of the CNS, and disease. There has also been immense interest in understanding how these molecules work. In this chapter I will highlight some of the key advances in our understanding of the function of glutamate receptor ion channels which have arisen from structural work, and address some of the most important issues which remain unanswered. The results discussed focus almost entirely on AMPA and kainate receptors, while NMDA receptors are discussed in the chapter by Yuan et al.

The granule cells (GC) of the dentate gyrus (DG) are very exciting cells, although it seems that they can also be very inhibiting cells. They contain and release the excitatory amino acid glutamate, the metal Zn++, the peptides Y, and dynorphin, brain derived neurotrophic factor (BDNF), and it has recently been well established that they also have the necessary machinery to synthesize, vesiculate and release the inhibitory amino acid GABA.6, 12 Among all these chemical messengers, glutamate is the only one with fast-acting excitatory actions. There is no doubt on the excitatory nature of the GC, but when they fire at high frequencies, peptides Y14 and dynorphin35 are released to produce inhibition and glutamate reaches an extracellular concentration that, by spilling over, activates presynaptic mGlu and kainate receptors21, 28 to inhibit further release. To complicate things further, GABA can be released from the mossy fibers (MF) under certain conditions.

In addition to being actively involved in the general metabolism of the central nervous system, the amino acids glutamate and Chapter 16(173,175–176)-aminobutyrate (GABA) serve as respectively excitatory and inhibitory neurotransmitters (Schousboe, 1990). This function is mediated by pharmacologically distinct receptors for each of the two amino acids. Receptors for glutamate are generally subdivided into 3 major classes exhibiting different pharmacological profiles, i.e. N-methyl-D-aspartate-(NMDA), kainate-, and quisqualate/AMPA (RS-α-amino-3-hydraxy-5-methyl-4-isoxazolo-propionate)- receptors (Watkins and Olverman, 1987). Likewise, GABA receptors are subdivided into two major classes referred to as GABAA and GABAB receptors (Johnston et al., 1984). It should, however, be emphasized that diversities exist within these respective classes of receptors as binding sites with different affinities for the amino acids have been observed (Johnston et al., 1984; Drejer & Honore, 1988). Such a diversity is consistent with the discovery of a large number of subunits of these receptors with different amino acid sequences (Schofield et al., 1987; Hollmann et al., 1989). This, in turn, may be reflected by the multiple functions of these receptors such as involvement in modulation of neuro-transmitter release and mediation of trophic and toxic actions. These functional aspects will be discussed below.

Stroke or cerebrovascular accident reduces blood flow and decreases oxygen supply (ischemia) in brain tissue. This may be resulted from vascular obstruction when a blood vessel is blocked or by hemorrhage when bleeding occurs into the brain tissue. Decrease in oxygen supply shifts pH to acidosis and increases extracellular K+ concentration, which depolarizes neural cell membrane. Anoxic depolarization leads to excessive release of glutamate, which then activates various glutamate receptors in the synapse or the extrasynaptic space. Opening of ionotropic glutamate receptors (NMDA, AMPA and kainate receptors) causes influx of Na+ through the activated glutamate-gated ion channels. In response to anoxia, Ca2+ also enters the cells in excessive amounts via activated NMDA receptors and Ca2+-permeable AMPA receptors. This will lead to activation of several Ca2+-dependent intracellular signal transduction pathways (proteases, kinases, endonucleases, lipoxygeneses and nitric oxide synthase), which ultimately leads to neural death (Vizi et al., 1996; Parsons et al., 1998).

In this chapter we begin to explore the function of dopaminestimulated PLM, especially as it relates to neuronal mechanisms that are central to attention. Under normal conditions D4R-mediated PLM activity is limited by the available concentration of dopamine, which is required for changing the receptor to its R* shape and initiating PLM. Nature designed the PLM system in this manner to be responsive to changes in the level of dopamine. Dopamine-releasing nerves can therefore bring about a localized change in membrane fluidity within D4R-containing target nerves across the synapse by causing the dopamine concentration to build up in the synaptic cleft. In other words the design features of this system allow the biophysical character of the post-synaptic membrane to be modulated whenever dopamine is released from the presynaptic nerve. Effects of dopamine-induced PLM on the post-synaptic nerve reflect D4R-mediated solid-state signaling.

Glutamate is considered to be a major excitatory neurotransmitter in the mammalian nervous system. As a neurotransmitter, glutamate plays important roles in cellular function through the activation of ionotropic glutamate receptors (iGluRs), such as N-methyl-D-aspartate (NMDA) receptors, α-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) receptors, and kainate (KA) receptors. In addition to its function in neuronal differentiation, migration, and survival in the developing brain (Hack and Balazs, 1994), glutamate is necessary for learning and memory acquisition (Steele et al., 1995). Alternatively, glutamate also can be a principal mediator of cellular injury. Following ionotropic receptor activation, glutamate can result in excitotoxicity that may be a critical component for the development of neurodegenerative diseases (Beal, 1992).

In this chapter we review the interaction of glutamate and dopamine in striatum and nucleus accumbens of the conscious rat during aging. We studied the effects of increasing concentrations of endogenous glutamate (obtained by blocking its reuptake) on the extracellular concentrations of dopamine in striatum and nucleus accumbens in the young rat. It was found that increasing concentrations of glutamate correlated significantly with increasing concentrations of dopamine in both structures. The increase of dopamine were significantly reduced after blockade of NMDA and AMPA/kainate glutamate receptors, suggesting that the increase of dopamine was mediated by glutamate. The interaction glutamate/dopamine showed a significant age-related decrease in nucleus accumbens but not in striatum. It is suggested that the interaction glutamate-dopamine represents a balanced input to the GABA neuron in the striatum and nucleus accumbens and that during aging this balance is disrupted.

The physiological roles of the pineal hormone melatonin are still not completely clarified. Recently it has been shown that melatonin is a potent, endogenous scavenger of reactive oxygen species suggesting that it might interfere with neurodegenerative processing involving free-radical formation and excitatory aminoacid release. These neuroprotective effects of melatonin may result, at least in part, from a sparing of glutathione reductase, which is decreased following administration of the neurotoxic agent kainate (KA) in rats. Moreover, KA causes a rapid decrease in glutathione (GSH) content of cultured cerebellar granule neurons but not in astrocytes. These cell types both express functional KA receptors, but only the former is sensitive to reactive oxygen species-dependent KA injury. Melatonin counteracts the changes in GSH, induced by KA, in cultured cerebellar granule neurons.

Increasing evidence indicates that nitric oxide (NO), a gaseous intra- and extracellular messenger, is involved in various neurotoxic and neurodegenerative processes. Inappropriate formation of NO is emerging as an important factor in the neurotoxicity associated with a variety of central nervous system disorders. Enhancement of NO production occurs in the central nervous system during stroke, seizures and acute and chronic inflammatory and neurodegenerative disorders (for reviews see Dawson and Dawson 1996; Scabo 1996). NO appears to be an important mediator of neuronal injury following activation of NMDA receptors (Dawson et al., 1991, 1993; Lipton et al., 1993), but also in MPTP, methamphetamine and ammonia neurotoxicity (Spencer Smith et al., 1994; Kosenko et al., 1995; Hantraye et al., 1996; Przedborski et al., 1996; Di Monte et al., 1996). NO formed by increased expression of neuronal NO synthase (NOS) may also play a role in capsaicin-induced neurotoxicity (Vizzard et al. 1995). Considerable differences in the contribution of the various isoenzymes of NOS to neuronal damage have been observed. Excessive activation of the isoform located in neurons (nNOS) appears to significantly contribute to hypoxic-ischemic brain damage (Ferriero et al., 1996) and to glutamate neurotoxicity, whereas the inducible isoform of NOS (iNOS) can be expressed under inflammatory conditions (Scabo 1996). Recent evidence in NOS mutant mice indicates that neonatal mice lacking nNOS are less vulnerable to hypoxic-ischemic injury (Ferriero et al., 1996; Panahian et al., 1996) and that malonate striatal lesions were significantly attenuated in the nNOS mutant mice but were significantly increased in the endothelial NOS (eNOS) mutant mice (Schulz et al., 1996). Cortical cultures from transgenic mice lacking nNOS are relatively resistant to NMDA neurotoxicity, but not to kainate neurotoxicity (Dawson et al., 1996).

Glutamate (or a related excitatory amino acid) is a major excitatory neurotransmitter in the mammalian cochlear nucleus (for reviews, see Wenthold. 1991; Hunter et al., 1992; Wenthold et al., 1993). Two main excitatory pathways which terminate in the cochlear nucleus, the auditory nerve and the parallel fiber pathway, both use glutamate as a neurotransmitter, and, although the evidence is less compelling, most other excitatory synapses in the cochlear nucleus probably also use glutamate as a neurotransmitter. The dominant role of glutamate in the cochlear nucleus is consistent with its role elsewhere in the CNS where it is thought to be a neurotransmitter at most excitatory synapses (Monaghan et al., 1989). In addition to this major role in neurotransmission, two other properties have also contributed to the intense interest in glutamate: 1) Glutamate and glutamate analogs are potent neurotoxins, and this neurotoxicity is mediated through the same receptors which are involved in neurotransmission (for review, see Choi and Rothman, 1990). Human disorders, such as stroke, may involve a release of glutamate which leads to further neuronal loss through excitotoxicity. Therefore, blocking glutamate receptors would be expected to lessen the damage, and animal studies have indicated this may be a promising approach to treatment. Furthermore, neuronal loss that occurs with some neurodegenerative disorders may involve excitotoxicity suggesting that a similar treatment may be promising. 2) Glutamate receptors are involved in synaptic plasticity such as in learning and memory or synapse formation. In one model it is proposed that long-term potentiation (LTP) results from an increase in functional AMPA receptors at the postsynaptic membrane (Liao et al., 1995). Therefore, knowledge of the mechanisms which regulate the expression of synaptic glutamate receptors will give insights into the general area of synaptic plasticity.