Glutamate

C H A P T E R

36 Glutamate JONAS BROMAN Department of Physiological Sciences, Lund University Lund, Sweden

ERIC RINVIK, MARCO SASSOE-POGNETTO, HOSSEIN KHALKHALI SHANDIZ and OLE PETTER OTTERSEN Centre for Molecular Biology and Neuroscience, Institute of Basic Medical Sciences University of Oslo, Blindern, Oslo, Norway Dipartimento di Anatomia, Farmacologia e Medicina Legale University of Turin, Italy

Glutamate (Glu) is undoubtedly the most prevalent transmitter in the brain. This amino acid is probably being used as a signaling substance in a majority of synapses, alone or along with peptides or other neuroactive compounds that colocalize with Glu. The excitatory effect of Glu was recognized in the early 1950s (Hayashi, 1954; Curtis and Watkins, 1960), but it took a long time until Glu was generally accepted as a neurotransmitter (Krnjevic, 1986; Watkins, 1986). Notably, the high concentration and relatively even distribution of Glu among brain regions were difficult to reconcile with a transmitter role. By the mid-1980s (Fonnum, 1984), Glu largely fulfilled the four main criteria for classification as a neurotransmitter: presynaptic localization, release by physiological stimuli, identical action with naturally occurring transmitter, and mechanism for rapid termination of transmitter action. Later investigations have strengthened a neurotransmitter role for Glu by demonstrating an ATPdependent selective transport of Glu into purified synaptic vesicles (Naito and Ueda, 1985; Maycox et al., 1990; Fykse et al., 1989; Winther and Ueda, 1993), the presence of high concentrations of Glu in synaptic vesicles isolated from the brain (Riveros et al., 1986; Burger et al., 1989; Orrego and Villanueva, 1993), and Ca2+-dependent exocytotic release of Glu from isolated nerve terminals (Nicholls, 1995). However, the

The Rat Nervous System, Third Edition

molecular basis for vesicular accumulation of Glu was long unknown. This has changed with the discovery of a family of vesicular Glu transporters (VGLUT1-3; Bellocchio et al., 2000; Takamori et al., 2000, 2001, 2002; Fremeau et al., 2001; Gras et al., 2002). As to the postsynaptic effect of Glu, rapid application of Glu to neuronal membrane patches at a concentration similar to that estimated to be present in the synaptic cleft following exocytotic release mimics the response that is obtained following the activation of excitatory synapses (Colquhoun et al., 1992; Clements et al., 1992; Bergles et al., 1999). Extensive molecular studies during the recent decade have provided detailed knowledge on the subunit proteins and gene families of Glu receptors (for reviews, see Blackstone and Huganir, 1995; Scannevin and Huganir, 2000). This chapter deals with anatomical aspects of transmitter Glu and provides an overview of the neuronal populations that use Glu as a neurotransmitter. The wide distribution of Glu precludes a comprehensive analysis of the topic within the available space and, in many cases, we have had to cite reviews rather than the original publications. Before describing the putative Glu-ergic projections in the brain, it is necessary to discuss the techniques that are available for the identification of neurons that use Glu as a transmitter. Biochemical procedures,

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detecting reduced content or uptake of Glu or Glu analogues following lesions, have proved useful in investigations of major projections (e.g., corticofugal fiber tracts; Fonnum, 1984; Storm-Mathisen and Ottersen, 1988), but poor sensitivity hampers analysis of smaller pathways. Identification of many minor Glu-ergic projections was made possible by the use of the metabolically inert Glu analogue D-[3H]aspartate as a transmitter-specific retrograde tracer (Baughman and Gilbert, 1980; Streit, 1980). It is a problem that 3 D-[ H]aspartate does not differentiate between putative Glu-ergic and aspartergic projections. Further, a number of fiber tracts likely to use Glu as a neurotransmitter are poorly labeled or unlabeled by D-[3H]aspartate, possibly due to the low presynaptic Glu uptake capacity of the terminals of such pathways (Ottersen, 1991). Analyses of the detailed anatomical distribution of Glu became possible with the development of antibodies to aldehyde-fixed amino acids (Storm-Mathisen et al., 1983). Early studies based on amino acid immunocytochemistry (Ottersen and Storm-Mathisen, 1984a, 1984b; Somogyi et al., 1986; Wanaka et al., 1987; Yoshida et al., 1987; Hepler et al., 1988; Chagnaud et al., 1989; Liu et al., 1989; Pow and Crook, 1993) demonstrated that Glu is widely distributed in the brain and localized not only in presumed Glu-ergic neurons, but also in neurons with other transmitter signatures. This is in line with biochemical data pointing to the involvement of Glu in several metabolic functions (protein synthesis, intermediary metabolism, and as a precursor for GABA). Introduction of the postembedding immunogold technique to amino acid immunocytochemistry (Somogyi and Hodgson, 1985) made it possible to analyze the distribution of Glu at a quantitative level and at higher anatomical resolution. This helped distinguish transmitter Glu from other pools of Glu. Using the immunogold approach, Somogyi et al. (1986) demonstrated enrichment of Glu immunoreactivity in parallel and mossy fiber terminals in the cerebellum, and later studies showed that the strength of the immunogold signal in these terminals was strongly correlated to the density of synaptic vesicles (Ji et al., 1991). Based on the assumption that a vesicular enrichment of Glu is a hallmark of Glu-ergic synapses, the quantitative immunogold approach has been used extensively to identify such synapses in the mammalian brain. The usefulness of this approach has been further improved by the development of combinations of anterograde tracing and immunogold labeling (De Biasi and Rustioni, 1988; Broman et al., 1990). As many of the data reviewed here are based on immunogold labeling, a critical evaluation of this technique appears relevant.

The ubiquitous presence of Glu in the central nervous system (CNS) sets hurdles for the analysis of Glu immunogold-labeled preparations and calls for quantitative analyses. Thus, the mere presence of Glu does not necessarily indicate a transmitter role. It serves to illustrate this that low levels of Glu occur in terminals rich in GABA or glycine (e.g., Somogyi et al., 1986; Bramham et al., 1990; Broman et al., 1990, 1993; Todd et al., 1994; Örnung et al., 1998). As biochemical studies have demonstrated high levels of Glu in synaptic vesicles (see earlier discussion), Glu-ergic terminals should be rich in Glu. Data from immunogold studies support this notion. However, demonstration of an enrichment of Glu fulfills only the first of the four main criteria of a neurotransmitter. A critical question is whether Glu may be present in high concentrations also in terminals not using Glu for synaptic transmission. The levels of Glu in terminals with other transmitter signatures than GABA or glycine (e.g., monoaminergic fibers) are largely unknown (but see Torrealba and Müller, 1999). It is noteworthy that strong immunogold signals for Glu were detected in motor nerve terminals innervating fast-twitch (but not slow-twitch) muscle fibers in rats (Waerhaug and Ottersen, 1993), although it remains to be shown whether these terminals release Glu in addition to acetylcholine. More recently Clarke et al. (1997) reported that cholinergic terminals in the basal ganglia contained levels of Glu that were intermediate to those in terminals with asymmetric and symmetric synapses, respectively. A corelease of acetylcholine and Glu has been demonstrated from presumed cholinergic synaptosomes and from cholinergic terminals of the Torpedo electric organ (Docherty et al., 1987; Vyas and Bradford, 1987). Although a colocalization of Glu with another neuroactive compound may point to transmitter roles for both substances, one cannot rule out that significant levels of “metabolic” Glu are present in certain populations of terminals. We must conclude that an enrichment of Glu within nerve terminals speaks strongly in favor of a transmitter role for Glu, but that immunogold data, like data obtained with other techniques, must be interpreted with due caution and with reference to alternative techniques addressing different features of Glu-ergic synapses. One such feature is vesicular Glu uptake. The discovery of a family of vesicular Glu transporters (VGLUT1-3; Bellocchio et al., 2000; Takamori et al., 2000, 2001, 2002; Fremeau et al., 2001; Gras et al., 2002) has opened up new possibilities for the identification of putative Glu-ergic neurons. Antibodies to these transporters have provided selective labeling of vesicle clusters in well-characterized Glu-ergic path-

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ways (e.g., Fremeau et al., 2001). However, with this approach, any negative results must be interpreted with caution as there might be VGLUT isoforms that remain to be discovered. For references to early studies of Glu pathways, the reader may consult previous reviews (e.g., Ottersen and Storm-Mathisen, 1984b; Fonnum, 1984; Ottersen, 1991; Storm-Mathisen et al., 1995; Broman et al., 2000). In keeping with the scope of the present volume, we have largely ignored analyses in species other than rat.

ANATOMICAL SYSTEMS Neocortex Being easily amenable to biochemical analysis, the massive corticofugal projections, especially corticostriatal projections, were among the first for which Glu was assigned a transmitter role (Divac et al., 1977; McGeer et al., 1977). Since then, biochemical, pharmacological, and immunocytochemical studies have implicated Glu as a neurotransmitter in a large number of neocortical output systems (for reviews, see Fonnum, 1984; Storm-Mathisen and Ottersen, 1988; Tsumoto, 1990; Ottersen, 1991; McCormick and von Krosigk, 1992; Broman et al., 2000). In addition to the corticostriatal path (Fonnum et al., 1981; Girault et al., 1986; Gundersen et al., 1996), putative Glu-ergic pathways include cortical projections to the thalamus (Lund-Karlsen and Fonnum, 1978; Baughman and Gilbert, 1980, 1981; Fonnum et al., 1981; Young et al., 1981; Montero and Wenthold, 1989; Montero, 1990; Broman and Ottersen, 1992; McCormick and von Krosigk, 1992; De Biasi et al., 1994a; Ericson et al., 1995; Blomqvist et al., 1996; Eaton and Salt, 1996), to several loci in the brain stem (Young et al., 1981; Rustioni and Cuenod, 1982; Matute and Streit, 1985; Azkue et al., 1995; Ortega et al., 1995; Mize and Butler, 1996; Torrealba and Müller, 1996, 1999), and to the spinal cord (Young et al., 1981; Rustioni and Cuenod, 1982; Potashner et al., 1988; Valtschanoff et al., 1993). Retrograde tracing with D-[3H]aspartate also supports Glu as a neurotransmitter in pyramidal neurons projecting to the ipsilateral or contralateral cortex (local, associational, and commissural connections; Streit, 1980; Barbaresi et al., 1987; Elberger, 1989; Kisvarday et al., 1989; Johnson and Burkhalter, 1992). Although the majority of cortical interneurons are inhibitory and immunoreactive for GABA (Somogyi et al., 1998), a special type of local circuit neuron in layer IV, the spiny stellate neuron, is excitatory and assumed to use Glu as a neurotransmitter (Saint Marie and Peters, 1985; Tsumoto, 1990; Anderson et al., 1994).

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Details are yet sparse as to what populations of cortical neurons express vesicular Glu transporters. VGLUT1 mRNA is expressed at high levels in neurons of all layers (except layer I), whereas VGLUT2 mRNA shows a more restricted distribution with a preference for small neurons of layer IV (Ni et al., 1994; Hisano et al., 2000; Fremeau et al., 2001; Herzog et al., 2001; Fremeau et al., 2002). Although further analyses are required, distribution patterns suggest that pyramidal projection neurons primarily use VGLUT1 to accumulate Glu into synaptic vesicles (this is also supported by the distribution and appearance of VGLUT1-immunoreactive terminals in subcortical areas; Bellocchio et al., 1998; Sakata-Haga et al., 2001; Kaneko et al., 2002; Varoqui et al., 2002), whereas excitatory cortical local circuit neurons may use VGLUT2 for this purpose. With respect to the recently described VGLUT3, data on cortical expression are sparse and partly conflicting (Fremeau et al., 2002; Gras et al., 2002; Schäfer et al., 2002; Takamori et al., 2002). In conclusion, there is strong and overwhelming evidence that Glu acts as a neurotransmitter in most, if not all, projection neurons of the cerebral cortex and presumably also in excitatory cortical local circuit neurons (Fig. 1). Although there is now strong evidence in support of Glu as a neurotransmitter in the thalamic inputs to the cerebral cortex, the evidence in favor of such a role has been less straightforward than for corticofugal projections. In some studies, cortical injections of 3 D-[ H]aspartate have resulted in no or only few retrogradely labeled neurons in the thalamus (Streit, 1980; Baughman and Gilbert, 1981; Barbaresi et al., 1987). Others have demonstrated retrograde D-[3H]aspartate transport from the cortex to a large number of neurons in the nonspecific groups of nuclei (e.g., midline and intralaminar nuclei; Ottersen et al., 1983), in the lateral geniculate nucleus (Johnson and Burkhalter, 1992), and in the mediodorsal and other medial and intralaminar nuclei (Pirot et al., 1994). In Glu immunogold studies, high levels of Glu have been detected in collateral terminals of geniculocortical neurons in cats (Montero, 1990) and in anterogradely labeled thalamocortical terminals in the somatic sensory, auditory, and visual cortices of rats (Kharazia and Weinberg, 1993, 1994). The latter authors also noted a significant positive correlation between the densities of Glu immunogold labeling and synaptic vesicles in thalamocortical terminals. Terminals of thalamocortical axons projecting from the anterior thalamic nuclei to the retrosplenial granular cortex are similarly rich in Glu (Wang et al., 2001). In situ hybridization reveals strong expression of VGLUT2 mRNA in numerous neurons throughout the

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thalamocortical input, i.e., most dense in layer IV but also evident in layers I and VI (Bellocchio et al., 1998; Fremeau et al., 2001, Fujiyama et al., 2001; Herzog et al., 2001; Sakata-Haga et al., 2001; Kaneko and Fujiyama,, 2002; Kaneko et al., 2002; Varoqui et al., 2002; Minelli et al., 2003). Further, kainic acid lesions of the thalamic ventrobasal complex result in almost complete disappearance of VGLUT2 immunoreactivity in the somatosensory cortices, with no apparent reduction of VGLUT1 immunoreactivity (Fujiyama et al., 2001). Thus, available data from D-[3H]aspartate tracing, Glu immunogold labeling, and detection of vesicular Glu transporters and their mRNAs concur with physiological and pharmacological observations (see, e.g., Tsumoto, 1990; Hicks et al., 1991; McCormick and von Krosigk, 1992) in providing strong support for Glu as a transmitter in most, if not all, thalamocortical neurons. The literature on excitatory connections in the hippocampus has been reviewed elsewhere (Ottersen and Storm-Mathisen, 2000) and is not dealt with here.

Sensory Systems Somatosensory Pathways

FIGURE 1 Glutamatergic projections originating in the neocortex are shown. The neocortex gives rise to glutamatergic projections to the ipsilateral (1) and contralateral (2) neocortices, as well as to a large number of subcortical structures (some target structures have been left out for the sake of clarity): ACb, accumbens nucleus; Amg, amygdala; CG, central gray; CPu, caudate putamen; Cu, cuneate nucleus; Gr, gracile nucleus; IC, inferior colliculus; Pn, pontine nuclei; R, nucleus ruber; SC, superior colliculus; SN, substantia nigra; Th, thalamus; Tu, olfactory tubercle; VTA, ventral tegmental area.

thalamus, whereas VGLUT1 mRNA is expressed at low levels in many nuclei, except in the medial habenula where VGLUT1 mRNA expression is high (Ni et al., 1994; Hisano et al., 2000; Fremeau et al., 2001; Herzog et al., 2001). While VGLUT1-immunoreactive terminals are distributed fairly homogeneously throughout all cortical layers, immunocytochemical detection of VGLUT2 reveals high densities of terminal staining primarily in cortical layers receiving

Glu has been regarded as a strong transmitter candidate in primary afferent neurons ever since its excitatory effect on spinal neurons was detected (Curtis and Watkins, 1960; Rustioni and Weinberg, 1989; Broman, 1994; Broman et al., 2000), although there have been uncertainties regarding the proportion and types of primary afferent fibers using Glu as a neurotransmitter (Salt and Hill, 1983; Schneider and Perl, 1988). Investigations during the recent decade have provided strong support for Glu as a neurotransmitter in all categories of primary afferent fibers terminating in the dorsal horn and in dorsal column nuclei. Primary afferent terminals in all laminae of the spinal cord dorsal horn are rich in Glu (Broman et al., 1993; Valtschanoff et al., 1994), as are those in the cuneate nucleus (De Biasi et al., 1994b). Enrichment of Glu has also been described in select populations of primary afferent terminals in the spinal or trigeminal dorsal horns (De Biasi and Rustioni, 1988; Maxwell et al., 1990b, 1993; Merighi et al., 1991; Rousselot et al., 1994; Iliakis et al., 1996) and in vagal afferents to the solitary tract nucleus (Saha et al., 1995; Sykes et al., 1997). There are also positive correlations between the density of synaptic vesicles and the Glu immunogold labeling density in different populations of dorsal horn primary afferent terminals, further supporting a vesicular localization and thus a transmitter role of Glu in these terminals (Broman and Ådahl, 1994;

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Larsson et al., 2001). In contrast to Glu, the levels of aspartate in lamina I–IV primary afferent terminals are low and not associated with synaptic vesicles (Larsson et al., 2001). Investigations on the localization of vesicular Glu transporters demonstrate that VGLUT1 is present in relatively large terminals located in the deep laminae of the dorsal horn (lamina III–VI) and less densely in parts of the ventral horn, including the motor nuclei. VGLUT2-immunoreactive terminals are, on average, smaller than VGLUT1-immunoreactive terminals and are distributed more evenly throughout the spinal gray matter with the highest density in laminae I and II (Kaneko et al., 2002; Varoqui et al., 2002; Li et al., 2003; Todd et al., 2003). Transganglionic labeling with cholera toxin subunit B (CTb, which selectively labels myelinated primary afferent fibers) demonstrates that all CTb-labeled primary afferent terminals in the deep dorsal horn contain VGLUT1 and that some also contain VGLUT2 (Todd et al., 2003). Most CTb-labeled terminals in lamina I (presumably A∂ nociceptor terminals) contain VGLUT2 but none contain VGLUT1. Of the examined primary afferent C-fiber terminals in lamina II (defined by isolectin B4 staining or immunolabeling for substance P + CGRP or somatostatin + CGRP), some displayed weak staining and others no staining for VGLUT2, whereas none were stained for VGLUT1 (however, see Li et al., 2003). The latter finding is somewhat surprising considering the high levels of Glu in primary afferent C-fiber terminals (Broman et al., 1993; Broman and Ådahl, 1994; Valtschanoff et al., 1994). A possible explanation is that vesicular Glu uptake in these terminals depends on VGLUT3 (the expression of which has been detected in dorsal root ganglia; Gras et al., 2002) or on a mechanism that remains to be characterized (Todd et al., 2003). Glu has been detected in sizable proportions of terminals contacting the cell bodies or dendrites of neurons that give rise to ascending somatosensory pathways, including the spinothalamic tract (Westlund et al., 1992; Lekan and Carlton, 1995), the spinocervical tract (Maxwell et al., 1992), and the postsynaptic dorsal column pathway (Maxwell et al., 1995). Such terminals may originate from primary afferents, from intrinsic spinal neurons, or from descending pathways (e.g., the corticospinal tract; Valtschanoff et al., 1993). Although their anatomic organization remains to be defined, there is considerable evidence for the presence of intrinsic excitatory circuits in the dorsal horn (Willis and Coggeshall, 1991). In the superficial dorsal horn, high levels of Glu have been detected in neurotensinimmunoreactive terminals, presumed to have an

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exclusive intraspinal origin (Todd et al., 1994). The majority of such terminals also label for VGLUT2, as do most enkephalin-immunoreactive terminals in the superficial dorsal horn (Todd et al., 2003). Further, virtually all substance P and somatostatinimmunoreactive terminals that lack CGRP (i.e., terminals that originate from other sources than the primary afferents) are immunopositive for VGLUT2 (Todd et al., 2003). Thus, current evidence supports the presence of several populations of excitatory local circuit neurons in the dorsal horn that influence the activity of ascending projection neurons through synaptic release of Glu. Negative findings following D-[3H]aspartate tracing from the thalamus initially argued against a role for excitatory amino acids as neurotransmitters in the ascending somatosensory pathways (Rustioni et al., 1983), although findings in electrophysiological/ pharmacological studies did support such a role (Salt, 1986; Klockgether, 1987; Salt and Eaton, 1996). However, since 1990, a series of studies using Glu immunogold labeling have provided strong evidence in support of Glu as a neurotransmitter in a number of ascending somatosensory pathways. The most comprehensive studies have been made in cats or primates, but available data from rats and mice (De Biasi and Rustioni, 1990; Hamori et al., 1990; De Biasi et al., 1994a; Hamlin et al., 1996; Azkue et al., 1998) are entirely consistent with findings in other species. Thus, enrichment of Glu has been detected in spinocervical tract terminals in the lateral cervical nucleus (Broman et al., 1990; Kechagias and Broman, 1994, 1995), in terminals from the lateral cervical and dorsal column nuclei in the thalamic ventral posterior lateral nucleus (VPL; Broman and Ottersen, 1992; De Biasi et al., 1994a; Kechagias and Broman, 1995), in spinothalamic tract terminals in the nucleus submedius and posterior region of the thalamus (Ericson et al., 1995; Blomqvist et al., 1996), and in spinomesencephalic terminals in the periaqueductal gray (Azkue et al., 1998). Further, in several of these terminal populations, significant positive correlations between synaptic vesicle and Glu immunogold labeling densities were evident, thus supporting a vesicular accumulation of Glu. A notable exception to the aforementioned findings is the report of relatively low levels of Glu in terminals of the postsynaptic dorsal column pathway (PSDC; De Biasi et al., 1995). Glu levels in PSDC terminals in the cuneate nucleus are significantly lower than those detected in primary afferent terminals and are about the same as those in inhibitory terminals. Thus, the transmitter of neurons projecting from the spinal cord to dorsal column nuclei remains to be identified.

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Little is known about the expression of vesicular Glu transporters in ascending somatosensory pathways. However, VGLUT2-immunoreactive terminals in the ventral posterior lateral nucleus of the thalamus display light and electron microscopic features typical of terminals derived from ascending somatosensory fibers (Sakata-Haga et al., 2001; Kaneko and Fujiyama, 2002; Kaneko et al., 2002). In conclusion, there is strong and, in some cases, overwhelming evidence that Glu serves a neurotransmitter role in perhaps all somatosensory primary afferent fibers and in at least most central somatosensory pathways, including the somatosensory thalamocortical connections (see Anatomical Systems, Neocortex; Fig. 2). Visual Pathways The initial part of visual pathways is situated in the retina, where photoreceptors form synaptic connections with bipolar cells, which in turn synapse with ganglion neurons in the inner plexiform layer. These connections are referred to as the “vertical” or “through” pathway of the retina, whereas horizontal cells and amacrine cells form the intraretinal lateral connections. There is strong support for Glu as a neurotransmitter in the “vertical” pathway (Ehinger and Dowling, 1987; Massey and Redburn, 1987; Daw et al., 1989). Glu is released from photoreceptors (Copenhagen and Jahr, 1989), and several immunocytochemical studies have reported high levels of Glu in photoreceptor cells, especially in their terminals (Davanger et al., 1991; Kalloniatis and Fletcher, 1993; Jojich and Pourcho, 1996; Huster et al., 1998; Davanger et al., 1994b). Also, bipolar cells are rich in Glu and their terminals contain higher levels of Glu than their parent cell bodies (Ehinger et al., 1988; Davanger et al., 1991; Martin and Grünert, 1992; Kalloniatis and Fletcher, 1993; Davanger et al., 1994a; Jojich and Pourcho, 1996). Although Glu is generally considered an excitatory transmitter, in the synapses between photoreceptors and on-center bipolar cells, Glu exerts an inhibitory action through metabotropic receptors (Copenhagen, 1991; Nakajima et al., 1993; Euler et al., 1996; Sasaki and Kaneko, 1996; Vardi and Morigiwa, 1997; Brandstatter et al., 1997; De Vries and Schwartz, 1999; Morigiwa and Vardi, 1999). Thus, light-induced hyperpolarization of photoreceptors, leading to a diminished release of Glu from their terminals, results in depolarization of on-center bipolar cells (reduced inhibition) and a hyperpolarization of off-center bipolar cells (reduced excitation). The patterns of mRNA expression and immunolabeling for vesicular Glu transporters in the rat retina suggest that VGLUT1 is used for vesicular accumulation of Glu in both photoreceptors and bipolar cells (Mimura et al., 2002).

FIGURE 2 Schematic drawing of somatosensory and visual glutamatergic fiber systems. CTT, cervicothalamic tract; DRG, dorsal root ganglion primary afferent fibers; Hy, retinal projection to the hypothalamus; LG, retinal projection to the lateral geniculate nucleus; ML, fibers in the medial lemniscus from cuneate and gracile nuclei; Ret, retinal photoreceptors and bipolar cells; SC, retinal projection to the superior colliculus; SCT, spinocervical tract; SpPAG, spinomesencephalic input to periaqueductal gray; STT, spinothalamic tract; TCss, somatosensory thalamocortical projections; TCv, visual thalamocortical projections.

Signals generated by photoreceptors are communicated to the brain through the axons of ganglion cells projecting through the optic nerve and tract. The main termination of the optic tract is located in the thalamic lateral geniculate nucleus, which relays the signals to the visual cortices. Pharmacological data support Glu as a transmitter of retinal terminals in the lateral geniculate nucleus of rats (Crunelli et al., 1987), and enucleation in rats results in a loss of Glu in this nucleus on the contralateral side (Sakurai and Okada, 1992). Further, retinal terminals in the lateral geniculate of both cats and monkeys are rich in Glu immunoreactivity (Montero and Wenthold, 1989;

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Montero, 1990). Ganglion neurons in the rat retina also express VGLUT2 mRNA and display VGLUT2 immunoreactivity (Mimura et al., 2002). VGLUT2immunoreactive terminals in the rat lateral geniculate correspond morphologically to retinal terminals, and contralateral enucleation results in a large decrease of VGLUT2 immunoreactivity in the lateral geniculate nucleus (Sakata-Haga et al., 2001; Kaneko and Fujiyama, 2002; Kaneko et al., 2002). Thus, several lines of evidence support a transmitter role for Glu in ganglion cell terminals in the latter nucleus. Studies using 3 D-[ H]aspartate tracing or Glu immunogold labeling also point to a neurotransmitter role of Glu in optic tract terminals in other regions, including the superior colliculus (Matute and Streit, 1985; Ortega et al., 1995; Mize and Butler, 1996), the pretectum (Nunes-Cardoso et al., 1991), and the hypothalamus (Castel et al., 1993; De Vries et al., 1993; Chen and Pourcho, 1995). As stated in the section on the neocortex, both 3 D-[ H]aspartate tracing and Glu immunogold labeling suggest that Glu acts as a transmitter in the thalamocortical connection of the lateral geniculate nucleus. Thus, Glu appears to mediate signal transfer in each step of the visual pathway, from photoreceptor synapses in the retina to geniculocortical synapses in the visual cortex (Fig. 2). Auditory Pathways Considerable evidence supports an excitatory amino acid as a neurotransmitter in the synapses between inner hair cells and cochlear afferent nerve fibers (reviewed by Usami et al., 2000). Hair cells are rich in Glu in comparison to most other cellular elements in the cochlea (Usami et al., 1992), and GluR2/3 and GluR4 AMPA receptor subunits have been detected in inner, but not outer, hair cell synapses (Matsubara et al., 1996). However, although immunogold particles signaling Glu are associated with synaptic vesicles in hair cells, it remains to be shown that hair cell synaptic vesicles are indeed rich in Glu (Usami et al., 2000). Thus, although Glu must be considered a very strong cochlear hair cell transmitter candidate, definitive evidence is pending. Several lines of evidence, including pharmacological data, also support a transmitter role for Glu in cochlear nerve terminals in the cochlear nuclei (reviewed by Parks, 2000). Further, quantitative immunogold studies have shown that type I cochlear afferent terminals are rich in Glu, display high Glu/glutamine ratios, and are depleted in Glu following K+-induced depolarization (Hackney et al., 1996; see also Alibardi, 2003). Among the other terminal populations in the auditory system that have been subjected to Glu immunogold analysis are the calyces of Held in the

FIGURE 3 Schematic drawing of auditory glutamatergic fiber systems. cH, calyces of Held in the medial nucleus of the trapezoid body; CN-LSO, cochlear nucleus inputs to the lateral superior olive; HC, cochlear hair cells; Pf, granule cell/parallel fibers in the dorsal cochlear nucleus; TCa, auditory thalamocortical projections; 8cn, cochlear primary afferent fibers.

medial nucleus of the trapezoid body. These large terminals, which originate in the ventral cochlear nucleus on the contralateral side, exhibit a strong Glu immunogold labeling that is concentrated over vesicle clusters and mitochondria (Grandes and Streit, 1989). Helfert et al. (1992) detected high levels of Glu in round vesicle-containing terminals, presumably originating from the ipsilateral cochlear nucleus, in the lateral superior olive. In the dorsal cochlear nucleus, parallel fiber terminals originating from granule cells are rich in Glu, which is depleted by depolarization with high [K+] (Osen et al., 1995). High levels of Glu have also been recorded in auditory nerve terminals and granule cell terminals, as well as in large “mossy” terminals in the dorsal cochlear nucleus (Rubio and Juiz, 1998). As indicated in the section on the neocortex, enrichment of Glu has also been detected in auditory thalamocortical axon terminals (Kharazia and Weinberg, 1994; Fig. 3).

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Additional excitatory projections in the brain stem auditory system include inputs to the nuclei of the lateral lemniscus from the cochlear nucleus and lateral superior olive contralaterally and from the medial superior olive ipsilaterally. The list of excitatory connections also comprises cochlear nuclei efferents to the bilateral medial superior olive, input through the lateral lemniscus to the inferior colliculus (including fibers from lateral lemniscal nuclei), commissural connections between the inferior colliculi, and the projection from the inferior colliculus to the medial geniculate body (the latter projection also includes an inhibitory component). That these fiber systems use an excitatory amino acid as a neurotransmitter receives support from pharmacological–physiological studies and studies of retrograde transport or uptake/release of D-[3H]aspartate (Schwarz and Schwarz, 1992; Suneja et al., 1995; Saint Marie, 1996; Moore et al., 1998; Wu, 1998; Parks, 2000; Bartlett and Smith, 2002). Glu is the most likely transmitter candidate of the aforementioned fiber systems. In agreement, auditory relay stations in the brain stem and thalamus contain an abundance of nerve terminals immunoreactive for VGLUT1 or 2 (Sakata-Haga et al., 2001; Kaneko et al., 2002; Varoqui et al., 2002). Olfactory Pathways The olfactory system comprises three hierarchically ordered subdivisions: the olfactory epithelium located in the nasal cavity, the olfactory bulb, and the olfactory cortex, an array of cortical areas that receive direct input from the olfactory bulb (Shipley and Ennis, 1996; Chapter 29). Evidence shows that Glu acts as a neurotransmitter in both primary sensory afferents to the olfactory bulb and in bulbar projections to higher order olfactory areas. The axons of sensory neurons that reach the olfactory bulb terminate in spheroid structures of neuropil called glomeruli, where they establish synapses with the dendrites of the output neurons (mitral and tufted cells) and one type of interneuron, the periglomerular cell. The Glu-ergic nature of these afferents is supported by immunocytochemical studies, showing that Glu occurs in high levels in axon terminals of olfactory sensory neurons (Liu et al., 1989; Sassoè-Pognetto et al., 1993). Significantly, Glu immunoreactivity is more elevated in nerve terminals compared with axons and with postsynaptic dendrites (Didier et al., 1994). Electrophysiological recordings also support this conclusion, as stimulation of the olfactory nerve evokes AMPA and NMDA responses in mitral cells (Berkowicz et al., 1994; Ennis et al., 1996). Olfactory neurons also contain taurine and carnosine, a dipeptide that has been proposed to have modu-

latory or neuroprotective functions (Margolis, 1974; Sassoè-Pognetto et al., 1993; Didier et al., 1994; Horning et al., 2000). There is extensive immunocytochemical and pharmacological evidence that the output neurons of the olfactory bulb release Glu (reviewed in Shepherd and Greer, 1998; Haberly, 1998). Mitral and tufted cells are strongly labeled with antibodies against Glu (Ottersen and Storm-Mathisen, 1984a, 1984b; Liu et al., 1989). These neurons also show immunoreactivity for N-acetyl-L-aspartyl-L-glutamic acid (NAAG) and aspartate (Anderson et al., 1986; Saito et al., 1986; Blakely et al., 1987), but a transmitter role of these substances is not supported by functional analyses (Whittemore and Koerner, 1989; Trombley and Shepherd, 1993). Substantial evidence shows that mitral and tufted cells release Glu both from their dendrites in the glomerular layer and external plexiform layer (where they establish dendrodendritic synapses with periglomerular cells and with granule cells, respectively) and from their axons in the olfactory cortex (Hennequet et al., 1998). Dendrodendritic synapses between mitral/tufted cells and granule cells are reciprocal pairs consisting of an asymmetric and a symmetric junction (Rall et al., 1966; Price and Powell, 1970a; Fig. 4). In these reciprocal connections, release of Glu from the principal neurons activates AMPA and NMDA receptors and triggers the release of GABA from granule cell spines (Nicoll, 1971a; Nowycky et al., 1981; Jahr and Nicoll, 1982; Trombley and Shepherd, 1992; Wellis and Kauer, 1993, 1994; Sassoè-Pognetto and Ottersen, 2000). In addition to activating postsynaptic receptors, Glu released by mitral cell dendrites can spread out of the synaptic cleft and activate receptors on the parent dendrite as well as on neighboring cells (Nicoll, 1971b; Aroniadou-Anderjaska et al., 1999; Isaacson, 1999; Friedman and Strowbridge, 2000; Salin et al., 2001). Immunogold and electrophysiological investigations indicate that some granule cell spines may also release Glu, although it is presently unknown whether individual granule cells can release both Glu and GABA (Didier et al., 2001). In conclusion, there is convincing evidence that Glu serves as a neurotransmitter in primary afferents and in local dendrodendritic circuits of the olfactory bulb. Other synapses that presumably use Glu are those formed by axon collaterals of mitral and tufted cells in the internal plexiform layer and granule cell layer. Afferent fibers from cerebral hemispheres also establish asymmetric synapses at various levels in the olfactory bulb (Price and Powell, 1970b; Pinching and Powell, 1972). The Glu-ergic nature of these synapses is supported indirectly by immunolabeling of axodendritic junctions with antibodies directed against Glu

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FIGURE 4 Dendrodendritic reciprocal synapses between a mitral cell (mc) and a granule cell spine (gc) in the rat olfactory bulb. The reciprocal synaptic arrangement consists of an asymmetric, mitral-to-granule synapse (thick arrow) and a symmetric, granule-to-mitral synapse (empty arrow), located side by side. This section was labeled with an antiserum against the NR1 subunit of NMDA receptors, and strong immunolabeling is visible over the asymmetric junction. Another asymmetric synapse with a different granule cell spine (lower left) is also labeled. Adapted from Sassoè-Pognetto and Ottersen (2000).

receptor subunits (Sassoè-Pognetto and Ottersen, 2000). Finally, external tufted cells and possibly other types of Glu-positive juxtaglomerular neurons may establish Glu-ergic synapses in the periglomerular neuropil (Pinching and Powell, 1971; Liu et al., 1989). Vomeronasal System The vomeronasal system is a chemosensory pathway that has evolved in many terrestrial vertebrates to detect nonvolatile pheromones associated primarily with social and reproductive behaviors. Pheromonal

information detected by the vomeronasal sensory organ is conveyed through the accessory olfactory bulb to the amygdala and then to the hypothalamus (Halpern, 1987; Mori, 1987; Liman, 1996; Bargmann, 1997). A convergence of immunocytochemical and electrophysiological data indicates that the basic synaptic organization of the accessory olfactory bulb is similar to that of the main olfactory bulb (Dudley and Moss, 1995; Jia et al., 1999; Quaglino et al., 1999). Thus, Glu appears to be the neurotransmitter used both by vomeronasal afferents and by output neurons. As in

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the main olfactory bulb, a punctate immunoreactivity for Glu is present in the periglomerular region and in the granule cell layer (Quaglino et al., 1999), suggesting that other types of synapse are also Glu-ergic.

Motor Pathways Motoneurons in the spinal cord and brain stem constitute the final common pathway for motor commands. The synaptic inputs to motoneuronal cell bodies and dendrites at different levels of the neuroaxis have been examined extensively in regard to neuroactive amino acid contents (Shupliakov et al., 1993; Murphy et al., 1996, Tai and Goshgarian, 1996; Yang et al., 1997; Örnung et al., 1998; Bae et al., 1999; Lindå et al., 2000; Somogyi, 2002). The proportion of premotoneuron terminals defined as Glu-ergic in immunogold studies varies from about 35% to over 50%. Thus, Glu seems to be a predominant excitatory transmitter in nerve terminals synapsing on motoneurons. The pattern of immunostaining for the vesicular Glu transporters VGLUT1 and VGLUT2 further underscores an essential role for transmitter Glu in the excitation of motoneurons and interneurons in the ventral horn (Kaneko et al., 2002; Varoqui et al., 2002; Todd et al., 2003). In the ventral horn of the rat spinal cord, a moderate density of relatively large VGLUT1-immunoreactive terminals is evident in lamina VII and in motor nuclei. The smaller VGLUT2immunoreactive terminals occur at moderate to high densities throughout the ventral horn. Glu-ergic terminals in the ventral horn may originate from fiber tracts descending from the cortex or the brain stem, from intraspinal neurons, or from primary afferent fibers. The only type of primary afferent fiber connecting directly with motoneurons are Ia fibers from muscle spindles. As for other primary afferent fibers (see section on somatosensory pathways), there is overwhelming evidence in favor of Glu as a transmitter in Ia afferent boutons. Transmission between Ia fibers and motoneurons is blocked by excitatory amino acid receptor antagonists (Jessel et al., 1986). Transganglionically labeled Ia afferent terminals in contact with motoneurons and neurons in the central cervical nucleus are also rich in Glu (Örnung et al., 1995), as are giant boutons (likely to originate from Ia fibers) in Clarke’s column (Maxwell et al., 1990a). Further, all primary afferent terminals (labeled by the transganglionic transport of choleragenoid) in the ventral horn contain VGLUT1 but not VGLUT2 immunoreactivity (Todd et al., 2003). Thus, all types of primary afferent-relayed reflex activity likely depend on Glu-ergic neurotransmission. In contrast to the well-established neurotransmitter role for Glu in primary afferent fibers contacting

motoneurons and ventral horn local circuit neurons, there is weak evidence for Glu neurotransmission in other types of inputs. A notable exception is the corticospinal tract, which is widely recognized to use Glu as a transmitter (Storm-Mathisen and Ottersen, 1988; Rustioni and Weinberg, 1989; Valtschanoff et al., 1993). However, because direct corticospinal input to motoneurons is sparse in rats (Terashima, 1995), most Glu-ergic terminals in this species on motoneurons (except those of Ia afferent origin) must originate from intraspinal neurons or descending tracts. Considerable evidence from physiological– pharmacological studies shows that excitatory amino acid receptors mediate several different inputs to motoneurons (e.g. McCrimmon et al., 1989; Floeter and Lev-Tov, 1993; Pinco and Lev-Tov, 1994; Chitravanshi and Sapru, 1996; Hori et al., 2002). Glu is likely to be the transmitter in these inputs, although conclusive evidence is lacking. Several studies have detected the presence of Glu in cell bodies of brain stem neurons projecting to the spinal cord (Beitz and Ecklund, 1988; Mooney et al., 1990; Nicholas et al., 1992; Liu et al., 1995), but cell body labeling for Glu is unreliable as a marker for Glu-ergic neurons (Broman et al., 2000). However, Stornetta et al. (2003) reported that bulbospinal neurons in the rostral ventral respiratory group express VGLUT2 mRNA and that their terminals in the cervical ventral horn are immunoreactive for the same transporter. These findings indicate that this bulbospinal projection is Glu-ergic.

Basal Ganglia The basal ganglia comprise the striatum (caudate nucleus and putamen), the nucleus accumbens, the globus pallidus, the entopeduncular nucleus, the subthalamic nucleus, and the substantia nigra. These structures have profuse and complex fiber connections with each other, as well as with several other regions of the CNS. In only a minority of the many fiber connections of the basal ganglia in the rat has the transmitter substance been established by means of combined tracing and immunocytochemical studies at the ultrastructural level. However, information gained from various types of investigations has led to the identification of strong transmitter candidates for a number of the basal ganglia connections. The massive corticostriatal projection serves to illustrate this point. Early electrophysiological (Kitai et al., 1976; Wilson, 1986) and neurochemical (Spencer, 1976; Divac et al., 1977; Kim et al., 1977; Streit, 1980; Fonnum et al., 1981) investigations were suggestive of a Glu-ergic excitatory input to the striatum from the cerebral cortex. A subsequent light microscopical immunohistochemical study documented

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that a very large number of fibers and bouton-like structures in striatum of the rat display Glu immunoreactivity (Ottersen and Storm-Mathisen, 1984a, 1984b). It was later shown that striatal boutons with the ultrastructural characteristics of cortical afferents were rich in Glu and that they also sustained a high-affinity uptake of aspartate (Gundersen et al., 1996). Thus, even though immunocytochemical analyses of anterogradely labeled corticostriatal boutons are pending, available data support the notion that cortocostriatal fibers use Glu as a transmitter. This would be in line with the many reports on the distribution in the striatum of the rat of various types of Glu receptors (Bernard et al., 1996; Wullner et al., 1997; Petralia et al., 2000; Shigemoto and Mizuno, 2000; Wisden et al., 2000). It should be noted, however, that organization of the corticostriatal projection is very complex, and it remains an open question whether Glu is a transmitter in all corticostriatal fibers or only in a subpopulation of them. A light microscopic investigation showed that 52–61% of retrogradely labeled corticostriatal neurons in the rat displayed Glu immunoreactivity (Bellomo et al., 1998). Up to 96% of these neurons were immunopositive when antisera against Glu and aspartate were used simultaneously, and Glu- and aspartate-immunopositive cortical neurons appeared to be largely segregated (Bellomo et al., 1998). These data should be interpreted with caution as the level of Glu (and aspartate) in cell bodies may reflect the size of the metabolic pools rather than the transmitter pools of the respective amino acids. The cerebral cortex also sends fibers to other basal ganglia than the striatum, although on a smaller scale. Thus, the subthalamic nucleus (STN) of the rat receives fibers from wide cortical areas (Afsharpour, 1985; Canteras et al., 1988). Electrophysiological investigations suggested that this input was excitatory (Kitai and Deniau, 1981; Rouzaire-Dubois and Scarnati, 1987; Feger and Mouroux, 1991; Fujimoto and Kita, 1993). In a combined tracing and immunocytochemical study in the rat, it was indeed shown that a considerable number of the corticosubthalamic boutons are rich in Glu (Bevan et al., 1995). In the rat the corticosubthalamic projection is accompanied by a more modest corticopallidal pathway (Naito and Kita, 1994). The corticopallidal boutons have an ultrastructural appearance similar to that of cortical efferents in other basal ganglia, suggesting that these afferents are Glu-ergic. Immunocytochemical evidence of this is pending. The thalamus represents the second largest source of afferents to the basal ganglia. In a combined tracing and immunocytochemical study in the rat, it was shown that axon terminals in the subthalamic nucleus

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that originate in the parafascicular nucleus of the thalamus are very rich in Glu (Bevan et al., 1995). This finding lends support to earlier physiological and pharmacological investigations (Mouroux and Féger, 1993; Féger et al., 1997). As far as the massive thalamostriatal projection is concerned, the identity of the transmitter substance remains to be determined (De las Heras et al., 1997), although electrophysiological studies point to an excitatory signal substance (Purpura and Malliani, 1967; Buchwald et al., 1973; Kitai et al., 1976). It is likely that at least part of the complex thalamostriatal projection is Glu-ergic. A light microscopical study has suggested that many afferents to the ventral striatum from the amygdala in the rat use Glu as a transmitter (McDonald, 1996). In recent years the subthalamic nucleus has taken central stage in attempts to explain the pathophysiology of Parkinson’s disease (Albin et al., 1989b). Although several observations clearly indicate that the original model was too simplified (Marsden and Obeso, 1994; Levy et al., 1997; Obeso et al., 1997, 2000; Wichmann and DeLong, 1998; Bar-Gad and Bergman, 2001), it remains unquestionable that hyperactivity of the subthalamic neurons is a prominent feature in Parkinson’s disease. The main efferent projections of the subthalamic nucleus terminate in the two segments of the globus pallidus (primate), the globus pallidus and entopeduncular nucleus (rodents), and in the pars compacta and pars reticulata of the substantia nigra. A smaller contingent of fibers extends to the tegmental pedunculopontine nucleus (PPN) (Parent and Hazrati, 1993). Light microscopical immunohistological studies have shown that practically all cells in STN display strong Glu immunoreactivity in the rat (Ottersen and Storm-Mathisen, 1984a, 1984b) as in other species (Smith and Parent, 1988; Albin et al., 1989a). Because the presence of Glu in neuronal cell bodies is not necessarily indicative of a transmitter role, these findings are not decisive. However, a transmitter role of Glu is supported by electrophysiological observations in the rat (Kitai and Kita, 1987) and by combined tracing and immunocytochemical studies in the cat (Rinvik and Ottersen, 1993). The latter immunogold analysis showed that boutons of subthalamonigral fibers are rich in Glu. Similar investigations have not been undertaken for the subthalamopallidal or subthalamoentopeduncular projections. However, in the rat there is ample evidence that the subthalamofugal fibers send branches to both the substantia nigra and the globus pallidus (van der Kooy and Hattori, 1980). When correlated with immunohistochemical and tracing studies in other species, as well as with electrophysiological investigations in the rat (Robledo and

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Féger, 1990), it appears highly likely that the subthalamic projections to the globus pallidus and entopeduncular nucleus are Glu-ergic. The nature of the transmitter substance in the projection from the subthalamic nucleus to the pedunculopontine nucleus (PPN) remains to be determined. However, some data are available on the reciprocal connection. In combined tracing and immunocytochemical studies in the rat it was shown that PPN sends Glu-enriched fibers to the STN (Bevan and Bolam, 1995) and entopeduncular nucleus (Clarke et al., 1997). The latter authors demonstrated that a significant portion of labeled axon terminals from PPN displayed high levels of immunoreactivity against both Glu and choline acetyltransferase, suggestive of a colocalization of Glu and acetylcholine.

Cerebellum The transmitter systems of the cerebellum have been reviewed by Voogd et al. (1996) and by Ottersen and Walberg (2000). These reviews should be consulted for a complete bibliography. Mossy and climbing fibers constitute the major afferent pathways to the cerebellar cortex (for references, see Palay and Chan-Palay, 1974). It has long been known that these pathways are excitatory and that they have separate origins; the most important ones being the pontine nuclei and the inferior olive, respectively. The mossy fiber system is by far the more massive and establishes contacts with dendritic digits of granule cells. The latter cells are themselves excitatory (see later) and constitute the second leg in a disynaptic excitatory input to Purkinje cells. In contrast, climbing fibers excite Purkinje cells directly through their synapses on Purkinje cell dendritic thorns. It is now well established that both of the major afferent pathways mediate fast excitation (Ito, 1984), and several lines of evidence point to Glu as the likely transmitter. Mossy fibers display a strong immunogold signal for Glu (Somogyi et al., 1986; Fig. 5B). The intensity of this signal is correlated positively to the packing density of

synaptic vesicles (Ji et al., 1991) and is abolished following depolarization of cerebellar slices with high [K+] (Ottersen et al., 1990). This implies that the immunolabeling is likely to represent a transmitter pool. The postsynaptic elements of mossy fibers express several types of Glu receptor (Cox et al., 1990; Gallo et al., 1992; Petralia and Wenthold, 1992), and pharmacological data are consistent with the idea that Glu acts as their endogenous ligand (Garthwaite and Brodbelt, 1990). Mossy fibers are also very rich in phosphateactivated glutaminase (Laake et al., 1999), which is a key enzyme in Glu synthesis. VGLUT1 and VGLUT2 have both been identified in mossy fibers (Fremeau et al., 2001), but there is still some uncertainty as to the degree of colocalization of these two vesicular Glu transporters. It must be emphasized that some mossy fibers may use other signal substances, instead of or in addition to Glu. Subpopulations of mossy fibers express cholinergic markers and neuroactive peptides with presumed modulatory functions. These data are reviewed by Voogd et al. (1996) and by Ottersen and Walberg (2000). Climbing fibers are now believed to use Glu as a transmitter, like the majority of mossy fibers. Climbing fibers are very rich in Glu (Ottersen et al., 1992), contain the vesicular Glu transporter VGLUT2 (Fremeau et al., 2001; Pahner et al., 2003), and face thorns that express high concentrations of AMPA receptors (Landsend et al., 1997). Early studies showed that climbing fibers take up and retrogradely transport 3 D-[ H]aspartate to their perikarya in the inferior olive (Wiklund et al., 1984). In regard to the latter finding, it should be noted that the tracer D-[3H]aspartate does not differentiate between transport of the endogenous substrates L-aspartate and L-Glu (Danbolt et al., 1994). In fact, L-aspartate was long held to be the most likely climbing fiber transmitter. Supporting this view were slice experiments showing that evoked release of endogenous aspartate from the cerebellar cortex could be reduced by lesions of the inferior olive by 3-acetylpyridine (Toggenburger et al., 1983; Vollenweider et al., 1990). However, quantitative immunogold

FIGURE 5 Electron micrographs showing the distribution of glutamate-like immunoreactivity (small gold particles) in the rat cerebellar cortex. The section was also labeled with antibodies to glutamine (an important glutamate precursor), which were visualized by the use of large gold particles. (A) Molecular layer. Parallel fiber terminals (pf) are labeled strongly for glutamate. Some glutamate immunoreactivity is also found in spines (s), reflecting the presence of a metabolic pool. Glial processes (g) contain little glutamate immunoreactivity but display significant glutamine immunolabeling. Some large particles (indicating glutamine) overlie the intercellular space (arrows). (B) Granule cell layer. A mossy fiber terminal (mf) is strongly glutamate immunoreactive and also appears to contain a sizable pool of glutamine. The Golgi (Go) cell terminal (probably GABAergic) is comparatively weakly labeled. Note the flat vesicles (arrows). Asterisks denote granule cell dendrites (adapted from Ottersen et al., 1992). Scale bars: 0.4 µm.

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analyses with specific antibodies demonstrated that the level of L-aspartate in climbing fiber terminals was low compared with the average tissue level and with the level of this amino acid in the parent cell bodies in the inferior olive (Zhang et al., 1990). It is possible that a relative shortage of oxygen and energy substrates during the preparation and incubation of brain slices leads to a buildup of L-aspartate in nerve terminals that contain only sparse amounts of this amino acid under physiological conditions (Gundersen et al., 1998). One must also consider the possibility that immunogold analyses fail to reveal the entire endogenous pool of transmitter L-aspartate, either because this pool is released during the preparation of the tissue or because it is inaccessible to immunogold detection. The latter explanations are unlikely but cannot be ruled out entirely. Another excitatory amino acid that has been implicated in climbing fiber neurotransmission is homocysteic acid (HCA; Cuénod et al., 1989). Like L-aspartate, this sulfur-containing amino acid is released in smaller quantities than normal following lesions of the inferior olive (Vollenweider et al., 1990). However, with the advent of specific antibodies it was shown that HCA-like immunoreactivity is largely confined to glial elements, including Bergmann fibers (Grandes et al., 1991; Zhang and Ottersen, 1992, 1993). This rules out a transmitter role of HCA in climbing fibers. The possibility remains that HCA is engaged in an unorthodox signaling process involving release from glial cells (Do et al., 1997). If a substrate of plasma membrane Glu transporters is responsible for signal transfer in climbing fiber–Purkinje cell synapses, one would expect an interference with Glu transport to affect the postsynaptic response to climbing fiber activation. To test this, Takahashi et al. (1996) injected D-aspartate into Purkinje cells and indeed demonstrated that this led to a prolonged excitatory postsynaptic current at the climbing fiber synapses. The most likely explanation of this finding is that the injected D-aspartate inhibits an excitatory amino acid transporter that normally contributes to the removal of transmitter from the synaptic cleft (also see Otis et al., 1997). Candidate transporters are EAAT4, which is concentrated at specific membrane domains in Purkinje cell spines (Dehnes et al., 1998), and EAAT3 (formerly EAAC1), which is distributed more generally in neuronal plasma membranes (Rothstein et al., 1994). Similar to mossy fibers, climbing fibers contain a number of neuroactive substances in addition to Glu, including peptides with possible modulatory functions (Voogd et al., 1996). Thus data reviewed earlier

must not be taken to indicate that Glu is the sole neuroactive compound released from climbing fibers. Parallel fibers are the axons of cerebellar granule cells and serve as the second leg of a disynaptic excitatory input to Purkinje cells. Parallel fibers also establish synapses with dendritic stems of interneurons (Palay and Chan-Palay, 1974). Compelling evidence points to Glu as a parallel fiber transmitter. One of the first pieces of evidence came with the study of Young et al. (1974), who observed that a granule cell loss (caused by virus infection) was accompanied by a decreased content of Glu and Glu/aspartate uptake in the cerebellar cortex. Subsequent investigations showed that the content and uptake, as well as the release of Glu, depend on intact granule cells (for reviews, see Ito, 1984; Ottersen and Storm-Mathisen, 1984b). Parallel fiber terminals display a strong immunogold signal for Glu (Somogyi et al., 1986; Ottersen, 1989; Fig. 5A) and this signal depends on a Glu pool that can be depleted by high [K+] (Ottersen et al., 1990). Immunogold analyses have also shown that the postsynaptic specializations of parallel fiber synapses express AMPA and δ2 receptors (Baude et al., 1994; Nusser et al., 1994; Landsend et al., 1997). Parallel fiber terminals contain the vesicular Glu transporter VGLUT1 (Fremeau et al., 2001; Pahner et al., 2003). Data supporting a transmitter role of Glu in parallel fibers are indeed overwhelming, but it remains to clarify how their transmitter pool is maintained. Whereas the major Glu-synthesizing enzyme phosphate-activated glutaminase (PAG) is abundant in mossy fiber terminals, it occurs at very low levels in terminals of parallel fibers (Laake et al., 1999). Thus the latter fibers probably depend on alternative sources for transmitter replenishment. Glu is also a strong transmitter candidate for one type of interneuron in the cerebellar cortex: the unipolar brush cell (Mugnaini et al., 1997). This cell is exceptional among cerebellar interneurons by showing an enrichment in Glu and being presynaptic to Glu receptors (Nunzi and Mugnaini, 1999). The unipolar brush cell contacts granule cells and other unipolar brush cells, and presumably also Golgi cells (Nunzi and Mugnaini, 1999).

CONCLUSION The present survey of putative Glu-ergic fiber systems emphasizes the predominant role of Glu as a transmitter of projection neurons in the central nervous system. The evidence is now compelling that Glu is involved in signal transfer in major sensory and

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motor pathways and in associational and commissural connections of the neocortex. Glu also appears to serve a transmitter role in all of the major fiber pathways in the cerebellum with the exception of the GABA-ergic Purkinje cell projection. In contrast, few types of local circuit neurons exhibit a Glu-ergic phenotype. The latter class of neurons largely depends on GABA or glycine for fast synaptic transmission. Glu is certainly a prevalent transmitter, but this must not be equated with uniformity at the level of synaptic transmission. As the present review has been focused on Glu, we have not elaborated on the issue of transmitter colocalization. The fact is that many of the fiber systems that contain Glu also contain other neuroactive compounds. These may be colocalized with Glu or occur in separate fibers. It is also clear that L-aspartate may rival Glu as a transmitter in certain fiber systems (Gundersen and Storm-Mathisen, 2000). Another level of complexity is added by the structural and molecular heterogeneity among Glu synapses. Some Glu synapses are ensheathed by glial processes, whereas other synapses lack glial investment (Chaudry et al., 1995). Such differences obviously affect transmitter diffusion and hence synaptic function, and the complement of glutamate receptors varies widely between synapses even within individual fiber pathways (e.g., Nusser et al., 1998; Takumi et al., 1999). As a result, Glu synapses exhibit a functional diversity that may be underestimated easily when the focus is restricted to the issue of transmitter phenotype. In fact, unraveling the heterogeneity of Glu synapses will be the next major challenge once the general map of Glu pathways has been established.

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