Introduction

W-Acetylaspartylglutamate (NAAG), the most abundant brain peptide,1 has been localized immunocytochemically in a variety of neurons throughout the nervous system. In many cases, NAAG's presence has been confirmed by HPLC, but these results will not be described here. NAAG is also sometimes found in glia,2, 3 which are partially responsible for its synthesis.4 In neurons, NAAG is seen in the cytoplasm around the nucleus, in proximal dendrites, and in axons and terminals, including synaptic vesicles. An overview of the neurons and systems in which NAAG has been found is given below. The pattern of labeled cells in subcortical structures is remarkably similar across species; in cerebral cortex however, rodents lack the pyramidal cell staining seen in cats, monkeys and humans. The overview starts with the visual system, which has been especially well studied.

2. VISUAL SYSTEM 2.1 Retina

NAAG labeling is seen in retinal ganglion cells, their dendrites in the inner plexiform layer (IPL) and their axons in the optic nerve fiber layer (ONFL) (Fig. 1a). Similar patterns are seen in turtle,5 chick,6 rat,7-9 cat,10 monkey11 and human.12, 13 but not frog.14, 15 Most, but not all, of the cells in the retinal ganglion cell layer are labeled.6, 9 10, 13, 16 Although this layer contains many "displaced" amacrine cells in addition to retinal ganglion cells,17, 18 several lines of evidence indicate that most of the labeled cells in this layer are retinal ganglion cells and most of the unlabeled ones are amacrine cells. The labeled cells in this layer are larger, on average, than the unlabeled cells,13 and, in

Suzannah Bliss Tieman, Center for Neuroscience Research and Department of Biological Sciences, The University at Albany, State University of New York, Albany, New York, 12222, email: [email protected].

Figure 1. Co-localization of NAAG (A) and AB5 (B) in retinal sections of a cat. Numerous cells in the ganglion cell layer (GCL) are stained by both antisera. However, one small cell (arrow in A) contains NAAG, but not the AB5 antigen. This is probably a displaced amacrine cell. PL, photoreceptor layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONFL, optic nerve fiber layer. Anti-NAAG, 1:1500; AB5, 1:7500. Scale bar, 20 ^m

Figure 1. Co-localization of NAAG (A) and AB5 (B) in retinal sections of a cat. Numerous cells in the ganglion cell layer (GCL) are stained by both antisera. However, one small cell (arrow in A) contains NAAG, but not the AB5 antigen. This is probably a displaced amacrine cell. PL, photoreceptor layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONFL, optic nerve fiber layer. Anti-NAAG, 1:1500; AB5, 1:7500. Scale bar, 20 ^m monkey, most of those in the nasal retina disappear following section of the optic chiasm.11 In cat, cells retrogradely labeled by Dil applied to the optic nerve also label for NAAG (Tieman, unpublished observations). In addition, NAAG co-localizes with AB516 (Fig. 1), a marker for retinal ganglion cells;19 over 90% of the AB5 positive cells contain NAAG, and about 75% of the NAAG positive cells co-label for AB5. Labeled ganglion cells include small, medium and large cells, and thus, presumably, W, X, and Y cells in cat and M and P cells in monkey. Furthermore, in parafoveal retina of monkey, virtually all cells throughout the depth of the ganglion cell layer were labeled, suggesting that all major classes of ganglion cells contain NAAG (i.e., M, P, midbrain projecting, On- and

Figure 2. NAAG-like immunoreactivity in the LGN of a chiasm-sectioned monkey. A. Low-power view. Note that the staining is largely confined to the layers receiving input from the ipsilateral eye (layers 2, 3, and 5). Anti-NAAG antiserum at 1:4000. Scale bar, 1 mm. B. NAAG immunoreactivity in the neuropil and cell bodies of the LGN. Note the punctate appearance of the staining in the neuropil, which probably represents the terminal arbors of retinogeniculate axons. Antiserum at 1:4000, staining enhanced with osmium. Scale bar, 20 ^m. Reprinted with permission from Tieman et al. 11

Figure 2. NAAG-like immunoreactivity in the LGN of a chiasm-sectioned monkey. A. Low-power view. Note that the staining is largely confined to the layers receiving input from the ipsilateral eye (layers 2, 3, and 5). Anti-NAAG antiserum at 1:4000. Scale bar, 1 mm. B. NAAG immunoreactivity in the neuropil and cell bodies of the LGN. Note the punctate appearance of the staining in the neuropil, which probably represents the terminal arbors of retinogeniculate axons. Antiserum at 1:4000, staining enhanced with osmium. Scale bar, 20 ^m. Reprinted with permission from Tieman et al. 11

Off-),11 since these various types occur at different depths of the ganglion cell layer.20 In cat, monkey and human, labeled cells include those having the morphological characteristics of ON-cells and OFF-cells (i.e., those with dendritic arborizations in sublaminae B and A of the inner plexiform layer21). Indeed, in parafoveal retina of monkey, the staining in the inner plexiform layer is concentrated into two bands, probably corresponding to sublaminae A and B. In addition, in chick and human, displaced ganglion cells are labeled.6, 13 Thus, in warm blooded vertebrates and turtles, but not frogs, the overwhelming majority of retinal ganglion cells contain NAAG.

NAAG is present in ganglion cell axons and terminals. The ONFL (see above) and

both the optic tract, , , and the accessory optic tract are labeled, as is the neuropil of retinal target areas such as the lateral geniculate nucleus (Fig. 2), pretectal nuclei, superior colliculus/optic tectum, suprachiasmatic nucleus and medial terminal nucleus,6-11, 24-26 and this label decreases sharply following section of the optic nerve.7-9, 25, 26 In monkeys, labeled neuropil is found in both magnocellular and parvicellular layers and in off-layers and o nlayers,11 reinforcing the idea that all major classes of retinal ganglion cells contain NAAG.

NAAG is also seen in amacrine cells; labeling in amacrine cell processes probably contributes to the labeling in the IPL. In frog, with few or no labeled ganglion cells, labeling in the IPL is intense,15 and, as in monkey parafoveal retina, is concentrated into distinct inner and outer bands. Amacrine cells are labeled in turtle,5 frog,15 cat,10

Figure 3. Patchy neuropil labeling in layer 4C of a chiasm-sectioned monkey. Shown are photographs of two adjacent tangential sections through area 17 taken near the edge of the section, where the plane of section runs through the layers at an angle. A. Section stained for NAAG, antiserum at 1:3000, osmium enhanced. B. Adjacent section reacted for cytochrome oxidase. In both A and B, the arrowheads at the side indicate the borders of layers 4C and 4CI3. The open arrows point to the ocular dominance stripes innervated by the intact, ipsilateral pathway. Scale bar, 500 ^m. Reprinted with permission from Tieman et a!11

Figure 3. Patchy neuropil labeling in layer 4C of a chiasm-sectioned monkey. Shown are photographs of two adjacent tangential sections through area 17 taken near the edge of the section, where the plane of section runs through the layers at an angle. A. Section stained for NAAG, antiserum at 1:3000, osmium enhanced. B. Adjacent section reacted for cytochrome oxidase. In both A and B, the arrowheads at the side indicate the borders of layers 4C and 4CI3. The open arrows point to the ocular dominance stripes innervated by the intact, ipsilateral pathway. Scale bar, 500 ^m. Reprinted with permission from Tieman et a!11

monkey11 and human.13 Furthermore, in frog, NAAG is found in the synaptic vesicles of these cells.14 In frog, cat, monkey and human, these include displaced amacrine cells, small cells in the ganglion cell layer with thin rims of cytoplasm that fail to label with AB5 in cat16 and survive in the nasal retina of chiasm-sectioned monkeys.11 These are more frequent in monkey and human than in cat or frog.

Other labeled retinal cells include a few bipolar cells in the frog,15 horizontal cells in cat10 and monkey,11 and interplexiform cells in monkey.11 In general, however, these cells are less heavily labeled than the amacrine and ganglion cells.

2.2 Lateral Geniculate Nucleus

NAAG positive neurons are found in the lateral geniculate nucleus, including the medial intralaminar nucleus, of rat,7, 9 26-28 cat10, 25, 29 and monkey11 (Fig. 2). These cells are easier to see following enucleation, which decreases the neuropil labeling in the denervated layers so that the labeled cell bodies stand out. Only the relay cells, which project to visual cortex, are positive for NAAG; the interneurons, which label for glutamic acid decarboxylase (GAD), the synthesizing enzyme for GABA, are not.9, 29

It is likely that the axon terminals of these relay cells also contain NAAG. A distinct

Figure 4. Both pyramidal and non-pyramidal cells are labeled for NAAG in the visual cortex of the cat. Layer 3, affinity-purified antiserum.

band of punctate label is seen in the geniculo-recipient layer of rat, monkey and human.11' 26, 30 In rats, labeling in this layer decreases in intensity ten days following section of the optic tract.26 Similarly, in the chiasm-sectioned monkeys, in which the cortex is deprived of input from the contralateral eye, the staining within layer 4C is patchy (Fig. 3); in tangential sections there are alternating bands of light and dark label that match the ocular dominance bands demonstrated by cytochrome oxidase histochemistry in adjacent sections.

2.3 Optic Tectum/Superior Colliculus

Most neurons in the upper, retinal receptive layers of the chick's optic tectum contain NAAG,6 as do some of the neurons in the deeper, efferent layers. Similarly, in rat, NAAG positive neurons occur throughout the layers of the superior colliculus.7, 9 26 In cat and monkey, in contrast, only cells in the deeper layers of the superior colliculus contain NAAG.10, 11

2.4 Visual Cortex

In cat, NAAG is seen in both nonpyramidal cells and the pyramidal cells of layers 3 and 5 in areas 17, 18 and 19 of the cerebral cortex10 (Fig. 4). In monkey, NAAG immunoreactivity is seen in some of the cell bodies in layers 2, 3A, 4B, 5 and 6 of area

17 and layers 3 and 5 of area 18. Stained cells in layer 6 of area 17 include the Meynert cells.11 Similar results were reported for human.30 In both cat and monkey, there are more immunoreactive neurons in areas 18 and 19 than in area 17; the layer 3 pyramidal cells of areas 18 and 19 are especially well stained. The situation appears to be different in rats: despite early reports of NAAG localization in pyramidal cells, studies with improved fixation and doubly purified antibodies found W-acetylaspartate (NAA) in pyramidal cells and NAAG mainly in interneurons, with only faint label in pyramidal cells.23,31 The authors suggest that reports of NAAG in rat pyramidal cells are due to cross-reactivity with NAA. In cat and monkey, however, pyramidal cell labeling could not be blocked by NAA-BSA at the highest levels tested, up to thirty times the concentration required for NAAG-BSA to block staining.10, 11 Furthermore, even using doubly purified antibodies and enhanced fixation, in cats, monkeys and humans, NAAG

antibodies label pyramidal cells. , ,

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