=

=

Fig. 4. Comparison of retinas 5 hours after a non-toxic compound (a), a gliotoxic compound (b) and neurotoxic compound (c). a. N-acetyl L-glutamic acid (12 mmoles/kg): the appearance of the infant retina is no different from that of an untreated animal (see illustration in Olney, 1969 a). Retinal layers as follows: U outer nuclear layer; V outer plexiform layer; W inner nuclear layer; X inner plexiform layer; Y ganglion cell layer; Z= Muller foot processes. b. Alpha aminoadipic acid (12 mmoles/kg); extreme edema of non-neuronal Muller cells is the only pathological manifestation. Swelling of nuclei and perikarya of Muller cells is evidenced by the light band running through middle of inner nuclear layer (W). Swollen foot processes (Z) of Muller cells cause widening of retina but note that row of ganglion cells (Y) surrounded by the Muller processes appear unaffected. c. L-cysteine (12 mmoles kg); reaction pattern is identical to that following L-glutamic acid (illustrated in Olney, 1969 a). Bipolar and amacrine neurons (arrows) in lower 1/3 of the inner nuclear layer, the ganglion cell layer (Y) and the neural processes comprising the inner plexiform layer (X) are degenerating. The extra thickness of this retina is due to edema of these inner retinal layers (all x 220)

of comparable size, N-methyl DL-aspartic acid was 100 times, DL-homocysteic acid 20 times and N-methyl DL-glutamic acid 5 times more potent than L-glutamic acid. By either light microscopy (Fig. 1f) or electron microscopy (Fig. 3) the cytopathological reaction pattern induced by these compounds appeared to be the same as that induced by L-glutamic acid and compounds of Table 1 A.

The third group of compounds (Table 1C), at a dose of 12 mmoles/kg, induced a pathological reaction identical in regional distribution but differing in cytological specificity from that following treatment with L-glutamic acid (MSG) or other compounds in Tables 1A and B. The compound studied most extensively in this group, DL-alpha aminoadipic acid, induced changes in only one cell type of the retina, the non-neuronal Muller cell (Fig. 4b). Ganglion, amacrine and bipolar cells, which comprise the inner retinal neural constituents necrosed by MSG (Olney, 1969 a) were not affected. Changes in the hypothalamus were restricted

Fig. 5. Light micrographic comparison of hypothalami 5 hours after a non-toxic compound (a) a gliotoxic compound (b) and neurotoxic compound (c). a. N-acetyl L-glutamic acid (12 mmoles/ kg); the arcuate region (ARC) of the infant hypothalamus appears essentially like that of an untreated animal (see illustrations in Olney, 1971 a). b. DL-alpha aminoadipic acid (12 mmoles/kg); marked edematous changes in glial (g) and ependymal (e) compartments distinguish this reaction pattern from the normal appearing hypothalamus (Fig. 5a) or from the pattern of rapid neuronal necrosis depicted in Fig. 5c. The fenestrated appearance of the arcuate region is caused by dilatation of glial processes surrounding the normal appearing neurons. c. L-glutamic acid (12 mmoles/kg); the numerous arcuate neurons (n) with dilated empty perikarya and shrunken dark nuclei beside an essentially normal ependymal wall give this reaction pattern a distinctive appearance which can be differentiated, even by light microscopy, from the glioependymal pattern in Fig. 5b (all x 300)

to the arcuate region, but appeared by light microscopy to be occurring around rather than within neurons (Fig. 5b). This reaction pattern was confirmed by electron microscopic examination at several intervals after treatment during which glial and ependymal cells manifested marked edema, progressing in some cells to organelle degeneration and cellular necrosis, while neurons retained a normal appearance (Figs. 6b, 7 and 8). Synaptic involvement is the most useful criterion available for distinguishing neuronal from non-neuronal cellular elements in acutely degenerating tissues of the central nervous system. This is particularly true in the arcuate nucleus, where neurons all fall within a size range only slightly larger than glia. Although we found many edematous and degenerate appearing structures in the arcuate region of animals treated with alpha aminoadipic acid, none was involved in a synapse.

The phosphonic acid analogues and the alpha methylated (methyl group on the alpha C rather than N atom) derivatives of DL-glutamic and aspartic acids (Table 1C) also tended to induce gliotoxic but neuron-sparing reactions, although the separation of effect between neuronal and non-neuronal compartments was most complete following alpha aminoadipic acid (other compounds in the group preferentially affected non-neuronal components but also affected neuronal components slightly, especially the alpha methylated compounds.

The final group (Table 1 D) comprised those compounds which were judged to have essentially no cytotoxic action on the infant hypothalamus (Figs. 1g-i, 5a and 6a) or retina (Fig. 4a). Each compound in this group was tested initially at 12 mmoles/kg, and when observed to be ineffective at this dose was tested also at