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The differential distribution of label following uptake of 3H-labeled amino acids in the dorsal cochlear nucleus of the cat
EXPERIMENTAL
NEUROLOGY
The Differential 3H-Labeled
73, 601-617 (1981)
Distribution of Label Following Uptake of Amino Acids in the Dorsal Cochlear Nucleus of the Cat An Autoradiographic
Study
ILSA R. SCHWARTZ’ Head
and Neck
Received
Surgery. School Los Angeles.
February
7. 1980:
of Medicine, University California 90024 revision
received
February
of California,
20. 1981
Light and electron microscopic autoradiography demonstrated a differential distribution of label in the dorsal cochlear nucleus (DCN) of the cat following the incubation of adjacent fresh brain slices with micromolar amounts of different tritiated amino acids in oxygenated Ringer’s bicarbonate solutions. After incubation with GABA, glutamic acid, or glycine, label was found primarily over synaptic terminals. It was most heavily concentrated in the outer molecular layer after GABA uptake, over the inner molecular layer after glutamic acid, and more evenly spread over the molecular layer, fusiform cell layer, and deep DCN after glycine. Labeling with aspartic acid was unimpressive. There was diffuse labeling over all of the DCN after incubation with taurine and alanine and, to a lesser extent, with aspartic acid. After incubation with taurine a few cells in the molecular layer were labeled; following alanine more and different cells (probably glia) were labeled. The distribution of labeled synaptic terminals can be correlated with the differential distribution of different classes of axonal input to the DCN.
INTRODUCTION A number of amino acids including glutamic acid, aspartic acid, glycine, and GABA have been suggested as putative neurotransmitters in the auAbbreviations: DCN-dorsal cochlear nucleus, RBS-Ringer’s bicarbonate solution, GABAT-aminobutyric acid. ’ This work was supported by grants NS 09823 and NS 14503 from the U.S. Public Health Service. The expert technical assistance of M. Rita Watson and Gary Fink is gratefully acknowledged. 601 0014-4886/81/090601-17$02.00/O Copyright 0 1981 by Academic Press. Inc. All rights of reproduction in any form reserved.
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ditory system ( 1,5,23,25,27,28). Previous approaches to the identification of transmitters in the auditory system utilized histochemical demonstrations of transmitter-related enzymes (26, 29), biochemical analyses of amino acid content and distribution in normal and pathologic systems (4, 5,24,25,27,28), morphological analysis of effects of transmitter analogues ( 1), and autoradiographic studies of differential distribution of label in the cochlea and cochlear nuclei after cochlear injection of labeled amino acids (6, 12). Most of these studies emphasized putative neurotransmitters associated with the primary cochlear afferent fibers which are most easily accessible through manipulations in the cochlea. Although each of those studies provided useful information, their interpretations are restricted in a number of ways. Enzymes related to putative transmitter amino acids may be involved with general cell metabolism as well as with specific transmitter properties. Biochemical analyses of microdissected tissue still refer to a homogenate of heterogeneous neural elements. In vivo uptake studies are limited in the number of controls which can be applied and by the fact that only one set of conditions can be tested in any individual animal. None of those studies explored the possibility of using chemical markers related to neurotransmitters to distinguish between morphologically similar groups of neuronal elements and to help clarify their anatomic relationships. Although neurons in fresh mammalian brain slices maintained in oxygenated salt solutions are routinely studied physiologically for several hours, postmortem changes in the tissue destroy many morphologic features. Therefore, any morphological study of slice preparations represents a compromise between “good preservation” and demonstration of relevant chemical and/or physiologic properties. Nevertheless, such preparations have been used to demonstrate the uptake of label from glycine, GABA, or glutamic acid into differing populations of neurons and synaptic terminals in spinal cord, cerebellum, hippocampus, and cortex (2, 3). We sought to develop a slice preparation which would allow us to compare, under controlled conditions, the properties of all classes of auditory neurons with regard to a variety of amino acids. Furthermore, we compared these properties in adjacent slices of tissue from individual animals and used autoradiographic methods which allow analysis at both the light microscopic and the ultrastructural levels. Such a preparation introduces the possibility of using a chemical marker to further the analysis of anatomic relationships between auditory neurons. We chose the cat dorsal cochlear nucleus for this analysis for several reasons: Its volume allows several adjacent slices through a single cell group to be compared, neurons and axonal endings are segregated in a laminated pattern, and considerable biochem-
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ical data about the amino acid content of these regions are available to aid in the interpretation of autoradiographic results. This paper reports the light microscopic autoradiographic evidence of a differential distribution of label after incubations of tissue slices from the dorsal cochlear nucleus (DCN) of normal adult cats with glutamic acid, aspartic acid, glycine, y-aminobutyric acid (GABA), taurine, and alanine. The material prepared for this study provides the basis for the electron microscopic analysis of these regions to be reported in detail in subsequent papers. METHODS Preparation of Tissue Slices. Nine normal adult cats were anesthetized with pentobarbital and perfused through the left ventricle with 200 ml saline-nitrite solution at room temperature to flush out the blood, followed by 500 ml at 0°C to rapidly chill the brain. The head was quickly removed and placed in the cold where the brain stem was rapidly exposed, removed, and immersed in ice-cold saline-nitrite (see Table 1 for slight procedural variations). The cochlear nuclei were dissected free, mounted in agar, immediately chilled on ice, and chopped in the transverse plane at 230 pm on a Sorval TC-2 tissue chopper in the cold. About 20 min elapsed from the start of the perfusion to the collection of sections. The rapid chilling of the tissue was intended to slow postmortem changes and render the tissue rigid enough to facilitate dissection and minimize mechanical damage from chopping (30). The sections were collected in the cold salinenitrite, selected and trimmed under the dissecting microscope, and transferred to ice-cold Ringer’s bicarbonate solution (RBS) containing 140 mM NaCl, 4 mM KCl, 1 mM MgS04.7H20, 2 mM CaCl,, 1 mM KH2P04, 12 mM NaHC03, and 11 mM dextrose. When all the sections were ready, they were transferred to fresh, oxygenated, room-temperature RBS and preincubated 20 min under a stream of O2 gas on a shaker table at room temperature (about 20°C). In some experiments alternating sections were separated into two to six groups as they were cut. In other experiments comparable sections were selected visually for the different experimental groups. Preliminary experiments utilizing conditions worked out for vertebrate retinas (19) determined that the zone of uniform penetration of tritiated amino acids extended into mammalian tissue blocks to a distance of about 200 pm after 15min incubations at room temperature. Thus, the choice of section thickness, 200 to 235 pm, represented a compromise between minimizing mechanical damage (and the maximum setting on the tissue
604
ILSA R. SCHWARTZ TABLE
1
Experimental Protocols Animal No.
Amino acid
IS-243
Asp GlY
IS-244
IS-245
IS-249
6.1 6.7
Experimental conditions
Incubation time (min)
Tissue blocks, not slices, no cold perfusion, dissected in cold 33 M sucrose
20-40
Asp Glu GlY
6.1 2.1 6.7
Cold saline perfusion, chopped, standard flat embedding
20
Asp Glu GIY
6.7 2.1 6.7
Cold saline perfusion, chopped, special flat embedding
20
Asp
6.1, 2.2 2.3 1.7, I.8
Glu GlY IS-250
Concentration (MM)
”
25
25
Glu GIY GABA
6.7, 4.5 1.4 6.7 2.8
IS-254
Asp Glu GIY GABA Tau Ala
5.8 2.3 6.7 2.8 4.3 4.4
20
IS-255
Tau Ala
4.3 4.3
IS-29 I
Asp Glu
5.8 2.3
With and without preincubation
l-20
GIY GABA
1.3 0.6
Hand sliced and chopped, gassed with 95% O,-5% co,
5,15
IS-320
Asp
I
20
chopper) and maximizing uniform penetration and exchange with the incubating solution. Preincubations in oxygenated salt solutions were used because of their beneficial effects on retina preparations.
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ACID LOCALIZATION
IN CAT DCN
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Incubation Protocols. After preincubations, sections were transferred into 300 to 600 ~1 RBS containing 100 @i/ml tritiated amino acid. The following compounds were obtained from New England Nuclear: [23H]glycine (15 Ci/mmol), L-[3,C3H]glutamic acid (43 and 46.15 Ci/ mmol), [2,3-3H]aspartic acid (15 and 17.3 Ci/mmol), [2,3-3H(N)]GABA (34.5 Ci/mmol), and [ 2-3H(N)]taurine (23.146 Ci/mmol). Samples containing 250 &i of the amino acids were dried under N2, dissolved in 1.25 ml distilled water, and stored at 4°C until mixed with an equal volume of RBS made up with twice the final concentrations of salts. Concentrations of amino acids in the final incubation solutions were 2.2 to 6.7 X 10e6 FM. The RBS solutions were prepared fresh and oxygenated by bubbling the gas through them just before use. Material from at least two animals was incubated with each amino acid. In each experiment adjacent sections from the same cochlear nucleus or from the other cochlear nucleus of the same animal were incubated in the different amino acids. The experiments are listed in Table 1. Internal controls were provided by the comparison of label patterns from different amino acids in adjacent sections and over the experimental series. Preparations for Microscopy. After incubations of 1 to 40 min under the same conditions as the preincubation, with the variations indicated in Table 1, the sections were transferred to fresh RBS for 2 to 5 min and then fixed 1 h in 1% paraformaldehyde-1.25% glutaraldehyde in 0.12 M phospate buffer at pH 7.2 at room temperature, then overnight at 4°C. The sections were given three 15-min rinses in the buffer, then brushed flat on the bottom of a vial with a convex bottom, and drops of 1% osmium tetroxide solution in 0.12 M phosphate buffer were introduced at the edges of the vial so that the sections were exposed only to the vapor. After 30 min at room temperature, additional osmium solution was introduced gently into the vial so that the hardened flat sections just floated. After an additional hour in the osmium the sections were rinsed briefly with phosphate buffer, block stained with uranyl acetate (15), dehydrated with increasing concentrations of ethanol and propylene oxide, and infiltrated with Epon. The sections are very fragile after osmication and must be treated with extreme care. After infiltration with Epon the sections were embedded in the cap end of a Beem capsule whose pointed end had been removed. A piece of nylon mesh was placed over the slice and the capsule snapped down over it to hold the slice flat against the cap. The capsule was filled with Epon and the plastic polymerized as usual. Procedures for light and electron microscopic autoradiography were reported previously (22). Light microscopic autoradiograms were exposed for 4 to 120 days; electron microscope autoradiograms were exposed 21 to 120 days.
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RESULTS Concentrations of label derived from glutamic acid, aspartic acid, GABA, glycine, taurine, and alanine were demonstrated to have differing distributions in the various layers, cell types, and subcellular areas in the dorsal cochlear nucleus of the cat with both light and electron microscopic autoradiography. Electron microscopic autoradiographic findings will be reported in detail in subsequent papers, but preliminary observations are included to demonstrate the specificity of labeling obtained in this study. For illustrations of the neuronal elements associated with each of the layers in the laminated DCN, refer to Figs. 2- 12,2-14, and 2-15 of Lorente de No ( 18) and Mugnaini et al. (20, 21). [JH]GABA (Figs. 1, 7). Silver grains occurred in small clusters with a size and distribution similar to those of synaptic boutons. Such clusters were found in all layers, but were present in greatest numbers in the superficial molecular layer (Fig. 7). Parallel fibers (arising from granule cells, some of which are intrinsic to this layer) are a major element of this layer. Granule cell bodies were not labeled nor were any other recognizable cell type aside from endothelial cells. Electron microscopic observations (Fig. 13) suggest that the labeled structures may be a subpopulation of the parallel fibers and their boutons en passant. [3H]Glutamic Acid (Figs. 2, 8). Clusters of silver grains with a size and distribution similar to those of synaptic boutons were present in all layers, but were most heavily concentrated in the inner molecular layer Axon arbors of several intrinsic neurons are found in this region, but the rather uniform distribution of labeled structures in this region does not suggest any particular type of axonal arborization. No cell bodies were labeled other than endothelial cells. The same pattern of labeling was seen both in chopped and hand-sliced sections and in sections incubated 1, 5, 10, 15, FIGS. 1-6. Light microscopic autoradiographs comparing the distribution of label in the cat dorsal cochlear nucleus (DCN) after incubation with the tritiated amino acids as indicated. Figures 1,4, 5, and 6 are from cat IS 254. Figures 2 and 3 are from cat IS 245. The sections shown in Figs. l-3 were exposed 28 or 29 days; the sections in Figs. 4-6 were exposed for 90 days. 125x. FIG. I. GABA incubation. Note the heavy concentration of small clumps of silver grains in the outer molecular layer (far right) and the large clumps over blood vessels most easily seen in the deep DCN (left half of figure). FIG. 2. Glutamic acid incubation. Small clumps of silver grains are concentrated over the inner molecular layer, and the deep DCN (left), the ependymal layer (at the surface of the section), and the outermost molecular layer are relatively free of label. FIG. 3. Glycine incubation. The ependymal layer and outermost molecular layer are relatively free of label, while small clumps of silver grains are fairly evenly distributed throughout the inner molecular and fusiform cell layers and deep DCN.
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ACID LOCALIZATION
IN CAT DCN
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20, or 25 min. Electron microscopic observations confirmed that the labeled structures were synaptic terminals and that they were slightly larger than the terminals and boutons en passant labeled with [3H]GABA (Fig. 14). [‘H]Glycine (Figs. 3, 9). Silver grain clusters with a size and distribution similar to those of synaptic boutons were spread fairly evenly through the molecular, fusiform cell, and deep layers. These clusters could be identified at the light microscopic level as being associated with fusiform cells, other neuronal somata, and large dendrites. No neuronal cell bodies were labeled and endothelial cells were only lightly labeled. Electron microscopic observation confirmed that the labeled structures were synaptic terminals and that they were contacting neuronal perikarya and large dendrites (Fig. 15). [3H]Aspartic Acid (Figs. 4, ZO). Compared with the localization of label from GABA, glutamic acid, and glycine, the localization of label from t3H]aspartic acid was unimpressive. After long exposure times a few discrete structures were labeled in the superficial molecular layer and a few in deeper areas. In general, light diffuse label was seen throughout the DCN. By contrast, in the same sections discrete clusters of silver grains were found in multipolar cell regions in the posterior ventral cochlear nucleus. Also, after much shorter exposure times, there was a heavy diffuse distribution of silver grains over the superficial molecular layer of adjacent cerebellar regions with labeling of cells and dendrites. After very short incubation times (1 to 2 min), discrete clusters of silver grains were accumulated in the outermost molecular layer of the DCN. After lo- or 20min incubations, the pattern was the same as that described above. [3H]Alanine (Figs. 5, II). A diffuse homogeneous distribution of silver grains was observed over the entire DCN after the same exposure times used to demonstrate localization with GABA, glycine, or glutamic acid. Endothelial cells were labeled. After longer exposure times concentrations of silver grains were noted over a few cells in the inner molecular layer. These cells were mostly small and had spherical somas. Electron microscopic observations suggest that these cells are glia. [:‘H]Tuurine (Figs. 6, 12). As with alanine, a diffuse homogeneous distribution of silver grains was observed over the entire DCN and endothelial FIG. 4. Aspartic acid incubation. The most striking feature of this figure is the lack of silver grains. See Fig. 10 for a higher-magnification view of another section of aspartic acid-incubated material. FIG. 5. Taurine incubation. At this magnification the low incidence of diffuse label is not apparent, although a labeled cell can be detected (arrowhead) in the molecular layer. The lower right corner is shown at a higher magnification in Fig. 11. FIG. 6. Alanine incubation. A few labeled cells (possibly glia) are observed in the inner molecular layer. The two cells shown at the upper right (arrowheads) are shown at a higher magnification in Fig. 12.
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LOCALIZATION
IN CAT
DCN
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cells were labeled. The few cells in the inner molecular layer labeled after longer incubation times appeared similar to those labeled after alanine incubations. Electron microscopic observations suggest that glial cells may be labeled. DISCUSSION This paper describes a differential distribution of label derived from different putative neurotransmitter amino acids in neural elements in the dorsal cochlear nucleus of the cat. These findings must be considered in terms of both what neural populations were labeled and what properties of neurons were demonstrated. Implications of Differential Patterns. The patterns of localization of label derived from [‘HIGABA, [3H]glycine, and [‘Hlglutamic acid differ markedly from one another and are suggestive of patterns of distribution of different classes of axons within the DCN. The pattern of labeling of synaptic terminals heaviest in the inner molecular layer seen after incubation with [3H]glutamic acid is coextensive with the axon arborizations of several different inputs to the DCN. The Golgi studies of Lorente de No (17, 18) showed that the arborization of Golgi type 2 axons are heavily concentrated in the inner molecular layer. Cells in the dorsal inferior colliculus also project to this region (9-l 1, 13, 14) as do other intrinsic neurons of the cochlear nucleus (7, 20, 21). Parallel fibers arising from different groups of neurons are also present throughout the molecular layer. The present data do not allow us to choose among the several possibilities. The diffuse pattern of synaptic terminal labeling in the molecular, fusiform, and deep DCN seen after incubation with [3H]glycine is also coextensive with several sets of inputs. In addition to intrinsic neurons of the DCN illustrated by Lorente de No (17, 18), descending inputs are known from the dorsal inferior colliculus to the inner molecular layer from cells in the ventral inferior colliculus to the fusiform cell layer, and from the nuclei of the lateral lemniscus to the deep DCN (9, 13, 14). A descending FIGS. 7-12. Details of the label distribution at a higher magnification. Each corresponds to the arrangement of animals and amino acids in Figs. 1-6. 495X. FIG. 7. GABA. The clumps of silver grains are concentrated in the outer molecular layer although a few are found in the inner molecular layer, fusiform cell layer, and deep DCN. FIG. 8. Glutamic acid. The clusters of silver grains in the inner molecular layer seem to be arranged in chains parallel to the surface of the DCN. FIG. 9. Glycine. Note the relative absence of label over blood vessels (arrowheads) compared with the density of silver grains over elements in the neuropile of the molecular, fusiform cell, and deep layers of the DCN.
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ACID LOCALIZATION
IN CAT DCN
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input from the superior olivary complex to the molecular and deep layers was also shown in the tree shrew (7). On the other hand, the primary auditory afferent fibers from the cochlea are most heavily concentrated in the deep DCN and fusiform cell layer (8). Although virtually all labeled synaptic terminals were found on dendrites or neuronal perikarya, many unlabeled terminals had the morphological features associated with the primary cochlear input. It seems unlikely that either glycine or glutamic acid labeled the primary afferent fibers. The present data do not allow us to choose among the several other possibilities. The pattern of labeling concentrated in the outer molecular layer after incubation with [3H]GABA is coextensive with the region containing large numbers of granule cell axons, the parallel fibers. Inputs descending from higher levels of the auditory system are found somewhat deeper in the molecular layer. Together with the electron microscopic observation of label in small unmyelinated axons and their associated boutons en passant, these observations suggest that the label derived from GABA is associated with the parallel fibers. The absence of a distinctive labeling pattern after incubation with [3H]aspartic acid suggests the absence of a high-affinity uptake system under the conditions of these experiments or the possibility that in the DCN the aspartic acid was rapidly metabolized to some other form with the tritiated label lost from the tissue, either in the metabolic process or in the subsequent processing. The labeling of glial cells after incubation with alanine is consistent with observations of glial labeling by alanine in other parts of the central nervous system. The diffuse labeling pattern seen after incubation with taurine may also be indicative of a nonspecific binding, or general breakdown, which could be consistent with the presence of label in some glial cells. Implications about Chemical Properties. If a specific group of neural elements is labeled after incubation with an amino acid, as our findings indicate, we have provided a marker for that group which can be used to assist in the analysis of its anatomic relationships. However, we must be careful in interpreting what the marker reflects about the properties of the labeled elements. FIG. 10. Aspartic acid. Note the presence of a few clumps of label (arrowheads) in the molecular and fusiform cell layers. FIG. Il. Taurine. The labeled cell from Fig. 5 is illustrated. A diffuse distribution of silver grains over the neuropile can also be detected, as can the silver grains over the large blood vessel at lower left. FIG. 12. Alanine. Two labeled cells from Fig. 6 are illustrated. A diffuse distribution of silver grains over the neuropile can also be detected.
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If the label localized within a synaptic terminal or cell is still associated with the original amino acid, we may infer either that the compound was selectively accumulated, bound, or not broken down by that element; that exchange diffusion has labeled a region with a high endogenous concentration of that amino acid (16); or that label has been selectively lost from other elements during incubation or processing. If all elements are equally healthy, a differential distribution can be interpreted as a special property of the labeled cell. However, if one population is selectively damaged by the slicing, we may be looking at an injury phenomenon not characteristic of a healthy cell. This might apply if extrinsic axons were affected while locally arborizing ones were not. In the findings reported here, the high level of label found over the synaptic terminals after incubation with tritiated glycine, GABA, or glutamic acid is much greater than that which could be accounted for by random binding and selective loss. The intensity of the labeling patterns seen in this study after incubation with micromolar amounts of labeled compounds suggests that active accumulation or endogenous exchange is occurring. For GABA, the distribution of the label is consistent with the biochemical data from analysis of microdissected regions of the cochlear nucleus showing high GABA concentrations in the molecular layer. For glycine, biochemical data showing even distributions over the molecular, fusiform, and deep layers correlate with the even label distributions. Thus, for these amino acids, exchange with high endogenous concentrations is a possibility. On the other hand, the absence of label concentrations in the regions shown to have higher concentrations of aspartic acid, the fusiform and deep layer, does not rule out the possibility that labeled compounds were taken up by elements in these regions. It is likely that labeled amino acids could be taken up and rapidly metabolized with the tritiated label split off and lost as water when the tissue is dehydrated. Alternatively, the label might be transferred to some compound which is rapidly eliminated from the neural elements or selectively lost in subsequent processing. If there is a localization of label no longer associated with the original amino acid,
FIGS. 13-15. Electron microscopic autoradiographs of synaptic terminals labeled after incubation with GABA, glutamic acid, and glycine, respectively. 16,100X. FIG. 13. GABA, cat IS 254, 21 days’ exposure. Note the localization of silver grains over longitudinally sectioned parallel fibers in the outer molecular layer. FIG. 14. Glutamic acid, cat IS 24525 days’ exposure. Note the localization of silver grains over two synaptic terminals in the outer molecular layer. FIG. 15. Glycine, cat IS 250, 25 days’ exposure. Note the heavy concentration of silver grains over a synaptic terminal contacting a dendrite in the deep DCN.
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its presence might reflect special metabolic properties of the labeled element which cause the [3H] to be transferred to another compound which is accumulated. This might account for the heavy concentration of label in the inner molecular layer after incubation with [3H]glutamic acid, where the biochemical data suggest a fairly uniform distribution of glutamic acid on a volume basis. Until the labeled compounds remaining after incubation are identified chromatographically these questions cannot be resolved. In conclusion, it is clear that different populations of synaptic terminals were labeled following incubations with different tritiated amino acids. Additional electron microscopic studies will be necessary to resolve features of these populations which can allow us to select among several candidate classes of axons. The correct chemical interpretation of these anatomic findings will require additional biochemical analyses. Nevertheless, these findings represent a promising first step in analyzing chemical properties of specific auditory neural elements. By providing markers for at least three different neuronal populations, this study has brought us closer to our ultimate goal of fully characterizing the connections and chemical and electrical properties of individual auditory neurons so that we may understand processing of auditory information. REFERENCES 1. BIRD, S. J.. R. L. GULLEY, R. J. WENTHOLD, AND J. FEX. 1978. Kainic acid injections result in degeneration of cochlear nucleus cells innervated by the auditory nerve. Science 208: 2.
1087-1089.
BLOOM, F., AND L. L. IVERSON. 1971. Localizing ‘H-GABA in nerve terminals of rat cerebral cortex by electron microscopic autoradiography. Nature (London) 229: 623630.
BLOOM, F., AND L. L. IVERSON. 1972. Studies of the uptake of ‘H-GABA and [‘Hlglycine in slices and homogenates of rat brain and spinal cord by electron microscopic autoradiography. Bruin Res. 41: 131-143. 4. GODFREY, D. A., AND F. M. MATSCHINSKY. 1976. Approach to three-dimensional mapping of quantitative histochemical measurements applied to studies of the cochlear nucleus. J. Histochem. Cytochem. 24: 697-712. 3.
5. GODFREY,
6.
7. 8. 9.
D.A.,
J. A. CARTER,
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0. H. LOWRY,
ANDF.
M. MATSCHINSKY.
1977. Quantitative histochemical mapping of candidate transmitter amino acids in cat cochlear nucleus. J. Hisrochem. Cytochem. 25: 417-43 1. JONES, D. R., AND J. H. CASSEDAY. 1979. Projections of auditory nerve in the cat as seen by anterograde transport methods. Neuroscience 4: 1299-l 3 13. JONES, D. R., AND J. H. CASSEDAY. 1979. Projections to laminae in dorsal cochlear nucleus in the tree shrew, Tupoia glis. Bruin Res. 160: 131-133. KANE, E. C. 1974. Patterns of degeneration in the caudal ccchlear nucleus of the cat after cochlear ablation. Anaf. Rec. 179: 67-92. KANE, E. C. 1976. Auditory neurons descending to caudal cochlear nucleus in cats: a horseradish peroxidase (HRP) study. Am. J. Anat. 146: 433442.
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10. KANE, E. C. 1976. Descending projections to specific regions of cat cochlear nucleus: a light microscopic study. Exp. Neural. 52: 372-388. 11. KANE, E. S. 1977. Origins of descending inputs to cat dorsal cochlear nucleus. Anar. Rec. 187: 619-620.
12. KANE, E. S. 1979. Central transport and distribution of labelled glutamic and aspartic acids to the cochlear nucleus in cats: an autoradiographic study. Neuroscience 4: 729743. 13. KANE, E. S., AND J. W. CONLEE. 1979. Descending inputs to the caudal cochlear nucleus of the cat: degeneration and autoradiographic studies. J. Camp. Neural. 18’1: 759-784. 14. KANE, E. S., AND R. C. FINN. 1977. Descending and intrinsic inputs to dorsal cochlear nucleus of cats: a horseradish peroxidase study. Neuroscience 2: 897-912. 15. KARNOVSKY, M. J. 1967. The ultrastructural basis of capillary permeability studied with peroxidase as a tracer. J. Cell Biol. 35: 213-236. 16. LEVI, G., AND M. RAITERI. 1974. Exchange of neurotransmitter amino acid at nerve endings can simulate high affinity uptake. Nature (London) 250~ 735-737. 17. LORENTE DE No, R. 1933. Anatomy of the eighth nerve. III. General plan of structure of the primary cochlear nuclei. Laryngoscope 43: 327-350. 18. LORENTE DE N& R. 1979. Central representation of the eighth nerve. Pages 64-86 in V. GOODHILL, Ed., Ear Diseases, Deafness and Dizziness. Harper and Row, Hagerstown, Maryland. 19. MARC, R. E., W. K. STELL. D. BOK, AND D. M. K. LAM. 1978. GABAergic pathways in the goldfish retina. J. Camp. Neural. 182: 221-246. 20. MUGNAINI, E., K. K. OSEN, A.-L. DAHL, V. L. FRIEDRICH, JR., ANDG. KORTE. 1980. Fine structure of granule cells and related interneurones (termed Golgi cells) in the cochlear nuclear complex of cat, rat and mouse. J. Neurocytol. 9: 537-570. 21. MUGNAINI, E., W. B. WARR, AND K. K. OSEN. 1980. Distribution and light microscopic features of granule cells in the cochlear nuclei of cat, rat and mouse. J. Comp. Neurol. 191: 581-606.
22. SCHWARTZ, I. R., AND D. BOK. 1979. Electron microscopic localization of [‘251]a-bungarotoxin binding sites in the outer plexiform layer of the goldfish retina. J. Neurocytol. 8: 53-66. 23. TACHIBANA, M., AND K. KIRUYAMA. 1974. Gamma-aminobutryic acid in the lower auditory pathway of the guinea pig. Brain Res. 69: 370-374. 24. THALMANN, R. 1975. Biochemical studies of the auditory system. Pages 31-44 in D. B. TOWER, Ed., The Nervous System. Vol. 3, Raven Press, New York. 25. WENTHOLD, R. J. 1978. Glutamic acid and aspartic acid in subdivisions of the cochlear nucleus after auditory nerve lesion. Brain Res. 143: 544-548. 26. WENTHOLD, R. J., AND J. FEX. 1976. Transmitter related enzymes in cochlea and cochlear nucleus of guinea pig. Trans. Am. Sot. Neurochem. 7: 193. 27. WENTHOLD, R. J., AND R. L. GULLEY. 1977. Aspartic acid and glutamic acid levels in the cochlear nucleus after auditory nerve lesion. Brain Res. 138: 11 l-l 23. 28. WENTHOLD, R. J., AND R. L. GULLEY. 1978. Glutamic acid and aspartic acid in the cochlear nucleus of the waltzing guinea pig. Bruin Res. 158: 295-302. 29. WENTHOLD, R. J., AND D. K. MOREST. 1976. Transmitter related enzymes in the guinea pig cochlear nucleus. Sot. Neurosci. Abstr. 2 28. 30. ZIGMOND, R. E., ANDY. BEN-ARI. 1976. A simple method for serial sectioning of fresh brain and the removal of identifiable nuclei from stained sections for biochemical analysis. J. Neurochem. 26: 1285-1287.
NEUROLOGY
The Differential 3H-Labeled
73, 601-617 (1981)
Distribution of Label Following Uptake of Amino Acids in the Dorsal Cochlear Nucleus of the Cat An Autoradiographic
Study
ILSA R. SCHWARTZ’ Head
and Neck
Received
Surgery. School Los Angeles.
February
7. 1980:
of Medicine, University California 90024 revision
received
February
of California,
20. 1981
Light and electron microscopic autoradiography demonstrated a differential distribution of label in the dorsal cochlear nucleus (DCN) of the cat following the incubation of adjacent fresh brain slices with micromolar amounts of different tritiated amino acids in oxygenated Ringer’s bicarbonate solutions. After incubation with GABA, glutamic acid, or glycine, label was found primarily over synaptic terminals. It was most heavily concentrated in the outer molecular layer after GABA uptake, over the inner molecular layer after glutamic acid, and more evenly spread over the molecular layer, fusiform cell layer, and deep DCN after glycine. Labeling with aspartic acid was unimpressive. There was diffuse labeling over all of the DCN after incubation with taurine and alanine and, to a lesser extent, with aspartic acid. After incubation with taurine a few cells in the molecular layer were labeled; following alanine more and different cells (probably glia) were labeled. The distribution of labeled synaptic terminals can be correlated with the differential distribution of different classes of axonal input to the DCN.
INTRODUCTION A number of amino acids including glutamic acid, aspartic acid, glycine, and GABA have been suggested as putative neurotransmitters in the auAbbreviations: DCN-dorsal cochlear nucleus, RBS-Ringer’s bicarbonate solution, GABAT-aminobutyric acid. ’ This work was supported by grants NS 09823 and NS 14503 from the U.S. Public Health Service. The expert technical assistance of M. Rita Watson and Gary Fink is gratefully acknowledged. 601 0014-4886/81/090601-17$02.00/O Copyright 0 1981 by Academic Press. Inc. All rights of reproduction in any form reserved.
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ditory system ( 1,5,23,25,27,28). Previous approaches to the identification of transmitters in the auditory system utilized histochemical demonstrations of transmitter-related enzymes (26, 29), biochemical analyses of amino acid content and distribution in normal and pathologic systems (4, 5,24,25,27,28), morphological analysis of effects of transmitter analogues ( 1), and autoradiographic studies of differential distribution of label in the cochlea and cochlear nuclei after cochlear injection of labeled amino acids (6, 12). Most of these studies emphasized putative neurotransmitters associated with the primary cochlear afferent fibers which are most easily accessible through manipulations in the cochlea. Although each of those studies provided useful information, their interpretations are restricted in a number of ways. Enzymes related to putative transmitter amino acids may be involved with general cell metabolism as well as with specific transmitter properties. Biochemical analyses of microdissected tissue still refer to a homogenate of heterogeneous neural elements. In vivo uptake studies are limited in the number of controls which can be applied and by the fact that only one set of conditions can be tested in any individual animal. None of those studies explored the possibility of using chemical markers related to neurotransmitters to distinguish between morphologically similar groups of neuronal elements and to help clarify their anatomic relationships. Although neurons in fresh mammalian brain slices maintained in oxygenated salt solutions are routinely studied physiologically for several hours, postmortem changes in the tissue destroy many morphologic features. Therefore, any morphological study of slice preparations represents a compromise between “good preservation” and demonstration of relevant chemical and/or physiologic properties. Nevertheless, such preparations have been used to demonstrate the uptake of label from glycine, GABA, or glutamic acid into differing populations of neurons and synaptic terminals in spinal cord, cerebellum, hippocampus, and cortex (2, 3). We sought to develop a slice preparation which would allow us to compare, under controlled conditions, the properties of all classes of auditory neurons with regard to a variety of amino acids. Furthermore, we compared these properties in adjacent slices of tissue from individual animals and used autoradiographic methods which allow analysis at both the light microscopic and the ultrastructural levels. Such a preparation introduces the possibility of using a chemical marker to further the analysis of anatomic relationships between auditory neurons. We chose the cat dorsal cochlear nucleus for this analysis for several reasons: Its volume allows several adjacent slices through a single cell group to be compared, neurons and axonal endings are segregated in a laminated pattern, and considerable biochem-
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ical data about the amino acid content of these regions are available to aid in the interpretation of autoradiographic results. This paper reports the light microscopic autoradiographic evidence of a differential distribution of label after incubations of tissue slices from the dorsal cochlear nucleus (DCN) of normal adult cats with glutamic acid, aspartic acid, glycine, y-aminobutyric acid (GABA), taurine, and alanine. The material prepared for this study provides the basis for the electron microscopic analysis of these regions to be reported in detail in subsequent papers. METHODS Preparation of Tissue Slices. Nine normal adult cats were anesthetized with pentobarbital and perfused through the left ventricle with 200 ml saline-nitrite solution at room temperature to flush out the blood, followed by 500 ml at 0°C to rapidly chill the brain. The head was quickly removed and placed in the cold where the brain stem was rapidly exposed, removed, and immersed in ice-cold saline-nitrite (see Table 1 for slight procedural variations). The cochlear nuclei were dissected free, mounted in agar, immediately chilled on ice, and chopped in the transverse plane at 230 pm on a Sorval TC-2 tissue chopper in the cold. About 20 min elapsed from the start of the perfusion to the collection of sections. The rapid chilling of the tissue was intended to slow postmortem changes and render the tissue rigid enough to facilitate dissection and minimize mechanical damage from chopping (30). The sections were collected in the cold salinenitrite, selected and trimmed under the dissecting microscope, and transferred to ice-cold Ringer’s bicarbonate solution (RBS) containing 140 mM NaCl, 4 mM KCl, 1 mM MgS04.7H20, 2 mM CaCl,, 1 mM KH2P04, 12 mM NaHC03, and 11 mM dextrose. When all the sections were ready, they were transferred to fresh, oxygenated, room-temperature RBS and preincubated 20 min under a stream of O2 gas on a shaker table at room temperature (about 20°C). In some experiments alternating sections were separated into two to six groups as they were cut. In other experiments comparable sections were selected visually for the different experimental groups. Preliminary experiments utilizing conditions worked out for vertebrate retinas (19) determined that the zone of uniform penetration of tritiated amino acids extended into mammalian tissue blocks to a distance of about 200 pm after 15min incubations at room temperature. Thus, the choice of section thickness, 200 to 235 pm, represented a compromise between minimizing mechanical damage (and the maximum setting on the tissue
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1
Experimental Protocols Animal No.
Amino acid
IS-243
Asp GlY
IS-244
IS-245
IS-249
6.1 6.7
Experimental conditions
Incubation time (min)
Tissue blocks, not slices, no cold perfusion, dissected in cold 33 M sucrose
20-40
Asp Glu GlY
6.1 2.1 6.7
Cold saline perfusion, chopped, standard flat embedding
20
Asp Glu GIY
6.7 2.1 6.7
Cold saline perfusion, chopped, special flat embedding
20
Asp
6.1, 2.2 2.3 1.7, I.8
Glu GlY IS-250
Concentration (MM)
”
25
25
Glu GIY GABA
6.7, 4.5 1.4 6.7 2.8
IS-254
Asp Glu GIY GABA Tau Ala
5.8 2.3 6.7 2.8 4.3 4.4
20
IS-255
Tau Ala
4.3 4.3
IS-29 I
Asp Glu
5.8 2.3
With and without preincubation
l-20
GIY GABA
1.3 0.6
Hand sliced and chopped, gassed with 95% O,-5% co,
5,15
IS-320
Asp
I
20
chopper) and maximizing uniform penetration and exchange with the incubating solution. Preincubations in oxygenated salt solutions were used because of their beneficial effects on retina preparations.
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Incubation Protocols. After preincubations, sections were transferred into 300 to 600 ~1 RBS containing 100 @i/ml tritiated amino acid. The following compounds were obtained from New England Nuclear: [23H]glycine (15 Ci/mmol), L-[3,C3H]glutamic acid (43 and 46.15 Ci/ mmol), [2,3-3H]aspartic acid (15 and 17.3 Ci/mmol), [2,3-3H(N)]GABA (34.5 Ci/mmol), and [ 2-3H(N)]taurine (23.146 Ci/mmol). Samples containing 250 &i of the amino acids were dried under N2, dissolved in 1.25 ml distilled water, and stored at 4°C until mixed with an equal volume of RBS made up with twice the final concentrations of salts. Concentrations of amino acids in the final incubation solutions were 2.2 to 6.7 X 10e6 FM. The RBS solutions were prepared fresh and oxygenated by bubbling the gas through them just before use. Material from at least two animals was incubated with each amino acid. In each experiment adjacent sections from the same cochlear nucleus or from the other cochlear nucleus of the same animal were incubated in the different amino acids. The experiments are listed in Table 1. Internal controls were provided by the comparison of label patterns from different amino acids in adjacent sections and over the experimental series. Preparations for Microscopy. After incubations of 1 to 40 min under the same conditions as the preincubation, with the variations indicated in Table 1, the sections were transferred to fresh RBS for 2 to 5 min and then fixed 1 h in 1% paraformaldehyde-1.25% glutaraldehyde in 0.12 M phospate buffer at pH 7.2 at room temperature, then overnight at 4°C. The sections were given three 15-min rinses in the buffer, then brushed flat on the bottom of a vial with a convex bottom, and drops of 1% osmium tetroxide solution in 0.12 M phosphate buffer were introduced at the edges of the vial so that the sections were exposed only to the vapor. After 30 min at room temperature, additional osmium solution was introduced gently into the vial so that the hardened flat sections just floated. After an additional hour in the osmium the sections were rinsed briefly with phosphate buffer, block stained with uranyl acetate (15), dehydrated with increasing concentrations of ethanol and propylene oxide, and infiltrated with Epon. The sections are very fragile after osmication and must be treated with extreme care. After infiltration with Epon the sections were embedded in the cap end of a Beem capsule whose pointed end had been removed. A piece of nylon mesh was placed over the slice and the capsule snapped down over it to hold the slice flat against the cap. The capsule was filled with Epon and the plastic polymerized as usual. Procedures for light and electron microscopic autoradiography were reported previously (22). Light microscopic autoradiograms were exposed for 4 to 120 days; electron microscope autoradiograms were exposed 21 to 120 days.
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RESULTS Concentrations of label derived from glutamic acid, aspartic acid, GABA, glycine, taurine, and alanine were demonstrated to have differing distributions in the various layers, cell types, and subcellular areas in the dorsal cochlear nucleus of the cat with both light and electron microscopic autoradiography. Electron microscopic autoradiographic findings will be reported in detail in subsequent papers, but preliminary observations are included to demonstrate the specificity of labeling obtained in this study. For illustrations of the neuronal elements associated with each of the layers in the laminated DCN, refer to Figs. 2- 12,2-14, and 2-15 of Lorente de No ( 18) and Mugnaini et al. (20, 21). [JH]GABA (Figs. 1, 7). Silver grains occurred in small clusters with a size and distribution similar to those of synaptic boutons. Such clusters were found in all layers, but were present in greatest numbers in the superficial molecular layer (Fig. 7). Parallel fibers (arising from granule cells, some of which are intrinsic to this layer) are a major element of this layer. Granule cell bodies were not labeled nor were any other recognizable cell type aside from endothelial cells. Electron microscopic observations (Fig. 13) suggest that the labeled structures may be a subpopulation of the parallel fibers and their boutons en passant. [3H]Glutamic Acid (Figs. 2, 8). Clusters of silver grains with a size and distribution similar to those of synaptic boutons were present in all layers, but were most heavily concentrated in the inner molecular layer Axon arbors of several intrinsic neurons are found in this region, but the rather uniform distribution of labeled structures in this region does not suggest any particular type of axonal arborization. No cell bodies were labeled other than endothelial cells. The same pattern of labeling was seen both in chopped and hand-sliced sections and in sections incubated 1, 5, 10, 15, FIGS. 1-6. Light microscopic autoradiographs comparing the distribution of label in the cat dorsal cochlear nucleus (DCN) after incubation with the tritiated amino acids as indicated. Figures 1,4, 5, and 6 are from cat IS 254. Figures 2 and 3 are from cat IS 245. The sections shown in Figs. l-3 were exposed 28 or 29 days; the sections in Figs. 4-6 were exposed for 90 days. 125x. FIG. I. GABA incubation. Note the heavy concentration of small clumps of silver grains in the outer molecular layer (far right) and the large clumps over blood vessels most easily seen in the deep DCN (left half of figure). FIG. 2. Glutamic acid incubation. Small clumps of silver grains are concentrated over the inner molecular layer, and the deep DCN (left), the ependymal layer (at the surface of the section), and the outermost molecular layer are relatively free of label. FIG. 3. Glycine incubation. The ependymal layer and outermost molecular layer are relatively free of label, while small clumps of silver grains are fairly evenly distributed throughout the inner molecular and fusiform cell layers and deep DCN.
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20, or 25 min. Electron microscopic observations confirmed that the labeled structures were synaptic terminals and that they were slightly larger than the terminals and boutons en passant labeled with [3H]GABA (Fig. 14). [‘H]Glycine (Figs. 3, 9). Silver grain clusters with a size and distribution similar to those of synaptic boutons were spread fairly evenly through the molecular, fusiform cell, and deep layers. These clusters could be identified at the light microscopic level as being associated with fusiform cells, other neuronal somata, and large dendrites. No neuronal cell bodies were labeled and endothelial cells were only lightly labeled. Electron microscopic observation confirmed that the labeled structures were synaptic terminals and that they were contacting neuronal perikarya and large dendrites (Fig. 15). [3H]Aspartic Acid (Figs. 4, ZO). Compared with the localization of label from GABA, glutamic acid, and glycine, the localization of label from t3H]aspartic acid was unimpressive. After long exposure times a few discrete structures were labeled in the superficial molecular layer and a few in deeper areas. In general, light diffuse label was seen throughout the DCN. By contrast, in the same sections discrete clusters of silver grains were found in multipolar cell regions in the posterior ventral cochlear nucleus. Also, after much shorter exposure times, there was a heavy diffuse distribution of silver grains over the superficial molecular layer of adjacent cerebellar regions with labeling of cells and dendrites. After very short incubation times (1 to 2 min), discrete clusters of silver grains were accumulated in the outermost molecular layer of the DCN. After lo- or 20min incubations, the pattern was the same as that described above. [3H]Alanine (Figs. 5, II). A diffuse homogeneous distribution of silver grains was observed over the entire DCN after the same exposure times used to demonstrate localization with GABA, glycine, or glutamic acid. Endothelial cells were labeled. After longer exposure times concentrations of silver grains were noted over a few cells in the inner molecular layer. These cells were mostly small and had spherical somas. Electron microscopic observations suggest that these cells are glia. [:‘H]Tuurine (Figs. 6, 12). As with alanine, a diffuse homogeneous distribution of silver grains was observed over the entire DCN and endothelial FIG. 4. Aspartic acid incubation. The most striking feature of this figure is the lack of silver grains. See Fig. 10 for a higher-magnification view of another section of aspartic acid-incubated material. FIG. 5. Taurine incubation. At this magnification the low incidence of diffuse label is not apparent, although a labeled cell can be detected (arrowhead) in the molecular layer. The lower right corner is shown at a higher magnification in Fig. 11. FIG. 6. Alanine incubation. A few labeled cells (possibly glia) are observed in the inner molecular layer. The two cells shown at the upper right (arrowheads) are shown at a higher magnification in Fig. 12.
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cells were labeled. The few cells in the inner molecular layer labeled after longer incubation times appeared similar to those labeled after alanine incubations. Electron microscopic observations suggest that glial cells may be labeled. DISCUSSION This paper describes a differential distribution of label derived from different putative neurotransmitter amino acids in neural elements in the dorsal cochlear nucleus of the cat. These findings must be considered in terms of both what neural populations were labeled and what properties of neurons were demonstrated. Implications of Differential Patterns. The patterns of localization of label derived from [‘HIGABA, [3H]glycine, and [‘Hlglutamic acid differ markedly from one another and are suggestive of patterns of distribution of different classes of axons within the DCN. The pattern of labeling of synaptic terminals heaviest in the inner molecular layer seen after incubation with [3H]glutamic acid is coextensive with the axon arborizations of several different inputs to the DCN. The Golgi studies of Lorente de No (17, 18) showed that the arborization of Golgi type 2 axons are heavily concentrated in the inner molecular layer. Cells in the dorsal inferior colliculus also project to this region (9-l 1, 13, 14) as do other intrinsic neurons of the cochlear nucleus (7, 20, 21). Parallel fibers arising from different groups of neurons are also present throughout the molecular layer. The present data do not allow us to choose among the several possibilities. The diffuse pattern of synaptic terminal labeling in the molecular, fusiform, and deep DCN seen after incubation with [3H]glycine is also coextensive with several sets of inputs. In addition to intrinsic neurons of the DCN illustrated by Lorente de No (17, 18), descending inputs are known from the dorsal inferior colliculus to the inner molecular layer from cells in the ventral inferior colliculus to the fusiform cell layer, and from the nuclei of the lateral lemniscus to the deep DCN (9, 13, 14). A descending FIGS. 7-12. Details of the label distribution at a higher magnification. Each corresponds to the arrangement of animals and amino acids in Figs. 1-6. 495X. FIG. 7. GABA. The clumps of silver grains are concentrated in the outer molecular layer although a few are found in the inner molecular layer, fusiform cell layer, and deep DCN. FIG. 8. Glutamic acid. The clusters of silver grains in the inner molecular layer seem to be arranged in chains parallel to the surface of the DCN. FIG. 9. Glycine. Note the relative absence of label over blood vessels (arrowheads) compared with the density of silver grains over elements in the neuropile of the molecular, fusiform cell, and deep layers of the DCN.
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input from the superior olivary complex to the molecular and deep layers was also shown in the tree shrew (7). On the other hand, the primary auditory afferent fibers from the cochlea are most heavily concentrated in the deep DCN and fusiform cell layer (8). Although virtually all labeled synaptic terminals were found on dendrites or neuronal perikarya, many unlabeled terminals had the morphological features associated with the primary cochlear input. It seems unlikely that either glycine or glutamic acid labeled the primary afferent fibers. The present data do not allow us to choose among the several other possibilities. The pattern of labeling concentrated in the outer molecular layer after incubation with [3H]GABA is coextensive with the region containing large numbers of granule cell axons, the parallel fibers. Inputs descending from higher levels of the auditory system are found somewhat deeper in the molecular layer. Together with the electron microscopic observation of label in small unmyelinated axons and their associated boutons en passant, these observations suggest that the label derived from GABA is associated with the parallel fibers. The absence of a distinctive labeling pattern after incubation with [3H]aspartic acid suggests the absence of a high-affinity uptake system under the conditions of these experiments or the possibility that in the DCN the aspartic acid was rapidly metabolized to some other form with the tritiated label lost from the tissue, either in the metabolic process or in the subsequent processing. The labeling of glial cells after incubation with alanine is consistent with observations of glial labeling by alanine in other parts of the central nervous system. The diffuse labeling pattern seen after incubation with taurine may also be indicative of a nonspecific binding, or general breakdown, which could be consistent with the presence of label in some glial cells. Implications about Chemical Properties. If a specific group of neural elements is labeled after incubation with an amino acid, as our findings indicate, we have provided a marker for that group which can be used to assist in the analysis of its anatomic relationships. However, we must be careful in interpreting what the marker reflects about the properties of the labeled elements. FIG. 10. Aspartic acid. Note the presence of a few clumps of label (arrowheads) in the molecular and fusiform cell layers. FIG. Il. Taurine. The labeled cell from Fig. 5 is illustrated. A diffuse distribution of silver grains over the neuropile can also be detected, as can the silver grains over the large blood vessel at lower left. FIG. 12. Alanine. Two labeled cells from Fig. 6 are illustrated. A diffuse distribution of silver grains over the neuropile can also be detected.
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If the label localized within a synaptic terminal or cell is still associated with the original amino acid, we may infer either that the compound was selectively accumulated, bound, or not broken down by that element; that exchange diffusion has labeled a region with a high endogenous concentration of that amino acid (16); or that label has been selectively lost from other elements during incubation or processing. If all elements are equally healthy, a differential distribution can be interpreted as a special property of the labeled cell. However, if one population is selectively damaged by the slicing, we may be looking at an injury phenomenon not characteristic of a healthy cell. This might apply if extrinsic axons were affected while locally arborizing ones were not. In the findings reported here, the high level of label found over the synaptic terminals after incubation with tritiated glycine, GABA, or glutamic acid is much greater than that which could be accounted for by random binding and selective loss. The intensity of the labeling patterns seen in this study after incubation with micromolar amounts of labeled compounds suggests that active accumulation or endogenous exchange is occurring. For GABA, the distribution of the label is consistent with the biochemical data from analysis of microdissected regions of the cochlear nucleus showing high GABA concentrations in the molecular layer. For glycine, biochemical data showing even distributions over the molecular, fusiform, and deep layers correlate with the even label distributions. Thus, for these amino acids, exchange with high endogenous concentrations is a possibility. On the other hand, the absence of label concentrations in the regions shown to have higher concentrations of aspartic acid, the fusiform and deep layer, does not rule out the possibility that labeled compounds were taken up by elements in these regions. It is likely that labeled amino acids could be taken up and rapidly metabolized with the tritiated label split off and lost as water when the tissue is dehydrated. Alternatively, the label might be transferred to some compound which is rapidly eliminated from the neural elements or selectively lost in subsequent processing. If there is a localization of label no longer associated with the original amino acid,
FIGS. 13-15. Electron microscopic autoradiographs of synaptic terminals labeled after incubation with GABA, glutamic acid, and glycine, respectively. 16,100X. FIG. 13. GABA, cat IS 254, 21 days’ exposure. Note the localization of silver grains over longitudinally sectioned parallel fibers in the outer molecular layer. FIG. 14. Glutamic acid, cat IS 24525 days’ exposure. Note the localization of silver grains over two synaptic terminals in the outer molecular layer. FIG. 15. Glycine, cat IS 250, 25 days’ exposure. Note the heavy concentration of silver grains over a synaptic terminal contacting a dendrite in the deep DCN.
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its presence might reflect special metabolic properties of the labeled element which cause the [3H] to be transferred to another compound which is accumulated. This might account for the heavy concentration of label in the inner molecular layer after incubation with [3H]glutamic acid, where the biochemical data suggest a fairly uniform distribution of glutamic acid on a volume basis. Until the labeled compounds remaining after incubation are identified chromatographically these questions cannot be resolved. In conclusion, it is clear that different populations of synaptic terminals were labeled following incubations with different tritiated amino acids. Additional electron microscopic studies will be necessary to resolve features of these populations which can allow us to select among several candidate classes of axons. The correct chemical interpretation of these anatomic findings will require additional biochemical analyses. Nevertheless, these findings represent a promising first step in analyzing chemical properties of specific auditory neural elements. By providing markers for at least three different neuronal populations, this study has brought us closer to our ultimate goal of fully characterizing the connections and chemical and electrical properties of individual auditory neurons so that we may understand processing of auditory information. REFERENCES 1. BIRD, S. J.. R. L. GULLEY, R. J. WENTHOLD, AND J. FEX. 1978. Kainic acid injections result in degeneration of cochlear nucleus cells innervated by the auditory nerve. Science 208: 2.
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BLOOM, F., AND L. L. IVERSON. 1971. Localizing ‘H-GABA in nerve terminals of rat cerebral cortex by electron microscopic autoradiography. Nature (London) 229: 623630.
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10. KANE, E. C. 1976. Descending projections to specific regions of cat cochlear nucleus: a light microscopic study. Exp. Neural. 52: 372-388. 11. KANE, E. S. 1977. Origins of descending inputs to cat dorsal cochlear nucleus. Anar. Rec. 187: 619-620.
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22. SCHWARTZ, I. R., AND D. BOK. 1979. Electron microscopic localization of [‘251]a-bungarotoxin binding sites in the outer plexiform layer of the goldfish retina. J. Neurocytol. 8: 53-66. 23. TACHIBANA, M., AND K. KIRUYAMA. 1974. Gamma-aminobutryic acid in the lower auditory pathway of the guinea pig. Brain Res. 69: 370-374. 24. THALMANN, R. 1975. Biochemical studies of the auditory system. Pages 31-44 in D. B. TOWER, Ed., The Nervous System. Vol. 3, Raven Press, New York. 25. WENTHOLD, R. J. 1978. Glutamic acid and aspartic acid in subdivisions of the cochlear nucleus after auditory nerve lesion. Brain Res. 143: 544-548. 26. WENTHOLD, R. J., AND J. FEX. 1976. Transmitter related enzymes in cochlea and cochlear nucleus of guinea pig. Trans. Am. Sot. Neurochem. 7: 193. 27. WENTHOLD, R. J., AND R. L. GULLEY. 1977. Aspartic acid and glutamic acid levels in the cochlear nucleus after auditory nerve lesion. Brain Res. 138: 11 l-l 23. 28. WENTHOLD, R. J., AND R. L. GULLEY. 1978. Glutamic acid and aspartic acid in the cochlear nucleus of the waltzing guinea pig. Bruin Res. 158: 295-302. 29. WENTHOLD, R. J., AND D. K. MOREST. 1976. Transmitter related enzymes in the guinea pig cochlear nucleus. Sot. Neurosci. Abstr. 2 28. 30. ZIGMOND, R. E., ANDY. BEN-ARI. 1976. A simple method for serial sectioning of fresh brain and the removal of identifiable nuclei from stained sections for biochemical analysis. J. Neurochem. 26: 1285-1287.