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The laminar distribution of amino acids in the caudal cerebellum and electrosensory lateral line lobe of weakly electric fish (Gymnotidae)
Brain Research, 425 (1987)218-224
218
Elsevier BRE 13027'
The laminar distribution of amino acids in the caudal cerebellum and electrosensory lateral line lobe of weakly electric fish
(Gymnotidae) Susan Nadi I and L e o n a r d Maler 2 iNIH-NINCDS, Bethesda, MD 20892 (U.S.A.) and 2Departmentof Anatomy, Facultyof Health Sciences, University of Ottawa, Ottawa, Ont. (Canada) (Accepted 28 April 1987)
Key words: Cerebellum; Electrosensory lateral line lobe; Amino acid; Glutamate; Aspartate; 7-Aminobutyric acid; Taurine; Glycine
We have studied the distribution of the putative amino acid neurotransmitters glutamate, aspartate, ~-aminobutyric acid (GABA), glycine, taurine and fl-alanine in the caudal cerebellar lobe and electrosensory lateral line lobe (ELL) of weakly electric gymnotid fish. In the caudal lobe of the cerebellum, the levels of the various amino acids in the granular and molecular layers are comparable to the levels in the rat cerebellum, with the exception of taurine which is present in greater amounts in the gymnotid. In the ELL, these amino acids are differentially distributed in the various layers of this structure. Glutamate and taurine are enriched in the molecular layer, whereas GABA, aspartate, and fl-alanine are enriched in the deep neuropil + granular layers. Glycine is slightly enriched in the pyramidal cell layer.
INTRODUCTION Several amino acids have been implicated as excitatory or inhibitory neurotransmitters of the vertebrate nervous system. The stereotyped organization of the cerebellum makes this structure especially suitable for cellular localization of putative neurotransmitters; glutamate, aspartate, ~-aminobutyric acid ( G A B A ) and taurine have all been suggested as amino acid neurotransmitters for various cell types and afferent fibers of the cerebellum. In the weakly electric gymnotid fish the caudal lobe of the cerebellum is inverted and its molecular layer is continuous with the molecular layer of an underlying structure - a hypertrophied primary electrosensory region, the electrosensory lateral line lobe ( E L L 26, in earlier publications this structure was referred to as the posterior lateral line lobe). The caudal lobe of the gymnotid cerebellum is morphologically similar to the cerebellum
of other vertebrates (Guest and Maler, unpublished observations). The E L L is a simple laminar region with afferent input and intrinsic circuitry very different from that of the cerebellum 8,17,24,29,41, but it does share parallel fibers with the overlying cerebellum 24,26,29. We have studied the distribution of various amino acids in the caudal lobe of the cerebellum and in the E L L in order to determine the extent to which the morphological similarities between these structures is paralleled by biochemical similarities. MATERIALS AND METHODS Six gymnotid fish of the species Apteronotus leptorhynchus (brown ghost knife fish) were sacrificed by decapitation and the brains removed over ice; vibratome sections o f the medulla were cut at 300 p m (4 °C) and then dissected over ice. The caudal lobe of the cerebellum was dissected into its granular portion
Correspondence: L. Maler, Department of Anatomy, Faculty of Health Sciences, University of Ottawa, 451 Smyth Road, Ottawa, Ontario K1H 8M5, Canada. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
219 (eminentia granularis posterioris3), and molecular portion. The E L L was dissected into its constituent laminae 24 using distinct layers of white matter as a guide (Fig. 1). The plexiform layer was used to separate deep neuropil plus granule cell layer and the stratum fibrosum was then used to separate pyramidal cell layer from the molecular layer. The division of the molecular layer into ventral and dorsal portions is not marked in unfixed vibratome sections and so the lowest fourth of the molecular layer was dissected and considered to comprise the ventral molecular layer, while the remaining tissue was considered to be dorsal molecular layer 26. The following tissue samples were therefore obtained (see Fig. 1 and ref. 24): (1) deep neuropil and granular layer - this lamina contains primary electroreceptor afferents, granular interneurons and terminal boutons arising from E L L interneurons, (2) pyramidal cell layer - this lamina contains the somata of the E L L efferent neurons and the terminal boutons of several types of E L L interneurons, (3) ventral molecular layer - this lamina is the recipient zone of descending input from an isthmic nucleus (n. praeeminentialis41), and also contains interneurons, (4) dorsal molecular layer - this lamina contains parallel fibers emanating from the granule cells of the caudal lobe of the cerebellum as well as stellate interneurons. The tissue samples were rapidly frozen and stored at - 8 0 °C until the biochemical analysis. The E L L is composed of 4 segments 8'17 one of which receives ampuilary electroreceptor input while the other 3 receive tuberous electroreceptor input ~7 (see ref. 7 for discussion of electroreceptors); the segments are morphologically similar and no attempt was made to segregate the tissue from the different segments. A m i n o acids from the microdissected cerebellar and E L L layers were extracted with 5% trichloroacetic acid and assayed with the dansyl chloride technique as previously described 42. Basically, a standard curve was established using the variation of the ratio of a constant amount of [3H]dansyl chloride/14C-ami no acid in the presence of an increasing amount of unlabeled amino acid. The unknown amino acid was determined off this curve. As each amino acid has a slightly different reaction pattern with the dansyl
Fig. 1. Photograph of a vibratome section through the medulla of Apteronotus leptorhynchus, using incident illumination to illustrate the appearance of the slices used for microdissection. The white matter laminae (st. fib. + pl. 1) were used as guides for separating the cellular and neuropil layers. Corp. cer., corpus cerebelli; d. mol., dorsal molecular layer; dn. 1., deep neuropil layer; EGp, eminentia granularis posterioris; gr. 1., granule cell layer; LC, caudal lobe of the cerebellum; mol., molecular layer of EGp; n. med., nucleus medialis; pl. 1., plexiform layer; pyr. 1., pyramidal cell layer; st. fib., stratum fibrosum; v. mol., ventral molecular layer.
chloride molecule, separate standard curves were determined for each of the amino acids reported. The unknown amino acid extract, dansyl chloride derivative, was separated by thin layer chromatography on micro-polyacrylamide sheets obtained from Schleicher and Schuell. Protein levels were determined by the Lowry assay. RESULTS AND DISCUSSION Levels of leucine were measured as a control for
220 TABLE I Distribution of amino acid in the caudal lobe of the cerebellum and ELL laminae. D. mol, dorsal molecular layer; V. mol, ventral molecular layer; Pyr, pyramidal cell layer; Gr. + D.N., granular interneuron + deep neuropil layer. Statistical comparisons (twotailed t-test) were done solely to compare the levels of amino acids within the laminae of the ELL. All comparisons are thus along columns and compare one amino acid between layers and not different amino acids in the same layer. The comparison is always done with respect to the dorsal molecular layer (except glycine) since this has an amino acid content similar to that of the cerebellar molecular layer; differences may therefore relate to the transmitters specific to ELL circuitry. ** Indicates the level of significance is P < 0.001; * or ~ indicates a level of significance of P < 0.01. Thus levels of glutamate and taurine are higher in dorsal molecular layer than in deep neuropil and granular layers (P < 0.001). GABA, aspartate and fl-alanine are higher in the deep neuropil and granular layers compared to the dorsal molecular layer (P < 0.001 for fl-alanine and P < 0.01 for GABA and aspartate). Glycine levels in the pyramidal cell layer are significantly greater than those in the ventral molecular layer (P < 0.01) but not significantly different from the other layers. Region
Cerebellum Mol. Gr. ELL D. tool V. tool Pyr. Gr. + D.N.
l~mol/mg Protein (mean _+S.D.) Glutamate
Taurine
GABA
Glycine
Aspartate
Alanine
Leucine
6.47 (_+0.78) 9.13 (_+0.39)
10.75 (_+0.96) 8.87 (_+1.13)
1.05 (_+0.1) 0.98 (_+0.28)
0.76 (_+0.11) 0.74 (_+0.11)
0.99 (_+0.12) 0.84 (+0.09)
0.22 (_+0.02) 0.19 (_+0.06)
0.11 (_+0.02) 0.13 (_+0.04)
7.58** (+0.53) 7.0 (+0.98) 6.81 (+0.88) 5.1 (_+0.62)
8.42** (_+0.99) 8.25 (+0.42) 5.93 (+1.18) 2.08 (_+0.35)
1.44 (_+0.26) 1.77 (+0.11) 1.86 (_+0.28) 2.07* (_+0.24)
1.01 (+0.21) 0.82 (_+0.13) 1.38 # (_+0.39) 1.11 (_+0.16)
0.99 (+0.11) 0.89 (_+0.06) 0.95 (_+0.23) 1.25" (_+0.13)
0.25 (+0.04) 0.33 (_+0.09) 0.33 (_+0.1) 0.66** (_+0.11)
0.08 (+0.02) 0.09 (_+0.02) 0.12 (_+0.03) 0.12 (_+0.05)
the consistency of o u r t e c h n i q u e . L e u c i n e c o n c e n t r a tion is fairly e v e n across l a m i n a e ; there is only 0.04 /~mol/mg p r o t e i n difference b e t w e e n the highest a n d lowest value. All o t h e r a m i n o acids assayed show l a m i n a r differences which might be related to a n e u r o t r a n s m i t t e r role ( T a b l e I). O u r results are expressed as a m i n o acid c o n t e n t per milligram p r o t e i n a n d are n o t directly c o m p a r able to similar studies o n the m a m m a l i a n c e r e b e l l u m . In both the m a m m a l i a n c e r e b e l l u m 36, a n d in the caudal lobe of the c e r e b e l l u m of these fish, g l u t a m a t e was f o u n d in greater q u a n t i t i e s t h a n the other a m i n o
acids studied (with one exception, see below). F o r this r e a s o n we expressed levels of each a m i n o acid as a fraction of the g l u t a m a t e c o n t e n t ( t a k e n as 1). A s Table II shows, the relative c o n c e n t r a t i o n s of most a m i n o acids are similar in the c e r e b e l l u m of g y m n o tids and rats36; this interspecific c o m p a r i s o n holds true for b o t h the g r a n u l e cell a n d m o l e c u l a r layers. The only exception is t a u r i n e ; the relative c o n c e n t r a tion of this a m i n o acid is a b o u t 5 times higher in the g y m n o t i d c e r e b e l l u m (caudal lobe) t h a n the m a m m a lian cerebellum. E v e n in this case the ratio of t a u r i n e c o n t e n t in the m o l e c u l a r layer to that of the g r a n u l a r
TABLE II Ratio in cerebellum of amino acid levels to the level of glutamate (taken as 1)
Values for the rat are given in parentheses and the raw data are taken from ref. 36. Abbreviations as in Table I. Region
Glutamate
Taurine
GABA
Glycine
Aspartate
Alanine
Cerebellum Mol. Gr.
1 (1) 1 (1)
1.63 (0.31) 0.98 (0.21)
0.17 (0.1) 0.11 (0.09)
0.12 (0.07) 0.08 (0.08)
0.15 (0.13) 0.09 (0.15)
0.03 (0.05) 0.02 (0.04)
221 cell layer is similar in rats (1.57) and gymnotids (1.21). The caudal lobe of the cerebellum is an archicerebellar region25'26; curiously, Chan-Palay and her colleagues9'1° have found that in the rat the archicerebellum (flocculus + nodulus) has the greatest number of cysteine sulphinic acid decarboxylase-immunoreactive neurons (CSAD - the biosynthetic enzyme for taurine) and thus would presumably have the highest taurine content. This suggests that the biochemistry of the cerebellar cortex is as conservative as its histology. For the ELL we consider separately the distribution of each amino acid.
Glutamate Glutamate is significantly enriched in the dorsal molecular layer compared to the deep neuropil + granular interneuron layers. Since the dominant axonal constituent of the dorsal molecular layer is parallel fibers arising from cerebellar granule cells24'26, this suggests that glutamate is the neurotransmitter of the cerebellar granule cell-parallel fiber system in these fish. Young et al. 48 first suggested that cerebellar granule cells might use glutamate as a transmitter and this hypothesis has been amply confirmed by subsequent biochemical 16,19,32,36,39,40,46and physiological studies43; our results offer further confirmation in a different species. The high levels of glutamate in the ventral molecular layer suggest that glutamate might also be the transmitter of the afferents from n. praeeminentialis; caution must be exercised in this case since the boundary between dorsal and ventral molecular layers is essentially arbitrary and we may have contaminated the ventral molecular layer with tissue from the glutamate-rich dorsal molecular layer. The lowest levels of glutamate are in the deep neuropil and granular interneuron layer; since this layer receives primary electroreceptive afferents, this result suggests that these afferents do not use glutamate as a neurotransmitter. Aspartate Aspartate is found at lower levels than glutamate in the ELL; unlike glutamate, it is significantly enriched in the deep neuropil + granular interneuron layer. The two major constituents of these layers are primary electroreceptor afferents and the granule type interneurons 24'28. The granule type interneu-
rons are unlikely to use aspartate as a neurotransmitter for two reasons: (a) their axons project to and ramify within the pyramidal cell layer24'28 which is not enriched in aspartate, and (b) both morphological28 and physiological5 studies suggest an inhibitory role for the granule type interneurons, while aspartate has excitatory actions in the vertebrate nervous system 14. We therefore propose aspartate as a candidate amino acid transmitter of primary electroreceptor afferents in the ELL. It is interesting that electroreceptors are probably evolutionarily derived from lateral line organs n (see also ref. 7). The auditory system may also be evolutionarily related to the lateral line receptors 45 and biochemical 47, and physiological3° studies have suggested that cochlear afferents might use aspartate as their transmitter. If our hypothesis is correct it would suggest that the acousticolateral system is very conservative in its use of neurotransmitters; physiological and pharmacological studies of primary afferent synaptic transmission in the ELL will be required to confirm our hypothesis.
GABA GABA is found in all layers of the ELL but it is significantly enriched in the deeper layers, especially the deep neuropil + granular interneuron layers. This result is consistent with the distribution of glutamic acid decarboxylase (GAD), the biosynthetic enzyme for GABA. GAD- and GABA-positive neurons and boutons are found in all layers of the ELL but are very dense in both the pyramidal cell layer and the deep neuropil + granular interneuron layers 27. G A B A is likely to be an important inhibitory neurotransmitter of the ELL, as it is in most parts of the central nervous system35; the potential role of GABAergic inhibition in the ELL is best discussed in relation to the cellular distribution of G A B A (Maler and Mugnaini, in preparation). Taurine Taurine is the amino acid which has the most uneven distribution in the ELL. Its concentration in the molecular layer (dorsal and ventral) is 4 times that of the deep neuropil + granular interneuron layers. By contrast there is only a two-and-a-half-fold difference in taurine concentration between the molecular
222 layer and white matter of the rat cerebellum 36. Extensive biochemical evidence suggests that taurine is associated with cerebellar stellate cells 32'33, and Okamoto and his colleagues have provided evidence that taurine is an inhibitory transmitter in the cerebellum and probably the transmitter of the stellate cells 37. There are still some difficulties with this idea. ChanPalay et al. 9'1° have suggested that taurine may be present in many cell types of the cerebellum and that it might not have a neurotransmitter role (see also refs. 21, 34); furthermore, it has been difficult to demonstrate sodium-independent (e.g. presumptive postsynaptic) binding of taurine in the cerebellum 2°23. The very uneven distribution of taurine in the ELL may make this a favorable structure in which to study the cellular distribution and physiological role of taurine.
Glycine Glycine has its highest concentration in the pyramidal cell layer of the ELL. Glycine is an inhibitory neurotransmitter mainly in the spinal cord and medulla1; since the pyramidal cell layer probably contains only inhibitory chemical synapses, it is an appropriate lamina for glycinergic synapses. The lack of unique markers for glycinergic synapses makes it difficult to correlate this biochemical finding with the structure of the ELL. Immunohistochemicai localization of glycine and its receptor 44, as well as binding studies on ELL tissue sections and physiological analysis, will be required to validate a transmitter role for glycine in the ELL.
fl-A lanine Alanine is only found in low amounts in the ELL but it is significantly concentrated in the deep neuropil + granular interneuron layers; we have no idea as to its cellular localization in these layers. Alanine has depressant actions in the nervous system 13, and is actively taken up by specific cell types L8'49. The pharmacology of fl-alanine is complex and earlier studies suggested that it might exert its action at a glycine receptor11"12; more recent work has supported the idea that fl-alanine acts at a receptor site distinct from that of G A B A or glycine 2'15. The differential laminar distribution of fl-alanine in the ELL might make this a useful preparation for investigating its putative transmitter role.
GENERAL DISCUSSION Although the mere presence of an amino acid does not necessarily entail its acting as a neurotransmitter, its local enrichment within one layer of a laminar neural structure suggests a neurotransmitter role which can be more rigorously tested with appropriate histological, pharmacological and physiological experiments. The biochemical data demonstrates that the deep neuropil and granule cell layer are enriched in aspartate, G A B A and fl-alanine. This suggests that primary electroreceptor afferents may use aspartate (or an aspartate-containing peptide) as an excitatory transmitter. G A B A is likely to be an inhibitory neurotransmitter in this lamina since recent immunohistochemical studies have shown numerous G A B A ergic boutons apparently making synaptic contact with granule cells and with dendrites of pyramidal and granule cells in the deep neuropil layer 27. The functional implications of these synapses will be discussed elsewhere. The role of fl-alanine in the deep neuropil layer is not known at this time. The pyramidal cell layer is enriched in G A B A , consistent with the immunohistochemical demonstration of numerous GABAergic boutons in this layer 27. These GABAergic boutons are derived from a variety of sources and their possible roles will be discussed elsewhere. This lamina is also slightly enriched in glycine, but this may of course have only a metabolic role in the large pyramidal neurons of this layer. The molecular layer is enriched in both glutamate and taurine. In the dorsal molecular layer the glutamate can certainly be associated with cerebellar parallel fiber. The exceptionally high content of taurine in this layer may make the ELL a useful system in which to study the role of this amino acid. Further immunohistochemical work will be required to define the cellular distribution of taurine before its function can be elucidated. The only neurotransmitters identified to date in the ELL are G A B A 27 and acetylcholine; acetylcholine probably acts via muscarinic receptors in the dorsal molecular layer 25,38. The laminar organization of the ELL, the fairly complete description of its circuitry 28 and the differential laminar distribution of amino acids within it make the ELL an excellent system in
223 which to study the physiology a n d p h a r m a c o l o g y of
mitters c o n t r i b u t e to the g e n e r a t i o n or m o d i f i c a t i o n
a m i n o acid n e u r o t r a n s m i t t e r s a n d their r e l a t i o n to cholinergic t r a n s m i s s i o n . T h e r e c e n t d e v e l o p m e n t of an E L L slice p r e p a r a t i o n will facilitate this w o r k 31.
of receptive field p r o p e r t i e s in the E L L .
T h e E L L is a first o r d e r e l e c t r o s e n s o r y r e g i o n a n d its cells can be f u n c t i o n a l l y c h a r a c t e r i z e d with respect to their receptive fields 4-6. It s h o u l d be possible
ACKNOWLEDGEMENTS
to c o m b i n e the p h a r m a c o l o g i c a l a n d f u n c t i o n a l analysis of the E L L a n d e x a m i n e how various n e u r o t r a n s -
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tors in the caudal cerebellum and electrosensory lateral line lobe of gymnotiform fish, Neurosci. Lett., 42 (1983) 137-143. Rhode, B.H., Rea, M.A., Simon, J.R. and McBride, W.J., Effects of X-irradiation induced loss of cerebellar granule cells on synaptosomal levels and high affinity uptake of amino acids, J. Neurochem., 32 (1979) 1431-1435. Sandoval, M.E. and Cotman, C.W., Evolution of glutamate as a neurotransmitter of cerebellar parallel fibers, Neuroscience, 3 (1978) 199-206. Sas, E. and Maler, L., The nucleus praeeminentialis: a Golgi study of a feedback center in the electrosensory system of a gymnotid fish, J. Comp. Neurol., 221 (1983) 127-144. Snodgrass, S.R. and Iversen, L.L., A sensitive double isotope derivative assay to measure release of amino acids from brain in vitro, Nature (New Biol.), 241 (1973) 154-156. Stone, T.W., Glutamate as the neurotransmitter of cerebellar cells in the rat: electrophysiological evidence, Br. J. Pharmacol., 66 (1979) 291-296. Triller, A., Cluzeaud, F., Pfeiffer, F., Betz, H. and Korn, H., Distribution of glycine at central synapses: an immunoelectron microscopy study, J. Cell Biol., 101 (1985) 683-688. Van Bergeijk, W.A., The evolution of vertebrate hearing. In Contributions to Sensory Physiology Vol. 2, Academic, New York, 1967, pp. 1-49. Vinh Tan Tran and Snyder, S.H., Amino acid neurotransmitter candidates in rat cerebellum: selective effects of kainic acid lesions, Brain Research, 167 (1979) 345-353. Wenthold, R.J., Glutamate and aspartate as neurotransmitters for the auditory nerve. In G. DiChiaro and G.L. Gessa (Eds.), Glutamate as a Neurotransmitter. Advances in Biochemical Psychopharmacology, Vol. 27, Raven, New York, 1981, pp. 69-78. Young, A.B., Oster-Granite, M.L., Herndon, R.M. and Snyder, S.H., Glutamic acid: selective depletion by viralinduced granule cell loss in hamster cerebellum, Brain Research, 73 (1974) 1-13. Zafra, F., Aragon, M.C., Valdivieso, F. and Giminez, C., fl-alanine transport into plasma membrane vesicles derived from rat brain synaptosomes, Neurochem. Res., 9 (1984) 695-707.
218
Elsevier BRE 13027'
The laminar distribution of amino acids in the caudal cerebellum and electrosensory lateral line lobe of weakly electric fish
(Gymnotidae) Susan Nadi I and L e o n a r d Maler 2 iNIH-NINCDS, Bethesda, MD 20892 (U.S.A.) and 2Departmentof Anatomy, Facultyof Health Sciences, University of Ottawa, Ottawa, Ont. (Canada) (Accepted 28 April 1987)
Key words: Cerebellum; Electrosensory lateral line lobe; Amino acid; Glutamate; Aspartate; 7-Aminobutyric acid; Taurine; Glycine
We have studied the distribution of the putative amino acid neurotransmitters glutamate, aspartate, ~-aminobutyric acid (GABA), glycine, taurine and fl-alanine in the caudal cerebellar lobe and electrosensory lateral line lobe (ELL) of weakly electric gymnotid fish. In the caudal lobe of the cerebellum, the levels of the various amino acids in the granular and molecular layers are comparable to the levels in the rat cerebellum, with the exception of taurine which is present in greater amounts in the gymnotid. In the ELL, these amino acids are differentially distributed in the various layers of this structure. Glutamate and taurine are enriched in the molecular layer, whereas GABA, aspartate, and fl-alanine are enriched in the deep neuropil + granular layers. Glycine is slightly enriched in the pyramidal cell layer.
INTRODUCTION Several amino acids have been implicated as excitatory or inhibitory neurotransmitters of the vertebrate nervous system. The stereotyped organization of the cerebellum makes this structure especially suitable for cellular localization of putative neurotransmitters; glutamate, aspartate, ~-aminobutyric acid ( G A B A ) and taurine have all been suggested as amino acid neurotransmitters for various cell types and afferent fibers of the cerebellum. In the weakly electric gymnotid fish the caudal lobe of the cerebellum is inverted and its molecular layer is continuous with the molecular layer of an underlying structure - a hypertrophied primary electrosensory region, the electrosensory lateral line lobe ( E L L 26, in earlier publications this structure was referred to as the posterior lateral line lobe). The caudal lobe of the gymnotid cerebellum is morphologically similar to the cerebellum
of other vertebrates (Guest and Maler, unpublished observations). The E L L is a simple laminar region with afferent input and intrinsic circuitry very different from that of the cerebellum 8,17,24,29,41, but it does share parallel fibers with the overlying cerebellum 24,26,29. We have studied the distribution of various amino acids in the caudal lobe of the cerebellum and in the E L L in order to determine the extent to which the morphological similarities between these structures is paralleled by biochemical similarities. MATERIALS AND METHODS Six gymnotid fish of the species Apteronotus leptorhynchus (brown ghost knife fish) were sacrificed by decapitation and the brains removed over ice; vibratome sections o f the medulla were cut at 300 p m (4 °C) and then dissected over ice. The caudal lobe of the cerebellum was dissected into its granular portion
Correspondence: L. Maler, Department of Anatomy, Faculty of Health Sciences, University of Ottawa, 451 Smyth Road, Ottawa, Ontario K1H 8M5, Canada. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
219 (eminentia granularis posterioris3), and molecular portion. The E L L was dissected into its constituent laminae 24 using distinct layers of white matter as a guide (Fig. 1). The plexiform layer was used to separate deep neuropil plus granule cell layer and the stratum fibrosum was then used to separate pyramidal cell layer from the molecular layer. The division of the molecular layer into ventral and dorsal portions is not marked in unfixed vibratome sections and so the lowest fourth of the molecular layer was dissected and considered to comprise the ventral molecular layer, while the remaining tissue was considered to be dorsal molecular layer 26. The following tissue samples were therefore obtained (see Fig. 1 and ref. 24): (1) deep neuropil and granular layer - this lamina contains primary electroreceptor afferents, granular interneurons and terminal boutons arising from E L L interneurons, (2) pyramidal cell layer - this lamina contains the somata of the E L L efferent neurons and the terminal boutons of several types of E L L interneurons, (3) ventral molecular layer - this lamina is the recipient zone of descending input from an isthmic nucleus (n. praeeminentialis41), and also contains interneurons, (4) dorsal molecular layer - this lamina contains parallel fibers emanating from the granule cells of the caudal lobe of the cerebellum as well as stellate interneurons. The tissue samples were rapidly frozen and stored at - 8 0 °C until the biochemical analysis. The E L L is composed of 4 segments 8'17 one of which receives ampuilary electroreceptor input while the other 3 receive tuberous electroreceptor input ~7 (see ref. 7 for discussion of electroreceptors); the segments are morphologically similar and no attempt was made to segregate the tissue from the different segments. A m i n o acids from the microdissected cerebellar and E L L layers were extracted with 5% trichloroacetic acid and assayed with the dansyl chloride technique as previously described 42. Basically, a standard curve was established using the variation of the ratio of a constant amount of [3H]dansyl chloride/14C-ami no acid in the presence of an increasing amount of unlabeled amino acid. The unknown amino acid was determined off this curve. As each amino acid has a slightly different reaction pattern with the dansyl
Fig. 1. Photograph of a vibratome section through the medulla of Apteronotus leptorhynchus, using incident illumination to illustrate the appearance of the slices used for microdissection. The white matter laminae (st. fib. + pl. 1) were used as guides for separating the cellular and neuropil layers. Corp. cer., corpus cerebelli; d. mol., dorsal molecular layer; dn. 1., deep neuropil layer; EGp, eminentia granularis posterioris; gr. 1., granule cell layer; LC, caudal lobe of the cerebellum; mol., molecular layer of EGp; n. med., nucleus medialis; pl. 1., plexiform layer; pyr. 1., pyramidal cell layer; st. fib., stratum fibrosum; v. mol., ventral molecular layer.
chloride molecule, separate standard curves were determined for each of the amino acids reported. The unknown amino acid extract, dansyl chloride derivative, was separated by thin layer chromatography on micro-polyacrylamide sheets obtained from Schleicher and Schuell. Protein levels were determined by the Lowry assay. RESULTS AND DISCUSSION Levels of leucine were measured as a control for
220 TABLE I Distribution of amino acid in the caudal lobe of the cerebellum and ELL laminae. D. mol, dorsal molecular layer; V. mol, ventral molecular layer; Pyr, pyramidal cell layer; Gr. + D.N., granular interneuron + deep neuropil layer. Statistical comparisons (twotailed t-test) were done solely to compare the levels of amino acids within the laminae of the ELL. All comparisons are thus along columns and compare one amino acid between layers and not different amino acids in the same layer. The comparison is always done with respect to the dorsal molecular layer (except glycine) since this has an amino acid content similar to that of the cerebellar molecular layer; differences may therefore relate to the transmitters specific to ELL circuitry. ** Indicates the level of significance is P < 0.001; * or ~ indicates a level of significance of P < 0.01. Thus levels of glutamate and taurine are higher in dorsal molecular layer than in deep neuropil and granular layers (P < 0.001). GABA, aspartate and fl-alanine are higher in the deep neuropil and granular layers compared to the dorsal molecular layer (P < 0.001 for fl-alanine and P < 0.01 for GABA and aspartate). Glycine levels in the pyramidal cell layer are significantly greater than those in the ventral molecular layer (P < 0.01) but not significantly different from the other layers. Region
Cerebellum Mol. Gr. ELL D. tool V. tool Pyr. Gr. + D.N.
l~mol/mg Protein (mean _+S.D.) Glutamate
Taurine
GABA
Glycine
Aspartate
Alanine
Leucine
6.47 (_+0.78) 9.13 (_+0.39)
10.75 (_+0.96) 8.87 (_+1.13)
1.05 (_+0.1) 0.98 (_+0.28)
0.76 (_+0.11) 0.74 (_+0.11)
0.99 (_+0.12) 0.84 (+0.09)
0.22 (_+0.02) 0.19 (_+0.06)
0.11 (_+0.02) 0.13 (_+0.04)
7.58** (+0.53) 7.0 (+0.98) 6.81 (+0.88) 5.1 (_+0.62)
8.42** (_+0.99) 8.25 (+0.42) 5.93 (+1.18) 2.08 (_+0.35)
1.44 (_+0.26) 1.77 (+0.11) 1.86 (_+0.28) 2.07* (_+0.24)
1.01 (+0.21) 0.82 (_+0.13) 1.38 # (_+0.39) 1.11 (_+0.16)
0.99 (+0.11) 0.89 (_+0.06) 0.95 (_+0.23) 1.25" (_+0.13)
0.25 (+0.04) 0.33 (_+0.09) 0.33 (_+0.1) 0.66** (_+0.11)
0.08 (+0.02) 0.09 (_+0.02) 0.12 (_+0.03) 0.12 (_+0.05)
the consistency of o u r t e c h n i q u e . L e u c i n e c o n c e n t r a tion is fairly e v e n across l a m i n a e ; there is only 0.04 /~mol/mg p r o t e i n difference b e t w e e n the highest a n d lowest value. All o t h e r a m i n o acids assayed show l a m i n a r differences which might be related to a n e u r o t r a n s m i t t e r role ( T a b l e I). O u r results are expressed as a m i n o acid c o n t e n t per milligram p r o t e i n a n d are n o t directly c o m p a r able to similar studies o n the m a m m a l i a n c e r e b e l l u m . In both the m a m m a l i a n c e r e b e l l u m 36, a n d in the caudal lobe of the c e r e b e l l u m of these fish, g l u t a m a t e was f o u n d in greater q u a n t i t i e s t h a n the other a m i n o
acids studied (with one exception, see below). F o r this r e a s o n we expressed levels of each a m i n o acid as a fraction of the g l u t a m a t e c o n t e n t ( t a k e n as 1). A s Table II shows, the relative c o n c e n t r a t i o n s of most a m i n o acids are similar in the c e r e b e l l u m of g y m n o tids and rats36; this interspecific c o m p a r i s o n holds true for b o t h the g r a n u l e cell a n d m o l e c u l a r layers. The only exception is t a u r i n e ; the relative c o n c e n t r a tion of this a m i n o acid is a b o u t 5 times higher in the g y m n o t i d c e r e b e l l u m (caudal lobe) t h a n the m a m m a lian cerebellum. E v e n in this case the ratio of t a u r i n e c o n t e n t in the m o l e c u l a r layer to that of the g r a n u l a r
TABLE II Ratio in cerebellum of amino acid levels to the level of glutamate (taken as 1)
Values for the rat are given in parentheses and the raw data are taken from ref. 36. Abbreviations as in Table I. Region
Glutamate
Taurine
GABA
Glycine
Aspartate
Alanine
Cerebellum Mol. Gr.
1 (1) 1 (1)
1.63 (0.31) 0.98 (0.21)
0.17 (0.1) 0.11 (0.09)
0.12 (0.07) 0.08 (0.08)
0.15 (0.13) 0.09 (0.15)
0.03 (0.05) 0.02 (0.04)
221 cell layer is similar in rats (1.57) and gymnotids (1.21). The caudal lobe of the cerebellum is an archicerebellar region25'26; curiously, Chan-Palay and her colleagues9'1° have found that in the rat the archicerebellum (flocculus + nodulus) has the greatest number of cysteine sulphinic acid decarboxylase-immunoreactive neurons (CSAD - the biosynthetic enzyme for taurine) and thus would presumably have the highest taurine content. This suggests that the biochemistry of the cerebellar cortex is as conservative as its histology. For the ELL we consider separately the distribution of each amino acid.
Glutamate Glutamate is significantly enriched in the dorsal molecular layer compared to the deep neuropil + granular interneuron layers. Since the dominant axonal constituent of the dorsal molecular layer is parallel fibers arising from cerebellar granule cells24'26, this suggests that glutamate is the neurotransmitter of the cerebellar granule cell-parallel fiber system in these fish. Young et al. 48 first suggested that cerebellar granule cells might use glutamate as a transmitter and this hypothesis has been amply confirmed by subsequent biochemical 16,19,32,36,39,40,46and physiological studies43; our results offer further confirmation in a different species. The high levels of glutamate in the ventral molecular layer suggest that glutamate might also be the transmitter of the afferents from n. praeeminentialis; caution must be exercised in this case since the boundary between dorsal and ventral molecular layers is essentially arbitrary and we may have contaminated the ventral molecular layer with tissue from the glutamate-rich dorsal molecular layer. The lowest levels of glutamate are in the deep neuropil and granular interneuron layer; since this layer receives primary electroreceptive afferents, this result suggests that these afferents do not use glutamate as a neurotransmitter. Aspartate Aspartate is found at lower levels than glutamate in the ELL; unlike glutamate, it is significantly enriched in the deep neuropil + granular interneuron layer. The two major constituents of these layers are primary electroreceptor afferents and the granule type interneurons 24'28. The granule type interneu-
rons are unlikely to use aspartate as a neurotransmitter for two reasons: (a) their axons project to and ramify within the pyramidal cell layer24'28 which is not enriched in aspartate, and (b) both morphological28 and physiological5 studies suggest an inhibitory role for the granule type interneurons, while aspartate has excitatory actions in the vertebrate nervous system 14. We therefore propose aspartate as a candidate amino acid transmitter of primary electroreceptor afferents in the ELL. It is interesting that electroreceptors are probably evolutionarily derived from lateral line organs n (see also ref. 7). The auditory system may also be evolutionarily related to the lateral line receptors 45 and biochemical 47, and physiological3° studies have suggested that cochlear afferents might use aspartate as their transmitter. If our hypothesis is correct it would suggest that the acousticolateral system is very conservative in its use of neurotransmitters; physiological and pharmacological studies of primary afferent synaptic transmission in the ELL will be required to confirm our hypothesis.
GABA GABA is found in all layers of the ELL but it is significantly enriched in the deeper layers, especially the deep neuropil + granular interneuron layers. This result is consistent with the distribution of glutamic acid decarboxylase (GAD), the biosynthetic enzyme for GABA. GAD- and GABA-positive neurons and boutons are found in all layers of the ELL but are very dense in both the pyramidal cell layer and the deep neuropil + granular interneuron layers 27. G A B A is likely to be an important inhibitory neurotransmitter of the ELL, as it is in most parts of the central nervous system35; the potential role of GABAergic inhibition in the ELL is best discussed in relation to the cellular distribution of G A B A (Maler and Mugnaini, in preparation). Taurine Taurine is the amino acid which has the most uneven distribution in the ELL. Its concentration in the molecular layer (dorsal and ventral) is 4 times that of the deep neuropil + granular interneuron layers. By contrast there is only a two-and-a-half-fold difference in taurine concentration between the molecular
222 layer and white matter of the rat cerebellum 36. Extensive biochemical evidence suggests that taurine is associated with cerebellar stellate cells 32'33, and Okamoto and his colleagues have provided evidence that taurine is an inhibitory transmitter in the cerebellum and probably the transmitter of the stellate cells 37. There are still some difficulties with this idea. ChanPalay et al. 9'1° have suggested that taurine may be present in many cell types of the cerebellum and that it might not have a neurotransmitter role (see also refs. 21, 34); furthermore, it has been difficult to demonstrate sodium-independent (e.g. presumptive postsynaptic) binding of taurine in the cerebellum 2°23. The very uneven distribution of taurine in the ELL may make this a favorable structure in which to study the cellular distribution and physiological role of taurine.
Glycine Glycine has its highest concentration in the pyramidal cell layer of the ELL. Glycine is an inhibitory neurotransmitter mainly in the spinal cord and medulla1; since the pyramidal cell layer probably contains only inhibitory chemical synapses, it is an appropriate lamina for glycinergic synapses. The lack of unique markers for glycinergic synapses makes it difficult to correlate this biochemical finding with the structure of the ELL. Immunohistochemicai localization of glycine and its receptor 44, as well as binding studies on ELL tissue sections and physiological analysis, will be required to validate a transmitter role for glycine in the ELL.
fl-A lanine Alanine is only found in low amounts in the ELL but it is significantly concentrated in the deep neuropil + granular interneuron layers; we have no idea as to its cellular localization in these layers. Alanine has depressant actions in the nervous system 13, and is actively taken up by specific cell types L8'49. The pharmacology of fl-alanine is complex and earlier studies suggested that it might exert its action at a glycine receptor11"12; more recent work has supported the idea that fl-alanine acts at a receptor site distinct from that of G A B A or glycine 2'15. The differential laminar distribution of fl-alanine in the ELL might make this a useful preparation for investigating its putative transmitter role.
GENERAL DISCUSSION Although the mere presence of an amino acid does not necessarily entail its acting as a neurotransmitter, its local enrichment within one layer of a laminar neural structure suggests a neurotransmitter role which can be more rigorously tested with appropriate histological, pharmacological and physiological experiments. The biochemical data demonstrates that the deep neuropil and granule cell layer are enriched in aspartate, G A B A and fl-alanine. This suggests that primary electroreceptor afferents may use aspartate (or an aspartate-containing peptide) as an excitatory transmitter. G A B A is likely to be an inhibitory neurotransmitter in this lamina since recent immunohistochemical studies have shown numerous G A B A ergic boutons apparently making synaptic contact with granule cells and with dendrites of pyramidal and granule cells in the deep neuropil layer 27. The functional implications of these synapses will be discussed elsewhere. The role of fl-alanine in the deep neuropil layer is not known at this time. The pyramidal cell layer is enriched in G A B A , consistent with the immunohistochemical demonstration of numerous GABAergic boutons in this layer 27. These GABAergic boutons are derived from a variety of sources and their possible roles will be discussed elsewhere. This lamina is also slightly enriched in glycine, but this may of course have only a metabolic role in the large pyramidal neurons of this layer. The molecular layer is enriched in both glutamate and taurine. In the dorsal molecular layer the glutamate can certainly be associated with cerebellar parallel fiber. The exceptionally high content of taurine in this layer may make the ELL a useful system in which to study the role of this amino acid. Further immunohistochemical work will be required to define the cellular distribution of taurine before its function can be elucidated. The only neurotransmitters identified to date in the ELL are G A B A 27 and acetylcholine; acetylcholine probably acts via muscarinic receptors in the dorsal molecular layer 25,38. The laminar organization of the ELL, the fairly complete description of its circuitry 28 and the differential laminar distribution of amino acids within it make the ELL an excellent system in
223 which to study the physiology a n d p h a r m a c o l o g y of
mitters c o n t r i b u t e to the g e n e r a t i o n or m o d i f i c a t i o n
a m i n o acid n e u r o t r a n s m i t t e r s a n d their r e l a t i o n to cholinergic t r a n s m i s s i o n . T h e r e c e n t d e v e l o p m e n t of an E L L slice p r e p a r a t i o n will facilitate this w o r k 31.
of receptive field p r o p e r t i e s in the E L L .
T h e E L L is a first o r d e r e l e c t r o s e n s o r y r e g i o n a n d its cells can be f u n c t i o n a l l y c h a r a c t e r i z e d with respect to their receptive fields 4-6. It s h o u l d be possible
ACKNOWLEDGEMENTS
to c o m b i n e the p h a r m a c o l o g i c a l a n d f u n c t i o n a l analysis of the E L L a n d e x a m i n e how various n e u r o t r a n s -
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