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Demonstration of glutamate-positive axon terminals forming asymmetric synapses in cat neocortex
162
Brain Research, 455 (1988) 162-165 Elsevier
BRE 22979
Demonstration of glutamate-positive axon terminals forming asymmetric synapses in cat neocortex Javier DeFelipe 2, Fiorenzo Conti 1, Susan L. Van Eyck 3 and Tullio Manzoni 1 tlnstitute of Human Physiology, University of Ancona, Ancona (Italy), 2lnstituto Cajal, CS1C, Madrid (Spain) and 3Department of Anatomy and Curriculum in Neurobiology, University of North Carolina, Chapel Hill, NC27514 (U.S.A.)
(Accepted 29 March 1988) Key words: Glutamate; Asymmetric synapse; Somatic sensory cortex; Visual cortex; Cat; Immunocytochemistry
Electron microscopic examination of sections immunocytochemically processed with an anti-glutamate serum reveals that many asymmetric synapses in the cat neocortex contain elevated levels of immunodetectabte glutamate. These labelled axon terminals are likely to use glutamate as neurotransmitter. Axon terminals forming symmetric contacts were never labelled. Since glutamate is known to exert potent excitatory effects on neocortical neurons, the present finding gives immunocytochemical evidence that asymmetric synapses are excitatory. y-Aminobutyric acid ( G A B A ) and glutamate (Giu) thought to be the major inhibitory and excitatory neurotransmitters, respectively, of the cerebral cortex 2°'21. Two major morphological types of synapses are present in the neocortex: one is characterized by a prominent postsynaptic density (asymmetric synapses) and the other by a very thin postsynaptic density (symmetric synapses) 2'12. Axon terminals forming synapses of the first type contain round vescicles and are supposed to be excitatory, while those forming synapses of the second type have flattened vescicles and are probably inhibitory 3. With the introduction of immunocytochemical techniques this hypothesis was supported by the observation that only axon terminals forming symmetric synapses contain the synthesizing enzyme for G A B A 14'28. Recent observations 4'6 indicate that numerous pyramidal neurons known to form asymmetric synapses 25'33 are immunoreactive for an antiGlu serum 15 in the neocortex. Some Glu-positive dot-like elements, presumably axon terminals, were also observed 4. However, conclusive evidence that Glu is contained in axon terminals forming asymmetric synapses has not been provided. In the present study correlative light and electron microscopic immunocytochemical
are
methods were employed to demonstrate that a particular population of asymmetric synapses in the cerebral cortex contains high levels of immunodetectable Glu. Experiments were carried out on the first somatic sensory (SI) and primary visual (area 17) areas of adult cats. Under deep barbiturate anaesthesia (Nembutal; 33 mg/kg, i.p.) animals were perfused transcardially with saline followed by a fixative solution containing 4% carbodiimide, 0.2% picric acid and 0.2% glutaraldehyde. Selected blocks from SI and from area 17 were post-fixed in 4% paraformaldehyde for 24-48 h and cut into 50-~m-thick sections with a Vibratome. The primary antibody had been previously characterized ~5 and does not cross-react with aspartate and G A B A 4'15. The immunocytochemical procedure and controls used in this study have already been published 4. Processed sections were osmicated (1% in 0.1 M phosphate buffer for 1 h), dehydrated in ethanol and then flat-embedded in thin sheets of Araldite resin. Sections were then cut with an ultramicrotome at 2/~m, examined at light microscope, photographed, then resectioned serially8 at 65-70 nm, and finally examined with a Jeol100B electron microscope. To overcome the possibil-
Correspondence: F. Conti, Istituto di Fisiologia Umana, Universit~ di Ancona, Via Ranieri-Monte d'Ago, 1-60131Ancona, ItaLy.
0006-8993/88/$03.50 (~ 1988 Elsevier Science Publishers B.V. (Biomedical Division)
163 ity that the limited penetration of the antibody could affect the interpretation of unlabelled elements, ultrathin sections were taken only from the first 4/zm from the block surface (see Fig. lb). Some sections were processed by omitting the primary antiserum of the staining sequence after which specific staining did not occur.
Fig. 1. Glu-positive neurons and puncta in area 17. In a (layers II-III) numerous pyramidal neurons are visible while punctate immunoreactive profiles are partially masked by the brownish background of osmicated material. In the semi-thin section (b), Glu-positivepuncta are well evident. The seeming non-homogeneity of Glu-positive puncta staining is due to the limited penetration of the antibody and to the inclination of the semi-thin section with regard to the tissue block surface. The zone characterized by more numerous puncta (asterisk) extends for about 4gin deep from the surface; labelled cells can be seen up to 8 #m deep from the surface (arrows) where few or no labelled puncta are visible. Scale bars: 50/++m.
In osmicated vibratome sections numerous immunoreactive neurons were observed in all cortical layers except layer I of both SI and area 17. Glu-positive neurons were more numerous in layers II, III, V and VI and were mostly pyramidal (Fig. la); some labelled non-pyramidal neurons were also identified, particularly in layer IV. Numerous lightly stained punctate profiles were observed in all cortical layers (Fig. la). In 2-/~m-thick plastic sections Glu-positive puncta were clearly visible (Fig. lb) and were mostly found in the neuropil. Only occasional puncta were observed around unlabelled neuronal somata. Electron microscopic examination of 60- to 70-nm-thick sections showed that Glu-positive puncta were immunoreactive axon terminals (Fig. 2a) or small dendritic processes (Fig. 2b). All synaptic contacts formed by Glu-positive axon terminals were of the asymmetric type (Fig. 2a). As predicted by light microscopic examination, the vast majority of postsynaptic targets of Glu-positive axon terminals were elements of the neuropil, identified as small and medium caliber dendritic shafts and dendritic spines (Fig. 2a). Most of these postsynaptic targets were unlabelled. A few Glu-positive synaptic terminals contacted unlabelled cell bodies. Within the zone of penetration of the antibody not all asymmetric contacts were labelled (Fig. 2b). The morphology of unlabelled axon terminals resembled that of adjacent immunoreactive terminals. In all ultrathin sections examined no axon terminals forming symmetric contacts were labelled by the antibody. The present results show that the antiGlu serum used in this study selectively stains a fraction of asymmetric synapses in the cerebral cortex and provide additional evidence for the assumption that the Glu immunoreactivity observed with this antibody in the cerebral cortex is likely to reflect the neurotransmitter pool of Glu 4. This finding gives strong experimental support to the classical correlation between morphology and function of cortical synapses 3 (see refs. 7, 31 for a similar correlation in hippocampal and cerebellar axon terminals). In our material all Glu-positive synapses were of the asymmetric type, i.e. the type considered excitatory, and since Glu is known to exert excitatory effects on cortical neurons el, Glu-positive synapses are presumably Glu-ergic excitatory synapses. The lack of Glu-positive symmetric synapses is in line with previous immuno-
164
Fig. 2. Electron micrographs of Glu-immunoreactive profiles after resectioning of the semi-thin section shown in Fig. lb. a: Glu-positive axon terminal (T) making an asymmetriccontact with an unlabelled dendrite, b: unlabelled axon terminal (A) forming an asymmetric contact with a small immunoreactive dendritic profile. Scale bars: 0.3 pm. cytochemical observations on the cerebral cortex which showed that most symmetric contacts are GABAergic ~3'14'ls'2s and, therefore, inhibitory. The clear difference between the present results and those on GABAergic terminals indicates that immunocytochemical methods can selectively distinguish at the electron microscopic level neuronal populations using synaptic transmitters with closely related metabolisms 24. Glu is involved in G A B A synthesis and breakdown but the relatively low concentration of Glu in GABAergic synapses 26 may account for the lack of staining of symmetric contacts. GABAergic synapses have been observed characteristically around the somata and proximal dendrites of pyramidal neurons and in the neuropil)3'1a'28, They are formed by terminals of non-spiny non-pyramidal neurons ~4aT.2s. Glu-positive synapses were mostly distributed in the neuropil and only occasionally around unlabelled cell bodies. Their origin has not yet been determined. The Glu-positive terminals observed in different layers of SI and area 17 may originate from different sources. The most probable are cortical pyramidal neurons. Axons of pyramidal cells form asymmetrical contacts with dendritic shafts and spines 19,25,33and pyramidal neurons represent the most common type of Glu-positive neurons
in the cerebral cortex 4,6. Another probable source of Glu-positive synapses are layer IV spiny stellate neurons which are considered excitatory 1~, form asymmetric contacts 22,3° and probably are the non-pyramidal neurons labelled by the anti-Glu serum in this layer a. The hypothesis that some Glu-positive axon terminals originate in thalamocortical projecting neurons cannot be ruled out -'9. Unlabelled terminals forming asymn:~tric synapses were consistently seen adjacent to Glu-positive asymmetric contacts. Although failure of staining cannot be completely excluded because of methodological limitations, unlabelled terminals are likely to contain a transmitter other than Glu. These terminals could originate in both cortical and subcortical neurons which can use as excitatory neurotransmitters several compounds, such as aspartate 1,5.23, Nacetylaspartylglutamate32, acetylcholine m'~6 and monoamines 927. We are grateful to Drs. A. Rustioni and E.G. Jones for critical reading of the manuscript, to Miss S. Modena for editorial assistance and to Miss C. Ferri for typing. This work was supported by funds from Regione Marche (Grant 031110), C.N.R., C.S.I.C. (Grant 603/070) and F.I.S. (Grant 87/1700).
165 1 Campistron, G., Buijs, R.M. and Geffard, M., Specific antibodies against aspartate and their immunocytochemical application in the rat brain, Brain Research, 365 (1986) 179-184. 2 Colonnier, M,, Synaptic patterns on different cell types in the different laminae of the cat visual cortex. An electron microscope study, Brain Research, 9 (1968) 268-287. 3 Colonnier, M., The electron-microscopic analysis of the neuronal organization of the cerebral cortex. In F.O. Schmitt, F.G. Worden and S.G. Dennis (Eds.), The Organization of the Cerebral Cortex, MIT, Cambridge, 1981, pp. 125-152. 4 Conti, F., Rustioni, A., Petrusz, P. and Towle, A.C., Glutamate-positive neurons in the somatic sensory cortex of rats and monkeys, J. Neurosci,, 7 (1987) 1887-1901. 5 Conti, F., Rustioni, A. and Petrusz, P., Co-localization of glutamate and aspartate immunoreactivity in neurons of the rat somatic sensory cortex. In T.P. Hicks, D. Lodge and H. McLennan (Eds.), Excitatory Amino Acid Transmission, Liss, New York, 1987, pp. 169-172. 6 Conti, F., Fabri, M. and Manzoni, T., Glutamate-positive cortico-cortical neurons in the somatic sensory areas I and If of cats, J. Neurosci., in press. 7 Cotman, C.W., Monaghan, D.T., Ottersen, O.P. and Storm-Mathisen, J., Anatomical organization of excitatory amino acid receptors and their pathways, Trends Neurosci., 10 (1987) 273-280. 8 DeFelipe, J. and Fairen, A., A type of basket cell in superficial layers of the cat visual cortex: a Golgi-electron microscope study, Brain Research, 244 (1982) 9-16. 9 DeFelipe, J. and Jones, E.G., A light and electron microscopic study of serotonin-immunoreactive fibers and terminals in the monkey sensory-motor cortex, Exp, Brain Res., in press. 10 De Lima, A.D. and Singer, W., Cholinergic innervation of the cat striate cortex: a choline acetyl transferase immunocytochemical analysis, J. Comp. Neurol., 250 (1986) 324-338. 11 Gilbert, C.D., Microcircuity of the visual cortex, Annu. Rev. Neurosci., 6 (1983)217-247. 12 Gray, E.G., Axo-somatic and axo-dendritic synapses of the cerebral cortex: an electron microscopic study, J. Anat., 93 (1959) 420-433. 13 Hendrickson, A.E., Hunt, S.P. and Wu, J.Y., Immunocytochemical localization of glutamic acid decarboxylase in monkey striate cortex, Nature (Lond.), 292 (1981) 605-607. 14 Hendry, S.H.C., Houser, C.R., Jones, E.G. and Vaughn, J.E., Synaptic organization of immunocytochemically identified G A B A neurons in the monkey sensory-motor cortex, J. Neurocytol., 12 (1983) 639-660. 15 Hepler, J.R., Toomin, C., McCarthy, K.D., Conti, F., Battaglia, G., Rustioni, A. and Petrusz, P., Characterization of antisera to glutamate and aspartate, J. Histochem. Cytochem., 36 (1988) 13-22. 16 Houser, C.R., Crawford, G.D., Salvaterra, P.M. and Vaughn, J.E., Immunocytochemical localization of choline acetyltransferase in rat cerebral cortex: a study of cholinergic neurons and synapses, J. Comp. Neurol., 234 (1985) 17-34.
17 Houser, C.R., Vaughn, J.E., Hendry, S.H.C., Jones, E.G. and Peters, A., G A B A neurons in the cerebral cortex. In E.G. Jones and A. Peters (Eds.), Cerebral Cortex, Vol. 2, Functional Properties of Cortical Cells, Plenum, New York, 1984, pp. 63-89. 18 Jones, E.G., Neurotransmitters in the cerebral cortex, J. Neurosurg., 65 (1986) 135-153. 19 Kisvarday, Z.F., Martin, K.A.C., Freund, T.F., Magl6czky, Zs, Whitteridge, D. and Somogy, P., Synaptic targets of HRP-filled layer Ill pyramidal cells in the cat striate cortex, Exp. Brain Res., 64 (1986) 541-552. 20 Krnj6vic, K., Chemical nature of synaptic transmission in vertebrates, Physiol. Rev., 54 (1974) 318-450. 21 Krnj6vic, K., Iontophoretic studies of neurons in the mammalian cerebral cortex, J. Physiol. (Lond.), 165 (1963) 274-304. 22 Le Vay, S., Synaptic patterns in the visual cortex of the cat and monkey. Electron microscopy of Golgi preparations, J. Comp. Neurol., 150 (1973) 53-86. 23 Madl, J.E., Beitz, A.J., Johnson, R.L. and Larson, A.A., Monoclonal antibodies specific for fixative-modified aspartare: immunocytochemical localization in the rat CNS, J. Neurosci., 7 (1987) 2639-2650. 24 McGeer, P.L., Eccles, J.C. and McGeer, E., Molecular Neurobiology of the Mammalian Brain, 2nd edn., Plenum, New York, 1987, 744 pp. 25 McGuire, B.A., Hornung, J.P., Gilbert, C.D. and Wiesel, T.N., Patterns of synaptic input to layer 4 of cat striate cortex, J. Neurosci., 4 (1984) 3021-3033. 26 Ottersen, O.P. and Storm-Mathisen, J., Glutamate and GABA-containing neurons in the mouse and rat brain, as demonstrated with a new immunocytochemical technique, J. Comp. Neurol., 229 (1984) 374-392. 27 Papadopoulos, G.C., Parnavelas, J.C. and Buijs, R., Monoaminergic fibers form conventional synapses in the cerebral cortex, Neurosci. Lett., 76 (1987) 275-279. 28 Ribak, C.E., Aspinous and sparsely-spinous stellate neurons in the visual cortex of rats contain glutamic acid decarboxylase, J. Neurocytol., 7 (1978) 461-478. 29 Rustioni, A., Battaglia, G., De Biasi, S. and Giuffrida, R., Neuromediators in somatosensory thalamus: an immunocytochemical overview. In M. Bentivoglio and R. Spreafico (Eds.), Cellular Thalamic Mechanisms, Elsevier, in press. 30 Saint Marie, R.L. and Peters, A., The morphology and synaptic connections of spiny stellate neurons in monkey visual cortex (Area 17). A Golgi-electron microscopic study, J. Comp. Neurol., 233 (1985) 213-235. 31 Somogy, P., Halasy, H., Somogy, J., Storm-Mathisen, J. and Ottersen, O.P., Quantification of immunogold labelling reveals enrichment of glutamate in mossy and parallel fibre terminals in cat cerebellum, Neuroscience, 19 (1986) 1045-1050. 32 Tieman, S.B., Cangro, C.B. and Neale, J.H., N-Acetylaspartylglutamate immunoreactivity in neurons of cat's visual system, Brain Research, 420 (1987) 188-193. 33 Wienfield, D.A., Brooke, R.N.L., Sloper, J.J. and Powell, T.P.S., A combined Golgi-electron microscopic study of the synapses made by the proximal axon and recurrent collaterals of a pyramidal cell in the somatic sensory cortex of the monkey, Neuroscience, 6 (1981) 1217-1230.
Brain Research, 455 (1988) 162-165 Elsevier
BRE 22979
Demonstration of glutamate-positive axon terminals forming asymmetric synapses in cat neocortex Javier DeFelipe 2, Fiorenzo Conti 1, Susan L. Van Eyck 3 and Tullio Manzoni 1 tlnstitute of Human Physiology, University of Ancona, Ancona (Italy), 2lnstituto Cajal, CS1C, Madrid (Spain) and 3Department of Anatomy and Curriculum in Neurobiology, University of North Carolina, Chapel Hill, NC27514 (U.S.A.)
(Accepted 29 March 1988) Key words: Glutamate; Asymmetric synapse; Somatic sensory cortex; Visual cortex; Cat; Immunocytochemistry
Electron microscopic examination of sections immunocytochemically processed with an anti-glutamate serum reveals that many asymmetric synapses in the cat neocortex contain elevated levels of immunodetectabte glutamate. These labelled axon terminals are likely to use glutamate as neurotransmitter. Axon terminals forming symmetric contacts were never labelled. Since glutamate is known to exert potent excitatory effects on neocortical neurons, the present finding gives immunocytochemical evidence that asymmetric synapses are excitatory. y-Aminobutyric acid ( G A B A ) and glutamate (Giu) thought to be the major inhibitory and excitatory neurotransmitters, respectively, of the cerebral cortex 2°'21. Two major morphological types of synapses are present in the neocortex: one is characterized by a prominent postsynaptic density (asymmetric synapses) and the other by a very thin postsynaptic density (symmetric synapses) 2'12. Axon terminals forming synapses of the first type contain round vescicles and are supposed to be excitatory, while those forming synapses of the second type have flattened vescicles and are probably inhibitory 3. With the introduction of immunocytochemical techniques this hypothesis was supported by the observation that only axon terminals forming symmetric synapses contain the synthesizing enzyme for G A B A 14'28. Recent observations 4'6 indicate that numerous pyramidal neurons known to form asymmetric synapses 25'33 are immunoreactive for an antiGlu serum 15 in the neocortex. Some Glu-positive dot-like elements, presumably axon terminals, were also observed 4. However, conclusive evidence that Glu is contained in axon terminals forming asymmetric synapses has not been provided. In the present study correlative light and electron microscopic immunocytochemical
are
methods were employed to demonstrate that a particular population of asymmetric synapses in the cerebral cortex contains high levels of immunodetectable Glu. Experiments were carried out on the first somatic sensory (SI) and primary visual (area 17) areas of adult cats. Under deep barbiturate anaesthesia (Nembutal; 33 mg/kg, i.p.) animals were perfused transcardially with saline followed by a fixative solution containing 4% carbodiimide, 0.2% picric acid and 0.2% glutaraldehyde. Selected blocks from SI and from area 17 were post-fixed in 4% paraformaldehyde for 24-48 h and cut into 50-~m-thick sections with a Vibratome. The primary antibody had been previously characterized ~5 and does not cross-react with aspartate and G A B A 4'15. The immunocytochemical procedure and controls used in this study have already been published 4. Processed sections were osmicated (1% in 0.1 M phosphate buffer for 1 h), dehydrated in ethanol and then flat-embedded in thin sheets of Araldite resin. Sections were then cut with an ultramicrotome at 2/~m, examined at light microscope, photographed, then resectioned serially8 at 65-70 nm, and finally examined with a Jeol100B electron microscope. To overcome the possibil-
Correspondence: F. Conti, Istituto di Fisiologia Umana, Universit~ di Ancona, Via Ranieri-Monte d'Ago, 1-60131Ancona, ItaLy.
0006-8993/88/$03.50 (~ 1988 Elsevier Science Publishers B.V. (Biomedical Division)
163 ity that the limited penetration of the antibody could affect the interpretation of unlabelled elements, ultrathin sections were taken only from the first 4/zm from the block surface (see Fig. lb). Some sections were processed by omitting the primary antiserum of the staining sequence after which specific staining did not occur.
Fig. 1. Glu-positive neurons and puncta in area 17. In a (layers II-III) numerous pyramidal neurons are visible while punctate immunoreactive profiles are partially masked by the brownish background of osmicated material. In the semi-thin section (b), Glu-positivepuncta are well evident. The seeming non-homogeneity of Glu-positive puncta staining is due to the limited penetration of the antibody and to the inclination of the semi-thin section with regard to the tissue block surface. The zone characterized by more numerous puncta (asterisk) extends for about 4gin deep from the surface; labelled cells can be seen up to 8 #m deep from the surface (arrows) where few or no labelled puncta are visible. Scale bars: 50/++m.
In osmicated vibratome sections numerous immunoreactive neurons were observed in all cortical layers except layer I of both SI and area 17. Glu-positive neurons were more numerous in layers II, III, V and VI and were mostly pyramidal (Fig. la); some labelled non-pyramidal neurons were also identified, particularly in layer IV. Numerous lightly stained punctate profiles were observed in all cortical layers (Fig. la). In 2-/~m-thick plastic sections Glu-positive puncta were clearly visible (Fig. lb) and were mostly found in the neuropil. Only occasional puncta were observed around unlabelled neuronal somata. Electron microscopic examination of 60- to 70-nm-thick sections showed that Glu-positive puncta were immunoreactive axon terminals (Fig. 2a) or small dendritic processes (Fig. 2b). All synaptic contacts formed by Glu-positive axon terminals were of the asymmetric type (Fig. 2a). As predicted by light microscopic examination, the vast majority of postsynaptic targets of Glu-positive axon terminals were elements of the neuropil, identified as small and medium caliber dendritic shafts and dendritic spines (Fig. 2a). Most of these postsynaptic targets were unlabelled. A few Glu-positive synaptic terminals contacted unlabelled cell bodies. Within the zone of penetration of the antibody not all asymmetric contacts were labelled (Fig. 2b). The morphology of unlabelled axon terminals resembled that of adjacent immunoreactive terminals. In all ultrathin sections examined no axon terminals forming symmetric contacts were labelled by the antibody. The present results show that the antiGlu serum used in this study selectively stains a fraction of asymmetric synapses in the cerebral cortex and provide additional evidence for the assumption that the Glu immunoreactivity observed with this antibody in the cerebral cortex is likely to reflect the neurotransmitter pool of Glu 4. This finding gives strong experimental support to the classical correlation between morphology and function of cortical synapses 3 (see refs. 7, 31 for a similar correlation in hippocampal and cerebellar axon terminals). In our material all Glu-positive synapses were of the asymmetric type, i.e. the type considered excitatory, and since Glu is known to exert excitatory effects on cortical neurons el, Glu-positive synapses are presumably Glu-ergic excitatory synapses. The lack of Glu-positive symmetric synapses is in line with previous immuno-
164
Fig. 2. Electron micrographs of Glu-immunoreactive profiles after resectioning of the semi-thin section shown in Fig. lb. a: Glu-positive axon terminal (T) making an asymmetriccontact with an unlabelled dendrite, b: unlabelled axon terminal (A) forming an asymmetric contact with a small immunoreactive dendritic profile. Scale bars: 0.3 pm. cytochemical observations on the cerebral cortex which showed that most symmetric contacts are GABAergic ~3'14'ls'2s and, therefore, inhibitory. The clear difference between the present results and those on GABAergic terminals indicates that immunocytochemical methods can selectively distinguish at the electron microscopic level neuronal populations using synaptic transmitters with closely related metabolisms 24. Glu is involved in G A B A synthesis and breakdown but the relatively low concentration of Glu in GABAergic synapses 26 may account for the lack of staining of symmetric contacts. GABAergic synapses have been observed characteristically around the somata and proximal dendrites of pyramidal neurons and in the neuropil)3'1a'28, They are formed by terminals of non-spiny non-pyramidal neurons ~4aT.2s. Glu-positive synapses were mostly distributed in the neuropil and only occasionally around unlabelled cell bodies. Their origin has not yet been determined. The Glu-positive terminals observed in different layers of SI and area 17 may originate from different sources. The most probable are cortical pyramidal neurons. Axons of pyramidal cells form asymmetrical contacts with dendritic shafts and spines 19,25,33and pyramidal neurons represent the most common type of Glu-positive neurons
in the cerebral cortex 4,6. Another probable source of Glu-positive synapses are layer IV spiny stellate neurons which are considered excitatory 1~, form asymmetric contacts 22,3° and probably are the non-pyramidal neurons labelled by the anti-Glu serum in this layer a. The hypothesis that some Glu-positive axon terminals originate in thalamocortical projecting neurons cannot be ruled out -'9. Unlabelled terminals forming asymn:~tric synapses were consistently seen adjacent to Glu-positive asymmetric contacts. Although failure of staining cannot be completely excluded because of methodological limitations, unlabelled terminals are likely to contain a transmitter other than Glu. These terminals could originate in both cortical and subcortical neurons which can use as excitatory neurotransmitters several compounds, such as aspartate 1,5.23, Nacetylaspartylglutamate32, acetylcholine m'~6 and monoamines 927. We are grateful to Drs. A. Rustioni and E.G. Jones for critical reading of the manuscript, to Miss S. Modena for editorial assistance and to Miss C. Ferri for typing. This work was supported by funds from Regione Marche (Grant 031110), C.N.R., C.S.I.C. (Grant 603/070) and F.I.S. (Grant 87/1700).
165 1 Campistron, G., Buijs, R.M. and Geffard, M., Specific antibodies against aspartate and their immunocytochemical application in the rat brain, Brain Research, 365 (1986) 179-184. 2 Colonnier, M,, Synaptic patterns on different cell types in the different laminae of the cat visual cortex. An electron microscope study, Brain Research, 9 (1968) 268-287. 3 Colonnier, M., The electron-microscopic analysis of the neuronal organization of the cerebral cortex. In F.O. Schmitt, F.G. Worden and S.G. Dennis (Eds.), The Organization of the Cerebral Cortex, MIT, Cambridge, 1981, pp. 125-152. 4 Conti, F., Rustioni, A., Petrusz, P. and Towle, A.C., Glutamate-positive neurons in the somatic sensory cortex of rats and monkeys, J. Neurosci,, 7 (1987) 1887-1901. 5 Conti, F., Rustioni, A. and Petrusz, P., Co-localization of glutamate and aspartate immunoreactivity in neurons of the rat somatic sensory cortex. In T.P. Hicks, D. Lodge and H. McLennan (Eds.), Excitatory Amino Acid Transmission, Liss, New York, 1987, pp. 169-172. 6 Conti, F., Fabri, M. and Manzoni, T., Glutamate-positive cortico-cortical neurons in the somatic sensory areas I and If of cats, J. Neurosci., in press. 7 Cotman, C.W., Monaghan, D.T., Ottersen, O.P. and Storm-Mathisen, J., Anatomical organization of excitatory amino acid receptors and their pathways, Trends Neurosci., 10 (1987) 273-280. 8 DeFelipe, J. and Fairen, A., A type of basket cell in superficial layers of the cat visual cortex: a Golgi-electron microscope study, Brain Research, 244 (1982) 9-16. 9 DeFelipe, J. and Jones, E.G., A light and electron microscopic study of serotonin-immunoreactive fibers and terminals in the monkey sensory-motor cortex, Exp, Brain Res., in press. 10 De Lima, A.D. and Singer, W., Cholinergic innervation of the cat striate cortex: a choline acetyl transferase immunocytochemical analysis, J. Comp. Neurol., 250 (1986) 324-338. 11 Gilbert, C.D., Microcircuity of the visual cortex, Annu. Rev. Neurosci., 6 (1983)217-247. 12 Gray, E.G., Axo-somatic and axo-dendritic synapses of the cerebral cortex: an electron microscopic study, J. Anat., 93 (1959) 420-433. 13 Hendrickson, A.E., Hunt, S.P. and Wu, J.Y., Immunocytochemical localization of glutamic acid decarboxylase in monkey striate cortex, Nature (Lond.), 292 (1981) 605-607. 14 Hendry, S.H.C., Houser, C.R., Jones, E.G. and Vaughn, J.E., Synaptic organization of immunocytochemically identified G A B A neurons in the monkey sensory-motor cortex, J. Neurocytol., 12 (1983) 639-660. 15 Hepler, J.R., Toomin, C., McCarthy, K.D., Conti, F., Battaglia, G., Rustioni, A. and Petrusz, P., Characterization of antisera to glutamate and aspartate, J. Histochem. Cytochem., 36 (1988) 13-22. 16 Houser, C.R., Crawford, G.D., Salvaterra, P.M. and Vaughn, J.E., Immunocytochemical localization of choline acetyltransferase in rat cerebral cortex: a study of cholinergic neurons and synapses, J. Comp. Neurol., 234 (1985) 17-34.
17 Houser, C.R., Vaughn, J.E., Hendry, S.H.C., Jones, E.G. and Peters, A., G A B A neurons in the cerebral cortex. In E.G. Jones and A. Peters (Eds.), Cerebral Cortex, Vol. 2, Functional Properties of Cortical Cells, Plenum, New York, 1984, pp. 63-89. 18 Jones, E.G., Neurotransmitters in the cerebral cortex, J. Neurosurg., 65 (1986) 135-153. 19 Kisvarday, Z.F., Martin, K.A.C., Freund, T.F., Magl6czky, Zs, Whitteridge, D. and Somogy, P., Synaptic targets of HRP-filled layer Ill pyramidal cells in the cat striate cortex, Exp. Brain Res., 64 (1986) 541-552. 20 Krnj6vic, K., Chemical nature of synaptic transmission in vertebrates, Physiol. Rev., 54 (1974) 318-450. 21 Krnj6vic, K., Iontophoretic studies of neurons in the mammalian cerebral cortex, J. Physiol. (Lond.), 165 (1963) 274-304. 22 Le Vay, S., Synaptic patterns in the visual cortex of the cat and monkey. Electron microscopy of Golgi preparations, J. Comp. Neurol., 150 (1973) 53-86. 23 Madl, J.E., Beitz, A.J., Johnson, R.L. and Larson, A.A., Monoclonal antibodies specific for fixative-modified aspartare: immunocytochemical localization in the rat CNS, J. Neurosci., 7 (1987) 2639-2650. 24 McGeer, P.L., Eccles, J.C. and McGeer, E., Molecular Neurobiology of the Mammalian Brain, 2nd edn., Plenum, New York, 1987, 744 pp. 25 McGuire, B.A., Hornung, J.P., Gilbert, C.D. and Wiesel, T.N., Patterns of synaptic input to layer 4 of cat striate cortex, J. Neurosci., 4 (1984) 3021-3033. 26 Ottersen, O.P. and Storm-Mathisen, J., Glutamate and GABA-containing neurons in the mouse and rat brain, as demonstrated with a new immunocytochemical technique, J. Comp. Neurol., 229 (1984) 374-392. 27 Papadopoulos, G.C., Parnavelas, J.C. and Buijs, R., Monoaminergic fibers form conventional synapses in the cerebral cortex, Neurosci. Lett., 76 (1987) 275-279. 28 Ribak, C.E., Aspinous and sparsely-spinous stellate neurons in the visual cortex of rats contain glutamic acid decarboxylase, J. Neurocytol., 7 (1978) 461-478. 29 Rustioni, A., Battaglia, G., De Biasi, S. and Giuffrida, R., Neuromediators in somatosensory thalamus: an immunocytochemical overview. In M. Bentivoglio and R. Spreafico (Eds.), Cellular Thalamic Mechanisms, Elsevier, in press. 30 Saint Marie, R.L. and Peters, A., The morphology and synaptic connections of spiny stellate neurons in monkey visual cortex (Area 17). A Golgi-electron microscopic study, J. Comp. Neurol., 233 (1985) 213-235. 31 Somogy, P., Halasy, H., Somogy, J., Storm-Mathisen, J. and Ottersen, O.P., Quantification of immunogold labelling reveals enrichment of glutamate in mossy and parallel fibre terminals in cat cerebellum, Neuroscience, 19 (1986) 1045-1050. 32 Tieman, S.B., Cangro, C.B. and Neale, J.H., N-Acetylaspartylglutamate immunoreactivity in neurons of cat's visual system, Brain Research, 420 (1987) 188-193. 33 Wienfield, D.A., Brooke, R.N.L., Sloper, J.J. and Powell, T.P.S., A combined Golgi-electron microscopic study of the synapses made by the proximal axon and recurrent collaterals of a pyramidal cell in the somatic sensory cortex of the monkey, Neuroscience, 6 (1981) 1217-1230.