- Email: [email protected]
High affinity uptake of D-aspartate in the barrel subfield of the mouse somatic sensory cortex
Brain Research, 201 (1980) 427-430
427
© Elsevier/North-Holland Biomedical Press
High affinity uptake of D-aspartate in the barrel subfield of the mouse somatic sensory cortex
ARVID J. SOREIDE and FRODE FONNUM Norwegian Defence Research Establishment, Division for Toxtcology, N-2007 Kjeller (Norway)
(Accepted July 10th, 1980) Key words: barrel subfield - - mouse sensory cortex - - high affinity uptake D-aspartate
The neurons of layer IV of the mouse first somatic sensory cortex (SmI) are arranged in a remarkably constant pattern of multicellular cytoarchitectonic units called barrels 24. Each barrel is known to be related to a single whisker vibrissa in a oneto-one manner16,18. The barrels consist of 'sides' of densely packed small nerve cell bodies and 'hollows' with relatively few perikarya 24. During the last years, much work has been carried out to define the structure and function of these barrels (for references see: 5, 9, 11, 12, 23). In the present paper autoradiography was used to demonstrate the high affinity uptake of D-[aH]aspartate (D-Asp) in the barrel subfield of the mouse SmI. o-Asp is concentrated in nerve terminals by the same high affinity uptake mechanism as Lglutamate and L-Asp. This has been demonstrated both kmeticallyl, 2 and by lesion studies6,14. Several studies on different neo-and allo-corticofugal projections all show that this high affinity uptake mechanism is a valuable marker for glutamergic or aspartergic nerve terminalsa,14,17. For incubation over longer periods, the metabolically inert D-Asp is preferred to L-glutamate and L-Asp which would be metabolized. Altogether 4 adult albino mice were used in this study. The animals were decapitated and the brains were quickly removed. The tissue was chilled in ice-cold Krebs phosphate buffer (pH 7.40) and cut into slices of 400/zm on a Sorvall TC-2 tissue chopper. A total of 16 slices containing different parts of the cerebral cortex were selected and incubated for 20 min at room temperature in 0.5 ml of freshly made oxygenated Krebs phosphate buffer (pH 7.40) containing 2/~M of D-[2,3-aH]Asp (specific activity 16.8 Ci/mmol). After the incubation, the slices were rinsed several times in Krebs buffer, fixed in 2.5 ~o glutaraldehyde, postfixed in 3 . 5 ~ formalin, dehydrated and embedded in paraffin. Serial sections of 5 #m were cut from the paraffin embedded slices. Every tenth section was mounted on microscopic slides and prepared for autoradiography using Kodak NTB2 and standard dipping procedures. The slides were kept in light-tight boxes at 4 °C for 7-28 days and were then developed in Kodak D19. The autoradiograms were not stained, but relevant sections from the same series were stained with Luxol fast blue and cresyl violet for light microscopy.
428
Fig. 1. Unstained autoradlogram of the mouse SmI cerebral cortex after incubation with 2/~M of t)-13H]Asp
Fig. 1 shows an autoradlogram from the mouse Sml after incubation with D-[3H] Asp. A laminar pattern was readily recogmzed in the autoradlogram and the pattern could be correlated to the individual cortical layers in the stained sections. The highest uptake was seen in layer I. Since the dark labehng was restricted to the precise thickness of the layer, we do not believe that it represents a diffusion artefact. The subsequent layers II and Ill were relatively pale and could not be distinguished from one another in the autoradlograms. Layer IV is characterized by a large number of relatively small neurons. In the cell-stained sections of SmI, aggregates of small nerve cells were found at regular intervals extending from the deep part of layer IV towards the surface m agreement with the location and appearance of the barrel sides described by Woolsey and van der Loos 24. In the autoradiograms there were darkly labeled stripes corresponding to the cell aggregates separated by lighter areas corresponding to the barrel hollows (Fig. 1). Thus, the barrel sides showed a high uptake of D-Asp whale the uptake was low m the barrel hollows. The barrel hollows seemed to have the lowest uptake in the Sml cortex. High power light microscopy indicated that the silver grams were not localized to cell bodies or to axons in the septum, but presumably to synaptlc terminals. Layers V and VI were relatively heavily labeled. The superficial part of layer V was darker than the surroundings and stood out as a marked hne. The border between layers V and VI could be identified m stained sections by the presence of large pyramidal
429 cells in layer V. Although layer VI was slightly darker than layer V, there was no marked difference between the two layers in the autoradiograms. Taken together the present study confirms a previous report that neocortex has a high uptake o f Glu/Asp a. It also shows that this uptake is mainly localized to layers I, V and VI. There are m a n y candidates a m o n g efferent fibers which could, in part, be responsible for the staining pattern. The most important efferent fibres are the thalamocortical fibers which mainly end in layers I, IV (hollows), V 22 and the commissural fibers in layers I (deep part), I I I and V (superficial part) 21,22. Also, the small excitatory intracortlcal neurons, which have recently been reviewed by Szenfftgothai 15, are candidates. Further autoradiographlc studies after specific lesions m a y discriminate between these candidates. It should be stressed, however, that m a n y o f the corticofugal fibres are t h o u g h t to be either glutamerglc or asparterglc 3,4, and that recurrent collaterals from these fibers probably also contrlbute to the high uptake in the deep cortical layers. The localization o f D-Asp uptake in the barrels o f layers IV is o f special interest since this cortical region has been examined in some detail by others. The uptake was low in the barrel hollows which receive the specific thalamocortical fibers 7,1°,1a,2°. In contrast to the barrel hollows, the barrel sides had a h~gh uptake o f D-Asp. The barrel sides contain small neurons, large dendrites, synaptic terminals and axonsT,lO, la. The large dendrites are t h o u g h t to belong to pyramidal cells o f layer V 19, while the axon and the terminals originate f r o m cortical neurons outside SmI 5. Studies by others have shown a low activity o f A C h E 8 and catecholamines 9 in this region, and the present findings indicate that m a n y o f the terminals m a y be glutamergic or aspartergic. In conclusion the pattern o f high affinity uptake o f D-Asp, a marker for glutamergic/ aspartergic nerve terminals, c o n f o r m to the barrel pattern described in layer IV o f somatosensory cortex.
1 Balcar, V. J. and Johnston, G. A. R., The structural specificity of the high affinity uptake of Lglutamate and L-aspartate by rat brain shces, J. Neurochem, 19 (1972) 2657-2666. 2 Dawes, L. P. and Johnston, G. A. R., Uptake and release ofo- and L-aspartate by rat brain slices, J. Neurochem., 26 (1976) 1007-1014. 3 Fonnum, F., Storm-Mathlsen, J and D~vac, I., Biochemical ewdence for glutamate as neurotransmltter in cort~co-strmtal and corUco-thalamic fibres in rat brain, Neuroscience (submitted). 4 Fonnum, F., Karlsen, R. L, Malthe-Sorenssen, D., Skrede, K. K. and Walaas, I., Locahzation of neurotransmitters, particularly glutamate, in h~ppocampus, septum, nucleus accumbens and superior colliculus, Progr. Bram Res., 51 (1979) 167-191. 5 Hmnchsen, C. F. L. and Stevens, G. E., Free structure of cortical barrels in the mouse, Aust. J. Zool., 25 (1977) 1-7. 6 Karlsen, R. L. and Fonnum, F., Evidence for glutamate as a neurotransmitter in the corticofugal fibres to the dorsal lateral gemculate body, Brain Research, 151 (1978) 457~,67. 7 Killackey, H. P. and Leshin, D., The organization of specific thalamocort~cal projections to the posteromedml barrel subfield of the rat somatic sensory cortex, Brain Research, 86 (1975) 469472. 8 Kristt, D A.~Deve~pment~fne~c~rtica~c~rcujtry:h~st~chem~ca~cahzat~n~facety~ch~hnesterasem relation to the cell layers of rat somato sensory cortex, J comp Neurol., 186 (1979) 1-16. 9 Lldov, H. G. W., Rice, F. L. and Molhver, M. E., The orgamzatlon of catecholamme mnervation of somatosensory cortex: the barrel field of the mouse, Brain Research, 153 (1978) 577-584 10 Petrovick3~, P and Druga, R , Pecuharities of the cytoarchitectonlcs and some afferent systems of the parietal cortex, Folia Morph. (Warszawa), 20 (1972) 161-163
430 11 Slmons, D. J., Response properties of vlbrissa units in rat SI somatosensory neocortex, J Neurophysiol., 41 (1978) 798-820. 12 Simons, D. J. and Woolsey, T. A., Functional orgamzatlon of mouse barrel cortex, Brain Research, 165 (1979) 327-332. 13 Steffen, H., Golgl-stamed barrel neurons m the somato-sensory region of the mouse cerebral cortex, Neurosci Lett., 2 (1976) 57-59. 14 Storm-Mathlsen, J. and Woxen Opsahl, M., Aspartate and/or glutamate may be transmitters in hippocampal efferents to septum and hypothalamus, Neurosci Lett., 9 (1978) 65-70. 15 Szent~gothai, J., The neuron network of the cerebral cortex: a functional interpretation, Proc. roy. Soc. B, 201 (1978) 219-248. 16 Van der Loos, H and Woolsey, T. A., Somatosensory cortex" structural alterations following early injury to sense organs, Science, 179 (1973) 395-398. 17 Walaas, I. and Fonnum, F., Blochem~cal evidence for glutamate as a transmitter m hJppocampal efferents to the basal forebrain and hypothalamus m the rat brain, Neuroscience, (submitted) 1980. 18 Welker, C., Microelectrode delineation of fine gram somatotoplc organtzation of SmI cerebral neocortex in albino rat, Brain Reseat oh, 26 (1971) 259-275 19 White, E. L., Ultrastructure and synaptzc contacts in barrels of mouse SI COltex, Brain Research, 105 (1976) 229-251. 20 White, E. L , Identified neurons of mouse SmI cortex which are postsynaptic to thaIamocort~cal axon terminals: a combined Golgi-electron microscopic and degeneration study, J comp Neurol, 181 (1978) 627-662. 21 Wise, S. P. and Jones, E. G., The orgamzat~on and the postnatal development of the comm~ssural pro lectlon of the somatic sensory cortex of the rat, J. comp. Neurol., 168 (1976) 313-343. 22 Wise, S. P. and Jones, E. G., Developmental studies of thalamocortlcal and commissural connect~ons in the rat somatic sensory cortex, J. eomp. Neurol., 178 (1978) 187-208. 23 Woolsey, T. A., Dierker, M L. and Wann, D. F., SmI cortex: quahtative and quantitative classification of Golgi-impregnated barrel neurons, Proc. nat Acad. Sci, ( W a s h ) , 72 (1975) 2165-2169. 24 Woolsey, T. A. and van der Loos, H., The structural orgamzation of layer IV in the somatic sensory region of mouse cerebral cortex: the description of a cortical field composed of discrete cytoarchJtectomc umts, Bram Resealch, 17 (1970) 205 242.
427
© Elsevier/North-Holland Biomedical Press
High affinity uptake of D-aspartate in the barrel subfield of the mouse somatic sensory cortex
ARVID J. SOREIDE and FRODE FONNUM Norwegian Defence Research Establishment, Division for Toxtcology, N-2007 Kjeller (Norway)
(Accepted July 10th, 1980) Key words: barrel subfield - - mouse sensory cortex - - high affinity uptake D-aspartate
The neurons of layer IV of the mouse first somatic sensory cortex (SmI) are arranged in a remarkably constant pattern of multicellular cytoarchitectonic units called barrels 24. Each barrel is known to be related to a single whisker vibrissa in a oneto-one manner16,18. The barrels consist of 'sides' of densely packed small nerve cell bodies and 'hollows' with relatively few perikarya 24. During the last years, much work has been carried out to define the structure and function of these barrels (for references see: 5, 9, 11, 12, 23). In the present paper autoradiography was used to demonstrate the high affinity uptake of D-[aH]aspartate (D-Asp) in the barrel subfield of the mouse SmI. o-Asp is concentrated in nerve terminals by the same high affinity uptake mechanism as Lglutamate and L-Asp. This has been demonstrated both kmeticallyl, 2 and by lesion studies6,14. Several studies on different neo-and allo-corticofugal projections all show that this high affinity uptake mechanism is a valuable marker for glutamergic or aspartergic nerve terminalsa,14,17. For incubation over longer periods, the metabolically inert D-Asp is preferred to L-glutamate and L-Asp which would be metabolized. Altogether 4 adult albino mice were used in this study. The animals were decapitated and the brains were quickly removed. The tissue was chilled in ice-cold Krebs phosphate buffer (pH 7.40) and cut into slices of 400/zm on a Sorvall TC-2 tissue chopper. A total of 16 slices containing different parts of the cerebral cortex were selected and incubated for 20 min at room temperature in 0.5 ml of freshly made oxygenated Krebs phosphate buffer (pH 7.40) containing 2/~M of D-[2,3-aH]Asp (specific activity 16.8 Ci/mmol). After the incubation, the slices were rinsed several times in Krebs buffer, fixed in 2.5 ~o glutaraldehyde, postfixed in 3 . 5 ~ formalin, dehydrated and embedded in paraffin. Serial sections of 5 #m were cut from the paraffin embedded slices. Every tenth section was mounted on microscopic slides and prepared for autoradiography using Kodak NTB2 and standard dipping procedures. The slides were kept in light-tight boxes at 4 °C for 7-28 days and were then developed in Kodak D19. The autoradiograms were not stained, but relevant sections from the same series were stained with Luxol fast blue and cresyl violet for light microscopy.
428
Fig. 1. Unstained autoradlogram of the mouse SmI cerebral cortex after incubation with 2/~M of t)-13H]Asp
Fig. 1 shows an autoradlogram from the mouse Sml after incubation with D-[3H] Asp. A laminar pattern was readily recogmzed in the autoradlogram and the pattern could be correlated to the individual cortical layers in the stained sections. The highest uptake was seen in layer I. Since the dark labehng was restricted to the precise thickness of the layer, we do not believe that it represents a diffusion artefact. The subsequent layers II and Ill were relatively pale and could not be distinguished from one another in the autoradlograms. Layer IV is characterized by a large number of relatively small neurons. In the cell-stained sections of SmI, aggregates of small nerve cells were found at regular intervals extending from the deep part of layer IV towards the surface m agreement with the location and appearance of the barrel sides described by Woolsey and van der Loos 24. In the autoradiograms there were darkly labeled stripes corresponding to the cell aggregates separated by lighter areas corresponding to the barrel hollows (Fig. 1). Thus, the barrel sides showed a high uptake of D-Asp whale the uptake was low m the barrel hollows. The barrel hollows seemed to have the lowest uptake in the Sml cortex. High power light microscopy indicated that the silver grams were not localized to cell bodies or to axons in the septum, but presumably to synaptlc terminals. Layers V and VI were relatively heavily labeled. The superficial part of layer V was darker than the surroundings and stood out as a marked hne. The border between layers V and VI could be identified m stained sections by the presence of large pyramidal
429 cells in layer V. Although layer VI was slightly darker than layer V, there was no marked difference between the two layers in the autoradiograms. Taken together the present study confirms a previous report that neocortex has a high uptake o f Glu/Asp a. It also shows that this uptake is mainly localized to layers I, V and VI. There are m a n y candidates a m o n g efferent fibers which could, in part, be responsible for the staining pattern. The most important efferent fibres are the thalamocortical fibers which mainly end in layers I, IV (hollows), V 22 and the commissural fibers in layers I (deep part), I I I and V (superficial part) 21,22. Also, the small excitatory intracortlcal neurons, which have recently been reviewed by Szenfftgothai 15, are candidates. Further autoradiographlc studies after specific lesions m a y discriminate between these candidates. It should be stressed, however, that m a n y o f the corticofugal fibres are t h o u g h t to be either glutamerglc or asparterglc 3,4, and that recurrent collaterals from these fibers probably also contrlbute to the high uptake in the deep cortical layers. The localization o f D-Asp uptake in the barrels o f layers IV is o f special interest since this cortical region has been examined in some detail by others. The uptake was low in the barrel hollows which receive the specific thalamocortical fibers 7,1°,1a,2°. In contrast to the barrel hollows, the barrel sides had a h~gh uptake o f D-Asp. The barrel sides contain small neurons, large dendrites, synaptic terminals and axonsT,lO, la. The large dendrites are t h o u g h t to belong to pyramidal cells o f layer V 19, while the axon and the terminals originate f r o m cortical neurons outside SmI 5. Studies by others have shown a low activity o f A C h E 8 and catecholamines 9 in this region, and the present findings indicate that m a n y o f the terminals m a y be glutamergic or aspartergic. In conclusion the pattern o f high affinity uptake o f D-Asp, a marker for glutamergic/ aspartergic nerve terminals, c o n f o r m to the barrel pattern described in layer IV o f somatosensory cortex.
1 Balcar, V. J. and Johnston, G. A. R., The structural specificity of the high affinity uptake of Lglutamate and L-aspartate by rat brain shces, J. Neurochem, 19 (1972) 2657-2666. 2 Dawes, L. P. and Johnston, G. A. R., Uptake and release ofo- and L-aspartate by rat brain slices, J. Neurochem., 26 (1976) 1007-1014. 3 Fonnum, F., Storm-Mathlsen, J and D~vac, I., Biochemical ewdence for glutamate as neurotransmltter in cort~co-strmtal and corUco-thalamic fibres in rat brain, Neuroscience (submitted). 4 Fonnum, F., Karlsen, R. L, Malthe-Sorenssen, D., Skrede, K. K. and Walaas, I., Locahzation of neurotransmitters, particularly glutamate, in h~ppocampus, septum, nucleus accumbens and superior colliculus, Progr. Bram Res., 51 (1979) 167-191. 5 Hmnchsen, C. F. L. and Stevens, G. E., Free structure of cortical barrels in the mouse, Aust. J. Zool., 25 (1977) 1-7. 6 Karlsen, R. L. and Fonnum, F., Evidence for glutamate as a neurotransmitter in the corticofugal fibres to the dorsal lateral gemculate body, Brain Research, 151 (1978) 457~,67. 7 Killackey, H. P. and Leshin, D., The organization of specific thalamocort~cal projections to the posteromedml barrel subfield of the rat somatic sensory cortex, Brain Research, 86 (1975) 469472. 8 Kristt, D A.~Deve~pment~fne~c~rtica~c~rcujtry:h~st~chem~ca~cahzat~n~facety~ch~hnesterasem relation to the cell layers of rat somato sensory cortex, J comp Neurol., 186 (1979) 1-16. 9 Lldov, H. G. W., Rice, F. L. and Molhver, M. E., The orgamzatlon of catecholamme mnervation of somatosensory cortex: the barrel field of the mouse, Brain Research, 153 (1978) 577-584 10 Petrovick3~, P and Druga, R , Pecuharities of the cytoarchitectonlcs and some afferent systems of the parietal cortex, Folia Morph. (Warszawa), 20 (1972) 161-163
430 11 Slmons, D. J., Response properties of vlbrissa units in rat SI somatosensory neocortex, J Neurophysiol., 41 (1978) 798-820. 12 Simons, D. J. and Woolsey, T. A., Functional orgamzatlon of mouse barrel cortex, Brain Research, 165 (1979) 327-332. 13 Steffen, H., Golgl-stamed barrel neurons m the somato-sensory region of the mouse cerebral cortex, Neurosci Lett., 2 (1976) 57-59. 14 Storm-Mathlsen, J. and Woxen Opsahl, M., Aspartate and/or glutamate may be transmitters in hippocampal efferents to septum and hypothalamus, Neurosci Lett., 9 (1978) 65-70. 15 Szent~gothai, J., The neuron network of the cerebral cortex: a functional interpretation, Proc. roy. Soc. B, 201 (1978) 219-248. 16 Van der Loos, H and Woolsey, T. A., Somatosensory cortex" structural alterations following early injury to sense organs, Science, 179 (1973) 395-398. 17 Walaas, I. and Fonnum, F., Blochem~cal evidence for glutamate as a transmitter m hJppocampal efferents to the basal forebrain and hypothalamus m the rat brain, Neuroscience, (submitted) 1980. 18 Welker, C., Microelectrode delineation of fine gram somatotoplc organtzation of SmI cerebral neocortex in albino rat, Brain Reseat oh, 26 (1971) 259-275 19 White, E. L., Ultrastructure and synaptzc contacts in barrels of mouse SI COltex, Brain Research, 105 (1976) 229-251. 20 White, E. L , Identified neurons of mouse SmI cortex which are postsynaptic to thaIamocort~cal axon terminals: a combined Golgi-electron microscopic and degeneration study, J comp Neurol, 181 (1978) 627-662. 21 Wise, S. P. and Jones, E. G., The orgamzat~on and the postnatal development of the comm~ssural pro lectlon of the somatic sensory cortex of the rat, J. comp. Neurol., 168 (1976) 313-343. 22 Wise, S. P. and Jones, E. G., Developmental studies of thalamocortlcal and commissural connect~ons in the rat somatic sensory cortex, J. eomp. Neurol., 178 (1978) 187-208. 23 Woolsey, T. A., Dierker, M L. and Wann, D. F., SmI cortex: quahtative and quantitative classification of Golgi-impregnated barrel neurons, Proc. nat Acad. Sci, ( W a s h ) , 72 (1975) 2165-2169. 24 Woolsey, T. A. and van der Loos, H., The structural orgamzation of layer IV in the somatic sensory region of mouse cerebral cortex: the description of a cortical field composed of discrete cytoarchJtectomc umts, Bram Resealch, 17 (1970) 205 242.