132
Brain Research, 122 (1977) 132 136 ?~ Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands
Axonal transport of pH]adenosine in visual and somatosensory pathways
LAWRENCE KRUGER and SAMUEL SAPORTA Department of Anatomy and Brain Research Institute, U.C.L.A. Center for Health Sciences, Los Angeles, Cali]~ 90024 (U.S.A.)
(Accepted November 8th, 1976)
A variety of labeled substances have been used in an attempt to trace the axonal trajectory of neurons to their terminations 1,4,6. Some evidence has also been adduced demonstrating transsynaptic transport of certain tritiated amino acids, peptides, monosaccharides and nucleosides 1,3,7-9, although it is not yet known whether labeled macromolecules manufactured from these precursors or some specific labeled breakdown products cross the synapse to the next neuron 5,7,8. The search for transneuronal transfer of substances whose release might be affected by synaptic activity and whose uptake may reflect metabolic alterations in the postsynaptic neuron led Schubert and Kreutzberg 7,8 to explore the transport of nucleosides because of their incorporation in RNA and DNA, and their metabolic roles as coenzymes and precursors for cyclic nucleotide production. Their experiments showed that tritiated uridine or adenosine injected into the rabbit visual cortex led to intense labeling of neuropil in a sector of the ipsilateral lateral geniculate nucleus, the thalamic nucleus known to be reciprocally related to the cortical injection site. In addition, a greater quantity of label was observed over the perikaryon of some neurons which was interpreted as probably due to transneuronal rather than retrograde transport. This interpretation was supported by similar observations in other thalamocortical systems 1°,11 and in the labeling of neuron perikarya in the cortex homotopically related to the injection site. However, as with the original observations, these neurons are known to be reciprocally connected to the injection site, thus failing to eliminate the possibility of retrograde transport. The apparent absence of silver grains in neuropil accompanied by pronounced perikaryal label in neurons known to project via the corpus callosum s led to the suspicion that retrograde transport of [3H]adenosine is possible 1°. In order to critically test whether postsynaptic transfer of an adenosine labeled substance can occur it seemed logical to study this possibility in sensory pathways where only anterograde transport might be expected. We selected two models for this purpose: the projection of the retina to the lateral geniculate nucleus and of the trigeminal ganglion to the medullary sensory trigeminal nuclear complex. In the first experiment, 50 #1 of [3H]adenosine ([2,8-all]adenosine, 21 Ci/mmole) in a concentration of 6 #Ci//A was injected into the posterior ocular chamber of an
133 adult monkey (Macaca fuscata) through a 25-gauge needle attached to a 50 #1 Hamilton syringe. The animal was perfused 36 h later with a mixture of 1 ~ paraformaldehyde and 1.25 ~ glutaraldehyde. The brain was blocked and immersed in 4 ~ paraformaldehyde. Paraffin sections cut a 7/~m were prepared for autoradiography according to the method of Cowan et al. 1 and were exposed for 6 weeks. The lateral geniculate nucleus reveals intense labeling in the appropriate laminae; a fairly uniform high grain density throughout layers 2, 3 and 5 of the ipsilateral nucleus is shown in Fig. 1. The distribution suggests a diffuse labeling of neuropil probably reflecting accumulation located in axon terminals. At higher magnification with either bright- or dark-field illumination (Figs. 2 and 3), there is no evidence of neuron perikaryal accumulation suggestive of transneuronal transport. Retrograde labeling would not be expected since there are no cells in the lateral geniculate body known to project upon the retina. The second test was to inject 3/~1 (50 pCi/ml) of the same specific activity of [all]adenosine used in the above experiment, into the Gasserian ganglion of 5 cats. The injection site was determined by inserting a 15-gauge spinal needle stereotaxically into Meckel's cave. Removal of the stylet reveals a slow flow of cerebrospinal fluid. An insulated steel wire microelectrode with a large tip of 20-50 #m calibrated to extend a specific length past the lumen of the spinal needle was inserted and adjusted to reveal multi-unit activity driven from receptive fields in the second division of the trigeminal nerve. The electrode was then removed and the needle of a 10 #1 microsyringe inserted through the needle cannula so that the tip of the needle projected the same distance past the lumen of the spinal needle as the electrode. The animals were sacrificed at 1-7 days and the tissue was processed in the same manner as noted above. The injection site revealed intense neuron perikaryon concentration of silver grains with widespread diffuse label throughout the ganglion. Transported nucleosides (or its derivates) were apparent throughout the ipsilateral sensory trigeminal nuclear complex with highest density in the principal nucleus and oralis portion of the spinal V nucleus. These experiments demonstrate the extensive, dense distribution of trigeminal afferents in the brain stem (Fig. 4), but maximum neuron perikaryal aggregation of silver grains (Fig. 5) does not appear to be more concentrated than in background neuropil. The apparent absence of transneuronal transport is presumably not related to survival period since the present experiments cover the range of previous experiments in which neuron perikarya were clearly labeled. It is conceivable that larger concentrations of labeled material would reveal postsynaptic label, as shown in the lateral geniculate only after multiple injection of 3 mCi of tritiated proline and fucose 9 but such quantities are at least in an order of magnitude greater than required for neuronal label with [3H]adenosineT,S,10,11. The possibility of retrograde transport of label derived from [3H]adenosine has been considered in some detail by Wise and Jones10,11 who noted that the sites of neuronal labeling also project to the cortical injection site, with two exceptions: the thalamic reticular complex and a few pontine neurons were labeled after cortical injections. The minimal number of grains in neuropil with pronounced label of
| s
135 neurons in these two sites might be most parsimoniously explained as retrograde transport, t h o u g h evidence for a direct pontocortical or thalamic reticulocortical projection is doubtful. The same argument is applicable to cell label o f the opposite h o m o t o p i c cortex where pyramidal neurons are heavily labeled in the absence o f extensive neuropil grains, as shown by Schubert and Kreutzberg 8. If any o f these examples constitute transsynaptic transport, there presumably should be significant signs o f presynaptic label. Further support for the existence of retrograde transport is given in an a d d e n d u m footnote by Wise and Jones 1° reporting retrograde label in chicken o c u l o m o t o r nucleus neurons after [3H]adenosine injection into the extraocular muscles. The present experiments reveal an apparent absence of transneuronal adenosinederived label in two sensory pathways over a range o f survival times that were useful for displaying neuron perikaryal label in thalamic and cortical neurons. It is possible that there is selectivity in those pathways where transneuronal label can be found. Alternatively, it might be argued that the evidence for synaptic transfer o f label is open to question and that neuron labeling with the adenosine axonal transport method m a y be largely or solely attributed to retrograde transport. The latter alternative m a y seem disappointing for the application o f this new technique in some neural systems, but it also m a y open a fruitful means of studying both anterograde and retrograde transport with a single method. Supported by Grants NB-5685 and EY-571 and a fellowship MH57934 (S.S.) from the National Institute of Health. We are indebted to Ms. Sharon S a m p o g n a and Ms. Jennifer Willies for expert histological assistance, and to Dr. Peter Schubert, whose suggestions enabled us to initiate these studies.
Fig. 1. Autoradiograph of a coronal section through the lateral geniculate body of a macaque in which the ipsilateral eye was injected with [3H]adenosine 36 h earlier. Heavy label can be seen throughout the neuropil of layers 2, 3 and 5 in the lateral geniculate on this side. Discontinuities in the labeled layers are blood vessels. Dark-field. × 55. Fig. 2. Higher magnification of a portion of layer 5 indicated in Fig. 1 by arrows for alignment illustrating the relatively diffuse distribution of silver grains with few clusters suggestive of neuron perikaryal label. Dark-field. × 112. Fig. 3. Higher magnification autoradiograph from the same sector of lateral geniculate, layer 5 shown in Fig. 2, but with bright-field illumination it is evident that neuronal label is not more pronounced than is neuropil. × 1040. Fig. 4. Autoradiograph of a horizontal section through the spinal trigeminal nucleus, pars caudalis of the cat. The ipsilateral Gasserian ganglion was injected with [3Hladenosine 7 days prior to sacrifice. The neuropil throughout the sensory trigeminal complex is heavily labeled except for the sparsely labeled layer (substantia gelatinosa) underlying the marginal zone. Note that the spinal tract of V (t) is less intensely labeled than the neuropil. Dark-field. × 180. Fig. 5. Higher magnification of the densely labeled medial sector (nucleus proprius) of the spinal V nucleus shown in Fig. 4. With bright-field illumination it is evident that despite some labeling of neurons, the grains are broadly dispersed and fail to display a pattern of distinct perikaryal label. × 640.
136 Note added in proof. Two recent brief reports offer additional evidence for retrograde axonal t r a n s p o r t of adenosine, as well as evidence for transfer in both directions. ( H u n t , S. P. and Ktinzle, H., Bidirectional m o v e m e n t of label and t r a n s n e u r o n a l transport p h e n o m e n a after injection of [3H]adenosine into the central nervous system, Brain Research, 112 (1976) 127-132. Wise, S. P., Retrograde axonal t r a n s p o r t of aden osine derivatives in the central nervous system, Neurosci. Abstr., 2 (1976) 43.)
1 Cowan, W. M., Gottlieb, D. K., Hendrickson, A. E., Price, J. L. and Woolsey, T. A., The autcradiographic demonstration of axonal connections in the central nervous system, Brain Research, 37 (1972) 21-51. 2 Droz, B., Koenig, H. L. and Di Giamberardino, L., Axonal migration of protein in nerve endings. 1. Radioautographic analysis of the renewal of protein in nerve endings of chicken ciliary ganglion after intracerebral injection of [3H]lysine, Brain Research, 60 (1973) 93-127. 3 Grafstein, B., Transneuronal transfer of radioactivity in the central nervous system, Science, 172 (1971) 177-179. 4 Hendrickson, A. E., Electron microscopic distribution of axoplasmic transport, J. comp. Neurol., 144 (1972) 381-398. 5 lngoglia, N. A., Grafstein, B., McEwen, B. S. and McQuorrie, I. G., Axonal transport of radioactivity in the goldfish optic system following intraocular injection of labeled RNA precursors, J. Neurochem., 20 (1973) 1605-1615. 6 Lasek, R. J., Protein transport in neurons, Int. Rev. Neurobiol., 13 (1970) 289-324. 7 Schubert, P. and Kreutzberg, G. W., Axonal transport of adenosine and uridine derivatives and transfer to postsynaptic neurons, Brain Research, 76 (1974) 525-530. 8 Schubert, P. and Kreutzberg, G. W., [3H]Adenosine, a tracer for neuronal connectivity, Brain Research, 85 (1975) 317-320. 9 Wiesel, T. N., Hubel, D. H. and Lam, D., Autoradiographic demonstration of ocular dominance columns in the monkey striate cortex by means of transsynaptic transport, Brain Research, 79 (1974) 273-279. 10 Wise, S. P. and Jones, E. G., Transneuronal or retrograde transport of [all]adenosine in the rat somatic sensory system, Brain Research, 107 (1976) 127 131. 11 Wise, S. P. and Jones, E. G., The organization and postnatal development of the commissural projection of the rat somatic sensory cortex, Brain Research, 168 (1976) 313-344.
Brain Research, 122 (1977) 132 136 ?~ Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands
Axonal transport of pH]adenosine in visual and somatosensory pathways
LAWRENCE KRUGER and SAMUEL SAPORTA Department of Anatomy and Brain Research Institute, U.C.L.A. Center for Health Sciences, Los Angeles, Cali]~ 90024 (U.S.A.)
(Accepted November 8th, 1976)
A variety of labeled substances have been used in an attempt to trace the axonal trajectory of neurons to their terminations 1,4,6. Some evidence has also been adduced demonstrating transsynaptic transport of certain tritiated amino acids, peptides, monosaccharides and nucleosides 1,3,7-9, although it is not yet known whether labeled macromolecules manufactured from these precursors or some specific labeled breakdown products cross the synapse to the next neuron 5,7,8. The search for transneuronal transfer of substances whose release might be affected by synaptic activity and whose uptake may reflect metabolic alterations in the postsynaptic neuron led Schubert and Kreutzberg 7,8 to explore the transport of nucleosides because of their incorporation in RNA and DNA, and their metabolic roles as coenzymes and precursors for cyclic nucleotide production. Their experiments showed that tritiated uridine or adenosine injected into the rabbit visual cortex led to intense labeling of neuropil in a sector of the ipsilateral lateral geniculate nucleus, the thalamic nucleus known to be reciprocally related to the cortical injection site. In addition, a greater quantity of label was observed over the perikaryon of some neurons which was interpreted as probably due to transneuronal rather than retrograde transport. This interpretation was supported by similar observations in other thalamocortical systems 1°,11 and in the labeling of neuron perikarya in the cortex homotopically related to the injection site. However, as with the original observations, these neurons are known to be reciprocally connected to the injection site, thus failing to eliminate the possibility of retrograde transport. The apparent absence of silver grains in neuropil accompanied by pronounced perikaryal label in neurons known to project via the corpus callosum s led to the suspicion that retrograde transport of [3H]adenosine is possible 1°. In order to critically test whether postsynaptic transfer of an adenosine labeled substance can occur it seemed logical to study this possibility in sensory pathways where only anterograde transport might be expected. We selected two models for this purpose: the projection of the retina to the lateral geniculate nucleus and of the trigeminal ganglion to the medullary sensory trigeminal nuclear complex. In the first experiment, 50 #1 of [3H]adenosine ([2,8-all]adenosine, 21 Ci/mmole) in a concentration of 6 #Ci//A was injected into the posterior ocular chamber of an
133 adult monkey (Macaca fuscata) through a 25-gauge needle attached to a 50 #1 Hamilton syringe. The animal was perfused 36 h later with a mixture of 1 ~ paraformaldehyde and 1.25 ~ glutaraldehyde. The brain was blocked and immersed in 4 ~ paraformaldehyde. Paraffin sections cut a 7/~m were prepared for autoradiography according to the method of Cowan et al. 1 and were exposed for 6 weeks. The lateral geniculate nucleus reveals intense labeling in the appropriate laminae; a fairly uniform high grain density throughout layers 2, 3 and 5 of the ipsilateral nucleus is shown in Fig. 1. The distribution suggests a diffuse labeling of neuropil probably reflecting accumulation located in axon terminals. At higher magnification with either bright- or dark-field illumination (Figs. 2 and 3), there is no evidence of neuron perikaryal accumulation suggestive of transneuronal transport. Retrograde labeling would not be expected since there are no cells in the lateral geniculate body known to project upon the retina. The second test was to inject 3/~1 (50 pCi/ml) of the same specific activity of [all]adenosine used in the above experiment, into the Gasserian ganglion of 5 cats. The injection site was determined by inserting a 15-gauge spinal needle stereotaxically into Meckel's cave. Removal of the stylet reveals a slow flow of cerebrospinal fluid. An insulated steel wire microelectrode with a large tip of 20-50 #m calibrated to extend a specific length past the lumen of the spinal needle was inserted and adjusted to reveal multi-unit activity driven from receptive fields in the second division of the trigeminal nerve. The electrode was then removed and the needle of a 10 #1 microsyringe inserted through the needle cannula so that the tip of the needle projected the same distance past the lumen of the spinal needle as the electrode. The animals were sacrificed at 1-7 days and the tissue was processed in the same manner as noted above. The injection site revealed intense neuron perikaryon concentration of silver grains with widespread diffuse label throughout the ganglion. Transported nucleosides (or its derivates) were apparent throughout the ipsilateral sensory trigeminal nuclear complex with highest density in the principal nucleus and oralis portion of the spinal V nucleus. These experiments demonstrate the extensive, dense distribution of trigeminal afferents in the brain stem (Fig. 4), but maximum neuron perikaryal aggregation of silver grains (Fig. 5) does not appear to be more concentrated than in background neuropil. The apparent absence of transneuronal transport is presumably not related to survival period since the present experiments cover the range of previous experiments in which neuron perikarya were clearly labeled. It is conceivable that larger concentrations of labeled material would reveal postsynaptic label, as shown in the lateral geniculate only after multiple injection of 3 mCi of tritiated proline and fucose 9 but such quantities are at least in an order of magnitude greater than required for neuronal label with [3H]adenosineT,S,10,11. The possibility of retrograde transport of label derived from [3H]adenosine has been considered in some detail by Wise and Jones10,11 who noted that the sites of neuronal labeling also project to the cortical injection site, with two exceptions: the thalamic reticular complex and a few pontine neurons were labeled after cortical injections. The minimal number of grains in neuropil with pronounced label of
| s
135 neurons in these two sites might be most parsimoniously explained as retrograde transport, t h o u g h evidence for a direct pontocortical or thalamic reticulocortical projection is doubtful. The same argument is applicable to cell label o f the opposite h o m o t o p i c cortex where pyramidal neurons are heavily labeled in the absence o f extensive neuropil grains, as shown by Schubert and Kreutzberg 8. If any o f these examples constitute transsynaptic transport, there presumably should be significant signs o f presynaptic label. Further support for the existence of retrograde transport is given in an a d d e n d u m footnote by Wise and Jones 1° reporting retrograde label in chicken o c u l o m o t o r nucleus neurons after [3H]adenosine injection into the extraocular muscles. The present experiments reveal an apparent absence of transneuronal adenosinederived label in two sensory pathways over a range o f survival times that were useful for displaying neuron perikaryal label in thalamic and cortical neurons. It is possible that there is selectivity in those pathways where transneuronal label can be found. Alternatively, it might be argued that the evidence for synaptic transfer o f label is open to question and that neuron labeling with the adenosine axonal transport method m a y be largely or solely attributed to retrograde transport. The latter alternative m a y seem disappointing for the application o f this new technique in some neural systems, but it also m a y open a fruitful means of studying both anterograde and retrograde transport with a single method. Supported by Grants NB-5685 and EY-571 and a fellowship MH57934 (S.S.) from the National Institute of Health. We are indebted to Ms. Sharon S a m p o g n a and Ms. Jennifer Willies for expert histological assistance, and to Dr. Peter Schubert, whose suggestions enabled us to initiate these studies.
Fig. 1. Autoradiograph of a coronal section through the lateral geniculate body of a macaque in which the ipsilateral eye was injected with [3H]adenosine 36 h earlier. Heavy label can be seen throughout the neuropil of layers 2, 3 and 5 in the lateral geniculate on this side. Discontinuities in the labeled layers are blood vessels. Dark-field. × 55. Fig. 2. Higher magnification of a portion of layer 5 indicated in Fig. 1 by arrows for alignment illustrating the relatively diffuse distribution of silver grains with few clusters suggestive of neuron perikaryal label. Dark-field. × 112. Fig. 3. Higher magnification autoradiograph from the same sector of lateral geniculate, layer 5 shown in Fig. 2, but with bright-field illumination it is evident that neuronal label is not more pronounced than is neuropil. × 1040. Fig. 4. Autoradiograph of a horizontal section through the spinal trigeminal nucleus, pars caudalis of the cat. The ipsilateral Gasserian ganglion was injected with [3Hladenosine 7 days prior to sacrifice. The neuropil throughout the sensory trigeminal complex is heavily labeled except for the sparsely labeled layer (substantia gelatinosa) underlying the marginal zone. Note that the spinal tract of V (t) is less intensely labeled than the neuropil. Dark-field. × 180. Fig. 5. Higher magnification of the densely labeled medial sector (nucleus proprius) of the spinal V nucleus shown in Fig. 4. With bright-field illumination it is evident that despite some labeling of neurons, the grains are broadly dispersed and fail to display a pattern of distinct perikaryal label. × 640.
136 Note added in proof. Two recent brief reports offer additional evidence for retrograde axonal t r a n s p o r t of adenosine, as well as evidence for transfer in both directions. ( H u n t , S. P. and Ktinzle, H., Bidirectional m o v e m e n t of label and t r a n s n e u r o n a l transport p h e n o m e n a after injection of [3H]adenosine into the central nervous system, Brain Research, 112 (1976) 127-132. Wise, S. P., Retrograde axonal t r a n s p o r t of aden osine derivatives in the central nervous system, Neurosci. Abstr., 2 (1976) 43.)
1 Cowan, W. M., Gottlieb, D. K., Hendrickson, A. E., Price, J. L. and Woolsey, T. A., The autcradiographic demonstration of axonal connections in the central nervous system, Brain Research, 37 (1972) 21-51. 2 Droz, B., Koenig, H. L. and Di Giamberardino, L., Axonal migration of protein in nerve endings. 1. Radioautographic analysis of the renewal of protein in nerve endings of chicken ciliary ganglion after intracerebral injection of [3H]lysine, Brain Research, 60 (1973) 93-127. 3 Grafstein, B., Transneuronal transfer of radioactivity in the central nervous system, Science, 172 (1971) 177-179. 4 Hendrickson, A. E., Electron microscopic distribution of axoplasmic transport, J. comp. Neurol., 144 (1972) 381-398. 5 lngoglia, N. A., Grafstein, B., McEwen, B. S. and McQuorrie, I. G., Axonal transport of radioactivity in the goldfish optic system following intraocular injection of labeled RNA precursors, J. Neurochem., 20 (1973) 1605-1615. 6 Lasek, R. J., Protein transport in neurons, Int. Rev. Neurobiol., 13 (1970) 289-324. 7 Schubert, P. and Kreutzberg, G. W., Axonal transport of adenosine and uridine derivatives and transfer to postsynaptic neurons, Brain Research, 76 (1974) 525-530. 8 Schubert, P. and Kreutzberg, G. W., [3H]Adenosine, a tracer for neuronal connectivity, Brain Research, 85 (1975) 317-320. 9 Wiesel, T. N., Hubel, D. H. and Lam, D., Autoradiographic demonstration of ocular dominance columns in the monkey striate cortex by means of transsynaptic transport, Brain Research, 79 (1974) 273-279. 10 Wise, S. P. and Jones, E. G., Transneuronal or retrograde transport of [all]adenosine in the rat somatic sensory system, Brain Research, 107 (1976) 127 131. 11 Wise, S. P. and Jones, E. G., The organization and postnatal development of the commissural projection of the rat somatic sensory cortex, Brain Research, 168 (1976) 313-344.
