Brain Research, 85 (1975) 249-253 C~ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
249
POSSIBLE DETERMINANTS OF THE DEGREE OF RETROGRADE NEURONAL LABELING WITH HORSERADISH PEROXIDASE
E. G. JONES
Department of Anatomy, Washington University School of Medicine, St. Louis, Mo. 63110 (U.S.A.)
The introduction of techniques which utilize the anterograde and retrograde axonal flow of proteins for tracing neuronal pathwaysl, 7-9 has permitted certain new observations on the relationships of various types of thalamic cell to the cerebral cortex. In the course of these investigations certain methodological considerations, especially relating to the retrograde horseradish peroxidase technique have arisen. Localized injections of type VI (Sigma) horseradish peroxidase (0.1-0.3 #l in a concentration of 500 #g/#l) made in single cortical areas of rats, cats and monkeys lead to intense, retrograde labeling of many cells in the topographically related part of the appropriate thalamic relay nucleus and to relatively much lighter labeling of a few cells in the intralaminar complex of nuclei4,5,11. The differential intensity of labeling, which is reflected in the size and number of horseradish peroxidase positive granules found in the cell soma and proximal dendrites (Figs. 1 and 2), is maintained irrespective of the survival time and of the amount of horseradish peroxidase injected. Cells of a single intralaminar nucleus can be lightly labeled from injections in quite widely separated areas of the cerebral cortex in the frontal, parietal and cingulate regions 5. On the other hand, multiple injections of horseradish peroxidase in the postcentral gyrus, though appearing to coalesce, still lead to labeling of thalamic relay cells in bands, each band corresponding to an injection site and with intervening bands of unlabeled cells4,5,7,11. At the center of each band some cells, in addition to containing horseradish peroxidase positive granules, show a diffuse brown staining of the perikaryal and dendritic membranes (Fig. 3) and it is thought that the axons of these cells may have been damaged by the injection needle. Complementary experiments conducted with the anterograde, autoradiographic technique 3, suggest that the degree of concentration of horseradish peroxidase transported in a retrograde manner to the thalamic cells is dependent upon the density of the terminal axonal field available for incorporation of the enzyme at the injection site. Injections of tritiated proline and/or leucine involving the intralaminar nuclei of cats 6, and after survival periods (24-36 h) in which labeled material transported in the rapid phase of axoplasmic transport is used as the marker, demonstrate a small, diffuse field widely scattered across the frontal, parietal and medial cortex and with the autoradiographic grains especially concentrated in the superficial part of layer
t'~
251 I (Fig. 4). This contrasts with the intense terminal axonal field, localized to specific cortical areas and concentrated in layers IV and IIIb, demonstrable 8,6 after injections of tritiated amino acids in the main thalamic relay nuclei (Fig. 5). As the number of silver grains observed at these short survival periods is probably related in some way to the number and size of the axon terminals and possibly to the number o f preterminal axon branches present, then these factors are probably the main determinants of a cell's capacity to accumulate horseradish peroxidase in the retrograde manner. This proposal is reinforced by the fact that it is possible to label the intralaminar cells heavily following injections o f horseradish peroxidase in the caudate nucleus and putamenS, 10. Here the retrograde labeling is always intense and many cells are labeled, contrasting with the situation after injections o f the cortex. This is probably determined by the density of the terminal axonal field of the intralaminar cell's axons in the caudate nucleus and putamen as indicated by the intensity o f terminal labeling in these nuclei following injections of tritiated amino acids in the intralaminar nuclei (Fig. 6). Other studies currently in progress demonstrate that thalamic cells that are undergoing retrograde shrinkage as the result of ablation of one cortical area may still be labeled by horseradish peroxidase transported in a retrograde manner from an injection site in another cortical area. Six months after ablation of the first somatic sensory area (SI) in cats, the ventrobasal complex undergoes severe retrograde degeneration, characterized by shrinkage of nerve cells and severe gliosis (Fig. 7). But most o f the remaining shrunken neurons can be labeled in a retrograde manner following injections of horseradish peroxidase in the intact second somatic sensory area (SII) 24-48 h prior to killing the animal (Figs. 7 and 8). These neurons are much smaller than those which are labeled after injections of horseradish peroxidase in animals in which both SI and SII are intact (Fig. 8). Although there are a number of possible explanations as to why the neurons projecting to SII should shrink following destruction of SI, one likely interpretation is that they normally send axon branches to both cortical areas. Supported by G r a n t number 5RO1 NS10526 from the National Institutes of Health, U.S. Public Health Service. I am indebted to Ms. Bertha McClure for tech-
Figs. 1 and 2. Photomicrographs showing differential retrograde labeling of cells in an intralaminar nucleus (Fig. 1) and in a relay nucleus (Fig. 2) following an injection of horseradish peroxidase in the cerebral cortex of a squirrel monkey. Thionin counterstain, x 500. Fig. 3. Diffuse retrograde labeling with horseradish peroxidase (see text). Thionin counterstain, x 500. Fig. 4. Bright-field photomicrograph of an autoradiograph showing a small but definite concentration of silver grains lying in the most superficial portion of layer I of the somatic sensory cortex of a cat following an injection of tritiated proline 5 days previously in the caudal intralaminar nuclei. Thionin counterstain, x 800. Fig. 5. Dark-field photomicrograph of an autoradiograph showing the dense concentration of silver grains in layers IIIb and IV of the somatic sensory cortex of a squirrel monkey following an injection of tritiated proline 5 days previously in the ventrobasal complex, x 600.
252
Fig. 6. Dark-held photomicrograph of an autoradiograph showing a portion of the internal capsule (IC) and the intense aggregation of silver grains in the caudate nucleus (right) in the same brain from which Fig. 4 was taken. 700. Fig. 7. Photomicrograph showing shrunken neurons labeled in the intensely gliotic ventrobasal complex (see inset) of a cat, 6 months alter ablation of the ipsilateral SI cortex and 36 h after injection of horseradish peroxidase in the ipsilateral SII cortex. Thionin counterstain. 230, inset, 20.
253
40'
401
30'
30-
SI
20.
~ 20-
Normal
,~i '". . . . .
E 14S ~ 10"
10'
0
160
320
480
I
640
Cross-sectional
160 area
of
cell
soma
|
320
480
W
| 640
(p2)
Fig. 8. Graphs showing the size-distribution of cells in the ipsilateral ventrobasal complex labeled by horseradish peroxidase (stipple) following an injection of SII, compared with the size-distribution of cells in the opposite ventrobasal complex (hatching). On the left, where SII was injected 6 months after ablation of the ipsilateral SI, the labeled cells are all greatly shrunken. On the right, where SI1 was injected in the presence of an intact SI, the labeled cells fall within the normal size range. Cell areas measured using a computer program described by Cowan and Wann 2.
nical assistance. This paper was presented at the meeting on 'The Use of Axonal Transport for Studies of Neuronal Connectivity' held at Gwatt-Thun, Switzerland, July 2-4, 1974. 1 COWAN, W. M., GOTTLIEB, D. I., HENDRICKSON,A. I., PRICE, J. L., AND WOOLSEY, T. A., The autoradiographic demonstration of axonal connections in the central nervous system, Brain Research, 37 (1972) 21-51. 2 COWAN,W. M., AND WANN, D. F., A computer system for the measurement of cell and nuclear sizes, J. Microsc., 99 (1973) 331-348. 3 JONES, E. G., Lamination and differential distribution of thalamic afferents within the sensorymotor cortex of the squirrel monkey, J. comp. Neurol., (1974) in press. 4 JONES, E. G., AND LEAVITT, R. Y., Demonstration of thalamo-cortical connectivity in the cat somatic-sensory system by retrograde axonal transport of horseradish peroxidase, Brain Research, 63 (1973) 414-418. 5 JONES, E. G., AND LEAV1TT, R. Y., Retrograde axonal transport and the demonstration of nonspecific projections to the cerebral cortex and striatum from thalamic intralaminar nuclei in the rat, cat and monkey, J. comp. Neurol., 154 (1974) 349-378. 6 KRETTEK, J. E., JR., JONES, E. G., PRICE, J. L., AND BURTON, H., Unpublished observations. 7 KRISTENSSON, K., OLSSON, Y., AND SJOSTRAND, J., Axonal uptake and retrograde transport of exogenous proteins in the hypoglossal nerve, Brain Research, 32 (1971) 399-406. 8 LASEK,R., JOSEPH, J. S., AND WHITLOCK,O. G., Evaluation o f a radioautographic neuroanatomical tracing method, Brain Research, 8 (1968) 319-336. 9 LAVAIL, J. M., WINSTON, K. R., AND TISH, A., A method based on retrograde intraaxonal transport of protein for identification of cell bodies of axons terminating within the CNS, Brain Research, 58 (1973) 470-477. ]0 NAUTA, n . J. W., PRITZ, M. B., AND LASEK, R. J., Afferents to the rat caudatoputamen studied with horseradish peroxidase. An evaluation of a retrograde neuroanatomical research method, Brain Research, 67 (1974) 219-238. 11 RALSTON,H. J., III, AND SHARP, P. V., The identification of thalamocortical relay cells in the adult cat by means of retrograde axonal transport of horseradish peroxidase, Brain Research, 62 (1973) 273-278.
249
POSSIBLE DETERMINANTS OF THE DEGREE OF RETROGRADE NEURONAL LABELING WITH HORSERADISH PEROXIDASE
E. G. JONES
Department of Anatomy, Washington University School of Medicine, St. Louis, Mo. 63110 (U.S.A.)
The introduction of techniques which utilize the anterograde and retrograde axonal flow of proteins for tracing neuronal pathwaysl, 7-9 has permitted certain new observations on the relationships of various types of thalamic cell to the cerebral cortex. In the course of these investigations certain methodological considerations, especially relating to the retrograde horseradish peroxidase technique have arisen. Localized injections of type VI (Sigma) horseradish peroxidase (0.1-0.3 #l in a concentration of 500 #g/#l) made in single cortical areas of rats, cats and monkeys lead to intense, retrograde labeling of many cells in the topographically related part of the appropriate thalamic relay nucleus and to relatively much lighter labeling of a few cells in the intralaminar complex of nuclei4,5,11. The differential intensity of labeling, which is reflected in the size and number of horseradish peroxidase positive granules found in the cell soma and proximal dendrites (Figs. 1 and 2), is maintained irrespective of the survival time and of the amount of horseradish peroxidase injected. Cells of a single intralaminar nucleus can be lightly labeled from injections in quite widely separated areas of the cerebral cortex in the frontal, parietal and cingulate regions 5. On the other hand, multiple injections of horseradish peroxidase in the postcentral gyrus, though appearing to coalesce, still lead to labeling of thalamic relay cells in bands, each band corresponding to an injection site and with intervening bands of unlabeled cells4,5,7,11. At the center of each band some cells, in addition to containing horseradish peroxidase positive granules, show a diffuse brown staining of the perikaryal and dendritic membranes (Fig. 3) and it is thought that the axons of these cells may have been damaged by the injection needle. Complementary experiments conducted with the anterograde, autoradiographic technique 3, suggest that the degree of concentration of horseradish peroxidase transported in a retrograde manner to the thalamic cells is dependent upon the density of the terminal axonal field available for incorporation of the enzyme at the injection site. Injections of tritiated proline and/or leucine involving the intralaminar nuclei of cats 6, and after survival periods (24-36 h) in which labeled material transported in the rapid phase of axoplasmic transport is used as the marker, demonstrate a small, diffuse field widely scattered across the frontal, parietal and medial cortex and with the autoradiographic grains especially concentrated in the superficial part of layer
t'~
251 I (Fig. 4). This contrasts with the intense terminal axonal field, localized to specific cortical areas and concentrated in layers IV and IIIb, demonstrable 8,6 after injections of tritiated amino acids in the main thalamic relay nuclei (Fig. 5). As the number of silver grains observed at these short survival periods is probably related in some way to the number and size of the axon terminals and possibly to the number o f preterminal axon branches present, then these factors are probably the main determinants of a cell's capacity to accumulate horseradish peroxidase in the retrograde manner. This proposal is reinforced by the fact that it is possible to label the intralaminar cells heavily following injections o f horseradish peroxidase in the caudate nucleus and putamenS, 10. Here the retrograde labeling is always intense and many cells are labeled, contrasting with the situation after injections o f the cortex. This is probably determined by the density of the terminal axonal field of the intralaminar cell's axons in the caudate nucleus and putamen as indicated by the intensity o f terminal labeling in these nuclei following injections of tritiated amino acids in the intralaminar nuclei (Fig. 6). Other studies currently in progress demonstrate that thalamic cells that are undergoing retrograde shrinkage as the result of ablation of one cortical area may still be labeled by horseradish peroxidase transported in a retrograde manner from an injection site in another cortical area. Six months after ablation of the first somatic sensory area (SI) in cats, the ventrobasal complex undergoes severe retrograde degeneration, characterized by shrinkage of nerve cells and severe gliosis (Fig. 7). But most o f the remaining shrunken neurons can be labeled in a retrograde manner following injections of horseradish peroxidase in the intact second somatic sensory area (SII) 24-48 h prior to killing the animal (Figs. 7 and 8). These neurons are much smaller than those which are labeled after injections of horseradish peroxidase in animals in which both SI and SII are intact (Fig. 8). Although there are a number of possible explanations as to why the neurons projecting to SII should shrink following destruction of SI, one likely interpretation is that they normally send axon branches to both cortical areas. Supported by G r a n t number 5RO1 NS10526 from the National Institutes of Health, U.S. Public Health Service. I am indebted to Ms. Bertha McClure for tech-
Figs. 1 and 2. Photomicrographs showing differential retrograde labeling of cells in an intralaminar nucleus (Fig. 1) and in a relay nucleus (Fig. 2) following an injection of horseradish peroxidase in the cerebral cortex of a squirrel monkey. Thionin counterstain, x 500. Fig. 3. Diffuse retrograde labeling with horseradish peroxidase (see text). Thionin counterstain, x 500. Fig. 4. Bright-field photomicrograph of an autoradiograph showing a small but definite concentration of silver grains lying in the most superficial portion of layer I of the somatic sensory cortex of a cat following an injection of tritiated proline 5 days previously in the caudal intralaminar nuclei. Thionin counterstain, x 800. Fig. 5. Dark-field photomicrograph of an autoradiograph showing the dense concentration of silver grains in layers IIIb and IV of the somatic sensory cortex of a squirrel monkey following an injection of tritiated proline 5 days previously in the ventrobasal complex, x 600.
252
Fig. 6. Dark-held photomicrograph of an autoradiograph showing a portion of the internal capsule (IC) and the intense aggregation of silver grains in the caudate nucleus (right) in the same brain from which Fig. 4 was taken. 700. Fig. 7. Photomicrograph showing shrunken neurons labeled in the intensely gliotic ventrobasal complex (see inset) of a cat, 6 months alter ablation of the ipsilateral SI cortex and 36 h after injection of horseradish peroxidase in the ipsilateral SII cortex. Thionin counterstain. 230, inset, 20.
253
40'
401
30'
30-
SI
20.
~ 20-
Normal
,~i '". . . . .
E 14S ~ 10"
10'
0
160
320
480
I
640
Cross-sectional
160 area
of
cell
soma
|
320
480
W
| 640
(p2)
Fig. 8. Graphs showing the size-distribution of cells in the ipsilateral ventrobasal complex labeled by horseradish peroxidase (stipple) following an injection of SII, compared with the size-distribution of cells in the opposite ventrobasal complex (hatching). On the left, where SII was injected 6 months after ablation of the ipsilateral SI, the labeled cells are all greatly shrunken. On the right, where SI1 was injected in the presence of an intact SI, the labeled cells fall within the normal size range. Cell areas measured using a computer program described by Cowan and Wann 2.
nical assistance. This paper was presented at the meeting on 'The Use of Axonal Transport for Studies of Neuronal Connectivity' held at Gwatt-Thun, Switzerland, July 2-4, 1974. 1 COWAN, W. M., GOTTLIEB, D. I., HENDRICKSON,A. I., PRICE, J. L., AND WOOLSEY, T. A., The autoradiographic demonstration of axonal connections in the central nervous system, Brain Research, 37 (1972) 21-51. 2 COWAN,W. M., AND WANN, D. F., A computer system for the measurement of cell and nuclear sizes, J. Microsc., 99 (1973) 331-348. 3 JONES, E. G., Lamination and differential distribution of thalamic afferents within the sensorymotor cortex of the squirrel monkey, J. comp. Neurol., (1974) in press. 4 JONES, E. G., AND LEAVITT, R. Y., Demonstration of thalamo-cortical connectivity in the cat somatic-sensory system by retrograde axonal transport of horseradish peroxidase, Brain Research, 63 (1973) 414-418. 5 JONES, E. G., AND LEAV1TT, R. Y., Retrograde axonal transport and the demonstration of nonspecific projections to the cerebral cortex and striatum from thalamic intralaminar nuclei in the rat, cat and monkey, J. comp. Neurol., 154 (1974) 349-378. 6 KRETTEK, J. E., JR., JONES, E. G., PRICE, J. L., AND BURTON, H., Unpublished observations. 7 KRISTENSSON, K., OLSSON, Y., AND SJOSTRAND, J., Axonal uptake and retrograde transport of exogenous proteins in the hypoglossal nerve, Brain Research, 32 (1971) 399-406. 8 LASEK,R., JOSEPH, J. S., AND WHITLOCK,O. G., Evaluation o f a radioautographic neuroanatomical tracing method, Brain Research, 8 (1968) 319-336. 9 LAVAIL, J. M., WINSTON, K. R., AND TISH, A., A method based on retrograde intraaxonal transport of protein for identification of cell bodies of axons terminating within the CNS, Brain Research, 58 (1973) 470-477. ]0 NAUTA, n . J. W., PRITZ, M. B., AND LASEK, R. J., Afferents to the rat caudatoputamen studied with horseradish peroxidase. An evaluation of a retrograde neuroanatomical research method, Brain Research, 67 (1974) 219-238. 11 RALSTON,H. J., III, AND SHARP, P. V., The identification of thalamocortical relay cells in the adult cat by means of retrograde axonal transport of horseradish peroxidase, Brain Research, 62 (1973) 273-278.
