- Email: [email protected]
A horseradish peroxidase study of the cortico-olivary projection in the cat
306
Brain Research, 116 (1976) 306-311 © Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands
Short Communications
A horseradish peroxidase study of the cortico-olivary projection in the cat
G. A. BISHOP, R. A. McCREA and S. T. K1TAI Morin Memorial Laboratory, Department of Anatomy, School of Medicine, Wayne State University, Detroit, Mich. 48201 (U.S.A.)
(Accepted July 27th, 1976)
The existence of a direct projection from the cerebral cortex to the inferior olive (IO) has been shown in the past with both anatomical and electrophysiological techniques. Degeneration studies 12-14 have shown that the majority, if not all of this projection, arises from the primary motor area bilaterally, although Walberg 14 describes other cortical areas as also giving rise to olivary afferents. Sousa-Pinto and Broda113 found that there was a somatotopic pattern in the distribution of these endings in the dorsal and medial accessory olive and the rostral part of the ventral lamella of the principle olive. Electrophysiological studies ~,2,a° have demonstrated a projection almost exclusively from the ipsilateral sensorimotor cortex, which, based on the long latency of the response (8-9.4 msec) is considered to be mediated by slow pyramidal tract (PT) fibers 5. In order to identify the specific cortical areas and layer, and the type of neuron giving rise to this projection, we have used the technique of retrograde transport of the enzyme horseradish peroxidase (H RP). H R P (Sigma Type VI) was pressure injected through the needle of a 1 #1 Hamilton syringe into the IO of 6 young adult cats in volumes ranging from 0.25 to 1 ~l often in multiple injections, over periods of 1-2 h. In 4 of the cats, the IO was approached from a dorsal direction. In the remaining 2 animals, a ventral approach was made to control for spread of the enzyme to the reticular formation. Following survivals of 25-68 h, the animals were anesthetized and perfused intracardially with a buffered fixative of 1 ~ glutaraldehyde and 3 ~o paraformaldehyde. Transverse sections of the brain stem and sagittal sections of the cerebral cortex were cut in 50/~m frozen sections. Following reaction with diaminobenzidine and HzO2, alternate sections were mounted and lightly counterstained with cresyl violet. Complete details of the histological procedures have been described elsewhere 4. Fig. 1A is a series of transverse sections through the IO approximately 1.5 m m apart showing the extent of the injection in cat IO-2 in which 0.65/A were injected over 2 h in multiple injections. The dark reaction product covered the IO over most of it's rostrocaudal extent except for the most lateral aspect of the principle olive and the
307
P
/ .
"~
-
iiiiiiiiii~:~::
/"
"9, ":,
~:
....,.,.,. .:.....,.....
~:!iiii:iiiiiiiiii!
=E 15z
.,.....:.:.:.:,.... :.:.;.:.:.;,:.;.:,:
::::::::::::::;:::;
i:!:~:!:!:!:E:E:i i:!i!!i!i!i!iii~i: i:i:i:i:i:!:!:E:!.
5-
i:i:::::~u:iiiili;!iil;iiii!; ....................
. / 10-15 16-20 21-25 26-30 31-35 36-40 41-45 46-50 SIZE IN /4
Fig. 1. A: series of transverse sections through the 10 approximately 1.5 mm apart showing the extent of the injection in cat |0-2. B : schematic representation of the sensorimotor cortex and adjacent areas with the cruciate sulcus split apart showing the distribution of labeled neurons following the injection in A. Each dot represents one neuron. C: histogram of size distribution of labeled neurons found in the ASG (unshaded) and PSG (shaded).
caudalmost aspect of the medial accessory olive. A light brown reaction was present in the reticular formation immediately dorsal to the IO, primarily along the electrode tract. There was light spread of the injection across the midline which did not extend to the contralateral IO. In some cats, the injections were larger, spreading to the contralateral IO and more heavily to the reticular formation dorsal to the IO. When alternate 50 # m sagittal sections of the pericruciate cortex were examined, uptake was found bilaterally in the anterior sigmoid gyrus (ASG), coronal gyrus (CG), and posterior sigmoid gyrus (PSG). Fig. 1B is a schematic drawing of the sensorimotor cortex and adjacent cortical areas, with the cruciate sulcus split apart, illustrating the distribution of labeled neurons in cat IO-2. This drawing was constructed by locating labeled cells on alternate sagittal sections. These were placed at the appropriate laterali-
308
Fig. 2A. For legend see facing page. ty on the schematic drawing, each dot representing one neuron. As seen in Fig. 1B, the labeled neurons are scattered diffusely throughout the entire mediolateral extent of the pericruciate cortex. Since only alternate sections were examined, the actual number of neurons may be greater than those illustrated. Both distribution and number of labeled neurons on both sides appeared to be the same. Laterally, they were found in the anterior portions of both the CG and the ASG. Medially, labeled cells were found more posterior in the ASG, with the majority being located within or very near to the cruciate sulcus. In the most medial sections, labeled neurons were found closely associated to the cruciate sulcus (corresponding to area 6 of Hassler-Muhs-Clement3). Scattered neurons were labeled in the PSG mainly in the area of the cruciate sulcus. No labeled neurons were found in any other cortical area. Following the larger injections of H R P
309
Fig. 2. A: photomicrograph of pyramidal cell (arrow) located in layer V of the ASG. B: pyramidal cell from A in the ASG. Calibrations: A, 100/~m; B, 10/~m. into the IO, the contralateral A S G was more heavily labeled. In these cases, neurons were found throughout the entire mediolateral extent of the cruciate sulcus, distributing to it's deepest aspects. Several cells were found at the transition point between the A S G and PSG. In the PSG, there did not appear to be a signifcant increase in the number o f labeled neurons. In all cases, the labeled neurons, regardless of the cortical area in which they were found, were located in layer V and were either pyramidal or fusiform in shape. Fig. 2A is a photomicrograph showing a labeled neuron in layer V of the ASG. Fig. 2B shows this neuron at a higher magnification. Uniform brown granules can be seen to fill the neuron, except for the area of its nucleus, and extend out into the proximal parts of the apical and basal dendrites.
310 Pyramidal cells of the cortex have been classified as small (10-12/zm), medium (20-25 #m), large (30-35/~m) and giant (45-96/zm)6, s. The majority of labeled ceils in the ASG (Fig. 1C unshaded), have soma diameters of 21-30/zm which would include them in the classification of medium pyramidal cells. A second large group is found in the 31 40 # m range placing them in the large pyramidal cell category, in the PSG (Fig. 1C shaded), the majority of labeled cells have diameters of 21-25 # m which would include them in the classification of medium pyramidal cells. No giant cells were found to be labeled in any area. As stated previously, it has been demonstrated electrophysiologically that the corticofugal projection to the IO is mediated by the slow PT fibers. Naito et al. 7 recorded intracellularly from pyramidal neurons and subsequently stained them with methylene blue. They found that fast PT cells had soma diameters of 40-60 .um, while slow PT cells were smaller in size being 20-30 ~m in diameter. The vast majority of neurons in the pericruciate cortex, labeled following H R P injections in the IO, had soma diameters less than 40/~m, which would indicate that they were probably slow PT cells. Our findings in general confirm the degeneration studies of Sousa-Pinto and Broda113, except for the projection from the PSG to the IO found in our study. Diffuse distribution and scanty number of labeled neurons found in the PSG in this study would indicate that it would be extremely difficult to define PSG-IO connections by degeneration studies. On the other hand, Sousa-Pinto 11 has shown that the paramedian reticular nucleus (PRN) receives afferents from the PSG. In addition, Rossi and Brodal 9 have shown that the nucleus reticularis gigantocellularis (NRG) receives afferents from the sensorimotor cortex. It cannot be ruled out, therefore, that the uptake we have found in the PSG is due to spread to the PRN or NRG. However, the spread to this latter nucleus is very faint and is confined for the most part to its extreme caudal end. The spread to the PRN is likewise very faint and confined to the ventral subdivision, in which Sousa.Pinto reports only very scanty degeneration following lesions of the PSG. Also, in support of the existence of the projection from the PSG indicated in our study, Armstrong et al. 2 observed in their electrophysiological study that the stimulation of the ipsilateral PSG cortical areas would elicit responses in the IO (text Fig. 10). Thus it appears that the direct projection from the cerebral cortex to the IO arises bilaterally from the medium pyramidal cells of layer V of the primary motor area (ASG) with contributions from both the secondary motor area (area 6 of Hassler-MuhsClement) and the anterior part of the coronal gyrus. In addition there is a possibility that a small projection from the PSG is also present. This work was supported by USPHS NS00405 and RR 5384.
1 Armstrong, D. M., Functional significanceof the inferior olive, Physiol. Rev., 54 (1974) 358-417. 2 Armstrong, D. M. and Harvey, R. J., Responses in the inferior olive to stimulation of the cerebellar and cerebral cortices in the cat, J. Physiol. (Lond.), 187 (1966) 553-574. 3 Hassler, R. und Muhs-Clement, K., Architektonischer Aufbau des sensorimotorischen und parietalen Cortex der Katze, J. Hirnforseh., 6 (1964) 377-420.
311 4 Kitai, S. T., McCrea, R. A., Preston, R. J. and Bishop, G. A., Electrophysiological and horseradish peroxidase studies of precerebellar afferents to the nucleus interpositus anterior. I. Climbing fiber system, Brain Research, (1977) in press. 5 Kitai, S. T., Oshima, T., Provini, L. and Tsukahara, N., Cerebrocerebellar connections mediated by fast and slow conducting pyramidal tract fibres of the cat, Brain Research, 15 (1969) 267-271. 6 Lewis, B. and Clarke, H., The cortical lamination of the motor area of the brain, Proc. roy. Sac. B, 27 (1878) 38M9. 7 Naito, H., Nakamura, K., Kurosaki, T. and Tamura, Y., Precise location of fast and slow pyramidal tract cells in cat sensorimotor cortex, Brain Research, 14 (1969) 237-239. 8 Ram6n y Cajal, S., Histologie du Syst~me Nerveux de rHomme et des Fertdbrds, Maloine, Paris, 1909-1911. 9 Rossi, G. F. and Brodal, A., Corticofugal fibers to the brainstem reticular formation. An experimental study in the cat, J. Anat. (Land.j, 90 (1956) 42-62. 10 Sedgwick, E. M. and Williams, T. D., Responses of single units in the inferior olive to stimulation of the limb nerves, peripheral skin receptors, cerebellum, caudate nucleus, and motor cortex, J. Physiol. (Land.j, 189 (1967) 261-279. 11 Sousa-Pinto, A., The cortical projection onto the paramedian reticular and perihypoglossal nuclei (nucleus praepositus hypoglossi, nucleus intercalatus and nucleus of Roller) of the medulla oblongata of the cat. An experimental-anatomical study, Brain Research, 18 (1970) 77-91. 12 Sousa-Pinto, A., Experimental anatomical demonstration ofa cortico-olivary projection from area 6 (supplementary motor area?) in the cat, Brain Research, 16 (1969) 73-83. 13 Sousa-Pinto, A. and Brodal, A., Demonstration of a somatotopical pattern in the cortico-olivary projection in the cat. An experimental-anatomical study, Exp. Brain Res., 8 (1969) 364-386. 14 Walberg, F., Descending connections to the inferior olive. An experimental study in the cat, J. camp. Neural., 104 (1956) 77-174.
Brain Research, 116 (1976) 306-311 © Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands
Short Communications
A horseradish peroxidase study of the cortico-olivary projection in the cat
G. A. BISHOP, R. A. McCREA and S. T. K1TAI Morin Memorial Laboratory, Department of Anatomy, School of Medicine, Wayne State University, Detroit, Mich. 48201 (U.S.A.)
(Accepted July 27th, 1976)
The existence of a direct projection from the cerebral cortex to the inferior olive (IO) has been shown in the past with both anatomical and electrophysiological techniques. Degeneration studies 12-14 have shown that the majority, if not all of this projection, arises from the primary motor area bilaterally, although Walberg 14 describes other cortical areas as also giving rise to olivary afferents. Sousa-Pinto and Broda113 found that there was a somatotopic pattern in the distribution of these endings in the dorsal and medial accessory olive and the rostral part of the ventral lamella of the principle olive. Electrophysiological studies ~,2,a° have demonstrated a projection almost exclusively from the ipsilateral sensorimotor cortex, which, based on the long latency of the response (8-9.4 msec) is considered to be mediated by slow pyramidal tract (PT) fibers 5. In order to identify the specific cortical areas and layer, and the type of neuron giving rise to this projection, we have used the technique of retrograde transport of the enzyme horseradish peroxidase (H RP). H R P (Sigma Type VI) was pressure injected through the needle of a 1 #1 Hamilton syringe into the IO of 6 young adult cats in volumes ranging from 0.25 to 1 ~l often in multiple injections, over periods of 1-2 h. In 4 of the cats, the IO was approached from a dorsal direction. In the remaining 2 animals, a ventral approach was made to control for spread of the enzyme to the reticular formation. Following survivals of 25-68 h, the animals were anesthetized and perfused intracardially with a buffered fixative of 1 ~ glutaraldehyde and 3 ~o paraformaldehyde. Transverse sections of the brain stem and sagittal sections of the cerebral cortex were cut in 50/~m frozen sections. Following reaction with diaminobenzidine and HzO2, alternate sections were mounted and lightly counterstained with cresyl violet. Complete details of the histological procedures have been described elsewhere 4. Fig. 1A is a series of transverse sections through the IO approximately 1.5 m m apart showing the extent of the injection in cat IO-2 in which 0.65/A were injected over 2 h in multiple injections. The dark reaction product covered the IO over most of it's rostrocaudal extent except for the most lateral aspect of the principle olive and the
307
P
/ .
"~
-
iiiiiiiiii~:~::
/"
"9, ":,
~:
....,.,.,. .:.....,.....
~:!iiii:iiiiiiiiii!
=E 15z
.,.....:.:.:.:,.... :.:.;.:.:.;,:.;.:,:
::::::::::::::;:::;
i:!:~:!:!:!:E:E:i i:!i!!i!i!i!iii~i: i:i:i:i:i:!:!:E:!.
5-
i:i:::::~u:iiiili;!iil;iiii!; ....................
. / 10-15 16-20 21-25 26-30 31-35 36-40 41-45 46-50 SIZE IN /4
Fig. 1. A: series of transverse sections through the 10 approximately 1.5 mm apart showing the extent of the injection in cat |0-2. B : schematic representation of the sensorimotor cortex and adjacent areas with the cruciate sulcus split apart showing the distribution of labeled neurons following the injection in A. Each dot represents one neuron. C: histogram of size distribution of labeled neurons found in the ASG (unshaded) and PSG (shaded).
caudalmost aspect of the medial accessory olive. A light brown reaction was present in the reticular formation immediately dorsal to the IO, primarily along the electrode tract. There was light spread of the injection across the midline which did not extend to the contralateral IO. In some cats, the injections were larger, spreading to the contralateral IO and more heavily to the reticular formation dorsal to the IO. When alternate 50 # m sagittal sections of the pericruciate cortex were examined, uptake was found bilaterally in the anterior sigmoid gyrus (ASG), coronal gyrus (CG), and posterior sigmoid gyrus (PSG). Fig. 1B is a schematic drawing of the sensorimotor cortex and adjacent cortical areas, with the cruciate sulcus split apart, illustrating the distribution of labeled neurons in cat IO-2. This drawing was constructed by locating labeled cells on alternate sagittal sections. These were placed at the appropriate laterali-
308
Fig. 2A. For legend see facing page. ty on the schematic drawing, each dot representing one neuron. As seen in Fig. 1B, the labeled neurons are scattered diffusely throughout the entire mediolateral extent of the pericruciate cortex. Since only alternate sections were examined, the actual number of neurons may be greater than those illustrated. Both distribution and number of labeled neurons on both sides appeared to be the same. Laterally, they were found in the anterior portions of both the CG and the ASG. Medially, labeled cells were found more posterior in the ASG, with the majority being located within or very near to the cruciate sulcus. In the most medial sections, labeled neurons were found closely associated to the cruciate sulcus (corresponding to area 6 of Hassler-Muhs-Clement3). Scattered neurons were labeled in the PSG mainly in the area of the cruciate sulcus. No labeled neurons were found in any other cortical area. Following the larger injections of H R P
309
Fig. 2. A: photomicrograph of pyramidal cell (arrow) located in layer V of the ASG. B: pyramidal cell from A in the ASG. Calibrations: A, 100/~m; B, 10/~m. into the IO, the contralateral A S G was more heavily labeled. In these cases, neurons were found throughout the entire mediolateral extent of the cruciate sulcus, distributing to it's deepest aspects. Several cells were found at the transition point between the A S G and PSG. In the PSG, there did not appear to be a signifcant increase in the number o f labeled neurons. In all cases, the labeled neurons, regardless of the cortical area in which they were found, were located in layer V and were either pyramidal or fusiform in shape. Fig. 2A is a photomicrograph showing a labeled neuron in layer V of the ASG. Fig. 2B shows this neuron at a higher magnification. Uniform brown granules can be seen to fill the neuron, except for the area of its nucleus, and extend out into the proximal parts of the apical and basal dendrites.
310 Pyramidal cells of the cortex have been classified as small (10-12/zm), medium (20-25 #m), large (30-35/~m) and giant (45-96/zm)6, s. The majority of labeled ceils in the ASG (Fig. 1C unshaded), have soma diameters of 21-30/zm which would include them in the classification of medium pyramidal cells. A second large group is found in the 31 40 # m range placing them in the large pyramidal cell category, in the PSG (Fig. 1C shaded), the majority of labeled cells have diameters of 21-25 # m which would include them in the classification of medium pyramidal cells. No giant cells were found to be labeled in any area. As stated previously, it has been demonstrated electrophysiologically that the corticofugal projection to the IO is mediated by the slow PT fibers. Naito et al. 7 recorded intracellularly from pyramidal neurons and subsequently stained them with methylene blue. They found that fast PT cells had soma diameters of 40-60 .um, while slow PT cells were smaller in size being 20-30 ~m in diameter. The vast majority of neurons in the pericruciate cortex, labeled following H R P injections in the IO, had soma diameters less than 40/~m, which would indicate that they were probably slow PT cells. Our findings in general confirm the degeneration studies of Sousa-Pinto and Broda113, except for the projection from the PSG to the IO found in our study. Diffuse distribution and scanty number of labeled neurons found in the PSG in this study would indicate that it would be extremely difficult to define PSG-IO connections by degeneration studies. On the other hand, Sousa-Pinto 11 has shown that the paramedian reticular nucleus (PRN) receives afferents from the PSG. In addition, Rossi and Brodal 9 have shown that the nucleus reticularis gigantocellularis (NRG) receives afferents from the sensorimotor cortex. It cannot be ruled out, therefore, that the uptake we have found in the PSG is due to spread to the PRN or NRG. However, the spread to this latter nucleus is very faint and is confined for the most part to its extreme caudal end. The spread to the PRN is likewise very faint and confined to the ventral subdivision, in which Sousa.Pinto reports only very scanty degeneration following lesions of the PSG. Also, in support of the existence of the projection from the PSG indicated in our study, Armstrong et al. 2 observed in their electrophysiological study that the stimulation of the ipsilateral PSG cortical areas would elicit responses in the IO (text Fig. 10). Thus it appears that the direct projection from the cerebral cortex to the IO arises bilaterally from the medium pyramidal cells of layer V of the primary motor area (ASG) with contributions from both the secondary motor area (area 6 of Hassler-MuhsClement) and the anterior part of the coronal gyrus. In addition there is a possibility that a small projection from the PSG is also present. This work was supported by USPHS NS00405 and RR 5384.
1 Armstrong, D. M., Functional significanceof the inferior olive, Physiol. Rev., 54 (1974) 358-417. 2 Armstrong, D. M. and Harvey, R. J., Responses in the inferior olive to stimulation of the cerebellar and cerebral cortices in the cat, J. Physiol. (Lond.), 187 (1966) 553-574. 3 Hassler, R. und Muhs-Clement, K., Architektonischer Aufbau des sensorimotorischen und parietalen Cortex der Katze, J. Hirnforseh., 6 (1964) 377-420.
311 4 Kitai, S. T., McCrea, R. A., Preston, R. J. and Bishop, G. A., Electrophysiological and horseradish peroxidase studies of precerebellar afferents to the nucleus interpositus anterior. I. Climbing fiber system, Brain Research, (1977) in press. 5 Kitai, S. T., Oshima, T., Provini, L. and Tsukahara, N., Cerebrocerebellar connections mediated by fast and slow conducting pyramidal tract fibres of the cat, Brain Research, 15 (1969) 267-271. 6 Lewis, B. and Clarke, H., The cortical lamination of the motor area of the brain, Proc. roy. Sac. B, 27 (1878) 38M9. 7 Naito, H., Nakamura, K., Kurosaki, T. and Tamura, Y., Precise location of fast and slow pyramidal tract cells in cat sensorimotor cortex, Brain Research, 14 (1969) 237-239. 8 Ram6n y Cajal, S., Histologie du Syst~me Nerveux de rHomme et des Fertdbrds, Maloine, Paris, 1909-1911. 9 Rossi, G. F. and Brodal, A., Corticofugal fibers to the brainstem reticular formation. An experimental study in the cat, J. Anat. (Land.j, 90 (1956) 42-62. 10 Sedgwick, E. M. and Williams, T. D., Responses of single units in the inferior olive to stimulation of the limb nerves, peripheral skin receptors, cerebellum, caudate nucleus, and motor cortex, J. Physiol. (Land.j, 189 (1967) 261-279. 11 Sousa-Pinto, A., The cortical projection onto the paramedian reticular and perihypoglossal nuclei (nucleus praepositus hypoglossi, nucleus intercalatus and nucleus of Roller) of the medulla oblongata of the cat. An experimental-anatomical study, Brain Research, 18 (1970) 77-91. 12 Sousa-Pinto, A., Experimental anatomical demonstration ofa cortico-olivary projection from area 6 (supplementary motor area?) in the cat, Brain Research, 16 (1969) 73-83. 13 Sousa-Pinto, A. and Brodal, A., Demonstration of a somatotopical pattern in the cortico-olivary projection in the cat. An experimental-anatomical study, Exp. Brain Res., 8 (1969) 364-386. 14 Walberg, F., Descending connections to the inferior olive. An experimental study in the cat, J. camp. Neural., 104 (1956) 77-174.