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Distribution of terminals of thalamocortical fibers originating from the ventrolateral nucleus of the cat thalamus
Ne,+rosciem'e Letters, 96 (1989) 163 167
163
Elsevier Scientific Publishers Ireland Ltd.
NSL 05827
Distribution of terminals of thalamocortical fibers originating from the ventrolateral nucleus of the cat thalamus Y. S h i n o d a and S. K a k e i Department <~["Physiology, School of Medicine, Tokyo Medical and Dental University, Tokyo (Japan) (Received 26 August 1988; Revised version received 22 September 1988; Accepted 23 September 1988)
Key words: Motor cortex; Thalamus; Ventrolateral nucleus; Phaseolus vulgaris leucoagglutinin Anterograde labelling following focal injections of Phaseolus vulgaris leucoagglutinin was used to identify the threedimensional cortical distribution of thalamocortical (TC) fibers from the ventrolateral nucleus of the thalamus of the cat. The labelled TC fibers were distributed usually in layers I and IIl of the motor cortex and the terminals in layer III tended to aggregate into patches about 1 1.5 m m wide in a mediolateral direction. These patches were arranged in longitudinal strips about 2 5 m m long in a rostrocaudal direction and were separated by gaps of terminal free area.
The intracortical organization of thalamocortical (TC) fibers has been analyzed extensively in the visual system and it is known that geniculocortical fibers are arranged in parallel slabs about 400/lm wide representing alternately the right and left eyes [14]. Much less is known about the TC projection from the ventroanterior and ventrolateral nuclei of the thalamus (VA-VL complex) to the motor cortex. We therefore analyzed the distribution patterns of TC terminals in the motor cortex, visualized after focal injections of Phaseolus vulgaris leucoagglutinin (PHA-L) [3] into the VA-VL complex. A preliminary note of this work has appeared previously [12]. Experiments were performed on 4 adult cats anesthetized with Nembutal (Abott, Switzerland, 35 mg/kg). A small hole over the parietal cortex on each side was made to introduce into the V A - V L complex a glass pipette filled with 2.5% Phaseolus vulgaris leucoagglutinin (PHA-L) in 35 mM sodium phosphate-buffered saline (pH = 8.0). PHA-L was injected iontophoretically by positive current of 4 10 ,uA (7 s duration) applied using a constant current generator every 14 s for 1 h. During the survival time of 2 3 weeks, the animals were given prophylactic doses of antibiotic
Corresponden<'e. Y. Shinoda, Department of Physiology, School of Medicine, Tokyo Medical and Dental University, [-5-45, Yushima, Bunkyo-ku, Tokyo, Japan. 0304-3940'89 $ 03 50 @ 1989 Elsevier Scientific Publishers Ireland Ltd.
164
(penicilline G procaine; Crysticilline, Squibb) and an analgesic (meperidine hydrochloride; Demerol, Winthrop) for the first week. These animals did not show any sign of discomfort. The animals were then reanesthetized and perfused with the method of Berod et al. [2]. Serial frontal sections of the cortex and the thalamus 75 or 100 /xm thick were cut the next day and treated for immunohistochemical localization of the transported lectin with the ABC method [3, 4]. Iontophoretic injection of PHA-L resulted in well-localized injection sites (the inset on the bottom left in Fig. 1). Several intensely stained glial cells at the center of the injection were surrounded by a number of neurons that appeared to be filled by the lectin. The injection sites were usually less than 1.0 mm, and sometimes as small as 400 Itm in diameter. PHA-L appeared to be transported preferentially in an anterograde direction, since no retrogradely labelled cortical cells were observed. The mor-
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Fig. 1. Camera lucida drawing of PHA-L-tabelted TC axons in the motor cortex. This drawing was made on a single frontal section (100 u m thick) and the location of this patch o f axon terminals is shown in the inset on the bottom right. Cells in layer V are large pyramidal cells. The broken line on the bottom shows the border between the cortical grey and white matter. R o m a n letters show the cortical layers. The injection site of P H A - L in the VA-VL complex is shown on the bottom left. cru, cruciate sulcus; presyl, presylvian sulcus; VA, ventroanterior nucleus; VM, ventromedial nucleus; AV, anteroventral nucleus; V L ventrolateral nucleus.
165
phology of the labelled fine axons, boutons en passant and various terminal specializations could be resolved (Fig. 1), but the labelled structures had a more dusty appearance than those visualized by intracellular injection of horseradish peroxidase (HRP) [11]. Fine axons and swellings en passant were found usually in cortical layers I and III (Fig. 1), but in some animals, these were restricted to layer ! or in layer III. Labelling of parent axons in the subcortical white matter was not observed. Labelling of fine collaterals and swellings en passant in layer III was well localized in a mediolateral direction and labelled fibers were grouped into discrete ~atcbes about 1-1.5 mm wide within layer III of the motor cortex. In contrast, when labelling
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Fig. 2. Two longitudinal strips of labelled TC fibers in the motor cortex. Representative sections (100 iLm thick) are separated by 600 Itm. Note the terminal free gap between two patches that are arranged in a rostrocaudal direction.
166
appeared in layer I or in both layers I and III, labelled fibers in layer I were not confined mediolaterally within the same vertical band as the patches in layer IlI but rather extended widely in a mediolateral direction beyond the area of the patches in layer III. To determine the pattern in which the patches of the labelled fibers were organized over a wide cortical area, we attempted to reconstruct patches from serial sections. We aligned each section with the appropriate region of the next section in a rostrocaudal direction, using cut ends of blood vessels as markers. In this reconstruction, it turned out that patches of the labelled fibers about I--1.5 mm wide on individual sections were aligned in a rostrocaudal direction, making a longitudinal strip as viewed from the surface. In representative frontal sections at 600/tm intervals (Fig. 2) the patches indicated by upward arrows continued on into the ventral bank of the cruciate sulcus, more-or-less in parallel to a sagittal plane in a rostrocaudal direction over a distance of 3.8 ram. Longitudinal strips ranged from 2 to 5 mm in length (n = 6). Their location was almost in parallel to a sagittal plane, but the strips ran slightly obliquely, rostromedially to caudolaterally. A small focal injection in the VAVL complex usually resulted in multiple strips of labelled fibers in the motor cortex. The number of longitudinal strips varied, depending on the amount of PHA-L injected. Two to 7 strips were differentiated in 4 cats. Roughly, parallel strips, each about 1-1.5 mm wide, were separated from each other by regions in which the label was much diminished or practically absent. In the example of Fig. 2, the patches indicated by arrow heads appeared lateral to the patches indicated by arrows and these two were clearly separated by a gap of label-free area. The laterally located patch continued further caudally from the ventral bank of the cruciate sulcus to its dorsal bank for a distance of about 4.5 ram. The distances between strips, as well as the widths and the lengths of individual strips, were irregular. When the injected PHA-L was large in amount, wide strips were found as if adjacent strips had fused. The present finding of the cortical distribution of TC axons from the VA-VL complex is in agreement with that of Strick and Sterling [I 3], in that terminals are distributed mainly in layers I and III. Sasaki et al. [10] suggested from electrophysiological data that there should be superficial and deep TC projections. These dual projections were demonstrated anatomically [7, 9]. More recently, Jinnai et al. [5] reported that TC neurons in the VA-VL complex which receive inputs from the globus pallidus, but not from the cerebellar nuclei project to layer I of the anterior sigmoid gyrus in the cat. However, a focal injection of PHA-L in the caudal portion of the VA-VL complex where the cerebellar projection exists resulted in labelling of TC fibers in both layers I and IlI in the present study. This discrepancy remains unresolved. One possibility is that small neurons projecting only to layer I may be involved in the caudal part of the VA-VL complex [9]. The intracortical morphology of single axons of thalamic neurons receiving monosynaptic inputs from the cerebellar nuclei has been analyzed recently, using intraaxonal injection of H R P and threedimensional reconstruction of the axonal trajectory from serial sections [6, 1 l]. Single TC axons originating from the VA-VL complex divided into several main branches, which further divided into numerous branches, forming multiple plexuses of terminals in the cerebral grey matter, mainly in layer
167
III and sparsely in layers I and VI. These multiple plexuses (2 6 for each axon and an average of 0.5 mm in diameter) were separated by terminal-free gaps and distributed mainly in a rostrocaudal direction for a distance of up to 6 mm in the motor cortex. Taking this morphology of single TC axons into account, the present result implies that a cluster of TC neurons in a focal area of the VA-VL complex, each with projection distributed over wide rostrocaudal region of the cortex, make up the longitudinal strips of axon terminals. The longitudinal strips of terminals that we have described are much larger than individual motor efferent columns or zones defined by intracortical microstimulation [1]. Therefore, they might control multiple efferent zones of different muscles, but they might still be involved in control of single muscles, if they were distributed to multiple cortical representations of single muscle [8]. The authors thank Dr. P. Zarzecki for critical reading o f the manuscript. This research was supported by a grant from the Japanese Ministry o f Education, Science and Culture for Scientific Research (63870008). I Asanuma, H. and Sakata. H., Functional organization of a cortical efferent system examined with focal depth stimulation in cats, J. Neurophysiol., 30 (1967) 35 54. 2 Berod, A., Hartman, B.K. and Pujol, J.F., Importance of fixation in immunohistochemistry: use of formaldehyde at variable pH for the localization of tyrosine hydroxylase, J. Histochem. Cytochem., 29 ( 1981 ) 844 850. 3 Gerfen, C.R. and Sawchenko, P.E., An anterograde neuroanatomical tracing method that shows the detailed morphology of neurons, their axons and terminals: immunohistochemical localization of an axonally transported plant lectin, Phaseolus vulgaris leucoagglutinin (PHA-L). Brain Res,, 290 (1984) 219 238. 4 Hsu, S.M.. Raine, L. and Fanger, H., Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures, J. Histochem. Cytochem., 29 (1981) 577 580. 5 Jinnai, K., Nambu, A. and Yoshida, S., Thalamic afferents to layer I of anterior sigmoid cortex originating from the VA-VL neurons with entopeduncular input, Exp. Brain Res., 69 (1987) 67 76. 6 Kakei, S., Futami, T. and Shinoda, Y., Projection patterns of single ventrolateral nucleus neurons in the motor cortex and surrounding cortical areas in the cat, Neurosci. Res., $7 (1988) 94. 7 Kawaguchi, S., Samejima, A. and Y a m a m o t o , T., Post-natal development of the cerebello-ccrcbral projection in kittens, J. Physiol. (Lond.), 343 (1983) 215 232. 8 Pappas. C.L. and Strick. P.L., Double representation of the distal forelimb in cat motor cortex, Brain Res.. 167 (1979)412 416. 9 Penny, G.R., ltoh, K. and Diamond, 1.1., Cells of different sizes in the ventral nuclei project to different layers of the somatic cortex in the cat, Brain Res., 242 (1982) 55 65. I0 Sasuki, K., Stanton, H.P. and Dieckman, G,, Characteristic features of augmenting and recruiting rcsponscs in the cerebral cortex, Exp. Neurol., 26 (1970) 369 392. I Shinoda. Y., General Discussion 3 in Motor Areas of the Cerebral Cortex, Ciba Foundation Symposium, 132. Wiley, New York, 1987, pp. 221 230. 2 Shinoda, Y., Kakei, S. and Ohgaki, T., Multiple anteroposteriorly-elongated rod-like aggregates of cerebral terminals of thalamocortical neurons in a small focal part of the VL nucleus, ENA Ahstr., (1988) 229. 3 Strick, P.I,. and Sterling, P., Synaptic termination of afl'erents from the ventrolateral nucleus of the thalamus in the cat motor cortex. A light and electron microscope study, J. Comp. Neurol., 153 (1974) 77 106. 14 Wiesel, T.N., tlubel, D.H. and Lam, D.M.K., Autoradiographic demonstration of ocular dominance columns in the monkey striate cortex by means of transneuronal transport, Brain Res., 79 (1974) 273 279.
163
Elsevier Scientific Publishers Ireland Ltd.
NSL 05827
Distribution of terminals of thalamocortical fibers originating from the ventrolateral nucleus of the cat thalamus Y. S h i n o d a and S. K a k e i Department <~["Physiology, School of Medicine, Tokyo Medical and Dental University, Tokyo (Japan) (Received 26 August 1988; Revised version received 22 September 1988; Accepted 23 September 1988)
Key words: Motor cortex; Thalamus; Ventrolateral nucleus; Phaseolus vulgaris leucoagglutinin Anterograde labelling following focal injections of Phaseolus vulgaris leucoagglutinin was used to identify the threedimensional cortical distribution of thalamocortical (TC) fibers from the ventrolateral nucleus of the thalamus of the cat. The labelled TC fibers were distributed usually in layers I and IIl of the motor cortex and the terminals in layer III tended to aggregate into patches about 1 1.5 m m wide in a mediolateral direction. These patches were arranged in longitudinal strips about 2 5 m m long in a rostrocaudal direction and were separated by gaps of terminal free area.
The intracortical organization of thalamocortical (TC) fibers has been analyzed extensively in the visual system and it is known that geniculocortical fibers are arranged in parallel slabs about 400/lm wide representing alternately the right and left eyes [14]. Much less is known about the TC projection from the ventroanterior and ventrolateral nuclei of the thalamus (VA-VL complex) to the motor cortex. We therefore analyzed the distribution patterns of TC terminals in the motor cortex, visualized after focal injections of Phaseolus vulgaris leucoagglutinin (PHA-L) [3] into the VA-VL complex. A preliminary note of this work has appeared previously [12]. Experiments were performed on 4 adult cats anesthetized with Nembutal (Abott, Switzerland, 35 mg/kg). A small hole over the parietal cortex on each side was made to introduce into the V A - V L complex a glass pipette filled with 2.5% Phaseolus vulgaris leucoagglutinin (PHA-L) in 35 mM sodium phosphate-buffered saline (pH = 8.0). PHA-L was injected iontophoretically by positive current of 4 10 ,uA (7 s duration) applied using a constant current generator every 14 s for 1 h. During the survival time of 2 3 weeks, the animals were given prophylactic doses of antibiotic
Corresponden<'e. Y. Shinoda, Department of Physiology, School of Medicine, Tokyo Medical and Dental University, [-5-45, Yushima, Bunkyo-ku, Tokyo, Japan. 0304-3940'89 $ 03 50 @ 1989 Elsevier Scientific Publishers Ireland Ltd.
164
(penicilline G procaine; Crysticilline, Squibb) and an analgesic (meperidine hydrochloride; Demerol, Winthrop) for the first week. These animals did not show any sign of discomfort. The animals were then reanesthetized and perfused with the method of Berod et al. [2]. Serial frontal sections of the cortex and the thalamus 75 or 100 /xm thick were cut the next day and treated for immunohistochemical localization of the transported lectin with the ABC method [3, 4]. Iontophoretic injection of PHA-L resulted in well-localized injection sites (the inset on the bottom left in Fig. 1). Several intensely stained glial cells at the center of the injection were surrounded by a number of neurons that appeared to be filled by the lectin. The injection sites were usually less than 1.0 mm, and sometimes as small as 400 Itm in diameter. PHA-L appeared to be transported preferentially in an anterograde direction, since no retrogradely labelled cortical cells were observed. The mor-
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dorsal
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.
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mm
Fig. 1. Camera lucida drawing of PHA-L-tabelted TC axons in the motor cortex. This drawing was made on a single frontal section (100 u m thick) and the location of this patch o f axon terminals is shown in the inset on the bottom right. Cells in layer V are large pyramidal cells. The broken line on the bottom shows the border between the cortical grey and white matter. R o m a n letters show the cortical layers. The injection site of P H A - L in the VA-VL complex is shown on the bottom left. cru, cruciate sulcus; presyl, presylvian sulcus; VA, ventroanterior nucleus; VM, ventromedial nucleus; AV, anteroventral nucleus; V L ventrolateral nucleus.
165
phology of the labelled fine axons, boutons en passant and various terminal specializations could be resolved (Fig. 1), but the labelled structures had a more dusty appearance than those visualized by intracellular injection of horseradish peroxidase (HRP) [11]. Fine axons and swellings en passant were found usually in cortical layers I and III (Fig. 1), but in some animals, these were restricted to layer ! or in layer III. Labelling of parent axons in the subcortical white matter was not observed. Labelling of fine collaterals and swellings en passant in layer III was well localized in a mediolateral direction and labelled fibers were grouped into discrete ~atcbes about 1-1.5 mm wide within layer III of the motor cortex. In contrast, when labelling
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i I
\
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~~
{~~
cr__
)~
dorsal
rostra; lateral
Fig. 2. Two longitudinal strips of labelled TC fibers in the motor cortex. Representative sections (100 iLm thick) are separated by 600 Itm. Note the terminal free gap between two patches that are arranged in a rostrocaudal direction.
166
appeared in layer I or in both layers I and III, labelled fibers in layer I were not confined mediolaterally within the same vertical band as the patches in layer IlI but rather extended widely in a mediolateral direction beyond the area of the patches in layer III. To determine the pattern in which the patches of the labelled fibers were organized over a wide cortical area, we attempted to reconstruct patches from serial sections. We aligned each section with the appropriate region of the next section in a rostrocaudal direction, using cut ends of blood vessels as markers. In this reconstruction, it turned out that patches of the labelled fibers about I--1.5 mm wide on individual sections were aligned in a rostrocaudal direction, making a longitudinal strip as viewed from the surface. In representative frontal sections at 600/tm intervals (Fig. 2) the patches indicated by upward arrows continued on into the ventral bank of the cruciate sulcus, more-or-less in parallel to a sagittal plane in a rostrocaudal direction over a distance of 3.8 ram. Longitudinal strips ranged from 2 to 5 mm in length (n = 6). Their location was almost in parallel to a sagittal plane, but the strips ran slightly obliquely, rostromedially to caudolaterally. A small focal injection in the VAVL complex usually resulted in multiple strips of labelled fibers in the motor cortex. The number of longitudinal strips varied, depending on the amount of PHA-L injected. Two to 7 strips were differentiated in 4 cats. Roughly, parallel strips, each about 1-1.5 mm wide, were separated from each other by regions in which the label was much diminished or practically absent. In the example of Fig. 2, the patches indicated by arrow heads appeared lateral to the patches indicated by arrows and these two were clearly separated by a gap of label-free area. The laterally located patch continued further caudally from the ventral bank of the cruciate sulcus to its dorsal bank for a distance of about 4.5 ram. The distances between strips, as well as the widths and the lengths of individual strips, were irregular. When the injected PHA-L was large in amount, wide strips were found as if adjacent strips had fused. The present finding of the cortical distribution of TC axons from the VA-VL complex is in agreement with that of Strick and Sterling [I 3], in that terminals are distributed mainly in layers I and III. Sasaki et al. [10] suggested from electrophysiological data that there should be superficial and deep TC projections. These dual projections were demonstrated anatomically [7, 9]. More recently, Jinnai et al. [5] reported that TC neurons in the VA-VL complex which receive inputs from the globus pallidus, but not from the cerebellar nuclei project to layer I of the anterior sigmoid gyrus in the cat. However, a focal injection of PHA-L in the caudal portion of the VA-VL complex where the cerebellar projection exists resulted in labelling of TC fibers in both layers I and IlI in the present study. This discrepancy remains unresolved. One possibility is that small neurons projecting only to layer I may be involved in the caudal part of the VA-VL complex [9]. The intracortical morphology of single axons of thalamic neurons receiving monosynaptic inputs from the cerebellar nuclei has been analyzed recently, using intraaxonal injection of H R P and threedimensional reconstruction of the axonal trajectory from serial sections [6, 1 l]. Single TC axons originating from the VA-VL complex divided into several main branches, which further divided into numerous branches, forming multiple plexuses of terminals in the cerebral grey matter, mainly in layer
167
III and sparsely in layers I and VI. These multiple plexuses (2 6 for each axon and an average of 0.5 mm in diameter) were separated by terminal-free gaps and distributed mainly in a rostrocaudal direction for a distance of up to 6 mm in the motor cortex. Taking this morphology of single TC axons into account, the present result implies that a cluster of TC neurons in a focal area of the VA-VL complex, each with projection distributed over wide rostrocaudal region of the cortex, make up the longitudinal strips of axon terminals. The longitudinal strips of terminals that we have described are much larger than individual motor efferent columns or zones defined by intracortical microstimulation [1]. Therefore, they might control multiple efferent zones of different muscles, but they might still be involved in control of single muscles, if they were distributed to multiple cortical representations of single muscle [8]. The authors thank Dr. P. Zarzecki for critical reading o f the manuscript. This research was supported by a grant from the Japanese Ministry o f Education, Science and Culture for Scientific Research (63870008). I Asanuma, H. and Sakata. H., Functional organization of a cortical efferent system examined with focal depth stimulation in cats, J. Neurophysiol., 30 (1967) 35 54. 2 Berod, A., Hartman, B.K. and Pujol, J.F., Importance of fixation in immunohistochemistry: use of formaldehyde at variable pH for the localization of tyrosine hydroxylase, J. Histochem. Cytochem., 29 ( 1981 ) 844 850. 3 Gerfen, C.R. and Sawchenko, P.E., An anterograde neuroanatomical tracing method that shows the detailed morphology of neurons, their axons and terminals: immunohistochemical localization of an axonally transported plant lectin, Phaseolus vulgaris leucoagglutinin (PHA-L). Brain Res,, 290 (1984) 219 238. 4 Hsu, S.M.. Raine, L. and Fanger, H., Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures, J. Histochem. Cytochem., 29 (1981) 577 580. 5 Jinnai, K., Nambu, A. and Yoshida, S., Thalamic afferents to layer I of anterior sigmoid cortex originating from the VA-VL neurons with entopeduncular input, Exp. Brain Res., 69 (1987) 67 76. 6 Kakei, S., Futami, T. and Shinoda, Y., Projection patterns of single ventrolateral nucleus neurons in the motor cortex and surrounding cortical areas in the cat, Neurosci. Res., $7 (1988) 94. 7 Kawaguchi, S., Samejima, A. and Y a m a m o t o , T., Post-natal development of the cerebello-ccrcbral projection in kittens, J. Physiol. (Lond.), 343 (1983) 215 232. 8 Pappas. C.L. and Strick. P.L., Double representation of the distal forelimb in cat motor cortex, Brain Res.. 167 (1979)412 416. 9 Penny, G.R., ltoh, K. and Diamond, 1.1., Cells of different sizes in the ventral nuclei project to different layers of the somatic cortex in the cat, Brain Res., 242 (1982) 55 65. I0 Sasuki, K., Stanton, H.P. and Dieckman, G,, Characteristic features of augmenting and recruiting rcsponscs in the cerebral cortex, Exp. Neurol., 26 (1970) 369 392. I Shinoda. Y., General Discussion 3 in Motor Areas of the Cerebral Cortex, Ciba Foundation Symposium, 132. Wiley, New York, 1987, pp. 221 230. 2 Shinoda, Y., Kakei, S. and Ohgaki, T., Multiple anteroposteriorly-elongated rod-like aggregates of cerebral terminals of thalamocortical neurons in a small focal part of the VL nucleus, ENA Ahstr., (1988) 229. 3 Strick, P.I,. and Sterling, P., Synaptic termination of afl'erents from the ventrolateral nucleus of the thalamus in the cat motor cortex. A light and electron microscope study, J. Comp. Neurol., 153 (1974) 77 106. 14 Wiesel, T.N., tlubel, D.H. and Lam, D.M.K., Autoradiographic demonstration of ocular dominance columns in the monkey striate cortex by means of transneuronal transport, Brain Res., 79 (1974) 273 279.