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Corticocortical connections of frontal oculomotor areas in the cat
Brain Research, 414 (1987) 91-98
91
Elsevier BRE 12648
Corticocortical connections of frontal oculomotor areas in the cat Mitsukazu Nakai, Yasuhiko Tamai and Eizo Miyashita Department of Physiology, Wakayama Medical College, Wakayama (Japan) (Accepted 11 November 1986)
Key words: Eye movement; Monocular movement; Frontal eye field; Oculomotor area; Coronal sulcus; Anterior ectosylvian sulcus; Cat
The corticocortical connections of the frontal 'oculomotor' areas related to eye movements of the cat were studied using the retrograde horseradish peroxidase (HRP) tracing method combined with electrophysiological techniques. The following results were obtained. (1) The medial wall of the hemisphere under the cruciate sulcus (CRU), where contralateral conjugate eye deviation was elicited, received fibers from the medial bank of the presylvian sulcus (PRE). The fundus of the coronal sulcus (COR), where monocular movement of the contralateral eye was evoked, received fibers from the lateral bank of the PRE. (2) All the frontal oculomotor areas, the medial wall of the hemisphere under the CRU, the fundus of the COR, and both banks of the PRE, received fibers from the ipsilateral ventral bank of the anterior ectosylvian sulcus (AES). (3) The ventral bank of the AES received fibers from the caudal part of the lateral suprasylvian visual areas. On the basis of the fiber connections, the frontal oculomotor areas can be subdivided into a 'medial' area, the medial wall of the hemisphere under the CRU and the medial bank of the PRE, and a 'lateral' area, the lateral bank of the PRE and the fundus of the COR. Moreover, we found evidence of fiber projections from the ventral bank of the AES to the frontal oculomotor areas that were physiologicallyidentified.
INTRODUCTION It is well known that electrical stimulation of specific regions in the h u m a n and simian frontal lobes, can elicit eye m o v e m e n t s 3'15'24'26'34. It has been suggested that the frontal 'oculomotor' areas responsible for eye movements in the cat are in the medial wall of the hemisphere u n d e r the cruciate sulcus ( C R U ) and the ventral and medial banks of the presylvian sulcus (PRE) 1°'11'25. O n the basis of the direc-
the contralateral eye could be evoked. In the research described here we studied, first, the corticocortical connections of the frontal oculomotor areas in the cat using retrograde H R P tracing combined with electrophysiological techniques. Secondly, we explored the cortical connections to these frontal oculomotor areas from other visually related cortices. MATERIALS AND METHODS
tion and the latency of evoked eye movements, Guitton and Mandl 1° subdivided the frontal oculomotor areas into two subregions, one 'medial' and one 'lateral'. The medial area included the medial wall of the hemisphere u n d e r the C R U and the medial b a n k of the P R E , and the lateral area included the lateral bank of the PRE. However, the anatomical relationships between these frontal oculomotor areas have not been clarified. Recently, we 32'33 demonstrated an additional oculomotor area in the fundus of the coronal sulcus ( C O R ) in which monocular movements of
Eleven adult cats weighing 2 . 5 - 3 . 5 kg were used. U n d e r inhalation anesthesia of halothane ( 1 - 2 % ) mixed with 40% nitrous oxide and 60% oxygen, a tracheostomy was performed and the head of the animal was set in a stereotaxic frame. The cranium was opened in the frontal or parietal region and an acryl round chamber was positioned over a cranial window and fixed to the skull with dental wax. After the spinal cord was transected at C 1 and all wounds infiltrated with 1% lidocaine, the inhalation anesthesia
Correspondence: Y. Tamai, Department of Physiology, Wakayama Medical College, 9-Kyubancho, Wakayama 640, Japan.
92 was discontinued and the animal was maintained with artificial respiration. The animal was then removed from the stereotaxic apparatus and its head was rigidly held by attaching a head-holding device to several screws implanted into the skull. The body temperature of the animal was kept at 37-38 °C by placing a warm pad under the torso. A tungsten-in-glass microelectrode 29 was inserted into the cortex through the chamber filled with warm, liquid paraffin. The exposed tip of the electrode was 15-20/~m in diameter, 30-50~tm in length and filled with a 20% horseradish peroxidase (HRP) solution. The tungsten wire was tightly fixed to the tip of the glass capillary to prevent the H R P solution from leaking. Initially, the Reinoso-Suarez atlas of the cat's brain 23 was used to guide the stimulating elec-
trode. After electrode placement, 10 consecutive pulses (pulse width 0.3 ms, frequency 400 Hz, intensity under 30etA) were delivered to evoke eye movement. The latency of the eye movement was measured by detecting the reflected light from the surface of the eye with a photoelement (Cds). When an eye movement was detected, the H R P solution in the stimulating electrode was injected iontophoretically (5 e t a for 3 min) into the cortex after the tungsten wire was pulled up several millimeters opening the tip of the glass capillary. The stimulating, recording and H R P delivery system is shown in Fig. 1A. Following a survival period of 2 - 3 days, the animal was anesthetized deeply with Nembutal (40 mg/kg) and perfused transcardially with 0.9% saline followed by a phosphate buffer mixture of 1.5% para-
A
B
C
R!
CRU
CO
J
Fig. 1. The experimental paradigm and the site of HRP injections. A: schematic drawing showing the experimental setup used for stimulating, recording and delivering HRP. B: locations of the frontal oculomotor areas, the medial wall of the hemisphere under the CRU (dotted area), the medial (horizontal hatched area) and the lateral (vertical hatched area) banks of the PRE, the fundus of the COR (crosshatched area) where eye movements were evoked by electrical microstimulation. C: low power photomicrograph of the coronal section of the HRP injection site (arrow) outlined in B. CRU, cruciate sulcus; PRE, presylvian sulcus; COR, coronal sulcus.
93 formaldehyde and 4% glutaraldehyde, and finally by a perfusion wash with 10% sucrose. The cerebral cortex was sliced into sections 50/~m thick, incubated in tetramethylbenzidine (TMB) and hydrogen peroxide solution, and processed according to the method of Mesulam 17. The HRP-labeled cells were detected after counterstaining with Neutral red.
A~
a
B~ b
%
RESULTS To identify the frontal 'oculomotor' areas, we stimulated the cortical tissue with each insertion of the microelectrode and confirmed evoked eye movements. We injected HRP into the medial wall of the hemisphere under the CRU (dotted area in Fig. 1B) after confirming the presence of contralateral conjugate eye deviation with a latency of 65 ms (range 60-80 ms). The location of the oculomotor area in the fundus of the C O R (crosshatched area in Fig. 1B) was detected by the presence of a characteristic monocular eye movement, a saccadic medial eye movement with a short latency of 19 ms (range 18-20 ms). The oculomotor areas in the medial (vertical hatched area in Fig. 1B) and the lateral (horizontal hatched area in Fig. 1B) banks of the PRE were respectively identified by the presence of a centering eye movement with a latency of 30 ms (range 20-40 ms) and an eye movement with a latency of 22 ms (range 16-22 ms). Overall, 9 HRP injections were made into the frontal oculomotor areas. Seven preparations, in which the HRP deposit was restricted to the corresponding cortex, were selected. Fig. 1C shows a low power photograph of a coronal section of one of the injection sites (arrow) in the medial wall of the hemisphere under the CRU.
Injection into the medial wall of the hemisphere under the CR U Following HRP injection into the medial wall of the hemisphere under the CRU, HRP-labeled cells were found in the medial bank of the PRE and in the ventral bank of the AES and the insular cortex (IC). A schematic representation, Fig. 2A, shows the typical distribution of the labeled cells in the medial bank of the PRE and the ventral bank of the AES (dotted area) following HRP injection into the medial wall of the hemisphere under the CRU (solid area). It is notable that the labeled cells were found in the third and
Fig. 2. Schematic representation of the distribution of HRPlabeled cells in the frontal oculomotor areas. The HRP injection sites (solid area) are shown in the frontal coronal section (a) of the medial wall of the hemisphere under the CRU (A), the medial (B) and the lateral (C) banks of the PRE and the fundus of the COR (D). Dotted areas in the coronal sections (a and b) indicate the locations of HRP-labeled cells observed in each preparation. CRU, cruciate sulcus; LS, lateral sulcus; SUPS, suprasylvian sulcus; AES, anterior ectosylvian sulcus; SIL, sulcus cerebri lateralis.
fifth layer of the medial bank of the PRE, and in the ventral bank of the AES. The labeled cells in the PRE zone extended widely throughout the medial bank, particularly in the rostral area. The labeled cells in the ventral bank of the AES and the IC were found in the third layer; they extended for about 4 mm along the sulcus. The photomicrographs Fig. 3A a and 3A b show HRP-containing cells (arrows) in the medial bank of the PRE and the ventral bank of the AES, respectively.
Injection into the fundus of the COR. Following HRP injection into the fundus of the COR (solid area in Fig. 2B), HRP-labeled cells were found in the lateral bank of the PRE, and in the fundus and the dorsal banks of the AES (dotted area in Fig. 2B). The labeled cells in the PRE zone (arrow in Fig. 3Ba) were distributed in the third layer of the lateral bank about 4 mm along the sulcus. The density of labeled cells in the lateral bank of the PRE following HRP injection into the fundus of the COR was lower than that in the medial bank of the PRE following HRP injection into the medial wall of the hemisphere
94
Fig. 3. High power photomicrographs of HRP-labeled cells (arrow) shown in Fig. 2. A a and A b indicate some of the HRP-labeled cells in the medial bank of the PRE (a in Fig. 2A) and the ventral bank of the AES (b in Fig. 2A) following HRP injection into the medial wall of the hemisphere under the CRU (solid area in Fig. 2A). The remaining pictures are represented in the same manner.
u n d e r the C R U . T h e l a b e l e d cells detected in the A E S (arrow in Fig. 3Bb) were distributed sparsely in the third layer of the f u n d u s a n d in the dorsal b a n k for 2 - 3 m m along the sulcus.
Injection into the medial and the lateral walls of the PRE. Following H R P i n j e c t i o n into the medial b a n k of the P R E (solid area in Fig. 2C), H R P - l a b e l e d cells
95 were distributed sparsely in the third layer of the ventral bank for 2 - 3 m m along the A E S ( d o t t e d area in Fig. 2Cb and arrow in Fig. 3C). W h e n H R P was injected into the lateral wall of the P R E (solid a r e a in Fig. 2D), the l a b e l e d cells were found widely dispersed in the third layer of the fundus of the A E S , and in the third and fifth layers of the IC ( d o t t e d area in Fig. 2D b and arrow in Fig. 3D). N o l a b e l e d cells were found in the medial wall of the h e m i s p h e r e u n d e r the C R U or the fundus of the C O R . Thus, it b e c a m e evident that neurons in the medial and the lateral banks of the P R E p r o j e c t e d to the medial wall of the h e m i s p h e r e under the C R U and to the
A
C
sops l
fundus of the C O R , respectively, and that all these frontal o c u l o m o t o r areas received fibers from the ventral b a n k of the A E S (Fig. 4C). The labeled cells were found consistently in the third layer of the cortex contralateral to the H R P injection site. No labeled cells were detected in the occipital region including areas 17, 18, 19, or in the lateral suprasylvian visual area following H R P injection into the frontal o c u l o m o t o r areas.
Injection into the A E S In two p r e p a r a t i o n s , H R P was injected into the ventral b a n k of the A E S where labeled cells had been
B
so~
Fig. 4. A: schematic representation of the distribution of HRP-labeled cells (dotted area) in the LS (the ventral bank of the MSS, and the dorsal bank of the PSS) following HRP injection into the AES (solid area). B: a high power photomicrograph of HRP-labeled cells (arrow) in the LS (dotted area) shown in A. C: summary of the corticocortical projections of the frontal oculomotor areas. Arrows indicate the directions of the projections. The neurons in the medial and the lateral banks of the PRE project, respectively, to the medial wall of the hemisphere under the CRU and to the fundus of the COR. All frontal oculomotor areas, the medial wall of the hemisphere under the CRU, the medial and the lateral banks of the PRE and the fundus of the COR receive fibers from the ipsilateral ventral bank of the AES; the AES receives fibers from the LS. SUPS, suprasylvian sulcus; AES, anterior ectosylvian sulcus; COR, coronal sulcus; PRE, presylvian sulcus; CRU, cruciate sulcus, MSS, middle suprasylvian sulcus; PSS, posterior suprasylvian sulcus.
96 found following H R P injection into the frontal oculomotor areas (solid area in Fig. 4A). The ventral bank of the AES contained the neurons characteristically responsive for visual stimuli, such as a rapidly moving target iri the visual field. The labeled cells were observed in the third layer of the cortex along the middle and posterior suprasylvian sulcus (dotted area in Fig. 4Ab and arrow in Fig. 4B). This cortical area was included by Palmer et al. in the caudal part of the lateral suprasylvian visual area (LS) corresponding closely to the posterolateral lateral suprasylvian visual areas (PLLS) and the dorsal lateral suprasylvian visual areas (DLS). A few cells were also detected in the contralateral caudal lateral suprasylvian visual area. No labeled cells could be detected in the frontal cortices responsible for eye movement. The projections from the ventral bank of the A E S to the frontal cortices did not appear to be reciprocal. It is, therefore, likely that the fibers arose from the caudal part of the LS, especially on the ipsilateral side, projected to the ventral bank of the AES, and that the ventral bank of the A E S sends fibers to all frontal oculomotor areas (Fig. 4C). DISCUSSION Information concerning the corticocortical connections for neurons involved in eye movement is scanty, though the afferent system from visual cortex to the frontal cortices has been reported 21314'3°. Hassler n and Shlag et al. 25 have reported that the medial wall of the hemisphere under the C R U and the cortex surrounding the PRE in the cat are responsible for eye movements. Guitton and Mand l° subdivided these frontal oculomotor areas into one medial and one lateral area on the basis of evoked eye movement direction and latency. The medial area consisted of the medial wall of the hemisphere under the C R U and the medial bank of the PRE, where conjugate eye deviation with a shorter latency was evoked. The lateral area consisted of the lateral bank of the PRE where a centering eye movement with longer latency than that in the medial area was induced. Our study using the HRP-tracing method combined with microstimulations strongly supported the existence of a neural relationship between the medial wall of the hemisphere under the C R U and the medial bank of the PRE. That is, the medial wall of the hemi-
sphere under the C R U received fibers from the medial bank of the PRE. On the other hand, the cortical fibers from the lateral bank of the P R E projected to the fundus of the C O R . Therefore, the fundus of the C O R which we have demonstrated 33 to be an additional frontal oculomotor area related to the characteristic monocular movement of the contralateral eye is involved in the lateral area described by Guitton and Mandl 1°. No significant connections were observed between the medial and the lateral areas. Further, it was interesting that the cortical connections, such as those from the presylvian cortex to the coronal sulcus zone or the medial wall of the hemisphere zone, were not reciprocated. The reason for this is that we used as small amount of H R P to match to the small stimulation area for eliciting the eye movements as possible. The possibility of reciprocal innervations cannot be denied in those cortices. We also demonstrated that fibers projected from the ventral bank of the A E S to all the frontal oculomotor areas in cats, while no direct projections from the classical visual cortex or the LS were observed. According to an autoradiographic study with tritiated proline 2~27, the ventral and dorsal banks of the C R U and the adjacent medial surface of the cat brain received direct projections from area 19 and the LS on the ipsilateral side. However, the results cannot be compared directly with ours because the termination sites in their study were not identified as the cortical areas responsible for eye movement. A classical degeneration study 14 and a recent study using axonal transport of H R P - W G A 4 suggested that the frontal superficial cortical area along the coronal sulcus received fibers from the superficial cortex along the A E S or from the secondary somatic sensory area. Our studies proved that the hurried cortices in the frontal cortices also received fibers from the cortex surrounding the AES. Moreover, the restricted area in the burried cortices was physiologically identified as a cortical area responsible for eye movement. Tberefore, all the frontal oculomotor areas may be controlled by the cortex surrounding the AES. In H R P studies in monkeys, the dorsal and the ventral portions of the cortex bound by the arcuate sulcus, including area 8, i.e. frontal eye field, have been reported to receive heavy inputs from the cortices within the superior temporal sulcus and from the inferior temporal cortex 2'5. It has been suggested that
97 the inferior temporal cortex of primate have an important role in the visual processing system 6'8 and might correspond to the cortex surrounding the A E S of the cat 5'6'21. By electrophysiological studies, the cortex surrounding the A E S has been reported to have a unique visual function, different from that of the classical visual area 9'16"t9-21 since the cortex has multimodal sensory receptor fields for visual, auditory and somesthetic input. Graybiel's early work 7 and recent studies with autoradiography 27'28 or various tracers such as H R P 19'2° and fluorescent dyes TM suggest that this cortical area receives convergent afferent input from area of the thalamic L P - p u l v i n a r complex and the bilateral LS. This study also demonstrated that the ipsilateral LS innervated the ventral bank of the A E S where labeled cells had been found following H R P injection into the frontal oculomotor
REFERENCES 1 Barbas, H. and Mesulam, M.-M., Organization of afferent input to subdivisions of area 8 in the Rhesus monkey, J. Comp. Neurol., 200 (1981) 407-431. 2 Battaglini, P.P., Squatrito, S., Gallei, C., Maioli, M.G. and Sanseverino, E.R., Autoradiographic evidence of visual cortical projections to the frontal cortex in the cat, Arch. ltal. Biol., 118 (1980) 189-195. 3 Bizzi, E., Discharge of frontal eye field neurons during saccadic and following eye movements in unanesthetized monkeys, Exp. Brain Res., 6 (1968) 69-80. 4 Burton, H. and Kopf, E.M., Ipsilateral cortical connections from the second and fourth somatic sensory areas in the cat, J. Comp. Neurol., 225 (1984) 527-553. 5 Desimone, R., Fleming, J. and Gross, C.G., Prestriate afferents to inferior temporal cortex: and HRP study, Brain Research, 184 (1980) 41-55. 6 Desimone, R. and Gross, C.G., Visual areas in the temporal cortex of the Macaque, Brain Research, 178 (1979) 363-380. 7 Graybiel, A.M., Some ascending connections of the pulvinar and nucleus lateralis posterior of the thalamus in the cat, Brain Research, 44 (1972) 99-125. 8 Gross, C.G., Rocha-Miranda, C.E. and Bender, D.B., Visual properties of neurons in inferotemporal cortex of the Macaque, J. Neurophysiol., 35 (1972) 96-111. 9 Guiloff, R.D., Lifschitz, W.S., Ormeno, G.O. and Adrian, H.A., Visual evoked potentials in cortical auditory and anterior ectosylvian areas of cat, Vision Res., Suppl., 3 (1971) 339-364. 10 Guitton, D. and Mandl, G., Frontal 'oculomotor' area in alert cat. I. Eye movements and neck muscle activity evoked by stimulation, Brain Research, 149 (1978) 295-312. 11 Guitton, D. and Mandl, G., Frontal 'oculomotor' area in alert cat. II. Unit discharges associated with eye move-
areas. The ventral bank of the A E S may correspond to the ectosylvian visual area (20) or the anterior ectosylvian visual area (19) as the ventral bank of the AES contained the n e u r o n s characteristically responsive for visual stimuli. Therefore, it appears that the ventral bank of the A E S may play an important role in integrating visual, acoustic and somatosensory information which is provided to all the frontal oculomotor areas in the cat. ACKNOWLEDGEMENTS The authors thank Dr. T. Tsujimoto for his support of this experiment, D. Mrozek for editing and T. Tsujinaka for typing this manuscript. This work was supported by grants from the Japanese Ministry of Education, Science and Culture to Y. Tamai.
ments and neck muscle activity, Brain Research, 149 (1978) 313-327. 12 Hassler, R., Extrapyramidal motor areas of cat's frontal lobe: their functional and architectonic differentiation, Int. J. Neurol., 5 (1966) 301-316. 13 Imbert, M., Bignall, K.E. and Buser, P., Neocortical interconnections in the cat, J. Neurophysiol., 29 (1966) 382-395. 14 Kawamura, K., Corticocortical fiber connections of the cat cerebrum. I. The temporal region, Brain Research, 51 (1973) 1-21. 15 Lemmen, J.L., Davis, J.S. and Radnor, L.L., Observations on stimulation of the human frontal eye field, J. Comp. Neurol., 112 (1959) 163-167. 16 Loe, R.R. and Benevento, L.A., Auditory-visual interaction in single units in the orbito-insular cortex of the cat, Electroencephalogr. Clin. Neurophysiol., 26 (1969) 395-398. 17 Mesulam, M.-M., Tetramethyl benzidine for Horseradish Peroxidase neurohistochemistry: a non-carcinogenic blue reaction product with superior sensitivity for visualizing neural afferents and efferents, J. Histochem. Cytochem., 26 (1978) 106-117. 18 Miceli, D., Reperant, J. and Ptito, M., Intracortical connections of the anterior ectosylvian and lateral suprasylvian visual areas in the cat, Brain Research, 347 (1985) 291-298. 19 Mucke, L., Norita, M., Benedek, G. and Creutzfeldt, O., Physiologic and anatomic investigation of a visual cortical area situated in the ventral bank of the anterior ectosylvian sulcus of the cat, Exp. Brain Res., 46 (1982) 1-11. 20 Olson, C.R. and Graybiel, A.M., A visual area in the anterior ectosylvian sulcus of the cat, Soc. Neurosci. Abstr., 7 (1981) 831. 21 Olson, C.R. and Graybiel, A.M., An outlying visual area in the cerebral cortex of the cat, Progr. Brain Res., 58 (1983) 239-245. 22 Palmer, L.A., Rosenquist, A.C. and Tusa, R.J., The reti-
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notopic organization of lateral suprasylvian visual areas in the cat, J. Comp. Neurol., 117 (1978) 237-256. Reinoso-Saurez, F., Topographischer Hirnatlas der Katze fur Experimental-Physiologische Untersuchungen, Merk, Darmstadt, 1961. Robinson, D.A. and Fuchs, A.F., Eye movements evoked by stimulation of frontal eye fields, J. Neurophysiol., 32 (1969) 637-648. Schlag, J. and Schlag-Rey, M., Induction of oculomotor responses by electrical stimulation of the prefrontal cortex in the cat, Brain Research, 22 (1970) 1-13. Smith, W.K., The frontal eye fields. In P.C. Bucy (Ed.), The precentral motor cortex, Univ. Illinois Press, 1949, 308-342. Squatrito, S., Galletti, C., Battaglini, P.P. and Sanseverino, E.R., An autoradiographic study of bilateral cortical projections from cat area 19 and lateral suprasylvian visual area, Arch. ltal. Biol., 119 (1981) 21-42. Squatrito, S., Galletti, C., Maioli, M.G. and Battaglini, P.P., Cortical visual input to the orbito-insular cortex in the cat, Brain Research, 221 (1981) 71-79. Stoney, S. D., Jr., Thompson, W.D. and Asanuma, H., Ex-
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citation of pyramidal tract cells by intracortical microstimulation: effective extent of stimulating current, J. Neurophysiol., 31 (1968) 659-669. Swadlow, H.A., Efferent systems of primary visual cortex: a review of structure and function, Brain Res. Rev., 6 (1983) 1-24. Symonds, L.L. and Rosenquist, A.C., Corticocortical connections among visual areas in the cat, J. Comp. Neurol., 229 (1984) 1-38. Tamai, Y., Fujii, T., Nakai, M., Komai, N. and Tsujimoto, T., Monocular movement of contralateral eye following cortical stimulation in the coronal sulcus of cat, Brain Research, 324 (1984) 138-141. Tamai, Y., Fujii, T., Nakai, M., Komai, N. and Tsujimoto, T., Eye movement evoked by electrical stimulation of the frontal cortex in the coronary sulcus of the cat, Jap. J. Physiol., 33 (1983) 305-308. Wagman, I.H., Krieger, H. P., Papatheodorou, C.A. and Bender, M.B., Eye movements elicited by surface and depth stimulation of the frontal lobe of Macaque mulatta, J. Comp. Neurol., 117 (1961) 179-188.
91
Elsevier BRE 12648
Corticocortical connections of frontal oculomotor areas in the cat Mitsukazu Nakai, Yasuhiko Tamai and Eizo Miyashita Department of Physiology, Wakayama Medical College, Wakayama (Japan) (Accepted 11 November 1986)
Key words: Eye movement; Monocular movement; Frontal eye field; Oculomotor area; Coronal sulcus; Anterior ectosylvian sulcus; Cat
The corticocortical connections of the frontal 'oculomotor' areas related to eye movements of the cat were studied using the retrograde horseradish peroxidase (HRP) tracing method combined with electrophysiological techniques. The following results were obtained. (1) The medial wall of the hemisphere under the cruciate sulcus (CRU), where contralateral conjugate eye deviation was elicited, received fibers from the medial bank of the presylvian sulcus (PRE). The fundus of the coronal sulcus (COR), where monocular movement of the contralateral eye was evoked, received fibers from the lateral bank of the PRE. (2) All the frontal oculomotor areas, the medial wall of the hemisphere under the CRU, the fundus of the COR, and both banks of the PRE, received fibers from the ipsilateral ventral bank of the anterior ectosylvian sulcus (AES). (3) The ventral bank of the AES received fibers from the caudal part of the lateral suprasylvian visual areas. On the basis of the fiber connections, the frontal oculomotor areas can be subdivided into a 'medial' area, the medial wall of the hemisphere under the CRU and the medial bank of the PRE, and a 'lateral' area, the lateral bank of the PRE and the fundus of the COR. Moreover, we found evidence of fiber projections from the ventral bank of the AES to the frontal oculomotor areas that were physiologicallyidentified.
INTRODUCTION It is well known that electrical stimulation of specific regions in the h u m a n and simian frontal lobes, can elicit eye m o v e m e n t s 3'15'24'26'34. It has been suggested that the frontal 'oculomotor' areas responsible for eye movements in the cat are in the medial wall of the hemisphere u n d e r the cruciate sulcus ( C R U ) and the ventral and medial banks of the presylvian sulcus (PRE) 1°'11'25. O n the basis of the direc-
the contralateral eye could be evoked. In the research described here we studied, first, the corticocortical connections of the frontal oculomotor areas in the cat using retrograde H R P tracing combined with electrophysiological techniques. Secondly, we explored the cortical connections to these frontal oculomotor areas from other visually related cortices. MATERIALS AND METHODS
tion and the latency of evoked eye movements, Guitton and Mandl 1° subdivided the frontal oculomotor areas into two subregions, one 'medial' and one 'lateral'. The medial area included the medial wall of the hemisphere u n d e r the C R U and the medial b a n k of the P R E , and the lateral area included the lateral bank of the PRE. However, the anatomical relationships between these frontal oculomotor areas have not been clarified. Recently, we 32'33 demonstrated an additional oculomotor area in the fundus of the coronal sulcus ( C O R ) in which monocular movements of
Eleven adult cats weighing 2 . 5 - 3 . 5 kg were used. U n d e r inhalation anesthesia of halothane ( 1 - 2 % ) mixed with 40% nitrous oxide and 60% oxygen, a tracheostomy was performed and the head of the animal was set in a stereotaxic frame. The cranium was opened in the frontal or parietal region and an acryl round chamber was positioned over a cranial window and fixed to the skull with dental wax. After the spinal cord was transected at C 1 and all wounds infiltrated with 1% lidocaine, the inhalation anesthesia
Correspondence: Y. Tamai, Department of Physiology, Wakayama Medical College, 9-Kyubancho, Wakayama 640, Japan.
92 was discontinued and the animal was maintained with artificial respiration. The animal was then removed from the stereotaxic apparatus and its head was rigidly held by attaching a head-holding device to several screws implanted into the skull. The body temperature of the animal was kept at 37-38 °C by placing a warm pad under the torso. A tungsten-in-glass microelectrode 29 was inserted into the cortex through the chamber filled with warm, liquid paraffin. The exposed tip of the electrode was 15-20/~m in diameter, 30-50~tm in length and filled with a 20% horseradish peroxidase (HRP) solution. The tungsten wire was tightly fixed to the tip of the glass capillary to prevent the H R P solution from leaking. Initially, the Reinoso-Suarez atlas of the cat's brain 23 was used to guide the stimulating elec-
trode. After electrode placement, 10 consecutive pulses (pulse width 0.3 ms, frequency 400 Hz, intensity under 30etA) were delivered to evoke eye movement. The latency of the eye movement was measured by detecting the reflected light from the surface of the eye with a photoelement (Cds). When an eye movement was detected, the H R P solution in the stimulating electrode was injected iontophoretically (5 e t a for 3 min) into the cortex after the tungsten wire was pulled up several millimeters opening the tip of the glass capillary. The stimulating, recording and H R P delivery system is shown in Fig. 1A. Following a survival period of 2 - 3 days, the animal was anesthetized deeply with Nembutal (40 mg/kg) and perfused transcardially with 0.9% saline followed by a phosphate buffer mixture of 1.5% para-
A
B
C
R!
CRU
CO
J
Fig. 1. The experimental paradigm and the site of HRP injections. A: schematic drawing showing the experimental setup used for stimulating, recording and delivering HRP. B: locations of the frontal oculomotor areas, the medial wall of the hemisphere under the CRU (dotted area), the medial (horizontal hatched area) and the lateral (vertical hatched area) banks of the PRE, the fundus of the COR (crosshatched area) where eye movements were evoked by electrical microstimulation. C: low power photomicrograph of the coronal section of the HRP injection site (arrow) outlined in B. CRU, cruciate sulcus; PRE, presylvian sulcus; COR, coronal sulcus.
93 formaldehyde and 4% glutaraldehyde, and finally by a perfusion wash with 10% sucrose. The cerebral cortex was sliced into sections 50/~m thick, incubated in tetramethylbenzidine (TMB) and hydrogen peroxide solution, and processed according to the method of Mesulam 17. The HRP-labeled cells were detected after counterstaining with Neutral red.
A~
a
B~ b
%
RESULTS To identify the frontal 'oculomotor' areas, we stimulated the cortical tissue with each insertion of the microelectrode and confirmed evoked eye movements. We injected HRP into the medial wall of the hemisphere under the CRU (dotted area in Fig. 1B) after confirming the presence of contralateral conjugate eye deviation with a latency of 65 ms (range 60-80 ms). The location of the oculomotor area in the fundus of the C O R (crosshatched area in Fig. 1B) was detected by the presence of a characteristic monocular eye movement, a saccadic medial eye movement with a short latency of 19 ms (range 18-20 ms). The oculomotor areas in the medial (vertical hatched area in Fig. 1B) and the lateral (horizontal hatched area in Fig. 1B) banks of the PRE were respectively identified by the presence of a centering eye movement with a latency of 30 ms (range 20-40 ms) and an eye movement with a latency of 22 ms (range 16-22 ms). Overall, 9 HRP injections were made into the frontal oculomotor areas. Seven preparations, in which the HRP deposit was restricted to the corresponding cortex, were selected. Fig. 1C shows a low power photograph of a coronal section of one of the injection sites (arrow) in the medial wall of the hemisphere under the CRU.
Injection into the medial wall of the hemisphere under the CR U Following HRP injection into the medial wall of the hemisphere under the CRU, HRP-labeled cells were found in the medial bank of the PRE and in the ventral bank of the AES and the insular cortex (IC). A schematic representation, Fig. 2A, shows the typical distribution of the labeled cells in the medial bank of the PRE and the ventral bank of the AES (dotted area) following HRP injection into the medial wall of the hemisphere under the CRU (solid area). It is notable that the labeled cells were found in the third and
Fig. 2. Schematic representation of the distribution of HRPlabeled cells in the frontal oculomotor areas. The HRP injection sites (solid area) are shown in the frontal coronal section (a) of the medial wall of the hemisphere under the CRU (A), the medial (B) and the lateral (C) banks of the PRE and the fundus of the COR (D). Dotted areas in the coronal sections (a and b) indicate the locations of HRP-labeled cells observed in each preparation. CRU, cruciate sulcus; LS, lateral sulcus; SUPS, suprasylvian sulcus; AES, anterior ectosylvian sulcus; SIL, sulcus cerebri lateralis.
fifth layer of the medial bank of the PRE, and in the ventral bank of the AES. The labeled cells in the PRE zone extended widely throughout the medial bank, particularly in the rostral area. The labeled cells in the ventral bank of the AES and the IC were found in the third layer; they extended for about 4 mm along the sulcus. The photomicrographs Fig. 3A a and 3A b show HRP-containing cells (arrows) in the medial bank of the PRE and the ventral bank of the AES, respectively.
Injection into the fundus of the COR. Following HRP injection into the fundus of the COR (solid area in Fig. 2B), HRP-labeled cells were found in the lateral bank of the PRE, and in the fundus and the dorsal banks of the AES (dotted area in Fig. 2B). The labeled cells in the PRE zone (arrow in Fig. 3Ba) were distributed in the third layer of the lateral bank about 4 mm along the sulcus. The density of labeled cells in the lateral bank of the PRE following HRP injection into the fundus of the COR was lower than that in the medial bank of the PRE following HRP injection into the medial wall of the hemisphere
94
Fig. 3. High power photomicrographs of HRP-labeled cells (arrow) shown in Fig. 2. A a and A b indicate some of the HRP-labeled cells in the medial bank of the PRE (a in Fig. 2A) and the ventral bank of the AES (b in Fig. 2A) following HRP injection into the medial wall of the hemisphere under the CRU (solid area in Fig. 2A). The remaining pictures are represented in the same manner.
u n d e r the C R U . T h e l a b e l e d cells detected in the A E S (arrow in Fig. 3Bb) were distributed sparsely in the third layer of the f u n d u s a n d in the dorsal b a n k for 2 - 3 m m along the sulcus.
Injection into the medial and the lateral walls of the PRE. Following H R P i n j e c t i o n into the medial b a n k of the P R E (solid area in Fig. 2C), H R P - l a b e l e d cells
95 were distributed sparsely in the third layer of the ventral bank for 2 - 3 m m along the A E S ( d o t t e d area in Fig. 2Cb and arrow in Fig. 3C). W h e n H R P was injected into the lateral wall of the P R E (solid a r e a in Fig. 2D), the l a b e l e d cells were found widely dispersed in the third layer of the fundus of the A E S , and in the third and fifth layers of the IC ( d o t t e d area in Fig. 2D b and arrow in Fig. 3D). N o l a b e l e d cells were found in the medial wall of the h e m i s p h e r e u n d e r the C R U or the fundus of the C O R . Thus, it b e c a m e evident that neurons in the medial and the lateral banks of the P R E p r o j e c t e d to the medial wall of the h e m i s p h e r e under the C R U and to the
A
C
sops l
fundus of the C O R , respectively, and that all these frontal o c u l o m o t o r areas received fibers from the ventral b a n k of the A E S (Fig. 4C). The labeled cells were found consistently in the third layer of the cortex contralateral to the H R P injection site. No labeled cells were detected in the occipital region including areas 17, 18, 19, or in the lateral suprasylvian visual area following H R P injection into the frontal o c u l o m o t o r areas.
Injection into the A E S In two p r e p a r a t i o n s , H R P was injected into the ventral b a n k of the A E S where labeled cells had been
B
so~
Fig. 4. A: schematic representation of the distribution of HRP-labeled cells (dotted area) in the LS (the ventral bank of the MSS, and the dorsal bank of the PSS) following HRP injection into the AES (solid area). B: a high power photomicrograph of HRP-labeled cells (arrow) in the LS (dotted area) shown in A. C: summary of the corticocortical projections of the frontal oculomotor areas. Arrows indicate the directions of the projections. The neurons in the medial and the lateral banks of the PRE project, respectively, to the medial wall of the hemisphere under the CRU and to the fundus of the COR. All frontal oculomotor areas, the medial wall of the hemisphere under the CRU, the medial and the lateral banks of the PRE and the fundus of the COR receive fibers from the ipsilateral ventral bank of the AES; the AES receives fibers from the LS. SUPS, suprasylvian sulcus; AES, anterior ectosylvian sulcus; COR, coronal sulcus; PRE, presylvian sulcus; CRU, cruciate sulcus, MSS, middle suprasylvian sulcus; PSS, posterior suprasylvian sulcus.
96 found following H R P injection into the frontal oculomotor areas (solid area in Fig. 4A). The ventral bank of the AES contained the neurons characteristically responsive for visual stimuli, such as a rapidly moving target iri the visual field. The labeled cells were observed in the third layer of the cortex along the middle and posterior suprasylvian sulcus (dotted area in Fig. 4Ab and arrow in Fig. 4B). This cortical area was included by Palmer et al. in the caudal part of the lateral suprasylvian visual area (LS) corresponding closely to the posterolateral lateral suprasylvian visual areas (PLLS) and the dorsal lateral suprasylvian visual areas (DLS). A few cells were also detected in the contralateral caudal lateral suprasylvian visual area. No labeled cells could be detected in the frontal cortices responsible for eye movement. The projections from the ventral bank of the A E S to the frontal cortices did not appear to be reciprocal. It is, therefore, likely that the fibers arose from the caudal part of the LS, especially on the ipsilateral side, projected to the ventral bank of the AES, and that the ventral bank of the A E S sends fibers to all frontal oculomotor areas (Fig. 4C). DISCUSSION Information concerning the corticocortical connections for neurons involved in eye movement is scanty, though the afferent system from visual cortex to the frontal cortices has been reported 21314'3°. Hassler n and Shlag et al. 25 have reported that the medial wall of the hemisphere under the C R U and the cortex surrounding the PRE in the cat are responsible for eye movements. Guitton and Mand l° subdivided these frontal oculomotor areas into one medial and one lateral area on the basis of evoked eye movement direction and latency. The medial area consisted of the medial wall of the hemisphere under the C R U and the medial bank of the PRE, where conjugate eye deviation with a shorter latency was evoked. The lateral area consisted of the lateral bank of the PRE where a centering eye movement with longer latency than that in the medial area was induced. Our study using the HRP-tracing method combined with microstimulations strongly supported the existence of a neural relationship between the medial wall of the hemisphere under the C R U and the medial bank of the PRE. That is, the medial wall of the hemi-
sphere under the C R U received fibers from the medial bank of the PRE. On the other hand, the cortical fibers from the lateral bank of the P R E projected to the fundus of the C O R . Therefore, the fundus of the C O R which we have demonstrated 33 to be an additional frontal oculomotor area related to the characteristic monocular movement of the contralateral eye is involved in the lateral area described by Guitton and Mandl 1°. No significant connections were observed between the medial and the lateral areas. Further, it was interesting that the cortical connections, such as those from the presylvian cortex to the coronal sulcus zone or the medial wall of the hemisphere zone, were not reciprocated. The reason for this is that we used as small amount of H R P to match to the small stimulation area for eliciting the eye movements as possible. The possibility of reciprocal innervations cannot be denied in those cortices. We also demonstrated that fibers projected from the ventral bank of the A E S to all the frontal oculomotor areas in cats, while no direct projections from the classical visual cortex or the LS were observed. According to an autoradiographic study with tritiated proline 2~27, the ventral and dorsal banks of the C R U and the adjacent medial surface of the cat brain received direct projections from area 19 and the LS on the ipsilateral side. However, the results cannot be compared directly with ours because the termination sites in their study were not identified as the cortical areas responsible for eye movement. A classical degeneration study 14 and a recent study using axonal transport of H R P - W G A 4 suggested that the frontal superficial cortical area along the coronal sulcus received fibers from the superficial cortex along the A E S or from the secondary somatic sensory area. Our studies proved that the hurried cortices in the frontal cortices also received fibers from the cortex surrounding the AES. Moreover, the restricted area in the burried cortices was physiologically identified as a cortical area responsible for eye movement. Tberefore, all the frontal oculomotor areas may be controlled by the cortex surrounding the AES. In H R P studies in monkeys, the dorsal and the ventral portions of the cortex bound by the arcuate sulcus, including area 8, i.e. frontal eye field, have been reported to receive heavy inputs from the cortices within the superior temporal sulcus and from the inferior temporal cortex 2'5. It has been suggested that
97 the inferior temporal cortex of primate have an important role in the visual processing system 6'8 and might correspond to the cortex surrounding the A E S of the cat 5'6'21. By electrophysiological studies, the cortex surrounding the A E S has been reported to have a unique visual function, different from that of the classical visual area 9'16"t9-21 since the cortex has multimodal sensory receptor fields for visual, auditory and somesthetic input. Graybiel's early work 7 and recent studies with autoradiography 27'28 or various tracers such as H R P 19'2° and fluorescent dyes TM suggest that this cortical area receives convergent afferent input from area of the thalamic L P - p u l v i n a r complex and the bilateral LS. This study also demonstrated that the ipsilateral LS innervated the ventral bank of the A E S where labeled cells had been found following H R P injection into the frontal oculomotor
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