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Evidence for the existence of a substance P-containing pathway from the nucleus laterodorsalis tegmenti (Castaldi) to the lateral septal area of the rat
Brain Research, 230 (1981) 351-355
351
Elsevier/North-Holland Biomedical Press
Evidence for the existence of a substance P-containing pathway from the nucleus laterodorsalis tegmenti (Castaldi) to the lateral septal area of the rat
MASAHIRO SAKANAKA, SADAO SHIOSAKA, KENICHI TAKATSUKI, SHINOBU INAGAKI, HIROSHI TAKAGI, EMIKO SENBA, YURIKO KAWAI, YOSHINOBU HARA, HISASHI IIDA, HIDEO MINAGAWA, TAKASHI MATSUZAKI and MASAYA TOHYAMA Department of Neuroanatomy, Institute of'Higher Nervous Activity, Osaka University Medical School, 3-57 Nakanoshima 4-chome, Kitaku, Osaka (530) (Japan)
(Accepted September 10th, 1981) Key words: substance P - - n. laterodorsalis tegmenti - - septum
The existence of a long ascending substance P (SP)-containing neuron system from the nucleus laterodorsalis tegmenti of Castaidi to the lateral septal area was demonstrated in this study. The destruction of the n. laterodorsalis tegmenti resulted in a marked reduction of SP-positive fibers in the iosilateral lateral septal area. Recent immunohistochemical studies have shown that the lateral septal area (SL) contains numerous substance P (SP)-containing fibersS, 7. However, as to the origins of these fibers little is known. The present study describes the presence of a long ascending SP pathway from the n. laterodorsalis tegmenti (TLD) of Castaldi 1 to the SL. [It should be noted that although Castaldi included the locus coeruleus and Barrington's nucleus in the T L D 1, we excluded these regions from the T L D (see also ref. 17).] A total of 47 male rats (body weight 30-40 g) were used in an immunohistochemical study. In order to determine the origin of SP-positive fibers in the SL, several kinds of operations were made stereotaxically, such as unilateral or bilateral electrolytic destruction of the TLD, hemitransection of the brain stem just caudal to the T L D , and electrolytic lesions or hemitransectiort of the medial forebrain bundle (MFB) at various levels, respectively. Animals were kept alive for 3-7 days after operations. All animals were perfused via the ascending aorta with 50 cc of ice-cold saline followed by 200 cc of Zamboni's fixative is. The brain was removed stereotaxically with a stereotaxic apparatus and immersed in the same fixative for 6 h and then rinsed for 24-28 h in phosphate buffer containing 30 9/00 sucrose. Serial fiontal, sagittal or horizontal frozen sections were cut in a cryostat with a section thickness of 10 /~m, paying attention to the stereotaxic levels of the sections. After sectioning, the sections were immediately processed for the indirect immunofluorescent technique of Coons 2. Since absorption of antiserum by excessive synthetic SP completely eliminates immunostaining, the structures stained with SP antiserum are considered specific (as to specifi0006-8993/81/0000-0000/$02.75 © Elsevier/North-Holland Biomedical Press
352
IV
Fig. 1. A: fluorescence photomicrograph showing numerous SP-positive cells in the TLD. Frontal section. Case SL 7. ;~62. B and C: light-field photomicrographs showing an injection site of HRP in the SL and HRP-labeled cells in the TLD subsequent to HRP injection into the SL, respectively. Case SL 7. Frontal sections. Case SL 38. ~<13 (B), ~<115 (C). D: light-field photomicrograph showing the electrical lesion of the TLD (indicated by arrowheads). Frontal section. Case SL 29. 28. city see also refs. 5, 9 and 12). The positive structures identified in this study should correctly be described as SP-like immunoreactive, but in this study we shall use the simpler term SP-positive. A total o f 8 rats were used in a horseradish peroxidase ( H R P ) study. A solution o f 40 ~o H R P was i o n t o p h o r e t i c a l l y injected into the SL with the help o f a stereotaxic a p p a r a t u s . One or two days after injection, the rat brain was subjected to H R P treatment 10. T e r m i n o l o g y was based u p o n the atlas o f K 6 n i g a n d Klippel ~, Palkovits and J a c o b o w i t z 11, Swanson a n d C o w a n 15 a n d T o h y a m a et al. 17. A s shown in Fig. I A , n u m e r o u s SP-positive cells were f o u n d in the T L D . Unilateral destruction o f the T L D (Fig. 1D) resulted in a m a r k e d reduction o f SPpositive fibers in the SL on the o p e r a t e d side (Fig. 2A, B). It should be stressed that this a l t e r a t i o n was confined to the dorsal (d), intermediate (i) a n d ventral (v) parts o f the SL from the level o f A8380# to A6790# o f the atlas of K 6 n i g a n d K l i p p e l 6. However,
353
Fig. 2. A and B. destruction of the TLD resulted in a marked decrease of SP-positive fibers in the ipsilateral SL. Control side (A), operated side (B). Frontal sections. Case SL 29. x 50 (A), x 50 (B). C and D: destruction of the medial forebrain bundle caused much more pronounced reduction of SP-positive fibers in the SL on the operated side, suggesting the presence of other origins of SP-positive fibers in the SL in addition to the TLD SP cells. Control side (C), operated side (D). Frontal sections. Case SL 47. x 50 (A), x 50 (B).
354 no changes of SP-positive fibers were detected in other SL areas after destruction of the unilateral TLD. Bilateral destruction of the TLD also caused a marked decrease of SP-positive fibers in the bilateral SL at the levels mentioned above, as compared with normal rats. No alterations were identified in other SL areas subsequent to bilateral destruction of the TLD. In addition, transection of the lower brain stem just caudal to the T L D failed t6 demonstrate any changes of SP-positive fibers throughout tbe whole extent of the SL, even in areas where a marked decrease of SP-pesitive fibers was seen after destruction of the TLD. These facts strongly indicate that SP-positive cells situated in the TLD project ipsilaterally to the SL from the level of A8380# to A6790/~ of the atlas of K6nig and Klippel 6. In support of this, HRP injection into the SL (Fig. IB) resulted in demonstrating HRP-labeled cells in the T L D on the ipsilateral side. The locations of these labeled cells are very similar to the distribution of SP-positive cells in the TLD (cf. Fig. 1A and C). SP-positive fibers originating in the TLD which project to the SL seem to pass the MFB, because destruction of the MFB at the level of the n. ventromedialis hypothalami (VMH)resulted in a similar decrease pattern of SP-positive fibers in the ipsilateral SL (from the level of A8380# to A6790/~) to that found in the SL after destruction of the TLD. Furthermore, it should be noted that following destruction of the TLD, a significant number of SP-positive fibers was still found (Fig. 2B). These facts suggest that SL is also innervated by other SP-positive cells in addition to T L D SP-positive cells. In support of this suggestion, destruction of the MFB at the level of the anterior hypothalamic nucleus (AH) resulted in much more pronounced decrease of SPpositive fibers in the ipsilateral SL (Fig. 2C, D) than that found in the SL after T L D destruction (cf. Fig. 2B and D) or destruction of MFB at the VMH level. These findings suggest that (1) other origins of SP-positive fibers of the SL except for the T L D SP-positive cells, might exist in the MFB at a level between the AH and VMH or (2) they are located in the extra MFB area whose axons joined the MFB at a level between the AH and VMH to reach the SL. Thus, the present study clearly demonstrates the presence of the SP pathway from T L D to SL. It has been well known by means of H R P method that the nemons of the TLD give rise to long ascending projections to the forebrain via MFB 8,13,16A7, though their exact terminal fields have still been obscure. Based upon the present and previous findings 8,13,16,17, T L D is one of the major sources of septal SP and might play an important role in regulation of septal function such as motivational, emotional and associative processes 4,15 together with well-known other afferent sources to the septal area 14. In addition, the facts that SL is innervated by more than one SP neuron system (TLD and others) suggest complicated functions of SP in the SL, though exact functions of SP are now open to discussion.
1 Castaldi, L., Studi sulla struttura e sullo sviluppo del mesencefalo.Ricerche in Cavia cobaya, lIl, Arch. ital. Anat. EmbrioL, 23 (1926) 481-609. 2 Coons, A. H., Fluorescent antibody methods. In J. F. Danielli (Ed.), General Cytochemical Methods, Academic Press, New York, NY, 1958, pp. 399~,22.
355 3 Cuello, A. C. and Kanazawa, I., The distribution of substance P immunoreactive fibers in the rat central nervous system, J. comp. Neurol., 178 (1978) 129-156. 4 De France, J. F. (Ed.), The Septal Nuclei, Plenum, New York, NY, 1976. 5 Inagaki, S., Senba, E., Shiosaka, S., Takagi, H., Kawai, Y., Takatsuki, K., Sakanaka, M., Matsuzaki, T. and Tohyama, M., Regional distribution of substance P-like immunoreactivity in the frog brain and spinal cord: immunohistochemical analysis, J. comp. NeuroL, 201 (1981) 243-254. 6 K/~nig, J. F. R. and Klippel, R. A., The Rat Brain. A Stereotaxic Atlas of the Forebrain and Lower parts of the Brain Stem, Williams and Wilkins, Baltimore, MD, 1963. 7 Ljungdahl, A., Htikfelt, T. and Nilsson, G., Distribution of substance P-like immunoreactivity in the central nervous system of the rat. I. Cell bodies and nerve terminals, Neuroscience, 3 (1978) 861-943. 8 Llamas, A., Reinoso-Stiarez, F. and Martinez-Moreno, E., Projections to the gyrus proreus from the brain stem tegmentum (locus coeruleus, raphe nuclei) in the cat, demonstrated by retrograde transport of horseradish peroxidase, Brain Research, 89 (1975) 331-336. 9 Matsuzaki, T., Shiosaka, S., Inagaki, S., Sakanaka, M., Takatsuki, K., Takagi, H., Senba, E., Kawai, Y. and Tohyama, M., Distribution of neuropeptides in the dorsal pontine tegmental area of the rat, Cell. tool Biol., (1981) in press. 10 Mesulam, M.-M., Tetramethylbenzidine for horseradish peroxidase neurohistochemistry: a noncarcinogenic blue reaction product with superior sensitivity for visualizing neuronal afferents and efferents, J. Histochem. Cytochem., 26 (1978) 106-117. 11 Palkovits, M. and Jacobowitz, D. M., Topographic atlas of catecholamine and acetylcholinesterase-containing neurons in the rat brain. II. Hindbrain (mesencephalon, rhombencephalon), J. comp. NeuroL, 157 (1974) 2942. 12 Sakanaka, M., Shiosaka, S., Takatsuki, K., Inagaki, S., Takagi, H., Senba, E., Kawai, Y., Matsuzaki, T. and Tohyama, M., Experimental immunohistochemical studies on the amygdalofugal peptidergic (substance P and somatostatin) fibers in the stria terminalis of the rat, Brain Research, 221 (1981) 231-242. 13 Sakumoto, T., Tohyama, M., Satoh, K., Kimoto, Y., Kinugasa, T., Tanizawa, O., Kurachi, K. and Shimizu, N., Afferent fiber connections from lower brain stem to hypothalamus studied by the horseradish peroxidase method with special reference to noradrenaline innervation, Exp. Brain Res., 31 (1978) 81-94. 14 Segal, M. and Landis, S. C., Afferents to the septal area of the rat studied with the method of retrograde axonal transport of horseradish peroxidase, Brain Research, 82 (1974) 263-268. 15 Swanson, L. W. and Cowan, W. M., The connections of the septal region in the rat, J. comp. Neurol., 186 (1979) 621-656. 16 Takagi, H., Shiosaka, S., Tohyama, M., Senba, E. and Sakanaka, M., Ascending components of the medial forebrain bundle from the lower brain stem in the rat, with special reference to raphe and catecholamine cell groups. A study by the HRP method, Brain Research, 193 (1980) 315-337. 17 Tohyama, M., Satoh, K., Sakumoto, T., Kimoto, Y., Takahashi, Y., Yamamoto, K. and Itakura, T., Organization and projections of the neurons in the dorsal tegmental area of the rat, J. Hirnforsch., 19 (1978) 165-176. 18 Zamboni, L. and De Martino, C., Buffered picric-acid formaldehyde: a new rapid fixative for electron microscopy, J. Cell Biol., 35 (1967) 148A.
ABBREVIATIONS CC d i ntd ntm
corpus callosum dorsal part of the SL intermediate part of the SL n. tegmenti dorsalis (Gudden) n. tractus mesencephali nervi trigemini
PCS TLD v VL IV
pedunculus cerebellaris superior n. laterodorsalis tegmenti ventral part of the SL ventriculus lateralis ventriculus quartus
351
Elsevier/North-Holland Biomedical Press
Evidence for the existence of a substance P-containing pathway from the nucleus laterodorsalis tegmenti (Castaldi) to the lateral septal area of the rat
MASAHIRO SAKANAKA, SADAO SHIOSAKA, KENICHI TAKATSUKI, SHINOBU INAGAKI, HIROSHI TAKAGI, EMIKO SENBA, YURIKO KAWAI, YOSHINOBU HARA, HISASHI IIDA, HIDEO MINAGAWA, TAKASHI MATSUZAKI and MASAYA TOHYAMA Department of Neuroanatomy, Institute of'Higher Nervous Activity, Osaka University Medical School, 3-57 Nakanoshima 4-chome, Kitaku, Osaka (530) (Japan)
(Accepted September 10th, 1981) Key words: substance P - - n. laterodorsalis tegmenti - - septum
The existence of a long ascending substance P (SP)-containing neuron system from the nucleus laterodorsalis tegmenti of Castaidi to the lateral septal area was demonstrated in this study. The destruction of the n. laterodorsalis tegmenti resulted in a marked reduction of SP-positive fibers in the iosilateral lateral septal area. Recent immunohistochemical studies have shown that the lateral septal area (SL) contains numerous substance P (SP)-containing fibersS, 7. However, as to the origins of these fibers little is known. The present study describes the presence of a long ascending SP pathway from the n. laterodorsalis tegmenti (TLD) of Castaldi 1 to the SL. [It should be noted that although Castaldi included the locus coeruleus and Barrington's nucleus in the T L D 1, we excluded these regions from the T L D (see also ref. 17).] A total of 47 male rats (body weight 30-40 g) were used in an immunohistochemical study. In order to determine the origin of SP-positive fibers in the SL, several kinds of operations were made stereotaxically, such as unilateral or bilateral electrolytic destruction of the TLD, hemitransection of the brain stem just caudal to the T L D , and electrolytic lesions or hemitransectiort of the medial forebrain bundle (MFB) at various levels, respectively. Animals were kept alive for 3-7 days after operations. All animals were perfused via the ascending aorta with 50 cc of ice-cold saline followed by 200 cc of Zamboni's fixative is. The brain was removed stereotaxically with a stereotaxic apparatus and immersed in the same fixative for 6 h and then rinsed for 24-28 h in phosphate buffer containing 30 9/00 sucrose. Serial fiontal, sagittal or horizontal frozen sections were cut in a cryostat with a section thickness of 10 /~m, paying attention to the stereotaxic levels of the sections. After sectioning, the sections were immediately processed for the indirect immunofluorescent technique of Coons 2. Since absorption of antiserum by excessive synthetic SP completely eliminates immunostaining, the structures stained with SP antiserum are considered specific (as to specifi0006-8993/81/0000-0000/$02.75 © Elsevier/North-Holland Biomedical Press
352
IV
Fig. 1. A: fluorescence photomicrograph showing numerous SP-positive cells in the TLD. Frontal section. Case SL 7. ;~62. B and C: light-field photomicrographs showing an injection site of HRP in the SL and HRP-labeled cells in the TLD subsequent to HRP injection into the SL, respectively. Case SL 7. Frontal sections. Case SL 38. ~<13 (B), ~<115 (C). D: light-field photomicrograph showing the electrical lesion of the TLD (indicated by arrowheads). Frontal section. Case SL 29. 28. city see also refs. 5, 9 and 12). The positive structures identified in this study should correctly be described as SP-like immunoreactive, but in this study we shall use the simpler term SP-positive. A total o f 8 rats were used in a horseradish peroxidase ( H R P ) study. A solution o f 40 ~o H R P was i o n t o p h o r e t i c a l l y injected into the SL with the help o f a stereotaxic a p p a r a t u s . One or two days after injection, the rat brain was subjected to H R P treatment 10. T e r m i n o l o g y was based u p o n the atlas o f K 6 n i g a n d Klippel ~, Palkovits and J a c o b o w i t z 11, Swanson a n d C o w a n 15 a n d T o h y a m a et al. 17. A s shown in Fig. I A , n u m e r o u s SP-positive cells were f o u n d in the T L D . Unilateral destruction o f the T L D (Fig. 1D) resulted in a m a r k e d reduction o f SPpositive fibers in the SL on the o p e r a t e d side (Fig. 2A, B). It should be stressed that this a l t e r a t i o n was confined to the dorsal (d), intermediate (i) a n d ventral (v) parts o f the SL from the level o f A8380# to A6790# o f the atlas of K 6 n i g a n d K l i p p e l 6. However,
353
Fig. 2. A and B. destruction of the TLD resulted in a marked decrease of SP-positive fibers in the ipsilateral SL. Control side (A), operated side (B). Frontal sections. Case SL 29. x 50 (A), x 50 (B). C and D: destruction of the medial forebrain bundle caused much more pronounced reduction of SP-positive fibers in the SL on the operated side, suggesting the presence of other origins of SP-positive fibers in the SL in addition to the TLD SP cells. Control side (C), operated side (D). Frontal sections. Case SL 47. x 50 (A), x 50 (B).
354 no changes of SP-positive fibers were detected in other SL areas after destruction of the unilateral TLD. Bilateral destruction of the TLD also caused a marked decrease of SP-positive fibers in the bilateral SL at the levels mentioned above, as compared with normal rats. No alterations were identified in other SL areas subsequent to bilateral destruction of the TLD. In addition, transection of the lower brain stem just caudal to the T L D failed t6 demonstrate any changes of SP-positive fibers throughout tbe whole extent of the SL, even in areas where a marked decrease of SP-pesitive fibers was seen after destruction of the TLD. These facts strongly indicate that SP-positive cells situated in the TLD project ipsilaterally to the SL from the level of A8380# to A6790/~ of the atlas of K6nig and Klippel 6. In support of this, HRP injection into the SL (Fig. IB) resulted in demonstrating HRP-labeled cells in the T L D on the ipsilateral side. The locations of these labeled cells are very similar to the distribution of SP-positive cells in the TLD (cf. Fig. 1A and C). SP-positive fibers originating in the TLD which project to the SL seem to pass the MFB, because destruction of the MFB at the level of the n. ventromedialis hypothalami (VMH)resulted in a similar decrease pattern of SP-positive fibers in the ipsilateral SL (from the level of A8380# to A6790/~) to that found in the SL after destruction of the TLD. Furthermore, it should be noted that following destruction of the TLD, a significant number of SP-positive fibers was still found (Fig. 2B). These facts suggest that SL is also innervated by other SP-positive cells in addition to T L D SP-positive cells. In support of this suggestion, destruction of the MFB at the level of the anterior hypothalamic nucleus (AH) resulted in much more pronounced decrease of SPpositive fibers in the ipsilateral SL (Fig. 2C, D) than that found in the SL after T L D destruction (cf. Fig. 2B and D) or destruction of MFB at the VMH level. These findings suggest that (1) other origins of SP-positive fibers of the SL except for the T L D SP-positive cells, might exist in the MFB at a level between the AH and VMH or (2) they are located in the extra MFB area whose axons joined the MFB at a level between the AH and VMH to reach the SL. Thus, the present study clearly demonstrates the presence of the SP pathway from T L D to SL. It has been well known by means of H R P method that the nemons of the TLD give rise to long ascending projections to the forebrain via MFB 8,13,16A7, though their exact terminal fields have still been obscure. Based upon the present and previous findings 8,13,16,17, T L D is one of the major sources of septal SP and might play an important role in regulation of septal function such as motivational, emotional and associative processes 4,15 together with well-known other afferent sources to the septal area 14. In addition, the facts that SL is innervated by more than one SP neuron system (TLD and others) suggest complicated functions of SP in the SL, though exact functions of SP are now open to discussion.
1 Castaldi, L., Studi sulla struttura e sullo sviluppo del mesencefalo.Ricerche in Cavia cobaya, lIl, Arch. ital. Anat. EmbrioL, 23 (1926) 481-609. 2 Coons, A. H., Fluorescent antibody methods. In J. F. Danielli (Ed.), General Cytochemical Methods, Academic Press, New York, NY, 1958, pp. 399~,22.
355 3 Cuello, A. C. and Kanazawa, I., The distribution of substance P immunoreactive fibers in the rat central nervous system, J. comp. Neurol., 178 (1978) 129-156. 4 De France, J. F. (Ed.), The Septal Nuclei, Plenum, New York, NY, 1976. 5 Inagaki, S., Senba, E., Shiosaka, S., Takagi, H., Kawai, Y., Takatsuki, K., Sakanaka, M., Matsuzaki, T. and Tohyama, M., Regional distribution of substance P-like immunoreactivity in the frog brain and spinal cord: immunohistochemical analysis, J. comp. NeuroL, 201 (1981) 243-254. 6 K/~nig, J. F. R. and Klippel, R. A., The Rat Brain. A Stereotaxic Atlas of the Forebrain and Lower parts of the Brain Stem, Williams and Wilkins, Baltimore, MD, 1963. 7 Ljungdahl, A., Htikfelt, T. and Nilsson, G., Distribution of substance P-like immunoreactivity in the central nervous system of the rat. I. Cell bodies and nerve terminals, Neuroscience, 3 (1978) 861-943. 8 Llamas, A., Reinoso-Stiarez, F. and Martinez-Moreno, E., Projections to the gyrus proreus from the brain stem tegmentum (locus coeruleus, raphe nuclei) in the cat, demonstrated by retrograde transport of horseradish peroxidase, Brain Research, 89 (1975) 331-336. 9 Matsuzaki, T., Shiosaka, S., Inagaki, S., Sakanaka, M., Takatsuki, K., Takagi, H., Senba, E., Kawai, Y. and Tohyama, M., Distribution of neuropeptides in the dorsal pontine tegmental area of the rat, Cell. tool Biol., (1981) in press. 10 Mesulam, M.-M., Tetramethylbenzidine for horseradish peroxidase neurohistochemistry: a noncarcinogenic blue reaction product with superior sensitivity for visualizing neuronal afferents and efferents, J. Histochem. Cytochem., 26 (1978) 106-117. 11 Palkovits, M. and Jacobowitz, D. M., Topographic atlas of catecholamine and acetylcholinesterase-containing neurons in the rat brain. II. Hindbrain (mesencephalon, rhombencephalon), J. comp. NeuroL, 157 (1974) 2942. 12 Sakanaka, M., Shiosaka, S., Takatsuki, K., Inagaki, S., Takagi, H., Senba, E., Kawai, Y., Matsuzaki, T. and Tohyama, M., Experimental immunohistochemical studies on the amygdalofugal peptidergic (substance P and somatostatin) fibers in the stria terminalis of the rat, Brain Research, 221 (1981) 231-242. 13 Sakumoto, T., Tohyama, M., Satoh, K., Kimoto, Y., Kinugasa, T., Tanizawa, O., Kurachi, K. and Shimizu, N., Afferent fiber connections from lower brain stem to hypothalamus studied by the horseradish peroxidase method with special reference to noradrenaline innervation, Exp. Brain Res., 31 (1978) 81-94. 14 Segal, M. and Landis, S. C., Afferents to the septal area of the rat studied with the method of retrograde axonal transport of horseradish peroxidase, Brain Research, 82 (1974) 263-268. 15 Swanson, L. W. and Cowan, W. M., The connections of the septal region in the rat, J. comp. Neurol., 186 (1979) 621-656. 16 Takagi, H., Shiosaka, S., Tohyama, M., Senba, E. and Sakanaka, M., Ascending components of the medial forebrain bundle from the lower brain stem in the rat, with special reference to raphe and catecholamine cell groups. A study by the HRP method, Brain Research, 193 (1980) 315-337. 17 Tohyama, M., Satoh, K., Sakumoto, T., Kimoto, Y., Takahashi, Y., Yamamoto, K. and Itakura, T., Organization and projections of the neurons in the dorsal tegmental area of the rat, J. Hirnforsch., 19 (1978) 165-176. 18 Zamboni, L. and De Martino, C., Buffered picric-acid formaldehyde: a new rapid fixative for electron microscopy, J. Cell Biol., 35 (1967) 148A.
ABBREVIATIONS CC d i ntd ntm
corpus callosum dorsal part of the SL intermediate part of the SL n. tegmenti dorsalis (Gudden) n. tractus mesencephali nervi trigemini
PCS TLD v VL IV
pedunculus cerebellaris superior n. laterodorsalis tegmenti ventral part of the SL ventriculus lateralis ventriculus quartus