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Evidence for the existence of a substance P-containing pathway from the nucleus laterodorsalis tegmenti (Castaldi) to the medial frontal cortex of the rat
123
Brain Research, 259 (1983) 123-126 Elsevier Biomedical Press
Evidence for the existence of a substance P-containing pathway from the nucleus laterodorsalis tegmenti (Castaldi) to the medial frontal cortex of the rat MASAHIRO SAKANAKA, SADAO SHIOSAKA, KENICHI TAKATSUKI and MASAYA TOHYAMA
Department of Neuroanatomy, Institute of Higher Nervous Activity, Osaka University Medical School, 3-5 7 Nakanoshima 4-Chome, Kitaku Osaka 530 (Japan) (Accepted August 31st, 1982)
Key words: substance P - n. laterodorsalis tegmenti - medial frontal cortex
A long ascending substance P (SP)-containing pathway from the nucleus laterodorsalis tegmenti (TLD) to the medial frontal cortex (MFC) has been demonstrated by means of experimental immunohistochemicalprocedures. The unilateral destruction of the TLD, which contains numerous SP-like immunoreactive (SPLI) cells, resulted in a marked ipsilateral decrease in the SPLI fibers in the MFC.
Recent immunohistochemical studies have revealed that the medial frontal cortex (MFC) contains a number of substance P-like immunoreactive (SPLI) fibers4,6,8. However, little is known of the origins of these SPLI fibers in the MFC, though a suggestion has been made by Paxinos et al) 2. The present study describes the existence of a substance P (SP)-containing pathway from the nucleus laterodorsalis tegmenti (TLD) to the MFC. (It should be noted that although Castaldi included the locus coeruleus and Barrington's nucleus in the TLD 2, we exclude these regions from the TLD in our studyl4.) Twenty-eight male rats (body weight 40-60 g) were used in an immunohistochemical study. In order to determine the origins of the SPLI fibers in the MFC, several kinds of operations were made stereotaxically, such as unilateral destruction of the TLD, bilateral destruction of the TLD and hemi- or total transection of the brain just caudal to the TLD, respectively. The animals were kept alive for 4-7 days after the operations. Each animal for the immunohistochemistry was perfused via the ascending aorta with 50 cc of ice-cold saline followed by 300 cc Zamboni's fluid ~s. The brain was quickly removed and 0006-8993/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press
immersed in the same fixative for 48 h at 4 °C, and then rinsed for 24-48 h in 0.1 M phosphate buffer containing 30% sucrose. Serial frontal, sagittal and horizontal sections were cut on 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 3. The specificity of the antiserum against SP was tested by the radioimmunoassay system. The cross-reactivity of this antibody compared with structurally similar peptides, eledoisin, and physalemin, was less than 0.02%, and compared with other related peptides (somatostatin, glucagon, bradykinin, enkephalin and endorphin) was less than 0.01%. Specificity was also tested by the absorption test. Since the structures stained with SP antiserum were not seen in the sections stained with the control serum (for which SP antiserum absorbed by excess SP (80/tg/cc) was used), the structures stained with SP antiserum were considered specific. Six male rats were used in a horseradish peroxidase (HRP) study. A solution of 40% HRP was~ iontophoretically injected into the MFC with the help of stereotaxic apparatus. One or two
124
Fig. 1. A: the unilateral destruction of the TLD caused a marked ipsilateral reduction in the coarse SPLI fibers in the MFC, while the fine terminals remain intact in laminae II and IV even after this lesion. Left side: control. Right side: operated side. Frontal section, case 24, x 70. B: fluorescent photomicrograph showing a higher magnification of the area indicated by the rectangle in A. The very fine terminals in lamina II, which remained intact following the destruction of the TLD, can be recognized under magnification. Left side: control. Right side: operated side. Frontal section, case 24, x 140. Abbreviations: 1I, lamina II; III, lamina III; IV, lamina IV.
days after being injected, the rat brains were subjected to H R P treatment 9. Termimology was based upon the atlas of K6nig and Klippel 7, Palkovits and Jacobowitz 1~, and Tohyama et al. j4. As seen in Fig. 1, a moderate number of SPLI fibers were observed in the M F C ranging from the level o f A 12760 ~tm to A 8920/zm in the atlas of K6nig and Klippel 7, though they did not show a uniform distribution. These SPLI fibers appeared to be composed of two types: one of relatively coarse varicose fibers in laminae II, III and IV often forming bands perpendicular to the brain surface, and the other of very fine terminals with a perineuronal appearance especially in laminae II and IV (Fig. 1). It should be stressed that the coarse fibers were affected by the destruction of the TLD, but the fine termi-
nals were not affected by this lesion (see precisely below). The unilateral destruction of the TLD (Fig. 2A), which is very rich in SPLI cells (Fig. 2B), resuited in a marked ipsilateral decrease in the coarse SPLI fibers in the M F C at the levels mentioned above (Fig. 1), while this operation failed to cause any changes in the fine SPLI terminals in the M F C (Fig. 1). The bilateral destruction of the TLD also caused a marked reduction in the coarse SPLI fibers bilaterally in the M F C similar to that found ipsilaterally in the M F C subsequent to the unilateral destruction of the TLD, compared to the number of coarse SPLI fibers in normal rats. On the other hand, the hemi- or total transection of the brain just caudal to the TLD failed to demonstrate any alterations in the SPLI fibers in the MFC. These facts strongly
125
!! !il ill ~i
c i I t g
~'j
I
!D Fig. 2. A: fight-field photomicrograph showing the electric lesion of the TLD (indicated by arrowheads). Frontal section. Case 24, X 31. B: fluorescent photomicrograph showing numerous SPLI cells in the TLD. Frontal section. Case 3, x 85. C and D: light-field photomicrograph showing an injection site of HRP into the MFC (C) and HRP-labeled cells occurring in the TLD subsequent to HRP injection into the MFC (D), respectively. The same case. Frontal sections. Case 5; C, x 10; D, x 125. Abbreviations: ntd, n. tegmenti dorsalis (Gudden); PCS, pedunculus cerebellaris superior; TLD, n. laterodorsalis tegmenti.
suggest that the SPLI cells located in the T L D send their long ascending fibers to innervate the ipsilateral MFC. In support of this, many HRPlabeled cells were identified in the T L D following H R P injection into the M F C (Fig. 2C,D). The locations of these labeld cells were very similar to those of SPLI cells in the T L D (Fig. 2B,D). Thus, the present study demonstrated that the coarse SPLI fibers in the M F C arise ipsilaterally from the SPLI cells in the TLD. Furthermore, it should be noted that the fine terminals remain intact in the M F C after the destruction of the TLD. This fact indicates that this type of SPLI terminals is innervated by other origins apart from the TLD.
It has been reported previously by means of the H R P method that the neurons of the TLD give rise to long ascending fibers to the M F C 14. The present study has clearly confirmed this projection and further shown that the SP-containing pathway is a major component of this T L D M F C tract. On the basis of the previous study ~3, SPLI cells in the TLD also appear to project to the lateral septal area (LS). Although it is not clear from the present study whether or not a single SPLI cell projects simultaneously to the LS and to the MFC, the SPLI cells in the TLD might be related to the various functions of the forebrain, particularly to delayed alternation, and motivational, emotional and associative larocesses 1,5,10.
126 1 Brito, G. N. O., Thomas, G. J., Davis, B. J. and Gingold, S. I., Prelimbic cortex, mediodorsal thalamus, septum, and delayed alternation in rats, Exp. Brain Res., 46 (1982) 52-58. 2 Castaldi, L., Studi sulla struttura e sullo sviluppo del mesencephalo. Ricerche in Cavia cobaya. III, Arch. ital. A nato. Embriol., 23 (1926) 481 ~09. 3 Coons, A. H., Fluorescent antibody methods. In J. F. Danielli (Ed.), General Cytochemical Methods, Academic Press, New York, NY, 1958, pp. 399-422. 4 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. 5 De France, J. F. (Ed.), The Septal Nuclei, Plenum, New York, 1976. 6 Inagaki, S., Sakanaka, M., Shiosaka, S., Senba, E., Takatsuki, K., Takagi, H., Kawai, Y., Minagawa, H. and Tohyama, M., Ontogeny of substance P-containing neuron system of the rat: Immunohistochemical analysis - I. Forebrain and upper brain stem, Neuroscience, 7 (1982) 251-277. 7 KOnig, 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. 8 Ljungdahl, A., Hrkfelt, 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.
9 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. 10 Miles, R. C. and Blomquist, A. J., Frontal lesions and behavioral deficits, J. Neurophysiol., 23 (1960) 471-484. 11 Palkovits, M. and Jacobowitz, D. M., Topographic atlas of catecholamine and acetylcholinesterase-codtaining neurons in the rat brain. II. Hindbrain (mesencephalon, rhombencephalon), J. comp. Neurol., 157 (1974) 29-42. 12 Paxinos, G., Emson, P. C. and Cuello, A. C., The substance P projections to the frontal cortex and the substantia nigra, Neurosci. Lett., 7 (1978) 127-131. 13 Sakanaka, M., Shiosaka, S., Takatsuki, K., Inagaki, S., Takagi, H., Senba, E., Kawai, Y., Hara, Y., Iida, H., Minagawa, H., Matsuzaki, T. and Tohyama, M., 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 (I 981) 351-355. 14 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. 15 Zamboni, L. and De Martino, C., Buffered picric-acid formaldehyde: a new rapid fixative for electron microscopy, J. CellBiol., 35 (1967) 148A.
Brain Research, 259 (1983) 123-126 Elsevier Biomedical Press
Evidence for the existence of a substance P-containing pathway from the nucleus laterodorsalis tegmenti (Castaldi) to the medial frontal cortex of the rat MASAHIRO SAKANAKA, SADAO SHIOSAKA, KENICHI TAKATSUKI and MASAYA TOHYAMA
Department of Neuroanatomy, Institute of Higher Nervous Activity, Osaka University Medical School, 3-5 7 Nakanoshima 4-Chome, Kitaku Osaka 530 (Japan) (Accepted August 31st, 1982)
Key words: substance P - n. laterodorsalis tegmenti - medial frontal cortex
A long ascending substance P (SP)-containing pathway from the nucleus laterodorsalis tegmenti (TLD) to the medial frontal cortex (MFC) has been demonstrated by means of experimental immunohistochemicalprocedures. The unilateral destruction of the TLD, which contains numerous SP-like immunoreactive (SPLI) cells, resulted in a marked ipsilateral decrease in the SPLI fibers in the MFC.
Recent immunohistochemical studies have revealed that the medial frontal cortex (MFC) contains a number of substance P-like immunoreactive (SPLI) fibers4,6,8. However, little is known of the origins of these SPLI fibers in the MFC, though a suggestion has been made by Paxinos et al) 2. The present study describes the existence of a substance P (SP)-containing pathway from the nucleus laterodorsalis tegmenti (TLD) to the MFC. (It should be noted that although Castaldi included the locus coeruleus and Barrington's nucleus in the TLD 2, we exclude these regions from the TLD in our studyl4.) Twenty-eight male rats (body weight 40-60 g) were used in an immunohistochemical study. In order to determine the origins of the SPLI fibers in the MFC, several kinds of operations were made stereotaxically, such as unilateral destruction of the TLD, bilateral destruction of the TLD and hemi- or total transection of the brain just caudal to the TLD, respectively. The animals were kept alive for 4-7 days after the operations. Each animal for the immunohistochemistry was perfused via the ascending aorta with 50 cc of ice-cold saline followed by 300 cc Zamboni's fluid ~s. The brain was quickly removed and 0006-8993/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press
immersed in the same fixative for 48 h at 4 °C, and then rinsed for 24-48 h in 0.1 M phosphate buffer containing 30% sucrose. Serial frontal, sagittal and horizontal sections were cut on 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 3. The specificity of the antiserum against SP was tested by the radioimmunoassay system. The cross-reactivity of this antibody compared with structurally similar peptides, eledoisin, and physalemin, was less than 0.02%, and compared with other related peptides (somatostatin, glucagon, bradykinin, enkephalin and endorphin) was less than 0.01%. Specificity was also tested by the absorption test. Since the structures stained with SP antiserum were not seen in the sections stained with the control serum (for which SP antiserum absorbed by excess SP (80/tg/cc) was used), the structures stained with SP antiserum were considered specific. Six male rats were used in a horseradish peroxidase (HRP) study. A solution of 40% HRP was~ iontophoretically injected into the MFC with the help of stereotaxic apparatus. One or two
124
Fig. 1. A: the unilateral destruction of the TLD caused a marked ipsilateral reduction in the coarse SPLI fibers in the MFC, while the fine terminals remain intact in laminae II and IV even after this lesion. Left side: control. Right side: operated side. Frontal section, case 24, x 70. B: fluorescent photomicrograph showing a higher magnification of the area indicated by the rectangle in A. The very fine terminals in lamina II, which remained intact following the destruction of the TLD, can be recognized under magnification. Left side: control. Right side: operated side. Frontal section, case 24, x 140. Abbreviations: 1I, lamina II; III, lamina III; IV, lamina IV.
days after being injected, the rat brains were subjected to H R P treatment 9. Termimology was based upon the atlas of K6nig and Klippel 7, Palkovits and Jacobowitz 1~, and Tohyama et al. j4. As seen in Fig. 1, a moderate number of SPLI fibers were observed in the M F C ranging from the level o f A 12760 ~tm to A 8920/zm in the atlas of K6nig and Klippel 7, though they did not show a uniform distribution. These SPLI fibers appeared to be composed of two types: one of relatively coarse varicose fibers in laminae II, III and IV often forming bands perpendicular to the brain surface, and the other of very fine terminals with a perineuronal appearance especially in laminae II and IV (Fig. 1). It should be stressed that the coarse fibers were affected by the destruction of the TLD, but the fine termi-
nals were not affected by this lesion (see precisely below). The unilateral destruction of the TLD (Fig. 2A), which is very rich in SPLI cells (Fig. 2B), resuited in a marked ipsilateral decrease in the coarse SPLI fibers in the M F C at the levels mentioned above (Fig. 1), while this operation failed to cause any changes in the fine SPLI terminals in the M F C (Fig. 1). The bilateral destruction of the TLD also caused a marked reduction in the coarse SPLI fibers bilaterally in the M F C similar to that found ipsilaterally in the M F C subsequent to the unilateral destruction of the TLD, compared to the number of coarse SPLI fibers in normal rats. On the other hand, the hemi- or total transection of the brain just caudal to the TLD failed to demonstrate any alterations in the SPLI fibers in the MFC. These facts strongly
125
!! !il ill ~i
c i I t g
~'j
I
!D Fig. 2. A: fight-field photomicrograph showing the electric lesion of the TLD (indicated by arrowheads). Frontal section. Case 24, X 31. B: fluorescent photomicrograph showing numerous SPLI cells in the TLD. Frontal section. Case 3, x 85. C and D: light-field photomicrograph showing an injection site of HRP into the MFC (C) and HRP-labeled cells occurring in the TLD subsequent to HRP injection into the MFC (D), respectively. The same case. Frontal sections. Case 5; C, x 10; D, x 125. Abbreviations: ntd, n. tegmenti dorsalis (Gudden); PCS, pedunculus cerebellaris superior; TLD, n. laterodorsalis tegmenti.
suggest that the SPLI cells located in the T L D send their long ascending fibers to innervate the ipsilateral MFC. In support of this, many HRPlabeled cells were identified in the T L D following H R P injection into the M F C (Fig. 2C,D). The locations of these labeld cells were very similar to those of SPLI cells in the T L D (Fig. 2B,D). Thus, the present study demonstrated that the coarse SPLI fibers in the M F C arise ipsilaterally from the SPLI cells in the TLD. Furthermore, it should be noted that the fine terminals remain intact in the M F C after the destruction of the TLD. This fact indicates that this type of SPLI terminals is innervated by other origins apart from the TLD.
It has been reported previously by means of the H R P method that the neurons of the TLD give rise to long ascending fibers to the M F C 14. The present study has clearly confirmed this projection and further shown that the SP-containing pathway is a major component of this T L D M F C tract. On the basis of the previous study ~3, SPLI cells in the TLD also appear to project to the lateral septal area (LS). Although it is not clear from the present study whether or not a single SPLI cell projects simultaneously to the LS and to the MFC, the SPLI cells in the TLD might be related to the various functions of the forebrain, particularly to delayed alternation, and motivational, emotional and associative larocesses 1,5,10.
126 1 Brito, G. N. O., Thomas, G. J., Davis, B. J. and Gingold, S. I., Prelimbic cortex, mediodorsal thalamus, septum, and delayed alternation in rats, Exp. Brain Res., 46 (1982) 52-58. 2 Castaldi, L., Studi sulla struttura e sullo sviluppo del mesencephalo. Ricerche in Cavia cobaya. III, Arch. ital. A nato. Embriol., 23 (1926) 481 ~09. 3 Coons, A. H., Fluorescent antibody methods. In J. F. Danielli (Ed.), General Cytochemical Methods, Academic Press, New York, NY, 1958, pp. 399-422. 4 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. 5 De France, J. F. (Ed.), The Septal Nuclei, Plenum, New York, 1976. 6 Inagaki, S., Sakanaka, M., Shiosaka, S., Senba, E., Takatsuki, K., Takagi, H., Kawai, Y., Minagawa, H. and Tohyama, M., Ontogeny of substance P-containing neuron system of the rat: Immunohistochemical analysis - I. Forebrain and upper brain stem, Neuroscience, 7 (1982) 251-277. 7 KOnig, 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. 8 Ljungdahl, A., Hrkfelt, 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.
9 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. 10 Miles, R. C. and Blomquist, A. J., Frontal lesions and behavioral deficits, J. Neurophysiol., 23 (1960) 471-484. 11 Palkovits, M. and Jacobowitz, D. M., Topographic atlas of catecholamine and acetylcholinesterase-codtaining neurons in the rat brain. II. Hindbrain (mesencephalon, rhombencephalon), J. comp. Neurol., 157 (1974) 29-42. 12 Paxinos, G., Emson, P. C. and Cuello, A. C., The substance P projections to the frontal cortex and the substantia nigra, Neurosci. Lett., 7 (1978) 127-131. 13 Sakanaka, M., Shiosaka, S., Takatsuki, K., Inagaki, S., Takagi, H., Senba, E., Kawai, Y., Hara, Y., Iida, H., Minagawa, H., Matsuzaki, T. and Tohyama, M., 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 (I 981) 351-355. 14 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. 15 Zamboni, L. and De Martino, C., Buffered picric-acid formaldehyde: a new rapid fixative for electron microscopy, J. CellBiol., 35 (1967) 148A.