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Identification of thalamic neurons with vasoactive intestinal polypeptide-like immunoreactivity in the rat
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BRE 21172
Identification of thalamic neurons with vasoactive intestinal polypeptide-like immunoreactivity in the rat TAKESHI KANEKO t , KEI TASHIRO ~. "I'EI'SUO SUGIMO'I'OE, AKIRA KONISHI2 and NOBORU MIZUNO E
1Department of Anatomy (lst Division), Faculty of Medicine and ~'College of Medical Technology, Kyoto UniversiO', Kyoto 606 (Japan) (Accepted June 2hth, 1985)
Key words: vasoactive intestinal polypeptide (VIP) - - thalamus - - ventrolateral thalamic nucleus - thalamic reticular nucleus ..... rat - - immunohistochemistry
Cell bodies with vasoactive intestinal polypeptide-like immunoreactivity (VIP-LI) were found in the thalamus of the rat. They were distributed throughout the ventrolateral nucleus (VL) and in the whole extent of the thalamic reticular nucleus (R) except for its most rostral part. On the basis of soma diameters, VIP-LI cells in the VL and R were assumed to be projection neurons.
Vasoactive intestinal p o l y p e p t i d e (VIP) was first isolated from porcine intestine 13,14. Subsequent studies using r a d i o i m m u n o a s s a y and i m m u n o h i s t o c h e m ical techniques have revealed a wide distribution of VIP-like immunoreacfivity (V1P-LI) in the central and peripheral nervous structures of various species (for reviews, refs. 3, 11, 12). H o w e v e r , neuronal cell bodies with VIP-LI has not yet been r e p o r t e d in the thalamus. The present r e p o r t therefore describes the localization of VIP-LI cells in the thalamus of the rat. Nineteen male Wistar rats, weighing 150-250 g, were anesthetized with sodium p e n t o b a r b i t a l (120 mg/kg b.wt., i.p.) and perfused transcardially with 200 ml of K r e b s - R i n g e r solution, followed by 200 ml of Z a m b o n i ' s solution (2% p a r a f o r m a l d e h y d e and 0.2% picric acid in 0.1 M p h o s p h a t e buffer, p H 7.2) t7 for 30 rain at room t e m p e r a t u r e . The brain was quickly r e m o v e d , cut into several blocks and placed in the same fixative for 4 h at 4 °C. A f t e r rinsing in ice-cold 10 mM p h o s p h a t e - b u f f e r e d 0.85% saline (PBS) containing 10% sucrose and then in the same PBS containing 20% sucrose, the blocks were frozen on dry ice and cut into frontal sections of 40ktm thickness. The sections were collected in the PBS; of two consecutive sections, one was processed for VIP immunohistochemistry and the o t h e r for the Nissl stain (toluidine blue). F o r VIP immunohistochemistry, the
sections were soaked for 1 h in the PBS containing 10% normal goat serum and 0.3% Triton X-100 and then exposed to antiserum against VIP ( A n t i b o d y Vt28, Lot 8403015; Immunonuclear) diluted 1:2000. Sites of a n t i g e n - a n t i b o d y reaction were revealed by the unlabeled antibody p e r o x i d a s e - a n t i peroxidase ( P A P ) methodlS; goat antibody for rabbit lgG (Cappel; 1:50) and P A P ( D a k o ; 1:200) were conjugated to the primary antibody at room temperature over 1 h for each incubation and the peroxidase was visualized by the incubation at r o o m t e m p e r a ture for 20 rain with 0.02% diaminobenzidine tetrahydrochloride and 0 . 0 0 4 5 ~ hydrogen peroxidase in 50 mM Tris-HCl buffer (pH 7.2). Preincubation of the diluted antiserum (1:2000) with porcine VIP (Peptide Institute) at a concentration of 10 j,g/ml resulted in elimination of all immunohistochemical staining. The thalamic nuclei were n a m e d according to Bold et al. 2. I11 the thalamus, cell bodies with intense V I P - L I were detected in the ventrolateral (VL) and reticular nuclei (R; Fig. 1). They were distributed throughout the VL and in the whole extent of the R except for its most rostral part. The cell bodies with intense V I P - L I in the VL and R were as large as those of large neurons in the VL and R which were seen in Nisslstained preparations. The largest and smallest celt
Correspondence." N. Mizuno, Department of Anatomy ( 1st Division k, Faculty of Medicine, Kyoto University, Kyoto 606, Japan. 00ll6-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
391 body diameters of V I P - L I cells selected r a n d o m l y were m e a s u r e d ; the largest d i a m e t e r was the longest axis and the smallest d i a m e t e r was t a k e n at a right angle to a point halfway on the longest axis. These were 19.0 _+ 2 . 7 ~ m ( m e a n + S . D . ) and 11.5 +_ 1.6/~m in the VL (n = 100; Fig. 2a) and 20.3 +_ 3.4/~m and 7.4 + 1.9/~m in the R (n = 100; Fig. 2b). Some cell bodies in the v e n t r o m e d i a l nucleus ( V M ) , submedius nucleus (Sin) and intralaminar and v e n t r o b a s a l (VB) nuclear regions adjacent to the V L also showed immunohistochemical staining (Fig. 1), although these were small in n u m b e r and, in most instances, their V I P - L I was w e a k e r than that of V L and R neurons. The V I P content in the thalamus has been r e p o r t e d to be 1 0 % - 2 5 % of that in the cerebral cortexa,6, ~0. This is one reason why no previous studies described V I P - L I in the thalamus. H o w e v e r , a m o r e i m p o r t a n t reason may be suppression of V I P - L I during the process of fixation. In fact, V I P - L I in the thalamus
began to be suppressed after placing tissue blocks in 4% p a r a f o r m a l d e h y d e solution for 16 h at 4 °C. Therefore, in the present study, tissue blocks were placed in Z a m b o n i ' s fixative (containing 2% paraf o r m a l d e h y d e and 0.2% picric acid) 17 for only 4 h at 4 °C and cell bodies with V I P - L I were found in the thalamus. On the basis of soma size, V I P - L I cells identified in the present study in the V L and R were p r e s u m e d to be relay neurons. V I P - L I of these neurons were confined to the cell bodies; neither dendrites nor axons showed VIP-LI. On the other hand, VIP-L1 neurons which have so far been described in o t h e r regions of the central nervous system, such as the cerebral cortex, amygdala, suprachiasmatic nucleus and spinal cord 4-7,16, a p p e a r to be smaller in size than V L and R neurons with V I P - L I and show V I P - L I often in their dendrites and occasionally in their axons, as well as in their cell bodies. Large V I P - L I neurons in the V L
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Fig. 1. Photomicrograph of a frontal section through the middle level of the thalamus, showing cell bodies with VIP-LI in the VL and R. Some cell bodies in the Sin, VM, VB, and intralaminar nuclei show weak immunohistochemical staining. LD, laterodorsal nucleus; MD, mediodorsal nucleus; rot, mammillothalamic tract. Scale bar, 0.5 mm.
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[-'if_.2. Cell bodies with intense V1p-I,l in the V t. l a~ agd P- IbL ScaLebar. 51)kma. The authors are grateful to Drs, Yasuyoshi Watanabe and Yoshihiro Urade of the Hayaish~ Biointorand R may constitute a functional group which is difmation Transfer Project of the Research Develferent from that of the parvicellular VIP-LI neurons. opment Corporation, Japan, for their valuabie adNeurons in the R of the rat have been known to vice and discussions. The technical assistance of Miss contain },,aminobutyric acid ( G A B A ) and are asKimiko A m a n o , the photographic help of Mr. Akira sumed to be inhibitory neur°nsS" On the other hand, Uesugi and the support of the Niwa Medical FoundaVIP has been reported to exert potent excitatory eftion are also gratefully acknowledged-'This work has fects upon corticospinal neur°ns9 and neurons in the also been supported in part by Grant-in-Aid for Scicaudal spinal trigeminal nucleus~5" Thus, it is interentific Research 59480095 from the Ministry of Eduesting and important to knoW if VIP may be cocation, Science and Culture of Japan. located with G A B A in neurons of the R.
l Besson, 3,, Rotsztejn, W., Laburthe, M., Epelbaum, !'" Beaudet, A., Kordon, C. and Rosselin, G., Vasoactive intestinal peptide (VIP): brain distribution, subcellular local)zation and effect of deafferentation of the hypothatamus in male rats, Brain Research, 165 (1979) 79-85. 2 Bold, E.L., Castro, A.J. and Neafsey, E.J., Cytoarchitecture of the dorsal thalamus of tt~e rat, Brain Res, Bull., 12 52l-Y,, 527.Chayvialle, ].-A.. Said, S.I. and Dubois, 3 (1984) Charnay, P.M., Localization of vasoactive intestinal peptide immunoreactivity in human foetus and newborn infant spinal cord, Neuroscience, 14 (1985) 195-205. 4 Connor, J.R. and Peters, A.. Vasoactive intestinal polypeptide.immunoreactive neurons in rat visual cortex. Neuroscience, 12 11984) 1027-1044. M., Vasoac5 Fuji, K,, Senba,. .E., . . . .Ueda, . . . . Y. and Tohyama, taining neurons m the • .:_,, ,,~,lvne~tide t vlr)-~U" . x~,,ro~ci Left., tire m t e S t m a , v ~ at.~ * u o ; r n r o i e c t l o n S , ~'~ • " spinal cord of the rat anu m,,- v- , 37 (1983} 51-55.
6 Lor6n, 1., Emson, P.C-, Fahrenkrug, j , Bj6rk|und, A., Alj. H~kanson, R. and Sunder, F., Distribution ot lumets, , l~p~Plt;d; i~;;he rat and mouse vasoactive intestinal4P brain Neuroscience, ( - " and Bloom, 7 Morrison, J.H., Magistretti, P.J., Benoit, RF.E., The distribution and morphological characteristics ~f the intracortical ViP-positive cell: an immunohistochemlcBrain Research, 292 11984) 269-282alanalysis, Storm-Mathisen, J'" Glutamate- and 80tterson, O.P. andneurons in the mouse and rat brain, as GABA-containing demonstrated with a new immunocytochemical technique, J. Comp. NeuroL, 22911984) 374-392. 9 Phillis, J.W. and Kirkpatrick, J .R., The actions of motilin, luteinizing hormone releasing hormone, choiecystokinim somatostatin, vasoactive ntestinal peptide, and other peptides on rat cerebral cc~tical neurons, can. J. physiol. Pharmacol., 58 11980) 612-623. 10 Roberts, G.W., Woodahams, P.L., Bryan, M.G., Crow, T.J., Bl0o~, S.R. and Polak, J M , VIP in the rat brain: ev-
393 idence for a major pathway linking the amygdala and hypothalamus via the stria terminalis, Histochemistry, 65 (1980) 103-119. 11 Rost~ne, W.H., Neurobiological and neuroendocrine functions of the vasoactive intestinal peptide (VIP), Prog. Neurobiol., 22 (1984) 103-129. 12 Said, S.I. (Ed.), Vasoactive Intestinal Peptide, Raven, New York, 1982. 13 Said, S.I. and Mutt, V., Polypeptide with broad biological activity: isolation from small intestine, Science, 169 (1970) 1217-1218. 14 Said, S.I. and Mutt, V., Isolation from porcine intestinal wall of a vasoactive octacosapeptide related to secretin and to glucagon, Eur. J. Biochem., 28 (1972) 199-204.
15 Salt, T.E. and Hill, R.G., Vasoactive intestinal polypeptide (VIP) applied by microiontophoresis excites single neurons in the trigeminal nucleus caudalis of the rat, Neuropeptides, 1 (1981) 403-408. 16 Sims, K.B., Hoffman, D.L., Said, S.I. and Zimmerman. E.A., Vasoactive intestinal polypeptide (VIP) in mouse and rat brain: an immunocytochemical study. Brain Research, 186 (1980) 165-183. I7 Stephani, M., DeMartinio, C. and Zamboni. L., Fixation of ejaculated spermatozoa for electron microscopy. Nature (London), 216 (1967) 173-174. 18 Sternberger, L.A.. lrnmunocytochemistry, 2nd edn., Wiley, New York, 1979.
[~ralh ~(e~earch
:;4:~
BRE 21172
Identification of thalamic neurons with vasoactive intestinal polypeptide-like immunoreactivity in the rat TAKESHI KANEKO t , KEI TASHIRO ~. "I'EI'SUO SUGIMO'I'OE, AKIRA KONISHI2 and NOBORU MIZUNO E
1Department of Anatomy (lst Division), Faculty of Medicine and ~'College of Medical Technology, Kyoto UniversiO', Kyoto 606 (Japan) (Accepted June 2hth, 1985)
Key words: vasoactive intestinal polypeptide (VIP) - - thalamus - - ventrolateral thalamic nucleus - thalamic reticular nucleus ..... rat - - immunohistochemistry
Cell bodies with vasoactive intestinal polypeptide-like immunoreactivity (VIP-LI) were found in the thalamus of the rat. They were distributed throughout the ventrolateral nucleus (VL) and in the whole extent of the thalamic reticular nucleus (R) except for its most rostral part. On the basis of soma diameters, VIP-LI cells in the VL and R were assumed to be projection neurons.
Vasoactive intestinal p o l y p e p t i d e (VIP) was first isolated from porcine intestine 13,14. Subsequent studies using r a d i o i m m u n o a s s a y and i m m u n o h i s t o c h e m ical techniques have revealed a wide distribution of VIP-like immunoreacfivity (V1P-LI) in the central and peripheral nervous structures of various species (for reviews, refs. 3, 11, 12). H o w e v e r , neuronal cell bodies with VIP-LI has not yet been r e p o r t e d in the thalamus. The present r e p o r t therefore describes the localization of VIP-LI cells in the thalamus of the rat. Nineteen male Wistar rats, weighing 150-250 g, were anesthetized with sodium p e n t o b a r b i t a l (120 mg/kg b.wt., i.p.) and perfused transcardially with 200 ml of K r e b s - R i n g e r solution, followed by 200 ml of Z a m b o n i ' s solution (2% p a r a f o r m a l d e h y d e and 0.2% picric acid in 0.1 M p h o s p h a t e buffer, p H 7.2) t7 for 30 rain at room t e m p e r a t u r e . The brain was quickly r e m o v e d , cut into several blocks and placed in the same fixative for 4 h at 4 °C. A f t e r rinsing in ice-cold 10 mM p h o s p h a t e - b u f f e r e d 0.85% saline (PBS) containing 10% sucrose and then in the same PBS containing 20% sucrose, the blocks were frozen on dry ice and cut into frontal sections of 40ktm thickness. The sections were collected in the PBS; of two consecutive sections, one was processed for VIP immunohistochemistry and the o t h e r for the Nissl stain (toluidine blue). F o r VIP immunohistochemistry, the
sections were soaked for 1 h in the PBS containing 10% normal goat serum and 0.3% Triton X-100 and then exposed to antiserum against VIP ( A n t i b o d y Vt28, Lot 8403015; Immunonuclear) diluted 1:2000. Sites of a n t i g e n - a n t i b o d y reaction were revealed by the unlabeled antibody p e r o x i d a s e - a n t i peroxidase ( P A P ) methodlS; goat antibody for rabbit lgG (Cappel; 1:50) and P A P ( D a k o ; 1:200) were conjugated to the primary antibody at room temperature over 1 h for each incubation and the peroxidase was visualized by the incubation at r o o m t e m p e r a ture for 20 rain with 0.02% diaminobenzidine tetrahydrochloride and 0 . 0 0 4 5 ~ hydrogen peroxidase in 50 mM Tris-HCl buffer (pH 7.2). Preincubation of the diluted antiserum (1:2000) with porcine VIP (Peptide Institute) at a concentration of 10 j,g/ml resulted in elimination of all immunohistochemical staining. The thalamic nuclei were n a m e d according to Bold et al. 2. I11 the thalamus, cell bodies with intense V I P - L I were detected in the ventrolateral (VL) and reticular nuclei (R; Fig. 1). They were distributed throughout the VL and in the whole extent of the R except for its most rostral part. The cell bodies with intense V I P - L I in the VL and R were as large as those of large neurons in the VL and R which were seen in Nisslstained preparations. The largest and smallest celt
Correspondence." N. Mizuno, Department of Anatomy ( 1st Division k, Faculty of Medicine, Kyoto University, Kyoto 606, Japan. 00ll6-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
391 body diameters of V I P - L I cells selected r a n d o m l y were m e a s u r e d ; the largest d i a m e t e r was the longest axis and the smallest d i a m e t e r was t a k e n at a right angle to a point halfway on the longest axis. These were 19.0 _+ 2 . 7 ~ m ( m e a n + S . D . ) and 11.5 +_ 1.6/~m in the VL (n = 100; Fig. 2a) and 20.3 +_ 3.4/~m and 7.4 + 1.9/~m in the R (n = 100; Fig. 2b). Some cell bodies in the v e n t r o m e d i a l nucleus ( V M ) , submedius nucleus (Sin) and intralaminar and v e n t r o b a s a l (VB) nuclear regions adjacent to the V L also showed immunohistochemical staining (Fig. 1), although these were small in n u m b e r and, in most instances, their V I P - L I was w e a k e r than that of V L and R neurons. The V I P content in the thalamus has been r e p o r t e d to be 1 0 % - 2 5 % of that in the cerebral cortexa,6, ~0. This is one reason why no previous studies described V I P - L I in the thalamus. H o w e v e r , a m o r e i m p o r t a n t reason may be suppression of V I P - L I during the process of fixation. In fact, V I P - L I in the thalamus
began to be suppressed after placing tissue blocks in 4% p a r a f o r m a l d e h y d e solution for 16 h at 4 °C. Therefore, in the present study, tissue blocks were placed in Z a m b o n i ' s fixative (containing 2% paraf o r m a l d e h y d e and 0.2% picric acid) 17 for only 4 h at 4 °C and cell bodies with V I P - L I were found in the thalamus. On the basis of soma size, V I P - L I cells identified in the present study in the V L and R were p r e s u m e d to be relay neurons. V I P - L I of these neurons were confined to the cell bodies; neither dendrites nor axons showed VIP-LI. On the other hand, VIP-L1 neurons which have so far been described in o t h e r regions of the central nervous system, such as the cerebral cortex, amygdala, suprachiasmatic nucleus and spinal cord 4-7,16, a p p e a r to be smaller in size than V L and R neurons with V I P - L I and show V I P - L I often in their dendrites and occasionally in their axons, as well as in their cell bodies. Large V I P - L I neurons in the V L
f
a~
7~ d
Fig. 1. Photomicrograph of a frontal section through the middle level of the thalamus, showing cell bodies with VIP-LI in the VL and R. Some cell bodies in the Sin, VM, VB, and intralaminar nuclei show weak immunohistochemical staining. LD, laterodorsal nucleus; MD, mediodorsal nucleus; rot, mammillothalamic tract. Scale bar, 0.5 mm.
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[-'if_.2. Cell bodies with intense V1p-I,l in the V t. l a~ agd P- IbL ScaLebar. 51)kma. The authors are grateful to Drs, Yasuyoshi Watanabe and Yoshihiro Urade of the Hayaish~ Biointorand R may constitute a functional group which is difmation Transfer Project of the Research Develferent from that of the parvicellular VIP-LI neurons. opment Corporation, Japan, for their valuabie adNeurons in the R of the rat have been known to vice and discussions. The technical assistance of Miss contain },,aminobutyric acid ( G A B A ) and are asKimiko A m a n o , the photographic help of Mr. Akira sumed to be inhibitory neur°nsS" On the other hand, Uesugi and the support of the Niwa Medical FoundaVIP has been reported to exert potent excitatory eftion are also gratefully acknowledged-'This work has fects upon corticospinal neur°ns9 and neurons in the also been supported in part by Grant-in-Aid for Scicaudal spinal trigeminal nucleus~5" Thus, it is interentific Research 59480095 from the Ministry of Eduesting and important to knoW if VIP may be cocation, Science and Culture of Japan. located with G A B A in neurons of the R.
l Besson, 3,, Rotsztejn, W., Laburthe, M., Epelbaum, !'" Beaudet, A., Kordon, C. and Rosselin, G., Vasoactive intestinal peptide (VIP): brain distribution, subcellular local)zation and effect of deafferentation of the hypothatamus in male rats, Brain Research, 165 (1979) 79-85. 2 Bold, E.L., Castro, A.J. and Neafsey, E.J., Cytoarchitecture of the dorsal thalamus of tt~e rat, Brain Res, Bull., 12 52l-Y,, 527.Chayvialle, ].-A.. Said, S.I. and Dubois, 3 (1984) Charnay, P.M., Localization of vasoactive intestinal peptide immunoreactivity in human foetus and newborn infant spinal cord, Neuroscience, 14 (1985) 195-205. 4 Connor, J.R. and Peters, A.. Vasoactive intestinal polypeptide.immunoreactive neurons in rat visual cortex. Neuroscience, 12 11984) 1027-1044. M., Vasoac5 Fuji, K,, Senba,. .E., . . . .Ueda, . . . . Y. and Tohyama, taining neurons m the • .:_,, ,,~,lvne~tide t vlr)-~U" . x~,,ro~ci Left., tire m t e S t m a , v ~ at.~ * u o ; r n r o i e c t l o n S , ~'~ • " spinal cord of the rat anu m,,- v- , 37 (1983} 51-55.
6 Lor6n, 1., Emson, P.C-, Fahrenkrug, j , Bj6rk|und, A., Alj. H~kanson, R. and Sunder, F., Distribution ot lumets, , l~p~Plt;d; i~;;he rat and mouse vasoactive intestinal4P brain Neuroscience, ( - " and Bloom, 7 Morrison, J.H., Magistretti, P.J., Benoit, RF.E., The distribution and morphological characteristics ~f the intracortical ViP-positive cell: an immunohistochemlcBrain Research, 292 11984) 269-282alanalysis, Storm-Mathisen, J'" Glutamate- and 80tterson, O.P. andneurons in the mouse and rat brain, as GABA-containing demonstrated with a new immunocytochemical technique, J. Comp. NeuroL, 22911984) 374-392. 9 Phillis, J.W. and Kirkpatrick, J .R., The actions of motilin, luteinizing hormone releasing hormone, choiecystokinim somatostatin, vasoactive ntestinal peptide, and other peptides on rat cerebral cc~tical neurons, can. J. physiol. Pharmacol., 58 11980) 612-623. 10 Roberts, G.W., Woodahams, P.L., Bryan, M.G., Crow, T.J., Bl0o~, S.R. and Polak, J M , VIP in the rat brain: ev-
393 idence for a major pathway linking the amygdala and hypothalamus via the stria terminalis, Histochemistry, 65 (1980) 103-119. 11 Rost~ne, W.H., Neurobiological and neuroendocrine functions of the vasoactive intestinal peptide (VIP), Prog. Neurobiol., 22 (1984) 103-129. 12 Said, S.I. (Ed.), Vasoactive Intestinal Peptide, Raven, New York, 1982. 13 Said, S.I. and Mutt, V., Polypeptide with broad biological activity: isolation from small intestine, Science, 169 (1970) 1217-1218. 14 Said, S.I. and Mutt, V., Isolation from porcine intestinal wall of a vasoactive octacosapeptide related to secretin and to glucagon, Eur. J. Biochem., 28 (1972) 199-204.
15 Salt, T.E. and Hill, R.G., Vasoactive intestinal polypeptide (VIP) applied by microiontophoresis excites single neurons in the trigeminal nucleus caudalis of the rat, Neuropeptides, 1 (1981) 403-408. 16 Sims, K.B., Hoffman, D.L., Said, S.I. and Zimmerman. E.A., Vasoactive intestinal polypeptide (VIP) in mouse and rat brain: an immunocytochemical study. Brain Research, 186 (1980) 165-183. I7 Stephani, M., DeMartinio, C. and Zamboni. L., Fixation of ejaculated spermatozoa for electron microscopy. Nature (London), 216 (1967) 173-174. 18 Sternberger, L.A.. lrnmunocytochemistry, 2nd edn., Wiley, New York, 1979.