194
Neuroscience Letters, 120 (1990) 194~196 Elsevier Scientific Publishers Ireland Ltd.
NSL 07353
Substance P-like immunoreactive neurons in the nucleus tractus solitarii of the rat send their axons to the nucleus accumbens Y u n - Q i n g Li, Z h i - R e n R a o a n d J i - W u Shi Department of Anatomy, The Fourth Military Medical University, Xi'an. Shaanxi (People's Republic qf China)
(Received 5 July 1990; Revised version received 17 August 1990; Accepted 23 August 1990) Key word~: Nucleus tractus solitarii; Nucleus accumbens; Horseradish peroxidase; lmmunocytochemistry;Substance P; Double-labeling; Rat
By a double-labelingmethod combining the retrograde tracing of horseradish peroxidasewith an immunocytochemicaltechnique, substance P-like immunoreactive neurons in the medial and commissural subnuclei of the nucleus tractus solitarii (NTS) of the rat were found to send axons to the nucleus accumbens.
The nucleus tractus solitarii (NTS), a major site of termination of visceral afferent fibers, has been suggested to be reciprocally connected with the nucleus accumbens (ACB), an important structure of the limbic system [7]. It has been reported that NTS contains many neuronal elements with substance P-like immunoreactivity (SPLI) [4]. Hence, in the present work, we examined whether SP-LI neurons in NTS of the rat might send their axons to ACB. In 10 rats anesthetized with sodium pentobarbital (40 mg/kg, i.p.), 0.1/11 of a solution of 30% horseradish peroxidase (HRP, Sigma VI) dissolved in 0.9% saline was stereotaxically injected into ACB unilaterally through a micropipette (tip diameter 45/~m) by aquatic pressure. After 2 days, the rats were injected with colchicine (6080/zg, in 0.9 % saline) into the lateral ventricle. The rats were allowed to survive for 1-2 days, and then perfused through the ascending aorta with 0.9% saline, followed by a fixative containing 4% paraformaldehyde and 0.05 % glutaraldehyde in 0.1 M phosphate buffer (PB, pH 7.4). The brains were removed immediately after the perfusion and put into 30% sucrose in 0.1 M PB (pH 7.4) at 4°C until they sank to the bottom of the container. Subsequently, the brains were cut into coronal frozen sections of 30 /~m thickness. For the histochemical demonstration of H R P , the sections were treated with tetramethylbenzidine (TMB) [5], sodium tungstate (ST) was used as a stabilizer [1]. Then, the sections were Correspondence." Y-Q. Li, Department of Anatomy, The Fourth Military Medical University, Xi'an, Shaanxi 710032, People's Republic of China.
0304-3940/90/$ 03.50 ,~ 1990 ElsevierScientific Publishers Ireland Ltd.
treated with diaminobenzidine (DAB) and cobalt chloride (CoCt2) to enhance H R P reaction products [6]. Subsequently the sections were processed for immunocytochemistry using an antibody raised against SP (1:8000, INC) diluted with 0.01 M PBS (pH 7.4). After incubation in the primary antiserum for 2 days at 4°C, the sections were processed using the avidin-biotin method (Vector ABC Staining Kit) [2] and reacted only with DAB and H202. H R P reaction products were seen as black punctate granules in cytoplasm (Fig. 2B), whereas cytoplasm of SP-LI neurons was homogeneously immunostained in brown. Thus, cytoplasm of double-labelled neurons were homogeneous brown and contained black punctate granules (Fig. 2C, D). All of the H R P injection sites were restricted within the confines of the ACB (Fig. 2A). HRP-labelled neuronal cell bodies in NTS were distributed bilaterally in NTS with a predominant ipsilateral distribution. HRP-labelled neuronal cell bodies in NTS were seen mainly in the medial and commissural subnuclei of NTS at the levels of the obex (Figs. 1 and 2B). Other subnuclei of NTS also contained a few HRP-labeled neuronal cell bodies. At the levels rostral and caudal to the obex, only a few HRP-labelled neurons were found (Fig. 1). HRP-labelled neuronal cell bodies in NTS were oval or fusiform in shape with diameters ranging from 10 to 15/tm. SP-LI neurons were chiefly observed in the medial and commissural subnuclei of NTS at the obex levels. Only a few SP-LI neuronal cell bodies were scattered in other subnuclei of NTS at the levels between the rostral part of the obex and the hypoglossal nucleus. Many SP-LI
195
I
Fig. 1. Projecting drawings of the coronal sections, showing the distributions of HRP-labelled (O), SP-LI (&) and SP-HRP double-labelled (am) neuronal cell bodies in the nucleus tractus solitarii (NTS). Each symbol .represents ! neuron. AP, area postrema; CC, central canal; CON, commissural subnucleus of NTS; CU, cuneate nucleus; D, dorsal subnucleus of NTS; GR, gracile nucleus; IN, intermediate subnucleus of NTS; L, lateral area of NTS; M, medial area of NTS; MDD, dorsal medullary reticular field; MDV, ventral medullary reticular nucleus; MN, medial subnucleus of NTS; PY, pyramidal tract; TS, tractus solitarii; VLN, ventrolateral subnucleus of NTS; VN, ventral subnucleus of NTS; 10, dorsal nucleus of the vagus nerve; 12, hypoglossal nucleus; 12N, root of the hypoglossal nerve.
n e u r o n s were also f o u n d in the c o n t r a l a t e r a l N T S , especially in the m e d i a l a n d c o m m i s s u r a l subnuclei at the levels o f the obex. S P - H R P d o u b l e - l a b e l l e d n e u r o n s were o b s e r v e d in the m e d i a l a n d c o m m i s s u r a l subnuclei o f N T S at the levels o f the obex (Fig. 1). In a rat, a t o t a l o f 32 S P - H R P d o u b l e - l a b e l l e d n e u r o n s were seen in N T S ; 27 o f t h e m
were o b s e r v e d ipsilaterally, a n d 5 c o n t r a l a t e r a l l y . O u t o f 27 S P - H R P d o u b l e - l a b e l l e d n e u r o n s in the ipsilateral N T S , 21 were l o c a t e d in the m e d i a l subnucleus (Fig. 2C) a n d 6 in the c o m m i s s u r a l subnucleus (Fig. 2D). These S P - H R P d o u b l e - l a b e l l e d n e u r o n s c o n s t i t u t e 9 % (32/350) o f the t o t a l H R P - l a b e l l e d N T S neurons, a n d 13% (32/ 243) o f S P - L I N T S neurons. S P - H R P d o u b l e - l a b e l l e d
196
~c
B
cc
Fig. 2. A: a site of HRP injection in the nucleus accumbens (ACB). Bar = 400/lm. B: HRP-labelled neurons in the medial and commissural subnuclei of the nucleus tractus solitarius (NTS). Bar = 120/~m. C: an SP HRP double-labelled neuron in the medial subnucleus of NTS. Bar = 15 pm. D: an SP HRP double-labelled neuron in the commissural subnucleus of NTS. Bar = 15/lm. AC, anterior commissure; BV, blood vessel; CC, central canal; LV, lateral ventricle.
neurons were oval or fusiform in shape with diameters ranging from 10 to 15 pm (Fig. 2C, D). The primary afferent fibers of the vagus nerve have been reported to make synaptic contacts with SP-LI dendrites and neuronal cell bodies [3]. The results of the present study indicate that some SP-LI neurons in NTS send their axons to ACB, thereby mediating visceral information from the primary afferent fibers of the vagus nerve directly to ACB.
4
5
6 1 Gu, Y.M., The Studies on the Dynamics and Histochemistry of the HRP-TMB Reaction, Doctoral Dissertation, Sun Yat-Sen University of Medical Sciences, Guangzhou, 1989, pp. 61-63. 2 Hsu, S.M., Raine, I. and Fanger, H., Use of avidin-biotin peroxidase complex (ABC) in immuno-peroxidase techniques, a comparison between ABC and unlabelled antibody (PAP) procedures, J. Histochem. Cytochem., 29 (1981) 577-580. 3 Kawai, Y., Mori, S. and Takagi, H., Vagal afferents interact with
7
substance P-immunoreactive structures in the nucleus of the tractus solitarius: immunoelectron microscopy combined with an anterograde degeneration study, Neurosci. Lett., 101 (1989) ~10. Ljungdahl, A.O., H6kfelt, 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) 861943. 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) 10(~117. Rye, D.B., Saper, C.B. and Wainer, B.H., Stabilization of the tetramethylbenzidine (TMB) reaction product: application for retrograde and anterograde tracing, and combination with immunohistochemistry, J. Histochem. Cytochem. 32 (1984) 1145-1153. Sofroniew, M.V., Direct reciprocal connection between bed nucleus of the stria terminalis and dorsomedial medulla oblongata: evidence from immunohistochemical detection of tracer proteins, J. Comp. Neurol., 213 (1983) 399~,05.
Neuroscience Letters, 120 (1990) 194~196 Elsevier Scientific Publishers Ireland Ltd.
NSL 07353
Substance P-like immunoreactive neurons in the nucleus tractus solitarii of the rat send their axons to the nucleus accumbens Y u n - Q i n g Li, Z h i - R e n R a o a n d J i - W u Shi Department of Anatomy, The Fourth Military Medical University, Xi'an. Shaanxi (People's Republic qf China)
(Received 5 July 1990; Revised version received 17 August 1990; Accepted 23 August 1990) Key word~: Nucleus tractus solitarii; Nucleus accumbens; Horseradish peroxidase; lmmunocytochemistry;Substance P; Double-labeling; Rat
By a double-labelingmethod combining the retrograde tracing of horseradish peroxidasewith an immunocytochemicaltechnique, substance P-like immunoreactive neurons in the medial and commissural subnuclei of the nucleus tractus solitarii (NTS) of the rat were found to send axons to the nucleus accumbens.
The nucleus tractus solitarii (NTS), a major site of termination of visceral afferent fibers, has been suggested to be reciprocally connected with the nucleus accumbens (ACB), an important structure of the limbic system [7]. It has been reported that NTS contains many neuronal elements with substance P-like immunoreactivity (SPLI) [4]. Hence, in the present work, we examined whether SP-LI neurons in NTS of the rat might send their axons to ACB. In 10 rats anesthetized with sodium pentobarbital (40 mg/kg, i.p.), 0.1/11 of a solution of 30% horseradish peroxidase (HRP, Sigma VI) dissolved in 0.9% saline was stereotaxically injected into ACB unilaterally through a micropipette (tip diameter 45/~m) by aquatic pressure. After 2 days, the rats were injected with colchicine (6080/zg, in 0.9 % saline) into the lateral ventricle. The rats were allowed to survive for 1-2 days, and then perfused through the ascending aorta with 0.9% saline, followed by a fixative containing 4% paraformaldehyde and 0.05 % glutaraldehyde in 0.1 M phosphate buffer (PB, pH 7.4). The brains were removed immediately after the perfusion and put into 30% sucrose in 0.1 M PB (pH 7.4) at 4°C until they sank to the bottom of the container. Subsequently, the brains were cut into coronal frozen sections of 30 /~m thickness. For the histochemical demonstration of H R P , the sections were treated with tetramethylbenzidine (TMB) [5], sodium tungstate (ST) was used as a stabilizer [1]. Then, the sections were Correspondence." Y-Q. Li, Department of Anatomy, The Fourth Military Medical University, Xi'an, Shaanxi 710032, People's Republic of China.
0304-3940/90/$ 03.50 ,~ 1990 ElsevierScientific Publishers Ireland Ltd.
treated with diaminobenzidine (DAB) and cobalt chloride (CoCt2) to enhance H R P reaction products [6]. Subsequently the sections were processed for immunocytochemistry using an antibody raised against SP (1:8000, INC) diluted with 0.01 M PBS (pH 7.4). After incubation in the primary antiserum for 2 days at 4°C, the sections were processed using the avidin-biotin method (Vector ABC Staining Kit) [2] and reacted only with DAB and H202. H R P reaction products were seen as black punctate granules in cytoplasm (Fig. 2B), whereas cytoplasm of SP-LI neurons was homogeneously immunostained in brown. Thus, cytoplasm of double-labelled neurons were homogeneous brown and contained black punctate granules (Fig. 2C, D). All of the H R P injection sites were restricted within the confines of the ACB (Fig. 2A). HRP-labelled neuronal cell bodies in NTS were distributed bilaterally in NTS with a predominant ipsilateral distribution. HRP-labelled neuronal cell bodies in NTS were seen mainly in the medial and commissural subnuclei of NTS at the levels of the obex (Figs. 1 and 2B). Other subnuclei of NTS also contained a few HRP-labeled neuronal cell bodies. At the levels rostral and caudal to the obex, only a few HRP-labelled neurons were found (Fig. 1). HRP-labelled neuronal cell bodies in NTS were oval or fusiform in shape with diameters ranging from 10 to 15/tm. SP-LI neurons were chiefly observed in the medial and commissural subnuclei of NTS at the obex levels. Only a few SP-LI neuronal cell bodies were scattered in other subnuclei of NTS at the levels between the rostral part of the obex and the hypoglossal nucleus. Many SP-LI
195
I
Fig. 1. Projecting drawings of the coronal sections, showing the distributions of HRP-labelled (O), SP-LI (&) and SP-HRP double-labelled (am) neuronal cell bodies in the nucleus tractus solitarii (NTS). Each symbol .represents ! neuron. AP, area postrema; CC, central canal; CON, commissural subnucleus of NTS; CU, cuneate nucleus; D, dorsal subnucleus of NTS; GR, gracile nucleus; IN, intermediate subnucleus of NTS; L, lateral area of NTS; M, medial area of NTS; MDD, dorsal medullary reticular field; MDV, ventral medullary reticular nucleus; MN, medial subnucleus of NTS; PY, pyramidal tract; TS, tractus solitarii; VLN, ventrolateral subnucleus of NTS; VN, ventral subnucleus of NTS; 10, dorsal nucleus of the vagus nerve; 12, hypoglossal nucleus; 12N, root of the hypoglossal nerve.
n e u r o n s were also f o u n d in the c o n t r a l a t e r a l N T S , especially in the m e d i a l a n d c o m m i s s u r a l subnuclei at the levels o f the obex. S P - H R P d o u b l e - l a b e l l e d n e u r o n s were o b s e r v e d in the m e d i a l a n d c o m m i s s u r a l subnuclei o f N T S at the levels o f the obex (Fig. 1). In a rat, a t o t a l o f 32 S P - H R P d o u b l e - l a b e l l e d n e u r o n s were seen in N T S ; 27 o f t h e m
were o b s e r v e d ipsilaterally, a n d 5 c o n t r a l a t e r a l l y . O u t o f 27 S P - H R P d o u b l e - l a b e l l e d n e u r o n s in the ipsilateral N T S , 21 were l o c a t e d in the m e d i a l subnucleus (Fig. 2C) a n d 6 in the c o m m i s s u r a l subnucleus (Fig. 2D). These S P - H R P d o u b l e - l a b e l l e d n e u r o n s c o n s t i t u t e 9 % (32/350) o f the t o t a l H R P - l a b e l l e d N T S neurons, a n d 13% (32/ 243) o f S P - L I N T S neurons. S P - H R P d o u b l e - l a b e l l e d
196
~c
B
cc
Fig. 2. A: a site of HRP injection in the nucleus accumbens (ACB). Bar = 400/lm. B: HRP-labelled neurons in the medial and commissural subnuclei of the nucleus tractus solitarius (NTS). Bar = 120/~m. C: an SP HRP double-labelled neuron in the medial subnucleus of NTS. Bar = 15 pm. D: an SP HRP double-labelled neuron in the commissural subnucleus of NTS. Bar = 15/lm. AC, anterior commissure; BV, blood vessel; CC, central canal; LV, lateral ventricle.
neurons were oval or fusiform in shape with diameters ranging from 10 to 15 pm (Fig. 2C, D). The primary afferent fibers of the vagus nerve have been reported to make synaptic contacts with SP-LI dendrites and neuronal cell bodies [3]. The results of the present study indicate that some SP-LI neurons in NTS send their axons to ACB, thereby mediating visceral information from the primary afferent fibers of the vagus nerve directly to ACB.
4
5
6 1 Gu, Y.M., The Studies on the Dynamics and Histochemistry of the HRP-TMB Reaction, Doctoral Dissertation, Sun Yat-Sen University of Medical Sciences, Guangzhou, 1989, pp. 61-63. 2 Hsu, S.M., Raine, I. and Fanger, H., Use of avidin-biotin peroxidase complex (ABC) in immuno-peroxidase techniques, a comparison between ABC and unlabelled antibody (PAP) procedures, J. Histochem. Cytochem., 29 (1981) 577-580. 3 Kawai, Y., Mori, S. and Takagi, H., Vagal afferents interact with
7
substance P-immunoreactive structures in the nucleus of the tractus solitarius: immunoelectron microscopy combined with an anterograde degeneration study, Neurosci. Lett., 101 (1989) ~10. Ljungdahl, A.O., H6kfelt, 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) 861943. 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) 10(~117. Rye, D.B., Saper, C.B. and Wainer, B.H., Stabilization of the tetramethylbenzidine (TMB) reaction product: application for retrograde and anterograde tracing, and combination with immunohistochemistry, J. Histochem. Cytochem. 32 (1984) 1145-1153. Sofroniew, M.V., Direct reciprocal connection between bed nucleus of the stria terminalis and dorsomedial medulla oblongata: evidence from immunohistochemical detection of tracer proteins, J. Comp. Neurol., 213 (1983) 399~,05.
