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A morphological analysis of neurons in the lateral parabrachial nucleus: An intracellular horseradish peroxidase study in the rat
Neuroscience Letters, 112 (1990) 133 136 Elsevier Scientific Publishers Ireland Ltd.
133
NSL 06841
A morphological analysis of neurons in the lateral parabrachial nucleus: an intracellular horseradish peroxidase study in the rat Pifu Luo, Jishuo Li and Zhiren Rao Department of ,4natomy, Fourth Military Medical University, Shaanxi (People's Republic of China) (Received 13 November 1989; Revised version received 16 January 1990; Accepted 18 January 1990) Key words." Parabrachial nucleus; Intracellular injection; Horseradish peroxidase; Rat Neurons in the lateral parabrachial nucleus (LPB) of the rat were analyzed by the intracellular horseradish peroxidase method. Sixteen LPB neurons were successfully labeled with HRP injected intracellularly. HRP-labeled LPB neurons were divided into type I (7 neurons) and type II (9 neurons) LPB neurons. Type I LPB neurons, which were activated antidromically by stimulation of the ipsilateral posteromedial ventral nucleus of the thalamus, had long dendrites and a long axon. Type II LPB neurons, which were not activated antidromically by the stimulation of the thalamus, had short dendrites and a short axon. It was concluded that type I LPB neurons were projection neurons, while type II LPB neurons were local circuit neurons.
T h e afferent a n d efferent c o n n e c t i o n s o f the lateral p a r a b r a c h i a l nucleus (LPB) have been well s t u d i e d [2-7], b u t the d a t a c o n c e r n i n g the m o r p h o l o g y o f i n d i v i d u a l LPB n e u r o n s a r e scanty [8]. In the present study, therefore, an a t t e m p t was m a d e to observe i n d i v i d u a l LPB n e u r o n s o f the rat b y intracellular injection o f h o r s e r a d i s h p e r o x i d a s e ( H R P ) into i n d i v i d u a l LPB neurons. A d u l t S p r a g u e - D a w l e y rats o f either sex (200-250 g) were anesthetized with s o d i u m p e n t o b a r b i t a l (4 mg/100 g b.wt.), a n d p a r a l y z e d with gallamine t r i e t h i o d i d e (5 mg/100 g b.wt.). T h e n the rats were artificially ventilated on a stereotaxic a p p a r a tus. G l a s s - m i c r o e l e c t r o d e s with tip d i a m e t e r o f 0.5-1.0 p m were filled with 5-10% H R P (Sigma T y p e VI) in 0.05 M T r i s - H C l buffer ( p H 7.6) a n d 0.25 M KC1 were used for i n t r a c e l l u l a r recording. A n t i d r o m i c o r o r t h o d r o m i c responses were e v o k e d by s t i m u l a t i n g the ipsilateral p o s t e r o m e d i a l ventral t h a l a m i c nucleus o r the ipsilateral nucleus o f the solitary tract, respectively. A f t e r successful i n t r a c e l l u l a r recording, H R P was i o n t o p h o r e t i c a l l y injected into
Correspondence." P. Luo, Department of Anatomy, Fourth Military Medical University, Xi'an 710032, Shaanxi, People's Republic of China. 0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
134
LL , . , C
Fig. 1. Camera lucida drawing of a type I LPB neuron with spiny, long dendrites and a long axon (ax), which was identified by antidromic responses following stimulation of the ipsilateral posteromedial ventral thalamic nucleus. The latency was 2.9 ms. The photomicrographs of the cell body, and dendritic regions b and c of this neuron are shown in Fig. 3A-C. L, lateral; C, caudal. Scale= 50/~m.
soma of the LPB neurons through the recording electrode by passing 5 20 nA depolarizing rectangular current pulses of 200 ms duration at 2-3 Hz for 1 5 min. After 2-12 h, the rats were re-anesthetized deeply, and then perfused through the ascending aorta with 100 ml of 0.9% saline, followed by 300 ml of a mixture of 1.0% paraformaldehyde and 1.25% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). The brains were removed, and serial frozen sections of 60/~m thickness were cut transversely, horizontally, or parasagittally. For the histochemical demonstration of HRP, the sections were placed in a 5% cobaltous chloride solution for intensification of the H R P reaction product [1] prior to incubation with diaminobenzidine tetrahydrochloride and hydrogen peroxide. The reconstruction of labeled neurons was done by using a drawing tube with an objective of x 40 or x 100. A total of 16 neurons were labeled successfully in different regions of the LPB. These LPB neurons were divided into type I neurons (7 neurons) and type II neurons (9 neurons). Type I neurons were identified by antidromic responses following stimulation of the ipsilateral posteromedial ventral thalamic nucleus. The antidromic spike potentials with an amplitude of 40-50 mV were activated after a constant latency of 2.6-3.8 ms (3.1 +0.56 ms, n = 7 ) , followed by a 100 or 200 Hz stimulus frequency. These neurons were large or medium-sized, and had a long axon and 2-5 primary dendrites which further branched 3-5 times (Fig. 1). The length of dendrites varied from 300 to 750/tm. Most of the dendrites extended rostrocaudally along the longitu-
135
5ms
Fig. 2. Camera lucida drawing of a type II LPB neuron with short dendrites and a short axon (ax). An orthodromic response was evoked by stimulation of the ipsilateral nucleus of the solitary tract. The latency was 18 ms. The photomicrographsof the cell body of this neuron is shownin Fig. 3D. D, dorsal; L, lateral. Scale= 50 gin. dinal axis of the brainstem. Many spines were densely distributed on the dendrites (Figs. 1 and 3B,C); the distance between the dendritic spines varied from 2 to 6 pm. Somatic spines were also observed occasionally (Fig. 3A). Most of the dendrites of type I neurons extended out of the confines of the LPB to distribute in the medial parabrachial nucleus, locus coeruleus, locus subcoeruleus, cerebellum, cuneiform nucleus, pedunculopontine tegmental nucleus, and reticular formation of the midbrain. In 3 of the 7 type I neurons, an axon originated from the primary dendrites and extended rostromedially to ascend in the dorsal tegmental bundle. Axon collaterals arising from these axons appeared to terminate in the dorsal raphe nucleus and the nucleus of Edinger-Westphal. In 1 of the 7 type I neurons, an axon was observed to descend in the Probst's tract. Type II neurons could not be activated antidromically. In 3 of the 9 type II neurons, orthodromic responses were evoked by stimulating the nucleus of the solitary tract with increasing stimulus strength. The response latencies varied from 15 to 30 ms. They were bipolar or multipolar neurons of medium or small size, and had a short axon and 2~, primary dendrites (Fig. 2). In type II neurons, dendritic spines were distributed far less densely than in type I neurons (Figs. 2 and 3D), the distance between the dendritic spines varied from 10 to 20/zm. The axons and dendrites of type II neurons were distributed within the confines of the LPB. The present study revealed 2 types of neurons in the LPB. Type I LPB neurons which sent their axons to the ipsilateral posteromedial ventral nucleus of the thalamus had long dendrites, through which various afferent fibers appeared to converge upon type I LPB neurons. It has also been reported in the cat that LPB neurons sending their axons to the amygdala were provided with long dendrites [8]. On the other hand, in type II LPB neurons, both axon and dendrites were distributed within the confines of the LPB; type II LPB neurons were considered to be local circuit neurons.
136
Fig. 3, Photomicrographs of a type l LPB neuron (A C) shown in Fig. l, and a type II LPB neuron (D) shown in Fig. 2. A: the type I neuron which is shown in Fig. 1, arrows indicate somatic spines, ax, axon. Scale = 20 Ftm. B: a portion of spiny dendrite of the type l LPB neurons, which is shown in Fig. 1 (b region). Arrowheads indicate filopodia-like dendritic processes. Scale = 10 pm. C: a portion of a distal dendrite with bead varicosities of the type I LPB neuron, which is shown in Fig. 1 (c region). Scale = 10 pro. D: a type II LPB neuron with a short axon (ax), shown in Fig. 2, arrowheads point to dendritic spines. Scale ==20/~m. 1 Adams, J.C., Technical considerations on the use of horseradish peroxidase as a neural marker, Neuroscience, 2 (1977) 141 145. 2 Fulwiler, C. and Saper, C.B.~ Subnuclear organization of the efferent connections of the parabrachial nucleus in the rat. Brain Res. Rev., 7 (1984) 229 259. 3 Kapp, B.S., Markgrat, C.G., Schwaber, J.S. and Bilyk-Spafford, T., The organization of dorsal medullary projections to the central amygdaloid nucleus and parabrachial nuclei in the rabbit, Neuroscience, 30(1989) 717 732. 4 Mizuno~ N., Nomura, S. and Takeuchi, Y., The parabrachial nucleus as an intermediate relay station of the visceral afferent pathways in the cat. In M. lto~ et al. (Eds.), Integrative Control Functions of the Brain, Vol. III, Kokansha, Tokyo/Elsevier, Amsterdam, 1980, pp. 51 64. 5 0 g a w a , H., Hayama, T. and Ito, S., Response properties of the parabrachio-thalamic taste and mechanoreceptive neurons in rats, Exp. Brain Res., 68 (1987) 449~,57. 6 Saper, C.B. and Loewy, A.D., Efferent connections of the parabrachial nucleus in the rat, Brain Res., 197 (1980) 291 317. 7 Shimada, S., Shiosaka, S., Emson, P.C., Hillyard, C.J., Girgis, S., MacIntyre, I. and Tohyama, M., Calcitonic gene-related peptidergic projection from the parabranchial area to the forebrain and diencephalon in the rat: an immunohistochemical analysis, Neuroscience, 16 (1985) 607 616. 8 Takeuchi, Y., Takahashi, O., Satoda~ T. and Matsushima, R.M., Long dendrites of the parabrachial nucleus neurons projecting to the amygdala. A horseradish peroxidase and Golgi study in the cat, Exp. Neurol., 96 (1987) 203 -207.
133
NSL 06841
A morphological analysis of neurons in the lateral parabrachial nucleus: an intracellular horseradish peroxidase study in the rat Pifu Luo, Jishuo Li and Zhiren Rao Department of ,4natomy, Fourth Military Medical University, Shaanxi (People's Republic of China) (Received 13 November 1989; Revised version received 16 January 1990; Accepted 18 January 1990) Key words." Parabrachial nucleus; Intracellular injection; Horseradish peroxidase; Rat Neurons in the lateral parabrachial nucleus (LPB) of the rat were analyzed by the intracellular horseradish peroxidase method. Sixteen LPB neurons were successfully labeled with HRP injected intracellularly. HRP-labeled LPB neurons were divided into type I (7 neurons) and type II (9 neurons) LPB neurons. Type I LPB neurons, which were activated antidromically by stimulation of the ipsilateral posteromedial ventral nucleus of the thalamus, had long dendrites and a long axon. Type II LPB neurons, which were not activated antidromically by the stimulation of the thalamus, had short dendrites and a short axon. It was concluded that type I LPB neurons were projection neurons, while type II LPB neurons were local circuit neurons.
T h e afferent a n d efferent c o n n e c t i o n s o f the lateral p a r a b r a c h i a l nucleus (LPB) have been well s t u d i e d [2-7], b u t the d a t a c o n c e r n i n g the m o r p h o l o g y o f i n d i v i d u a l LPB n e u r o n s a r e scanty [8]. In the present study, therefore, an a t t e m p t was m a d e to observe i n d i v i d u a l LPB n e u r o n s o f the rat b y intracellular injection o f h o r s e r a d i s h p e r o x i d a s e ( H R P ) into i n d i v i d u a l LPB neurons. A d u l t S p r a g u e - D a w l e y rats o f either sex (200-250 g) were anesthetized with s o d i u m p e n t o b a r b i t a l (4 mg/100 g b.wt.), a n d p a r a l y z e d with gallamine t r i e t h i o d i d e (5 mg/100 g b.wt.). T h e n the rats were artificially ventilated on a stereotaxic a p p a r a tus. G l a s s - m i c r o e l e c t r o d e s with tip d i a m e t e r o f 0.5-1.0 p m were filled with 5-10% H R P (Sigma T y p e VI) in 0.05 M T r i s - H C l buffer ( p H 7.6) a n d 0.25 M KC1 were used for i n t r a c e l l u l a r recording. A n t i d r o m i c o r o r t h o d r o m i c responses were e v o k e d by s t i m u l a t i n g the ipsilateral p o s t e r o m e d i a l ventral t h a l a m i c nucleus o r the ipsilateral nucleus o f the solitary tract, respectively. A f t e r successful i n t r a c e l l u l a r recording, H R P was i o n t o p h o r e t i c a l l y injected into
Correspondence." P. Luo, Department of Anatomy, Fourth Military Medical University, Xi'an 710032, Shaanxi, People's Republic of China. 0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
134
LL , . , C
Fig. 1. Camera lucida drawing of a type I LPB neuron with spiny, long dendrites and a long axon (ax), which was identified by antidromic responses following stimulation of the ipsilateral posteromedial ventral thalamic nucleus. The latency was 2.9 ms. The photomicrographs of the cell body, and dendritic regions b and c of this neuron are shown in Fig. 3A-C. L, lateral; C, caudal. Scale= 50/~m.
soma of the LPB neurons through the recording electrode by passing 5 20 nA depolarizing rectangular current pulses of 200 ms duration at 2-3 Hz for 1 5 min. After 2-12 h, the rats were re-anesthetized deeply, and then perfused through the ascending aorta with 100 ml of 0.9% saline, followed by 300 ml of a mixture of 1.0% paraformaldehyde and 1.25% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). The brains were removed, and serial frozen sections of 60/~m thickness were cut transversely, horizontally, or parasagittally. For the histochemical demonstration of HRP, the sections were placed in a 5% cobaltous chloride solution for intensification of the H R P reaction product [1] prior to incubation with diaminobenzidine tetrahydrochloride and hydrogen peroxide. The reconstruction of labeled neurons was done by using a drawing tube with an objective of x 40 or x 100. A total of 16 neurons were labeled successfully in different regions of the LPB. These LPB neurons were divided into type I neurons (7 neurons) and type II neurons (9 neurons). Type I neurons were identified by antidromic responses following stimulation of the ipsilateral posteromedial ventral thalamic nucleus. The antidromic spike potentials with an amplitude of 40-50 mV were activated after a constant latency of 2.6-3.8 ms (3.1 +0.56 ms, n = 7 ) , followed by a 100 or 200 Hz stimulus frequency. These neurons were large or medium-sized, and had a long axon and 2-5 primary dendrites which further branched 3-5 times (Fig. 1). The length of dendrites varied from 300 to 750/tm. Most of the dendrites extended rostrocaudally along the longitu-
135
5ms
Fig. 2. Camera lucida drawing of a type II LPB neuron with short dendrites and a short axon (ax). An orthodromic response was evoked by stimulation of the ipsilateral nucleus of the solitary tract. The latency was 18 ms. The photomicrographsof the cell body of this neuron is shownin Fig. 3D. D, dorsal; L, lateral. Scale= 50 gin. dinal axis of the brainstem. Many spines were densely distributed on the dendrites (Figs. 1 and 3B,C); the distance between the dendritic spines varied from 2 to 6 pm. Somatic spines were also observed occasionally (Fig. 3A). Most of the dendrites of type I neurons extended out of the confines of the LPB to distribute in the medial parabrachial nucleus, locus coeruleus, locus subcoeruleus, cerebellum, cuneiform nucleus, pedunculopontine tegmental nucleus, and reticular formation of the midbrain. In 3 of the 7 type I neurons, an axon originated from the primary dendrites and extended rostromedially to ascend in the dorsal tegmental bundle. Axon collaterals arising from these axons appeared to terminate in the dorsal raphe nucleus and the nucleus of Edinger-Westphal. In 1 of the 7 type I neurons, an axon was observed to descend in the Probst's tract. Type II neurons could not be activated antidromically. In 3 of the 9 type II neurons, orthodromic responses were evoked by stimulating the nucleus of the solitary tract with increasing stimulus strength. The response latencies varied from 15 to 30 ms. They were bipolar or multipolar neurons of medium or small size, and had a short axon and 2~, primary dendrites (Fig. 2). In type II neurons, dendritic spines were distributed far less densely than in type I neurons (Figs. 2 and 3D), the distance between the dendritic spines varied from 10 to 20/zm. The axons and dendrites of type II neurons were distributed within the confines of the LPB. The present study revealed 2 types of neurons in the LPB. Type I LPB neurons which sent their axons to the ipsilateral posteromedial ventral nucleus of the thalamus had long dendrites, through which various afferent fibers appeared to converge upon type I LPB neurons. It has also been reported in the cat that LPB neurons sending their axons to the amygdala were provided with long dendrites [8]. On the other hand, in type II LPB neurons, both axon and dendrites were distributed within the confines of the LPB; type II LPB neurons were considered to be local circuit neurons.
136
Fig. 3, Photomicrographs of a type l LPB neuron (A C) shown in Fig. l, and a type II LPB neuron (D) shown in Fig. 2. A: the type I neuron which is shown in Fig. 1, arrows indicate somatic spines, ax, axon. Scale = 20 Ftm. B: a portion of spiny dendrite of the type l LPB neurons, which is shown in Fig. 1 (b region). Arrowheads indicate filopodia-like dendritic processes. Scale = 10 pm. C: a portion of a distal dendrite with bead varicosities of the type I LPB neuron, which is shown in Fig. 1 (c region). Scale = 10 pro. D: a type II LPB neuron with a short axon (ax), shown in Fig. 2, arrowheads point to dendritic spines. Scale ==20/~m. 1 Adams, J.C., Technical considerations on the use of horseradish peroxidase as a neural marker, Neuroscience, 2 (1977) 141 145. 2 Fulwiler, C. and Saper, C.B.~ Subnuclear organization of the efferent connections of the parabrachial nucleus in the rat. Brain Res. Rev., 7 (1984) 229 259. 3 Kapp, B.S., Markgrat, C.G., Schwaber, J.S. and Bilyk-Spafford, T., The organization of dorsal medullary projections to the central amygdaloid nucleus and parabrachial nuclei in the rabbit, Neuroscience, 30(1989) 717 732. 4 Mizuno~ N., Nomura, S. and Takeuchi, Y., The parabrachial nucleus as an intermediate relay station of the visceral afferent pathways in the cat. In M. lto~ et al. (Eds.), Integrative Control Functions of the Brain, Vol. III, Kokansha, Tokyo/Elsevier, Amsterdam, 1980, pp. 51 64. 5 0 g a w a , H., Hayama, T. and Ito, S., Response properties of the parabrachio-thalamic taste and mechanoreceptive neurons in rats, Exp. Brain Res., 68 (1987) 449~,57. 6 Saper, C.B. and Loewy, A.D., Efferent connections of the parabrachial nucleus in the rat, Brain Res., 197 (1980) 291 317. 7 Shimada, S., Shiosaka, S., Emson, P.C., Hillyard, C.J., Girgis, S., MacIntyre, I. and Tohyama, M., Calcitonic gene-related peptidergic projection from the parabranchial area to the forebrain and diencephalon in the rat: an immunohistochemical analysis, Neuroscience, 16 (1985) 607 616. 8 Takeuchi, Y., Takahashi, O., Satoda~ T. and Matsushima, R.M., Long dendrites of the parabrachial nucleus neurons projecting to the amygdala. A horseradish peroxidase and Golgi study in the cat, Exp. Neurol., 96 (1987) 203 -207.