0 1992 Gordon and Breach Science Publishers S.A.
Intern. J . Neuroscience, 1992. Vol. 67, pp. 111-117
Printed in the United States of America
Reprints available directly horn the publisher Photocopying permitted by license only
Brief Communication
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ELECTROACUPUNCTURE ACCELERATED THE EXPRESSION OF CFOS PROTOONCOGENE IN SEROTONERGIC NEURONS OF NUCLEUS RAPHE DORSALIS QING-PING MA, YAN ZHOU, YING-XIN YU, JI-SHENG HAN Department of Physiology, Beijing Medical University, Beijing 1ooO83 (People's Republic of China) (Received March 25. 1992)
It has been proposed that the FOS protein encoded by c-fos protooncogene functions as a nuclear 'third messenger" molecule that couples short-term extracellular signals to long-term alterations in cell function, by regulating the expression of specific target genes. In the present study, immunocytochemical double staining technique was used to investigate the effects of electroacupuncture on the expression of c-fos oncogene in the serotonergic neurons in the nucleus raphe dorsalis (NRD) that has been known to play an important role in the endogenous analgesic system of the brain. The number of FOS positive serotonergic cells in the NRD increased significantly after the electroacupuncture stimulation. These results indicate that electroacupuncture can activate central serotonergic neurons at gene expression level. Keywords: Electroacupuncture; Serotonin: C-Fos protooncogene; Nucleus raphe dorsalis.
The c-fos protooncogene is the mammalian homolog of the v-fos oncogene found in 2 murine osteogenic sarcoma viruses. C-fos encodes a nuclear phosphoprotein, Fos, that binds to DNA (Curran, 1985; Curran, Van Beveren, Ling & verma, 1985; Sambucetti, & Curran, 1986). It has been proposed that the Fos protein functions as a nuclear 'third messenger" molecule that couples short-term extracellular signals to long-term alteration in cell function, by regulating the expression of specific target gene (Curran, & Morgan, 1985; Goelet, Castellucci, Schacher, & Kandel, 1986). The nucleus raphe dorsalis in the brainstem is the largest serotonergic nucleus (Wiklund, Leger, & Persson, 1981), and has been implicated in the endogenous antinociceptive system by many experimental findings (Andersen & Dafny , 1983; Fardin, Oliveras, & Besson, 1984; Goelet et al., 1986; Mayer, Wolfle, Akil, Carde & Liebeskind, 1974). It has been shown that electroacupuncture can affect the spontaneous firing of the NRD neurons (Wu, Xu, Li, Wu, Yu, Liu & Zhong, 1979), but whether the electroacupuncture stimulation can cause long-term alterations in the dorsal raphe neurons is still unclear. The aim of the present study was to determine if electroacupuncture (as a means to activate endogenous analgesic system) can induce the expression of c-fos oncogene in the serotonergic neurons of the NRD, with immunocytochemical double staining techniques. METHODS Male Wistar rats (250-300 g) were divided into 2 groups, each group containing 6-8 animals. The animals were kept in a plastic holder with hind legs and tail *All correspondences should be addressed to Dr.Qing-Ping Ma 111
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extending. Focused light from a 12.5 W projection bulb was applied to the lower 1/3 of the tail and the tail flick latency (TFL) was recorded as pain threshold. Values from the first two measurements, with an interval of 5 min, were averaged as the basal TFL. Subsequent values were expressed as percentage of the basal TFL with +150% as the cut-off level to avoid damage to the skin. For electroacupuncture stimulation, two stainless steel needles of .25 mm diameter were inserted into each hind leg, one in the point Zusanli (stomach 36), 5 mm lateral to anterior tubercles of the tibia, and the other in the point Sanyinjiao (spleen 6) which was located in front of the Archilles tendon. The needles were connected to a stimulator (WQ-SB HAN ACUTENS, Aeron, Beijing). The animals in the electroacupuncture group received a series of stimuli of biphasic pulses with a positive rectangular pulse of . 3 ms duration followed by a negative spike. The frequency of the pulse was 15 Hz. The amplitude of the pulses was increased stepwise from 1 mA to 3 mA in a period of 30 min. The animals in the control group did not receive electrical stirnulation. Two hours after the completion of the electroacupuncture stimulation, the animals were anesthetized with 10% chloral hydrate (4OOmg/kg, i.p.) and perfused through the ascending aorta with 200 ml warm (about 30-37°C) saline, followed by 500 m l 4 % paraformaldehyde fixative in .1 M phosphate buffer (PB, pH 7.4). After perfusion, the brains were removed and postfixed in 4% paraformaldehyde for 12 h and then cryoprotected overnight in 30% sucrose in .1 M PB. Thirty micron frontal brainstem sections were cut in a cryostat. The sections were then immunostained for Fos protein by the avidin-biotin-peroxidase (ABC) method of Hsu et al. (Hsu, Raine, & Fanger, 1981). The tissue sections were washed with 3% bovine serum albumin (BSA) and .25% Triton-X 100 in .01 M phosphate buffered saline (PBS, pH 7.4), and then incubated in the Fos antibody raised in rabbits against a synthetic peptide which corresponded to the N-terminal (residues 4-17) of the Fos protein (1:2OOO,Oncogene Science, Uniondale, NY) at 4°C for 48 h. The sections were then incubated in biotinylated goat antirabbit IgG and avidin-biotin-peroxidasecomplex (Vector labs, Burlingame, CA). The reaction product was visualized with .Ol% hydrogen peroxidase, .05% diaminobenzidine (DAB)and .01% cobalt chloride. After the immunostaining procedure for Fos protein, the sections were washed in .01 M PBS and then incubated in rabbit anti-5-hydroxytryptamine (5-HT)serum (1 :8OOO, Immunonuclear) at 4°C for 48 h. The sections were then incubated in biotinylated goat anti-rabbit IgG and avidin-biotin-peroxidasecomplex. The reaction product was visualized with .01% hydrogen peroxidase and .05% DAB. Two procedures had been used to determine the specificity of the imrnunostaining for Fos protein. One was omission of the Fos antiserum from the immunostaining protocol which completely abolished labelling. The other was to incubate tissue sections with Fos antibody preabsorbed with the synthetic peptide (Oncogene Science, Uniondale, NY); the immunostaining was also completely abolished.
RESULTS Consistent with the known nuclear location of the Fos protein (Curran, 1984). Fosimmunoreactive neurons were easily recognized by their diffusely stained black nuclei and unlabelled nucleoli, whereas the cytoplasm of 5-HT-positive neurons was homogeneously stained in light brown. Hence, double labelled cells could be easily identified by their black nuclei and brown cytoplasm (Fig. 1). In the control group, almost all labelled cells in the NRD were 5-HT-single-positive, and only a few Fos
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ELECTROCUPUNCI'URE AND PROTOONCOGENE
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FIGURE 1 The Fos and 5-HTdouble-labelledcells were recognized by black-stained nuclei and brownish-stained cytoplasm (indicated by arrows). Bar. 50 pm.
FIGURE 2 In
the. control group, very few serotonergic neurons had a Fos-immunoreactive nucleus, most cells were serotonin single-positive. Bar, 100 pm.
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FIGURE 3 In the electroacupuncture group, numerous serotonergic neurons were Fos-irnmunoreactive. Bar, 100 pn.
and 5-HT double-labelled cells were scattered along the entire rostrocaudal extent of the NRD (Fig. 2). In the electroacupuncture group, many double-labelled cells were observed in the NRD (Fig. 3). Double-labelled cells were round, fusiform or triangular in shape with diameters ranging from 15 p m to 35 pm. The distribution of double-labelled neurons is shown schematically in Fig. 4. In order to get an idea on the effects of electroacupuncture on the number of double-labelled cells, 5 sections of the mesencephalic part were taken randomly from each group. Only 10.2% of 5-HT-positive cells in the NRD of the control group were Fos-immunoreactive, whereas more than one fourth (26.5%) of 5-HT-positive cells in the electroacupuncture group were Fos-immunoreactive, which was significantly greater than that of the control group (x2 test, p < .01).
DISCUSSION Current research suggests that c-fos expression may be related to the neuron’s ability to convert short-term synaptic stimulation into long-term responses and may thus contribute to the adaptive alterations involved in neuronal plasticity and memory formation (Goelet et al., 1986). It has been found that c-Fos and c-Jun proteins are involved in the upregulation of the proenkephalin (Sonnenberg, Rauscher, Morgan, & Curran, 1989) and of the nerve growth factor (Hengerer, Lindholm, Heumann, Ruther, Wagner, & Thoenen, 1990). Cutaneous inflammation induces an increased transcription rate of proenkephalin which is preceded by expression of c-fos gene (Draisci, & Iadarola, 1989). Since endogenous opioid has been suggested to subserve
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HGURE 4 The distribution of Fos and 5-HT double-labelled neurons in the NRD of the electroacupuncture group. Each solid circle represents 5 double-labelled cells. FL,fasciculus longitudinalis; FLM, fasciculus longitudinalis medialis; LM, lemniscus medialis; ncs, nucleus centralis superior; P, tractus corticospinalis; PCS, pedunculus cerebellaris superior; rtp, nucleus reticularis tagmenti pontis, TTS, tractus tectospinalis.
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the analgesic action of brain stimulation (Mayer et al., 1974) and electroacupuncture (Han, & Terenius, 1982), we hypothesize that expression of c-fos may also translate electroacupuncture stimulation into long-term nuclear responses. Experimental data have implicated the NRD in the endogenous antinociceptive system (Fardin et al., 1984; Mayer et al., 1974). Previous studies from our laboratory suggested that serotonergic projections from the NRD to the nucleus accumbens and amygdala may mediate the analgesic action of morphine and electroacupuncture (Han, & Xuan, 1986; Xuan, Shi, Zhou, & Han, 1986; Yu, Shi, & Han, 1989). In addition, we have demonstrated that morphine injected into the periaqueductal gray (PAG, including the NRD) could increase the release of enkephalins in the N. accumbens and amygdala (Ma, & Han, 1991; Ma, Shi, & Han, 1991). Therefore, it is worthwhile to investigate the c-fos expression in the NRD. Since electroacupuncture can affect the firing activities of the neurons in the NRD (Wu et al., 1979), electroacupuncture stimulation will possibly induce c-fos expression in the NRD, especially its serotonergic neurons. This was confirmed in the present study. Electroacupuncture significantly increased the number of Fos-immunoreactive serotonergic neurons in the NRD. These results indicate that electroacupuncture causes not only transient changes of electrical activities, but also long-term changes in gene transcription. Since most NRD neurons are serotonergic (Wiklund et al., 1981), it is understandable that the Fos-immunoreactive neurons in the NRD were principally serotonergic. The preproenkephalin gene has been identified as one of the possible target genes for c-fos (Sonnenberg et al., 1989). How can the Fos protein be related to the function of serotonin? At present there is no indication about c-fos regulation of the transcription of serotonin synthesizing enzymes. Since the results of the present study demonstrated definitely that c-fos was involved in the activity of serotonergic neurons, c-fos must play a role in regulating the function of serotonergic neurons. Further studies are required to elucidate possible roles of the c-fos expression in the serotonergic neurons.
REFERENCES Andersen, E. & Dafny, N., (1983). An ascending serotonergic pain modulation pathway from the dorsal raphe nucleus to the parafascicular nucleus of the thalamus. Brain Research, 269. 57-67. Curran, T. (1984). Viral and cellular fos proteins. Cell, 36, 259-268. Curran, T. & Morgan, J. I., (1985). Superinduction of c-fos by nerve growth factor in the presence of peripherally active benzodiazepines. Science. 229. 1265- 1268. Curran, T., Van Beveren, C., Ling, N. & Verma, 1. M.,(1985). Viral and cellular fos proteins are complexed with a 39,000-Dalton cellular protein. Molecular Cell Biology, 5 . 167-172. Draisci, G. & Iadarola, M. I . , (1989). Temporal analysis of increases in c-fos, prepdynorphin and in rat spinal cord. Molecular Brain Research, 6 , 31-37. preproenkephalin -As Fardin. V . . Oliveras, J. L. & Besson, J. M., (1984). A reinvestigation of the periaqueductal gray matter in the cat, 11. Differential characteristics of the analgesia induced by ventral and dorsal PAC stimulation. Bruin Research, 306. 125-139. Goelet, P., Castellucci, V. F., Schacher, S. & Kandel, E. R., (1986). The long and the short of longterm memory-a molecular framework. Nature. 322, 419-422. Han, J. S. & Terenius, L., (1982). Neurochemical basis of acupuncture analgesia. Annual Review of Pharmacology and Toxicology. 22. 193-220. Han, J. S . & Xuan, Y.T., (1986). A mesolimbic loop of analgesia. I. Activation by morphine of a serotonergic pathway from periaqueductd gray to nucleus accumbens. InternationaI Journal of Neuroscience, 29, 109-1 18. Hengerer, B., Lindholm, D., Heurnann, R.,Ruther, U.,Wagner, E. F. & Thoenen, H.,(1990). Lesioninduced increase in nerve growth factor mRNA is mediated by c-fos. Proceedings of National Academy of Science USA, 87, 3899-3903.
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Hsu, S. M., Raine. L. & Fanger, H., (1981). Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabelled antibody (PAP)procedures. Journul of Histochemisrry and Cytochemistry, 29, 577-580. Ma, Q. P. & Han, J. S . . (1991).Neurochemical studies on the mesolimbic circuitry of antinociception. Brain Research. 566, 95-102. Ma, Q. P., Shi. Y.S. & Han, J. S.. (1991).The interaction between the periaqueductal gray and N. accumbens in accelerating the release of enkephalins and p-endorphin. Chinese Science Bulleh’ne, 36, 855-858. Mayer, D. J., Wolfle, L.. Akil, H., Carder, B. & Liebeskind, J. C.. (1974).Analgesia from electrical stimulation in the brainstem of the rat. Science, 174, 1351-1354. Sambucetti. L. C. & Curran, T., (1986). The Fos protein complex is associated with DNA in isolated nuclei and binds to DNA cellulose. Science, 234, 1417-1419. Sonnenberg, J. L.,Rauscher, J. F., Morgan, J. I. & Curran, T.. (1989). Regulation of proenkephalin by Fos and Jun. Science, 246. 1622-1625. Wiklund, L., Leger, L. & Persson, M.,(1981). Monoamine cell distribution in the cat brain stem. A fluorescence histochemical study with quantification of indolaminergic and locus coeruleus cell groups. Journal of Compararive Neurology. 203, 613-647. Wu, B. J., Xu, J. L., Li, D. Z., Wu. F. J., Yu, Y. X., Liu, Y. C. & Zhong, G. G., (1979). The influences of electmacupuncture and noxious stimulation on the single-unit f ~ n g of s the dorsal raphe nucleus in the rat. Chinese Science Bulletine, 24, 570-573. Xuan, Y.T.,Shi, Y. S., Zhou, Z. F. & Han, J. S., (1986). Studies on the mesolimbic loop of antinociception. II. A serotonin-enkephalin interaction in the nucleus accumbens. Neuroscience, 19.403-
409. Yu. L. C., Shi, Y. S. & Han, J. S., (1989). A neural pathway from periaqueductal gray involved in antinociception. Chinese Science Bulletine. 34, 68-7 1.
to amygdala
Intern. J . Neuroscience, 1992. Vol. 67, pp. 111-117
Printed in the United States of America
Reprints available directly horn the publisher Photocopying permitted by license only
Brief Communication
Int J Neurosci Downloaded from informahealthcare.com by Chinese University of Hong Kong on 02/08/15 For personal use only.
ELECTROACUPUNCTURE ACCELERATED THE EXPRESSION OF CFOS PROTOONCOGENE IN SEROTONERGIC NEURONS OF NUCLEUS RAPHE DORSALIS QING-PING MA, YAN ZHOU, YING-XIN YU, JI-SHENG HAN Department of Physiology, Beijing Medical University, Beijing 1ooO83 (People's Republic of China) (Received March 25. 1992)
It has been proposed that the FOS protein encoded by c-fos protooncogene functions as a nuclear 'third messenger" molecule that couples short-term extracellular signals to long-term alterations in cell function, by regulating the expression of specific target genes. In the present study, immunocytochemical double staining technique was used to investigate the effects of electroacupuncture on the expression of c-fos oncogene in the serotonergic neurons in the nucleus raphe dorsalis (NRD) that has been known to play an important role in the endogenous analgesic system of the brain. The number of FOS positive serotonergic cells in the NRD increased significantly after the electroacupuncture stimulation. These results indicate that electroacupuncture can activate central serotonergic neurons at gene expression level. Keywords: Electroacupuncture; Serotonin: C-Fos protooncogene; Nucleus raphe dorsalis.
The c-fos protooncogene is the mammalian homolog of the v-fos oncogene found in 2 murine osteogenic sarcoma viruses. C-fos encodes a nuclear phosphoprotein, Fos, that binds to DNA (Curran, 1985; Curran, Van Beveren, Ling & verma, 1985; Sambucetti, & Curran, 1986). It has been proposed that the Fos protein functions as a nuclear 'third messenger" molecule that couples short-term extracellular signals to long-term alteration in cell function, by regulating the expression of specific target gene (Curran, & Morgan, 1985; Goelet, Castellucci, Schacher, & Kandel, 1986). The nucleus raphe dorsalis in the brainstem is the largest serotonergic nucleus (Wiklund, Leger, & Persson, 1981), and has been implicated in the endogenous antinociceptive system by many experimental findings (Andersen & Dafny , 1983; Fardin, Oliveras, & Besson, 1984; Goelet et al., 1986; Mayer, Wolfle, Akil, Carde & Liebeskind, 1974). It has been shown that electroacupuncture can affect the spontaneous firing of the NRD neurons (Wu, Xu, Li, Wu, Yu, Liu & Zhong, 1979), but whether the electroacupuncture stimulation can cause long-term alterations in the dorsal raphe neurons is still unclear. The aim of the present study was to determine if electroacupuncture (as a means to activate endogenous analgesic system) can induce the expression of c-fos oncogene in the serotonergic neurons of the NRD, with immunocytochemical double staining techniques. METHODS Male Wistar rats (250-300 g) were divided into 2 groups, each group containing 6-8 animals. The animals were kept in a plastic holder with hind legs and tail *All correspondences should be addressed to Dr.Qing-Ping Ma 111
Int J Neurosci Downloaded from informahealthcare.com by Chinese University of Hong Kong on 02/08/15 For personal use only.
I12
Q.P. M A , et a/.
extending. Focused light from a 12.5 W projection bulb was applied to the lower 1/3 of the tail and the tail flick latency (TFL) was recorded as pain threshold. Values from the first two measurements, with an interval of 5 min, were averaged as the basal TFL. Subsequent values were expressed as percentage of the basal TFL with +150% as the cut-off level to avoid damage to the skin. For electroacupuncture stimulation, two stainless steel needles of .25 mm diameter were inserted into each hind leg, one in the point Zusanli (stomach 36), 5 mm lateral to anterior tubercles of the tibia, and the other in the point Sanyinjiao (spleen 6) which was located in front of the Archilles tendon. The needles were connected to a stimulator (WQ-SB HAN ACUTENS, Aeron, Beijing). The animals in the electroacupuncture group received a series of stimuli of biphasic pulses with a positive rectangular pulse of . 3 ms duration followed by a negative spike. The frequency of the pulse was 15 Hz. The amplitude of the pulses was increased stepwise from 1 mA to 3 mA in a period of 30 min. The animals in the control group did not receive electrical stirnulation. Two hours after the completion of the electroacupuncture stimulation, the animals were anesthetized with 10% chloral hydrate (4OOmg/kg, i.p.) and perfused through the ascending aorta with 200 ml warm (about 30-37°C) saline, followed by 500 m l 4 % paraformaldehyde fixative in .1 M phosphate buffer (PB, pH 7.4). After perfusion, the brains were removed and postfixed in 4% paraformaldehyde for 12 h and then cryoprotected overnight in 30% sucrose in .1 M PB. Thirty micron frontal brainstem sections were cut in a cryostat. The sections were then immunostained for Fos protein by the avidin-biotin-peroxidase (ABC) method of Hsu et al. (Hsu, Raine, & Fanger, 1981). The tissue sections were washed with 3% bovine serum albumin (BSA) and .25% Triton-X 100 in .01 M phosphate buffered saline (PBS, pH 7.4), and then incubated in the Fos antibody raised in rabbits against a synthetic peptide which corresponded to the N-terminal (residues 4-17) of the Fos protein (1:2OOO,Oncogene Science, Uniondale, NY) at 4°C for 48 h. The sections were then incubated in biotinylated goat antirabbit IgG and avidin-biotin-peroxidasecomplex (Vector labs, Burlingame, CA). The reaction product was visualized with .Ol% hydrogen peroxidase, .05% diaminobenzidine (DAB)and .01% cobalt chloride. After the immunostaining procedure for Fos protein, the sections were washed in .01 M PBS and then incubated in rabbit anti-5-hydroxytryptamine (5-HT)serum (1 :8OOO, Immunonuclear) at 4°C for 48 h. The sections were then incubated in biotinylated goat anti-rabbit IgG and avidin-biotin-peroxidasecomplex. The reaction product was visualized with .01% hydrogen peroxidase and .05% DAB. Two procedures had been used to determine the specificity of the imrnunostaining for Fos protein. One was omission of the Fos antiserum from the immunostaining protocol which completely abolished labelling. The other was to incubate tissue sections with Fos antibody preabsorbed with the synthetic peptide (Oncogene Science, Uniondale, NY); the immunostaining was also completely abolished.
RESULTS Consistent with the known nuclear location of the Fos protein (Curran, 1984). Fosimmunoreactive neurons were easily recognized by their diffusely stained black nuclei and unlabelled nucleoli, whereas the cytoplasm of 5-HT-positive neurons was homogeneously stained in light brown. Hence, double labelled cells could be easily identified by their black nuclei and brown cytoplasm (Fig. 1). In the control group, almost all labelled cells in the NRD were 5-HT-single-positive, and only a few Fos
Int J Neurosci Downloaded from informahealthcare.com by Chinese University of Hong Kong on 02/08/15 For personal use only.
ELECTROCUPUNCI'URE AND PROTOONCOGENE
1 I3
FIGURE 1 The Fos and 5-HTdouble-labelledcells were recognized by black-stained nuclei and brownish-stained cytoplasm (indicated by arrows). Bar. 50 pm.
FIGURE 2 In
the. control group, very few serotonergic neurons had a Fos-immunoreactive nucleus, most cells were serotonin single-positive. Bar, 100 pm.
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FIGURE 3 In the electroacupuncture group, numerous serotonergic neurons were Fos-irnmunoreactive. Bar, 100 pn.
and 5-HT double-labelled cells were scattered along the entire rostrocaudal extent of the NRD (Fig. 2). In the electroacupuncture group, many double-labelled cells were observed in the NRD (Fig. 3). Double-labelled cells were round, fusiform or triangular in shape with diameters ranging from 15 p m to 35 pm. The distribution of double-labelled neurons is shown schematically in Fig. 4. In order to get an idea on the effects of electroacupuncture on the number of double-labelled cells, 5 sections of the mesencephalic part were taken randomly from each group. Only 10.2% of 5-HT-positive cells in the NRD of the control group were Fos-immunoreactive, whereas more than one fourth (26.5%) of 5-HT-positive cells in the electroacupuncture group were Fos-immunoreactive, which was significantly greater than that of the control group (x2 test, p < .01).
DISCUSSION Current research suggests that c-fos expression may be related to the neuron’s ability to convert short-term synaptic stimulation into long-term responses and may thus contribute to the adaptive alterations involved in neuronal plasticity and memory formation (Goelet et al., 1986). It has been found that c-Fos and c-Jun proteins are involved in the upregulation of the proenkephalin (Sonnenberg, Rauscher, Morgan, & Curran, 1989) and of the nerve growth factor (Hengerer, Lindholm, Heumann, Ruther, Wagner, & Thoenen, 1990). Cutaneous inflammation induces an increased transcription rate of proenkephalin which is preceded by expression of c-fos gene (Draisci, & Iadarola, 1989). Since endogenous opioid has been suggested to subserve
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HGURE 4 The distribution of Fos and 5-HT double-labelled neurons in the NRD of the electroacupuncture group. Each solid circle represents 5 double-labelled cells. FL,fasciculus longitudinalis; FLM, fasciculus longitudinalis medialis; LM, lemniscus medialis; ncs, nucleus centralis superior; P, tractus corticospinalis; PCS, pedunculus cerebellaris superior; rtp, nucleus reticularis tagmenti pontis, TTS, tractus tectospinalis.
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the analgesic action of brain stimulation (Mayer et al., 1974) and electroacupuncture (Han, & Terenius, 1982), we hypothesize that expression of c-fos may also translate electroacupuncture stimulation into long-term nuclear responses. Experimental data have implicated the NRD in the endogenous antinociceptive system (Fardin et al., 1984; Mayer et al., 1974). Previous studies from our laboratory suggested that serotonergic projections from the NRD to the nucleus accumbens and amygdala may mediate the analgesic action of morphine and electroacupuncture (Han, & Xuan, 1986; Xuan, Shi, Zhou, & Han, 1986; Yu, Shi, & Han, 1989). In addition, we have demonstrated that morphine injected into the periaqueductal gray (PAG, including the NRD) could increase the release of enkephalins in the N. accumbens and amygdala (Ma, & Han, 1991; Ma, Shi, & Han, 1991). Therefore, it is worthwhile to investigate the c-fos expression in the NRD. Since electroacupuncture can affect the firing activities of the neurons in the NRD (Wu et al., 1979), electroacupuncture stimulation will possibly induce c-fos expression in the NRD, especially its serotonergic neurons. This was confirmed in the present study. Electroacupuncture significantly increased the number of Fos-immunoreactive serotonergic neurons in the NRD. These results indicate that electroacupuncture causes not only transient changes of electrical activities, but also long-term changes in gene transcription. Since most NRD neurons are serotonergic (Wiklund et al., 1981), it is understandable that the Fos-immunoreactive neurons in the NRD were principally serotonergic. The preproenkephalin gene has been identified as one of the possible target genes for c-fos (Sonnenberg et al., 1989). How can the Fos protein be related to the function of serotonin? At present there is no indication about c-fos regulation of the transcription of serotonin synthesizing enzymes. Since the results of the present study demonstrated definitely that c-fos was involved in the activity of serotonergic neurons, c-fos must play a role in regulating the function of serotonergic neurons. Further studies are required to elucidate possible roles of the c-fos expression in the serotonergic neurons.
REFERENCES Andersen, E. & Dafny, N., (1983). An ascending serotonergic pain modulation pathway from the dorsal raphe nucleus to the parafascicular nucleus of the thalamus. Brain Research, 269. 57-67. Curran, T. (1984). Viral and cellular fos proteins. Cell, 36, 259-268. Curran, T. & Morgan, J. I., (1985). Superinduction of c-fos by nerve growth factor in the presence of peripherally active benzodiazepines. Science. 229. 1265- 1268. Curran, T., Van Beveren, C., Ling, N. & Verma, 1. M.,(1985). Viral and cellular fos proteins are complexed with a 39,000-Dalton cellular protein. Molecular Cell Biology, 5 . 167-172. Draisci, G. & Iadarola, M. I . , (1989). Temporal analysis of increases in c-fos, prepdynorphin and in rat spinal cord. Molecular Brain Research, 6 , 31-37. preproenkephalin -As Fardin. V . . Oliveras, J. L. & Besson, J. M., (1984). A reinvestigation of the periaqueductal gray matter in the cat, 11. Differential characteristics of the analgesia induced by ventral and dorsal PAC stimulation. Bruin Research, 306. 125-139. Goelet, P., Castellucci, V. F., Schacher, S. & Kandel, E. R., (1986). The long and the short of longterm memory-a molecular framework. Nature. 322, 419-422. Han, J. S. & Terenius, L., (1982). Neurochemical basis of acupuncture analgesia. Annual Review of Pharmacology and Toxicology. 22. 193-220. Han, J. S . & Xuan, Y.T., (1986). A mesolimbic loop of analgesia. I. Activation by morphine of a serotonergic pathway from periaqueductd gray to nucleus accumbens. InternationaI Journal of Neuroscience, 29, 109-1 18. Hengerer, B., Lindholm, D., Heurnann, R.,Ruther, U.,Wagner, E. F. & Thoenen, H.,(1990). Lesioninduced increase in nerve growth factor mRNA is mediated by c-fos. Proceedings of National Academy of Science USA, 87, 3899-3903.
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to amygdala
