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Colocalization of NADPH-diaphorase with neuropeptides in the intrapancreatic neurons of the chicken
Neuroscience Letters 182 (1994) 37-40
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NEUROSCIENC[ lETTERS
Colocalization of NADPH-diaphorase with neuropeptides in the intrapancreatic neurons of the chicken Kohzy Hiramatsu*, Koji Ohshima Laboratory of Functional Anatomy, Faculty of Agriculture, Shinshu University, Minamiminowa, Nagano-ken 399-45, Japan
Received 27 July 1994; Revised version received26 September 1994; Accepted 26 September 1994
Abstract Colocalization of nitric oxide with neuropeptides was investigated in the chicken pancreas by use of double staining combined with the indirect immunofluorescence technique and histochemistry for NADPH-diaphorase, a specific marker for neural nitric oxide synthase. NADPH-diaphorase positive ganglia were easily detected in the interlobular connective tissue. Many NADPH-diaphorase positive ganglion cells also showed immunoreactivity for VIP (80.9%) or galanin (76.2%). Some ganglion cells showed enzyme activity only (about 20%). Very few neurons were NADPH-diaphorase negative, but immunopositive for VIP (2.0%) or galanin (3.7%). The present study provides evidence that nitric oxide colocates with VIP and galanin in the chicken pancreas. Key words." NADPH-diaphorase; Vasoactive intestinal polypeptide; Galanin; Colocalization; Pancreas; Innervation; Domestic fowl
Nitric oxide (NO), a free radical gas, is considered as one of the neurotransmitters in the mammalian central and peripheral nervous system [4]. NO is converted from L-arginine by the enzyme nitric oxide synthase (NOS). In the mammalian brain, there are some populations of neurons which show nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity [20]. Recent studies revealed that neural N A D P H - d is identical to NOS in the central and peripheral nervous system [7,11]. Thus histochemistry of N A D P H - d is used as a marker for neuronal NOS [9]. Studies on the distribution of nerves showing N A D P H - d activity were carried out in the enteric nervous system [2,3:6] and the pancreas [8,13,17]. These studies carried out in mammals revealed the colocalization of N A D P H - d activity and neuropeptide immunoreactivity in the same neuron and suggested interactions of N O and neuropeptides. Vasoactive intestinal polypeptide (VIP)- and galanincontaining neurons are two major types of neurons in the chicken pancreas [10,15]. The present study aimed at clarifying the probability of colocalization of NO with
*Corresponding author. Fax: (81) (265) 72-5259. 0304-3940194l$7.00© 1994 Elsevier ScienceIreland Ltd. All rights reserved S S D I 0304-3940(94)00752-7
these neuropeptides in the intrapancreatic neurons of the chicken. Five adult White Leghorn chickens of both sexes, weighing 1.5-2.2 kg, were used in this study. Animals were perfused with 0.75% NaC1 solution, and followed by a mixture of 4% paraformaldehyde, 0.1% glutaraldehyde and 0.21% picric acid in 0.1 M phosphate buffer (pH 7.4). Pancreata were immediately removed after perfusion and dissected into small blocks. Tissue blocks were immersed in the same perfusate for 6 h at 4°C and then transferred to 20% phosphate-buffered sucrose for cryoprotection at 4°C overnight. Frozen sections were serially cut at 16/2m thickness in the cryostat. Every fifth section was mounted on gelatin-coated glass slides. The indirect immunofluorescence technique [5] was applied to demonstrate immunoreactivity for neuropeptides. Frozen sections treated with 10% normal goat serum (Cedarlane, Canada) were incubated with antiserum against porcine VIP (diluted to 1:500; Incstar, USA) or porcine galanin (1:250; Chemicon, USA) at room temperature overnight. After several washes with PBS, the sections were incubated with FITC (fluorescein isothiocyanate)-conjugated goat anti-rabbit Ig G (1:100; E-Y Laboratories, USA) for 1 h at room temperature. They were then rinsed with phosphate
38
K. Hiramatsu, K. Ohshima/Neuroscienee Letters 182 (1994) 37 40
Fig. 1. Paired micrographs (a and b, c and d) of the intrapancreatic ganglia. Three types of ganglion cells are identified in an intrapancreatic ganglion. Arrowheads indicate ganglion cells showing both NADPH-d activity (a,c) and immunoreactivity for VIP (b) or galanin (d). Some neurons show only NADPH-d activity (small arrows) or neuropeptide immunoreactivity (large arrows), x 540.
buffer saline (PBS) and coverslipped with a mixture of glycerol and PBS (3: 1). All ganglia were photographed under a fluorescence microscope, and then the coverslips were removed in PBS. The sections were subsequently processed for histochemistry for NADPH-d. NADPH-d activity was demonstrated according to the method of Scherer-Singler et al. [16] slightly modified by Shimosegawa et al. [18]. Frozen sections were incubated in the medium which contained 1.0 mM beta-NADPH (Oriental Yeast Co., LTD., Tokyo, Japan) as a substrate, 0.2 mM nitroblue tetrazolium (Wako Pure Chemicals Industries, LTD., Osaka, Japan) and 0.2% Triton X-100 in 0.1 M Tris-HCl buffer (pH 8.0). Incubation was carried out at 37°C for 60 rain. After several rinses with PBS, sections were coverslipped with a mixture of glycerol and PBS (3:1). All ganglia corresponding to those in the fluorescence photomicrographs were photographed under a light microscope. Colocalization was evaluated by comparing the fluorescence photomicrographs for neuropeptide-immunoreactivity with the photomicrographs for NADPH-d activity. The percentage of ganglion cells showing NADPH-d activity and/or immunoreactivity for neuropeptides was determined from 36 sections for each bird, and the mean value was calculated for each type of ganglion cell.
NADPH-d activity was detected in nerve fibers and ganglion cells of the chicken pancreas. NADPH-d positive ganglion cells were observed in the interlobular connective tissue (Fig. lb,d) of the ventral and the dorsal lobes, but not in the splenic lobe. Double staining showed the coexistence of NADPH-d and the neuropeptides VIP and galanin (Fig. 1). Intrapancreatic neurons were divided into at least three groups on basis of the results of double staining. Neurons of the first group showed both NADPH-d activity and neuropeptide immunoreactivity (Fig. 1, arrowheads). This group makes up the largest proportion of intrapancreatic neurons. The second group consisted of neurons showing only NADPH-d activity (Fig. l b,d, small arrows). Neurons of the third group were infrequent and only showed immunoreactivity for neuropeptides (Fig. la,c, large arrows). Table 1 summarizes the proportion of these three groups. Many ganglion cells showed both NADPH-d activity and immunoreactivity for VIP or galanin (respectively 80.9% and 76.2%). Few ganglion cells showed only immunoreactivity for VIP or galanin (respectively 2.0% and 3.7%). In this study, ganglion cells showing neither NADPH-d activity nor immunoreactivity for neuropeptides were not detected. At first, NO was noticed as endothelium-derived relaxing factor [12,14]. Some studies, however, mentioned its
K. Hiramatsu, K. Ohshima/Neuroscience Letters 182 (1994) 37~10
role as neurotransmitter in the central nervous system [4]. Recently, NADPH-d and NOS have been shown to be the same molecule [7,11]. In the peripheral nervous system, NADPH-d positive nerves were also demonstrated and NO is considered as one of neurotransmitters by many investigators [2,3,6-9,13,17-19]. The present study demonstrated the existence of NADPH-d positive neurons in the chicken pancreas. This result indicates the postganglionic, nitrergic innervation of the chicken pancreas and it is probable to regulate the pancreatic secretion by neuronal NO. Several studies indicated that NADPH-d positive neurons coexpressed immunoreactivity for neuropeptides in the central and peripheral nervous system. NADPH-d positive neurons within the rat striatum also contained both somatostatin and avian pancreatic polypeptide [20]. In the rat visceral afferent neurons, NADPH-d colocated with substance P and calcitonin gene-related peptide [1]. Nitrergic neurons of the mammalian digestive tract also showed VIP immunoreactivity [2,3,6,19]. In the rat enteric nervous system, all galanin-containing neurons showed NADPH-d activity [13]. It is suggested that NO and VIP or galanin may be co-transmitters in the mammalian enteric nervous system [2]. Also in the rat pancreas, most NADPH-d positive neurons showed immunoreactivity for VIP [13,18]. But the colocalization of NADPH-d with galanin has not been identified in spite of the projection of the enteric neurons to the pancreas [13]. The present study revealed that NADPH-d positive neurons of the chicken pancreas contained high ratios of VIP or galanin (80.9% and 76.2% respectively). VIP- and galanin-containing nerves are the prevailing nerve types and innervate both the endocrine and the exocrine parts of the chicken pancreas [10,15]. Moreover, VIP colocates with galanin in the intrapancreatic neurons [15]. These data suggest that the
Table 1 Proportions of neurons showing NADPH-diaphorase activity and/or immunoreactivity for neuropeptides Types of neurons
Total number of counted neurons
Percentage (%)*
NADPH-d+/VIP+ NADPH-d+/VIPNADPH-d-/VIP+
909 180 26
80.9 + 6.0 17.1 + 6.2 2.0 + 0.8
NADPH-d+/GAL+ NADPH-d+/GALNADPH-d-/GAL+
713 187 32
76.2 _+4.7 20.1 + 4.6 3.7 + 1.6
1115 and 932 neurons from 5 chickens were counted to evaluate the colocalization of NADPH-d with VIP or galanin, respectively. Intrapancreatic neurons were divided into three types; NADPH-d+/neuropeptide+, NADPH-d+/neuropeptide-, and NADPH-d-/neuropeptide+. VIP, vasoactive intestinal polypeptide; GAL, galanin; +, positive; - , negative. *Values are expressed with mean _+S.D. from five chickens.
39
neuronal messengers, VIE galanin and NO, interact with each other in the intrapancreatic neuron of the chicken. This study was supported in part by Grant-in-Aid (05760222) from the Ministry of Education, Science and Culture of Japan to K.H. [1] Aimi, Y., Fujimura, M., Vincent, S.R. and Kimura, H., Localization of NADPH-diaphorase-containing neurons in sensory ganglia of the rat, J. Comp. Neurol., 306 (1991) 382-392. [2] Aimi, Y., Kimura, H., Kinoshita, T., Minami, Y., Fujimura, M. and Vincent, S.R., Histochemical localization of nitric oxide synthase in rat enteric nervous system, Neuroscience, 53 (1993) 553560. [3] Barbiers, M., Timmermans, J.-P., Scheuermann, D.W., Adriaensen, D., Mayer, B. and De Groodt-Lasseel, M.H.A., Distribution and morphological features of nitergic neurons in the porcine large intestine, Histochemistry, 100 (1993) 27 34. [4] Bredt, D.S. and Snyder, S.H., Nitric oxide, a novel neuronal messenger, Neuron, 8 (1992) 3 11. [5] Coons, A.H., Leduc, E.H. and Connolly, J.M., Studies on antibody production. I. A method for the histochemical demonstration of specific antibody and its application to a study of the hyperimmune rabbit, J. Exp. Med., 102 (1955) 42-60. [6] Costa, M., Furness, J.B., Pompolo, S., Brookes, S.J.H., Bornstein, J.C., Bredt, D.S. and Snyder, S.H., Projections and chemical coding of neurons with immunoreactivity for nitric oxide synthase in the guinea-pig small intestine, Neurosci. Lett., 148 (1992) 121-125. [7] Dawson, T.M., Bredt, D.S., Fotuhi, M., Hwang, P.M. and Snyder, S.H., Nitric oxide synthase and neural NADPH diaphorase are identical in brain and peripheral tissues, Proc. Natl. Acad. Sci. USA, 88 (1991) 7797-7801. [8] De Giorgio, R., Parodi, J.E., Brecha, N.C., Brunicardi, F.C., Brecker, J.M., Go, V.L.W. and Sternini, C., Nitric oxide producing neurons in the monkey and human digestive system, J. Comp. Neurol., 342 (1994) 619 627. [9] Grozdanovic, Z., Baumgarten, H.G. and Brtining, G., Histochemistry of NADPH-diaphorase, a marker for neuronal nitric oxide synthase, in the peripheral autonomic nervous system of the mouse, Neuroscience, 48 (1992) 225-235. [10] Hiramatsu, K. and Watanabe, T., Immunohistochemical study on the distribution of vasoactive intestinal polypeptide (VIP) containing nerve fibers in the chicken pancreas, Z. mikrosk.-anat. Forsch., 103 (1989) 689-699. [11] Hope, B.T., Michael, G.J., Knigge, K.M. and Vincent, S.R., Neural NADPH diaphorase is a nitric oxide synthase, Proc. Natl. Acad. Sci. USA, 88 (1991) 2811-2814. [12] Ignarro, L.J., Ross, G. and Tillisch, J., Pharmacology of endothelium-derived nitric oxide and nitrovasodilators, West J. Med., 154 (1991) 51-62. [13] Kirchgessner, A.L., Liu, M.T. and Gershon, M.D., NADPH diaphorase (nitric oxide synthase)-containing nerves in the enteropancreatic innervation: sources, co-stored neuropeptides, and pancreatic function, J. Comp. Neurol., 342 (1994) 115 130. [14] Palmer, R.M.J., Ferrige, A.G. and Moncada, S., Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor, Nature, 327 (1987) 524~526. [15] Salakij, C., Watanabe, T., Takahashi, S., Ohmori, Y. and Nagatsu, I., Immunohistochemical studies on the intrinsic pancreatic nerves in the chicken, J. Auton. Nerv. Syst., 40 (1992) 131-140. [16] Scherer-Singler, U., Vincent, S.R., Kimura, H. and McGeer, E.G., Demonstration of a unique population of neurons with NADPHdiaphorase histochemistry, J. Neurosci. Methods, 9 (1983) 229234.
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K. Hiramatsu, K. Ohshima/Neuroscience Letters 182 (1994) 37-40
[17] Shimosegawa, T., Abe, T., Satoh, A., Asakura, T., Yoshida, K., Koizumi, M. and Toyota, T., Histochemical demonstration of NADPH-diaphorase activity, a marker for nitric oxide synthase, in neurons of the rat pancreas, Neurosci. Lett., 148 (1992) 67-70. [18] Shimosegawa, T., Abe, T., Satoh, A., Abe, R., Kikuchi, Y., Koizumi, M. and Toyota, T., NADPH-diaphorase activity in neurons of the mammalian pancreas: coexpression with vasoactive intestinal polypeptide, Gastroenterology, 105 (1993) 999-1008. [19] Timmermans, J.-E, Barbiers, M., Scheuermann, D.W., Bogers,
J.J., Adriaensen, D., Fekete, E., Mayer, B., Van Marck, E. A. and De Groodt-Lasseel, M.H.A., Nitric oxide synthase immunoreactivity in the enteric nervous system of the developing human digestive tract, Cell Tissue Res., 275 (1994) 235-245. [20] Vincent, S.R., Johansson~ O., H6kfelt, T., Skirboll, L., Elde, R.E, Terenius, L., Kimmel, J. and Goldstein, M., NADPH-diaphorase: a selective histochemical marker for striatal neurons containing both somatostatin- and avian pancreatic polypeptide (APP)-like immunoreactivities, J. Comp. Neurol., 217 (1983) 252-263.
ELSEVIER
NEUROSCIENC[ lETTERS
Colocalization of NADPH-diaphorase with neuropeptides in the intrapancreatic neurons of the chicken Kohzy Hiramatsu*, Koji Ohshima Laboratory of Functional Anatomy, Faculty of Agriculture, Shinshu University, Minamiminowa, Nagano-ken 399-45, Japan
Received 27 July 1994; Revised version received26 September 1994; Accepted 26 September 1994
Abstract Colocalization of nitric oxide with neuropeptides was investigated in the chicken pancreas by use of double staining combined with the indirect immunofluorescence technique and histochemistry for NADPH-diaphorase, a specific marker for neural nitric oxide synthase. NADPH-diaphorase positive ganglia were easily detected in the interlobular connective tissue. Many NADPH-diaphorase positive ganglion cells also showed immunoreactivity for VIP (80.9%) or galanin (76.2%). Some ganglion cells showed enzyme activity only (about 20%). Very few neurons were NADPH-diaphorase negative, but immunopositive for VIP (2.0%) or galanin (3.7%). The present study provides evidence that nitric oxide colocates with VIP and galanin in the chicken pancreas. Key words." NADPH-diaphorase; Vasoactive intestinal polypeptide; Galanin; Colocalization; Pancreas; Innervation; Domestic fowl
Nitric oxide (NO), a free radical gas, is considered as one of the neurotransmitters in the mammalian central and peripheral nervous system [4]. NO is converted from L-arginine by the enzyme nitric oxide synthase (NOS). In the mammalian brain, there are some populations of neurons which show nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity [20]. Recent studies revealed that neural N A D P H - d is identical to NOS in the central and peripheral nervous system [7,11]. Thus histochemistry of N A D P H - d is used as a marker for neuronal NOS [9]. Studies on the distribution of nerves showing N A D P H - d activity were carried out in the enteric nervous system [2,3:6] and the pancreas [8,13,17]. These studies carried out in mammals revealed the colocalization of N A D P H - d activity and neuropeptide immunoreactivity in the same neuron and suggested interactions of N O and neuropeptides. Vasoactive intestinal polypeptide (VIP)- and galanincontaining neurons are two major types of neurons in the chicken pancreas [10,15]. The present study aimed at clarifying the probability of colocalization of NO with
*Corresponding author. Fax: (81) (265) 72-5259. 0304-3940194l$7.00© 1994 Elsevier ScienceIreland Ltd. All rights reserved S S D I 0304-3940(94)00752-7
these neuropeptides in the intrapancreatic neurons of the chicken. Five adult White Leghorn chickens of both sexes, weighing 1.5-2.2 kg, were used in this study. Animals were perfused with 0.75% NaC1 solution, and followed by a mixture of 4% paraformaldehyde, 0.1% glutaraldehyde and 0.21% picric acid in 0.1 M phosphate buffer (pH 7.4). Pancreata were immediately removed after perfusion and dissected into small blocks. Tissue blocks were immersed in the same perfusate for 6 h at 4°C and then transferred to 20% phosphate-buffered sucrose for cryoprotection at 4°C overnight. Frozen sections were serially cut at 16/2m thickness in the cryostat. Every fifth section was mounted on gelatin-coated glass slides. The indirect immunofluorescence technique [5] was applied to demonstrate immunoreactivity for neuropeptides. Frozen sections treated with 10% normal goat serum (Cedarlane, Canada) were incubated with antiserum against porcine VIP (diluted to 1:500; Incstar, USA) or porcine galanin (1:250; Chemicon, USA) at room temperature overnight. After several washes with PBS, the sections were incubated with FITC (fluorescein isothiocyanate)-conjugated goat anti-rabbit Ig G (1:100; E-Y Laboratories, USA) for 1 h at room temperature. They were then rinsed with phosphate
38
K. Hiramatsu, K. Ohshima/Neuroscienee Letters 182 (1994) 37 40
Fig. 1. Paired micrographs (a and b, c and d) of the intrapancreatic ganglia. Three types of ganglion cells are identified in an intrapancreatic ganglion. Arrowheads indicate ganglion cells showing both NADPH-d activity (a,c) and immunoreactivity for VIP (b) or galanin (d). Some neurons show only NADPH-d activity (small arrows) or neuropeptide immunoreactivity (large arrows), x 540.
buffer saline (PBS) and coverslipped with a mixture of glycerol and PBS (3: 1). All ganglia were photographed under a fluorescence microscope, and then the coverslips were removed in PBS. The sections were subsequently processed for histochemistry for NADPH-d. NADPH-d activity was demonstrated according to the method of Scherer-Singler et al. [16] slightly modified by Shimosegawa et al. [18]. Frozen sections were incubated in the medium which contained 1.0 mM beta-NADPH (Oriental Yeast Co., LTD., Tokyo, Japan) as a substrate, 0.2 mM nitroblue tetrazolium (Wako Pure Chemicals Industries, LTD., Osaka, Japan) and 0.2% Triton X-100 in 0.1 M Tris-HCl buffer (pH 8.0). Incubation was carried out at 37°C for 60 rain. After several rinses with PBS, sections were coverslipped with a mixture of glycerol and PBS (3:1). All ganglia corresponding to those in the fluorescence photomicrographs were photographed under a light microscope. Colocalization was evaluated by comparing the fluorescence photomicrographs for neuropeptide-immunoreactivity with the photomicrographs for NADPH-d activity. The percentage of ganglion cells showing NADPH-d activity and/or immunoreactivity for neuropeptides was determined from 36 sections for each bird, and the mean value was calculated for each type of ganglion cell.
NADPH-d activity was detected in nerve fibers and ganglion cells of the chicken pancreas. NADPH-d positive ganglion cells were observed in the interlobular connective tissue (Fig. lb,d) of the ventral and the dorsal lobes, but not in the splenic lobe. Double staining showed the coexistence of NADPH-d and the neuropeptides VIP and galanin (Fig. 1). Intrapancreatic neurons were divided into at least three groups on basis of the results of double staining. Neurons of the first group showed both NADPH-d activity and neuropeptide immunoreactivity (Fig. 1, arrowheads). This group makes up the largest proportion of intrapancreatic neurons. The second group consisted of neurons showing only NADPH-d activity (Fig. l b,d, small arrows). Neurons of the third group were infrequent and only showed immunoreactivity for neuropeptides (Fig. la,c, large arrows). Table 1 summarizes the proportion of these three groups. Many ganglion cells showed both NADPH-d activity and immunoreactivity for VIP or galanin (respectively 80.9% and 76.2%). Few ganglion cells showed only immunoreactivity for VIP or galanin (respectively 2.0% and 3.7%). In this study, ganglion cells showing neither NADPH-d activity nor immunoreactivity for neuropeptides were not detected. At first, NO was noticed as endothelium-derived relaxing factor [12,14]. Some studies, however, mentioned its
K. Hiramatsu, K. Ohshima/Neuroscience Letters 182 (1994) 37~10
role as neurotransmitter in the central nervous system [4]. Recently, NADPH-d and NOS have been shown to be the same molecule [7,11]. In the peripheral nervous system, NADPH-d positive nerves were also demonstrated and NO is considered as one of neurotransmitters by many investigators [2,3,6-9,13,17-19]. The present study demonstrated the existence of NADPH-d positive neurons in the chicken pancreas. This result indicates the postganglionic, nitrergic innervation of the chicken pancreas and it is probable to regulate the pancreatic secretion by neuronal NO. Several studies indicated that NADPH-d positive neurons coexpressed immunoreactivity for neuropeptides in the central and peripheral nervous system. NADPH-d positive neurons within the rat striatum also contained both somatostatin and avian pancreatic polypeptide [20]. In the rat visceral afferent neurons, NADPH-d colocated with substance P and calcitonin gene-related peptide [1]. Nitrergic neurons of the mammalian digestive tract also showed VIP immunoreactivity [2,3,6,19]. In the rat enteric nervous system, all galanin-containing neurons showed NADPH-d activity [13]. It is suggested that NO and VIP or galanin may be co-transmitters in the mammalian enteric nervous system [2]. Also in the rat pancreas, most NADPH-d positive neurons showed immunoreactivity for VIP [13,18]. But the colocalization of NADPH-d with galanin has not been identified in spite of the projection of the enteric neurons to the pancreas [13]. The present study revealed that NADPH-d positive neurons of the chicken pancreas contained high ratios of VIP or galanin (80.9% and 76.2% respectively). VIP- and galanin-containing nerves are the prevailing nerve types and innervate both the endocrine and the exocrine parts of the chicken pancreas [10,15]. Moreover, VIP colocates with galanin in the intrapancreatic neurons [15]. These data suggest that the
Table 1 Proportions of neurons showing NADPH-diaphorase activity and/or immunoreactivity for neuropeptides Types of neurons
Total number of counted neurons
Percentage (%)*
NADPH-d+/VIP+ NADPH-d+/VIPNADPH-d-/VIP+
909 180 26
80.9 + 6.0 17.1 + 6.2 2.0 + 0.8
NADPH-d+/GAL+ NADPH-d+/GALNADPH-d-/GAL+
713 187 32
76.2 _+4.7 20.1 + 4.6 3.7 + 1.6
1115 and 932 neurons from 5 chickens were counted to evaluate the colocalization of NADPH-d with VIP or galanin, respectively. Intrapancreatic neurons were divided into three types; NADPH-d+/neuropeptide+, NADPH-d+/neuropeptide-, and NADPH-d-/neuropeptide+. VIP, vasoactive intestinal polypeptide; GAL, galanin; +, positive; - , negative. *Values are expressed with mean _+S.D. from five chickens.
39
neuronal messengers, VIE galanin and NO, interact with each other in the intrapancreatic neuron of the chicken. This study was supported in part by Grant-in-Aid (05760222) from the Ministry of Education, Science and Culture of Japan to K.H. [1] Aimi, Y., Fujimura, M., Vincent, S.R. and Kimura, H., Localization of NADPH-diaphorase-containing neurons in sensory ganglia of the rat, J. Comp. Neurol., 306 (1991) 382-392. [2] Aimi, Y., Kimura, H., Kinoshita, T., Minami, Y., Fujimura, M. and Vincent, S.R., Histochemical localization of nitric oxide synthase in rat enteric nervous system, Neuroscience, 53 (1993) 553560. [3] Barbiers, M., Timmermans, J.-P., Scheuermann, D.W., Adriaensen, D., Mayer, B. and De Groodt-Lasseel, M.H.A., Distribution and morphological features of nitergic neurons in the porcine large intestine, Histochemistry, 100 (1993) 27 34. [4] Bredt, D.S. and Snyder, S.H., Nitric oxide, a novel neuronal messenger, Neuron, 8 (1992) 3 11. [5] Coons, A.H., Leduc, E.H. and Connolly, J.M., Studies on antibody production. I. A method for the histochemical demonstration of specific antibody and its application to a study of the hyperimmune rabbit, J. Exp. Med., 102 (1955) 42-60. [6] Costa, M., Furness, J.B., Pompolo, S., Brookes, S.J.H., Bornstein, J.C., Bredt, D.S. and Snyder, S.H., Projections and chemical coding of neurons with immunoreactivity for nitric oxide synthase in the guinea-pig small intestine, Neurosci. Lett., 148 (1992) 121-125. [7] Dawson, T.M., Bredt, D.S., Fotuhi, M., Hwang, P.M. and Snyder, S.H., Nitric oxide synthase and neural NADPH diaphorase are identical in brain and peripheral tissues, Proc. Natl. Acad. Sci. USA, 88 (1991) 7797-7801. [8] De Giorgio, R., Parodi, J.E., Brecha, N.C., Brunicardi, F.C., Brecker, J.M., Go, V.L.W. and Sternini, C., Nitric oxide producing neurons in the monkey and human digestive system, J. Comp. Neurol., 342 (1994) 619 627. [9] Grozdanovic, Z., Baumgarten, H.G. and Brtining, G., Histochemistry of NADPH-diaphorase, a marker for neuronal nitric oxide synthase, in the peripheral autonomic nervous system of the mouse, Neuroscience, 48 (1992) 225-235. [10] Hiramatsu, K. and Watanabe, T., Immunohistochemical study on the distribution of vasoactive intestinal polypeptide (VIP) containing nerve fibers in the chicken pancreas, Z. mikrosk.-anat. Forsch., 103 (1989) 689-699. [11] Hope, B.T., Michael, G.J., Knigge, K.M. and Vincent, S.R., Neural NADPH diaphorase is a nitric oxide synthase, Proc. Natl. Acad. Sci. USA, 88 (1991) 2811-2814. [12] Ignarro, L.J., Ross, G. and Tillisch, J., Pharmacology of endothelium-derived nitric oxide and nitrovasodilators, West J. Med., 154 (1991) 51-62. [13] Kirchgessner, A.L., Liu, M.T. and Gershon, M.D., NADPH diaphorase (nitric oxide synthase)-containing nerves in the enteropancreatic innervation: sources, co-stored neuropeptides, and pancreatic function, J. Comp. Neurol., 342 (1994) 115 130. [14] Palmer, R.M.J., Ferrige, A.G. and Moncada, S., Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor, Nature, 327 (1987) 524~526. [15] Salakij, C., Watanabe, T., Takahashi, S., Ohmori, Y. and Nagatsu, I., Immunohistochemical studies on the intrinsic pancreatic nerves in the chicken, J. Auton. Nerv. Syst., 40 (1992) 131-140. [16] Scherer-Singler, U., Vincent, S.R., Kimura, H. and McGeer, E.G., Demonstration of a unique population of neurons with NADPHdiaphorase histochemistry, J. Neurosci. Methods, 9 (1983) 229234.
40
K. Hiramatsu, K. Ohshima/Neuroscience Letters 182 (1994) 37-40
[17] Shimosegawa, T., Abe, T., Satoh, A., Asakura, T., Yoshida, K., Koizumi, M. and Toyota, T., Histochemical demonstration of NADPH-diaphorase activity, a marker for nitric oxide synthase, in neurons of the rat pancreas, Neurosci. Lett., 148 (1992) 67-70. [18] Shimosegawa, T., Abe, T., Satoh, A., Abe, R., Kikuchi, Y., Koizumi, M. and Toyota, T., NADPH-diaphorase activity in neurons of the mammalian pancreas: coexpression with vasoactive intestinal polypeptide, Gastroenterology, 105 (1993) 999-1008. [19] Timmermans, J.-E, Barbiers, M., Scheuermann, D.W., Bogers,
J.J., Adriaensen, D., Fekete, E., Mayer, B., Van Marck, E. A. and De Groodt-Lasseel, M.H.A., Nitric oxide synthase immunoreactivity in the enteric nervous system of the developing human digestive tract, Cell Tissue Res., 275 (1994) 235-245. [20] Vincent, S.R., Johansson~ O., H6kfelt, T., Skirboll, L., Elde, R.E, Terenius, L., Kimmel, J. and Goldstein, M., NADPH-diaphorase: a selective histochemical marker for striatal neurons containing both somatostatin- and avian pancreatic polypeptide (APP)-like immunoreactivities, J. Comp. Neurol., 217 (1983) 252-263.