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Immunohistochemical evidence for the presence of calbindin containing neurones in the myenteric plexus of the guinea-pig stomach
Neuroscience Letters 270 (1999) 71±74
Immunohistochemical evidence for the presence of calbindin containing neurones in the myenteric plexus of the guinea-pig stomach Dania Reiche, Helga Pfannkuche, Klaus Michel, Susanne Hoppe, Michael Schemann* Department of Physiology, School of Veterinary Medicine, Bischofsholer Damm 15, D-30173 Hannover, Germany Received 19 April 1999; received in revised form 25 May 1999; accepted 25 May 1999
Abstract Using immunohistochemistry we studied the presence of calbindin in myenteric neurones of the guinea-pig stomach. A rabbit anti recombinant rat calbindin-D28k (CALB) stained 12, 12 and 25% of all myenteric neurones in the fundus, corpus and antrum, respectively. A rabbit anti recombinant human CALB stained 4, 4 and 16%, respectively. A mouse monoclonal antibody against the chicken intestinal CALB showed no labelling. In all regions most calbindin neurones were additionally choline acetyltransferase (ChAT) positive while only a small proportion exhibited nicotinamide adenosine dinucleatide phosphate (NADPH)-diaphorase-activity. Numerous calbindin -positive varicose nerve ®bres were present within myenteric ganglia, rarely detectable in the muscle layers and virtually absent in the mucosa. This study demonstrated that a supopulation of cholinergic myenteric neurones in the stomach contain calbindin and suggested that many of these neurones ful®l interneuronal tasks. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Myenteric plexus; Enteric nervous system; Calcium-binding proteins; Acetylcholine; Nitric oxide; Interneurones; Calbindin; Stomach
Calbindin-D28k (CALB) belongs to the superfamily of EF-hand helix-loop-helix Ca 21-binding proteins, representing a large group of subfamilies with a highly homologous structure of an EF-hand Ca 21-binding site [2]. CALB has originally been discovered in the chicken small intestine [23]. In the gastrointestinal tract CALB has been demonstrated in enteric neurones and certain neuroendocrine epithelial cells in several species [1,6,22]. CALB immunoreactivity in myenteric neurones of the guinea-pig small and large intestine has been used as a valuable tool to identify a population of intrinsic primary afferent neurones [7,13]. In the guinea-pig small intestine about 20±30% of myenteric neurones are CALB-positive [5,8,20]. In contrast, comparable studies in the guinea-pig stomach reported either a total lack [17] or very few CALB-positive neurones [6]. Both studies, however, used antibodies which readily labelled CALB-positive neurones in the guinea-pig small intestine. In the present study we were able to detect and quantify CALB-positive enteric cell bodies in the guinea-pig * Corresponding author. Tel.: 149-511-856-7452; fax: 149-511856-7687. E-mail address: [email protected] (M. Schemann)
stomach with two polyclonal antibodies raised in rabbits against recombinant rat and recombinant human CALB. The additional presence of choline acetyltransferase (ChAT) or nicotinamide adenosine dinucleatide phosphate-diaphorase (NADPH-d) was investigated, the latter identi®es nitric oxide synthesizing neurones [17]. All methods were previously described in detail [17]. Brie¯y, adult guinea pigs of either sex (300±600 g) were killed by cervical dislocation and their stomach was removed, opened and pinned out in a Petri dish. After ®xation for 4 h at room temperature in 0.1 M phosphate buffer containing 4% paraformaldehyde and 0.2% picric acid the tissues were repeatedly rinsed in phosphate buffer. Whole mount preparations of the myenteric plexus were obtained by removing mucosa and circular muscle. For the immunohistochemistry all preparations were permeabilized for 1 h in PBS containing 0.5% Triton X-100, 4% horse serum and 0.1% NaN3 followed by a 12±16 h incubation with the following primary antibodies: goat anti ChAT (1:100; AB144P, Chemicon, Hofheim, Germany), rabbit anti CALB (1:1000, AB1778, Chemicon; raised against human recombinant CALB), rabbit anti CALB (1:3000, CB 38, SWant, Bellinzona, Switzerland; raised against rat recom-
0304-3940/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 9 9) 00 47 1- 1
72
D. Reiche et al. / Neuroscience Letters 270 (1999) 71±74
teric neurones in the stomach. Counts of CALB-immunoreactive neurones were obtained separately from the fundus, corpus and antrum (based on 20 myenteric ganglia in each region, three animals; Table 1). The rabbit anti rat CALB stained in the fundus and corpus 12% and in the antrum 25% of all myenteric neurones. The corresponding data for the rabbit anti human CALB was 4% in fundus and corpus and 16% in the antrum. Thus, in all gastric regions the rabbit anti rat CALB stained signi®cantly more myenteric neurones than the rabbit anti human CALB. For both antisera the relative proportion of CALB-positive neurones was signi®cantly higher in the antrum. Using the rabbit anti rat CALB the majority of CALB-positive neurones were additionally ChAT-positive (Fig. 1 and Table 1); signi®cantly more CALB-neurones colocalized ChAT in the antrum (89%) as compared with the fundus or corpus (70%). Labelling with the rabbit anti human CALB revealed that 88% of CALB-neurones in the antrum contained ChAT but only 45 and 31% in the fundus and corpus, respectively (Table 1). Remaining cells exhibited NADPH-d-activity or were ChAT- and NADPH-d-negative (Fig. 1 and Table 1). However, the CALB/NADPH-d and CALB/2 populations represented only about 1±2% of gastric myenteric neurones. Only in the antrum a very small population of myenteric neurones (,1%) was CALB-positive and additionally contained ChAT and NADPH-d. The colocalization patterns clearly indicated that, between the two antisera, the rabbit anti human CALB stained less ChAT-containing CALBneurones whereas the other populations were of comparable size (Table 1). For most CALB-labelled neurones it was not possible to identify their detailed morphology. In the antrum, however, some of the brightly labelled neurones had multiple long
binant CALB), mouse anti CALB (1:200, 300 17-F, SWant; raised against chicken intestinal CALB). Subsequently, the preparations were washed and incubated for 1±2 h with species-speci®c secondary antibodies raised in donkeys labelled with one of the following ¯uorophores: AMCA (7-amino-4-methylcoumarin-3-acetate, 1:50), DTAF (dichlorotriazinyl amino¯uorescin, 1:200), Cy3 (carboxymethylindocyanine, 1:500) (all: Dianova, Hamburg, Germany). The preparations were examined with an epi¯uorescence microscope (IX70, Olympus, Japan) ®tted with appropriate ®lter blocks [12]. In whole mount preparations the NADPH-d acitivity was visualized by incubating the tissues for 2 h at 378C in 0.1 M phosphate buffer (pH 8) containing 0.05 mg/ml b-NADPH, 0.1 mg/ml nitro-blue tetrazolium and 0.5% Triton X-100. As the NADPH-d staining interferes with the ¯uorescent labelling, CALB-immunoreactive neurones were documented and counted prior to NADPH-d histochemistry [17]. To evaluate the speci®city of the three antibodies the following tests were employed. CALB-staining of myenteric neurones was abolished by preadsorption with CALB (1 mM, recombinant rat, SWant). Preadsorption with calretinin (1±10 mM, recombinant human, SWant) did not affect the CALBimmunoreactivity. In addition, all antibodies stained identical myenteric neurones with comparable intensity in the guineapig colon where CALB-immunoreactivity has been studied previously [14]. Data were statistically analyzed by the Mann±Whitney rank sum test or chi-square-test. P-values ,0.05 were taken as statistically signi®cant. In whole mount preparations of the stomach the mouse anti-CALB antibody gave a very faint staining of only few neurones; the staining was too faint to be reliably evaluated. In contrast, both rabbit anti-CALB antisera labelled myen-
Table 1 Number of CALB-positive gastric myenteric neurones (cells per ganglion, mean ^ SD, n 60 ganglia, three animals) a
Total number of myenteric neurones Rabbit anti recombinant rat CALB: CALB (all) b CALB/ChAT b CALB/NADPH-d CALB/± CALB/ChAT/NADPH-d Rabbit anti recombinant human CALB: CALB (all) CALB/ChAT CALB/NADPH-d CALB/± CALB/ChAT/NADPH-d a
Fundus
Corpus
Antrum
25.2 ^ 11.5*
32.7 ^ 18.4**
90.2 ^ 69.9***
3.0 ^ 2.3 2.1 ^ 1.9 0.4 ^ 0.7 0.5 ^ 0.8 0
4.0 2.8 0.4 0.8 0
^ 3.1 ^ 2.3 ^ 0.8 ^ 0.9
22.9 ^ 18.0 c 20.3 ^ 16.1 c 1.6 ^ 2.4 0.6 ^ 1.1 0.4 ^ 0.7
1.1 ^ 1.5 0.5 ^ 0.9 0.1 ^ 0.4 0.5 ^ 1.0 0
1.3 0.4 0.3 0.6 0
^ 1.7 ^ 0.9 ^ 0.6 ^ 0.8
14.6 ^ 11.2 c 12.8 ^ 9.6 c 1.0 ^ 1.5 0.6 ^ 1.1 0.3 ^ 0.6
*Based on data from [15]; **based on data from [17]; ***since virtually all gastric myenteric neurones in the guinea-pig are either ChAT- or NADPH-d-positive [15,17] the overall number of myenteric neurones per ganglion in the antrum was evaluated by counting ChAT- and NADPH-d-positive cells. bIn all regions signi®cantly more neurones were labelled than with the rabbit anti recombinant human CALB. cSignifcantly higher proportion (within the plexus) than in fundus and corpus.
D. Reiche et al. / Neuroscience Letters 270 (1999) 71±74
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within myenteric ganglia (Fig. 1). Only a few immunoreactive ®bres were seen in the muscle layers and CALB-positive ®bres were virtually absent in the mucosa (results from cryostat sections). The results of this study indicated the presence of CALBpositive myenteric neurones in the guinea-pig stomach. The relative lack of CALB neurones in the guinea-pig stomach reported previously [6,17] is very likely due to the antibodies used. Whereas all three antibodies tested in our study stained enteric neurones in the small and large intestine only two antisera were able to detect CALB-positive neurones in the stomach. Whether different af®nities of the antibodies or region-speci®c conformational differences of the calbindin protein are responsible remains to be investigated. In the myenteric plexus of the small intestine CALBpositive myenteric neurones made up about 20±30% of the entire population and were virtually all cholinergic [4,5,8,11,20]. Most, if not all, had projections to the mucosa [4,8,19] and the mucosal terminals as well as the cell body appeared to code sensory stimuli [7,13]. A comparable proportion of CALB-neurones were found in the antrum (25% with the rabbit anti rat CALB) and like in the intestine those were mainly ChAT-positive. However, signi®cantly
Fig. 1. Photomicrographs illustrating the immunoreacitvity of gastric myenteric neurones using antisera against recombinant rat calbindin-D28k (CALB) in the corpus (A). Most CALB-positive neurones additionally contained choline acteyltranferase (ChAT, B) and not NADPH-diaphorase (C) (arrow in (A±C)). However, a small population also colocalized CALB and NADPH-diaphorase (arrowhead in (A±C)). In (A) abundant varicose CALB-positive nerve ®bres encircling ganglion cells are visible.
processes (Fig. 2). These multipolar neurones made up 4% of the CALB-positive cells in the antrum; their peculiar morphology is not comparable with the CALB neurones in the guinea-pig intestine, which exhibit smooth, round to ovoid cell bodies with typical Dogiel type II morphology [6,8,14]. Occasionally, we also observed CALB-neurones with short paddle-shaped processes and Dogiel type I morphology. With both rabbit antisera we observed in all gastric regions numerous CALB-positive varicose ®bres
Fig. 2. Illustration of one CALB-positive myenteric neurone in the gastric antrum with a multipolar morphology.
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D. Reiche et al. / Neuroscience Letters 270 (1999) 71±74
less neurones were stained in the proximal stomach (corpus and fundus). CALB-neurones in the stomach and intestine differ in several aspects. Thus, CALB neurones in the stomach seemed not to project to the mucosa and hence are probably not involved in transduction of luminal stimuli. Furthermore, CALB-positive intestinal myenteric neurones belong to the class of AH neurones named after their typical long lasting after-hyperpolarization [10,13]. Although 24% of myenteric neurones in the antrum were AH-neurones [21] AH-neurones were virtually absent in the corpus [18]. This indicates that there is no clear relation between CALBimmunoreactivity and AH-properties in gastric myenteric neurones. The generation of action potentials in AH-cells, most of which are CALB-positive, depend on calcium and sodium in¯ux [9]. Thus, the need for an intracellular calcium buffering system appears plausible although it is unlikely that this is the sole function of calcium binding proteins in enteric neurones. Even though gastric myenteric neurones normally have tetrodotoxin sensitive sodium action potentials, robust calcium spikes occur in gastric neurones after blockade of sodium and potassium channels by tetrodotoxin and tetraethylammonium [18]. One common feature of CALB-positive neurones in the ileum and the stomach is that they give rise to varicose endings in myenteric ganglia and therefore very likely ful®l interneuronal tasks [3,5,6,8,20]. Although intracellular dye ®lls did not reveal multipolar neurones in the gastric corpus [12,16], multi-targeted neurones with numerous axon-collaterals projecting within the plexus and to the muscle layers have been described [16]. Whether CALB-immunoreactivity is present in these multi-targeted neurones and whether they are involved in primary afferent and/or interneuronal functions remains to be investigated. Their projections and morphology would certainly favour such a suggestion. [1] Buffa, R., Mare, P., Salvadore, M., Solcia, E., Furness, J.B. and Lawson, D.E., Calbindin 28kDa in endocrine cells of known or putative calcium-regulating function. Thyro-parathyroid C cells, gastric ECL cells, intestinal secretin and enteroglucagon cells, pancreatic glucagon, insulin and PP cells, adrenal medullary NA cells and some pituitary (TSH?) cells. Histochemistry, 91 (1989) 107±113. [2] Celio, M.R., Pauls, T.L. and Schwaller, B., Introduction to EF-hand calcium-binding proteins. In M.R. Celio (Ed.), Guidebook to the Calcium-Binding Proteins, Oxford University Press, Oxford, 1996, pp. 15±20. [3] Clerc, N., Furness, J.B., Bornstein, J.C. and Kunze, W.A., Correlation of electrophysiological and morphological characteristics of myenteric neurons of the duodenum in the guinea-pig. Neuroscience, 82 (1998) 899±914. [4] Clerc, N., Furness, J.B., Li, Z.S., Bornstein, J.C. and Kunze, W.A., Morphological and immunohistochemical identi®cation of neurons and their targets in the guinea-pig duodenum. Neuroscience, 86 (1998) 679±694. [5] Costa, M., Brookes, S.J., Steele, P.A., Gibbins, I., Burcher, E. and Kandiah, C.J., Neurochemical classi®cation of myenteric neurons in the guinea-pig ileum. Neuroscience, 75 (1996) 949±967. [6] Furness, J.B., Keast, J.R., Pompolo, S., Bornstein, J.C., Costa, M., Emson, P.C. and Lawson, D.E., Immunohisto-
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chemical evidence for the presence of calcium-binding proteins in enteric neurons. Cell Tissue Res., 252 (1988) 79±87. Furness, J.B., Kunze, W.A., Bertrand, P.P., Clerc, N. and Bornstein, J.C., Intrinsic primary afferent neurons of the intestine. Prog. Neurobiol., 54 (1998) 1±18. Furness, J.B., Trussell, D.C., Pompolo, S., Bornstein, J.C. and Smith, T.K., Calbindin neurons of the guinea-pig small intestine: quantitative analysis of their numbers and projections. Cell Tissue Res., 260 (1990) 261±272. Hirst, G.D.S., Holman, M.E. and Spence, I., Two types of neurones in the myenteric plexus of duodenum in the guinea-pig. J. Physiol., 236 (1974) 303±326. Iyer, V., Bornstein, J.C., Costa, M., Furness, J.B., Takahashi, Y. and Iwanaga, T., Electrophysiology of guinea-pig myenteric neurons correlated with immunoreactivity for calcium binding proteins. J. Auton. Nerv. Syst., 22 (1988) 141±150. Li, Z.S. and Furness, J.B., Immunohistochemical localisation of cholinergic markers in putative intrinsic primary afferent neurons of the guinea-pig small intestine. Cell Tissue Res., 294 (1998) 35±43. Michel, K., Sann, H., Schaaf, C. and Schemann, M., Subpopulations of gastric myenteric neurons are differentially activated via distinct serotonin receptors: projection, neurochemical coding, and functional implications. J. Neurosci., 17 (1997) 8009±8017. Neunlist, M., Dobreva, G. and Schemann, M., Characteristics of mucosally-projecting myenteric neurones in the guinea-pig proximal colon. J. Physiol. (Lond.), (1999) in press. Neunlist, M. and Schemann, M., Projections and neurochemical coding of myenteric neurons innervating the mucosa of the guinea pig proximal colon. Cell Tissue Res., 287 (1997) 119±125. Pfannkuche, H., Reiche, D., Firzlaff, U., Sann, H. and Schemann, M., Enkephalin-immunoreactive subpopulations in the myenteric plexus of the guinea-pig fundus project primarily to the muscle and not to the mucosa. Cell Tissue Res., 294 (1998) 45±55. Schemann, M. and Schaaf, C., Differential projection of cholinergic and nitroxidergic neurons in the myenteric plexus of guinea pig stomach. Am. J. Physiol., 269 (1995) G186±G195. Schemann, M., Schaaf, C. and Mader, M., Neurochemical coding of enteric neurons in the guinea pig stomach. J. Comp. Neurol., 353 (1995) 161±178. Schemann, M. and Wood, J.D., Electrical behaviour of myenteric neurones in the gastric corpus of the guineapig. J. Physiol. (Lond.), 417 (1989) 501±518. Song, Z.M., Brookes, S.J. and Costa, M., All calbindinimmunoreactive myenteric neurons project to the mucosa of the guinea-pig small intestine. Neurosci. Lett., 180 (1994) 219±222. Steele, P.A., Brookes, S.J. and Costa, M., Immunohistochemical identi®cation of cholinergic neurons in the myenteric plexus of guinea-pig small intestine. Neuroscience, 45 (1991) 227±239. Tack, J.F. and Wood, J.D., Electrical behaviour of myenteric neurones in the gastric antrum of the guinea-pig. J. Physiol. (Lond.), 447 (1992) 49±66. Walters, J.R., Bishop, A.E., Facer, P., Lawson, E.M., Rogers, J.H. and Polak, J.M., Calretinin and calbindin-D28k immunoreactivity in the human gastrointestinal tract. Gastroenterology, 104 (1993) 1381±1389. Wasserman, R.H. and Taylor, A.N., Vitamin D3-induced calcium-binding protein in chick intestinal mucosa. Science, 152 (1966) 791±793.
Immunohistochemical evidence for the presence of calbindin containing neurones in the myenteric plexus of the guinea-pig stomach Dania Reiche, Helga Pfannkuche, Klaus Michel, Susanne Hoppe, Michael Schemann* Department of Physiology, School of Veterinary Medicine, Bischofsholer Damm 15, D-30173 Hannover, Germany Received 19 April 1999; received in revised form 25 May 1999; accepted 25 May 1999
Abstract Using immunohistochemistry we studied the presence of calbindin in myenteric neurones of the guinea-pig stomach. A rabbit anti recombinant rat calbindin-D28k (CALB) stained 12, 12 and 25% of all myenteric neurones in the fundus, corpus and antrum, respectively. A rabbit anti recombinant human CALB stained 4, 4 and 16%, respectively. A mouse monoclonal antibody against the chicken intestinal CALB showed no labelling. In all regions most calbindin neurones were additionally choline acetyltransferase (ChAT) positive while only a small proportion exhibited nicotinamide adenosine dinucleatide phosphate (NADPH)-diaphorase-activity. Numerous calbindin -positive varicose nerve ®bres were present within myenteric ganglia, rarely detectable in the muscle layers and virtually absent in the mucosa. This study demonstrated that a supopulation of cholinergic myenteric neurones in the stomach contain calbindin and suggested that many of these neurones ful®l interneuronal tasks. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Myenteric plexus; Enteric nervous system; Calcium-binding proteins; Acetylcholine; Nitric oxide; Interneurones; Calbindin; Stomach
Calbindin-D28k (CALB) belongs to the superfamily of EF-hand helix-loop-helix Ca 21-binding proteins, representing a large group of subfamilies with a highly homologous structure of an EF-hand Ca 21-binding site [2]. CALB has originally been discovered in the chicken small intestine [23]. In the gastrointestinal tract CALB has been demonstrated in enteric neurones and certain neuroendocrine epithelial cells in several species [1,6,22]. CALB immunoreactivity in myenteric neurones of the guinea-pig small and large intestine has been used as a valuable tool to identify a population of intrinsic primary afferent neurones [7,13]. In the guinea-pig small intestine about 20±30% of myenteric neurones are CALB-positive [5,8,20]. In contrast, comparable studies in the guinea-pig stomach reported either a total lack [17] or very few CALB-positive neurones [6]. Both studies, however, used antibodies which readily labelled CALB-positive neurones in the guinea-pig small intestine. In the present study we were able to detect and quantify CALB-positive enteric cell bodies in the guinea-pig * Corresponding author. Tel.: 149-511-856-7452; fax: 149-511856-7687. E-mail address: [email protected] (M. Schemann)
stomach with two polyclonal antibodies raised in rabbits against recombinant rat and recombinant human CALB. The additional presence of choline acetyltransferase (ChAT) or nicotinamide adenosine dinucleatide phosphate-diaphorase (NADPH-d) was investigated, the latter identi®es nitric oxide synthesizing neurones [17]. All methods were previously described in detail [17]. Brie¯y, adult guinea pigs of either sex (300±600 g) were killed by cervical dislocation and their stomach was removed, opened and pinned out in a Petri dish. After ®xation for 4 h at room temperature in 0.1 M phosphate buffer containing 4% paraformaldehyde and 0.2% picric acid the tissues were repeatedly rinsed in phosphate buffer. Whole mount preparations of the myenteric plexus were obtained by removing mucosa and circular muscle. For the immunohistochemistry all preparations were permeabilized for 1 h in PBS containing 0.5% Triton X-100, 4% horse serum and 0.1% NaN3 followed by a 12±16 h incubation with the following primary antibodies: goat anti ChAT (1:100; AB144P, Chemicon, Hofheim, Germany), rabbit anti CALB (1:1000, AB1778, Chemicon; raised against human recombinant CALB), rabbit anti CALB (1:3000, CB 38, SWant, Bellinzona, Switzerland; raised against rat recom-
0304-3940/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 9 9) 00 47 1- 1
72
D. Reiche et al. / Neuroscience Letters 270 (1999) 71±74
teric neurones in the stomach. Counts of CALB-immunoreactive neurones were obtained separately from the fundus, corpus and antrum (based on 20 myenteric ganglia in each region, three animals; Table 1). The rabbit anti rat CALB stained in the fundus and corpus 12% and in the antrum 25% of all myenteric neurones. The corresponding data for the rabbit anti human CALB was 4% in fundus and corpus and 16% in the antrum. Thus, in all gastric regions the rabbit anti rat CALB stained signi®cantly more myenteric neurones than the rabbit anti human CALB. For both antisera the relative proportion of CALB-positive neurones was signi®cantly higher in the antrum. Using the rabbit anti rat CALB the majority of CALB-positive neurones were additionally ChAT-positive (Fig. 1 and Table 1); signi®cantly more CALB-neurones colocalized ChAT in the antrum (89%) as compared with the fundus or corpus (70%). Labelling with the rabbit anti human CALB revealed that 88% of CALB-neurones in the antrum contained ChAT but only 45 and 31% in the fundus and corpus, respectively (Table 1). Remaining cells exhibited NADPH-d-activity or were ChAT- and NADPH-d-negative (Fig. 1 and Table 1). However, the CALB/NADPH-d and CALB/2 populations represented only about 1±2% of gastric myenteric neurones. Only in the antrum a very small population of myenteric neurones (,1%) was CALB-positive and additionally contained ChAT and NADPH-d. The colocalization patterns clearly indicated that, between the two antisera, the rabbit anti human CALB stained less ChAT-containing CALBneurones whereas the other populations were of comparable size (Table 1). For most CALB-labelled neurones it was not possible to identify their detailed morphology. In the antrum, however, some of the brightly labelled neurones had multiple long
binant CALB), mouse anti CALB (1:200, 300 17-F, SWant; raised against chicken intestinal CALB). Subsequently, the preparations were washed and incubated for 1±2 h with species-speci®c secondary antibodies raised in donkeys labelled with one of the following ¯uorophores: AMCA (7-amino-4-methylcoumarin-3-acetate, 1:50), DTAF (dichlorotriazinyl amino¯uorescin, 1:200), Cy3 (carboxymethylindocyanine, 1:500) (all: Dianova, Hamburg, Germany). The preparations were examined with an epi¯uorescence microscope (IX70, Olympus, Japan) ®tted with appropriate ®lter blocks [12]. In whole mount preparations the NADPH-d acitivity was visualized by incubating the tissues for 2 h at 378C in 0.1 M phosphate buffer (pH 8) containing 0.05 mg/ml b-NADPH, 0.1 mg/ml nitro-blue tetrazolium and 0.5% Triton X-100. As the NADPH-d staining interferes with the ¯uorescent labelling, CALB-immunoreactive neurones were documented and counted prior to NADPH-d histochemistry [17]. To evaluate the speci®city of the three antibodies the following tests were employed. CALB-staining of myenteric neurones was abolished by preadsorption with CALB (1 mM, recombinant rat, SWant). Preadsorption with calretinin (1±10 mM, recombinant human, SWant) did not affect the CALBimmunoreactivity. In addition, all antibodies stained identical myenteric neurones with comparable intensity in the guineapig colon where CALB-immunoreactivity has been studied previously [14]. Data were statistically analyzed by the Mann±Whitney rank sum test or chi-square-test. P-values ,0.05 were taken as statistically signi®cant. In whole mount preparations of the stomach the mouse anti-CALB antibody gave a very faint staining of only few neurones; the staining was too faint to be reliably evaluated. In contrast, both rabbit anti-CALB antisera labelled myen-
Table 1 Number of CALB-positive gastric myenteric neurones (cells per ganglion, mean ^ SD, n 60 ganglia, three animals) a
Total number of myenteric neurones Rabbit anti recombinant rat CALB: CALB (all) b CALB/ChAT b CALB/NADPH-d CALB/± CALB/ChAT/NADPH-d Rabbit anti recombinant human CALB: CALB (all) CALB/ChAT CALB/NADPH-d CALB/± CALB/ChAT/NADPH-d a
Fundus
Corpus
Antrum
25.2 ^ 11.5*
32.7 ^ 18.4**
90.2 ^ 69.9***
3.0 ^ 2.3 2.1 ^ 1.9 0.4 ^ 0.7 0.5 ^ 0.8 0
4.0 2.8 0.4 0.8 0
^ 3.1 ^ 2.3 ^ 0.8 ^ 0.9
22.9 ^ 18.0 c 20.3 ^ 16.1 c 1.6 ^ 2.4 0.6 ^ 1.1 0.4 ^ 0.7
1.1 ^ 1.5 0.5 ^ 0.9 0.1 ^ 0.4 0.5 ^ 1.0 0
1.3 0.4 0.3 0.6 0
^ 1.7 ^ 0.9 ^ 0.6 ^ 0.8
14.6 ^ 11.2 c 12.8 ^ 9.6 c 1.0 ^ 1.5 0.6 ^ 1.1 0.3 ^ 0.6
*Based on data from [15]; **based on data from [17]; ***since virtually all gastric myenteric neurones in the guinea-pig are either ChAT- or NADPH-d-positive [15,17] the overall number of myenteric neurones per ganglion in the antrum was evaluated by counting ChAT- and NADPH-d-positive cells. bIn all regions signi®cantly more neurones were labelled than with the rabbit anti recombinant human CALB. cSignifcantly higher proportion (within the plexus) than in fundus and corpus.
D. Reiche et al. / Neuroscience Letters 270 (1999) 71±74
73
within myenteric ganglia (Fig. 1). Only a few immunoreactive ®bres were seen in the muscle layers and CALB-positive ®bres were virtually absent in the mucosa (results from cryostat sections). The results of this study indicated the presence of CALBpositive myenteric neurones in the guinea-pig stomach. The relative lack of CALB neurones in the guinea-pig stomach reported previously [6,17] is very likely due to the antibodies used. Whereas all three antibodies tested in our study stained enteric neurones in the small and large intestine only two antisera were able to detect CALB-positive neurones in the stomach. Whether different af®nities of the antibodies or region-speci®c conformational differences of the calbindin protein are responsible remains to be investigated. In the myenteric plexus of the small intestine CALBpositive myenteric neurones made up about 20±30% of the entire population and were virtually all cholinergic [4,5,8,11,20]. Most, if not all, had projections to the mucosa [4,8,19] and the mucosal terminals as well as the cell body appeared to code sensory stimuli [7,13]. A comparable proportion of CALB-neurones were found in the antrum (25% with the rabbit anti rat CALB) and like in the intestine those were mainly ChAT-positive. However, signi®cantly
Fig. 1. Photomicrographs illustrating the immunoreacitvity of gastric myenteric neurones using antisera against recombinant rat calbindin-D28k (CALB) in the corpus (A). Most CALB-positive neurones additionally contained choline acteyltranferase (ChAT, B) and not NADPH-diaphorase (C) (arrow in (A±C)). However, a small population also colocalized CALB and NADPH-diaphorase (arrowhead in (A±C)). In (A) abundant varicose CALB-positive nerve ®bres encircling ganglion cells are visible.
processes (Fig. 2). These multipolar neurones made up 4% of the CALB-positive cells in the antrum; their peculiar morphology is not comparable with the CALB neurones in the guinea-pig intestine, which exhibit smooth, round to ovoid cell bodies with typical Dogiel type II morphology [6,8,14]. Occasionally, we also observed CALB-neurones with short paddle-shaped processes and Dogiel type I morphology. With both rabbit antisera we observed in all gastric regions numerous CALB-positive varicose ®bres
Fig. 2. Illustration of one CALB-positive myenteric neurone in the gastric antrum with a multipolar morphology.
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less neurones were stained in the proximal stomach (corpus and fundus). CALB-neurones in the stomach and intestine differ in several aspects. Thus, CALB neurones in the stomach seemed not to project to the mucosa and hence are probably not involved in transduction of luminal stimuli. Furthermore, CALB-positive intestinal myenteric neurones belong to the class of AH neurones named after their typical long lasting after-hyperpolarization [10,13]. Although 24% of myenteric neurones in the antrum were AH-neurones [21] AH-neurones were virtually absent in the corpus [18]. This indicates that there is no clear relation between CALBimmunoreactivity and AH-properties in gastric myenteric neurones. The generation of action potentials in AH-cells, most of which are CALB-positive, depend on calcium and sodium in¯ux [9]. Thus, the need for an intracellular calcium buffering system appears plausible although it is unlikely that this is the sole function of calcium binding proteins in enteric neurones. Even though gastric myenteric neurones normally have tetrodotoxin sensitive sodium action potentials, robust calcium spikes occur in gastric neurones after blockade of sodium and potassium channels by tetrodotoxin and tetraethylammonium [18]. One common feature of CALB-positive neurones in the ileum and the stomach is that they give rise to varicose endings in myenteric ganglia and therefore very likely ful®l interneuronal tasks [3,5,6,8,20]. Although intracellular dye ®lls did not reveal multipolar neurones in the gastric corpus [12,16], multi-targeted neurones with numerous axon-collaterals projecting within the plexus and to the muscle layers have been described [16]. Whether CALB-immunoreactivity is present in these multi-targeted neurones and whether they are involved in primary afferent and/or interneuronal functions remains to be investigated. Their projections and morphology would certainly favour such a suggestion. [1] Buffa, R., Mare, P., Salvadore, M., Solcia, E., Furness, J.B. and Lawson, D.E., Calbindin 28kDa in endocrine cells of known or putative calcium-regulating function. Thyro-parathyroid C cells, gastric ECL cells, intestinal secretin and enteroglucagon cells, pancreatic glucagon, insulin and PP cells, adrenal medullary NA cells and some pituitary (TSH?) cells. Histochemistry, 91 (1989) 107±113. [2] Celio, M.R., Pauls, T.L. and Schwaller, B., Introduction to EF-hand calcium-binding proteins. In M.R. Celio (Ed.), Guidebook to the Calcium-Binding Proteins, Oxford University Press, Oxford, 1996, pp. 15±20. [3] Clerc, N., Furness, J.B., Bornstein, J.C. and Kunze, W.A., Correlation of electrophysiological and morphological characteristics of myenteric neurons of the duodenum in the guinea-pig. Neuroscience, 82 (1998) 899±914. [4] Clerc, N., Furness, J.B., Li, Z.S., Bornstein, J.C. and Kunze, W.A., Morphological and immunohistochemical identi®cation of neurons and their targets in the guinea-pig duodenum. Neuroscience, 86 (1998) 679±694. [5] Costa, M., Brookes, S.J., Steele, P.A., Gibbins, I., Burcher, E. and Kandiah, C.J., Neurochemical classi®cation of myenteric neurons in the guinea-pig ileum. Neuroscience, 75 (1996) 949±967. [6] Furness, J.B., Keast, J.R., Pompolo, S., Bornstein, J.C., Costa, M., Emson, P.C. and Lawson, D.E., Immunohisto-
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