Distribution of GABA, glutamate decarboxylase 67 (GAD67), Vesicular GABA transporter (VGAT) and GABA B -receptor Immunoreactivities in the Rat Esophagus

Distribution of GABA, glutamate decarboxylase 67 (GAD67), Vesicular GABA transporter (VGAT) and GABA B -receptor Immunoreactivities in the Rat Esophagus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Distribution of GABA, glutamate decarboxylase 67 (GAD67), Vesicular GABA transporter (VGAT) and GABA B -receptor Immunoreactivities in the Rat Esophagus Hiroshi Murabayashi, Yoshiki Hira, Hitoshi Kawano, Yukio Oomori This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9132607/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 10 You are reading this latest preprint version Abstract We investigated the distributions of gamma-aminobutyric acid (GABA), vesicular GABA transporter (VGAT), GABA B -receptor (GABA B -R) and glutamate decarboxylase 67 (GAD67), immunoreactivities in the rat esophagus. GABA immunoreactivity was found in the nerve fibers of the esophagus, but not in the neurons. A few GABA-immunoreactive nerve fibers ran along the muscularis mucosae and some GABA-immunoreactive nerve bundles and fibers ran along and contacted clusters of myenteric neurons. Numerous GABA-immunoreactive nerve terminals ran along the striated muscles and formed motor endplates on the muscles while GABA-immunoreactive nerve fibers contacted nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d)-positive or choline acetyltransferase (ChAT)-immunopositive neurons in the myenteric plexus. NADPH-d-positive nerve fibers often intermingled with GABA-immunoreactive motor endplates in the striated muscles. The GABA-immunoreactive nerve fibers contacting the myenteric neurons were also ChAT-immunopositive. The GABA-immunoreactive nerve fibers that formed motor endplates corresponding to α-bungarotoxin (BTX)-positive areas on the striated muscles were also ChAT-immunopositive. The average percentage of GABA-immunoreactive motor endplates to total ChAT-immunoreactive motor endplates in the upper, middle, and lower portions of the esophagus was 24.8%. VGAT immunoreactivity was seen in almost all motor endplates on the esophageal striated muscles, but not in the neurons of the myenteric plexus. Some VGAT-immunoreactive nerve terminals contacted clusters of myenteric neurons. GABA B -R immunoreactivity was observed in numerous myenteric ganglia and in the proximal-to-distal part of the axons of the ganglia, but not in the striated and smooth muscles. GABA B -R-immunoreactive neurons were brain nitric oxide synthase (bNOS)- or ChAT-immunopositive. The present study suggests that GABA and GABA B -R may be present in the neuronal elements of the striated muscle of the rat esophagus and may play an important role in the local inhibitory system of rat esophageal motility. No GAD67 immunoreactivity was found in the nerve fibers and neurons of the esophagus. In the nucleus ambiguus and in the dorsal motor nucleus of the vagal nerves, GAD67 and VGAT immunoreactivities were shown in numerous nerve fibers on the surface of the cell bodies, but not in the cell bodies of the vesicular acetylcholine transporter-immunoreactive neurons. The present study suggests that GABA may be taken up and accumulated in the synaptic vesicles by VGAT in the nerve terminals, but not in the cell bodies of the neurons of the brainstem. GABA GABA receptor Esophagus Brainstem Immunohistochemistry Rat Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction In the rat esophagus, the muscularis externa consists almost entirely of striated muscle fibers and only partly of smooth muscle fibers at the esophagogastric junction. Esophageal motility is mainly controlled by vagal efferents from the motor neurons of the nucleus ambiguus (for review see Cunningham and Sawchenko 1990 ; Roman and Gonella1987). These efferents directly innervate the striated muscle via the motor endplates and neurons of the dorsal motor nucleus of the vagal nerves and indirectly regulate the smooth muscle via myenteric intrinsic neurons. On the other hand, vagal afferents are derived from sensory neurons in the nodose ganglion whose terminals perceive various stimulations from the esophagus and relay the sensory information to the medulla oblongata (Roman 1982 ; for review see Cunningham and Sawchenko 1990 ; Conklin and Christensen 1994 ; Lu and Bieger 1998 ). In addition, the esophagus is innervated by the sensory neurons of the dorsal root ganglia and the intrinsic neurons (Conklin and Christensen 1994 ). The striated muscle of the rat esophagus are co-innervated via their motor endplates by both cholinergic nerve endings from the neurons of the nucleus ambiguus and nitric oxide synthase (NOS)-positive nerve terminals from intrinsic neurons containing nitric oxide (Neuhuber et al. 1994 , 1998 ; Wörl et al. 1994 ; Kuramoto et al. 1999 ) and neuropeptides such as galanin, vasoactive intestinal polypeptide, neuropeptide Y, calcitonin gene-related peptide (CGRP), pituitary adenylate cyclase-activating polypeptide (PACAP) and enkephalin (Uddman et al. 1980 ; Kuramoto and Endo 1995 ; Kuramoto et al. 1996 ; Neuhuber et al. 2001 ; Wu et al. 2003 ; Kuramoto et al. 2024 ). Nitric oxide and these neuropeptides act on smooth muscle as inhibitory mediators in the intestine (Bult et al. 1990 ; Gibson et al. 1990 ; Tøttrup et al. 1991 ; for review see Furness 2000 ; for review see Brookes 2001 ). However, it is unclear whether or how these mediators are involved in the inhibition of esophageal striated muscles. Thus, the local inhibitory neuronal system involved in esophageal motility remains poorly established, although the central control of esophageal motility is remarkably well-understood (for review see Hornby and Abrahams 2000 ). Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the central and peripheral nervous systems. Previous studies found both GABA- and glutamic acid decarboxylase (GAD)-immunoreactive nerve fibers and neurons in the gastrointestinal tract (Jessen et al. 1986 ; Saito and Tanaka 1986 ; Davanger et al. 1987 ; Hills et al. 1987 ; Furness et al. 1989 ). GABA exerts its inhibitory actions through 3 distinct types of receptors, GABA A -receptor (GABA A -R), GABA B -receptor (GABA B -R), and GABA C -receptor (GABA C -R) (for review see Bowery et al. 2002 ; for review see Feigenspan and Bormann 1998 ). Previous molecular and immunohistochemical studies found GABA A -R, GABA B -R, and GABA C -R on enteric neurons (Krantis et al. 1995 ; Nakajima et al. 1996 ; Zeiter et al. 1996 ; Fletcher et al. 2001 ). As well as in the brain, GABA B -Rs are also abundantly expressed in the gastrointestinal tract (Hyland and Cryan 2010 ). GABA can directly and indirectly stimulate the enteric excitatory and inhibitory neurons projecting to the muscle (Krantis et al. 1980 ; Maggi et al. 1984 ). GABA A -R elicits contraction through an excitatory action on cholinergic postganglionic neurons, whereas, in the small and large intestine, GABA B -R induces relaxation through an inhibitory presynaptic action on cholinergic postganglionic neurons (Giotti et al. 1983 ; Gentilini et al. 1992 ; Minocha and Galligan 1993 ). Nonetheless, it remains unclear whether GABA, GABA B -R, VGAT exist in the inhibitory neuronal system of the rat esophagus and, if so, what functional significance they play. To clarify these issues, we immunohistochemically examined the presence and distribution of GABA, GABA-B-R, GAD67 and VGAT in the neuronal elements of the rat esophagus and discuss their possible involvement in the inhibition of esophageal motility. Furthermore, we examined the presence of VGAT, GAD67 in the cholinergic neurons of the nucleus ambiguus and in the dorsal motor nucleus of the vagal nerves. Materials and Methods Twenty-five male Wistar rats (Japan SLC, Shizuoka, Japan; 8 weeks old; body weight 180–200 g) were used in this study. The animals received commercial food pellets and water ad libitum. All experimental procedures were performed according to the Guidelines for Animal Care by the Japanese Red Cross Hokkaido College of Nursing (Permit Number: 33, 34, 41). The animals were deeply anesthetized by inhalation with sevoflurane and perfused through the heart with 200 ml of physiological saline and 200 ml of 4% paraformaldehyde or 0.3% glutaraldehyde plus 4% paraformaldehyde in 0.01 M phosphate-buffered saline (PBS) pH 7.4. The esophagus, brainstem and cerebellum were removed and immersed in the same fixative for 2 h at 4°C. For whole-mount preparations of esophagus, both the oral end of the esophagus and the proximal part of the stomach were ligated with a cotton thread, and the lumen was distended with a fixative solution, which was introduced with a needle. After fixation in the same fixative for 1 h, the esophagus was opened longitudinally, and the mucosal and submucosal layers were stripped away from the esophagus; the outer striated muscle layer attached to the myenteric plexus was then isolated by dissecting away the inner striated muscle layer. To prepare the cryostat sections, the esophagus was immersed overnight in PBS containing 30% sucrose at 4°C. The esophagus was divided into 3 portions (upper, middle, and lower), cut into 12 µm thick sections using a cryostat, and mounted on glass slides coated with poly-L-lysine (Sigma, St. Louis, MO, USA). For immunohistochemistry (Table 1 ), the whole-mount preparations and sections of the esophagus were incubated with primary antibodies overnight at 4°C, followed by incubation for 2 h with a secondary antibody conjugated to indocarbocyanine (Cy3) or Alexa Fluor® 488. For double immunostaining, the whole-mount preparations and sections were incubated for 12 h at 4°C with a mixture of 2 primary antisera raised against different species. The immunoreacted whole-mount preparations and sections were rinsed in PBS and then incubated with a mixture of secondary antibodies conjugated to Cy3 or Alexa Fluor® 488. After PBS washes, some whole-mount preparations and sections were additionally incubated with fluorescein (FITC)-conjugated α-bungarotoxin (BTX) (1:1000; PK-CA707-00011; PromoKine, Heidelberg, Germany) as a marker of nicotinic acetylcholine receptor at the neuromuscular junctions of striated muscles and were incubated free-floating for NADPH-d as a marker of NOS (Ward et al. 1992 ) for 1 h at room temperature in 0.1 M PBS containing 0.2 mg/ml nitroblue tetrazolium (N-6876; Sigma), 1.0 mg/ml β-NADPH (309-50471; Oriental Yeast Co. Ltd., Tokyo, Japan), and 0.3% Triton X-100. Table 1 List of primary antisera and secondary fluorescence conjugated antisera used for immunohistochemistry in the present study. Primary antisera Host animals Dilution Catalogue No. Source bNOS Rabbit 1:4000 N31030 Transduction Laboratories, KY, USA ChAT Goat 1:100 AB144P Chemicon International Inc, Temecula, CA, USA VAChT Goat 1:400 ABN100 Chemicon International Inc GABA Rabbit 1:2000 A-2052 SIGMA BIO SCIENCES, MO, USA GAD67 Mouse 1:500 MAB5406 Millipore Corporation, Temecula, CA, USA VGAT Rabbit 1:600 131 002 Synaptic Systems, Goettingen, Germany GABA B -R Guinea pig 1:4000 AB1531 Chemicon International Inc PGP9.5 Rabbit 1:5000 RA95101 UltraClone Ltd, Isle of Wight, UK bNOS: brain nitric oxide synthase, ChAT: choline acetyltransferase, VAChT: vesicular acetylcholine transporter, GABA: γ-aminobutyric acid, GAD: glutamate decarboxylase, GABA B -R: GABA B -receptor, PGP9.5: protein gene product 9.5, Cy3: indocarbocyanine Secondary antisera Conjugates Dilution Catalogue No. Source Anti-goat IgG Alexa Fluor® 488 1:100 705-545-147 Jackson ImmunoResearch, West Grove, PA, USA Anti-guinea pig IgG Cy3 1:250 706-165-148 Jackson ImmunoResearch Anti-rabbit IgG Alexa Fluor® 488 1:100 711-545-152 Jackson ImmunoResearch Anti-rabbit IgG Cy3 1:250 711-165-152 Jackson ImmunoResearch Anti-goat IgG biotin 1:500 705-065-147 Jackson ImmunoResearch Anti-rabbit IgG biotin 1:500 BA-1000 VECTOR LABORATORIES, Burlingame, CA, USA Anti-mouse IgG biotin 1:500 BA-2000 VECTOR LABORATORIES To examine the colocalization of GABA-/ ChAT-immunoreactive nerve terminals in the neuromuscular junctions of the striated muscle, 20 sections were randomly selected from the upper, middle, and lower portions of the esophagus obtained from each of 10 animals. The number of GABA-/ ChAT-immunoreactive nerve terminals on the motor endplates of the striated muscles were counted. Furthermore, the colocalization ratio of GABA B -R/ protein gene product 9.5 (PGP9.5), brain nitric oxide synthase (bNOS)/ GABA B -R- or ChAT-/ GABA B -R-immunoreactive neurons in the myenteric plexus was examined. Five areas of 2.5 mm × 2.5 mm (= 6.25 mm 2 ) in the whole-mount preparations were randomly selected 100 GABA B -R-immunoreactive nerve cells from the 3 portions of the esophagus of 3 rats, the numbers of GABA B -R-/PGP9.5-, bNOS-/ GABA B -R- or ChAT-/ GABA B -R-immunoreactive neurons in the myenteric plexus were counted. The data obtained from each portion of five rats were shown as mean ± S.E. and were analyzed by the non-parametric U-test. To prepare the cryostat sections, the brainstem and cerebellum were left overnight in PBS containing 30% sucrose at 4°C. These tissues were cut about 50 µm thick or serial 20 µm thick using a cryostat and mounted on glass slides coated with poly-L-lysine (Sigma, St. Louis, Mo, USA). We examined the cryostat sections immunohistochemically by using VGAT and GAD67 antibodies whether the cholinergic neurons in the nucleus ambiguus and in the dorsal motor nucleus of the vagal nerves contained GABA or not. For immunohistochemistry, the immunofluorescent method and avidin-biotin-peroxidase complex (ABC) method (Hsu et al. 1981 ) was used. Some brain sections were with a staining kit purchased from Vector Laboratories (Burlingame, Calif.,USA). The tissues were incubated for 48 h at room temperature with antibodies described above, followed by incubation in biotinylated secondary antibodies and ABC for 1 h respectively at room temperature. The antigen-antibody reaction sites were visualized by incubating the section s for 15 min at room temperature with diaminobenzidine tetrahydrochloride and 0.01% hydrogen peroxide in Tris-HCL buffer (25mM, pH 7.6). The specificity of the immunohistochemical staining was confirmed by replacing the primary antibody with normal serum and by using diluted antiserum pretreated with a GABA (G-012;10 µg/ml; Research Biochemicals, Natick, MA., USA) for 24 h at 4°C. No immunostaining was observed under these conditions. Results In the cryostat sections, a few GABA-immunoreactive nerve fibers were found near and in the muscularis mucosae (Fig. 1 a) and the submucosal tissue of the rat esophagus. Numerous GABA-immunoreactive nerve fibers ran along the striated muscle fibers and terminated on them. The nerve fibers formed motor endplates on the striated muscle fibers (Fig. 1 b). Thick GABA-immunoreactive nerve bundles and fibers ran among the ganglia in the myenteric plexus. Fine GABA-immunoreactive nerve fibers were seen on the neurons in the myenteric plexus and were in close contact with myenteric neurons (Fig. 1 c). No GABA immunoreactivity was observed in the mucous epithelial cells, in the lamina propria, or in the neurons of the myenteric plexus of the rat esophagus. GABA B -R immunoreactivity was seen only in the neurons of the myenteric plexus in the rat esophagus (Fig. 1 d). The size of the GABA B -R-immunoreactive neurons ranged from small to large. The immunoreactivity was intense at the cell surface membrane and as granular structures in the cytoplasm. No GABA B -R immunoreactivity was observed in the nerve terminals, in the striated muscles or in the smooth muscle cells of the muscularis mucosae of the esophagus. In the whole mount preparations, GABA immunoreactivity was observed in numerous nerve fibers, but not in the neurons of the rat esophagus. Thick GABA-immunoreactive nerve bundles and fibers ran along the clusters of neurons in the myenteric plexus (Fig. 2 ) and ramified into one or more branches that projected to a few myenteric ganglia in the myenteric plexus and to the striated muscles. Numerous GABA-immunoreactive nerve terminals formed the motor endplates on the striated muscle fibers (Fig. 3 ). Double staining showed that GABA-immunoreactive nerve fibers were sometimes in close contact with NADPH-d-positive or -negative neurons in the myenteric plexus (Fig. 4 a, b). NADPH-d-positive nerve fibers from myenteric neurons often intermingled with the GABA-immunoreactive motor endplates in the striated muscles. Furthermore, GABA-immunoreactive nerve fibers were sometimes in close contact with ChAT-immunoreactive or ChAT-immunonegative neurons in the myenteric plexus and were ChAT-immunopositive (Fig. 4 c, d). Some GABA-immunoreactive nerve fibers eventually formed motor endplates on the striated muscles. Double immunostaining showed that the numerous GABA-immunoreactive nerve terminals forming motor endplates on the striated muscles were both ChAT-immunoreactive (Fig. 5 a, b) and BTX-positive (Fig. 5 c, d). Some of the ChAT-immunoreactive or BTX-positive motor endplates were GABA-immunonegative (Fig. 5 a–d). Triple staining showed that NADPH-d-positive nerve fibers intermingled with GABA-immunopositive nerve terminals and were located at BTX-positive areas (Fig. 5 e–g). The percentages of ChAT-immunoreactive motor endplates with GABA immunoreactivity to total ChAT-immunopositive endplates in the upper, middle, and lower portions of the esophagus were 33.7%, 17.0%, and 23.7%, respectively (average, 24.8%) (Table 2). The differences among the number of GABA- or ChAT-positive neuromuscular junctions in the three portions of the esophagus were not significantly different. However, no GABA immunoreactivity was seen in nerve cell bodies in the myenteric plexus of the esophagus. In the control, immunohistochemical staining was confirmed by replacing the primary antibody with absorbed with 10µg GABA. No GABA-immunostaining was observed in in the ChAT-immunoreactive motor endplates of the control sections of the rat esophagus (Fig. 5 h, i). GABA B -R immunoreactivity was observed in numerous myenteric neurons of the rat esophagus. The GABA B -R-immunoreactive neurons ranged in size from small to large, and the immunoreactivity was localized intensely to the cell surface membrane and to granular structures in the cytoplasm (Fig. 6 ). The immunoreactivity was often detectable in the proximal-to-distal part of the axons, but not in the terminals. Double immunostaining showed that the percentage of GABA B -R-immunoreactive neurons to myenteric neurons with PGP9.5 immunoreactivity was 100% in the upper, middle, and lower portions of the rat esophageal myenteric plexus. The PGP9.5 antibody was used for the neurons in the rat esophageal myenteric plexus (Kuramoto et al. 2019 ). Double immunostaining in the whole-mount preparations showed that numerous GABA B -R-immunoreactive neurons were NOS-immunopositive (Fig. 7 a, b) or ChAT-immunopositive (Fig. 7 c, d). The percentages of NOS-/GABA B -R-immunoreactive neurons in the upper, middle, and lower portions of the rat esophageal myenteric plexus were 64.0%, 68.6%, and 63.6% (average, 65.4%), respectively (Table 3). The percentages of ChAT-/GABA B -R-immunoreactive neurons were 14.0%, 13.6%, and 9.6% (average, 12.4%) in the upper, middle, and lower portions of the rat esophageal myenteric plexus (Table 3). The differences among the number of NOS-/GABA B -R- or ChAT-/GABA B -R-immunopositive neuromuscular junctions in the three portions of the esophagus were not significantly different. However, no GABA B -R immunoreactivity was found in the nerve terminals and neuromuscular junctions in the striated muscles, in the striated muscle cells, or in the smooth muscle cells of the muscularis mucosae. In the whole-mount preparation of esophagus, VGAT immunoreactivity was shown mainly in the motor endplates on the striated muscles of the esophagus. The VGAT immunoreactivity was fine granular in the motor endplates (Fig. 8 a, b). However, no or weak immunoreactivity was found in the nerve fibers. VGAT immunoreactivity was seen as small dots within nerve fibers and in the periphery of the nerve cells in the myenteric plexus. In the double staining with ChAT and VGAT antibodies, almost all ChAT-immunoreactive motor endplates were VGAT-immunopositive. Some VGAT-immunoreactive nerve fibers contacted the soma of the ChAT-immunoreactive neurons in the myenteric plexus. No VGAT-immunoreactive neurons were seen in the myenteric plexus. No GAD67 immunoreactivity was observed in the neuronal elements of the esophagus. In the double staining with vesicular acetylcholine transporter (VAChT), VGAT and GAD67 antibodies, GAD67 and VGAT immunoreactivities were not seen in the VAChT-immunoreactive neurons in the nucleus ambiguus and in the dorsal motor nucleus of vagal nerves (Fig. 9 a–f). GAD67 (Fig. 9 f) and VGAT (Fig. 9 b, d) immunoreactivities were observed in numerous nerve fibers and in nerve terminals on the surface of the neurons, but not in the dendrites and in the cell bodies of VAChT-immunoreactive neurons (Fig. 9 a, c, e) in the rat nucleus ambiguus and in the dorsal motor nucleus of vagal nerves. In the rat cerebellum as the positive control, the GABA, GAD67 and VGAT immunoreactivities of the Purkinje cells were observed on the surface and in the periphery of the cell bodies, and in numerous axons, but not in the cell bodies and in the dendrites (Fig. 10 a–d) which was in accordance with the previous reports (McLaughlin et al. 1975 ; Oertel et al.1981; Ottersen and Strom-Mathisen 1984 ; Chaundhry et al. 1998; Takayama and Inoue 2004 ). Discussion We first revealed that GABA-immunoreactive nerve fibers were ChAT-immunopositive and formed motor endplates corresponding to the BTX-positive areas of the striated muscles of the adult rat esophagus. This suggests that GABA and acetylcholine are colocalized in the motor endplates corresponding to the nicotinic acetylcholine receptor-rich portion and that GABA in the motor endplates may exert an inhibitory effect on the motility of the striated muscles of the rat esophagus. GABA in the motor endplates on the striated muscles of the vertebrates has not previously been seen. In addition, the present study showed that VGAT immunoreactivity was found in almost all motor endplates of the esophageal striated muscles. GABA is localized in the membrane of GABAergic vesicles and loads synthesized GABA into the synaptic vesicles by VGAT at the axon terminals (McIntire et al. 1997 ; Chaudhry et al. 1998 ; Fon and Edwards, 2001 ). The present study suggests that GABA may be present in the neuronal elements of the striated muscles of the rat esophagus, and synthesized GABA may be taken up and accumulated in the synaptic vesicles by VGAT in the nerve terminals. Furthermore, we were unable to observe GABA-R immunoreactivity on the striated muscles of the rat esophagus. The role that GABA plays in transmission to the esophageal striated muscles is unknown. However, GABA does not directly affect the contraction of the skeletal muscle of mice (Chiou and Chang, 1994 ) and of the smooth muscle in several regions of the intestine of the guinea pig (Krantis et al. 1980 ; Krantis and Kerr 1981 ; Kleinrock and Kilbinger 1983). Thus, it may be unlikely that GABA directly influences the striated muscles or smooth muscles in the esophagus. Further gene and protein analyses are required to confirm the expression of other GABA-Rs in the striated muscles of esophagus. A previous study showed that GABA had inhibitory effects on the secretion of acetylcholine at cholinergic synaptic contacts with the striated muscles by cultured retinal neurons (Agardh et al. 1985 ). GABA depresses excitatory cholinergic transmission in the gut by inhibiting transmitter release, an effect mediated through presynaptic GABA B -Rs (Fargeas et al. 1988 ; Kerr et al. 1990 ; Gentilini et al. 1992 ) and modifies transmission from enteric neurons (Ong and Kerr 1983 , 1984 ; Ohkawa 1987 ). However, we failed to demonstrate GABA B -R immunoreactivity on pre- and postsynaptic sites in the neuromuscular junctions of the rat esophageal striated muscle because of the negligible quantity of GABA B -R proteins in the nerve terminals or because of other GABA-Rs. Taken together, one possibility is that GABA from nerve terminals may have inhibitory effects on the release of GABA, acetylcholine via presynaptic GABA B -R or other types of GABA-Rs. The present study showed that NADPH-d (as a marker of NOS)-positive nerve fibers from intrinsic neurons intermingled with GABA-immunoreactive motor endplates in the esophageal striated muscles. Previous immunohistochemical studies revealed the dual innervation of motor endplates by axons that were immunoreactive for both NOS and galanin (Kuramoto et al. 1999 ), vasoactive intestinal polypeptide, neuropeptide tyrosine, galanin, or enkephalin (Kuramoto and Endo 1995 , Kuramoto et al. 1996 ; Neuhuber et al. 2001 ; for review see Wörl and Neuhuber 2005 ). These neuropeptides act as inhibitory transmitters in the enteric nervous system of the intestine (for review see Furness 2000 ; for review see Brookes, 2001 ) and are found in myenteric neurons and nerve terminals on the motor endplates in the esophagus of various mammals (Wörl and Neuhuber 2005 ). When the endplates were orthogradely labeled by injection of DiI into the brainstem, none had NADPH-d reactivity (Neuhuber et al. 1994 ) and none had galanin immunoreactivity (Wörl et al. 1998 ). Unilateral vagotomy did not reduce the number of NOS-immunoreactive terminals innervating the striated muscle, although more than half of the CGRP-immunoreactive motor endplates were lost (Kuramoto et al., 1999 ). These data suggest that the dual innervation on the motor endplates may be derived from intrinsic neurons and may inhibit the motility of esophageal striated muscles. Furthermore, we observed GABA B -R immunoreactivity in the neuronal cell bodies and in the proximal-to-distal part of the axons, but not in the terminals. Our immunohistochemical method may not have been sensitive enough to detect GABA B -R in the nerve terminals due to their low levels of GABA B -R protein. GABA from the motor endplates may have a local inhibitory effect on the dual-innervated nerve terminals of the motor endplates on the striated muscle fibers because these esophageal myenteric neurons dually innervated the motor endplates and were positive for GABA B -R. The present study revealed that GABA-immunoreactive nerve fibers were in contact with a few NADPH-d-positive or ChAT-immunoreactive neurons in the myenteric plexus of the rat esophagus and that GABA B -R-immunoreactive neurons in the myenteric plexus were NOS- or ChAT-immunopositive. This infers that GABA from the nerve fibers may have inhibitory effects on the release of neurons via postsynaptic GABA B -R. Previous immunohistochemical studies demonstrated the presence of GABA or GAD (Jessen et al. 1986 ; Saito and Tanaka 1986 ; Davanger et al. 1987 ; Hills et al. 1987 ; Furness et al. 1989 ), GABA A -R (Krantis et al. 1995 ), and GABA B -receptor (Nakajima et al. 1996 ) in the neuronal elements of the intestine. GABA depresses excitatory cholinergic transmission in the gut by inhibiting transmitter release, an effect mediated through presynaptic GABA B -R (Fargeas et al. 1988 ; Kerr et al. 1990 ; Gentilini et al. 1992 ). The inhibition of GABA B -R in the neurons was mainly achieved via modulation of neurotransmitter release from presynaptic terminals and hyperpolarization of postsynaptic membranes (for review see Bowery et al. 2002 ). However, the present immunohistochemical method may not have been able to detect the presynaptic GABA B -R in the nerve terminals due to low levels of GABA B -R. Taken together, GABA from the nerve fibers contacting the esophageal myenteric neurons may have inhibitory effects on the release of GABA and acetylcholine via presynaptic GABA B -R and on the release of nitric oxide or acetylcholine from intrinsic neurons via postsynaptic GABA B -R. We found no GABA-, GAD67- and VGAT-immunoreactive neurons in the rat esophageal myenteric plexus. This infers that GABA-immunoreactive nerve fibers in the rat esophagus may be extrinsic in origin. Previous studies showed innervation of dorsal motor nucleus of vagal nerves in the intrinsic neurons of the myenteric plexus and innervation of nucleus ambiguus in the striated muscles in the esophagus (Roman 1982 ; for review see Cunningham and Sawchenko 1990 ; Conklin and Christensen 1994 ). Because the present findings indicate that GABA-immunopositive nerve terminals are present on motor endplates that are immunopositive for ChAT, these seem to originate from the nucleus ambiguus. However, the present study showed that GAD67 and VGAT immunoreactivities were found in numerous nerve fibers on the cell bodies, but not in the dendrites and cell bodies of the VAChT-immunoreactive neurons in the rat nucleus ambiguus and in the dorsal motor nucleus of vagal nerves. However, No GAD67-green fluorescent protein was shown in the ChAT-immunoreactive neurons of the nucleus ambiguus and the dorsal motor nucleus of vagal nerves of the transgenic mice (Gotts et al. 2015 ). Also, the neurons of the nucleus ambiguus and the dorsal motor nucleus of vagal nerves did not contain GAD and GABA immunoreactivity (Blessing 1990 ; Maqbool et al. 1991 ). These results suggest that GABA may be synthesized and taken up and accumulated in the synaptic vesicles by VGAT in the nerve terminals, but not in the cell bodies of the neurons. During development, GABA disappeared from the cell bodies and dendrites of Purkinje cells and dendrites and axons of Golgi cells, became confined to the terminals as reported in previous studies (Takayama and Inoue, 2004 ). In the adult cerebellum, GABA is exclusively transported into the synaptic vesicles by VGAT at the axon terminals (Fon and Edwards, 2001 ). It is possible that the GABA expression phenomenon in Purkinje cells of the cerebellum during development also occurs in the VAChT-positive neurons in the nucleus ambiguus and dorsal nucleus of the vagal nerves. We showed that thick GABA-immunoreactive nerve fibers divided into thinner nerve fibers and that these fine nerve fibers were in close contact with myenteric ganglia on the way to the striated muscle fibers. Tracer experiments showed that the nerve fibers derived from the nucleus ambiguus innervated the striated muscles and ramified into collateral branches that projected to one or more myenteric ganglia (Powley et al. 2013 ). These data suggest that the nerve fibers from the nucleus ambiguus mainly innervate striated muscles and partly innervate the myenteric ganglia. In conclusion, we revealed the distribution of GABA, VGAT and GABA B -R immunoreactivities in the rat esophagus. However, the present study showed that GAD67 and VGAT immunoreactivities were found in numerous nerve fibers on the surface of the cell bodies, but not in the dendrites and cell bodies of the VAChT-immunoreactive neurons in the rat nucleus ambiguus and in the dorsal motor nucleus of vagal nerves. These results suggest that GABA and GABA B -R may be related to the local control of the inhibitory nervous system of rat esophageal motility, and that GABA may be synthesized and taken up and accumulated in the synaptic vesicles by VGAT in the nerve terminals, but not in the cell bodies of the neurons. The present study sheds new light on the local inhibitory neuronal system involved in esophageal motility. Declarations Acknowledgments The authors dedicate this paper to the memory of our late friend of Dr. Hirofumi Kuramoto, whose insight and encouragement were invaluable to this work. Conflict of Interest: The authors declare that they have no conflict of interest. References Agardh E, Yeh HH, Herrmann R, Puro DG (1985) gamma-aminobutyric acid-mediated inhibition at cholinergic synapses formed by cultured retinal neurons. 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Neurosci Lett 23:263–268 Krantis A, Shabnavard L, Nichols K, de Blas AL, Staines W (1995) Localization of GABA A receptor immunoreactivity in NO synthase positive myenteric neurons. J Auton Nerv Syst 53:157–165 Kuramoto H, Endo Y (1995) Galanin-immunoreactive nerve terminals innervating the striated muscle fibers of the rat esophagus. Neurosci Lett 188:171–174 Kuramoto H, Kato Y, Sakaoto H, EndoY (1996) Galanin-containing nerve terminals that are involved in a dual innervation of the striated muscles of the rat esophagus. Brain Res 734:186–192 Kuramoto H, Kawano H, Sakamoto H, Furness JB (1999) Motor innervation by enteric nerve fibers containing both nitric oxide synthase and galanin immunoreactivities in the striated muscle of the rat esophagus. Cell Tissue Res 295:241–245 Kuramoto H, Yoshimura R, Sakamoto H, Kadowaki M (2019) Reginal variations in the number distribution of intrinsic myenteric neurons and coinnervated motor endplates on the striated muscles in the rat esophagus. 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J Auton Pharmacol 4:77–85 McIntire SL, Reimer RJ, Schuske K, Edwards RH, Jorgensen EM (1997) Identification and characterization of the vesicular GABA transporter. Nature 389:870–876 McLaughlin BJ, Wood JG, Saito K, Roberts E, Wu J-Y (1975) The fine structural localization of glutamate decarboxylase in developing axonal processes and presynaptic terminals of rodent cerebellum. 85:355–371 Minocha A, Galligan JJ (1993) Excitatory and inhibitory responses mediated by GABAA and GABAB receptors in guinea pig distal colon. Eur J Pharmacol 230:187–193 Nakajima K, Tooyama I, Kuriyama K, Kimura H (1996) Immunohistochemical demonstration of GABA B receptors in the rat gastrointestinal tract. Neurochem Res 21:211–215 Neuhuber WL, Wörl J, Berthoud H-R, Conte B (1994) NADPH-diaphorase-positive nerve fibers associated with motor endplates in the rat esophagus. new evidence for co-innervation of striated muscle by enteric neurons. 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Brain Res 376:78–84 Takayama C, Inoue Y (2004) Extrasynaptic localization of GABA in the developing mouse cerebellum. Neurosci Res 50:447–458 Tøttrup A, Svane D, Forman A (1991) Nitric oxide mediating NANC inhibition of opossum lower esophageal sphincter. Am J Physiol 260:G385–389 Uddman R, Alumets J, Håkanson R, Sundler F, Walles B (1980) Peptidergic (enkephalin) innervation of the mammalian esophagus. Gastroenterology 78:732–737 Ward SM, Xue C, Shuttleworth CW, Bredt DS, Snyder SH, Sanders KM (1992) NADPH diaphorase and nitric oxide synthase colocalization in enteric neurons of canine proximal colon. Am J Physiol 263:G277–284 Wörl J, Mayer B, Neuhuber WL (1994) Nitrergic innervation of the rat esophagus: focus on motor endplates. J Auton Nerv Syst 49:227–233 Wörl J, Fischer J, Neuhuber WL (1998) Nonvagal origin galanin-containing nerve terminals innervating striated muscle fibers of the rat esophagus. Cell Tissue Res 292:453–461 Wörl J, Neuhuber WL (2005) Enteric co-innervation of motor endplates in the esophagus: state of the art ten years after. Histochem Cell Biol 123:117–130 Wu M, Majewski M, Wojtkiewicz J, Vanderwinden J-M, Adriaensen D, Timmermans J-P (2003) Anatomical and neurochemical features of the extrinsic and intrinsic innervation of the striated muscle in the porcine esophagus: evidence for regional and species differences. Cell Tissue Res 311:289–297 Zeiter DK, Li X, Broussard (1996) Identification of the GABA A receptor alpha subunit mRNA in rat intestine. Brain Res Mol Brain Res 39:241–244 Table 2 and 3 Table 2 and 3 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table23.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 08 May, 2026 Reviews received at journal 08 May, 2026 Reviewers agreed at journal 28 Apr, 2026 Reviewers agreed at journal 27 Apr, 2026 Reviews received at journal 29 Mar, 2026 Reviewers agreed at journal 19 Mar, 2026 Reviewers invited by journal 19 Mar, 2026 Editor assigned by journal 17 Mar, 2026 Submission checks completed at journal 17 Mar, 2026 First submitted to journal 15 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9132607","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":608954282,"identity":"4e46f7ac-7327-42dd-92c1-5fe29f56d4d6","order_by":0,"name":"Hiroshi Murabayashi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8UlEQVRIiWNgGAWjYLCCDwwMCQw8bECSDS7Ghkc9AwPjDJK1MPPAtBBQCAG6M3IffrbdY5fHz3Ms8cODMhsG/tkNjB9+MPDl4dJidiPdWDrnWXKxZG/bYYmEc2kMEncOMEv2MLAV49aSxiCdc+BA4obz7A0SiW2H6zdIJDBIA52Y2IBbC/NvC4iW5h9ALQwGEgnMvwloYZNmAGk523ZMAqqFDb8tZ56xWfYcSE6c2XMszQLslxuJbZY9Bnj8cjyN+caPA3aJ/Txpxjd/gEJsRvLhGz8qjuEMMWyAEegkg2MJpGgBgxrStYyCUTAKRsFwBQB+cVXIDRF44QAAAABJRU5ErkJggg==","orcid":"","institution":"Japanese Red Cross Hokkaido College of Nursing","correspondingAuthor":true,"prefix":"","firstName":"Hiroshi","middleName":"","lastName":"Murabayashi","suffix":""},{"id":608954283,"identity":"db6559c3-0a91-49a1-be91-b70f13b3d841","order_by":1,"name":"Yoshiki Hira","email":"","orcid":"","institution":"Asahikawa Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yoshiki","middleName":"","lastName":"Hira","suffix":""},{"id":608954284,"identity":"056863f1-1ec5-4bc6-8c51-28c79d4d13cb","order_by":2,"name":"Hitoshi Kawano","email":"","orcid":"","institution":"Saga University Faculty of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hitoshi","middleName":"","lastName":"Kawano","suffix":""},{"id":608954285,"identity":"9146c206-6883-4e5b-ba9e-6ef4fa7ed5d1","order_by":3,"name":"Yukio Oomori","email":"","orcid":"","institution":"Obihiro Otani Junior College","correspondingAuthor":false,"prefix":"","firstName":"Yukio","middleName":"","lastName":"Oomori","suffix":""}],"badges":[],"createdAt":"2026-03-16 03:39:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9132607/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9132607/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105564394,"identity":"95eaa7a6-d929-42bd-ab2a-17262c6421f9","added_by":"auto","created_at":"2026-03-27 12:49:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":12380265,"visible":true,"origin":"","legend":"\u003cp\u003ea–d. GABA (a-c) and GABAB-R (d) immunoreactivities in cryostat sections of rat esophagus. A GABA-immunoreactive nerve fiber runs in the muscularis mucosae (a). GABA-immunoreactive nerve fiber terminates and forms motor endplate on the striated muscles (b). GABA-immunoreactive varicose nerve fibers are seen in myenteric plexus and contact intrinsic neurons (c). GABAB-R- immunoreactive neurons are observed in the myenteric plexus (d). ME: Mucous Epithelium. MM: Muscularis Mucosae. IML: Inner Muscle Layer. OML: Outer Muscle layer. MP: Myenteric Plexus. Bar: a: 50µm, b–d: 20µm\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-9132607/v1/f73618d4f2a51c91cf27837f.png"},{"id":105239169,"identity":"ef8c0977-b432-4e2e-b3ca-c8cab9dabe22","added_by":"auto","created_at":"2026-03-23 21:27:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":10030970,"visible":true,"origin":"","legend":"\u003cp\u003eGABA immunoreactivity in whole mount preparations of rat esophageal myenteric plexus. GABA-immunoreactive nerve fibers closely contact and run along the intrinsic immunonegative neurons in the myenteric plexus. Bar: 20µm.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-9132607/v1/3eb6e4b607daf13353f504ba.png"},{"id":105564064,"identity":"5e7f9231-8eb4-4367-8d93-2b613b98ee87","added_by":"auto","created_at":"2026-03-27 12:48:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2521240,"visible":true,"origin":"","legend":"\u003cp\u003eGABA immunoreactivity in whole mount preparations of rat esophageal striated muscles. GABA-immunoreactive nerve fiber forms motor endplate in the striated muscles. Bar: 20 µm.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-9132607/v1/345e48a2c458b19e759d67e0.png"},{"id":105239172,"identity":"115428d8-e838-4b00-8919-1918745f5bcf","added_by":"auto","created_at":"2026-03-23 21:27:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":6951314,"visible":true,"origin":"","legend":"\u003cp\u003ea–d. Double immunostaining for GABA (a, c), NADPH-d (b) and ChAT (d) in whole mount preparation of rat esophageal myenteric plexus. GABA immunoreactive nerve fibers (a) contact NADPH-d-reactive neurons (asterisks) in the myenteric plexus. Some NADPH-d-positive neurons (asterisks) were seen in the myenteric plexus (b). GABA- immunoreactive nerve fibers closely contact ChAT-immunoreactive neuron\u003cdel\u003es\u003c/del\u003e (asterisk\u003cdel\u003es\u003c/del\u003e) in the myenteric plexus (c, d). Many of GABA-immunoreactive nerve fibers were ChAT-immunoreactive (arrows of c, d). Bar: 20 µm.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-9132607/v1/e725b6b9d8ad1bb29ddd5721.png"},{"id":105239174,"identity":"4c098939-2bea-4c28-b249-e786759e9244","added_by":"auto","created_at":"2026-03-23 21:27:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":16253171,"visible":true,"origin":"","legend":"\u003cp\u003ea–i. Double or triple staining for GABA (a, c, e), ChAT (b), α-bungarotoxin (BTX) (d, g), NADPH-d (f) in whole mount preparations and control sections of ChAT (h) and GABA absorbed with 10 µg GABA (i) of the rat esophageal striated muscles. GABA-/ChAT- (arrowhead) or only ChAT (arrow)-immunoreactive fibers and motor endplates are seen in the striated muscles (a, b). GABA-/BTX- (arrowhead) or only BTX (arrow)-positive motor endplates are seen in the striated muscles (c, d). NADPH-d-positive nerve fibers (arrows) (f) intermingle with GABA-immunoreactive motor endplates (e) and are ramified on the BTX-positive area (g). No GABA immunoreactivity is seen in the ChAT-immunoreactive motor endplate (h, i). Bar: 20 µm.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-9132607/v1/fe4be400582b77a608807b7a.png"},{"id":105564497,"identity":"77b53517-0335-43bb-baf8-d688353477ab","added_by":"auto","created_at":"2026-03-27 12:49:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":7750692,"visible":true,"origin":"","legend":"\u003cp\u003eGABA\u003csub\u003eB\u003c/sub\u003e-R immunoreactivity in whole mount preparations of rat esophageal myenteric plexus. GABA\u003csub\u003eB\u003c/sub\u003e-R- immunoreactive neurons are seen in the myenteric plexus. GABA\u003csub\u003eB\u003c/sub\u003e-R- immunoreactive neurons extend single long axons to the distal direction (arrows). Bar: 20 µm.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-9132607/v1/a84c8080d3644d05078016ee.png"},{"id":105239175,"identity":"dc65fa74-f9bc-4b2c-ae42-45e2d43a68ae","added_by":"auto","created_at":"2026-03-23 21:27:41","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":13348775,"visible":true,"origin":"","legend":"\u003cp\u003ea–d. Double immunostaining for GABA\u003csub\u003eB\u003c/sub\u003e-R (a, c), ChAT (b) and bNOS (d) in whole mount preparations of the neurons in the rat esophageal myenteric plexus. Some GABA\u003csub\u003eB\u003c/sub\u003e-R-immunoreactive neurons are immunoreactive for ChAT or bNOS (asterisks of a and b, c and d) while a part of GABA\u003csub\u003eB\u003c/sub\u003e-R-immunoreactive neurons is ChAT- or bNOS-immunonegative (arrow of a and b, c and d). Bar: 20 µm.\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-9132607/v1/d84a8fd30a58d0363b25f68a.png"},{"id":105239176,"identity":"43dd68b4-88fc-4f21-9783-5b88f47e528a","added_by":"auto","created_at":"2026-03-23 21:27:41","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":13476662,"visible":true,"origin":"","legend":"\u003cp\u003ea–b. Double immunostaining for ChAT (a) and VGAT (b) in whole mount preparations of the nerve terminals in the rat esophageal striated muscles. VGAT immunoreactivity is seen in all ChAT-immunoreactive motor endplates. The VGAT immunoreactivity is found strongly in the nerve terminals rather than in the nerve fibers and is fine granular in the motor endplates. Bar: 20 µm.\u003c/p\u003e","description":"","filename":"Fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-9132607/v1/5bb8aebd7ba168b36dcb6969.png"},{"id":105239177,"identity":"c97df227-84c6-4fcd-94b2-043258b135fd","added_by":"auto","created_at":"2026-03-23 21:27:41","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":56264300,"visible":true,"origin":"","legend":"\u003cp\u003ea–f. Double immunostaining for VAChT (a) and VGAT (b) in same section in the nucleus ambiguus of the rat medulla oblongata. Immunostaining for VAChT (c, e), VGAT (d) and GAD67 (f) using serial sections in the nucleus ambiguus of the rat medulla oblongata. VGAT (b, d) immunoreactivity is seen in the nerve fibers on the surface of and in the periphery of cell bodies (a, b asterisk), but not in the cell bodies of the VAChT-immunoreactive neurons (a, c asterisk) in the the nucleus ambiguus. GAD67 immunoreactivity (f) is found in the nerve fibers on the surface of and in the periphery of, but not in the cell bodies of the VAChT-immunoreactive neurons (e) of the nucleus ambiguus. NAB: nucleus ambiguus of the vagal nerves. Bars: 50 µm.\u003c/p\u003e","description":"","filename":"fig9.png","url":"https://assets-eu.researchsquare.com/files/rs-9132607/v1/1cff610ebb21414d2e2ee36d.png"},{"id":105239178,"identity":"e1b785cd-f7f8-404f-ba36-a27c6ba4db34","added_by":"auto","created_at":"2026-03-23 21:27:41","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":48093762,"visible":true,"origin":"","legend":"\u003cp\u003ea–d. Double immunostaining for VAChT (a, c) and VGAT (b) or GAD67 (d) in same section in the dorsal motor nucleus of the vagal nerves of the rat medulla oblongata. VGAT- or GAD67 immunoreactivity is seen in the nerve fibers on the surface of and in the periphery nerve fibers of, but not in the cell bodies (b, d) of the VAChT-immunoreactive neurons of the dorsal motor nucleus of the vagal nerves (a, c). DMV: dorsal motor nucleus of the vagal nerves Bar: 50 µm.\u003c/p\u003e","description":"","filename":"fig10.png","url":"https://assets-eu.researchsquare.com/files/rs-9132607/v1/fe910132da1a5af35ff23206.png"},{"id":105903839,"identity":"45acd04e-2cab-47b5-9783-72b0f7cecc59","added_by":"auto","created_at":"2026-04-01 09:54:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":95861725,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9132607/v1/844c2224-cf02-4200-abc8-0e5bb5889d49.pdf"},{"id":105239167,"identity":"d2faa44b-d874-4b38-8df6-f147c4e68e6f","added_by":"auto","created_at":"2026-03-23 21:27:40","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16310,"visible":true,"origin":"","legend":"","description":"","filename":"Table23.docx","url":"https://assets-eu.researchsquare.com/files/rs-9132607/v1/9b69b48c7acf58fef687be03.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Distribution of GABA, glutamate decarboxylase 67 (GAD67), Vesicular GABA transporter (VGAT) and GABA B -receptor Immunoreactivities in the Rat Esophagus","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn the rat esophagus, the muscularis externa consists almost entirely of striated muscle fibers and only partly of smooth muscle fibers at the esophagogastric junction. Esophageal motility is mainly controlled by vagal efferents from the motor neurons of the nucleus ambiguus (for review see Cunningham and Sawchenko \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Roman and Gonella1987). These efferents directly innervate the striated muscle via the motor endplates and neurons of the dorsal motor nucleus of the vagal nerves and indirectly regulate the smooth muscle via myenteric intrinsic neurons. On the other hand, vagal afferents are derived from sensory neurons in the nodose ganglion whose terminals perceive various stimulations from the esophagus and relay the sensory information to the medulla oblongata (Roman \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1982\u003c/span\u003e; for review see Cunningham and Sawchenko \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Conklin and Christensen \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Lu and Bieger \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). In addition, the esophagus is innervated by the sensory neurons of the dorsal root ganglia and the intrinsic neurons (Conklin and Christensen \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). The striated muscle of the rat esophagus are co-innervated via their motor endplates by both cholinergic nerve endings from the neurons of the nucleus ambiguus and nitric oxide synthase (NOS)-positive nerve terminals from intrinsic neurons containing nitric oxide (Neuhuber et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1994\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; W\u0026ouml;rl et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Kuramoto et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) and neuropeptides such as galanin, vasoactive intestinal polypeptide, neuropeptide Y, calcitonin gene-related peptide (CGRP), pituitary adenylate cyclase-activating polypeptide (PACAP) and enkephalin (Uddman et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e1980\u003c/span\u003e; Kuramoto and Endo \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Kuramoto et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Neuhuber et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Wu et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Kuramoto et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Nitric oxide and these neuropeptides act on smooth muscle as inhibitory mediators in the intestine (Bult et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Gibson et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; T\u0026oslash;ttrup et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; for review see Furness \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; for review see Brookes \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). However, it is unclear whether or how these mediators are involved in the inhibition of esophageal striated muscles. Thus, the local inhibitory neuronal system involved in esophageal motility remains poorly established, although the central control of esophageal motility is remarkably well-understood (for review see Hornby and Abrahams \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2000\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the central and peripheral nervous systems. Previous studies found both GABA- and glutamic acid decarboxylase (GAD)-immunoreactive nerve fibers and neurons in the gastrointestinal tract (Jessen et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1986\u003c/span\u003e; Saito and Tanaka \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1986\u003c/span\u003e; Davanger et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Hills et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Furness et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). GABA exerts its inhibitory actions through 3 distinct types of receptors, GABA\u003csub\u003eA\u003c/sub\u003e-receptor (GABA\u003csub\u003eA\u003c/sub\u003e-R), GABA\u003csub\u003eB\u003c/sub\u003e-receptor (GABA\u003csub\u003eB\u003c/sub\u003e-R), and GABA\u003csub\u003eC\u003c/sub\u003e-receptor (GABA\u003csub\u003eC\u003c/sub\u003e-R) (for review see Bowery et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; for review see Feigenspan and Bormann \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Previous molecular and immunohistochemical studies found GABA\u003csub\u003eA\u003c/sub\u003e-R, GABA\u003csub\u003eB\u003c/sub\u003e-R, and GABA\u003csub\u003eC\u003c/sub\u003e-R on enteric neurons (Krantis et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Nakajima et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Zeiter et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Fletcher et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). As well as in the brain, GABA\u003csub\u003eB\u003c/sub\u003e-Rs are also abundantly expressed in the gastrointestinal tract (Hyland and Cryan \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). GABA can directly and indirectly stimulate the enteric excitatory and inhibitory neurons projecting to the muscle (Krantis et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1980\u003c/span\u003e; Maggi et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1984\u003c/span\u003e). GABA\u003csub\u003eA\u003c/sub\u003e-R elicits contraction through an excitatory action on cholinergic postganglionic neurons, whereas, in the small and large intestine, GABA\u003csub\u003eB\u003c/sub\u003e-R induces relaxation through an inhibitory presynaptic action on cholinergic postganglionic neurons (Giotti et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1983\u003c/span\u003e; Gentilini et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Minocha and Galligan \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1993\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNonetheless, it remains unclear whether GABA, GABA\u003csub\u003eB\u003c/sub\u003e-R, VGAT exist in the inhibitory neuronal system of the rat esophagus and, if so, what functional significance they play. To clarify these issues, we immunohistochemically examined the presence and distribution of GABA, GABA-B-R, GAD67 and VGAT in the neuronal elements of the rat esophagus and discuss their possible involvement in the inhibition of esophageal motility. Furthermore, we examined the presence of VGAT, GAD67 in the cholinergic neurons of the nucleus ambiguus and in the dorsal motor nucleus of the vagal nerves.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eTwenty-five male Wistar rats (Japan SLC, Shizuoka, Japan; 8 weeks old; body weight 180\u0026ndash;200 g) were used in this study. The animals received commercial food pellets and water ad libitum. All experimental procedures were performed according to the Guidelines for Animal Care by the Japanese Red Cross Hokkaido College of Nursing (Permit Number: 33, 34, 41).\u003c/p\u003e \u003cp\u003eThe animals were deeply anesthetized by inhalation with sevoflurane and perfused through the heart with 200 ml of physiological saline and 200 ml of 4% paraformaldehyde or 0.3% glutaraldehyde plus 4% paraformaldehyde in 0.01 M phosphate-buffered saline (PBS) pH 7.4. The esophagus, brainstem and cerebellum were removed and immersed in the same fixative for 2 h at 4\u0026deg;C. For whole-mount preparations of esophagus, both the oral end of the esophagus and the proximal part of the stomach were ligated with a cotton thread, and the lumen was distended with a fixative solution, which was introduced with a needle. After fixation in the same fixative for 1 h, the esophagus was opened longitudinally, and the mucosal and submucosal layers were stripped away from the esophagus; the outer striated muscle layer attached to the myenteric plexus was then isolated by dissecting away the inner striated muscle layer. To prepare the cryostat sections, the esophagus was immersed overnight in PBS containing 30% sucrose at 4\u0026deg;C. The esophagus was divided into 3 portions (upper, middle, and lower), cut into 12 \u0026micro;m thick sections using a cryostat, and mounted on glass slides coated with poly-L-lysine (Sigma, St. Louis, MO, USA).\u003c/p\u003e \u003cp\u003eFor immunohistochemistry (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), the whole-mount preparations and sections of the esophagus were incubated with primary antibodies overnight at 4\u0026deg;C, followed by incubation for 2 h with a secondary antibody conjugated to indocarbocyanine (Cy3) or Alexa Fluor\u0026reg; 488. For double immunostaining, the whole-mount preparations and sections were incubated for 12 h at 4\u0026deg;C with a mixture of 2 primary antisera raised against different species. The immunoreacted whole-mount preparations and sections were rinsed in PBS and then incubated with a mixture of secondary antibodies conjugated to Cy3 or Alexa Fluor\u0026reg; 488. After PBS washes, some whole-mount preparations and sections were additionally incubated with fluorescein (FITC)-conjugated α-bungarotoxin (BTX) (1:1000; PK-CA707-00011; PromoKine, Heidelberg, Germany) as a marker of nicotinic acetylcholine receptor at the neuromuscular junctions of striated muscles and were incubated free-floating for NADPH-d as a marker of NOS (Ward et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e1992\u003c/span\u003e) for 1 h at room temperature in 0.1 M PBS containing 0.2 mg/ml nitroblue tetrazolium (N-6876; Sigma), 1.0 mg/ml β-NADPH (309-50471; Oriental Yeast Co. Ltd., Tokyo, Japan), and 0.3% Triton X-100.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eList of primary antisera and secondary fluorescence conjugated antisera used for immunohistochemistry in the present study.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrimary antisera\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHost animals\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDilution\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCatalogue No.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSource\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ebNOS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:4000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN31030\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTransduction Laboratories, KY, USA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChAT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGoat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAB144P\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eChemicon International Inc, Temecula, CA, USA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVAChT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGoat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eABN100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eChemicon International Inc\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGABA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:2000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA-2052\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSIGMA BIO SCIENCES, MO, USA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGAD67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMAB5406\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMillipore Corporation, Temecula, CA, USA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVGAT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e131 002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSynaptic Systems, Goettingen, Germany\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGABA\u003csub\u003eB\u003c/sub\u003e-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGuinea pig\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:4000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAB1531\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eChemicon International Inc\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePGP9.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:5000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRA95101\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eUltraClone Ltd, Isle of Wight, UK\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003ebNOS: brain nitric oxide synthase, ChAT: choline acetyltransferase, VAChT: vesicular acetylcholine transporter, GABA: γ-aminobutyric acid, GAD: glutamate decarboxylase, GABA\u003csub\u003eB\u003c/sub\u003e-R: GABA \u003csub\u003eB\u003c/sub\u003e-receptor, PGP9.5: protein gene product 9.5, Cy3: indocarbocyanine\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSecondary antisera\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eConjugates\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDilution\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCatalogue No.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSource\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnti-goat IgG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAlexa Fluor\u0026reg; 488\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e705-545-147\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eJackson ImmunoResearch, West Grove, PA, USA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnti-guinea pig IgG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCy3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e706-165-148\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eJackson ImmunoResearch\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnti-rabbit IgG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAlexa Fluor\u0026reg; 488\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e711-545-152\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eJackson ImmunoResearch\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnti-rabbit IgG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCy3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e711-165-152\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eJackson ImmunoResearch\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnti-goat IgG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ebiotin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e705-065-147\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eJackson ImmunoResearch\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnti-rabbit IgG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ebiotin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBA-1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eVECTOR LABORATORIES, Burlingame, CA, USA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnti-mouse IgG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ebiotin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBA-2000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eVECTOR LABORATORIES\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTo examine the colocalization of GABA-/ ChAT-immunoreactive nerve terminals in the neuromuscular junctions of the striated muscle, 20 sections were randomly selected from the upper, middle, and lower portions of the esophagus obtained from each of 10 animals. The number of GABA-/ ChAT-immunoreactive nerve terminals on the motor endplates of the striated muscles were counted. Furthermore, the colocalization ratio of GABA\u003csub\u003eB\u003c/sub\u003e-R/ protein gene product 9.5 (PGP9.5), brain nitric oxide synthase (bNOS)/ GABA\u003csub\u003eB\u003c/sub\u003e-R- or ChAT-/ GABA\u003csub\u003eB\u003c/sub\u003e-R-immunoreactive neurons in the myenteric plexus was examined.\u003c/p\u003e \u003cp\u003eFive areas of 2.5 mm \u0026times; 2.5 mm (=\u0026thinsp;6.25 mm\u003csup\u003e2\u003c/sup\u003e) in the whole-mount preparations were randomly selected 100 GABA\u003csub\u003eB\u003c/sub\u003e-R-immunoreactive nerve cells from the 3 portions of the esophagus of 3 rats, the numbers of GABA\u003csub\u003eB\u003c/sub\u003e-R-/PGP9.5-, bNOS-/ GABA\u003csub\u003eB\u003c/sub\u003e-R- or ChAT-/ GABA\u003csub\u003eB\u003c/sub\u003e-R-immunoreactive neurons in the myenteric plexus were counted. The data obtained from each portion of five rats were shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E. and were analyzed by the non-parametric U-test.\u003c/p\u003e \u003cp\u003eTo prepare the cryostat sections, the brainstem and cerebellum were left overnight in PBS containing 30% sucrose at 4\u0026deg;C. These tissues were cut about 50 \u0026micro;m thick or serial 20 \u0026micro;m thick using a cryostat and mounted on glass slides coated with poly-L-lysine (Sigma, St. Louis, Mo, USA). We examined the cryostat sections immunohistochemically by using VGAT and GAD67 antibodies whether the cholinergic neurons in the nucleus ambiguus and in the dorsal motor nucleus of the vagal nerves contained GABA or not. For immunohistochemistry, the immunofluorescent method and avidin-biotin-peroxidase complex (ABC) method (Hsu et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1981\u003c/span\u003e) was used. Some brain sections were with a staining kit purchased from Vector Laboratories (Burlingame, Calif.,USA). The tissues were incubated for 48 h at room temperature with antibodies described above, followed by incubation in biotinylated secondary antibodies and ABC for 1 h respectively at room temperature. The antigen-antibody reaction sites were visualized by incubating the section s for 15 min at room temperature with diaminobenzidine tetrahydrochloride and 0.01% hydrogen peroxide in Tris-HCL buffer (25mM, pH 7.6).\u003c/p\u003e \u003cp\u003eThe specificity of the immunohistochemical staining was confirmed by replacing the primary antibody with normal serum and by using diluted antiserum pretreated with a GABA (G-012;10 \u0026micro;g/ml; Research Biochemicals, Natick, MA., USA) for 24 h at 4\u0026deg;C. No immunostaining was observed under these conditions.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eIn the cryostat sections, a few GABA-immunoreactive nerve fibers were found near and in the muscularis mucosae (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) and the submucosal tissue of the rat esophagus. Numerous GABA-immunoreactive nerve fibers ran along the striated muscle fibers and terminated on them. The nerve fibers formed motor endplates on the striated muscle fibers (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Thick GABA-immunoreactive nerve bundles and fibers ran among the ganglia in the myenteric plexus. Fine GABA-immunoreactive nerve fibers were seen on the neurons in the myenteric plexus and were in close contact with myenteric neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). No GABA immunoreactivity was observed in the mucous epithelial cells, in the lamina propria, or in the neurons of the myenteric plexus of the rat esophagus.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGABA\u003csub\u003eB\u003c/sub\u003e-R immunoreactivity was seen only in the neurons of the myenteric plexus in the rat esophagus (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). The size of the GABA\u003csub\u003eB\u003c/sub\u003e-R-immunoreactive neurons ranged from small to large. The immunoreactivity was intense at the cell surface membrane and as granular structures in the cytoplasm. No GABA\u003csub\u003eB\u003c/sub\u003e-R immunoreactivity was observed in the nerve terminals, in the striated muscles or in the smooth muscle cells of the muscularis mucosae of the esophagus.\u003c/p\u003e \u003cp\u003eIn the whole mount preparations, GABA immunoreactivity was observed in numerous nerve fibers, but not in the neurons of the rat esophagus. Thick GABA-immunoreactive nerve bundles and fibers ran along the clusters of neurons in the myenteric plexus (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and ramified into one or more branches that projected to a few myenteric ganglia in the myenteric plexus and to the striated muscles. Numerous GABA-immunoreactive nerve terminals formed the motor endplates on the striated muscle fibers (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDouble staining showed that GABA-immunoreactive nerve fibers were sometimes in close contact with NADPH-d-positive or -negative neurons in the myenteric plexus (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b). NADPH-d-positive nerve fibers from myenteric neurons often intermingled with the GABA-immunoreactive motor endplates in the striated muscles. Furthermore, GABA-immunoreactive nerve fibers were sometimes in close contact with ChAT-immunoreactive or ChAT-immunonegative neurons in the myenteric plexus and were ChAT-immunopositive (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, d). Some GABA-immunoreactive nerve fibers eventually formed motor endplates on the striated muscles.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDouble immunostaining showed that the numerous GABA-immunoreactive nerve terminals forming motor endplates on the striated muscles were both ChAT-immunoreactive (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b) and BTX-positive (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, d). Some of the ChAT-immunoreactive or BTX-positive motor endplates were GABA-immunonegative (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea\u0026ndash;d). Triple staining showed that NADPH-d-positive nerve fibers intermingled with GABA-immunopositive nerve terminals and were located at BTX-positive areas (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee\u0026ndash;g). The percentages of ChAT-immunoreactive motor endplates with GABA immunoreactivity to total ChAT-immunopositive endplates in the upper, middle, and lower portions of the esophagus were 33.7%, 17.0%, and 23.7%, respectively (average, 24.8%) (Table\u0026nbsp;2). The differences among the number of GABA- or ChAT-positive neuromuscular junctions in the three portions of the esophagus were not significantly different. However, no GABA immunoreactivity was seen in nerve cell bodies in the myenteric plexus of the esophagus.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the control, immunohistochemical staining was confirmed by replacing the primary antibody with absorbed with 10\u0026micro;g GABA. No GABA-immunostaining was observed in in the ChAT-immunoreactive motor endplates of the control sections of the rat esophagus (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh, i).\u003c/p\u003e \u003cp\u003eGABA\u003csub\u003eB\u003c/sub\u003e-R immunoreactivity was observed in numerous myenteric neurons of the rat esophagus. The GABA\u003csub\u003eB\u003c/sub\u003e-R-immunoreactive neurons ranged in size from small to large, and the immunoreactivity was localized intensely to the cell surface membrane and to granular structures in the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The immunoreactivity was often detectable in the proximal-to-distal part of the axons, but not in the terminals. Double immunostaining showed that the percentage of GABA\u003csub\u003eB\u003c/sub\u003e-R-immunoreactive neurons to myenteric neurons with PGP9.5 immunoreactivity was 100% in the upper, middle, and lower portions of the rat esophageal myenteric plexus. The PGP9.5 antibody was used for the neurons in the rat esophageal myenteric plexus (Kuramoto et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Double immunostaining in the whole-mount preparations showed that numerous GABA\u003csub\u003eB\u003c/sub\u003e-R-immunoreactive neurons were NOS-immunopositive (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea, b) or ChAT-immunopositive (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec, d). The percentages of NOS-/GABA\u003csub\u003eB\u003c/sub\u003e-R-immunoreactive neurons in the upper, middle, and lower portions of the rat esophageal myenteric plexus were 64.0%, 68.6%, and 63.6% (average, 65.4%), respectively (Table\u0026nbsp;3). The percentages of ChAT-/GABA\u003csub\u003eB\u003c/sub\u003e-R-immunoreactive neurons were 14.0%, 13.6%, and 9.6% (average, 12.4%) in the upper, middle, and lower portions of the rat esophageal myenteric plexus (Table\u0026nbsp;3). The differences among the number of NOS-/GABA\u003csub\u003eB\u003c/sub\u003e-R- or ChAT-/GABA\u003csub\u003eB\u003c/sub\u003e-R-immunopositive neuromuscular junctions in the three portions of the esophagus were not significantly different. However, no GABA\u003csub\u003eB\u003c/sub\u003e-R immunoreactivity was found in the nerve terminals and neuromuscular junctions in the striated muscles, in the striated muscle cells, or in the smooth muscle cells of the muscularis mucosae.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the whole-mount preparation of esophagus, VGAT immunoreactivity was shown mainly in the motor endplates on the striated muscles of the esophagus. The VGAT immunoreactivity was fine granular in the motor endplates (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea, b). However, no or weak immunoreactivity was found in the nerve fibers. VGAT immunoreactivity was seen as small dots within nerve fibers and in the periphery of the nerve cells in the myenteric plexus.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the double staining with ChAT and VGAT antibodies, almost all ChAT-immunoreactive motor endplates were VGAT-immunopositive. Some VGAT-immunoreactive nerve fibers contacted the soma of the ChAT-immunoreactive neurons in the myenteric plexus. No VGAT-immunoreactive neurons were seen in the myenteric plexus. No GAD67 immunoreactivity was observed in the neuronal elements of the esophagus.\u003c/p\u003e \u003cp\u003eIn the double staining with vesicular acetylcholine transporter (VAChT), VGAT and GAD67 antibodies, GAD67 and VGAT immunoreactivities were not seen in the VAChT-immunoreactive neurons in the nucleus ambiguus and in the dorsal motor nucleus of vagal nerves (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea\u0026ndash;f). GAD67 (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ef) and VGAT (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eb, d) immunoreactivities were observed in numerous nerve fibers and in nerve terminals on the surface of the neurons, but not in the dendrites and in the cell bodies of VAChT-immunoreactive neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea, c, e) in the rat nucleus ambiguus and in the dorsal motor nucleus of vagal nerves.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the rat cerebellum as the positive control, the GABA, GAD67 and VGAT immunoreactivities of the Purkinje cells were observed on the surface and in the periphery of the cell bodies, and in numerous axons, but not in the cell bodies and in the dendrites (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ea\u0026ndash;d) which was in accordance with the previous reports (McLaughlin et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1975\u003c/span\u003e; Oertel et al.1981; Ottersen and Strom-Mathisen \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1984\u003c/span\u003e; Chaundhry et al. 1998; Takayama and Inoue \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe first revealed that GABA-immunoreactive nerve fibers were ChAT-immunopositive and formed motor endplates corresponding to the BTX-positive areas of the striated muscles of the adult rat esophagus. This suggests that GABA and acetylcholine are colocalized in the motor endplates corresponding to the nicotinic acetylcholine receptor-rich portion and that GABA in the motor endplates may exert an inhibitory effect on the motility of the striated muscles of the rat esophagus. GABA in the motor endplates on the striated muscles of the vertebrates has not previously been seen. In addition, the present study showed that VGAT immunoreactivity was found in almost all motor endplates of the esophageal striated muscles. GABA is localized in the membrane of GABAergic vesicles and loads synthesized GABA into the synaptic vesicles by VGAT at the axon terminals (McIntire et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Chaudhry et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Fon and Edwards, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). The present study suggests that GABA may be present in the neuronal elements of the striated muscles of the rat esophagus, and synthesized GABA may be taken up and accumulated in the synaptic vesicles by VGAT in the nerve terminals.\u003c/p\u003e \u003cp\u003eFurthermore, we were unable to observe GABA-R immunoreactivity on the striated muscles of the rat esophagus. The role that GABA plays in transmission to the esophageal striated muscles is unknown. However, GABA does not directly affect the contraction of the skeletal muscle of mice (Chiou and Chang, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1994\u003c/span\u003e) and of the smooth muscle in several regions of the intestine of the guinea pig (Krantis et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1980\u003c/span\u003e; Krantis and Kerr \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1981\u003c/span\u003e; Kleinrock and Kilbinger 1983). Thus, it may be unlikely that GABA directly influences the striated muscles or smooth muscles in the esophagus. Further gene and protein analyses are required to confirm the expression of other GABA-Rs in the striated muscles of esophagus.\u003c/p\u003e \u003cp\u003eA previous study showed that GABA had inhibitory effects on the secretion of acetylcholine at cholinergic synaptic contacts with the striated muscles by cultured retinal neurons (Agardh et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1985\u003c/span\u003e). GABA depresses excitatory cholinergic transmission in the gut by inhibiting transmitter release, an effect mediated through presynaptic GABA\u003csub\u003eB\u003c/sub\u003e-Rs (Fargeas et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Kerr et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Gentilini et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1992\u003c/span\u003e) and modifies transmission from enteric neurons (Ong and Kerr \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1983\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1984\u003c/span\u003e; Ohkawa \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). However, we failed to demonstrate GABA\u003csub\u003eB\u003c/sub\u003e-R immunoreactivity on pre- and postsynaptic sites in the neuromuscular junctions of the rat esophageal striated muscle because of the negligible quantity of GABA\u003csub\u003eB\u003c/sub\u003e-R proteins in the nerve terminals or because of other GABA-Rs. Taken together, one possibility is that GABA from nerve terminals may have inhibitory effects on the release of GABA, acetylcholine via presynaptic GABA\u003csub\u003eB\u003c/sub\u003e-R or other types of GABA-Rs.\u003c/p\u003e \u003cp\u003eThe present study showed that NADPH-d (as a marker of NOS)-positive nerve fibers from intrinsic neurons intermingled with GABA-immunoreactive motor endplates in the esophageal striated muscles. Previous immunohistochemical studies revealed the dual innervation of motor endplates by axons that were immunoreactive for both NOS and galanin (Kuramoto et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1999\u003c/span\u003e), vasoactive intestinal polypeptide, neuropeptide tyrosine, galanin, or enkephalin (Kuramoto and Endo \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1995\u003c/span\u003e, Kuramoto et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Neuhuber et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; for review see W\u0026ouml;rl and Neuhuber \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). These neuropeptides act as inhibitory transmitters in the enteric nervous system of the intestine (for review see Furness \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; for review see Brookes, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) and are found in myenteric neurons and nerve terminals on the motor endplates in the esophagus of various mammals (W\u0026ouml;rl and Neuhuber \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). When the endplates were orthogradely labeled by injection of DiI into the brainstem, none had NADPH-d reactivity (Neuhuber et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1994\u003c/span\u003e) and none had galanin immunoreactivity (W\u0026ouml;rl et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Unilateral vagotomy did not reduce the number of NOS-immunoreactive terminals innervating the striated muscle, although more than half of the CGRP-immunoreactive motor endplates were lost (Kuramoto et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). These data suggest that the dual innervation on the motor endplates may be derived from intrinsic neurons and may inhibit the motility of esophageal striated muscles. Furthermore, we observed GABA\u003csub\u003eB\u003c/sub\u003e-R immunoreactivity in the neuronal cell bodies and in the proximal-to-distal part of the axons, but not in the terminals. Our immunohistochemical method may not have been sensitive enough to detect GABA\u003csub\u003eB\u003c/sub\u003e-R in the nerve terminals due to their low levels of GABA\u003csub\u003eB\u003c/sub\u003e-R protein. GABA from the motor endplates may have a local inhibitory effect on the dual-innervated nerve terminals of the motor endplates on the striated muscle fibers because these esophageal myenteric neurons dually innervated the motor endplates and were positive for GABA\u003csub\u003eB\u003c/sub\u003e-R.\u003c/p\u003e \u003cp\u003eThe present study revealed that GABA-immunoreactive nerve fibers were in contact with a few NADPH-d-positive or ChAT-immunoreactive neurons in the myenteric plexus of the rat esophagus and that GABA\u003csub\u003eB\u003c/sub\u003e-R-immunoreactive neurons in the myenteric plexus were NOS- or ChAT-immunopositive. This infers that GABA from the nerve fibers may have inhibitory effects on the release of neurons via postsynaptic GABA\u003csub\u003eB\u003c/sub\u003e-R. Previous immunohistochemical studies demonstrated the presence of GABA or GAD (Jessen et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1986\u003c/span\u003e; Saito and Tanaka \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1986\u003c/span\u003e; Davanger et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Hills et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Furness et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1989\u003c/span\u003e), GABA\u003csub\u003eA\u003c/sub\u003e-R (Krantis et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1995\u003c/span\u003e), and GABA\u003csub\u003eB\u003c/sub\u003e-receptor (Nakajima et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1996\u003c/span\u003e) in the neuronal elements of the intestine. GABA depresses excitatory cholinergic transmission in the gut by inhibiting transmitter release, an effect mediated through presynaptic GABA\u003csub\u003eB\u003c/sub\u003e-R (Fargeas et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Kerr et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Gentilini et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). The inhibition of GABA\u003csub\u003eB\u003c/sub\u003e-R in the neurons was mainly achieved via modulation of neurotransmitter release from presynaptic terminals and hyperpolarization of postsynaptic membranes (for review see Bowery et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). However, the present immunohistochemical method may not have been able to detect the presynaptic GABA\u003csub\u003eB\u003c/sub\u003e-R in the nerve terminals due to low levels of GABA\u003csub\u003eB\u003c/sub\u003e-R. Taken together, GABA from the nerve fibers contacting the esophageal myenteric neurons may have inhibitory effects on the release of GABA and acetylcholine via presynaptic GABA\u003csub\u003eB\u003c/sub\u003e-R and on the release of nitric oxide or acetylcholine from intrinsic neurons via postsynaptic GABA\u003csub\u003eB\u003c/sub\u003e-R.\u003c/p\u003e \u003cp\u003eWe found no GABA-, GAD67- and VGAT-immunoreactive neurons in the rat esophageal myenteric plexus. This infers that GABA-immunoreactive nerve fibers in the rat esophagus may be extrinsic in origin. Previous studies showed innervation of dorsal motor nucleus of vagal nerves in the intrinsic neurons of the myenteric plexus and innervation of nucleus ambiguus in the striated muscles in the esophagus (Roman \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1982\u003c/span\u003e; for review see Cunningham and Sawchenko \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Conklin and Christensen \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Because the present findings indicate that GABA-immunopositive nerve terminals are present on motor endplates that are immunopositive for ChAT, these seem to originate from the nucleus ambiguus. However, the present study showed that GAD67 and VGAT immunoreactivities were found in numerous nerve fibers on the cell bodies, but not in the dendrites and cell bodies of the VAChT-immunoreactive neurons in the rat nucleus ambiguus and in the dorsal motor nucleus of vagal nerves.\u003c/p\u003e \u003cp\u003eHowever, No GAD67-green fluorescent protein was shown in the ChAT-immunoreactive neurons of the nucleus ambiguus and the dorsal motor nucleus of vagal nerves of the transgenic mice (Gotts et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Also, the neurons of the nucleus ambiguus and the dorsal motor nucleus of vagal nerves did not contain GAD and GABA immunoreactivity (Blessing \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Maqbool et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). These results suggest that GABA may be synthesized and taken up and accumulated in the synaptic vesicles by VGAT in the nerve terminals, but not in the cell bodies of the neurons.\u003c/p\u003e \u003cp\u003eDuring development, GABA disappeared from the cell bodies and dendrites of Purkinje cells and dendrites and axons of Golgi cells, became confined to the terminals as reported in previous studies (Takayama and Inoue, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). In the adult cerebellum, GABA is exclusively transported into the synaptic vesicles by VGAT at the axon terminals (Fon and Edwards, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). It is possible that the GABA expression phenomenon in Purkinje cells of the cerebellum during development also occurs in the VAChT-positive neurons in the nucleus ambiguus and dorsal nucleus of the vagal nerves.\u003c/p\u003e \u003cp\u003eWe showed that thick GABA-immunoreactive nerve fibers divided into thinner nerve fibers and that these fine nerve fibers were in close contact with myenteric ganglia on the way to the striated muscle fibers. Tracer experiments showed that the nerve fibers derived from the nucleus ambiguus innervated the striated muscles and ramified into collateral branches that projected to one or more myenteric ganglia (Powley et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). These data suggest that the nerve fibers from the nucleus ambiguus mainly innervate striated muscles and partly innervate the myenteric ganglia.\u003c/p\u003e \u003cp\u003eIn conclusion, we revealed the distribution of GABA, VGAT and GABA\u003csub\u003eB\u003c/sub\u003e-R immunoreactivities in the rat esophagus. However, the present study showed that GAD67 and VGAT immunoreactivities were found in numerous nerve fibers on the surface of the cell bodies, but not in the dendrites and cell bodies of the VAChT-immunoreactive neurons in the rat nucleus ambiguus and in the dorsal motor nucleus of vagal nerves. These results suggest that GABA and GABA\u003csub\u003eB\u003c/sub\u003e-R may be related to the local control of the inhibitory nervous system of rat esophageal motility, and that GABA may be synthesized and taken up and accumulated in the synaptic vesicles by VGAT in the nerve terminals, but not in the cell bodies of the neurons. The present study sheds new light on the local inhibitory neuronal system involved in esophageal motility.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors dedicate this paper to the memory of our late friend of Dr. Hirofumi Kuramoto, whose insight and encouragement were invaluable to this work.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Conflict of Interest: The authors declare that they have no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAgardh E, Yeh HH, Herrmann R, Puro DG (1985) gamma-aminobutyric acid-mediated inhibition at cholinergic synapses formed by cultured retinal neurons. 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Cell Tissue Res 292:453\u0026ndash;461\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eW\u0026ouml;rl J, Neuhuber WL (2005) Enteric co-innervation of motor endplates in the esophagus: state of the art ten years after. Histochem Cell Biol 123:117\u0026ndash;130\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu M, Majewski M, Wojtkiewicz J, Vanderwinden J-M, Adriaensen D, Timmermans J-P (2003) Anatomical and neurochemical features of the extrinsic and intrinsic innervation of the striated muscle in the porcine esophagus: evidence for regional and species differences. Cell Tissue Res 311:289\u0026ndash;297\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZeiter DK, Li X, Broussard (1996) Identification of the GABA\u003csub\u003eA\u003c/sub\u003e receptor alpha subunit mRNA in rat intestine. Brain Res Mol Brain Res 39:241\u0026ndash;244\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Table 2 and 3","content":"\u003cp\u003eTable 2 and 3 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-and-tissue-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ctre","sideBox":"Learn more about [Cell and Tissue Research](https://link.springer.com/journal/441)","snPcode":"441","submissionUrl":"https://submission.springernature.com/new-submission/441/3","title":"Cell and Tissue Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"GABA, GABA receptor, Esophagus, Brainstem, Immunohistochemistry, Rat","lastPublishedDoi":"10.21203/rs.3.rs-9132607/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9132607/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWe investigated the distributions of gamma-aminobutyric acid (GABA), vesicular GABA transporter (VGAT), GABA\u003csub\u003eB\u003c/sub\u003e-receptor (GABA\u003csub\u003eB\u003c/sub\u003e-R) and glutamate decarboxylase 67 (GAD67), immunoreactivities in the rat esophagus. GABA immunoreactivity was found in the nerve fibers of the esophagus, but not in the neurons. A few GABA-immunoreactive nerve fibers ran along the muscularis mucosae and some GABA-immunoreactive nerve bundles and fibers ran along and contacted clusters of myenteric neurons. Numerous GABA-immunoreactive nerve terminals ran along the striated muscles and formed motor endplates on the muscles while GABA-immunoreactive nerve fibers contacted nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d)-positive or choline acetyltransferase (ChAT)-immunopositive neurons in the myenteric plexus. NADPH-d-positive nerve fibers often intermingled with GABA-immunoreactive motor endplates in the striated muscles. The GABA-immunoreactive nerve fibers contacting the myenteric neurons were also ChAT-immunopositive. The GABA-immunoreactive nerve fibers that formed motor endplates corresponding to α-bungarotoxin (BTX)-positive areas on the striated muscles were also ChAT-immunopositive. The average percentage of GABA-immunoreactive motor endplates to total ChAT-immunoreactive motor endplates in the upper, middle, and lower portions of the esophagus was 24.8%. VGAT immunoreactivity was seen in almost all motor endplates on the esophageal striated muscles, but not in the neurons of the myenteric plexus. Some VGAT-immunoreactive nerve terminals contacted clusters of myenteric neurons. GABA\u003csub\u003eB\u003c/sub\u003e-R immunoreactivity was observed in numerous myenteric ganglia and in the proximal-to-distal part of the axons of the ganglia, but not in the striated and smooth muscles. GABA\u003csub\u003eB\u003c/sub\u003e-R-immunoreactive neurons were brain nitric oxide synthase (bNOS)- or ChAT-immunopositive. The present study suggests that GABA and GABA\u003csub\u003eB\u003c/sub\u003e-R may be present in the neuronal elements of the striated muscle of the rat esophagus and may play an important role in the local inhibitory system of rat esophageal motility. No GAD67 immunoreactivity was found in the nerve fibers and neurons of the esophagus. In the nucleus ambiguus and in the dorsal motor nucleus of the vagal nerves, GAD67 and VGAT immunoreactivities were shown in numerous nerve fibers on the surface of the cell bodies, but not in the cell bodies of the vesicular acetylcholine transporter-immunoreactive neurons. The present study suggests that GABA may be taken up and accumulated in the synaptic vesicles by VGAT in the nerve terminals, but not in the cell bodies of the neurons of the brainstem.\u003c/p\u003e","manuscriptTitle":"Distribution of GABA, glutamate decarboxylase 67 (GAD67), Vesicular GABA transporter (VGAT) and GABA B -receptor Immunoreactivities in the Rat Esophagus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-23 21:27:35","doi":"10.21203/rs.3.rs-9132607/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-08T13:16:38+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-08T12:08:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"93533062064727052384519925082770721012","date":"2026-04-28T20:14:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"65940587700121961690569929900599568367","date":"2026-04-27T11:01:06+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-29T21:55:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"94050675246987889496808103940329954274","date":"2026-03-19T14:18:41+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-19T11:34:17+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-17T13:28:38+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-17T13:28:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell and Tissue Research","date":"2026-03-16T03:28:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-and-tissue-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ctre","sideBox":"Learn more about [Cell and Tissue Research](https://link.springer.com/journal/441)","snPcode":"441","submissionUrl":"https://submission.springernature.com/new-submission/441/3","title":"Cell and Tissue Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b409e633-5e2d-4ad2-bac7-7ffb8051bade","owner":[],"postedDate":"March 23rd, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-08T13:16:38+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-08T12:08:36+00:00","index":21,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-08T13:27:11+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-23 21:27:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9132607","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9132607","identity":"rs-9132607","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}