Cell Tissue Res DOI 10.1007/s00441-014-1921-x
SHORT COMMUNICATION
Vesicular glutamate transporter 2-immunoreactive afferent nerve terminals in the carotid body of the rat Takuya Yokoyama & Nobuaki Nakamuta & Tatsumi Kusakabe & Yoshio Yamamoto
Received: 6 January 2014 / Accepted: 15 May 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract The carotid body is a peripheral chemoreceptor that detects decreases in arterial pO2 and subsequently activates the carotid sinus nerve. The hypoxia-evoked activity of the carotid sinus nerve has been suggested to be modulated by glutamate. In the present study, we investigate the immunohistochemical localization of vesicular glutamate transporters in the carotid body of the rat. Vesicular glutamate transporter 2 (VGLUT2) labeling was closely associated with glomus cells immunoreactive to tyrosine hydroxylase but was not in the cytoplasm of these cells. The VGLUT2 immunoreactivity was observed within nerve endings that were immunoreactive to P2X3 and densely localized inside P2X3-immunoreactive axon terminals. These results suggest that VGLUT2 is localized in the afferent nerve terminals of the carotid body. Glutamate may be released from afferent nerve terminals to modulate the chemosensory activity of the carotid body. Keywords Carotid body . Afferent nerve . Glutamate . Vesicular glutamate transporter . Immunohistochemistry The carotid body (CB) is a peripheral chemoreceptor that is responsible for monitoring the arterial blood levels of pO2, pCO2 and pH. Hypoxia has been shown to stimulate glomus T. Yokoyama : N. Nakamuta : Y. Yamamoto (*) Laboratory of Veterinary Anatomy and Cell Biology, Faculty of Agriculture, Iwate University, 18-8 Ueda 3-chome, Morioka, Iwate 020-8550, Japan e-mail: [email protected] T. Yokoyama : N. Nakamuta : Y. Yamamoto Department of Basic Veterinary Science, United Graduate School of Veterinary Science, Gifu University, Gifu, Japan T. Kusakabe Laboratory for Anatomy and Physiology, Department of Sport and Medical Science, Kokushikan University, Tama, Japan
cells within the CB to release ATP and triggers chemosensory discharges in the afferent nerve terminals of the carotid sinus nerve via P2X2/P2X3 purinoreceptors (Prasad et al. 2001; Buttigieg and Nurse 2004). Afferent signals from the CB are carried to the nucleus solitary tract, leading to an increase in ventilation (Lahiri et al. 2006). Hypoxic ventilatory responses were previously shown to be reduced by the N-methyl-Daspartate (NMDA) glutamate receptor antagonist MK801 and this effect was abolished by CB denervation in the rat (Ohtake et al. 1998). Thus, the activity of the carotid sinus nerves may be enhanced by glutamate in the CB. Recent immunohistochemical studies have demonstrated that vesicular glutamate transporters (VGLUTs), the vesicle loading protein for glutamate neurotransmission (Özkan and Ueda 1998), are localized in afferent nerve terminals. VGLUTs comprise three isoforms: VGLUT1, VGLUT2 and VGLUT3 (Takamori et al. 2000; Fremeau et al. 2002; Kaneko et al. 2002). Immunohistochemical studies have shown that VGLUT1 and/or VGLUT2 are widely expressed in afferent sensory nerve endings, such as intraganglionic laminar endings in the myenteric ganglia of the esophagus (Ewald et al. 2006), muscle spindle afferent fibers (Wu et al. 2004), periodontal Ruffini endings in the incisor (Honma et al. 2012) and pulmonary neuroepithelial bodies and airway smooth muscle (Brouns et al. 2006). Therefore, VGLUTs are also expected to be distributed in the afferent nerve terminals of the CB. On the other hand, glutamate immunoreactivity has also been demonstrated in the glomus cells of the cat and guinea pig (Kummer 1996; Torrealba et al. 1996). In the present study, we examine the immunohistochemical localization of VGLUTs in the CB of the rat. Male Wistar rats (8–10 weeks old; 180–200 g) were purchased from Japan SLC (Hamamatsu, Japan). All animal experiments in the present study were approved by the Local Animal Ethics Committee of Iwate University (accession number #A201326).
Cell Tissue Res
Animals were anesthetized using pentobarbital (50 mg/kg; intraperitoneal injection) and transcardially perfused with Ringer’s solution (200 ml) followed by 4 % paraformaldehyde in 0.1 M phosphate buffer (pH 7.4, 200 ml). Bifurcations of the common carotid artery containing CB were removed under a dissecting microscope. Tissue samples were then immersed in the same fixative for an additional 3 h at 4 °C. After three washes in phosphate-buffered saline (PBS; pH 7.4) for 10 min, tissue samples were soaked in PBS containing 30 % sucrose and frozen with O.C.T. compound medium (Sakura Finetech, Tokyo, Japan). Frozen tissues were sectioned at 10 μm in thickness by use of a cryostat (CM1900; Leica, Wetzlar, Germany) and were then mounted on glass slides coated with chrome-alum gelatin. Frozen sections were stained by double or triple immunofluorescence using antibodies against VGLUT1, VGLUT2, or VGLUT3, together with antibodies against tyrosine hydroxylase (TH) and/or the P2X3 purinoreceptor. In the present study, TH and P2X3 were used as markers for glomus cells and afferent nerve terminals, respectively (Habeck and Kummer 1993; Prasad et al. 2001). Cryostat sections were rinsed with PBS and incubated with non-immune donkey serum (1:50 dilution) for 30 min at room temperature. After blocking nonspecific reactions, sections were incubated with a mixture of primary antibodies overnight at 4 °C. Sections were then rinsed with PBS and incubated for 120 min at room temperature with a mixture of secondary antibodies. Details of the antibodies used in the present study are summarized in Table 1. After rinsing with PBS, sections were counterstained with DAPI and coverslipped with mounting medium (Fluoromount, Diagnostic Biosystems, Pleasanton, CA, USA). Sections were examined with a confocal laser scanning microscope (C1; Nikon, Tokyo, Japan). Images of Alexa488, Cy-3 and Alexa647 were colored in green, red and white using computer software (NIS-element; Nikon). PBS or non-immune serum was used for immunohistochemical controls instead of primary or secondary antisera. We confirmed the
complete abolishment of specific labeling in negative controls. The characteristics of the antibodies used in the present study are described as follows: the guinea pig antiVGLUT1 (AB5905; Chemicon, Temecula, CA, USA) was raised against synthetic peptide from rat VGLUT1 and recognized the C-terminus region of the protein. Western blot of the rat cerebral cortex showed a single band at 60 kDa (Melone et al. 2005). The guinea pig antiVGLUT2 (AB2251; Chemicon) was raised against GSTtagged recombinant rat VGLUT2. Western blot of cell lysate from rat astrocytic cultures yielded a band at 55 kDa (Montana et al. 2004). The guinea pig anti-VGLUT3 (AB5421; Chemicon) was raised against a synthetic peptide corresponding to the 569–588 amino acid sequence of rat VGLUT3. Western blot of lysate from rat PC12 cells showed a single band at 65 kDa (manufacturer’s report). The guinea pig antibodies against VGLUT1, VGLUT2 and VGLUT3 have been used in immunohistochemical studies of the nervous system (Stornetta et al. 2003; Gabellec et al. 2007; Fattorini et al. 2009). In the present study, VGLUT2 immunoreactivity was observed in the CB (Fig. 1b), while that of VGLUT1 and VGLUT3 was not (Fig. 1a, c). In sections stained with VGLUT2 and TH, VGLUT2 immunoreactivity was distributed within and around clusters of TH-immunoreactive glomus cells (Fig. 1b). VGLUT2 immunoreactivity was not observed in thin fibers with TH immunoreactivity within the CB (Fig. 1b). At higher magnification, VGLUT2 immunoreactivity was closely associated with glomus cells with TH immunoreactivity but was not in the cytoplasm of these cells (Fig. 1d–f). Double immunofluorescence for VGLUT2 and P2X3 demonstrated that VGLUT2 immunoreactivity was observed within nerve endings immunoreactive to P2X3 (Fig. 2a–c) and densely localized inside P2X3-immunoreactive axon terminals on the glomus cells (Fig. 2c). Furthermore, the triple immunolabeling method confirmed that VGLUT2 immunoreactivity was observed in the axoplasm of P2X3-
Table 1 Antibodies used in the present study Primary antibodies VGLUT1 VGLUT2 VGLUT3 TH P2X3 Secondary Antibodies A Chemicon, Temecula, CA, USA; B Neuromics, Edina, MN, USA; C Jackson Immunoresearch, West Grove, PA, USA; D Invitrogen, Carlsbad, CA, USA
Alexa fluor 488 labeled anti-guinea pig IgG Alexa fluor 647 labeled anti-mouse IgG Cy3 labeled anti-mouse IgG Cy3 labeled anti-rabbit IgG
Code
Host
Dilution
Source
AB5905 AB2251 AB5421 MAB318 RA10109
Guinea pig Guinea pig Guinea pig Mouse Rabbit
1:4,000 1:4,000 1:5,000 1:2,000 1:5,000
A A A A B
706-545-148 A31571 715-165-151 711-175-152
Donkey Donkey Donkey Donkey
1:200 1:600 1:100 1:100
C D C C
Cell Tissue Res Fig. 1 Lower magnification view of sections stained with VGLUT1 (a), VGLUT2 (b), or VGLUT3 (c) with TH in the carotid body of the rat. a VGLUT1 immunoreactivity is not observed in either TH-immunoreactive glomus cells or nerve bundles in the periphery of the carotid body (arrow). b VGLUT2 immunoreactivity is observed within and around clustered glomus cells with TH immunoreactivity but is not in these cells or TH-immunoreactive nerve bundles within the carotid body (arrows). c No VGLUT3 immunoreactivity is detected in either TH-immunoreactive glomus cells or nerve bundles (arrow). d–f A higher magnification view shows that VGLUT2 immunoreactivity is found in close association with glomus cells immunoreactive to TH (arrows in d, f) but not in the perinuclear cytoplasm of these cells
immunoreactive nerve terminals closely adjacent to THimmunoreactive glomus cells (Fig. 2d–f). The present results demonstrated that VGLUT2 immunoreactivity was localized within P2X3-immunoreactive afferent nerve terminals in the CB. Previous studies used electron microscopy to show that afferent nerve terminals were connected to glomus cells by reciprocal synapses and clear Fig. 2 a–c Double immunofluorescence image of VGLUT2 and the P2X3 purinoreceptor. VGLUT2 immunoreactivity is densely localized inside P2X3immunoreactive axon terminals (arrows in a-c). d–f Triple immunolabeling for VGLUT2 (Alexa488, green), P2X3 (Cy3, red) and TH (Alexa647, white). VGLUT2 immunoreactivity is observed within the axoplasm of P2X3-immunoreactive nerve terminals (arrow in d–f) closely adjacent to glomus cells immunoreactive to TH
vesicles in the nerve terminals were shown to accumulate near the presynaptic region in the CB of the rat (McDonald and Mitchell 1975). Because VGLUT2 was shown to be expressed on clear vesicles in the axon terminals of the rat brain (Kaneko et al. 2002), VGLUT2 may be localized in the membrane of these vesicles in the reciprocal synapses of afferent nerve terminals in the CB. Glutamate may be the
Cell Tissue Res
efferent transmitter/modulator in the reciprocal synapses of sensory nerve terminals in the CB. Moreover, the NMDA receptor subunits NR1, NR2A and NR2B were shown to be expressed in glomus cells in the rat by RT-PCR, immunoblotting and immunohistochemistry (Liu et al. 2009). The NMDA receptor is a non-specific cation channel that is activated by glutamate and increases Ca2+ influx in the synaptic membrane (Edmonds et al. 1995). Thus, glutamate from afferent nerve terminals may elevate membrane potentials in the glomus cells via NMDA receptors to increase the chemosensitivity of CB during hypoxia. The NMDA receptor NR2A/2B is known to be expressed in the slowly adapting mechanoreceptors of the Merkel– neurite complex in the rat sinus hair follicle and mechanically evoked firing is depressed by the NMDA receptor blockers, ifenprodil and MK801 (Cahusac et al. 2005). In addition, slowly adapting afferent discharge during stretching of the muscle spindle is increased by glutamate (Bewick et al. 2005). These findings suggest that glutamate directly stimulates afferent nerves to involve the prolonged firing of slowly adapting receptors. Likewise, the carotid sinus nerve has been described as a slowly adapting type of sensory nerve based on its electrophysiological characteristics (Prabhakar 2000). Although it remains unknown whether NMDA receptors are expressed in afferent nerves of the CB, glutamate released from nerve terminals may directly stimulate the carotid sinus nerve in addition to glomus cells during hypoxia. The present results showed that VGLUTs immunoreactivities were not observed in glomus cells. Therefore, VGLUTs may not be localized in the membrane of vesicles in glomus cells. In the CB of the mouse, GABA and its biosynthetic enzyme glutamate decarboxylase (GAD) immunoreactivities were shown to be expressed in glomus cells (Oomori et al. 1994). Because GABA is synthesized from glutamate by GAD in central GABAergic neurons (Martin and Tobin 2000), it seems that glutamate in glomus cells serves as the substrate for GABA synthesis. In the present study, VGLUT2 immunoreactivity was localized in the afferent nerve terminals of the CB. Glutamate from afferent nerve terminals may enhance the chemosensory activity of the CB via the excitation of glomus cells and/or their direct effects on afferent nerve terminals. Acknowledgment This study was partly supported by JSPS KAKENHI Grant Number 25350823.
References Bewick GS, Reid B, Richardson C, Banks RW (2005) Autogenic modulation of mechanoreceptor excitability by glutamate release from synaptic-like vesicles: evidence from the rat muscle spindle primary sensory ending. J Physiol 562:381–394
Brouns I, Pintelon I, De Proost I, Alewaters R, Timmermans J-P, Adriaensen D (2006) Neurochemical characterisation of sensory receptors in airway smooth muscle: comparison with pulmonary neuroepithelial bodies. Histochem Cell Biol 125: 351–367 Buttigieg J, Nurse CA (2004) Detection of hypoxia-evoked ATP release from chemoreceptor cells of the rat carotid body. Biochem Biophys Res Commun 322:82–87 Cahusac PM, Senok SS, Hitchcock IS, Genever PG, Baumann KI (2005) Are unconventional NMDA receptors involved in slowly adapting type I mechanoreceptor responses? Neuroscience 133:763–773 Edmonds B, Gibb AJ, Colquhoun D (1995) Mechanisms of activation of glutamate receptors and the time course of excitatory synaptic currents. Annu Rev Physiol 57:495–519 Ewald P, Neuhuber WL, Raab M (2006) Vesicular glutamate transporter 1 immunoreactivity in extrinsic and intrinsic innervation of the rat esophagus. Histochem Cell Biol 125:377–395 Fattorini G, Verderio C, Melone M, Giovedì S, Benfenati F, Matteoli M, Conti F (2009) VGLUT1 and VGAT are sorted to the same population of synaptic vesicles in subsets of cortical axon terminals. J Neurochem 110:1538–1546 Fremeau RT Jr, Burman J, Qureshi T, Tran CH, Proctor J, Johnson J, Zhang H, Sulzer D, Copenhagen DR, Storm-Mathisen J, Reimer RJ, Chaudhry FA, Edwards RH (2002) The identification of vesicular glutamate transporter 3 suggests novel modes of signaling by glutamate. Proc Natl Acad Sci USA 99:14488–14493 Gabellec MM, Panzanelli P, Sassoè-Pognetto M, Lledo PM (2007) Synapse-specific localization of vesicular glutamate transporters in the rat olfactory bulb. Eur J Neurosci 25:1373– 1383 Habeck JO, Kummer W (1993) Neuronal and neuroendocrine markers in the human carotid body in health and disease. Adv Exp Med Biol 337:31–35 Honma S, Kato A, Shi L, Yatani H, Wakisaka S (2012) Vesicular glutamate transporter immunoreactivity in the periodontal ligament of the rat incisor. Anat Rec 295:160–166 Kaneko T, Fujiyama F, Hioki H (2002) Immunohistochemical localization of candidates for vesicular glutamate transporters in the rat brain. J Comp Neurol 444:39–62 Kummer W (1996) Innervation of paraganglia. In: Unsicker K (ed) Autonomic-endocrine Interactions. Harwood, Amsterdam, pp 257–287 Lahiri S, Roy A, Baby SM, Hoshi T, Semenza GL, Prabhakar NR (2006) Oxygen sensing in the body. Prog Biophys Mol Biol 91:249–286 Liu Y, Ji ES, Xiang S, Tamisier R, Tong J, Huang J, Weiss JW (2009) Exposure to cyclic intermittent hypoxia increases expression of functional NMDA receptors in the rat carotid body. J Appl Physiol 106:259–267 Martin DL, Tobin AJ (2000) Mechanisms controlling GABA synthesis and degradation in the brain. In: Martin DL, Olsen RW (eds) GABA in the nervous system: the view at fifty years. Lippincott Williams & Wilkins, Philadelphia, pp 25–41 McDonald DM, Mitchell RA (1975) The innervation of glomus cells, ganglion cells and blood vessels in the rat carotid body: a quantitative ultrastructural analysis. J Neurocytol 4:177–230 Melone M, Burette A, Weinberg RJ (2005) Light microscopic identification and immunocytochemical characterization of glutamatergic synapses in brain sections. J Comp Neurol 492:495–509 Montana V, Ni Y, Sunjara V, Hua X, Parpura V (2004) Vesicular glutamate transporter-dependent glutamate release from astrocytes. J Neurosci 24:2633–2642 Ohtake PJ, Torres JE, Gozal YM, Graff GR, Gozal D (1998) NMDA receptors mediate peripheral chemoreceptor afferent input in the conscious rat. J Appl Physiol 84:853–861
Cell Tissue Res Oomori Y, Nakaya K, Tanaka H, Iuchi H, Ishikawa K, Satoh Y, Ono K (1994) Immunohistochemical and histochemical evidence for the presence of noradrenaline, serotonin and gamma-aminobutyric acid in chief cells of the mouse carotid body. Cell Tissue Res 278:249–254 Özkan ED, Ueda T (1998) Glutamate transport and storage in synaptic vesicles. Jpn J Pharmacol 77:1–10 Prabhakar NR (2000) Oxygen sensing by the carotid body chemoreceptors. J Appl Physiol 88:2287–2295 Prasad M, Fearon IM, Zhang M, Laing M, Vollmer C, Nurse CA (2001) Expression of P2X2 and P2X3 receptor subunits in rat carotid body afferent neurones: role in chemosensory signalling. J Physiol (Lond) 537:667–677
Stornetta RL, Sevigny CP, Guyenet PG (2003) Inspiratory augmenting bulbospinal neurons express both glutamatergic and enkephalinergic phenotypes. J Comp Neurol 455:113–124 Takamori S, Rhee JS, Rosenmund C, Jahn R (2000) Identification of a vesicular glutamate transporter that defines a glutamatergic phenotype in neurons. Nature 407:189–194 Torrealba F, Bustos G, Montero VM (1996) Glutamate in the glomus cells of the cat carotid body: immunocytochemistry and in vitro release. Neurochem Int 28:625–631 Wu SX, Koshimizu Y, Feng YP, Okamoto K, Fujiyama F, Hioki H, Li YQ, Kaneko T, Mizuno N (2004) Vesicular glutamate transporter immunoreactivity in the central and peripheral endings of musclespindle afferents. Brain Res 1011:247–251
SHORT COMMUNICATION
Vesicular glutamate transporter 2-immunoreactive afferent nerve terminals in the carotid body of the rat Takuya Yokoyama & Nobuaki Nakamuta & Tatsumi Kusakabe & Yoshio Yamamoto
Received: 6 January 2014 / Accepted: 15 May 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract The carotid body is a peripheral chemoreceptor that detects decreases in arterial pO2 and subsequently activates the carotid sinus nerve. The hypoxia-evoked activity of the carotid sinus nerve has been suggested to be modulated by glutamate. In the present study, we investigate the immunohistochemical localization of vesicular glutamate transporters in the carotid body of the rat. Vesicular glutamate transporter 2 (VGLUT2) labeling was closely associated with glomus cells immunoreactive to tyrosine hydroxylase but was not in the cytoplasm of these cells. The VGLUT2 immunoreactivity was observed within nerve endings that were immunoreactive to P2X3 and densely localized inside P2X3-immunoreactive axon terminals. These results suggest that VGLUT2 is localized in the afferent nerve terminals of the carotid body. Glutamate may be released from afferent nerve terminals to modulate the chemosensory activity of the carotid body. Keywords Carotid body . Afferent nerve . Glutamate . Vesicular glutamate transporter . Immunohistochemistry The carotid body (CB) is a peripheral chemoreceptor that is responsible for monitoring the arterial blood levels of pO2, pCO2 and pH. Hypoxia has been shown to stimulate glomus T. Yokoyama : N. Nakamuta : Y. Yamamoto (*) Laboratory of Veterinary Anatomy and Cell Biology, Faculty of Agriculture, Iwate University, 18-8 Ueda 3-chome, Morioka, Iwate 020-8550, Japan e-mail: [email protected] T. Yokoyama : N. Nakamuta : Y. Yamamoto Department of Basic Veterinary Science, United Graduate School of Veterinary Science, Gifu University, Gifu, Japan T. Kusakabe Laboratory for Anatomy and Physiology, Department of Sport and Medical Science, Kokushikan University, Tama, Japan
cells within the CB to release ATP and triggers chemosensory discharges in the afferent nerve terminals of the carotid sinus nerve via P2X2/P2X3 purinoreceptors (Prasad et al. 2001; Buttigieg and Nurse 2004). Afferent signals from the CB are carried to the nucleus solitary tract, leading to an increase in ventilation (Lahiri et al. 2006). Hypoxic ventilatory responses were previously shown to be reduced by the N-methyl-Daspartate (NMDA) glutamate receptor antagonist MK801 and this effect was abolished by CB denervation in the rat (Ohtake et al. 1998). Thus, the activity of the carotid sinus nerves may be enhanced by glutamate in the CB. Recent immunohistochemical studies have demonstrated that vesicular glutamate transporters (VGLUTs), the vesicle loading protein for glutamate neurotransmission (Özkan and Ueda 1998), are localized in afferent nerve terminals. VGLUTs comprise three isoforms: VGLUT1, VGLUT2 and VGLUT3 (Takamori et al. 2000; Fremeau et al. 2002; Kaneko et al. 2002). Immunohistochemical studies have shown that VGLUT1 and/or VGLUT2 are widely expressed in afferent sensory nerve endings, such as intraganglionic laminar endings in the myenteric ganglia of the esophagus (Ewald et al. 2006), muscle spindle afferent fibers (Wu et al. 2004), periodontal Ruffini endings in the incisor (Honma et al. 2012) and pulmonary neuroepithelial bodies and airway smooth muscle (Brouns et al. 2006). Therefore, VGLUTs are also expected to be distributed in the afferent nerve terminals of the CB. On the other hand, glutamate immunoreactivity has also been demonstrated in the glomus cells of the cat and guinea pig (Kummer 1996; Torrealba et al. 1996). In the present study, we examine the immunohistochemical localization of VGLUTs in the CB of the rat. Male Wistar rats (8–10 weeks old; 180–200 g) were purchased from Japan SLC (Hamamatsu, Japan). All animal experiments in the present study were approved by the Local Animal Ethics Committee of Iwate University (accession number #A201326).
Cell Tissue Res
Animals were anesthetized using pentobarbital (50 mg/kg; intraperitoneal injection) and transcardially perfused with Ringer’s solution (200 ml) followed by 4 % paraformaldehyde in 0.1 M phosphate buffer (pH 7.4, 200 ml). Bifurcations of the common carotid artery containing CB were removed under a dissecting microscope. Tissue samples were then immersed in the same fixative for an additional 3 h at 4 °C. After three washes in phosphate-buffered saline (PBS; pH 7.4) for 10 min, tissue samples were soaked in PBS containing 30 % sucrose and frozen with O.C.T. compound medium (Sakura Finetech, Tokyo, Japan). Frozen tissues were sectioned at 10 μm in thickness by use of a cryostat (CM1900; Leica, Wetzlar, Germany) and were then mounted on glass slides coated with chrome-alum gelatin. Frozen sections were stained by double or triple immunofluorescence using antibodies against VGLUT1, VGLUT2, or VGLUT3, together with antibodies against tyrosine hydroxylase (TH) and/or the P2X3 purinoreceptor. In the present study, TH and P2X3 were used as markers for glomus cells and afferent nerve terminals, respectively (Habeck and Kummer 1993; Prasad et al. 2001). Cryostat sections were rinsed with PBS and incubated with non-immune donkey serum (1:50 dilution) for 30 min at room temperature. After blocking nonspecific reactions, sections were incubated with a mixture of primary antibodies overnight at 4 °C. Sections were then rinsed with PBS and incubated for 120 min at room temperature with a mixture of secondary antibodies. Details of the antibodies used in the present study are summarized in Table 1. After rinsing with PBS, sections were counterstained with DAPI and coverslipped with mounting medium (Fluoromount, Diagnostic Biosystems, Pleasanton, CA, USA). Sections were examined with a confocal laser scanning microscope (C1; Nikon, Tokyo, Japan). Images of Alexa488, Cy-3 and Alexa647 were colored in green, red and white using computer software (NIS-element; Nikon). PBS or non-immune serum was used for immunohistochemical controls instead of primary or secondary antisera. We confirmed the
complete abolishment of specific labeling in negative controls. The characteristics of the antibodies used in the present study are described as follows: the guinea pig antiVGLUT1 (AB5905; Chemicon, Temecula, CA, USA) was raised against synthetic peptide from rat VGLUT1 and recognized the C-terminus region of the protein. Western blot of the rat cerebral cortex showed a single band at 60 kDa (Melone et al. 2005). The guinea pig antiVGLUT2 (AB2251; Chemicon) was raised against GSTtagged recombinant rat VGLUT2. Western blot of cell lysate from rat astrocytic cultures yielded a band at 55 kDa (Montana et al. 2004). The guinea pig anti-VGLUT3 (AB5421; Chemicon) was raised against a synthetic peptide corresponding to the 569–588 amino acid sequence of rat VGLUT3. Western blot of lysate from rat PC12 cells showed a single band at 65 kDa (manufacturer’s report). The guinea pig antibodies against VGLUT1, VGLUT2 and VGLUT3 have been used in immunohistochemical studies of the nervous system (Stornetta et al. 2003; Gabellec et al. 2007; Fattorini et al. 2009). In the present study, VGLUT2 immunoreactivity was observed in the CB (Fig. 1b), while that of VGLUT1 and VGLUT3 was not (Fig. 1a, c). In sections stained with VGLUT2 and TH, VGLUT2 immunoreactivity was distributed within and around clusters of TH-immunoreactive glomus cells (Fig. 1b). VGLUT2 immunoreactivity was not observed in thin fibers with TH immunoreactivity within the CB (Fig. 1b). At higher magnification, VGLUT2 immunoreactivity was closely associated with glomus cells with TH immunoreactivity but was not in the cytoplasm of these cells (Fig. 1d–f). Double immunofluorescence for VGLUT2 and P2X3 demonstrated that VGLUT2 immunoreactivity was observed within nerve endings immunoreactive to P2X3 (Fig. 2a–c) and densely localized inside P2X3-immunoreactive axon terminals on the glomus cells (Fig. 2c). Furthermore, the triple immunolabeling method confirmed that VGLUT2 immunoreactivity was observed in the axoplasm of P2X3-
Table 1 Antibodies used in the present study Primary antibodies VGLUT1 VGLUT2 VGLUT3 TH P2X3 Secondary Antibodies A Chemicon, Temecula, CA, USA; B Neuromics, Edina, MN, USA; C Jackson Immunoresearch, West Grove, PA, USA; D Invitrogen, Carlsbad, CA, USA
Alexa fluor 488 labeled anti-guinea pig IgG Alexa fluor 647 labeled anti-mouse IgG Cy3 labeled anti-mouse IgG Cy3 labeled anti-rabbit IgG
Code
Host
Dilution
Source
AB5905 AB2251 AB5421 MAB318 RA10109
Guinea pig Guinea pig Guinea pig Mouse Rabbit
1:4,000 1:4,000 1:5,000 1:2,000 1:5,000
A A A A B
706-545-148 A31571 715-165-151 711-175-152
Donkey Donkey Donkey Donkey
1:200 1:600 1:100 1:100
C D C C
Cell Tissue Res Fig. 1 Lower magnification view of sections stained with VGLUT1 (a), VGLUT2 (b), or VGLUT3 (c) with TH in the carotid body of the rat. a VGLUT1 immunoreactivity is not observed in either TH-immunoreactive glomus cells or nerve bundles in the periphery of the carotid body (arrow). b VGLUT2 immunoreactivity is observed within and around clustered glomus cells with TH immunoreactivity but is not in these cells or TH-immunoreactive nerve bundles within the carotid body (arrows). c No VGLUT3 immunoreactivity is detected in either TH-immunoreactive glomus cells or nerve bundles (arrow). d–f A higher magnification view shows that VGLUT2 immunoreactivity is found in close association with glomus cells immunoreactive to TH (arrows in d, f) but not in the perinuclear cytoplasm of these cells
immunoreactive nerve terminals closely adjacent to THimmunoreactive glomus cells (Fig. 2d–f). The present results demonstrated that VGLUT2 immunoreactivity was localized within P2X3-immunoreactive afferent nerve terminals in the CB. Previous studies used electron microscopy to show that afferent nerve terminals were connected to glomus cells by reciprocal synapses and clear Fig. 2 a–c Double immunofluorescence image of VGLUT2 and the P2X3 purinoreceptor. VGLUT2 immunoreactivity is densely localized inside P2X3immunoreactive axon terminals (arrows in a-c). d–f Triple immunolabeling for VGLUT2 (Alexa488, green), P2X3 (Cy3, red) and TH (Alexa647, white). VGLUT2 immunoreactivity is observed within the axoplasm of P2X3-immunoreactive nerve terminals (arrow in d–f) closely adjacent to glomus cells immunoreactive to TH
vesicles in the nerve terminals were shown to accumulate near the presynaptic region in the CB of the rat (McDonald and Mitchell 1975). Because VGLUT2 was shown to be expressed on clear vesicles in the axon terminals of the rat brain (Kaneko et al. 2002), VGLUT2 may be localized in the membrane of these vesicles in the reciprocal synapses of afferent nerve terminals in the CB. Glutamate may be the
Cell Tissue Res
efferent transmitter/modulator in the reciprocal synapses of sensory nerve terminals in the CB. Moreover, the NMDA receptor subunits NR1, NR2A and NR2B were shown to be expressed in glomus cells in the rat by RT-PCR, immunoblotting and immunohistochemistry (Liu et al. 2009). The NMDA receptor is a non-specific cation channel that is activated by glutamate and increases Ca2+ influx in the synaptic membrane (Edmonds et al. 1995). Thus, glutamate from afferent nerve terminals may elevate membrane potentials in the glomus cells via NMDA receptors to increase the chemosensitivity of CB during hypoxia. The NMDA receptor NR2A/2B is known to be expressed in the slowly adapting mechanoreceptors of the Merkel– neurite complex in the rat sinus hair follicle and mechanically evoked firing is depressed by the NMDA receptor blockers, ifenprodil and MK801 (Cahusac et al. 2005). In addition, slowly adapting afferent discharge during stretching of the muscle spindle is increased by glutamate (Bewick et al. 2005). These findings suggest that glutamate directly stimulates afferent nerves to involve the prolonged firing of slowly adapting receptors. Likewise, the carotid sinus nerve has been described as a slowly adapting type of sensory nerve based on its electrophysiological characteristics (Prabhakar 2000). Although it remains unknown whether NMDA receptors are expressed in afferent nerves of the CB, glutamate released from nerve terminals may directly stimulate the carotid sinus nerve in addition to glomus cells during hypoxia. The present results showed that VGLUTs immunoreactivities were not observed in glomus cells. Therefore, VGLUTs may not be localized in the membrane of vesicles in glomus cells. In the CB of the mouse, GABA and its biosynthetic enzyme glutamate decarboxylase (GAD) immunoreactivities were shown to be expressed in glomus cells (Oomori et al. 1994). Because GABA is synthesized from glutamate by GAD in central GABAergic neurons (Martin and Tobin 2000), it seems that glutamate in glomus cells serves as the substrate for GABA synthesis. In the present study, VGLUT2 immunoreactivity was localized in the afferent nerve terminals of the CB. Glutamate from afferent nerve terminals may enhance the chemosensory activity of the CB via the excitation of glomus cells and/or their direct effects on afferent nerve terminals. Acknowledgment This study was partly supported by JSPS KAKENHI Grant Number 25350823.
References Bewick GS, Reid B, Richardson C, Banks RW (2005) Autogenic modulation of mechanoreceptor excitability by glutamate release from synaptic-like vesicles: evidence from the rat muscle spindle primary sensory ending. J Physiol 562:381–394
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