Acta Oto-Laryngologica
ISSN: 0001-6489 (Print) 1651-2251 (Online) Journal homepage: http://www.tandfonline.com/loi/ioto20
Coexistence of Substance P and Calcitonin Generelated Peptide-like Immunoreactivities in the Rat Vestibular Endorgans Shin-Ichi Usami, Jiro Hozawa & Jukka Ylikoski To cite this article: Shin-Ichi Usami, Jiro Hozawa & Jukka Ylikoski (1991) Coexistence of Substance P and Calcitonin Gene-related Peptide-like Immunoreactivities in the Rat Vestibular Endorgans, Acta Oto-Laryngologica, 111:sup481, 166-169, DOI: 10.3109/00016489109131372 To link to this article: http://dx.doi.org/10.3109/00016489109131372
Published online: 08 Jul 2009.
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Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ioto20 Download by: [McMaster University]
Date: 16 April 2016, At: 05:32
Acta Otolaryngol (Stockh) 1991; Suppl. 481: 166-169
Coexistence of Substance P and Calcitonin Gene-related Peptidelike Immunoreactivities in the Rat Vestibular Endorgans
'
SHIN-ICHI USAMI,'. JIRO HOZAWA' and JUKKA YLIKOSKI'
Downloaded by [McMaster University] at 05:32 16 April 2016
Fro: the 'Department of Otorhinolaryngology, Hirosaki University School of Medicine, Japan, and -Department of Anatomy and Pathology, University of Helsinki, Finland
The immunocytochemical distribution and coexistence of substance P (SP) and calcitonin gene-related peptide (CGRP) in the rat vestibular endorgans were investigated. SP-like immunoreactivity was found in nervous elements beneath and around hair cells. CGRPlike immunoreactivity was also abundantly distributed beneath and within the sensory epithelia. In the present study, double-staining immunocytochemistry revealed that three different types of immunoreactivities: SP-positive/CGRP-negative, SP-negative/CGRPpositive, and SP/CGRP-positive immunostaining can be distinguished. SP/CGRP-immunoreactive fibers were localized within as well as beneath the sensory epithelia. These fibers often penetrated the epithelia and nearly reached the surface. The present immunocytochemica1 evidence suggests that different types of peripheral nervous systems may exist in the vestibular periphery. Key words: coexistence, substance P , calcitonin gene-related peptide (CGRP), rat vestibular endorgan.
INTRODUCTION Recent immunocytochemical studies suggest that several neuroactive peptides with presumed neurotransmitterheuromodulator functions are distributed in the vestibular endorgans (1-6). As in the other sensory nervous system, it is considered that substance P (SP) and calcitonin gene related peptide (CGRP) play an important role in the vestibular endorgans. Since the peripheral nervous systems seem to be subdivided into different populations of neurons on the basis of existencekoexistence of specific substances, we have further investigated, by using double-staining immunocytochemistry, neuronal subpopulations of SP- and CGRP-immunoreactivity.
MATERIAL AND METHODS Wister rats (150-300 g) were deeply anesthesized with sodium pentobarbital (100 mg/kg) and perfused transcardiallj, with saline (I50ml), followed by 4 % paraformaldehyde in 0.1 M phosphate buffer (150-300 ml). The temporal bone was immediately removed and the vestibular endorgans taken under an operative microscope. The specimens were postfixed in the same fixative for 4h (4"C), then immersed in 0.1 M phosphate buffer containing 10% and 30% sucrose overnight at 4°C. Serial cryostat sections (20 ym thick) were made and put onto albumin coated slides. An immunocytochemical double staining of SP and CGRP by the indirect immunofluorescence method was performed. The sections were first incubated overnight (4°C) in a mixture containing both SP monoclonal antibody raised in a rat (Sera Lab.) and CGRP polyclonal antibody raised in a rabbit (Amersham Co. Ltd),. The sections were then incubated for 2h at room temperature with a mixture of the secondary antisera, biotinylated sheep anti-rat immunoglobulin G (IgG) and tetramethylrhodamine isothiocyanate (TR1TC)-conjugated swine anti-rabbit IgG (Dako). Finally, they were incubated with
Downloaded by [McMaster University] at 05:32 16 April 2016
Coexistence of SP and CGRP
Figs. I , 2. Double-immunofluorescent photomicrographs of the rat macula utriculi. SP/CGRP-positive vancous fibers (arrow heads) are distributed inbeneath the sensory layer. One of these fibers penetrating the sensory epithelia is seen (right upper arrow head). Bars = 20 pm. Figs. 3, 4. Double-immunofluorescent photomicrographs of the rat macula utriculi. SP-positive/ CGRP-negative structures (Fig. 3; arrows) and SP-negative/CGRP-positive dot-like immunostaining (Fig. 4; arrows) are also found inbeneath the sensory epithelia. Bars = 20 pm.
fluorescein isothiocyanate (F1TC)-conjugated streptoavidin (15 min, room temperature). Then the tissues were mounted in a glycerol-PBS (phosphate buffered saline) mixture, and examined by Zeiss fluorescence microscope equipped with the proper filter combinations. First and second antisera were diluted with PBS containing 0.25% Triton X-100and used at the following dilution: SP, 1 : 500; CGRP, 1 :4000, biotinylated sheep anti-rat IgG, 1 : 100; TRITC-conjugated anti-rabbit IgG, 1 : 40, and FITC-conjugated streptoavidin, 1: 100.
167
168 S-i Usami et al.
Downloaded by [McMaster University] at 05:32 16 April 2016
RESULTS In the rat vestibular endorgans, many SP- and CGRP-like immunoreactivies were observed within and beneath the sensory epithelium of all vestibular endorgans studied. By using double-staining immunocytochemistry, three types of immunoreactivies, SP/CGRPpositive, SP-positive/CGRP-negative, and SP-negative/CGRP-positive immunostaining could be distinguished in the same section (Figs. 1 4 ) . SP/CGRP-poSitive fine varicous fibers, was distributed within the sensory epithelia as well as beneath the epithelia. These SP/CGRP-positive fibers often penetrated the epithelia from below, and nearly reached the endolymphatic surface, and appeared among the stroma of the vestibular endorgans (Fig 1 , 2). SP-positive/CGRP-negative immunostaining was usually found around the hair cells (Fig 3). These SP-like immunoreactivities may correspond to the nerve chalice of type I hair cells. Furthermore, as in our previous report in the guinea pig vestibular endogans (4), SP-like immunoreactivity was distributed predominantly in the peripheral area of each endorgan. The third type of immunoreactivity, SP-negative/CGRP-positive immunostaining, usually detected as dot-like immunoreactivity, was also found beneath the sensory epithelium (Fig 4).
DISCUSSION Recently, several neuroactive peptides have been identified in the vestibular endorgans. SP-like immunoreactivity has been reported in the vestibular endorgans and the vestibular ganglion of the rabbit (1, 2) and guinea pig (3, 4). These SP-like immunoreactivties were considered to be present in the afferent system, since they were found in the nerve chalice (which is the afferent nerve terminal of type I hair cells) and the vestibular ganglion cells. CGRP-like immunoreactivity was reported, on the other hand, in the efferent fibers originating in the brain stem (5). Tanaka et al. demonstrated CGRP-immunoreactive fibers of the rat vestibular endorgans that originated bilaterally from the area dorsolateral to the genu of the facial nerve, and made synaptic contacts with the nerve chalice of type I cells (6). In the present study, double-staining immunocytochemistry revealed that three different types of SP and CGRP immunoreactive fibers distributed in the vestibular endorgans. With regard to the origin of SP/CGRP-positive fibers, the possibilities would seem to be: i) a part of afferent fibers contains’both SP and CGRP, ii) a part of efferent fibers contains both SP and CGRP. iii) other origins, for instance trigeminal ganglion, send SP/CGRP-positive fibers similar to the other sensory systems. Although the origin and the functional role of these fibers remain to be established, the present immunocytochemical evidence suggests that the morphologically homogenous nerve elements in the rat vestibular endorgans seems to contain at least three populations of SP and/or CGRP-immunoreactive fibers. We are currently investigating origins and pathways of these chemically distinct nerve fibers.
REFERENCES 1. Ylikoski J, Eranko L, Paivarinta H. Substance P-like immunoreactivity in the rabbit inner ear. J
Laryngol Otol 1984; 98: 759-65. 2. Ylikoski J , Paivarinta H, Eranko L, Mrena 1, Lehtosalo J. Is substance P the neurotransmitter in the vestibular endorgans? Acta Otolaryngol (Stockh) 1984; 97: 523-8. 3. Ylikoski J , Pirvola U, Happola 0, Panula P, Virtanen I. Immunohistochemical demonstration of neuroactive substances in the inner ear of rat and guinea pig. Acta Otolaryngol (Stockh) 1989; 107: 4 17-23. 4. Usami S, Hozawa J , Tazawa M, Jin H, Matsubara A, Fujita S. Localization of substance P-like
Coexistence of SP and CGRP immunoreactivity in the guinea pig vestibular endorgans and the vestibular ganglion. Submitted for publication. 5. Tanaka M. Takeda N , Senba E, Tohyama M , Kubo T, Matsunaga T. Localization of calcitonin gene-related peptide in the vestibular end-organs in the rat: an immunohistochemical study. Brain Res 1988; 447: 175-7. 6. Tanaka M, Takeda N, Senba E, Tohyama M, Kubo T, Matsunaga T. Localization, origin and fine structure of calcitonin gene-related peptide-containing fibers in the vestibular end-organs of the rat. Brain Res 1989; 504: 31-5.
Downloaded by [McMaster University] at 05:32 16 April 2016
Address for correspondence: S. Usami, Department Otorhinolaryngology, Hirosaki University School of Medicine, 5 Zaifu-cho, Horosaki, 036 Japan
169
ISSN: 0001-6489 (Print) 1651-2251 (Online) Journal homepage: http://www.tandfonline.com/loi/ioto20
Coexistence of Substance P and Calcitonin Generelated Peptide-like Immunoreactivities in the Rat Vestibular Endorgans Shin-Ichi Usami, Jiro Hozawa & Jukka Ylikoski To cite this article: Shin-Ichi Usami, Jiro Hozawa & Jukka Ylikoski (1991) Coexistence of Substance P and Calcitonin Gene-related Peptide-like Immunoreactivities in the Rat Vestibular Endorgans, Acta Oto-Laryngologica, 111:sup481, 166-169, DOI: 10.3109/00016489109131372 To link to this article: http://dx.doi.org/10.3109/00016489109131372
Published online: 08 Jul 2009.
Submit your article to this journal
Article views: 1
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ioto20 Download by: [McMaster University]
Date: 16 April 2016, At: 05:32
Acta Otolaryngol (Stockh) 1991; Suppl. 481: 166-169
Coexistence of Substance P and Calcitonin Gene-related Peptidelike Immunoreactivities in the Rat Vestibular Endorgans
'
SHIN-ICHI USAMI,'. JIRO HOZAWA' and JUKKA YLIKOSKI'
Downloaded by [McMaster University] at 05:32 16 April 2016
Fro: the 'Department of Otorhinolaryngology, Hirosaki University School of Medicine, Japan, and -Department of Anatomy and Pathology, University of Helsinki, Finland
The immunocytochemical distribution and coexistence of substance P (SP) and calcitonin gene-related peptide (CGRP) in the rat vestibular endorgans were investigated. SP-like immunoreactivity was found in nervous elements beneath and around hair cells. CGRPlike immunoreactivity was also abundantly distributed beneath and within the sensory epithelia. In the present study, double-staining immunocytochemistry revealed that three different types of immunoreactivities: SP-positive/CGRP-negative, SP-negative/CGRPpositive, and SP/CGRP-positive immunostaining can be distinguished. SP/CGRP-immunoreactive fibers were localized within as well as beneath the sensory epithelia. These fibers often penetrated the epithelia and nearly reached the surface. The present immunocytochemica1 evidence suggests that different types of peripheral nervous systems may exist in the vestibular periphery. Key words: coexistence, substance P , calcitonin gene-related peptide (CGRP), rat vestibular endorgan.
INTRODUCTION Recent immunocytochemical studies suggest that several neuroactive peptides with presumed neurotransmitterheuromodulator functions are distributed in the vestibular endorgans (1-6). As in the other sensory nervous system, it is considered that substance P (SP) and calcitonin gene related peptide (CGRP) play an important role in the vestibular endorgans. Since the peripheral nervous systems seem to be subdivided into different populations of neurons on the basis of existencekoexistence of specific substances, we have further investigated, by using double-staining immunocytochemistry, neuronal subpopulations of SP- and CGRP-immunoreactivity.
MATERIAL AND METHODS Wister rats (150-300 g) were deeply anesthesized with sodium pentobarbital (100 mg/kg) and perfused transcardiallj, with saline (I50ml), followed by 4 % paraformaldehyde in 0.1 M phosphate buffer (150-300 ml). The temporal bone was immediately removed and the vestibular endorgans taken under an operative microscope. The specimens were postfixed in the same fixative for 4h (4"C), then immersed in 0.1 M phosphate buffer containing 10% and 30% sucrose overnight at 4°C. Serial cryostat sections (20 ym thick) were made and put onto albumin coated slides. An immunocytochemical double staining of SP and CGRP by the indirect immunofluorescence method was performed. The sections were first incubated overnight (4°C) in a mixture containing both SP monoclonal antibody raised in a rat (Sera Lab.) and CGRP polyclonal antibody raised in a rabbit (Amersham Co. Ltd),. The sections were then incubated for 2h at room temperature with a mixture of the secondary antisera, biotinylated sheep anti-rat immunoglobulin G (IgG) and tetramethylrhodamine isothiocyanate (TR1TC)-conjugated swine anti-rabbit IgG (Dako). Finally, they were incubated with
Downloaded by [McMaster University] at 05:32 16 April 2016
Coexistence of SP and CGRP
Figs. I , 2. Double-immunofluorescent photomicrographs of the rat macula utriculi. SP/CGRP-positive vancous fibers (arrow heads) are distributed inbeneath the sensory layer. One of these fibers penetrating the sensory epithelia is seen (right upper arrow head). Bars = 20 pm. Figs. 3, 4. Double-immunofluorescent photomicrographs of the rat macula utriculi. SP-positive/ CGRP-negative structures (Fig. 3; arrows) and SP-negative/CGRP-positive dot-like immunostaining (Fig. 4; arrows) are also found inbeneath the sensory epithelia. Bars = 20 pm.
fluorescein isothiocyanate (F1TC)-conjugated streptoavidin (15 min, room temperature). Then the tissues were mounted in a glycerol-PBS (phosphate buffered saline) mixture, and examined by Zeiss fluorescence microscope equipped with the proper filter combinations. First and second antisera were diluted with PBS containing 0.25% Triton X-100and used at the following dilution: SP, 1 : 500; CGRP, 1 :4000, biotinylated sheep anti-rat IgG, 1 : 100; TRITC-conjugated anti-rabbit IgG, 1 : 40, and FITC-conjugated streptoavidin, 1: 100.
167
168 S-i Usami et al.
Downloaded by [McMaster University] at 05:32 16 April 2016
RESULTS In the rat vestibular endorgans, many SP- and CGRP-like immunoreactivies were observed within and beneath the sensory epithelium of all vestibular endorgans studied. By using double-staining immunocytochemistry, three types of immunoreactivies, SP/CGRPpositive, SP-positive/CGRP-negative, and SP-negative/CGRP-positive immunostaining could be distinguished in the same section (Figs. 1 4 ) . SP/CGRP-poSitive fine varicous fibers, was distributed within the sensory epithelia as well as beneath the epithelia. These SP/CGRP-positive fibers often penetrated the epithelia from below, and nearly reached the endolymphatic surface, and appeared among the stroma of the vestibular endorgans (Fig 1 , 2). SP-positive/CGRP-negative immunostaining was usually found around the hair cells (Fig 3). These SP-like immunoreactivities may correspond to the nerve chalice of type I hair cells. Furthermore, as in our previous report in the guinea pig vestibular endogans (4), SP-like immunoreactivity was distributed predominantly in the peripheral area of each endorgan. The third type of immunoreactivity, SP-negative/CGRP-positive immunostaining, usually detected as dot-like immunoreactivity, was also found beneath the sensory epithelium (Fig 4).
DISCUSSION Recently, several neuroactive peptides have been identified in the vestibular endorgans. SP-like immunoreactivity has been reported in the vestibular endorgans and the vestibular ganglion of the rabbit (1, 2) and guinea pig (3, 4). These SP-like immunoreactivties were considered to be present in the afferent system, since they were found in the nerve chalice (which is the afferent nerve terminal of type I hair cells) and the vestibular ganglion cells. CGRP-like immunoreactivity was reported, on the other hand, in the efferent fibers originating in the brain stem (5). Tanaka et al. demonstrated CGRP-immunoreactive fibers of the rat vestibular endorgans that originated bilaterally from the area dorsolateral to the genu of the facial nerve, and made synaptic contacts with the nerve chalice of type I cells (6). In the present study, double-staining immunocytochemistry revealed that three different types of SP and CGRP immunoreactive fibers distributed in the vestibular endorgans. With regard to the origin of SP/CGRP-positive fibers, the possibilities would seem to be: i) a part of afferent fibers contains’both SP and CGRP, ii) a part of efferent fibers contains both SP and CGRP. iii) other origins, for instance trigeminal ganglion, send SP/CGRP-positive fibers similar to the other sensory systems. Although the origin and the functional role of these fibers remain to be established, the present immunocytochemical evidence suggests that the morphologically homogenous nerve elements in the rat vestibular endorgans seems to contain at least three populations of SP and/or CGRP-immunoreactive fibers. We are currently investigating origins and pathways of these chemically distinct nerve fibers.
REFERENCES 1. Ylikoski J, Eranko L, Paivarinta H. Substance P-like immunoreactivity in the rabbit inner ear. J
Laryngol Otol 1984; 98: 759-65. 2. Ylikoski J , Paivarinta H, Eranko L, Mrena 1, Lehtosalo J. Is substance P the neurotransmitter in the vestibular endorgans? Acta Otolaryngol (Stockh) 1984; 97: 523-8. 3. Ylikoski J , Pirvola U, Happola 0, Panula P, Virtanen I. Immunohistochemical demonstration of neuroactive substances in the inner ear of rat and guinea pig. Acta Otolaryngol (Stockh) 1989; 107: 4 17-23. 4. Usami S, Hozawa J , Tazawa M, Jin H, Matsubara A, Fujita S. Localization of substance P-like
Coexistence of SP and CGRP immunoreactivity in the guinea pig vestibular endorgans and the vestibular ganglion. Submitted for publication. 5. Tanaka M. Takeda N , Senba E, Tohyama M , Kubo T, Matsunaga T. Localization of calcitonin gene-related peptide in the vestibular end-organs in the rat: an immunohistochemical study. Brain Res 1988; 447: 175-7. 6. Tanaka M, Takeda N, Senba E, Tohyama M, Kubo T, Matsunaga T. Localization, origin and fine structure of calcitonin gene-related peptide-containing fibers in the vestibular end-organs of the rat. Brain Res 1989; 504: 31-5.
Downloaded by [McMaster University] at 05:32 16 April 2016
Address for correspondence: S. Usami, Department Otorhinolaryngology, Hirosaki University School of Medicine, 5 Zaifu-cho, Horosaki, 036 Japan
169
