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Localization of substance P-like immunoreactivity in guinea pig vestibular endorgans and the vestibular ganglion
Brain Research, 555 (1991) 153-158 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 000689939124754S
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BRES 24754
Localization of substance P-like immunoreactivity in guinea pig vestibular endorgans and the vestibular ganglion Shin-ichi Usami, Jiro Hozawa, Masayuki Tazawa, Hitoshi Jin, Atsushi Matsubara and Shigetoshi Fujita Department of Otorhinolaryngology, Hirosaki University School of Medicine, Hirosaki (Japan) (Accepted 16 April 1991) Key words: Substance P; Guinea pig; Vestibular endorgan; Vestibular ganglion
The immunocytochemicaldistribution of substance P (SP) in guinea pig vestibular endorgans and the vestibular ganglion was investigated. Two kinds of SP-immunoreactive fibers were distinguished. Most were thick, and found around or beneath sensory hair cells. These SP-immunoreactive fibers were distributed predominantly on the slope of the crista and the peripheral region of the macula. By electron microscopy, we confirmed this type of SP-like immunoreactivity to be restricted within primary afferent neurons. Some vestibular ganglion cells also showed SP-like immunoreactivity, suggesting that SP is present in some primary afferent neurons, and is involved in afferent neurotransmission. The characteristic distribution of SP may indicate functional differences within each endorgan. The other group of immunoreactive nerve fibers, varicousthin fibers, could be found in the stroma of vestibular endorgans, nerve trunk, vestibular ganglion, and along blood vessels of the vestibular ganglion. These fibers may have a different origin, and have an influence on blood flow and certain other functions. Substance P, present within primary afferent neurons 5" 12,13 of the spinal cord and trigeminal system and their central and peripheral branches, may possibly be involved in afferent neurotransmission 22-24. There is little information on SP in the inner ear except a few works that report the presence of SP-immunoreactive fibers in the basal zones of the sensory epithelium and SPimmunoreactive ceils in the vestibular ganglia in the rabbit 3°'31. However, in these studies, since there was no detailed description of SP-like immunoreactivities in the endorgans, it was very difficult to determine the function of SP in the vestibular endorgans. Therefore, in the present study, an attempt was made to determine the localization of SP-immunoreactive fibers and special attention is directed to the sensory hair cells of the guinea pig vestibular endorgans. Normal guinea pigs (200-400 g) were used. 0.2-0.5 ml of colchicine (5 mg/ml of saline) was injected into the middle ear of some animals 48 h before being killed. The animals were anesthetized with sodium pentobarbital (Nembutal, 100 mg/kg, i.p.) and perfused through the heart with phosphate-buffered saline (PBS), followed by 4% paraformaldehyde + 0.1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2). The vestibular endorgans and vestibular ganglion were dissected out from the temporal bone immediately after perfusion, and postfixed at 4 °C for 3 h. They were then placed in 10% sucrose-phosphate
buffer at 4 °C for 3 h, and transferred to 30% sucrosephosphate buffer overnight. Serial cryostat sections (15 gm thick) were cut and placed on albumin-coated slides. Some endorgans were incubated as whole-mount specimens. The ABC method was used for the immunocytochemistry. Sections or whole-mount specimens were incubated in the following solutions: 2% normal goat serum (Cappel) in 0.3% Triton X-100 PBS for 30 min; antisera against substance P in 0.3% Triton X-100 PBS for 18-24 h (whole-mount: 5 days) at 4 °C; biotinylated goat antirabbit IgG (Vector Labs., Inc.) for 30 min; Vectastain reagent (Vector Labs., Inc.) for 30 min; D A B / H 2 0 2 for 10 min. After the peroxidase reaction, the sections were examined by light microscope. For electron microscopy, the animals were perfused with 4% paraformaldehyde + 1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2). The vestibular endorgans and vestibular ganglion were postfixed with 4% paraformaldehyde at 4 °C for 3 h, washed in PBS at 4 °C overnight, and immersed in 30% sucrose phosphate buffer at 4 °C overnight. They were then frozen in liquid nitrogen and soon thawed in 30% sucrose phosphate buffer. Incubation was carried out as above except that Triton X-100 was not used. The immunocytochemically labeled specimens were postflxed for 1 h in 1% osmium tetroxide in 0.1 M phosphate buffer at 4 °C and embedded in Epon. Ultrathin sections (80-100 nm) were
Correspondence: S. Usami, Department of Otorhinolaryngology,Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki 036, Japan.
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Fig. 1. Substance P-like immunoreactivity in guinea pig vestibular endorgans and the vestibular ganglion (cryostat sections). A: immunoreactivity (arrowheads) is predominantly present on the slope of the crista ampullaris. Bar = 50/~m. B: control section in which the primary antibody was pre-absorbed with substance P. Observe the absence of immunostaining. Bar = 50 ~m. C: higher magnification of the macula utriculi. SP-like immunoreactivity present around hair cells. Immunoreactive puncta (arrowheads) and the neural complex can be seen at the base of the hair cells. Continuity of immunoreactivity is found between the pericellular region and primary afferent neurons (arrows). Bar = 10/~m. D: substance-P like immunoreactivity in the guinea pig vestibular ganglion. Most ganglion cells show more or less SP-like immunoreactivity. Strong immunoreaetivity is preferentially located on small cell bodies. Note that large cells (asterisk) show no reactivity. Bar = 50/~m. E,F: a fine varicous fiber (arrowheads) in nerve trunk (E), and along a blood vessel (F, asterisk). Note the difference in size between the two kinds of immunoreactive fibers. Bars = 10 #m.
155 stained with lead citrate and examined by a J E O L 100CX electron microscope. Primary antisera against substance P were raised in rabbits and used at dilutions of 1/4000 (cryostat section)1/10000 (whole mount). The specificity of the antiserum had previously been verified 14'18. Control experiments were also carried out using primary antiserum preabsorbed with 100/zM synthetic substance P, or normal rabbit serum instead of the primary antisera. SP-like immunoreactivity was found in all the vestibular endorgans. Two kinds of SP-immunoreactive fibers could be distinguished on the basis of size. The first type were thick and found situated around or beneath sensory hair cells (Figs: 1A,C and 2). SP-like immunoreactivity around the hair cells corresponded well with the known morphology of the nerve chalice (afferent nerve endings of type I hair cells). SP-immunoreactive puncta, most probably afferent endings of type II hair cells, were also
A
found beneath hair cells (Figs. 1C and 2C). These SP-immunoreactive fibers formed a complex beneath sensory epithelia, and could be traced toward the vestibular ganglion as several immunoreactive fibers (Fig. 1C). Immunoreactivity was found predominantly in the peripheral area of each endorgan, in sharp contrast to the summit of cristae and the striola of maculae, where there was only minimal immunoreactivity (Figs. 1A and 2A,B). In whole-mount specimens, this distribution pattern was very clear (Fig. 2A,B). In both cristae and maculae, substance P appeared as ring-shaped or dot-like immunoreactivity, the former corresponding to the nerve chalice of type I cells, and the latter, to the nerve endings of type II cells (Fig. 2C). Immunoreactive nerve fibers were also located in the peripheral parts of endorgans. By electron microscopy, this SP-like immunoreactivity was clearly restricted within primary afferent neurons. In the peripheral area of each endorgan, the nerve chalice
I
Fig. 2. Substance P-like immunoreactivity in guinea pig vestibular endorgans (whole-mount specimens). A,B: immunoreactive fibers are predominantly distributed in the peripheral region of the crista ampnllaris (A) and macula sacculi (B), whereas there is only weak immunoreactivity in the central part of each endorgan. Bars = 100/~m. C: both ring-(arrows) and dot-(arrowheads)-like immunoreactivity is made clear by higher magnification. Bar = 20/~m. D: immunoreactive thick fibers (arrowheads) run throughout the stroma. Bar = 20/~m.
156
Fig. 3. Uitrastructure of SP-like immunoreactivity in guinea pig vestibular endorgans. SP-like immunoreactivity is restricted within the nerve chalice (N). No immunoreactivity was found in the hair cell (HC) or the supporting cell (S). Bar = 2 pm.
surrounding type I cells was filled with substance P-like immunoreactivity (Fig. 3). The other group of immunoreactive varicous thin nerve fibers were only minimally present in the stroma of vestibular endorgans, nerve trunk, vestibular ganglion, and along blood vessels around the vestibular ganglion (Fig. 1E,F). Although several cells were intensively immunostained with SP, most vestibular ganglion cells showed SP-like immunoreactivity to some degree (Fig. 1D). SP-immunoreactive cells were relatively small in size. SP-like immunoreactivity can be easily detected in colchicinetreated animals, but is relatively weak in untreated animals. No immunoreactivity was found when the antiserum was preabsorbed with synthetic substance P, or when normal rabbit serum was used as the primary antiserum (Fig. 1B). The present results demonstrate that substance P-like immunoreactivity is present in guinea pig vestibular endorgans, the vestibular ganglion and rabbit 3°'31. Immunoreactivity around some hair cells in cryostat sections and ring-shaped immunoreactivity in whole-mount specimens indicated that substance P is most likely to be present in the nerve chalice (the afferent nerve ending contacting type I hair cells). Dot-like immunoreactivity may correspond to bouton type nerve endings apposed to type II hair cells. Nerve fibers at the base of the sensory cell layer were also immunoreacted, and there was
continuity of immunoreactivity from the sensory cell layer. Vestibular ganglion cells also showed SP-like immunoreactivity, indicating that substance P is present within primary afferent nerves. This is confirmed by the presence of substance P-like immunoreactive terminals in the vestibular nucleus x9, into which primary afferent neurons send their terminals. By electron microscopy, we confirmed immunoreactivity to be restricted within primary afferent nerve endings of types I and II hair cells. SP, located in the primary sensory afferent, is generally considered to act as a neurotransmitter or neuromodulator 22-24. Since vestibular ganglion cells have certain common features with respect to the distribution of SP, SP may also possibly be involved in neurotransmission in the vestibular afferent system. The favorable distribution of SP in the peripheral region of endorgans indicates possible functional differences between the central and peripheral regions of the vestibular endorgans. Indeed, electrophysiological differences in response have been reported 1'8'2°'28. In the guitar-fish semicircular canal, the location of primary afferent nerve fibers in crista has been shown to be closely correlated to physiological characteristics2°. Yagi et al. 2s classified primary afferent neurons into 3 groups based on spontaneous discharge patterns: regular, intermediate and irregular. They speculate that thick fibers innervating the central region of the crista may exhibit irregular discharge and high sensitivity to angular acceleration, and
157 that thin fibers innervating the p e r i p h e r a l region of the crista exhibit regular discharge and low sensitivity to angular acceleration. G o l d b e r g and F e r n a n d e z s also found two different afferent units, regular and irregular. The f o r m e r m a y c o r r e s p o n d to the response of thick fibers located in the central region of the crista, and the latter, to the response of thin fibers in the peripheral region. Based on the present morphological results and electrophysiological data, a n e u r o t r a n s m i t t e r / n e u r o m o d u l a t o r such as substance P m a y possibly be the cause of differences in the physiological characteristics of primary afferent neurons. Recently, the release of SP not only by central terminals but also by p e r i p h e r a l terminals of primary neurons has b e e n r e p o r t e d 2'3A5'21. That 80% of peptides
terminals. By electron microscopy, microvesicles, morphologically similar to typical presynaptic vesicles, were found in the nerve chalice of type I hair cells27. Synapsin I and synaptophysin, the m a j o r c o m p o n e n t s of small synaptic vesicle m e m b r a n e , were also i m m u n o c y t o c h e m ically d e m o n s t r a t e d in the nerve chalice 7'27, supporting the view that a n e u r o t r a n s m i t t e r - l i k e substance is released from the nerve chalice. A recent computerassisted reconstruction study has shown the presence of a neural n e t w o r k in vestibular endorgans, suggesting that the nerve chalice and its collaterals possibly contribute to the complex, a local c o m m u n i c a t i o n n e t w o r k 25"26. In this study, o t h e r m i n o r fibers with SP-like immunoreactivity, fine varicous fibers, were also found in the stroma of vestibular endorgans, nerve trunk, vestibular ganglion and along b l o o d vessels of the vestibular ganglion. Since these vessels are innervated by various n e u r o p e p t i d e s 4'6A6'29, fibers m a y be related to t h e m and
p r o d u c e d in cell bodies are t r a n s p o r t e d to the p e r i p h e r y 11 suggests that SP m a y have o t h e r functions. Actually, t h e r e is now evidence that SP m a y have several peripheral functions including vasodilation and plasma extravasation 9'1°. In the airway system, local or axon reflex m a y occur when sensory afferent terminals are stim u l a t e d 17. A l t h o u g h we do not know whether such a local reflex exists in the vestibular periphery, there is the question as to why SP accumulates so much in afferent
We are grateful to Dr. M. Tohyama for kindly providing the SP antibody. We thank Dr. M. Tohyama and Dr. M. Igarashi for their helpful comments. The technical assistance of K. Yanai and T. Suzuki is gratefully acknowledged.
1 Baird, R.A., Desmadryl, G., Fernandez, C. and Goldberg, J.M., The vestibular nerve of the chinchilla. II. Relation between afferent response properties and peripheral innervation patterns in the semicircular canals, J. Neurophysiol., 60 (1988) 182-203. 2 Bill, A., Stjernschantz, J., Mandahl, A., Brodin, E. and Nilsson, G., Substance P: release on trigeminal nerve stimulation, effects in the eye, Acta Physiol. Scand., 106 (1979) 371-373. 3 Brodin, E., Gazelius, B., Olgart, L. and Nilsson, G., Tissue concentration and release of substance P-like immunoreactivity in the dental pulp, Acta Physiol. Scand., 111 (1981) 141-149. 4 Chan-Palay, V., Innervation of cerebral blood vessels by norepinephrine, indoleamine, substance P and neurotensin fibers and the ieptomeningeal indoleamine axons: their roles in vasomotor activity and alterations of brain blood compositions. In C. Owman and L. Edvinsson (Eds.), Neurogenic Control of Brain Circulation, Pergamon Press, Oxford, 1977, pp. 39-57. 5 Cuello, A.C., Del Fiacco, M. and Paxinos, G., The central and peripheral ends of the substance P-containing sensory neurons in the rat trigeminal system, Brain Research, 152 (1978) 499-509. 6 Edvinsson, L. and Uddman, R., Immunohistochemical localization and dilatory effect of substance P on human cerebral vessels, Brain Research, 232 (1982) 466-471. 7 Favre, D., Scarfone, E., Di Gioia, G., De Camilli, P. and Demdmes, D., Presence of synapsin I in afferent and efferent nerve endings of vestibular sensory epithelia, Brain Research, 384 (1986) 379-382. 8 Goldberg, J.M., Minor, L.B. and Fern~indez, C., The functional organization of the vestibular labyrinth and of some of its central pathways. In J.C. Hwang, N.G. Daunton and V.J. Wilson (Eds.), Basic and Applied Aspects of Vestibular Function, Hong Kong University Press, Hong Kong, 1988, pp. 3-12. 9 H~igermark, 0., H6kfelt, T. and Pernow, B., Flare and itch induced by substance P in human skin, J. Invest. Dermatol., 71 (1978) 233-235. 10 Hallberg, D. and Pernow, B., Effect of substance P on various vascular beds in the dog, Acta Physiol. Scand., 93 (1975)
277-285. 11 Harmar, A. and Keen, P., Synthesis, and central and peripheral axonal transport of substance P in a dorsal root ganglion-nerve preparation in vitro, Brain Research, 231 (1982) 379-385. 12 H6kfelt, T., Kellerth, J.-O., Nilsson, G. and Pernow, B., Experimental immunohistochemical studies on the localization and distribution of substance P in the cat primary sensory neurons, Brain Research, 100 (1975) 235-252. 13 H6kfelt, T., Elde, R., Johansson, O., Luft, R., Nilsson, G. and Arimura, A., Immunohistochemical evidence for separate populations of somatostatin-containing and substance P-containing primary afferent neurons in the rat, Neuroscience, 1 (1976) 131-136. 14 Inagaki, S., Sakanaka, M., Shiosaka, S., Senba, E., Takatsuki, K., Takagi, H., Kawai Y., Minagawa, H. and Tohyama, M., Ontogeny of substance P-containing neuron system of the rat: immunohistochemical analysis. 1. Forebrain and upper brainstem, Neuroscience, 7 (1982) 251-277. 15 Jonsson, C.-E., Brodin, E., Daisgaard, C.-J. and Haegerstrand, A., Release of substance-P-like immunoreactivity in dog paw lymph after scalding injury, Acta Physiol. Scand., 126 (1986) 21-24. 16 Liu-Chen, L.-Y., Mayberg, M.R. and Moskowitz, M., Immunohistochemical evidence for a substance P-containing trigeminovascular pathway to pial arteries in cats, Brain Research, 268 (1983) 162-166. 17 Lundblad, L., Lundberg, J.M., Brodin, E. and ,~ngg~trd, A., Origin and distribution of capsaicin-sensitive substance Pimmunoreactive nerves in the nasal mucosa, Acta Otolaryngol., 96 (1983) 485-493. 18 Nishimoto, T., Akai, M., Inagaki, S., Shiosaka, S., Shimizu, Y., Yamamoto, K., Senba, E., Sakanaka, M., Takatsuki, K., Hara, Y., Takagi, H., Kawai, Y. and Tohyama, M., On the distribution and origins of the substance P in the papillae of the rat tongue: an experimental and immunohistochemical study, J. Comp. Neurol., 207 (1982) 85-92. 19 Nomura, I., Senba, E., Kubo, T., Shiraishi, T., Matsunaga, T.,
influence b l o o d flow and/or vascular permeability.
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Tohyama, M., Shiotani, Y. and Wu, J.-Y., Neuropeptides and y-aminobutyric acid in the vestibular nuclei of the rat: an immunohistochemicai analysis. I. Distribution, Brain Research, 311 (1984) 109-118. O'Leary, D.P., Dunn, R.E and Honrubia, V., Analysis of afferent responses from isolated semicircular canal of the guitar-fish using rotational acceleration white-noise inputs. I. Correlation of response dynamics with receptor innervation, J. Neurophysiol., 39 (1976) 631-644. Olgerth, L., Gazerius, B., Brodin, E. and Nilsson, G., Release of substance P-like immunoreactivity from the dental pulp, Acta Physiol. Scand., 101 (1977) 510-512. Otsuka, M. and Konishi, S., Release of substance P-like immunoreactivity from isolated spinal cord of newborn rat, Nature, 264 (1976) 83-84. Otsuka, M. and Takahashi, T., Putative peptide neurotransmitters, Annu. Rev. Pharmacol., 17 (1977) 425-439. Pernow, B., Substance E, Pharmacol. Rev., 35 (1983) 85-141. Ross, M.D., Cutler, L., Meyer, G., Lam, T. and Vaziri, P., 3-D components of a biological neural network visualized in computer generated imagery. I. Macular receptive field organization, Acta Otolaryngol. (Stockh.), 109 (1990) 83-90. Ross, M.D., Meyer, G., Lain, T., Cutler, L. and Vaziri, P., 3-D
27
28
29
30 31
components of a biological neural network visualized in computer generated imagery. If. Macular neural network organization, Acta Otolaryngol. (Stockh.), 109 (1990) 235-244. Scarfone, E., Dem6mes, D., Jahn, R., De Camilli, P. and Sans, A., Secretory function of the vestibular nerve calyx suggested by presence of vesicles, synapsin I, and synaptophysin, J. Neurosci., 8 (1988) 4640-4645. Yagi, T., Shimpson, N.E. and Markham, C.H., The relationship of conduction velocity to other physiological properties of the cat's horizontal canal neurons, Exp. Brain Res., 30 (1977) 587-600. Yamamoto, K., Matsuyama, T., Shiosaka, S., Inagaki, S., Senba, E., Shimizu, Y., Ishimoto, I., Hayakawa, T., Matsumoto, M. and Tohyama, M., Overall distribution of substance P-containing nerves in the wall of the cerebral arteries of the guinea pig and its origins, J. Comp. Neurol., 215 (1983) 421-426. Ylikoski, J., P/iiv~irinta, H., Er/ink6, L., Merena, I. and Lehtosalo, J., Is substance P the neurotransmitter in the vestibular endorgans?, Acta OtolaryngoL, 97 (1984) 523-528. Ylikoski, J., Er/ink6, L. and P~iiv/irinta, H., Substance P-like immunoreactivity in the rabbit inner ear, J. Largyngol. Otol., 98 (1984) 759-765.
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Localization of substance P-like immunoreactivity in guinea pig vestibular endorgans and the vestibular ganglion Shin-ichi Usami, Jiro Hozawa, Masayuki Tazawa, Hitoshi Jin, Atsushi Matsubara and Shigetoshi Fujita Department of Otorhinolaryngology, Hirosaki University School of Medicine, Hirosaki (Japan) (Accepted 16 April 1991) Key words: Substance P; Guinea pig; Vestibular endorgan; Vestibular ganglion
The immunocytochemicaldistribution of substance P (SP) in guinea pig vestibular endorgans and the vestibular ganglion was investigated. Two kinds of SP-immunoreactive fibers were distinguished. Most were thick, and found around or beneath sensory hair cells. These SP-immunoreactive fibers were distributed predominantly on the slope of the crista and the peripheral region of the macula. By electron microscopy, we confirmed this type of SP-like immunoreactivity to be restricted within primary afferent neurons. Some vestibular ganglion cells also showed SP-like immunoreactivity, suggesting that SP is present in some primary afferent neurons, and is involved in afferent neurotransmission. The characteristic distribution of SP may indicate functional differences within each endorgan. The other group of immunoreactive nerve fibers, varicousthin fibers, could be found in the stroma of vestibular endorgans, nerve trunk, vestibular ganglion, and along blood vessels of the vestibular ganglion. These fibers may have a different origin, and have an influence on blood flow and certain other functions. Substance P, present within primary afferent neurons 5" 12,13 of the spinal cord and trigeminal system and their central and peripheral branches, may possibly be involved in afferent neurotransmission 22-24. There is little information on SP in the inner ear except a few works that report the presence of SP-immunoreactive fibers in the basal zones of the sensory epithelium and SPimmunoreactive ceils in the vestibular ganglia in the rabbit 3°'31. However, in these studies, since there was no detailed description of SP-like immunoreactivities in the endorgans, it was very difficult to determine the function of SP in the vestibular endorgans. Therefore, in the present study, an attempt was made to determine the localization of SP-immunoreactive fibers and special attention is directed to the sensory hair cells of the guinea pig vestibular endorgans. Normal guinea pigs (200-400 g) were used. 0.2-0.5 ml of colchicine (5 mg/ml of saline) was injected into the middle ear of some animals 48 h before being killed. The animals were anesthetized with sodium pentobarbital (Nembutal, 100 mg/kg, i.p.) and perfused through the heart with phosphate-buffered saline (PBS), followed by 4% paraformaldehyde + 0.1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2). The vestibular endorgans and vestibular ganglion were dissected out from the temporal bone immediately after perfusion, and postfixed at 4 °C for 3 h. They were then placed in 10% sucrose-phosphate
buffer at 4 °C for 3 h, and transferred to 30% sucrosephosphate buffer overnight. Serial cryostat sections (15 gm thick) were cut and placed on albumin-coated slides. Some endorgans were incubated as whole-mount specimens. The ABC method was used for the immunocytochemistry. Sections or whole-mount specimens were incubated in the following solutions: 2% normal goat serum (Cappel) in 0.3% Triton X-100 PBS for 30 min; antisera against substance P in 0.3% Triton X-100 PBS for 18-24 h (whole-mount: 5 days) at 4 °C; biotinylated goat antirabbit IgG (Vector Labs., Inc.) for 30 min; Vectastain reagent (Vector Labs., Inc.) for 30 min; D A B / H 2 0 2 for 10 min. After the peroxidase reaction, the sections were examined by light microscope. For electron microscopy, the animals were perfused with 4% paraformaldehyde + 1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2). The vestibular endorgans and vestibular ganglion were postfixed with 4% paraformaldehyde at 4 °C for 3 h, washed in PBS at 4 °C overnight, and immersed in 30% sucrose phosphate buffer at 4 °C overnight. They were then frozen in liquid nitrogen and soon thawed in 30% sucrose phosphate buffer. Incubation was carried out as above except that Triton X-100 was not used. The immunocytochemically labeled specimens were postflxed for 1 h in 1% osmium tetroxide in 0.1 M phosphate buffer at 4 °C and embedded in Epon. Ultrathin sections (80-100 nm) were
Correspondence: S. Usami, Department of Otorhinolaryngology,Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki 036, Japan.
154
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B
....
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Fig. 1. Substance P-like immunoreactivity in guinea pig vestibular endorgans and the vestibular ganglion (cryostat sections). A: immunoreactivity (arrowheads) is predominantly present on the slope of the crista ampullaris. Bar = 50/~m. B: control section in which the primary antibody was pre-absorbed with substance P. Observe the absence of immunostaining. Bar = 50 ~m. C: higher magnification of the macula utriculi. SP-like immunoreactivity present around hair cells. Immunoreactive puncta (arrowheads) and the neural complex can be seen at the base of the hair cells. Continuity of immunoreactivity is found between the pericellular region and primary afferent neurons (arrows). Bar = 10/~m. D: substance-P like immunoreactivity in the guinea pig vestibular ganglion. Most ganglion cells show more or less SP-like immunoreactivity. Strong immunoreaetivity is preferentially located on small cell bodies. Note that large cells (asterisk) show no reactivity. Bar = 50/~m. E,F: a fine varicous fiber (arrowheads) in nerve trunk (E), and along a blood vessel (F, asterisk). Note the difference in size between the two kinds of immunoreactive fibers. Bars = 10 #m.
155 stained with lead citrate and examined by a J E O L 100CX electron microscope. Primary antisera against substance P were raised in rabbits and used at dilutions of 1/4000 (cryostat section)1/10000 (whole mount). The specificity of the antiserum had previously been verified 14'18. Control experiments were also carried out using primary antiserum preabsorbed with 100/zM synthetic substance P, or normal rabbit serum instead of the primary antisera. SP-like immunoreactivity was found in all the vestibular endorgans. Two kinds of SP-immunoreactive fibers could be distinguished on the basis of size. The first type were thick and found situated around or beneath sensory hair cells (Figs: 1A,C and 2). SP-like immunoreactivity around the hair cells corresponded well with the known morphology of the nerve chalice (afferent nerve endings of type I hair cells). SP-immunoreactive puncta, most probably afferent endings of type II hair cells, were also
A
found beneath hair cells (Figs. 1C and 2C). These SP-immunoreactive fibers formed a complex beneath sensory epithelia, and could be traced toward the vestibular ganglion as several immunoreactive fibers (Fig. 1C). Immunoreactivity was found predominantly in the peripheral area of each endorgan, in sharp contrast to the summit of cristae and the striola of maculae, where there was only minimal immunoreactivity (Figs. 1A and 2A,B). In whole-mount specimens, this distribution pattern was very clear (Fig. 2A,B). In both cristae and maculae, substance P appeared as ring-shaped or dot-like immunoreactivity, the former corresponding to the nerve chalice of type I cells, and the latter, to the nerve endings of type II cells (Fig. 2C). Immunoreactive nerve fibers were also located in the peripheral parts of endorgans. By electron microscopy, this SP-like immunoreactivity was clearly restricted within primary afferent neurons. In the peripheral area of each endorgan, the nerve chalice
I
Fig. 2. Substance P-like immunoreactivity in guinea pig vestibular endorgans (whole-mount specimens). A,B: immunoreactive fibers are predominantly distributed in the peripheral region of the crista ampnllaris (A) and macula sacculi (B), whereas there is only weak immunoreactivity in the central part of each endorgan. Bars = 100/~m. C: both ring-(arrows) and dot-(arrowheads)-like immunoreactivity is made clear by higher magnification. Bar = 20/~m. D: immunoreactive thick fibers (arrowheads) run throughout the stroma. Bar = 20/~m.
156
Fig. 3. Uitrastructure of SP-like immunoreactivity in guinea pig vestibular endorgans. SP-like immunoreactivity is restricted within the nerve chalice (N). No immunoreactivity was found in the hair cell (HC) or the supporting cell (S). Bar = 2 pm.
surrounding type I cells was filled with substance P-like immunoreactivity (Fig. 3). The other group of immunoreactive varicous thin nerve fibers were only minimally present in the stroma of vestibular endorgans, nerve trunk, vestibular ganglion, and along blood vessels around the vestibular ganglion (Fig. 1E,F). Although several cells were intensively immunostained with SP, most vestibular ganglion cells showed SP-like immunoreactivity to some degree (Fig. 1D). SP-immunoreactive cells were relatively small in size. SP-like immunoreactivity can be easily detected in colchicinetreated animals, but is relatively weak in untreated animals. No immunoreactivity was found when the antiserum was preabsorbed with synthetic substance P, or when normal rabbit serum was used as the primary antiserum (Fig. 1B). The present results demonstrate that substance P-like immunoreactivity is present in guinea pig vestibular endorgans, the vestibular ganglion and rabbit 3°'31. Immunoreactivity around some hair cells in cryostat sections and ring-shaped immunoreactivity in whole-mount specimens indicated that substance P is most likely to be present in the nerve chalice (the afferent nerve ending contacting type I hair cells). Dot-like immunoreactivity may correspond to bouton type nerve endings apposed to type II hair cells. Nerve fibers at the base of the sensory cell layer were also immunoreacted, and there was
continuity of immunoreactivity from the sensory cell layer. Vestibular ganglion cells also showed SP-like immunoreactivity, indicating that substance P is present within primary afferent nerves. This is confirmed by the presence of substance P-like immunoreactive terminals in the vestibular nucleus x9, into which primary afferent neurons send their terminals. By electron microscopy, we confirmed immunoreactivity to be restricted within primary afferent nerve endings of types I and II hair cells. SP, located in the primary sensory afferent, is generally considered to act as a neurotransmitter or neuromodulator 22-24. Since vestibular ganglion cells have certain common features with respect to the distribution of SP, SP may also possibly be involved in neurotransmission in the vestibular afferent system. The favorable distribution of SP in the peripheral region of endorgans indicates possible functional differences between the central and peripheral regions of the vestibular endorgans. Indeed, electrophysiological differences in response have been reported 1'8'2°'28. In the guitar-fish semicircular canal, the location of primary afferent nerve fibers in crista has been shown to be closely correlated to physiological characteristics2°. Yagi et al. 2s classified primary afferent neurons into 3 groups based on spontaneous discharge patterns: regular, intermediate and irregular. They speculate that thick fibers innervating the central region of the crista may exhibit irregular discharge and high sensitivity to angular acceleration, and
157 that thin fibers innervating the p e r i p h e r a l region of the crista exhibit regular discharge and low sensitivity to angular acceleration. G o l d b e r g and F e r n a n d e z s also found two different afferent units, regular and irregular. The f o r m e r m a y c o r r e s p o n d to the response of thick fibers located in the central region of the crista, and the latter, to the response of thin fibers in the peripheral region. Based on the present morphological results and electrophysiological data, a n e u r o t r a n s m i t t e r / n e u r o m o d u l a t o r such as substance P m a y possibly be the cause of differences in the physiological characteristics of primary afferent neurons. Recently, the release of SP not only by central terminals but also by p e r i p h e r a l terminals of primary neurons has b e e n r e p o r t e d 2'3A5'21. That 80% of peptides
terminals. By electron microscopy, microvesicles, morphologically similar to typical presynaptic vesicles, were found in the nerve chalice of type I hair cells27. Synapsin I and synaptophysin, the m a j o r c o m p o n e n t s of small synaptic vesicle m e m b r a n e , were also i m m u n o c y t o c h e m ically d e m o n s t r a t e d in the nerve chalice 7'27, supporting the view that a n e u r o t r a n s m i t t e r - l i k e substance is released from the nerve chalice. A recent computerassisted reconstruction study has shown the presence of a neural n e t w o r k in vestibular endorgans, suggesting that the nerve chalice and its collaterals possibly contribute to the complex, a local c o m m u n i c a t i o n n e t w o r k 25"26. In this study, o t h e r m i n o r fibers with SP-like immunoreactivity, fine varicous fibers, were also found in the stroma of vestibular endorgans, nerve trunk, vestibular ganglion and along b l o o d vessels of the vestibular ganglion. Since these vessels are innervated by various n e u r o p e p t i d e s 4'6A6'29, fibers m a y be related to t h e m and
p r o d u c e d in cell bodies are t r a n s p o r t e d to the p e r i p h e r y 11 suggests that SP m a y have o t h e r functions. Actually, t h e r e is now evidence that SP m a y have several peripheral functions including vasodilation and plasma extravasation 9'1°. In the airway system, local or axon reflex m a y occur when sensory afferent terminals are stim u l a t e d 17. A l t h o u g h we do not know whether such a local reflex exists in the vestibular periphery, there is the question as to why SP accumulates so much in afferent
We are grateful to Dr. M. Tohyama for kindly providing the SP antibody. We thank Dr. M. Tohyama and Dr. M. Igarashi for their helpful comments. The technical assistance of K. Yanai and T. Suzuki is gratefully acknowledged.
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