- Email: [email protected]
Synaptic bodies and vesicles in the calix type synapse of chicken semicircular canal ampullae
43
Neuroscience Letters, 129 (1991)43-46
© 1991 ElsevierScientificPublishers Ireland Ltd. 0304-3940/91/$03.50 ADONIS 030439409100401X NSL 07909
Synaptic bodies and vesicles in the calix type synapse of chicken semicircular canal ampullae M. Y a m a s h i t a 2 a n d H. O h m o r i 1 1National Institute for Physiological Sciences, Okazaki (Japan) and 2Department of Physiology, Osaka University Medical School, Suita( Japan )
(Received 17 April 1991;Accepted 26 April 1991) Key words: Vestibularcalix synapse; Synapticbody; Semicircularcanal ampullae; Postsynapticpotential; Isolated preparation; Chicken
Calix afferent fibers generate depolarizing DC (direct current) and superimposedAC (alternating current) potentials in response to a vibrating stimulation of the hair bundle in an isolated preparation of a chicken semicircularcanal ampulla. The wave form of the postsyuaptic potential appears similar to the transduction potential of hair cells, which suggests electricaltransmission in the calix type synapse. However,synaptic bodies and vesicleswere found by EM observation instead of gap junctions in the hair cell presynaptic to the calix afferent.The number of synaptic body was 11-12/haircell. These structures support chemicaltransmissionin the calixtype synapse.
Two types of afferent synapse are known in vestibular hair cells [9]. The calix type afferent encloses the type I hair cell and the bouton type afferent makes endings on the type II hair cell. Yamashita and Ohmori [11] have demonstrated differences in the pattern of postsynaptic potentials to mechanical stimulation of the hair bundle between the two types o f afferent synapse in an isolated preparation of a chicken semicircular canal ampulla. The calix type afferent generates depolarizing DC (direct current) and superimposed AC (alternating current) potentials in response to a vibrating stimulation o f the hair bundle, while the bouton type afferent shows transiently depolarizing, apparently quantal postsynaptic potentials to the same stimulation. The hair cell generates depolarizing DC and superimposed AC potentials in response to the vibrating stimulation of the hair bundle, and the feature is basically the same as the postsynaptic potential recorded in the calix afferent. These 3 response patterns are illustrated in Fig. 1. Because of the similarity of the postsynaptic potential recorded in the calix type afferent to the transduction potential of the hair cell, the response o f calix afferents might be mediated with electrical transmission between the type I hair cell and the calix afferent. However, the electrical transmission seems unlikely, because the amplitude o f the postsynaptic potential was increased by Correspondence: M. Yamashita, Department of Physiology, Osaka University Medical School,Yamadaoka 2-2, Suita city, Osaka 565, Japan.
hyperpolarization of the postsynaptic membrane by current injection and because the fluorescence dye Lucifer Yellow did not pass through the synapse [11]. The electrical responses somewhat differ between the hair cell and the calix type afferent: the postsynaptic potential o f calix afferents decays more slowly than the transduction potential of hair cells (Fig. 1). This slow decay can not be explained by a simple charging kinetics of the postsynaptic membrane with a certain time constant alone [11]. There were no observations so far of gap junctions in the calix synapses either of a chick [3, 7] or of a guinea pig
[4]. The structure of the chemically transmitting synapse has been thought to have accumulation of synaptic vesicles around synaptic bodies [5]. These vesicular accumulation and synaptic bodies are found not only in the bouton synapse but also in the calix synapse [1, 5]. However, the numbers o f these structures have been reported to be very low in calix type synapses [2, 4, 8]. We studied the serial section of the type I hair cell electron microscopically to investigate synaptic bodies and vesicles. The type I hair cell was identified in the isolated preparation of the chicken semicircular canal organ by labeling calices with intracellular injection of the fluorescence dye Lucifer Yellow and by photo-oxidation reaction of diaminobenzidine (DAB) for electron microscopic observation. A preliminary report has been published in an abstract form [10]. Details o f the preparation, dye injection and photooxidation have been described previously [11]. After the
44
hair
cell
V
calix
°°l
aff.
40ms
bouton
aff.
Y 2mV
-mechanical
vVvvv stimulation
20ms
Fig. 1. Typical records of transduction potential of a hair cell and postsynaptic potentials of calix and bouton type afferents. The hair cell (the top trace) and the calix afferent (the middle trace with a trace indicating the mechanical stimulation) respond with depolarizing DC and AC potentials to a vibration stimulus (250 Hz) applied water coupled to hair bundles in an isolated preparation of the semicircular canal ampulla. The bouton afferent responds with transiently depolarizing potentials to the same kind of mechanical stimulation (the bottom two traces with a trace of the mechanical stimulation, note the difference of a time scale). The cells recorded were morphologically identified by intracellular injection of Lucifer Yellow. Details of the preparation and recordings were described previously [ 11].
injection o f Lucifer Yellow a n d p h o t o - o x i d a t i o n , the a m p u l l a was fixed in 1.25% g l u t a r a l d e h y d e in 0.1 M p h o s p h a t e buffer ( p H 7.3) for one night at 4°C, a n d was then postfixed in 2% OsO4 in the p h o s p h a t e buffer for 1.5 h. It was rinsed in w a t e r for 20 m i n a n d was stained en bloc with 1.5% u r a n y l acetate for 1 h. A f t e r d e h y d r a t i o n in a series o f e t h a n o l s with g r a d e d c o n c e n t r a t i o n s a n d p r o p y l e n e oxide, the tissue was e m b e d d e d in epon. Serial sections were cut h o r i z o n t a l l y to the labeled calix with a glass o r a d i a m o n d knife. E a c h section was m o u n t e d o n a c o p p e r grid so t h a t the labeled calix was l o c a t e d at
the center o f the grid. T h e sections were stained with uranyl a c e t a t e (2%) a n d lead citrate a n d were o b s e r v e d with a J E O L 100 CX. The labeled calix encloses a type I hair cell (Fig. 2A). S y n a p t i c b o d i e s were f o u n d in the b a s a l p a r t o f the h a i r cell (Fig. 2B). The synaptic b o d y is r o u n d o r o v a l in shape a n d the d i a m e t e r is 100-150 nm. O n e synaptic b o d y was o b s e r v e d e x t e n d i n g into one or two successive sections. The n u m b e r o f s y n a p t i c b o d i e s in a single type I hair cell was c o u n t e d in two identified hair cells with labeled calices. Eleven a n d 12 synaptic b o d i e s were
45
A
. . . . . . . . .
B
0.9
9 Fig. 2. A: a transverse section of a type I hair cell and a calix ending. The calix afferentwas labeledwith intracellularinjectionof LuciferYellow and photo-oxidation.Scale bar: 2 am. B: synapticbody and vesiclesin a type I hair cell. The calix was labeled by LuciferYellow injection and photo-oxidation.Bar = 0.2/tm.
found in the type I hair cells. These numbers were somewhat higher than the number estimated in the chick cristae (4~6 per type I hair cell) [7] and comparable to the observation in the chinchilla's cristae (10-20 per type I hair cell) [6]. We could not find gap junctions in the calix synapse. The difference of postsynaptic responses between calix and bouton afferents may not be due to a quantitative difference of synaptic bodies. The number of synaptic bodies is comparatively larger in the type II hair cell than in the type I hair cell [6]. The type II hair cell, however, makes synaptic contacts not only with bouton afferents but also with the outer surface of calices [6, 7 and our unpublished observation]. Although we don't have any evidence to suggest it yet, the difference could be a qualitative difference of the nature of the release of transmitter substances. Alternatively, the difference of PoStsynaptic responses could also be due to postsynaptic structures. In the bouton type synapse, the postsynaptic membrane density is clearly thicker than the presynaptic membrane density and the area of the postsynaptic
thickening is confined to the region of presynaptic vesicle accumulation around synaptic body, while the calix synapse does not show such asymmetry or the concentration of postsynaptic density of the membrane (Fig. 3). Peusner et al. [7] have pointed out that the postsynaptic membrane density usually extends further laterally than the synaptic body in the calix type synapse. Assuming that the transmission from type I hair cell to calix type afferent is chemically mediated, the transmitter may act diffusely on the surface of the postsynaptic membrane and the transmitter could be accumulated within the synaptic cleft, and the quantal nature might have been modulated by repeated excitation of the postsynaptic receptor channels. The authors thank Professor K. Hama for encouragement. We also thank Ms. M. Yoshitomo for technical assistance, Mr. Oba and Mr. Maehashi for technical advice. This work was supported by Grant 63770099 to M.Y. from the Japanese Ministry of Education, Science and Culture.
46
Calix
Bouton
200
nm
Fig. 3. Synaptic structures of a calix (left) and a bouton (right) synapse in non-labeled preparations.
1 Engstr6m, H. and Engstr6m, B., The structure of the vestibular sensory epithelia. In T. Gualtierotti (Ed.), The Vestibular System: Function and Morphology, Springer, New York, 1981, pp. 3-37. 2 Favre, D. and Sans, A., Morphological changes in afferent vestibular hair cell synapses during the postnatal development of the cat, J. Neurocytol., 8 (1979) 765-775. 3 Ginzberg, R.D., Freeze-fracture morphology of the vestibular hair cell-primary afferent synapse in the chick, J. Neurocytol., 13 (1984) 393-405. 4 Gulley, R.L. and Bagger-Sjfb/ick, D., Freeze-fracture studies on the synapse between the type I hair cell and the calyceal terminal in the guinea-pig vestibular system, J. Neurocytol., 8 (1979) 591603. 5 Hamilton, D.W., The calyceal synapse of type I vestibular hair cells, J. Ultrastructure Res., 23 (1968) 98-114. 6 Lysakowski, A. and Goldberg, J.M., Regional variations in the synaptic organization of the chinchilla cristae, Soc. Neurosci. Abstr., 15 (1989) 502. 7 Peusner, K.D., Lindberg, N.H. and Mansfield, P.F., Ultrastructural study of calycine synaptic endings of colossal vestibular fibers
8
9
10
11
in the cristae ampullares of the developing chick, Int. J. Dev. Neurosci., 6 (1988) 267-283. Smith, C.A. and Rasmussen, G.L., Nerve endings in the maculae and cristae of the chinchilla vestibule, with a special reference to the efferents. In Third Symposium on the Role of the Vestibular Organs in Space Exploration, NASA SP-152, USGPO, Washington, DC, 1967, pp. 183-201. Wersiill, J., Studies on the structure and innervation of the sensory epithelium of the cristae ampullares in the guinea pig: a light and electron microscopic investigation, Acta Oto-Laryngol., Suppl. 126 (1956) 1-85. Yamashita, M., Synaptic bodies and vesicles in vestibular calyceal synapses of the chick: an electron microscope study with intracellular injection of lucifer yellow and photo-oxidation reaction of DAB, Jpn. J. Physiol., 39 (1989) S168. Yamashita, M. and Ohmori, H., Synaptic responses to mechanical stimulation in calyceal and bouton type vestibular afferents studied in an isolated preparation of semicircular canal ampullae of chicken, Exp. Brain Res., 80 (1990) 475~88.
Neuroscience Letters, 129 (1991)43-46
© 1991 ElsevierScientificPublishers Ireland Ltd. 0304-3940/91/$03.50 ADONIS 030439409100401X NSL 07909
Synaptic bodies and vesicles in the calix type synapse of chicken semicircular canal ampullae M. Y a m a s h i t a 2 a n d H. O h m o r i 1 1National Institute for Physiological Sciences, Okazaki (Japan) and 2Department of Physiology, Osaka University Medical School, Suita( Japan )
(Received 17 April 1991;Accepted 26 April 1991) Key words: Vestibularcalix synapse; Synapticbody; Semicircularcanal ampullae; Postsynapticpotential; Isolated preparation; Chicken
Calix afferent fibers generate depolarizing DC (direct current) and superimposedAC (alternating current) potentials in response to a vibrating stimulation of the hair bundle in an isolated preparation of a chicken semicircularcanal ampulla. The wave form of the postsyuaptic potential appears similar to the transduction potential of hair cells, which suggests electricaltransmission in the calix type synapse. However,synaptic bodies and vesicleswere found by EM observation instead of gap junctions in the hair cell presynaptic to the calix afferent.The number of synaptic body was 11-12/haircell. These structures support chemicaltransmissionin the calixtype synapse.
Two types of afferent synapse are known in vestibular hair cells [9]. The calix type afferent encloses the type I hair cell and the bouton type afferent makes endings on the type II hair cell. Yamashita and Ohmori [11] have demonstrated differences in the pattern of postsynaptic potentials to mechanical stimulation of the hair bundle between the two types o f afferent synapse in an isolated preparation of a chicken semicircular canal ampulla. The calix type afferent generates depolarizing DC (direct current) and superimposed AC (alternating current) potentials in response to a vibrating stimulation o f the hair bundle, while the bouton type afferent shows transiently depolarizing, apparently quantal postsynaptic potentials to the same stimulation. The hair cell generates depolarizing DC and superimposed AC potentials in response to the vibrating stimulation of the hair bundle, and the feature is basically the same as the postsynaptic potential recorded in the calix afferent. These 3 response patterns are illustrated in Fig. 1. Because of the similarity of the postsynaptic potential recorded in the calix type afferent to the transduction potential of the hair cell, the response o f calix afferents might be mediated with electrical transmission between the type I hair cell and the calix afferent. However, the electrical transmission seems unlikely, because the amplitude o f the postsynaptic potential was increased by Correspondence: M. Yamashita, Department of Physiology, Osaka University Medical School,Yamadaoka 2-2, Suita city, Osaka 565, Japan.
hyperpolarization of the postsynaptic membrane by current injection and because the fluorescence dye Lucifer Yellow did not pass through the synapse [11]. The electrical responses somewhat differ between the hair cell and the calix type afferent: the postsynaptic potential o f calix afferents decays more slowly than the transduction potential of hair cells (Fig. 1). This slow decay can not be explained by a simple charging kinetics of the postsynaptic membrane with a certain time constant alone [11]. There were no observations so far of gap junctions in the calix synapses either of a chick [3, 7] or of a guinea pig
[4]. The structure of the chemically transmitting synapse has been thought to have accumulation of synaptic vesicles around synaptic bodies [5]. These vesicular accumulation and synaptic bodies are found not only in the bouton synapse but also in the calix synapse [1, 5]. However, the numbers o f these structures have been reported to be very low in calix type synapses [2, 4, 8]. We studied the serial section of the type I hair cell electron microscopically to investigate synaptic bodies and vesicles. The type I hair cell was identified in the isolated preparation of the chicken semicircular canal organ by labeling calices with intracellular injection of the fluorescence dye Lucifer Yellow and by photo-oxidation reaction of diaminobenzidine (DAB) for electron microscopic observation. A preliminary report has been published in an abstract form [10]. Details o f the preparation, dye injection and photooxidation have been described previously [11]. After the
44
hair
cell
V
calix
°°l
aff.
40ms
bouton
aff.
Y 2mV
-mechanical
vVvvv stimulation
20ms
Fig. 1. Typical records of transduction potential of a hair cell and postsynaptic potentials of calix and bouton type afferents. The hair cell (the top trace) and the calix afferent (the middle trace with a trace indicating the mechanical stimulation) respond with depolarizing DC and AC potentials to a vibration stimulus (250 Hz) applied water coupled to hair bundles in an isolated preparation of the semicircular canal ampulla. The bouton afferent responds with transiently depolarizing potentials to the same kind of mechanical stimulation (the bottom two traces with a trace of the mechanical stimulation, note the difference of a time scale). The cells recorded were morphologically identified by intracellular injection of Lucifer Yellow. Details of the preparation and recordings were described previously [ 11].
injection o f Lucifer Yellow a n d p h o t o - o x i d a t i o n , the a m p u l l a was fixed in 1.25% g l u t a r a l d e h y d e in 0.1 M p h o s p h a t e buffer ( p H 7.3) for one night at 4°C, a n d was then postfixed in 2% OsO4 in the p h o s p h a t e buffer for 1.5 h. It was rinsed in w a t e r for 20 m i n a n d was stained en bloc with 1.5% u r a n y l acetate for 1 h. A f t e r d e h y d r a t i o n in a series o f e t h a n o l s with g r a d e d c o n c e n t r a t i o n s a n d p r o p y l e n e oxide, the tissue was e m b e d d e d in epon. Serial sections were cut h o r i z o n t a l l y to the labeled calix with a glass o r a d i a m o n d knife. E a c h section was m o u n t e d o n a c o p p e r grid so t h a t the labeled calix was l o c a t e d at
the center o f the grid. T h e sections were stained with uranyl a c e t a t e (2%) a n d lead citrate a n d were o b s e r v e d with a J E O L 100 CX. The labeled calix encloses a type I hair cell (Fig. 2A). S y n a p t i c b o d i e s were f o u n d in the b a s a l p a r t o f the h a i r cell (Fig. 2B). The synaptic b o d y is r o u n d o r o v a l in shape a n d the d i a m e t e r is 100-150 nm. O n e synaptic b o d y was o b s e r v e d e x t e n d i n g into one or two successive sections. The n u m b e r o f s y n a p t i c b o d i e s in a single type I hair cell was c o u n t e d in two identified hair cells with labeled calices. Eleven a n d 12 synaptic b o d i e s were
45
A
. . . . . . . . .
B
0.9
9 Fig. 2. A: a transverse section of a type I hair cell and a calix ending. The calix afferentwas labeledwith intracellularinjectionof LuciferYellow and photo-oxidation.Scale bar: 2 am. B: synapticbody and vesiclesin a type I hair cell. The calix was labeled by LuciferYellow injection and photo-oxidation.Bar = 0.2/tm.
found in the type I hair cells. These numbers were somewhat higher than the number estimated in the chick cristae (4~6 per type I hair cell) [7] and comparable to the observation in the chinchilla's cristae (10-20 per type I hair cell) [6]. We could not find gap junctions in the calix synapse. The difference of postsynaptic responses between calix and bouton afferents may not be due to a quantitative difference of synaptic bodies. The number of synaptic bodies is comparatively larger in the type II hair cell than in the type I hair cell [6]. The type II hair cell, however, makes synaptic contacts not only with bouton afferents but also with the outer surface of calices [6, 7 and our unpublished observation]. Although we don't have any evidence to suggest it yet, the difference could be a qualitative difference of the nature of the release of transmitter substances. Alternatively, the difference of PoStsynaptic responses could also be due to postsynaptic structures. In the bouton type synapse, the postsynaptic membrane density is clearly thicker than the presynaptic membrane density and the area of the postsynaptic
thickening is confined to the region of presynaptic vesicle accumulation around synaptic body, while the calix synapse does not show such asymmetry or the concentration of postsynaptic density of the membrane (Fig. 3). Peusner et al. [7] have pointed out that the postsynaptic membrane density usually extends further laterally than the synaptic body in the calix type synapse. Assuming that the transmission from type I hair cell to calix type afferent is chemically mediated, the transmitter may act diffusely on the surface of the postsynaptic membrane and the transmitter could be accumulated within the synaptic cleft, and the quantal nature might have been modulated by repeated excitation of the postsynaptic receptor channels. The authors thank Professor K. Hama for encouragement. We also thank Ms. M. Yoshitomo for technical assistance, Mr. Oba and Mr. Maehashi for technical advice. This work was supported by Grant 63770099 to M.Y. from the Japanese Ministry of Education, Science and Culture.
46
Calix
Bouton
200
nm
Fig. 3. Synaptic structures of a calix (left) and a bouton (right) synapse in non-labeled preparations.
1 Engstr6m, H. and Engstr6m, B., The structure of the vestibular sensory epithelia. In T. Gualtierotti (Ed.), The Vestibular System: Function and Morphology, Springer, New York, 1981, pp. 3-37. 2 Favre, D. and Sans, A., Morphological changes in afferent vestibular hair cell synapses during the postnatal development of the cat, J. Neurocytol., 8 (1979) 765-775. 3 Ginzberg, R.D., Freeze-fracture morphology of the vestibular hair cell-primary afferent synapse in the chick, J. Neurocytol., 13 (1984) 393-405. 4 Gulley, R.L. and Bagger-Sjfb/ick, D., Freeze-fracture studies on the synapse between the type I hair cell and the calyceal terminal in the guinea-pig vestibular system, J. Neurocytol., 8 (1979) 591603. 5 Hamilton, D.W., The calyceal synapse of type I vestibular hair cells, J. Ultrastructure Res., 23 (1968) 98-114. 6 Lysakowski, A. and Goldberg, J.M., Regional variations in the synaptic organization of the chinchilla cristae, Soc. Neurosci. Abstr., 15 (1989) 502. 7 Peusner, K.D., Lindberg, N.H. and Mansfield, P.F., Ultrastructural study of calycine synaptic endings of colossal vestibular fibers
8
9
10
11
in the cristae ampullares of the developing chick, Int. J. Dev. Neurosci., 6 (1988) 267-283. Smith, C.A. and Rasmussen, G.L., Nerve endings in the maculae and cristae of the chinchilla vestibule, with a special reference to the efferents. In Third Symposium on the Role of the Vestibular Organs in Space Exploration, NASA SP-152, USGPO, Washington, DC, 1967, pp. 183-201. Wersiill, J., Studies on the structure and innervation of the sensory epithelium of the cristae ampullares in the guinea pig: a light and electron microscopic investigation, Acta Oto-Laryngol., Suppl. 126 (1956) 1-85. Yamashita, M., Synaptic bodies and vesicles in vestibular calyceal synapses of the chick: an electron microscope study with intracellular injection of lucifer yellow and photo-oxidation reaction of DAB, Jpn. J. Physiol., 39 (1989) S168. Yamashita, M. and Ohmori, H., Synaptic responses to mechanical stimulation in calyceal and bouton type vestibular afferents studied in an isolated preparation of semicircular canal ampullae of chicken, Exp. Brain Res., 80 (1990) 475~88.