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The olfactory bulb mitral cell responses in the gecko as studied by intracellular recording
8raitt Re.search, 2~i (~')~2; 204 2i~
204
Elsevier F;i~:mcdical Pres~
The olfactory bulb mitral cell responses in the gecko as studied by intracellular recording
KEI ICHI TONOSA KI *
Zoological Institute, Faculty of Science, Tok),o Kyoiku University 3-29-1, Ot.~ttka, BuHky~-ku, ]~)kv~ (Japan) (Accepted October 8th, 1981)
Key worcZ~': intracellular recording
membrane resistance-- mitral cell .... odor response
proci,~n
yellow
The mitral cell odor-induced intracellular responses and the accompanying membrane resistance changes were studied in the gecko olfactory bulb. The mitral cells responded to odor either with depolarization or with hyperpolarization. The depolarization responses were accompanied by a resistance decrease. The hyperpolarization responses were accompanied by a resistance increase. The responses were enhanced by hyperpolarization and suppressed by depolarization of the membrane current. Two types of mitral cells were identified by procion yellow dye injection. The mitral cells in the olfactory bulb are second o r d e r neurons, that is, they are p o s t s y n a p t i c to the olfactory receptor cells. Vertebrate olfactory receptor cells respond to o d o r by d e p o l a r i z a t i o n t,3.l~, l n t r a c e l l u l a r recordings o f mitral cells have been reported, but no mention has been m a d e o f o d o r responses:~,~!'~: Extracellular recordings o f responses to o d o r in the mitral cells o f the olfactory bulb have been r e p o r t e d by several investigators e,~ -sne,la,l,5,19 who all n o t i c e d that some mitral cells show increased impulse frequency in response to o d o r stimulation, while other cells show a suppression o f their o n g o i n g discharges. However, intracellular recordings o f o d o r - e v o k e d electrical responses o f mitrat cells have not been reported. A d u l t geckos (Gekko gecko) were anesthetized with urethane. After the head o f the gecko was fixed in a h61der the olfactory bulb was exposed. C o n v e n t i o n a l intracellular recordings were made with h i g h - i m p e d a n c e (100-200 M fit) glass microelectrodes filled with a 6 °,;I a q u e o u s solution o f procion yellow. The resistance change o f the mitral cell was recorded with a bridge circuit a n d negative current pulses in the o r d e r o f 2 .,~ 10 -'~ n A a n d 100 ms d u r a t i o n , which were passed f r o m the electrode it~to the cell just before (control c u r r e n t pulse) a n d d u r i n g the o d o r s t i m u l a t i o n (test current pulse). Thus, a decrease in the m a g n i t u d e o f the electrotonic potential p r o d u c e d by the test current pulse c o r r e s p o n d s to a decrease in m e m b r a n e resistance. In some experi-
* Present address: Dept. of Oral Physiology, Gifu College of Dentistry, Takanu, Hozumi-cho, Motosu-gun, Gifu, Japan. 0006-8993/82/0000-0000/$02.75 ~) Elsevier Biomedical Press
205 ment s, a steady polarizing extrinsic current was passed throughthis bridge circuit to change the resting membrane potential in order to test the effect of membrane polarization. The response was amplified through DC and AC amplifiers and displayed on an oscilloscope. Responses were recorded on FM magnetic tape and analyzed later. After recording, p~ocion yellow was electrophoretically injected, so that the location and shape of the cell could be identified. The negatively charged dye was injected flom the pipet by passing a steady current of 5-10 nA for l-2 min. The response was checked again after dye injection. If this response variation was unusually great, the preparation was discarded. The olfactory bulb was then embedded in Epon and cut into 20 # m serial sections which were examined with a fluorescence microscope. n-Amyl acetate was used for odor stimulation. Air from a regulated duct was cleaned with activated charcoal and humidified with distilled water. Purified air was mixed with odor-saturated air in different ratios to obtain concentrations of amyl acetate ranging from 10.4 to 10o of vapor saturation. The odorized air flow (3 l/rain) was directed with Teflon tubing across the nose of the gecko.
A
hyperpolarizing current injection
control
20mV
l-
isec
i
control
test
pulses
B control
hyperpolarlzing current injection
depolarizing current injection
iOmV Isec
--~ control
if,, test
pulses
Fig. I. Example of the intracellular recordings from the mitral cell responses to n-amyl acetate (10 1 concentration) odor stimulation. A: depolarization odor response. B: hyperpolarization odor response. In record A control, the bridge is unbalanced before odor stimulation but becomes almost balanced in the odor response, indicating that the response is accompanied by a resistance decrease. In record B control, the bridge is balanced before odor stimulation and becomes unbalanced in the odor response, indicating that the response is accompanied by a resistance increase. Both the depolarization and the hyperpolarization odor response are enhanced by extrinsic hyperpolarization of the membrane potential. The hyperpolarization odor response is suppressed by depolarization of the membrane potential. Horizontal lines below the record of the traces indicate the duration of odor stimulation. The lowest line under the control response monitored the negative current pulses through the electrode. Electrotonic potentials are indicated by arrows. The vertical and horizontal bars on the right in A and B show 20 mV and 1 s respectively. Each millivolt of this deflection -- 5 Mr2 of cell membrane resistance.
l,
A
I!F I
B Fig. 2. Mitral cells injected with procion 3ellox~. A : an example of the large sized mitrai cell which gives the depolarization odor response. Both the axon a n d the dendrite are stained. B: an e x a m p l e of the small sized mitral cell which gives the hyperpolarization odor response. T h e axon is stained. Calibration bar 2 0 / , m .
207 Twelve intracell ular recordings from mitral cells were obtained. Recordings were positively identified as being from the mitral cell by procion yellow dye marking. The membrane potentials ranged from --20 to --60 mV and the spike height from 10 to 50 inV. Eight out of 12 mitral cells gave depolarization and the other 4 hyperpolarization responses to amyl acetate (10 -1 concentration) odor stimtdation. A typical example of a depolarization response to odor obtained from a mitral cell is shown in Fig. IA. The membrane potential is --25 mV, and the depolarization response to odor is approximately 8 mV in amplitude. The depolarization response to odor is accompanied by a decrease in membrane resistance as seen in the bridge record of Fig. 1A (control). Both the depolarization response to odor and the impulse height are enhanced by hyperpolarization of the membrane potential by a steady extrinsic polarizing current. Another typical example of a hyperpolarization response to odor obtained from a mitral cell is shown in Fig. lB. The membrane potential is --40 mV, and the response is accompanied by a membrane resistance increase as judged from the bridge record (Fig. 1B, control). The hyperpolarization response to odor is enhanced by hyperpolarization of the membrane potential and suppressed by depolarization of the membrane potential by a steady extrinsic polarizing current. The height of the impulses is increased by hyperpolarization of the membrane potential and decreased by depolarization of the membrane potential by a steady extrinsic polarizing current. In the olfactory bulb, basically two types of mitra! cells were identified by dye injection. One cell type is typically miter shaped, is about 25 × 10 #m, and has a clear axon and dendrites. Another is oval shaped and about 18 x 7 # m in size. Depolarization responses to odor were mainly found from the large sized cells. Fig. 2A shows an example of a mitral cell which gave a depolarization odor response and Fig. 2B shows an example of a mitral cell which gave a hyperpolarization odor response. Since the olfactory receptor cells are depolarized by odor stimulation, the depolarization of the postsynaptic terminals should be related to the presynaptic depolarization by an EPSP type of response which was observed in the depolarization odor response of the mitral cell. However, the hyperpolarization odor response of the mitral cell cannot be accounted for by a postsynaptic hyperpolarization response of the lPSP type, because the hyperpolarization odor response of the mitral cell was accompanied by an increase in membrane resistance, and its amplitude was enhanced by the hyperpolarization of the membrane potential and suppressed by the depolarization of the membrane potential by a steady extrinsic polarizing current. In the bipolar cells in the carp retina, it is known that the hyperpolarization responses were accompanied by a resistance increase, and the responses were augmented by hyperpolarization and suppressed by depolarization of the membrane by a currenO z,z6-1s. They suggested that these phenomena depend on the conductance change of subsynaptic potassium and/or chloride channels. Those relationships will be investigated in more detail in further experiments. 1 Aoki, K. and Takagi, S. F., Intracellular recording of the olfactory cell activity, Proc. Jap. Acad., 44 (1968) 856-858. 2 D6ving, K. B., Studies of the relation between the frog's electro-olfactogram and single unit activity in the olfactory bulb, Actaphysiol. Scand., 60 (1964) 150-163.
208 3 Getchell, T. V., Analysis of intracellular recording flom salamander olfactory cpitlwtiurn, Brctit¢ Research, 123 (1977) 275 286. 4 Kauer, J. S., Response patterns of amphibian olfactory bulb neurones to odour stimulation, d. Physiol. (Lomt.), 243 (1974) 695 715. 5 Kauer, J. S. and Moulton, D. G., Response of olfactory bulb neurones to odour stimulation oi small nasal area in the salamander, ,I. Physiol. florid.), 243 (1974) 717337. 6 Kauer, J. S. and Shepherd, G. M., Analysis of the onset of olfactory bulb unit responses to odour pulses in the salamander, J. Physiol. (Lond.), 272 (1977) 495-.516. 7 Mancia, M., Baumgarten, R. yon and Green, J. P., Response pattern of olfactory bu]~ neurones, Arch. Ital. Biol., 100 (1962) 449-462. 8 Mathews, D. W., Response patterns of single neurones in the tortoise olfactory epithelium and olfactory bulb, J. gen. Physiol., 60 (1972) 166 180. 9 Mori, K. and Takagi, S. F., An intracellular study of dendrodendritic inhibitor5 ~qvnapses on mitral cells in the rabbit olfactory bulb, 7. Physiol. (Lond.), 279 (1978) 569 588. l0 Mori, K. and Shepherd, G. M., Synaptic excitation and long-lasting inhibition of rnilral cells in the in vitro turtle olfactory bulb, Brain Research, 172 (1979) 155 159. II Saito, T., Kondo, H. and Toyoda, J.-l., lonoc mechanisms o1" two types of on-cenlei hipoiar cells in the carp retina, J. gen. Physiol., 73 (1979) 73 90. 12 Shibuya, T., Ai, N. and Takagi, S. F., Response types of single cell in the ol|aclory bulb. Pt'~z~ Jap. Acad., 38 (1962) 231-233. 13 Shibuya, T., Aihara, Y. and Tonosaki, K., Single cell responses to odors in the reptilian olfactoly bulb. In Y. Katsuki et al. (Eds.), f'ood Intake and Chemical Senses, Japan Scientific Societies Pres~, Tokyo, 1977, pp. 23 32. 14 Suzuki, N., lntracellular responses of lamprey olfactory receptors to current and d~cmicat stimulation. In Y. Katsuki et al. (Eds.), lC)~od Intake and Chemical Senses, Japan Scientific Soceities Press, Tokyo, 1977, pp. 13 22. 15 Tonosaki, K. and Shibuya, T., Action of some drugs on gecko olfactory bulb mitrai ceil responses to oxor stimulation, Brain Research, t 67 (I 979) 180 184. 16 Toyoda, J.-l., Membrane resistance cbanges underlying the bipolar cell response in ihc cacp retina, Vision Reds., 13 (1973) 283 294. 17 Toyoda, J.-l. and Tonosaki, K., Effect of polarisation of horizontal cells on the on-cenl re bipolar cell of carp retina, Nature (Land.), 276 (1978) 399-400. 18 Toyoda, J.-I. and Tonosaki, K., Studies on the mechanisms underlying horizontal-bipolar interaction in the carp retina, Sensot3, Processes, 2 (1978) 359 365. 19 Walsh, R. R., Single cell spike activity in the olfactory bulb, Amer, J. Physhd., 186 (~956) 255 2 5 7
204
Elsevier F;i~:mcdical Pres~
The olfactory bulb mitral cell responses in the gecko as studied by intracellular recording
KEI ICHI TONOSA KI *
Zoological Institute, Faculty of Science, Tok),o Kyoiku University 3-29-1, Ot.~ttka, BuHky~-ku, ]~)kv~ (Japan) (Accepted October 8th, 1981)
Key worcZ~': intracellular recording
membrane resistance-- mitral cell .... odor response
proci,~n
yellow
The mitral cell odor-induced intracellular responses and the accompanying membrane resistance changes were studied in the gecko olfactory bulb. The mitral cells responded to odor either with depolarization or with hyperpolarization. The depolarization responses were accompanied by a resistance decrease. The hyperpolarization responses were accompanied by a resistance increase. The responses were enhanced by hyperpolarization and suppressed by depolarization of the membrane current. Two types of mitral cells were identified by procion yellow dye injection. The mitral cells in the olfactory bulb are second o r d e r neurons, that is, they are p o s t s y n a p t i c to the olfactory receptor cells. Vertebrate olfactory receptor cells respond to o d o r by d e p o l a r i z a t i o n t,3.l~, l n t r a c e l l u l a r recordings o f mitral cells have been reported, but no mention has been m a d e o f o d o r responses:~,~!'~: Extracellular recordings o f responses to o d o r in the mitral cells o f the olfactory bulb have been r e p o r t e d by several investigators e,~ -sne,la,l,5,19 who all n o t i c e d that some mitral cells show increased impulse frequency in response to o d o r stimulation, while other cells show a suppression o f their o n g o i n g discharges. However, intracellular recordings o f o d o r - e v o k e d electrical responses o f mitrat cells have not been reported. A d u l t geckos (Gekko gecko) were anesthetized with urethane. After the head o f the gecko was fixed in a h61der the olfactory bulb was exposed. C o n v e n t i o n a l intracellular recordings were made with h i g h - i m p e d a n c e (100-200 M fit) glass microelectrodes filled with a 6 °,;I a q u e o u s solution o f procion yellow. The resistance change o f the mitral cell was recorded with a bridge circuit a n d negative current pulses in the o r d e r o f 2 .,~ 10 -'~ n A a n d 100 ms d u r a t i o n , which were passed f r o m the electrode it~to the cell just before (control c u r r e n t pulse) a n d d u r i n g the o d o r s t i m u l a t i o n (test current pulse). Thus, a decrease in the m a g n i t u d e o f the electrotonic potential p r o d u c e d by the test current pulse c o r r e s p o n d s to a decrease in m e m b r a n e resistance. In some experi-
* Present address: Dept. of Oral Physiology, Gifu College of Dentistry, Takanu, Hozumi-cho, Motosu-gun, Gifu, Japan. 0006-8993/82/0000-0000/$02.75 ~) Elsevier Biomedical Press
205 ment s, a steady polarizing extrinsic current was passed throughthis bridge circuit to change the resting membrane potential in order to test the effect of membrane polarization. The response was amplified through DC and AC amplifiers and displayed on an oscilloscope. Responses were recorded on FM magnetic tape and analyzed later. After recording, p~ocion yellow was electrophoretically injected, so that the location and shape of the cell could be identified. The negatively charged dye was injected flom the pipet by passing a steady current of 5-10 nA for l-2 min. The response was checked again after dye injection. If this response variation was unusually great, the preparation was discarded. The olfactory bulb was then embedded in Epon and cut into 20 # m serial sections which were examined with a fluorescence microscope. n-Amyl acetate was used for odor stimulation. Air from a regulated duct was cleaned with activated charcoal and humidified with distilled water. Purified air was mixed with odor-saturated air in different ratios to obtain concentrations of amyl acetate ranging from 10.4 to 10o of vapor saturation. The odorized air flow (3 l/rain) was directed with Teflon tubing across the nose of the gecko.
A
hyperpolarizing current injection
control
20mV
l-
isec
i
control
test
pulses
B control
hyperpolarlzing current injection
depolarizing current injection
iOmV Isec
--~ control
if,, test
pulses
Fig. I. Example of the intracellular recordings from the mitral cell responses to n-amyl acetate (10 1 concentration) odor stimulation. A: depolarization odor response. B: hyperpolarization odor response. In record A control, the bridge is unbalanced before odor stimulation but becomes almost balanced in the odor response, indicating that the response is accompanied by a resistance decrease. In record B control, the bridge is balanced before odor stimulation and becomes unbalanced in the odor response, indicating that the response is accompanied by a resistance increase. Both the depolarization and the hyperpolarization odor response are enhanced by extrinsic hyperpolarization of the membrane potential. The hyperpolarization odor response is suppressed by depolarization of the membrane potential. Horizontal lines below the record of the traces indicate the duration of odor stimulation. The lowest line under the control response monitored the negative current pulses through the electrode. Electrotonic potentials are indicated by arrows. The vertical and horizontal bars on the right in A and B show 20 mV and 1 s respectively. Each millivolt of this deflection -- 5 Mr2 of cell membrane resistance.
l,
A
I!F I
B Fig. 2. Mitral cells injected with procion 3ellox~. A : an example of the large sized mitrai cell which gives the depolarization odor response. Both the axon a n d the dendrite are stained. B: an e x a m p l e of the small sized mitral cell which gives the hyperpolarization odor response. T h e axon is stained. Calibration bar 2 0 / , m .
207 Twelve intracell ular recordings from mitral cells were obtained. Recordings were positively identified as being from the mitral cell by procion yellow dye marking. The membrane potentials ranged from --20 to --60 mV and the spike height from 10 to 50 inV. Eight out of 12 mitral cells gave depolarization and the other 4 hyperpolarization responses to amyl acetate (10 -1 concentration) odor stimtdation. A typical example of a depolarization response to odor obtained from a mitral cell is shown in Fig. IA. The membrane potential is --25 mV, and the depolarization response to odor is approximately 8 mV in amplitude. The depolarization response to odor is accompanied by a decrease in membrane resistance as seen in the bridge record of Fig. 1A (control). Both the depolarization response to odor and the impulse height are enhanced by hyperpolarization of the membrane potential by a steady extrinsic polarizing current. Another typical example of a hyperpolarization response to odor obtained from a mitral cell is shown in Fig. lB. The membrane potential is --40 mV, and the response is accompanied by a membrane resistance increase as judged from the bridge record (Fig. 1B, control). The hyperpolarization response to odor is enhanced by hyperpolarization of the membrane potential and suppressed by depolarization of the membrane potential by a steady extrinsic polarizing current. The height of the impulses is increased by hyperpolarization of the membrane potential and decreased by depolarization of the membrane potential by a steady extrinsic polarizing current. In the olfactory bulb, basically two types of mitra! cells were identified by dye injection. One cell type is typically miter shaped, is about 25 × 10 #m, and has a clear axon and dendrites. Another is oval shaped and about 18 x 7 # m in size. Depolarization responses to odor were mainly found from the large sized cells. Fig. 2A shows an example of a mitral cell which gave a depolarization odor response and Fig. 2B shows an example of a mitral cell which gave a hyperpolarization odor response. Since the olfactory receptor cells are depolarized by odor stimulation, the depolarization of the postsynaptic terminals should be related to the presynaptic depolarization by an EPSP type of response which was observed in the depolarization odor response of the mitral cell. However, the hyperpolarization odor response of the mitral cell cannot be accounted for by a postsynaptic hyperpolarization response of the lPSP type, because the hyperpolarization odor response of the mitral cell was accompanied by an increase in membrane resistance, and its amplitude was enhanced by the hyperpolarization of the membrane potential and suppressed by the depolarization of the membrane potential by a steady extrinsic polarizing current. In the bipolar cells in the carp retina, it is known that the hyperpolarization responses were accompanied by a resistance increase, and the responses were augmented by hyperpolarization and suppressed by depolarization of the membrane by a currenO z,z6-1s. They suggested that these phenomena depend on the conductance change of subsynaptic potassium and/or chloride channels. Those relationships will be investigated in more detail in further experiments. 1 Aoki, K. and Takagi, S. F., Intracellular recording of the olfactory cell activity, Proc. Jap. Acad., 44 (1968) 856-858. 2 D6ving, K. B., Studies of the relation between the frog's electro-olfactogram and single unit activity in the olfactory bulb, Actaphysiol. Scand., 60 (1964) 150-163.
208 3 Getchell, T. V., Analysis of intracellular recording flom salamander olfactory cpitlwtiurn, Brctit¢ Research, 123 (1977) 275 286. 4 Kauer, J. S., Response patterns of amphibian olfactory bulb neurones to odour stimulation, d. Physiol. (Lomt.), 243 (1974) 695 715. 5 Kauer, J. S. and Moulton, D. G., Response of olfactory bulb neurones to odour stimulation oi small nasal area in the salamander, ,I. Physiol. florid.), 243 (1974) 717337. 6 Kauer, J. S. and Shepherd, G. M., Analysis of the onset of olfactory bulb unit responses to odour pulses in the salamander, J. Physiol. (Lond.), 272 (1977) 495-.516. 7 Mancia, M., Baumgarten, R. yon and Green, J. P., Response pattern of olfactory bu]~ neurones, Arch. Ital. Biol., 100 (1962) 449-462. 8 Mathews, D. W., Response patterns of single neurones in the tortoise olfactory epithelium and olfactory bulb, J. gen. Physiol., 60 (1972) 166 180. 9 Mori, K. and Takagi, S. F., An intracellular study of dendrodendritic inhibitor5 ~qvnapses on mitral cells in the rabbit olfactory bulb, 7. Physiol. (Lond.), 279 (1978) 569 588. l0 Mori, K. and Shepherd, G. M., Synaptic excitation and long-lasting inhibition of rnilral cells in the in vitro turtle olfactory bulb, Brain Research, 172 (1979) 155 159. II Saito, T., Kondo, H. and Toyoda, J.-l., lonoc mechanisms o1" two types of on-cenlei hipoiar cells in the carp retina, J. gen. Physiol., 73 (1979) 73 90. 12 Shibuya, T., Ai, N. and Takagi, S. F., Response types of single cell in the ol|aclory bulb. Pt'~z~ Jap. Acad., 38 (1962) 231-233. 13 Shibuya, T., Aihara, Y. and Tonosaki, K., Single cell responses to odors in the reptilian olfactoly bulb. In Y. Katsuki et al. (Eds.), f'ood Intake and Chemical Senses, Japan Scientific Societies Pres~, Tokyo, 1977, pp. 23 32. 14 Suzuki, N., lntracellular responses of lamprey olfactory receptors to current and d~cmicat stimulation. In Y. Katsuki et al. (Eds.), lC)~od Intake and Chemical Senses, Japan Scientific Soceities Press, Tokyo, 1977, pp. 13 22. 15 Tonosaki, K. and Shibuya, T., Action of some drugs on gecko olfactory bulb mitrai ceil responses to oxor stimulation, Brain Research, t 67 (I 979) 180 184. 16 Toyoda, J.-l., Membrane resistance cbanges underlying the bipolar cell response in ihc cacp retina, Vision Reds., 13 (1973) 283 294. 17 Toyoda, J.-l. and Tonosaki, K., Effect of polarisation of horizontal cells on the on-cenl re bipolar cell of carp retina, Nature (Land.), 276 (1978) 399-400. 18 Toyoda, J.-I. and Tonosaki, K., Studies on the mechanisms underlying horizontal-bipolar interaction in the carp retina, Sensot3, Processes, 2 (1978) 359 365. 19 Walsh, R. R., Single cell spike activity in the olfactory bulb, Amer, J. Physhd., 186 (~956) 255 2 5 7