- Email: [email protected]
Biphasic acetylcholine responses of molluscan neurones: their dependence on acetylcholine concentration
426
Brain Research, 167 (1979) 426-430 ri~) Elsevier/North-Holland Biomedical Press
Biphasic acetytcholine responses of molluscan neurones: their dependence on aceWIchotine concentration
B. A. GER, A. N. K A T C H M A N and ELLA V. ZEIMAL
Laboratory of Pharmacology, Sechenov hzstitute Academy of Sciences of the USSR, Leningrad 194223 (U.S.S.R.) (Accepted January I lth, 1979)
Some identified neurones in the central ganglia of gastropod molluscs have more than one type of cholinoreceptor (ChR) on their membrane and respond to acetylcholine (ACh) application by polyphasic potential changes s-10. However, the ACh response of these same neurones is found to be monophasic under certain conditions. For example, in Aplysia neurones, it has been noted that the ACh response changes in complexity as a function of the distance of the ACh pipette from the cell surface 1. No attempt has yet been made to understand why the early, rapid component of response disappears with increased distance between the membrane and the ACh electrode, or to understand, in a more general sense, what factors control variations in the form of a response when more than one receptor type is present on the membrane of a single cell. One likely explanation is that, with increasing distance, the concentration of ACh delivered to the cell surface becomes insufficient for activating one of the receptor types, though remaining sufficient for activating the other receptor type. If this be the explanation for the change in response form in the experiment by Ascher and Kehoe 1, one would expect that increasing the dose of ACh with the pipette distant from the cell surface would be adequate for causing a reappearance of the C1--dependent component, and that, even close to the cell membrane, ACh concentrations could be found that would selectively activate only the K+-dependent response. We have tested this hypothesis by studying the differential sensitivity of these same two receptor types (see refs. 3 and 4) on identified neurones of Planorbarius corneus. To avoid influences from the neighbouring cells on the neurone under investigation, the experiments were performed on the completely isolated neurones of the left pedal ganglion, LPed-3 and LPed-4. Double-barrelled microelectrodes were filled with 2.5 M KCI. One barrel was used to record the membrane potential, the other to polarize the membrane. Two means of ACh application were used: electrophoresis and superfusion. For the electrophoretic application, a micropipette with a tip diameter less than 1 ,um was filled with 1 M ACh (resistance about 50 Mfl). Braking currents never exceeded 5 × 10-gA. For superfusion experiments, pipettes 5-7 cm long with tip diameters of between 100 and 300/~m were filled with an ACh solution of
427
desired concentration prepared ex tempore in the physiological solution. For applying ACh, the pipette was introduced into the solution and brought quickly up to the neuronal surface with a quick movement of the micromanipulator screw. Due to the constant flow of the physiological solution through the experimental chamber (0.2 ml/sec) the hydrodynamic forces ensured that the ACh concentration at the surface of the cell approached that of the pipette within 0.1 sec. To stop ACh application, the pipette was pulled out of the solution. It should be noted that in these cells, in contrast with the medial cells of the pleural ganglion of Aplysia, Eel is maintained at a level positive with respect to resting potential; hence, activation of the receptor mediating an increase in CI- conductance, produces a depolarizing rather than hyperpolarizing response. B far near
far
(60 mcm)
I nC
(I20 mcm)
A
-)5 mv
2
n£
-~
~\ f - -
io
ACh C
/
L f
0.2"IO-7A,
IO0 ms
(2 n6)
~v L
ACh 3 ms
Io ~c
,_d
2S \
8'I0-7A, (2.4 nO) -'35 mV
far
f.r
.
A
~30 ,,c~
(IO0 mcm)
20 ms (I2 nO) 2mY
c~r
-45 mV
-55 mV
D
5~
(3 ~¢)
~
/'-/. . . .
-'b"--IO zV L 5s
Fig. 1. Dependence of ACh response on the distance of electrophoretic micropipette from the cell surface (A,C) and on the amount of ACh ejected (B,D). A and D: LPed-3 neurone; B and C: LPed-4 neurone (resting potentials were 30 mV and -35 mV, respectively). A: figures from the left - - the levels of membrane potential; at the top - - the distance of electrophoretic micropipette from the neurone surface. ACh electrophoretic current - - 6'10 7A, 20 msec (12 nC). Note that the depolarizing phase of the response is diminished and then disappears with the removing of micropipette from the neurone. B: applications of ACh from the distance about 2000 t~m from the cell. Note the appearence of the rapid phase with the increasing of ACh amounts (nC). C: from the left - - distance of ACh micropipette from the cell (mcm = /~m; at the top - - ACh current (A), its duration (msec) and the total charge (nC). Note that the patterns of responses are essentially the same with the prolonged ACh current of low value (left column) and with short current of greater value (right column) provided the same charge passes through the micropipette. D: figures from the left indicate the electrophoresis duration (msec) and the charge (nC) passed through the ACh micropipette (close to the cell). At the top - - membrane potential levels (mV). Note the increase of the amplitude and the duration of the depolarizing phase with the increase of the ACh amount ejected from the pipette; the greater and the longer is the first phase the later the second phase appears.
428 As can be seen in Fig. t A and C. moving the electrode away from the neu tonal surface has the same effect on the form of ACh response in Planorbarius corneus neurones as it had been shown to have in Aplysm neurones. With the electrophoretic microelectrode close to the neurone, biphasic responses are obtained: an early CI-dependent depolarization followed by a K+-dependent hyperpolarization. However. when the ACh microelectrode was pulled back from the cell, the first phase became smaller or disappeared (as at ....35 mV in Fig. IA), with no significant change occurring in the second phase of the response. A further increase in distance between the ACh pipette and the cell caused a complete disappearance of the first phase at all membrane potentials studied. Fig. I B shows that the early component of the response, lost by placing the ACh electrode far from the cell surface, can be recovered by increasing the amplitude of the ACh pulse. These data suggest that the change in ACh concentration with distance was indeed responsible for the change in form as a function of the pipette membrane separation. The changes in form of the ACh response are due to changes in the amount of ACh ejected, and not to the duration of the electrophoresis itself (Fig. 1C). As can be seen in Fig. I D. as the depolarizing phase becomes larger and of longer duration with the increasing amounts of ACh (nC), the concomitantly occurring slow component, which is revealed only after termination of the rapid component, is more and more effectively masked. Consequently, it is difficult to study the behaviour of the slow component as a function of ACh concentration. More revealing information was obtained about the relationship between ACh concentration and the form of the response when using the method of superfusion ~see above), which permitted a rapid onset of known concentration of ACh over a large area of the neuronal membrane. Fig. 2 shows that small ACh concentrations ~5.10 -s M - I • 10 -7 M) caused a pure hyperpolarization. With greater ACh concentrations, a rapid depolarization preceded the hyperpolarization. A 10 times greater ACh concentration was needed to activate the ChRs responsible for the CI- -dependent phase of ACh potential than was needed for activating the receptor mediating K T-dependent phase. With ACh concentrations large enough to elicit a biphasic response the same masking of the slow component occurred that had been seen in the iontophoresis experiments (compare Fig. 2B and Fig. 1D). The fact established by these experiments, that the sensitivity of the so-called 'muscarinic' receptor type 4 (i.e. the receptor mediating an increase in K~conduct ance) is much greater than of the nicotinic receptor type (Le. that mediating the increase in C 1- conductance) can easily account for the selective activation of the K ~dependent response when the ACh electrode is pulled away from the cell surface (see Fig. IA-C). The pure hyperpolarizing response obtained with a distant electrode is simply a reflection of the fact that the concentration of A C h at the cell surface, with the ACh pipette at a distance, falls under the threshold concentration required for activating the less sensitive nicotinic receptor. These same experiments throw light on our own previous findings observed when ACh was simply introduced into the perfusion system 4 where the hyperpol-
429 A
5' IO-8 M
I'Io -7 M
I~'I0-6 M _S "\'
~'~'Io -6 M
~-, I' 10 .5 M
%
l'IO -5 M
I'IO -5 M I%'I0 4 M IO mV L 5s
IO mV h
5s
Fig. 2. Dependence of the form of ACh potential on ACh concentration. LPed-3 neurone. Arrows indicate the beginning (upward) and the end (downward) of the superfusion with ACh concentrations shown under each curve. A : recordings at the resting potential (-35 mV), B : at the membrane potential displaced to -50 inV. Note that ACh caused hyperpolarization at low concentrations (5 × l0 sI x l0 7 M) but biphasic response at higher ones. With ACh 1 x 10 6 1 x 10 4 M the depolarization appears first; it is replaced by the hyperpolarization only after the superfusion is finished. The higher ACh concentration (A) or the longer superfusion lasts (B), the later hyperpolarizing phase appears. arizing phase always appeared first. Since the A C h concentration, under such experimental conditions, increased only gradually at the cell surface, there is inevitably an initial period during which the concentration is insufficient for activating the nicotinic receptor. Although differential sensitivity of receptor types m a y well not be the only reason for variability in response under different experimental (or physiological) conditions, it clearly is one that explains some changes in response complexity. An observation that probably falls into this category is that seen by Levitan and Tauc 8 in Navanax neurones, in which - - studying a multicomponent response mediated by receptors having a different p h a r m a c o l o g y than those studied here - - only one c o m p o n e n t of the response was elicited with low iontophoretic currents, whereas a biphasic response was obtained with higher iontophoretic currents. It can be anticipated, however, that in m a n y instances changes in response complexity with increasing ionophoretic (i.e. localized application) doses of transmitter might simply reflect differential localization or differential densities of different receptor types. This explanation does not seem to hold in the case studied here, however, since perfusion and superfusion methods, which permit the A C h to reach most or all of the cell surface, yield response patterns that are strictly correlated with A C h concentration.
430 1 Ascher, P. and Kehoe, J. S., Amine and amino acid receptors in gastropod neurones. In L. L. Iversen, S. D. lversen and S. H. Snyder (Eds.), Handbook ofPsychopharmacology, Vol. 4. Plenum. New York, t975, pp. 265-310. 2 Gapon, S. A., Katchman, A N. and Frolova, E. V.. Studies of the chemoreceptive membrane m identified neuron of the gastropod mollusc Planorbarius corneus by different methods of drug application, J. evolut. Biochem. PhysioL, 14 (1978) 259-265 (in Russian). 3 Ger, B. A. and Zeimal, E. V.. Two kinds of cholinoreceptors on the membrane of the completely isolated identified Planorbarius corneus neuron. Nature (Lond.). 259 (1976) 681-684. 4 Ger, B. A. and Zeimal, E. V.. Pharmacological study of two kinds of chotinoreceptors on the membrane of identified completely isolated neurone of Planorbarius corneus. Brcin Research, 121 (1977) 131-149. 5 Kehoe, J. S., Pharmacological characteristics and ionic bases of two-componem postsynaptic inhibition, Nature (Lond.j, 215 ~1967) 1503 1505. 6 Kehoe, J. S., Three acetylcholine receptors on Aplysia neurones, J. Physiol. (Lomt./, 225 (1972) 115-146. 7 Korobtsov, G. N. and Sakharov, D. A.. Effect of serotonin and acetylcholine on various neur¢ms of the snail nervous system,. Neurophysiology (Kiev), 6 (1974) 644-651 tin Russian). 8 Levitan, H. and Tauc, L., Acetylcholine receptors: tOl~ographic distribution and ~harrnacolog~cal properties of two receptor types on a single molluscan neurone. J. Physiol. "Lor,d.L 222 (1972) 537-558. 9 Wachtel, H. and Kandel, E. R.. A direcl synaptic connection mediating t:oth cxcitatlon and inhibition, Science, 158 (1967) 1206-1208. 10 Wachtel, H. and Kandel, E. R., Conversion of synaptic excitation to inhibition at a dual chemical synapse, J. NeurophysioL, 34 (1971) 56-68.
Brain Research, 167 (1979) 426-430 ri~) Elsevier/North-Holland Biomedical Press
Biphasic acetytcholine responses of molluscan neurones: their dependence on aceWIchotine concentration
B. A. GER, A. N. K A T C H M A N and ELLA V. ZEIMAL
Laboratory of Pharmacology, Sechenov hzstitute Academy of Sciences of the USSR, Leningrad 194223 (U.S.S.R.) (Accepted January I lth, 1979)
Some identified neurones in the central ganglia of gastropod molluscs have more than one type of cholinoreceptor (ChR) on their membrane and respond to acetylcholine (ACh) application by polyphasic potential changes s-10. However, the ACh response of these same neurones is found to be monophasic under certain conditions. For example, in Aplysia neurones, it has been noted that the ACh response changes in complexity as a function of the distance of the ACh pipette from the cell surface 1. No attempt has yet been made to understand why the early, rapid component of response disappears with increased distance between the membrane and the ACh electrode, or to understand, in a more general sense, what factors control variations in the form of a response when more than one receptor type is present on the membrane of a single cell. One likely explanation is that, with increasing distance, the concentration of ACh delivered to the cell surface becomes insufficient for activating one of the receptor types, though remaining sufficient for activating the other receptor type. If this be the explanation for the change in response form in the experiment by Ascher and Kehoe 1, one would expect that increasing the dose of ACh with the pipette distant from the cell surface would be adequate for causing a reappearance of the C1--dependent component, and that, even close to the cell membrane, ACh concentrations could be found that would selectively activate only the K+-dependent response. We have tested this hypothesis by studying the differential sensitivity of these same two receptor types (see refs. 3 and 4) on identified neurones of Planorbarius corneus. To avoid influences from the neighbouring cells on the neurone under investigation, the experiments were performed on the completely isolated neurones of the left pedal ganglion, LPed-3 and LPed-4. Double-barrelled microelectrodes were filled with 2.5 M KCI. One barrel was used to record the membrane potential, the other to polarize the membrane. Two means of ACh application were used: electrophoresis and superfusion. For the electrophoretic application, a micropipette with a tip diameter less than 1 ,um was filled with 1 M ACh (resistance about 50 Mfl). Braking currents never exceeded 5 × 10-gA. For superfusion experiments, pipettes 5-7 cm long with tip diameters of between 100 and 300/~m were filled with an ACh solution of
427
desired concentration prepared ex tempore in the physiological solution. For applying ACh, the pipette was introduced into the solution and brought quickly up to the neuronal surface with a quick movement of the micromanipulator screw. Due to the constant flow of the physiological solution through the experimental chamber (0.2 ml/sec) the hydrodynamic forces ensured that the ACh concentration at the surface of the cell approached that of the pipette within 0.1 sec. To stop ACh application, the pipette was pulled out of the solution. It should be noted that in these cells, in contrast with the medial cells of the pleural ganglion of Aplysia, Eel is maintained at a level positive with respect to resting potential; hence, activation of the receptor mediating an increase in CI- conductance, produces a depolarizing rather than hyperpolarizing response. B far near
far
(60 mcm)
I nC
(I20 mcm)
A
-)5 mv
2
n£
-~
~\ f - -
io
ACh C
/
L f
0.2"IO-7A,
IO0 ms
(2 n6)
~v L
ACh 3 ms
Io ~c
,_d
2S \
8'I0-7A, (2.4 nO) -'35 mV
far
f.r
.
A
~30 ,,c~
(IO0 mcm)
20 ms (I2 nO) 2mY
c~r
-45 mV
-55 mV
D
5~
(3 ~¢)
~
/'-/. . . .
-'b"--IO zV L 5s
Fig. 1. Dependence of ACh response on the distance of electrophoretic micropipette from the cell surface (A,C) and on the amount of ACh ejected (B,D). A and D: LPed-3 neurone; B and C: LPed-4 neurone (resting potentials were 30 mV and -35 mV, respectively). A: figures from the left - - the levels of membrane potential; at the top - - the distance of electrophoretic micropipette from the neurone surface. ACh electrophoretic current - - 6'10 7A, 20 msec (12 nC). Note that the depolarizing phase of the response is diminished and then disappears with the removing of micropipette from the neurone. B: applications of ACh from the distance about 2000 t~m from the cell. Note the appearence of the rapid phase with the increasing of ACh amounts (nC). C: from the left - - distance of ACh micropipette from the cell (mcm = /~m; at the top - - ACh current (A), its duration (msec) and the total charge (nC). Note that the patterns of responses are essentially the same with the prolonged ACh current of low value (left column) and with short current of greater value (right column) provided the same charge passes through the micropipette. D: figures from the left indicate the electrophoresis duration (msec) and the charge (nC) passed through the ACh micropipette (close to the cell). At the top - - membrane potential levels (mV). Note the increase of the amplitude and the duration of the depolarizing phase with the increase of the ACh amount ejected from the pipette; the greater and the longer is the first phase the later the second phase appears.
428 As can be seen in Fig. t A and C. moving the electrode away from the neu tonal surface has the same effect on the form of ACh response in Planorbarius corneus neurones as it had been shown to have in Aplysm neurones. With the electrophoretic microelectrode close to the neurone, biphasic responses are obtained: an early CI-dependent depolarization followed by a K+-dependent hyperpolarization. However. when the ACh microelectrode was pulled back from the cell, the first phase became smaller or disappeared (as at ....35 mV in Fig. IA), with no significant change occurring in the second phase of the response. A further increase in distance between the ACh pipette and the cell caused a complete disappearance of the first phase at all membrane potentials studied. Fig. I B shows that the early component of the response, lost by placing the ACh electrode far from the cell surface, can be recovered by increasing the amplitude of the ACh pulse. These data suggest that the change in ACh concentration with distance was indeed responsible for the change in form as a function of the pipette membrane separation. The changes in form of the ACh response are due to changes in the amount of ACh ejected, and not to the duration of the electrophoresis itself (Fig. 1C). As can be seen in Fig. I D. as the depolarizing phase becomes larger and of longer duration with the increasing amounts of ACh (nC), the concomitantly occurring slow component, which is revealed only after termination of the rapid component, is more and more effectively masked. Consequently, it is difficult to study the behaviour of the slow component as a function of ACh concentration. More revealing information was obtained about the relationship between ACh concentration and the form of the response when using the method of superfusion ~see above), which permitted a rapid onset of known concentration of ACh over a large area of the neuronal membrane. Fig. 2 shows that small ACh concentrations ~5.10 -s M - I • 10 -7 M) caused a pure hyperpolarization. With greater ACh concentrations, a rapid depolarization preceded the hyperpolarization. A 10 times greater ACh concentration was needed to activate the ChRs responsible for the CI- -dependent phase of ACh potential than was needed for activating the receptor mediating K T-dependent phase. With ACh concentrations large enough to elicit a biphasic response the same masking of the slow component occurred that had been seen in the iontophoresis experiments (compare Fig. 2B and Fig. 1D). The fact established by these experiments, that the sensitivity of the so-called 'muscarinic' receptor type 4 (i.e. the receptor mediating an increase in K~conduct ance) is much greater than of the nicotinic receptor type (Le. that mediating the increase in C 1- conductance) can easily account for the selective activation of the K ~dependent response when the ACh electrode is pulled away from the cell surface (see Fig. IA-C). The pure hyperpolarizing response obtained with a distant electrode is simply a reflection of the fact that the concentration of A C h at the cell surface, with the ACh pipette at a distance, falls under the threshold concentration required for activating the less sensitive nicotinic receptor. These same experiments throw light on our own previous findings observed when ACh was simply introduced into the perfusion system 4 where the hyperpol-
429 A
5' IO-8 M
I'Io -7 M
I~'I0-6 M _S "\'
~'~'Io -6 M
~-, I' 10 .5 M
%
l'IO -5 M
I'IO -5 M I%'I0 4 M IO mV L 5s
IO mV h
5s
Fig. 2. Dependence of the form of ACh potential on ACh concentration. LPed-3 neurone. Arrows indicate the beginning (upward) and the end (downward) of the superfusion with ACh concentrations shown under each curve. A : recordings at the resting potential (-35 mV), B : at the membrane potential displaced to -50 inV. Note that ACh caused hyperpolarization at low concentrations (5 × l0 sI x l0 7 M) but biphasic response at higher ones. With ACh 1 x 10 6 1 x 10 4 M the depolarization appears first; it is replaced by the hyperpolarization only after the superfusion is finished. The higher ACh concentration (A) or the longer superfusion lasts (B), the later hyperpolarizing phase appears. arizing phase always appeared first. Since the A C h concentration, under such experimental conditions, increased only gradually at the cell surface, there is inevitably an initial period during which the concentration is insufficient for activating the nicotinic receptor. Although differential sensitivity of receptor types m a y well not be the only reason for variability in response under different experimental (or physiological) conditions, it clearly is one that explains some changes in response complexity. An observation that probably falls into this category is that seen by Levitan and Tauc 8 in Navanax neurones, in which - - studying a multicomponent response mediated by receptors having a different p h a r m a c o l o g y than those studied here - - only one c o m p o n e n t of the response was elicited with low iontophoretic currents, whereas a biphasic response was obtained with higher iontophoretic currents. It can be anticipated, however, that in m a n y instances changes in response complexity with increasing ionophoretic (i.e. localized application) doses of transmitter might simply reflect differential localization or differential densities of different receptor types. This explanation does not seem to hold in the case studied here, however, since perfusion and superfusion methods, which permit the A C h to reach most or all of the cell surface, yield response patterns that are strictly correlated with A C h concentration.
430 1 Ascher, P. and Kehoe, J. S., Amine and amino acid receptors in gastropod neurones. In L. L. Iversen, S. D. lversen and S. H. Snyder (Eds.), Handbook ofPsychopharmacology, Vol. 4. Plenum. New York, t975, pp. 265-310. 2 Gapon, S. A., Katchman, A N. and Frolova, E. V.. Studies of the chemoreceptive membrane m identified neuron of the gastropod mollusc Planorbarius corneus by different methods of drug application, J. evolut. Biochem. PhysioL, 14 (1978) 259-265 (in Russian). 3 Ger, B. A. and Zeimal, E. V.. Two kinds of cholinoreceptors on the membrane of the completely isolated identified Planorbarius corneus neuron. Nature (Lond.). 259 (1976) 681-684. 4 Ger, B. A. and Zeimal, E. V.. Pharmacological study of two kinds of chotinoreceptors on the membrane of identified completely isolated neurone of Planorbarius corneus. Brcin Research, 121 (1977) 131-149. 5 Kehoe, J. S., Pharmacological characteristics and ionic bases of two-componem postsynaptic inhibition, Nature (Lond.j, 215 ~1967) 1503 1505. 6 Kehoe, J. S., Three acetylcholine receptors on Aplysia neurones, J. Physiol. (Lomt./, 225 (1972) 115-146. 7 Korobtsov, G. N. and Sakharov, D. A.. Effect of serotonin and acetylcholine on various neur¢ms of the snail nervous system,. Neurophysiology (Kiev), 6 (1974) 644-651 tin Russian). 8 Levitan, H. and Tauc, L., Acetylcholine receptors: tOl~ographic distribution and ~harrnacolog~cal properties of two receptor types on a single molluscan neurone. J. Physiol. "Lor,d.L 222 (1972) 537-558. 9 Wachtel, H. and Kandel, E. R.. A direcl synaptic connection mediating t:oth cxcitatlon and inhibition, Science, 158 (1967) 1206-1208. 10 Wachtel, H. and Kandel, E. R., Conversion of synaptic excitation to inhibition at a dual chemical synapse, J. NeurophysioL, 34 (1971) 56-68.