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Differential effects of alcohols on the spike threshold of an identified motor axon in a crab (Pachygrapsus crassipes)
Neuroscience Letters, 133 (I 991) 3~5
3
© 1991 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/91/$ 03.50
NSL 08177
Differential effects of alcohols on the spike threshold of an identified motor axon in a crab (Pachygrapsus crassipes) Philip J. Stephens Villanova University, Department of Biology, Villanova, PA 19085 (U.S.A.} (Received 17 May 1991; Revised version received 31 July 1991; Accepted 5 August 199 I)
Key words: Alcohol; Spike threshold; Crab motor axon Observations were made on the fast bender excitor (FBE) axon in autotomized crab limbs bathed in salines made up with different alcohols. It has been shown previously that the presence of ethanol at a certain level causes a single action potential to generate additional spikes in the peripheral axon branches. The present study examines the level of different alcohols required to induce peripheral spike generation. For primary alcohols, increasing the molecular weight decreased the level of alcohol required to produce peripheral spike generation. The threshold level of 2butanol was greater than 1-butanol, but less than tertiary-butanol. These results are explained in terms of the partition coefficient, so that an alcohol with a higher partition coefficient enters the lipid bilayer more readily, thus a lower threshold level of that alcohol is required in the saline to generate additional spikes.
Anesthetics are lipid-soluble compounds which effect the cell membrane, rather than intracellular processes [6, 24], by reacting with the lipid phase of the membrane [8, 22], the ionic channels [12], or both. Electrophysiological techniques have revealed that anesthetics such as ethanol, have little effect on the resting potential [2, 5, 11] but have dramatic effects on the action potential. Ethanol decreases the voltage-sensitive inward sodium current and the delayed outward potassium current, decreases the rise time of the action potential and, above a certain concentration, blocks the spike altogether [1, 2, 4, 10, 14, 18, 19]. While ethanol, like most anesthetics, generally has a depressing effect on membrane excitability, there are some neurons that exhibit hyperexcitability in the presence of ethanol [13, 15, 26, 29]. In certain crab motor neurons, for example, the presence of ethanol above a critical level causes a single action potential to produce one or more additional spikes [30]. In these neurons the action potential is composed of a spike followed by a depolarizing afterpotential (DAP); ethanol increases the amplitude of the DAP and causes single action potentials to generate additional spikes in the peripheral axon branches [27, 32]. While work has been done on the effects of ethanol, little is known about the effects of other alcohols on the hyperexcitability of these neurons. The
Correspondence: P.J. Stephens, Villanova University, Department of Biology, Villanova, PA 19085, U.S.A.
present study addresses this problem using the crab fast bender excitor (FBE) axon and examines the threshold level of different alcohols (listed in Table I) required to elicit peripheral spike generation. Autotomized second and third walking limbs of the crab Pachygrapsus crassipes were held to the base of a plastic dish using dental wax. Preparations were immersed in a crab saline of the following composition: 470 mM NaC1, 8 mM KC1, 20 mM CaC12, 10 mM MgC12, 5 mM HEPES, pH 7.2. Fresh saline was perfused at a rate of about 10 ml/min and suction was applied to maintain saline volume in the preparation dish (9-11 ml) and to provide circulation; all experiments were performed at 22°C ( + I°C). Brief electrical shocks were applied to the exposed limb nerve and evoked action potentials were recorded through two glass microelectrodes inserted into the exposed FBE axon [27]. The microelectrode tips were separated by about 0.5 mm; examination of the time delay between the stimulus and the spike recorded at each location along the axon permitted the direction of spike propagation to be determined (Fig. 1). Observations were made in normal saline and in salines made up with various alcohols (listed in Table I); initial perfusion with a new saline took place at a rate of about 25 ml/min. Data were collected during and after a 10 min perfusion period; ethanol-induced changes in resting membrane potential and intracellular potassium occur within about 7 min of perfusion [28]. If no addi-
13
A
P
C
d tt___
__3
p~---
Fig. 1. The effect of alcohol-saline on the FBE action potential. A: a brief electrical shock was applied to the axon to produce an action potential in normal saline and in saline made up with 80 mM propanol (arrows). The peaks of the two spikes are superimposed to show the smaller spike and larger DAP recorded in the presence of alcohol. B: an evoked action potential recorded from two locations along the axon in saline made up with 50 mM 1-butanol. The electrical shock produced a spike which was recorded first at the proximally (p) located electrode (arrow) and then at the distally (d) located electrode. The second, additional spike was generated peripherally and traveled antidromically down the axon, since the spike was recorded first distally and then proximally (arrow). C, D: a brief shock applied to the limb nerve of a preparation bathed in saline made up with 60 mM l-butanol resulted in an action potential and 5 additional spikes. The additional spikes traveled orthodromically since each was recorded proximally (p) and then distally (d) - this is evident in D, which shows the first 3 spikes in C at a faster sweep speed. The arrow points to the peak of the first spike recorded at the proximally located electrode. Calibration: 10 mV and 0.5 ms (A), 4 ms (C), 1 ms (B, D).
tional spikes were recorded during the 10 min period, the preparation was perfused with saline made up with an increased (usually by 10 mM) level of alcohol. This proTABLE I THE THRESHOLD
CONCENTRATION
(mM) OF ALCOHOL
cess of increasing the alcohol level in a step-wise fashion was continued until additional, peripherally generated spikes were recorded (Fig. 1B); the level of alcohol at which additional spikes were recorded was taken as the threshold level. Perfusion with alcohol-saline decreased the amplitude of the spike but increased the amplitude of the DAP (Fig. 1A). When the level of alcohol in the saline was above a certain threshold level, additional peripherally generated spikes were recorded (Fig. 1B). Perfusion with saline containing lower levels of alcohol reversibly abolished the additional spikes, while perfusion with higher levels of alcohol produced a prolonged burst of action potentials (Fig. I C). These spikes traveled orthodromically down the axon (Fig. 1D), indicating that they were not generated in the peripheral axon branches. Peripheral spike generation was provoked by all alcohols except methanol, but the threshold level required to produce additional action potentials depended upon the alcohol used (Table I). It is clear from the data obtained for the primary alcohols that increasing the molecular weight decreased the threshold level required to produce peripheral spike generation (Table I). While it has been established that the alcohol-induced increase in DAP amplitude is responsible for the production of additional spikes [27, 32], the ion currents responsible for the DAP and the effects of alcohol on the membrane are not known. It seems possible that alcohol may interact with membrane proteins [12], enter the lipid phase of the membrane [8, 22], or act through some combination of both. It is interesting that, for primary alcohols, the membrane/buffer partition coefficient is directly proportional to its molecular weight (Table I). The partition coefficient values and the present data (Table I) are consistent with the hypothesis that alcohol enters the lipid phase of the membrane to produce peripheral spike generation. The higher the molecular weight, the higher
([Alcohol]) R E Q U I R E D
TO P R O D U C E
PERIPHERAL
SPIKE
G E N E R A T I O N IN FBE AXONS. The values are means ( + S.D.) for the number of preparations (n) tested for each alcohol. Membrane/buffer partition coefficient (Pm/b) values for the primary alcohols were obtained from ref. 22.
Alcohol
Formula
Pm/b
[Alcohol]
n
Methanol Ethanol l-Propanol I-Butanol l-Pentanol
CH3 (OH) CH3CH2 (OH) CH~CH~CH2 (OH) CH3(CH2)2CH2 (OH) CH3(CH2)3CH 2 (OH)
0.0045 0.14 0.45 1.5 3.6
> 500 348 (12.9) 103 (9.6) 63 (7.1) 32 (4.5)
6 12 4 8 5
2-Butanol 3-Butanol
CH3CH2CH(OH)CH3 (CH3)3C (OH)
362 ( 15.1 ) 262 (21.0)
4 6
the partition coefficient and the more readily the alcohol enters the lipid phase of the membrane. Accordingly, the saline level of alcohol required to produce additional spikes decreases as the molecular weight (and partition coefficient) is increased (Table I). The saline level of 2-butanol and tertiary-butanol required to produce peripheral spike generation is higher than for 1-butanol (Table I). Furthermore, the partition coefficients of these structural isomers are lower than the primary alcohol [22, 25]. These results again demonstrate that the partition coefficient of an alcohol is inversely proportional to the saline level required to produce additional spikes, suggesting that alcohol acts through the lipid phase of the membrane to induce peripheral spike generation. Recently it has been shown that alcohols bind to the phosphate group rather than to the hydrocarbon chains of membrane phospholipids [8]. These hydrogen bonds weaken the overall binding between the membrane and water, with the result that the membrane becomes disordered. Further evidence for the hypothesis that alcohol acts on the lipid portion of the membrane involves studies on the affects of temperature and acclimation on the threshold for peripheral spike generation [27, 31]. Warming the preparation above a critical threshold temperature (in the absence of alcohol) produces peripheral spike generation, and the threshold depends upon the acclimation temperature. It is interesting that acclimation [21] and alcohol [8, 17, 20, 27] effect membrane fluidity, and the resultant disorder in the membrane has a profound influence on the membrane proteins [2, 4, 7, 14, 16, 18]. Thus, it seems possible that ethanol and warming generate additional action potentials by changing the fluidity of the membrane lipids, which alters the properties of the channels that produce the DAP. Further, it has been shown that ethanol and temperature have an additive effect in this preparation (and others [3, 9, 23]), since warming decreases the level of ethanol required to produce peripheral spike generation [30]. Thus it would seem that ethanol and temperature act on at least one common target within the membrane. While alcohol and warming may change membrane fluidity to produce additional action potentials, the situation is complicated by the presence of a glial sheath surrounding the axon [33]. To date, it has not been determined whether the observed alcohol-induced effects are produced by changes in the membrane of the axon, the glial cell, or both. However, a recent study has shown that ethanol increases the level of potassium in the FBE axon and hyperpolarizes the resting potential [28]. This suggests that, irrespective of the site of alcohol's action, fundamental changes do take place within the FBE axon.
This work was funded by a grant from the National Institutes of Health (1 R15 NS27254-01) and by the Research Corporation. 1 Arhem, P. and Van Helden, D., Effects of aliphatic alcohols on myelinated nerve membrane, Acta Physiol. Scand., 119 (1983) 105107. 2 Armstrong, C.M. and Binstock, L., The effects of several alcohols on the properties of the squid giant axon. J. Gen. Physiol., 48 (1964) 265-277. 3 Bejanian, M., Alkana, R.L., Von Hungen, K., Baxter, C.F. and Syapin, P.J., Temperature alter ethanol-induced fluidization of C57 mouse brain membranes, Alcohol, 8 (1991) 117 121. 4 Bergmann, M.C., Klee, M.R. and Faber, D.S., Different sensitivities to ethanol of three early transient voltage clamp currents of Aplysia neurons, Pflfigers Arch., 348 (1974) 139 153. 5 Berry, M.S. and Penreath, V.M., The neurophysiology of alcohol. In M. Sandler (Ed.), Psychopharmacology of Alcohol, Raven, New York, 1980, pp. 43-72. 6 Blaustein, M.P. and Goldman, D.E., Competitive action of calcium and procaine on lobster axon, J. Gen. Physiol., 49 (1966) 10431063. 7 Chan, J. and Greenberg, D.A., Intracellular calcium in NCB-20 cells: elevation by depolarization and ethanol but not by glutamate, Brain Res., 539 (1991) 328 331. 8 Chiou, J.-S., Kuo, C.-C, Lin S.H., Kamaya, H. and Ueda, I., Interracial dehydration by alcohols: hydrogen bonding of alcohols to phospholipids, Alcohol, 8 (1991) 143 150. 9 Colley, C.M., Metcalfe, S.M., Turner, B., Burgen, A.S.V. and Metcalfe, J.C., The binding of benzyl alcohol to erythrocyte membranes, Biochim. Biophys. Acta, 233 (1971) 720-729. 10 Elliot, J.R. and Haydon, D.A., The actions of neutral anaesthetics on ion conductances of nerve membranes, Biochim. Biophys. Acta, 988 (1989) 257 286. 11 Faber, D.S. and Klee, M.R., Actions of ethanol on neuronal membrane properties and synaptic transmission. In K. Blum (Ed.), Alcohol and Opiates: Neurochemical and Behavioral Mechanism, Academic Press, New York (1977) 41~3. 12 Hille, B., Theories of anesthesia: general perturbations versus specific receptors. In B.R. Fink (Ed.), Molecular Mechanisms of Anesthesia, Progress in Anesthesia, Vol. 2, Raven, New York, 1980, pp. 1-5. 13 Hochner, B. and Spira, M.E., Two distinct propagating potentials in a single ethanol-treated axon, Brain Res., 398 (1986) 164-168. 14 Houck, D.J., Effects of alcohols on potentials of lobster axons, Am. J. Physiol., 216 (1969) 364-367. 15 Lazarus, R.E., Stephens, P.J. and Mindrebo, N., The peripheral generation of action potentials in excitatory motor neurons of a crab, J. Exp. Zool., 222 (1982) 129-136. 16 Messing, R.O., Carpenter, C.L., Diamond, I. and Greenberg, D.A., Ethanol regulates calcium channels in clonal neural cells, Proc. Natl. Acad. Sci., 83 (1986) 6213~215. 17 Metcalfe, J.C., Seeman, P. and Burgen, A.S.V., The proton relaxation of benzyl alcohol in erythrocyte membranes, Mol. Pharmacol., 4 (1968) 87 95. 18 Moore, J.W., Ulbricht, W. and Takata, M., Effects of ethanol on the sodium and potassium conductances of the squid giant axon membrane, J. Gen. Physiol., 48 (1964) 279 295. 19 Pasternostre, M. and Pichon, Y., Effects of N-alcohols on potassium conductance in squid giant axons, Eur. Biophys. J., 14 (1987) 279~88, 20 Paterson, S.J., Butler, K.W., Huang, P., Labelle, J., Smith, I.C,P.
21
22
23
24 25
26
27
and Schneider, H., The effects of alcohols on lipid bilayers: a spin label study, Biochim. Biophys. Acta, 266 (1972) 597-602. Pruitt, N.L., Adaptations to temperature in the cellular membranes of crustacea; membrane structure and metabolism, J. Therm. Biol., 15 (1990) 1-8. Roth, S. and Seeman, P., The membrane concentrations of neutral and positive anesthetics (alcohols, chlorpromazine, morphine) fit the Meyer-Overton rule of anesthesia; negative narcotics do not, Biochim. Biophys. Acta, 255 (1972) 207-219. Seeman, P., Temperature dependence of erythrocyte membrane expansion by alcohol anesthetics. Possible support for the partition theory of anesthesia, Biochim. Biophys. Acta, 183 (1969) 520-529. Seeman, P., The membrane actions of anesthetics and tranquillizers, Pharmacol. Rev., 24 (1972) 583~55. Seeman, P., Roth, S. and Schneider, H., The membrane concentrations of alcohol anesthetics, Biochim. Biophys. Acta, 225 (1971) 171-184. Silver, L.H. and Triestman, S.N., Effects of alcohol upon pacemaker activity in neurons of Aplysia californica., Cell. Mol. Neurobiol., 2 (1982) 215-225. Stephens, P.J., The effects of temperature and ethanol on the prop-
28
29 30
31 32
33
erties of the fast excitor axon to the bender muscle, Comp. Biochem. Physiol., 90A (1988) 341-347. Stephens, P.J., The effects of ethanol on intracellular potassium and the membrane potential of an identified crab motor axon, Comp. Biochem. Physiol., in press. Stephens, P.J. and Lazarus, R.E., Ethanol and temperature modify motor axon firing patterns, Brain Res., 229 (1981) 260-263. Stephens, P.J. and Atwood, H.L., Peripheral generation and modulation of crustacean motor axon activity at high temperatures, J. Comp. Physiol., 142 (198l) 309-314. Stephens, P.J. and Atwood, H.L., Thermal acclimation in a crustacean neuromuscular system, J. Exp. Biol., 98 (1982) 39-47. Stephens, P.J., Frascella, P.A. and Mindrebo, N., Effects of ethanol and temperature on a crab motor axon action potential: a possible mechanism for peripheral spike generation, J. Exp. Biol., 103 (1983) 289-301. Stephens, P.J., diCola, L.P., Church, P.J. and Dollahon, N.R., Spike afterpotentials in single, identified fast and slow motor neurons in the crab Pachygrapsus crassipes, Cornp. Biochem. Physiol., 93A (1989) 511-518.
3
© 1991 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/91/$ 03.50
NSL 08177
Differential effects of alcohols on the spike threshold of an identified motor axon in a crab (Pachygrapsus crassipes) Philip J. Stephens Villanova University, Department of Biology, Villanova, PA 19085 (U.S.A.} (Received 17 May 1991; Revised version received 31 July 1991; Accepted 5 August 199 I)
Key words: Alcohol; Spike threshold; Crab motor axon Observations were made on the fast bender excitor (FBE) axon in autotomized crab limbs bathed in salines made up with different alcohols. It has been shown previously that the presence of ethanol at a certain level causes a single action potential to generate additional spikes in the peripheral axon branches. The present study examines the level of different alcohols required to induce peripheral spike generation. For primary alcohols, increasing the molecular weight decreased the level of alcohol required to produce peripheral spike generation. The threshold level of 2butanol was greater than 1-butanol, but less than tertiary-butanol. These results are explained in terms of the partition coefficient, so that an alcohol with a higher partition coefficient enters the lipid bilayer more readily, thus a lower threshold level of that alcohol is required in the saline to generate additional spikes.
Anesthetics are lipid-soluble compounds which effect the cell membrane, rather than intracellular processes [6, 24], by reacting with the lipid phase of the membrane [8, 22], the ionic channels [12], or both. Electrophysiological techniques have revealed that anesthetics such as ethanol, have little effect on the resting potential [2, 5, 11] but have dramatic effects on the action potential. Ethanol decreases the voltage-sensitive inward sodium current and the delayed outward potassium current, decreases the rise time of the action potential and, above a certain concentration, blocks the spike altogether [1, 2, 4, 10, 14, 18, 19]. While ethanol, like most anesthetics, generally has a depressing effect on membrane excitability, there are some neurons that exhibit hyperexcitability in the presence of ethanol [13, 15, 26, 29]. In certain crab motor neurons, for example, the presence of ethanol above a critical level causes a single action potential to produce one or more additional spikes [30]. In these neurons the action potential is composed of a spike followed by a depolarizing afterpotential (DAP); ethanol increases the amplitude of the DAP and causes single action potentials to generate additional spikes in the peripheral axon branches [27, 32]. While work has been done on the effects of ethanol, little is known about the effects of other alcohols on the hyperexcitability of these neurons. The
Correspondence: P.J. Stephens, Villanova University, Department of Biology, Villanova, PA 19085, U.S.A.
present study addresses this problem using the crab fast bender excitor (FBE) axon and examines the threshold level of different alcohols (listed in Table I) required to elicit peripheral spike generation. Autotomized second and third walking limbs of the crab Pachygrapsus crassipes were held to the base of a plastic dish using dental wax. Preparations were immersed in a crab saline of the following composition: 470 mM NaC1, 8 mM KC1, 20 mM CaC12, 10 mM MgC12, 5 mM HEPES, pH 7.2. Fresh saline was perfused at a rate of about 10 ml/min and suction was applied to maintain saline volume in the preparation dish (9-11 ml) and to provide circulation; all experiments were performed at 22°C ( + I°C). Brief electrical shocks were applied to the exposed limb nerve and evoked action potentials were recorded through two glass microelectrodes inserted into the exposed FBE axon [27]. The microelectrode tips were separated by about 0.5 mm; examination of the time delay between the stimulus and the spike recorded at each location along the axon permitted the direction of spike propagation to be determined (Fig. 1). Observations were made in normal saline and in salines made up with various alcohols (listed in Table I); initial perfusion with a new saline took place at a rate of about 25 ml/min. Data were collected during and after a 10 min perfusion period; ethanol-induced changes in resting membrane potential and intracellular potassium occur within about 7 min of perfusion [28]. If no addi-
13
A
P
C
d tt___
__3
p~---
Fig. 1. The effect of alcohol-saline on the FBE action potential. A: a brief electrical shock was applied to the axon to produce an action potential in normal saline and in saline made up with 80 mM propanol (arrows). The peaks of the two spikes are superimposed to show the smaller spike and larger DAP recorded in the presence of alcohol. B: an evoked action potential recorded from two locations along the axon in saline made up with 50 mM 1-butanol. The electrical shock produced a spike which was recorded first at the proximally (p) located electrode (arrow) and then at the distally (d) located electrode. The second, additional spike was generated peripherally and traveled antidromically down the axon, since the spike was recorded first distally and then proximally (arrow). C, D: a brief shock applied to the limb nerve of a preparation bathed in saline made up with 60 mM l-butanol resulted in an action potential and 5 additional spikes. The additional spikes traveled orthodromically since each was recorded proximally (p) and then distally (d) - this is evident in D, which shows the first 3 spikes in C at a faster sweep speed. The arrow points to the peak of the first spike recorded at the proximally located electrode. Calibration: 10 mV and 0.5 ms (A), 4 ms (C), 1 ms (B, D).
tional spikes were recorded during the 10 min period, the preparation was perfused with saline made up with an increased (usually by 10 mM) level of alcohol. This proTABLE I THE THRESHOLD
CONCENTRATION
(mM) OF ALCOHOL
cess of increasing the alcohol level in a step-wise fashion was continued until additional, peripherally generated spikes were recorded (Fig. 1B); the level of alcohol at which additional spikes were recorded was taken as the threshold level. Perfusion with alcohol-saline decreased the amplitude of the spike but increased the amplitude of the DAP (Fig. 1A). When the level of alcohol in the saline was above a certain threshold level, additional peripherally generated spikes were recorded (Fig. 1B). Perfusion with saline containing lower levels of alcohol reversibly abolished the additional spikes, while perfusion with higher levels of alcohol produced a prolonged burst of action potentials (Fig. I C). These spikes traveled orthodromically down the axon (Fig. 1D), indicating that they were not generated in the peripheral axon branches. Peripheral spike generation was provoked by all alcohols except methanol, but the threshold level required to produce additional action potentials depended upon the alcohol used (Table I). It is clear from the data obtained for the primary alcohols that increasing the molecular weight decreased the threshold level required to produce peripheral spike generation (Table I). While it has been established that the alcohol-induced increase in DAP amplitude is responsible for the production of additional spikes [27, 32], the ion currents responsible for the DAP and the effects of alcohol on the membrane are not known. It seems possible that alcohol may interact with membrane proteins [12], enter the lipid phase of the membrane [8, 22], or act through some combination of both. It is interesting that, for primary alcohols, the membrane/buffer partition coefficient is directly proportional to its molecular weight (Table I). The partition coefficient values and the present data (Table I) are consistent with the hypothesis that alcohol enters the lipid phase of the membrane to produce peripheral spike generation. The higher the molecular weight, the higher
([Alcohol]) R E Q U I R E D
TO P R O D U C E
PERIPHERAL
SPIKE
G E N E R A T I O N IN FBE AXONS. The values are means ( + S.D.) for the number of preparations (n) tested for each alcohol. Membrane/buffer partition coefficient (Pm/b) values for the primary alcohols were obtained from ref. 22.
Alcohol
Formula
Pm/b
[Alcohol]
n
Methanol Ethanol l-Propanol I-Butanol l-Pentanol
CH3 (OH) CH3CH2 (OH) CH~CH~CH2 (OH) CH3(CH2)2CH2 (OH) CH3(CH2)3CH 2 (OH)
0.0045 0.14 0.45 1.5 3.6
> 500 348 (12.9) 103 (9.6) 63 (7.1) 32 (4.5)
6 12 4 8 5
2-Butanol 3-Butanol
CH3CH2CH(OH)CH3 (CH3)3C (OH)
362 ( 15.1 ) 262 (21.0)
4 6
the partition coefficient and the more readily the alcohol enters the lipid phase of the membrane. Accordingly, the saline level of alcohol required to produce additional spikes decreases as the molecular weight (and partition coefficient) is increased (Table I). The saline level of 2-butanol and tertiary-butanol required to produce peripheral spike generation is higher than for 1-butanol (Table I). Furthermore, the partition coefficients of these structural isomers are lower than the primary alcohol [22, 25]. These results again demonstrate that the partition coefficient of an alcohol is inversely proportional to the saline level required to produce additional spikes, suggesting that alcohol acts through the lipid phase of the membrane to induce peripheral spike generation. Recently it has been shown that alcohols bind to the phosphate group rather than to the hydrocarbon chains of membrane phospholipids [8]. These hydrogen bonds weaken the overall binding between the membrane and water, with the result that the membrane becomes disordered. Further evidence for the hypothesis that alcohol acts on the lipid portion of the membrane involves studies on the affects of temperature and acclimation on the threshold for peripheral spike generation [27, 31]. Warming the preparation above a critical threshold temperature (in the absence of alcohol) produces peripheral spike generation, and the threshold depends upon the acclimation temperature. It is interesting that acclimation [21] and alcohol [8, 17, 20, 27] effect membrane fluidity, and the resultant disorder in the membrane has a profound influence on the membrane proteins [2, 4, 7, 14, 16, 18]. Thus, it seems possible that ethanol and warming generate additional action potentials by changing the fluidity of the membrane lipids, which alters the properties of the channels that produce the DAP. Further, it has been shown that ethanol and temperature have an additive effect in this preparation (and others [3, 9, 23]), since warming decreases the level of ethanol required to produce peripheral spike generation [30]. Thus it would seem that ethanol and temperature act on at least one common target within the membrane. While alcohol and warming may change membrane fluidity to produce additional action potentials, the situation is complicated by the presence of a glial sheath surrounding the axon [33]. To date, it has not been determined whether the observed alcohol-induced effects are produced by changes in the membrane of the axon, the glial cell, or both. However, a recent study has shown that ethanol increases the level of potassium in the FBE axon and hyperpolarizes the resting potential [28]. This suggests that, irrespective of the site of alcohol's action, fundamental changes do take place within the FBE axon.
This work was funded by a grant from the National Institutes of Health (1 R15 NS27254-01) and by the Research Corporation. 1 Arhem, P. and Van Helden, D., Effects of aliphatic alcohols on myelinated nerve membrane, Acta Physiol. Scand., 119 (1983) 105107. 2 Armstrong, C.M. and Binstock, L., The effects of several alcohols on the properties of the squid giant axon. J. Gen. Physiol., 48 (1964) 265-277. 3 Bejanian, M., Alkana, R.L., Von Hungen, K., Baxter, C.F. and Syapin, P.J., Temperature alter ethanol-induced fluidization of C57 mouse brain membranes, Alcohol, 8 (1991) 117 121. 4 Bergmann, M.C., Klee, M.R. and Faber, D.S., Different sensitivities to ethanol of three early transient voltage clamp currents of Aplysia neurons, Pflfigers Arch., 348 (1974) 139 153. 5 Berry, M.S. and Penreath, V.M., The neurophysiology of alcohol. In M. Sandler (Ed.), Psychopharmacology of Alcohol, Raven, New York, 1980, pp. 43-72. 6 Blaustein, M.P. and Goldman, D.E., Competitive action of calcium and procaine on lobster axon, J. Gen. Physiol., 49 (1966) 10431063. 7 Chan, J. and Greenberg, D.A., Intracellular calcium in NCB-20 cells: elevation by depolarization and ethanol but not by glutamate, Brain Res., 539 (1991) 328 331. 8 Chiou, J.-S., Kuo, C.-C, Lin S.H., Kamaya, H. and Ueda, I., Interracial dehydration by alcohols: hydrogen bonding of alcohols to phospholipids, Alcohol, 8 (1991) 143 150. 9 Colley, C.M., Metcalfe, S.M., Turner, B., Burgen, A.S.V. and Metcalfe, J.C., The binding of benzyl alcohol to erythrocyte membranes, Biochim. Biophys. Acta, 233 (1971) 720-729. 10 Elliot, J.R. and Haydon, D.A., The actions of neutral anaesthetics on ion conductances of nerve membranes, Biochim. Biophys. Acta, 988 (1989) 257 286. 11 Faber, D.S. and Klee, M.R., Actions of ethanol on neuronal membrane properties and synaptic transmission. In K. Blum (Ed.), Alcohol and Opiates: Neurochemical and Behavioral Mechanism, Academic Press, New York (1977) 41~3. 12 Hille, B., Theories of anesthesia: general perturbations versus specific receptors. In B.R. Fink (Ed.), Molecular Mechanisms of Anesthesia, Progress in Anesthesia, Vol. 2, Raven, New York, 1980, pp. 1-5. 13 Hochner, B. and Spira, M.E., Two distinct propagating potentials in a single ethanol-treated axon, Brain Res., 398 (1986) 164-168. 14 Houck, D.J., Effects of alcohols on potentials of lobster axons, Am. J. Physiol., 216 (1969) 364-367. 15 Lazarus, R.E., Stephens, P.J. and Mindrebo, N., The peripheral generation of action potentials in excitatory motor neurons of a crab, J. Exp. Zool., 222 (1982) 129-136. 16 Messing, R.O., Carpenter, C.L., Diamond, I. and Greenberg, D.A., Ethanol regulates calcium channels in clonal neural cells, Proc. Natl. Acad. Sci., 83 (1986) 6213~215. 17 Metcalfe, J.C., Seeman, P. and Burgen, A.S.V., The proton relaxation of benzyl alcohol in erythrocyte membranes, Mol. Pharmacol., 4 (1968) 87 95. 18 Moore, J.W., Ulbricht, W. and Takata, M., Effects of ethanol on the sodium and potassium conductances of the squid giant axon membrane, J. Gen. Physiol., 48 (1964) 279 295. 19 Pasternostre, M. and Pichon, Y., Effects of N-alcohols on potassium conductance in squid giant axons, Eur. Biophys. J., 14 (1987) 279~88, 20 Paterson, S.J., Butler, K.W., Huang, P., Labelle, J., Smith, I.C,P.
21
22
23
24 25
26
27
and Schneider, H., The effects of alcohols on lipid bilayers: a spin label study, Biochim. Biophys. Acta, 266 (1972) 597-602. Pruitt, N.L., Adaptations to temperature in the cellular membranes of crustacea; membrane structure and metabolism, J. Therm. Biol., 15 (1990) 1-8. Roth, S. and Seeman, P., The membrane concentrations of neutral and positive anesthetics (alcohols, chlorpromazine, morphine) fit the Meyer-Overton rule of anesthesia; negative narcotics do not, Biochim. Biophys. Acta, 255 (1972) 207-219. Seeman, P., Temperature dependence of erythrocyte membrane expansion by alcohol anesthetics. Possible support for the partition theory of anesthesia, Biochim. Biophys. Acta, 183 (1969) 520-529. Seeman, P., The membrane actions of anesthetics and tranquillizers, Pharmacol. Rev., 24 (1972) 583~55. Seeman, P., Roth, S. and Schneider, H., The membrane concentrations of alcohol anesthetics, Biochim. Biophys. Acta, 225 (1971) 171-184. Silver, L.H. and Triestman, S.N., Effects of alcohol upon pacemaker activity in neurons of Aplysia californica., Cell. Mol. Neurobiol., 2 (1982) 215-225. Stephens, P.J., The effects of temperature and ethanol on the prop-
28
29 30
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erties of the fast excitor axon to the bender muscle, Comp. Biochem. Physiol., 90A (1988) 341-347. Stephens, P.J., The effects of ethanol on intracellular potassium and the membrane potential of an identified crab motor axon, Comp. Biochem. Physiol., in press. Stephens, P.J. and Lazarus, R.E., Ethanol and temperature modify motor axon firing patterns, Brain Res., 229 (1981) 260-263. Stephens, P.J. and Atwood, H.L., Peripheral generation and modulation of crustacean motor axon activity at high temperatures, J. Comp. Physiol., 142 (198l) 309-314. Stephens, P.J. and Atwood, H.L., Thermal acclimation in a crustacean neuromuscular system, J. Exp. Biol., 98 (1982) 39-47. Stephens, P.J., Frascella, P.A. and Mindrebo, N., Effects of ethanol and temperature on a crab motor axon action potential: a possible mechanism for peripheral spike generation, J. Exp. Biol., 103 (1983) 289-301. Stephens, P.J., diCola, L.P., Church, P.J. and Dollahon, N.R., Spike afterpotentials in single, identified fast and slow motor neurons in the crab Pachygrapsus crassipes, Cornp. Biochem. Physiol., 93A (1989) 511-518.