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Effects of electrical stimulation on the efflux of l -glutamate from peripheral nerve in vitro
EXPERIMENTAL NEUROLOGY ~{}, 291-296 (1971)
Effects of Electrical Stimulation on the Efflux of t-Glutamate from Peripheral Nerve in Vitro FRANCIS V. DEFEuBIS 1 Department of Anaesthesia Research, McGill Universit2, Montreal, Canada Received October 5, 1970 Desheathed frog sciatic nerves, previously loaded with labeled L-glutamate, released greater amounts of radioactivity when stimulated electrically, at intensities ranging from juxtathreshold to twice that needed to evoke maximal ~, p-action potentials. Incubations of control and stimulated companion nerves were carried out simultaneously. When an increase in the efflux of glutamate was not evident from the general shapes of the efflux curves, it became clear by determining the percentage of total radioactivity released in 120 min. It seems that there exists a discrete compartment of glutamate in peripheral nerve which is a part of the total exchangeable compartment and which can be released by electrical stimulation. The extra glutamate release accounts for about one-tenth of the total glutamate efflux and may be related to the increase in membrane permeability which occurs during conduction.
Introduction Recent reports, p r o m p t e d by studies of amino acid distribution (1, 8 ) , have described an influx and efftux of g l u t a m a t e in desheathed frog sciatic nerves (5, 6, 12, 13). I t has been claimed that after loading desheathed nerves w i t h labeled g l u t a m a t e at 8 ~ for 24 hr, an e x t r a release of radioactivity can be elicited by electrical stimulation of the nerve, and that the mechanism responsible for this m i g h t serve as a model for the release of t r a n s m i t t e r agents f r o m p r e s y n a p t i c terminals d u r i n g excitation ( 1 3 ) . T h e effects of electrical stimulation on the efflux of g l u t a m a t e from desheathed nerves have been studied further a n d the data suggest that the e x t r a glutamate is from the intracellular c o m p a r t m e n t of n e r v e a n d that it is relatively independent of stimulus intensity.
Materials and Methods T h e sources of animals, chemicals and reagents, composition of frog R i n g e r ' s solution, methods for d e s h e a t h i n g frog sciatic nerves, p r o c e d u r e s 1 This study was supported by the Medical Research Council of Canada. The author's present address: Department of Psychology, Purdue University, Lafayette, Indiana 47907. I thank Prof. K. Krnjevid for many helpful discussions on this work, and Miss Mary Jo Wagner for typing the manuscript. 291
292
DEFEUDIS
used for radioactive assay, and the general design employed in studies of glutamate efflux from nerve are presented in detail in previous reports (3, 4, 6, 10, 11, 13). Three nerve preparations, sheathed, "pre- and post-desheathed," ~ were used in the present studies. Experiments were performed on 46 nerves at room temperature during the summer. Companion nerves were used in most studies to control the frog-to-frog variation in permeability. W h e n the concentration of K § was increased (as K C I ) isotonicity was maintained by changing the Na § concentration. Electrical stimulation and recording of action potentials were carried out using a Grass SD 5 stimulator, a Grass P4 preamplifier, and a Nagard oscilloscope. Stimulation of nerves was carried out in small plastic chambers from which Ringer's solution containing the released radioactivity was collected (Fig. 1). Stimulation was carried out continuously for 60 min during which six changes in the bathing solution were made; aliquots were analyzed. Stimulation was begun after the rapid component of efflux and efflux was allowed to continue for 2 hr. The tetanizing electrical stimulation was varied from juxtathreshold to twice that needed to evoke maximal ~, fi -action potentials, and it was maintained at its initial suprathreshold strength by constantly checking thresholds. Nerves were stimulated for 0.04 msec, 50 or 150 pulse/sec. Action potentials were recorded from Trigger Circuit (starts sweep)
.1....__.~~
Ground electr-'3
"Qe ........................
1
rding electrodes
Central end
Peripheral end of nerve
of nerve
1~_5mm ~i~ Stim. comp.
15mm Collection comportment
~1"--8 rnm----~ Recording compartment
FIG. l. Schematic diagram of a four compartment plastic chamber used in studies of efflux of radioactivity from frog nerves. Solutions in the various compartments are indicated. Ringer's solution in the collection compartment (second from left) was changed at 10-rain intervals. The chamber was anchored to a ring stand on which were mounted stimulating, recording, and ground electrodes and the pipette through which oxygen was supplied. Abbreviations: PA, preamplifier; CRO, oscilloscope; Stim., stimulator. 2 "Predesheathed" refers to nerves stripped to their epi- plus- perineuria before being placed in labeled medium; "post-desheathed" refers to those which were desheathed after they had absorbed labeled medium.
GLUTAMATE
293
RELEASE
24 postdesheathed, four sheathed, and five predesheathed nerves after preincubation for 13-72 hr at 4~ in Ringer's solution containing 2.2 )< 10-~ M labeled glutamate (specific activity, 186 mc/mmole; New England Nuclear Corp. ; purified by chromatography). A length of nerve, 25-30 mm, was fixed at both ends of the chamber (Fig. 1) and a 15-mm middle segment was exposed to the collection fluids. After collections, the content of radioactivity in each segment was assayed for determining the total radioactivity initially present. Chambers were sealed free from leaks with wax and Vaseline. Using a pair of silver electrodes, recordings were made across a partition (resistance) separating two compartments containing Ringer's solution (Fig. 1). The central end of each nerve was stimulated with a bipolar electrode assembly; both the electrode and stimulated portion of nerve were immersed in paraffin. Collection fluids were continually bubbled with 100% 02. After experiments, nerves were checked for their abilities to conduct impulses and the amplitudes and waveforms of the action potentials were noted. Semilogarithmic plots were constructed and least square regression lines were calculated for estimation of rate constants. Results
Evidence that postdesheathed nerves suffered no major loss of function was obtained by demonstrating an undiminished response of the a,, /? group of fibers to stimulation. In Table 1 it is shown that it usually took about seven times threshold voltage to evoke maximal ~, fi -action potentials and that the threshold usually increased after nerves had been stimulated for 60 rain. The amplitudes of action potentials were greater in postthan in predesheathed nerves, averaging 6.8 mv in the former case. Diphasic action potentials could be evoked in all incubated postdesheathed nerves while those in predesheathed nerves were always monophasic. (In no cases were nerves crushed at the site of the recording electrode.) It is presumed that these changes in amplitude and waveform in predesheathed nerves are the result of degeneration particularly near the cut ends of the nerves. "FABLE 1 THRESHOLD VOLTAGES, VOLTAGES USED TO EVOKE ~IAXI:~IAL ~, [J+ACTIOx DOTENTI3,LS AND AMPLITUDES OF ~, B-AcTION POTENTIALS a Thresholds Preparation a n d preineubation time (hr) Postdesheathed, 21 36 Predesheathed, 13, 24 Sheathed, 70
Initial (v) 0.33~0.03(17) + 0.45, 0 . 4 8 0.42
After 120 min (v) 1.45• -i .6
Initial intensity to evoke max. actioll potential (v) 2.4:t=0.2(18) 6.0, 8 . 0 5.2
Amplitude (my) Initial 6.8• .0(11) 1.1, 1 . 8
After i20 m i n 3.6• 0.f, 0.l
" All stimulations were carried o u t for 0.04 msec a t 50-150 pulse/sec. Measuren~ents of thresholds a n d amplitudes were made immediately after placing nerves into chambers a n d a g a i n a t 120 m i n ; all nerves had been sthnttlated for 6ll rain before taking the last measurements. b M e a n s • s t a n d a r d errors of the means, or individual values; iltlmbers of nerves hi parentheses.
294
DEFEUDIS
Table 2 gives values of rate constants of slow efflux of radioactivity and percentages of total radioactivity released in 120 min, in control and stimulated nerves. In all cases, larger amounts of radioactivity were released with stimulation. (In one postdesheathed nerve and in the sheathed nerve, the increases were negligible.) In four cases shown in the table and in three cases not shown (since companion nerves were not used) a "peaked" release o~ radioactivity occurred with stimulation. There was no obvious correlation between stimulus intensity and the amount of extra radioactivity released, but a positive correlation is suggested by the values shown in Table 2. No action potentials could be monitored in nerves depolarized by high K + (110 m ~ ) , but a peaked release of radioactivity occurred in the two nerves treated in this way (Table 2). With constant parameters of stimulation, the extra release of radioactivity from nerves can be peaked (Fig. 2), or can be more or less linear, with an increase or a decrease in the magnitude of the rate constant for slow elflux. In Fig. 2 the release during the first period of stimulation (period 5) was 310% that of the preceding control period (period 4). Plots of percentage loss of radioactivity versus time showed that the initial intracellular radioactivity was about 62% in the two populations of nerves (companions). A portion of the extracellular glutamate ( < 20%) could have been due to accumulation of swelling fluid during preincubation; this artifact was controlled by using companion nerves which were preincubated under identical conditions. When differences in the percentage of radioactivity released in 120 rain between control and stinmlated nerves were tested statistically by pairing the observations (i.e, by taking the difference between percentage for stimulated and control), it was
TABLE 2 FIRST-ORDER I'~.ATE CONSI'ANIS IOR SLOW Et, FLUX AND PERCEN'tA<;E IJ.EIAqASI~ OF RADIOACTIVITYFROM IPRO(; NERVFS I~tI,;RSED IN INACTIVE FROI; I/INliER'$ SOLUTIONa Control nerves
Prelncuhation time (hr) Postde~heathed 24 24 21 24 24 24 24 24 21 24 Meaa • st~ Predesheathed 44 Sheathed 68
K,, sec "a ( X 104)
% Reh h~ 120 rain
1.28 1.89 0.81 1.46 I . 19 1.46 1.89 2.37 1.59 0.25 1.4 • 0.2
56.1 53.7 49.0 71.6 53.1 52.0 74.8 71.1 821 22.6 88.6 • 5.4
S d m u i a t e d nerves Stim freq. and voltage
K,, sec -~ (X 104)
% Rel. in 120 rain
Change % reh in 120 mif~
150, thresh 150, thresh 150, 1/2 max 150, max 150, max 50, max 150, nmx 150, max 50, 2 X max High K +
0.18 0.54 2.53 2.35 0. i8 p 2.57 P 2.04 P 1.5 =h 0.4
61.0 58.7 52.8 72.4 59.6 73.7 87.7 90.7 88.1 37.8 6 8 3 • 5.5
+4.9 +5.0 +3.8 +0.8 +6.5 +21.7 +12.9 +19.6 +7.0 +15.2 +9.7 • 2.3
1.81
154
High K +
p
30.7
+15.3
0.35
31.7
150, max
0.42
32.0
+0.3
" All nerves are arranged as companion sets; all were preineubated at 4~ and efflux was studied at 22~ All stimulations were for 60 ill{ll continuously, except where changes were made in electrolyte as noted above and ill the text. P, peaked release of radloactivky.
GLUTAMATE
295
RELEASE
2000-
looo
Post- De~ec~hed
80O
500 i 400 30<3 .c 2 0 0
E ~5
Stimulated
Nerve
IOO 8O
4C .-
V "--.aT
V
"~'---O,.,~ ^
Contr
"6
v
71% RELEASED IN 120 MIN
o,
1o 8
K S = 2.37
X
10 -4
- -
"~'~7
sec -I
"6
~.
4 3 2 1
210
I<
Stimulation
410
60 810 Time (min)
>[
1()0
120
FIG. 2. Semilogarithmic plots of the release of radioactivity from companion frog nerves. One nerve was stimulated for 60 min at an intensity strong enough to evoke maximal a, /?-action potentials (150 pulse/sec) ; the other served as a control. Percentages of total dpm released in 120 min and the value for Ks (in the control nerve) are indicated. Symbols: V, control; O, stimulated nerve; Q, period of stimulation. shown that the mean of the differences was significantly greater than zero ( P < 0.01) by the t test. Discussion
It seems that an increase in the release of glutamate does indeed occm when the nerves are stimulated electrically or depolarized by high K + These results confirm those of Wheeler, Boyarsky, and Brooks (13) who showed that a peaked release can occur with electrical stimulation. Hox~ever, the release may also be linear, and this can be monitored by using companion nerves and by estimating changes in the percentage of total radioactivity released over a given time interval. The extra release seems to be from the intracellular space of nerves since it was promoted by stimulation during the period of slow efflux. Estimation of the mean -value for the amount of extra glutamate actually followed by the tracer method used, using the increased release with stimulation of 9.7% (Table 2) of the 28,700 dpm released per stimulated nerve
296
DEFEUDIS
aud a s s u m i n g that metabolism at 4 ~ was negligible, gave 0.2 • 0.01 p m o l e / g wet wt "sec (10, SE). T h e relationship between glutamate release and the n e r v o u s impulse is obviously not simple. J u d g i n g from the limited n u m b e r of observations available, the e x t r a release seems to be rather poorly related to the stimulus intensity, a n d it can occur either very rapidly or in continuous linear fashion. Also, since it can be blocked by azide (13) it coincides well with the " e x t r a " o x y g e n uptake ( 2 ) , with the increased heat production ( 7 ) , and with the increased release of Ca "~ (9) which have all been shown to occur in "active" frog nerve. I n any case, one may postulate that the increase in m e m b r a n e permeability which occurs d u r i n g conduction would allow for an increase in efflux of glutamate from the fibers, or perhaps there is a reduced uptake due to slower glutamate p u m p i n g while the N a + - K § p u m p is active. References 1. APRISON, M. H., and R. WERlk[AN. 1968. A combined neurochemical and neurophysiological approach to identification of central nervous system transmitters, pp. 143-174. In "Neurosciences Research," Vol. 1. S. Ehrenpreis, and O. C. Solnitzky led.]. Academic Press, New York. 2. BRINK, F. JR., D. W. BRONK F. D. CARLSON, and C. M. CONNELLY. 1952. The oxygen uptake of active axons. Cold Spring Harbor Syrup. Quant. Biol. 18: 53-67. 3. DAINTY, J., and K. KRNJEVId. 1955. The rate of exchange of Na 24 in cat nerves. J. Physiol. London 128 : 489-503. 4. DEFEUDIS, F. V. 1968. "7-Aminobutyric Acid, Other Amino Acids and Electrolytes in Relation to Physiologic and Metabolic Activities of Brain." Doctoral Thesis, McGill University, Montreal. 5. DEFEuDIs, F. V. 1969. Glutamate fluxes in peripheral nerves. Second International Meeting International Society of Neurochemistry, Milan, p. 147. 6. DEFEuDIs, F. V. 1970. Role of the perineural sheath of peripheral nerve on fluxes of L-glutamate in vitro. Nature London 227 : 854-855. 7. FENO, T. P., and A. V. HILL. 1933. The effect of frequency of stimulation on the heat production of frog's nerve. Proe. Roy. Soc. London Ser. B. 113: 366-393. 8. GRAHAM,L. T. JR., R. P. SHANK, R. gERMAN, and M. H. APRISON. 1967. Distribution of some synaptic transmitter suspects in cat spinal cord: gtutamic acid, aspartic acid, ,/-aminobutyric acid, glycine and glutamine. Y. Neurochem. 14 : 465472. 9. KOKETSU,K., and S. MIYAMOTO. 1961. Release of calcium-45 from frog nerves during electrical activity. Nature London 189 : 402-403. 10. KRNJEVId, K. 1955. The distribution of Na and K in cat nerves. ]. Physiol. London 128: 473-488. 11. SHANES, A. M., and M. D. BERiVIAN. 1955. Penetration of the desheathed toad sciatic nerve by ions and molecules. II. Kinetics. J. Cell. Comp. Physiol. 45: 199-240. 12. WHEELER, D. D., and L. L. BOYARSKY. 1968. Influx of glutamic acid in peripheral nerve--characteristics of influx, f. Neuroehem. 15: 1019-1031. 13. WI-IEELER, D. D., L. L. BOYARSKY, and W. H. BROOKS. 1966. The release of amino acids from nerve during stimulation. Y. Cell. Physiol. 67 : 141-418.
Effects of Electrical Stimulation on the Efflux of t-Glutamate from Peripheral Nerve in Vitro FRANCIS V. DEFEuBIS 1 Department of Anaesthesia Research, McGill Universit2, Montreal, Canada Received October 5, 1970 Desheathed frog sciatic nerves, previously loaded with labeled L-glutamate, released greater amounts of radioactivity when stimulated electrically, at intensities ranging from juxtathreshold to twice that needed to evoke maximal ~, p-action potentials. Incubations of control and stimulated companion nerves were carried out simultaneously. When an increase in the efflux of glutamate was not evident from the general shapes of the efflux curves, it became clear by determining the percentage of total radioactivity released in 120 min. It seems that there exists a discrete compartment of glutamate in peripheral nerve which is a part of the total exchangeable compartment and which can be released by electrical stimulation. The extra glutamate release accounts for about one-tenth of the total glutamate efflux and may be related to the increase in membrane permeability which occurs during conduction.
Introduction Recent reports, p r o m p t e d by studies of amino acid distribution (1, 8 ) , have described an influx and efftux of g l u t a m a t e in desheathed frog sciatic nerves (5, 6, 12, 13). I t has been claimed that after loading desheathed nerves w i t h labeled g l u t a m a t e at 8 ~ for 24 hr, an e x t r a release of radioactivity can be elicited by electrical stimulation of the nerve, and that the mechanism responsible for this m i g h t serve as a model for the release of t r a n s m i t t e r agents f r o m p r e s y n a p t i c terminals d u r i n g excitation ( 1 3 ) . T h e effects of electrical stimulation on the efflux of g l u t a m a t e from desheathed nerves have been studied further a n d the data suggest that the e x t r a glutamate is from the intracellular c o m p a r t m e n t of n e r v e a n d that it is relatively independent of stimulus intensity.
Materials and Methods T h e sources of animals, chemicals and reagents, composition of frog R i n g e r ' s solution, methods for d e s h e a t h i n g frog sciatic nerves, p r o c e d u r e s 1 This study was supported by the Medical Research Council of Canada. The author's present address: Department of Psychology, Purdue University, Lafayette, Indiana 47907. I thank Prof. K. Krnjevid for many helpful discussions on this work, and Miss Mary Jo Wagner for typing the manuscript. 291
292
DEFEUDIS
used for radioactive assay, and the general design employed in studies of glutamate efflux from nerve are presented in detail in previous reports (3, 4, 6, 10, 11, 13). Three nerve preparations, sheathed, "pre- and post-desheathed," ~ were used in the present studies. Experiments were performed on 46 nerves at room temperature during the summer. Companion nerves were used in most studies to control the frog-to-frog variation in permeability. W h e n the concentration of K § was increased (as K C I ) isotonicity was maintained by changing the Na § concentration. Electrical stimulation and recording of action potentials were carried out using a Grass SD 5 stimulator, a Grass P4 preamplifier, and a Nagard oscilloscope. Stimulation of nerves was carried out in small plastic chambers from which Ringer's solution containing the released radioactivity was collected (Fig. 1). Stimulation was carried out continuously for 60 min during which six changes in the bathing solution were made; aliquots were analyzed. Stimulation was begun after the rapid component of efflux and efflux was allowed to continue for 2 hr. The tetanizing electrical stimulation was varied from juxtathreshold to twice that needed to evoke maximal ~, fi -action potentials, and it was maintained at its initial suprathreshold strength by constantly checking thresholds. Nerves were stimulated for 0.04 msec, 50 or 150 pulse/sec. Action potentials were recorded from Trigger Circuit (starts sweep)
.1....__.~~
Ground electr-'3
"Qe ........................
1
rding electrodes
Central end
Peripheral end of nerve
of nerve
1~_5mm ~i~ Stim. comp.
15mm Collection comportment
~1"--8 rnm----~ Recording compartment
FIG. l. Schematic diagram of a four compartment plastic chamber used in studies of efflux of radioactivity from frog nerves. Solutions in the various compartments are indicated. Ringer's solution in the collection compartment (second from left) was changed at 10-rain intervals. The chamber was anchored to a ring stand on which were mounted stimulating, recording, and ground electrodes and the pipette through which oxygen was supplied. Abbreviations: PA, preamplifier; CRO, oscilloscope; Stim., stimulator. 2 "Predesheathed" refers to nerves stripped to their epi- plus- perineuria before being placed in labeled medium; "post-desheathed" refers to those which were desheathed after they had absorbed labeled medium.
GLUTAMATE
293
RELEASE
24 postdesheathed, four sheathed, and five predesheathed nerves after preincubation for 13-72 hr at 4~ in Ringer's solution containing 2.2 )< 10-~ M labeled glutamate (specific activity, 186 mc/mmole; New England Nuclear Corp. ; purified by chromatography). A length of nerve, 25-30 mm, was fixed at both ends of the chamber (Fig. 1) and a 15-mm middle segment was exposed to the collection fluids. After collections, the content of radioactivity in each segment was assayed for determining the total radioactivity initially present. Chambers were sealed free from leaks with wax and Vaseline. Using a pair of silver electrodes, recordings were made across a partition (resistance) separating two compartments containing Ringer's solution (Fig. 1). The central end of each nerve was stimulated with a bipolar electrode assembly; both the electrode and stimulated portion of nerve were immersed in paraffin. Collection fluids were continually bubbled with 100% 02. After experiments, nerves were checked for their abilities to conduct impulses and the amplitudes and waveforms of the action potentials were noted. Semilogarithmic plots were constructed and least square regression lines were calculated for estimation of rate constants. Results
Evidence that postdesheathed nerves suffered no major loss of function was obtained by demonstrating an undiminished response of the a,, /? group of fibers to stimulation. In Table 1 it is shown that it usually took about seven times threshold voltage to evoke maximal ~, fi -action potentials and that the threshold usually increased after nerves had been stimulated for 60 rain. The amplitudes of action potentials were greater in postthan in predesheathed nerves, averaging 6.8 mv in the former case. Diphasic action potentials could be evoked in all incubated postdesheathed nerves while those in predesheathed nerves were always monophasic. (In no cases were nerves crushed at the site of the recording electrode.) It is presumed that these changes in amplitude and waveform in predesheathed nerves are the result of degeneration particularly near the cut ends of the nerves. "FABLE 1 THRESHOLD VOLTAGES, VOLTAGES USED TO EVOKE ~IAXI:~IAL ~, [J+ACTIOx DOTENTI3,LS AND AMPLITUDES OF ~, B-AcTION POTENTIALS a Thresholds Preparation a n d preineubation time (hr) Postdesheathed, 21 36 Predesheathed, 13, 24 Sheathed, 70
Initial (v) 0.33~0.03(17) + 0.45, 0 . 4 8 0.42
After 120 min (v) 1.45• -i .6
Initial intensity to evoke max. actioll potential (v) 2.4:t=0.2(18) 6.0, 8 . 0 5.2
Amplitude (my) Initial 6.8• .0(11) 1.1, 1 . 8
After i20 m i n 3.6• 0.f, 0.l
" All stimulations were carried o u t for 0.04 msec a t 50-150 pulse/sec. Measuren~ents of thresholds a n d amplitudes were made immediately after placing nerves into chambers a n d a g a i n a t 120 m i n ; all nerves had been sthnttlated for 6ll rain before taking the last measurements. b M e a n s • s t a n d a r d errors of the means, or individual values; iltlmbers of nerves hi parentheses.
294
DEFEUDIS
Table 2 gives values of rate constants of slow efflux of radioactivity and percentages of total radioactivity released in 120 min, in control and stimulated nerves. In all cases, larger amounts of radioactivity were released with stimulation. (In one postdesheathed nerve and in the sheathed nerve, the increases were negligible.) In four cases shown in the table and in three cases not shown (since companion nerves were not used) a "peaked" release o~ radioactivity occurred with stimulation. There was no obvious correlation between stimulus intensity and the amount of extra radioactivity released, but a positive correlation is suggested by the values shown in Table 2. No action potentials could be monitored in nerves depolarized by high K + (110 m ~ ) , but a peaked release of radioactivity occurred in the two nerves treated in this way (Table 2). With constant parameters of stimulation, the extra release of radioactivity from nerves can be peaked (Fig. 2), or can be more or less linear, with an increase or a decrease in the magnitude of the rate constant for slow elflux. In Fig. 2 the release during the first period of stimulation (period 5) was 310% that of the preceding control period (period 4). Plots of percentage loss of radioactivity versus time showed that the initial intracellular radioactivity was about 62% in the two populations of nerves (companions). A portion of the extracellular glutamate ( < 20%) could have been due to accumulation of swelling fluid during preincubation; this artifact was controlled by using companion nerves which were preincubated under identical conditions. When differences in the percentage of radioactivity released in 120 rain between control and stinmlated nerves were tested statistically by pairing the observations (i.e, by taking the difference between percentage for stimulated and control), it was
TABLE 2 FIRST-ORDER I'~.ATE CONSI'ANIS IOR SLOW Et, FLUX AND PERCEN'tA<;E IJ.EIAqASI~ OF RADIOACTIVITYFROM IPRO(; NERVFS I~tI,;RSED IN INACTIVE FROI; I/INliER'$ SOLUTIONa Control nerves
Prelncuhation time (hr) Postde~heathed 24 24 21 24 24 24 24 24 21 24 Meaa • st~ Predesheathed 44 Sheathed 68
K,, sec "a ( X 104)
% Reh h~ 120 rain
1.28 1.89 0.81 1.46 I . 19 1.46 1.89 2.37 1.59 0.25 1.4 • 0.2
56.1 53.7 49.0 71.6 53.1 52.0 74.8 71.1 821 22.6 88.6 • 5.4
S d m u i a t e d nerves Stim freq. and voltage
K,, sec -~ (X 104)
% Rel. in 120 rain
Change % reh in 120 mif~
150, thresh 150, thresh 150, 1/2 max 150, max 150, max 50, max 150, nmx 150, max 50, 2 X max High K +
0.18 0.54 2.53 2.35 0. i8 p 2.57 P 2.04 P 1.5 =h 0.4
61.0 58.7 52.8 72.4 59.6 73.7 87.7 90.7 88.1 37.8 6 8 3 • 5.5
+4.9 +5.0 +3.8 +0.8 +6.5 +21.7 +12.9 +19.6 +7.0 +15.2 +9.7 • 2.3
1.81
154
High K +
p
30.7
+15.3
0.35
31.7
150, max
0.42
32.0
+0.3
" All nerves are arranged as companion sets; all were preineubated at 4~ and efflux was studied at 22~ All stimulations were for 60 ill{ll continuously, except where changes were made in electrolyte as noted above and ill the text. P, peaked release of radloactivky.
GLUTAMATE
295
RELEASE
2000-
looo
Post- De~ec~hed
80O
500 i 400 30<3 .c 2 0 0
E ~5
Stimulated
Nerve
IOO 8O
4C .-
V "--.aT
V
"~'---O,.,~ ^
Contr
"6
v
71% RELEASED IN 120 MIN
o,
1o 8
K S = 2.37
X
10 -4
- -
"~'~7
sec -I
"6
~.
4 3 2 1
210
I<
Stimulation
410
60 810 Time (min)
>[
1()0
120
FIG. 2. Semilogarithmic plots of the release of radioactivity from companion frog nerves. One nerve was stimulated for 60 min at an intensity strong enough to evoke maximal a, /?-action potentials (150 pulse/sec) ; the other served as a control. Percentages of total dpm released in 120 min and the value for Ks (in the control nerve) are indicated. Symbols: V, control; O, stimulated nerve; Q, period of stimulation. shown that the mean of the differences was significantly greater than zero ( P < 0.01) by the t test. Discussion
It seems that an increase in the release of glutamate does indeed occm when the nerves are stimulated electrically or depolarized by high K + These results confirm those of Wheeler, Boyarsky, and Brooks (13) who showed that a peaked release can occur with electrical stimulation. Hox~ever, the release may also be linear, and this can be monitored by using companion nerves and by estimating changes in the percentage of total radioactivity released over a given time interval. The extra release seems to be from the intracellular space of nerves since it was promoted by stimulation during the period of slow efflux. Estimation of the mean -value for the amount of extra glutamate actually followed by the tracer method used, using the increased release with stimulation of 9.7% (Table 2) of the 28,700 dpm released per stimulated nerve
296
DEFEUDIS
aud a s s u m i n g that metabolism at 4 ~ was negligible, gave 0.2 • 0.01 p m o l e / g wet wt "sec (10, SE). T h e relationship between glutamate release and the n e r v o u s impulse is obviously not simple. J u d g i n g from the limited n u m b e r of observations available, the e x t r a release seems to be rather poorly related to the stimulus intensity, a n d it can occur either very rapidly or in continuous linear fashion. Also, since it can be blocked by azide (13) it coincides well with the " e x t r a " o x y g e n uptake ( 2 ) , with the increased heat production ( 7 ) , and with the increased release of Ca "~ (9) which have all been shown to occur in "active" frog nerve. I n any case, one may postulate that the increase in m e m b r a n e permeability which occurs d u r i n g conduction would allow for an increase in efflux of glutamate from the fibers, or perhaps there is a reduced uptake due to slower glutamate p u m p i n g while the N a + - K § p u m p is active. References 1. APRISON, M. H., and R. WERlk[AN. 1968. A combined neurochemical and neurophysiological approach to identification of central nervous system transmitters, pp. 143-174. In "Neurosciences Research," Vol. 1. S. Ehrenpreis, and O. C. Solnitzky led.]. Academic Press, New York. 2. BRINK, F. JR., D. W. BRONK F. D. CARLSON, and C. M. CONNELLY. 1952. The oxygen uptake of active axons. Cold Spring Harbor Syrup. Quant. Biol. 18: 53-67. 3. DAINTY, J., and K. KRNJEVId. 1955. The rate of exchange of Na 24 in cat nerves. J. Physiol. London 128 : 489-503. 4. DEFEUDIS, F. V. 1968. "7-Aminobutyric Acid, Other Amino Acids and Electrolytes in Relation to Physiologic and Metabolic Activities of Brain." Doctoral Thesis, McGill University, Montreal. 5. DEFEuDIs, F. V. 1969. Glutamate fluxes in peripheral nerves. Second International Meeting International Society of Neurochemistry, Milan, p. 147. 6. DEFEuDIs, F. V. 1970. Role of the perineural sheath of peripheral nerve on fluxes of L-glutamate in vitro. Nature London 227 : 854-855. 7. FENO, T. P., and A. V. HILL. 1933. The effect of frequency of stimulation on the heat production of frog's nerve. Proe. Roy. Soc. London Ser. B. 113: 366-393. 8. GRAHAM,L. T. JR., R. P. SHANK, R. gERMAN, and M. H. APRISON. 1967. Distribution of some synaptic transmitter suspects in cat spinal cord: gtutamic acid, aspartic acid, ,/-aminobutyric acid, glycine and glutamine. Y. Neurochem. 14 : 465472. 9. KOKETSU,K., and S. MIYAMOTO. 1961. Release of calcium-45 from frog nerves during electrical activity. Nature London 189 : 402-403. 10. KRNJEVId, K. 1955. The distribution of Na and K in cat nerves. ]. Physiol. London 128: 473-488. 11. SHANES, A. M., and M. D. BERiVIAN. 1955. Penetration of the desheathed toad sciatic nerve by ions and molecules. II. Kinetics. J. Cell. Comp. Physiol. 45: 199-240. 12. WHEELER, D. D., and L. L. BOYARSKY. 1968. Influx of glutamic acid in peripheral nerve--characteristics of influx, f. Neuroehem. 15: 1019-1031. 13. WI-IEELER, D. D., L. L. BOYARSKY, and W. H. BROOKS. 1966. The release of amino acids from nerve during stimulation. Y. Cell. Physiol. 67 : 141-418.