Brain Research, 89 (1975) 187-191
187
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Microiontophoretic studies of the effects of L-proline on neurons in the mammalian central nervous system
PETER ZARZECKI*, PAUL S. BLUM**, GARY E. CORDINGLEY AND GEORGE G. SOMJEN Department of Physiology and Pharmacology, Duke University, Durham, N.C. 277/0 (U.S.A.)
(Accepted February 11th, 1975)
In studies aimed at deciding the function of glutamate in synaptic transmission 3,5-v it would be helpful to have available a selective pharmacological antagonist to support the notion of glutamate-mediated synaptic excitation. Several derivatives of the stimulant amino acids have been proposed as specific glutamate antagonists, but clear evidence for their effect is lacking v. Our attention was called to e-proline since Van Harreveld and Fitkov/O 1 have reported that it reversibly and apparently competitively prevents the glutamate-induced increase of the transparency of the chick retina. The experiments described here were designed to discover whether proline is able to block excitation of central nervous system neurons by glutamate. Particular attention was paid to regional differences in the effects. We found variable effects ranging from no effect at all, to proline reversibly blocking in some cases and potentiating in others the glutamate-induced activity of single neurons. While our work was carried out, Felix and Ktinzle 4 demonstrated a depressant action of proline upon neurons of the cerebellum. Some of our results have already been reported in abstract form 12. We have applied proline from microiontophoretic electrodes in attempts to modify glutamate-induced excitation of neurons in three areas of the CNS. Proline solutions (0.5-2.0 M) were usually adjusted to a pH of 7.5-9.0. Some difficulty was encountered in passing prolonged currents from proline-containing barrels, even with barrels of low resistance (less than 50 M[]). It was sometimes observed that electrodes able to carry up to 150 nA for 15-30 rain through the proline barrel into saline, would only allow the passage of 5-10 nA into brain. To determine the effect of proline, a continuous record of the firing frequency of single neurons was made. With each neuron the response to a standard application of glutamate (1.0 M, pH 7.3, 5-25 sec, 10-100 hA) or aspartate (I.0 M, pH 8.2) was recorded before, during and after a * Present address: The Rockefeller University, New York, N.Y., U.S.A. ** Present address: Columbia University College of Physicians and Surgeons, Dept. of Neurology, New York, N.Y., U.S.A.
188 A. Proline-,10 T
u~
15]
~o E
g a J
B
g
g o
o
g og
o
go
(
I rain. Proline -~30
T
~ 5 o/ .;'~ .' , ' ~
.
,;T
~
,,
J:
,~
~.~
,
,
,
,I
,,.~ ,~ /~, / . -
E
#
2 rain.
I
C.
T
P~oline-,40
F
'
~'~
2 rnin.
Fig. 1. The antagonistic effect of iontophoretically applied L-proline upon glutamate-induced excitation of neurons in: A, the cerebral cortex; B, the spinal cord; and its lack of effect in C, the cuneate nucleus. The records are ratemeter outputs displayed on an ink-writing polygraph. Vertical deflections of the pen correspond to the number of impulses occurring within successive time periods of predetermined length. The periods of drug application are indicated by horizontal lines, and the currents used are given in nA. The cortical neuron illustrated in A was stimulated by alternate
8 sec applications of 40 nA currents of glutamate (g) and aspartate (a). The spinal neuron of B was stimulated by repeated applications of 35 nA of glutamate; cuneate neurons in C by repeated iontophoresis of 40 nA of glutamate. Gap in tracing in A is of 0.5 rain duration.
period of proline application. Control currents from NaC1 solutions (1.0 M) had no effect comparable to those of proline or glutamate. These tests were carried out in the cerebral cortex, the spinal cord and the cuneate nucleus of cats. In animals to be used for the study of cerebral cortical neurons, the cortex was exposed under ether anesthesia. Electrical recordings were made after discontinuation of the ether, and locally anesthetizing wound edges with lidocaine. Electrocorticograms dominated by slow waves and sleep spindles demonstrated the effectiveness of the local anesthesia. Spinal cord neurons were studied in unanesthetized decapitate preparations and in intact animals under barbiturate anesthesia. Cuneate nucleus neurons were tested in cats with forebrains isolated by rostropontine transection, or under chtoralose anesthesia. Neuromuscular paralysis was maintained with gallamine. The effect of proline was determined on 18 neurons of the cerebral cortex which were stimulated by the application of glutamate. Our criterion for accepting an effect
189
A, _
,P,~o], ~0
E
,*.t
0
~
CI-,2O
La,("d.~
~
~
~-/
~.,~ ~
~
~
'
B,
Pr-30
C1"30 Pr- ~,0
Pr"_.20 Pr-lO i
Pr.~-5
Pr-_.O P~'-_-5
Pr-O
!
Imin.
Fig. 2. Examples of: A, the potentiation of glutamate-induced excitation by proline; and B, independent proline-induced excitation of spinal interneurons. Polygraph records of ratemeter outputs as in Fig. 1. A: the response to repeated application of 65 nA of glutamate is potentiated during proline iontophoresis. Similar current carried by chloride ion (CI , 20) had no effect, B: the excitatory responses of another neuron to varying doses of proline. Simply removing the retaining current (Pr-, 0) allowed the diffusional release of sufficient proline to cause excitation. A retaining current of 5 nA (Pr-, --5) was adequate to prevent this diffusional release. Also shown is the negative current control (CI , 30). Seven minutes of recording are deleted at gap in tracing in B.
as being induced by proline was a reversible change of firing rate of at least 50 ~o of the pretreatment control level. N o effect was registered if this criterion was not achieved in 5-12 rain of drug application. Proline applied by currents o f 5-65 nA (electrode tip negative) reversibly prevented or reduced the excitation of 9 cells (50 ~o). This proline-induced antagonism of glutamate had a latency from several seconds to 3 min on different cells and was repeatable u p o n individual neurons. N o t included in these numbers, are cells which were apparently lost before the reversibility o f the effect could be determined and cases where the proline-filled barrel failed to deriver prolonged current. Fig. 1A illustrates the antagonistic effect of proline against excitation by both glutamate and aspartate. Equal degrees of antagonism were seen whenever both excitant amino acids were tested. The onset of the effect had a latency varying from a few seconds to 3 rain and the effect persisted for 10-170 sec following cessation o f iontophoresis. Proline did not usually alter the firing o f 'spontaneously' active cells, but a slight enhancement of u n p r o v o k e d activity was seen twice, and a small depression also twice. Spike amplitudes did not change during proline application.
190 In the cerebral cortex, the excitation of cells by glutamate was never enhanced by proline. By contrast in the spinal cord we found several examples where proline potentiated tile effect of glutamate. Fig. 2A illustrates one such case, and also demonstrates that current carried by CI ~ had no comparable effect. In this case, ~iae effect of proline was purely one of potentiating glutamate, for proline alone did not excite this cell. In the case illustrated in Fig. 2B, proline applied by itself had an excitant action. The very great sensitivity of this cell to stimulation by proline is noteworthy. Of 24 interneurons in the spinal cord, proline antagonized the effect of glutamate in 5 cases (Fig. 1 B); potentiated glutamate in 6; excited 2 cells but did not influence their glutamate-evoked firing; had no effect by itself and did not interact with glutamate in 11. Proline excited 3 additional cells upon which its interaction with glutamate could not be tested for technical reasons. Unlike the cerebral cortex and the spinal cord, in the cuneate nucleus proline failed to influence the activity of 24 neurons, including both interneurons and relay cells. Identification of the two types was by criteria similar to those established by Anderson et al. 1. An example of this lack of effect is seen in Fig. lC. Neither the firing evoked by stimulation of the superficial radial nerve, nor spontaneous or glutamate-evoked activity were altered during application of proline up to 40 nA tbr 12 min. Glutamate diethyl ester applied in the same manner, and with similar current intensities, had the depressant effect on glutamate-induced excitation which was already described by others 2,1°. The effectiveness of the diethyl ester indicates that drug-induced inhibition was not hindered in these experiments by technical failure. These observations suggest that proline is an antagonist of glutamate in selected areas of the central nervous system of cats. The differences in effects on cells found in different parts of the CNS, and indeed on different cells within the same tissue, may be related to a selective affinity of this compound for specific subsynaptic sites. This claim has relevance in view of McLennan's 8,9 proposal that there may be two types of receptors for excitant amino acids, One unspecific (affected by any excitant amino acid and found on most neurons) and the other specifically activated by glutamate and perhaps identical with the excitatory synaptic receptor. Proline is clearly not a universal blocker of the effects of glutamate; however, proline in lhct may be an antagonist at sites where glutamate acts as a synaptic transmitter but not where it acts as a non-specific stimulant. Felix and Kfinzle 4 have suggested that naturally occurring proline may have a physiological role in some parts of the mammalian central nervous system. Our results are compatible with this proposal, although not directly supporting it. Supported by grants of the U.S.P.H;S., NS 05330 and NS 10507.
] ANDERSON,P., ECCLES, J. C., SCHM1DT,R. F., AND YOKOTA,T., Identification of relay cells and interneurons in the cuneate nucleus, J. Neurophysio/., 27 (1964) 1080-1095. 2 CURTIS,D. R., DUGGAN,A. W., FELIX,D., JOHNSTON,G. A. R., TEB~OS,A. K., AND WATKINS, J. C., Excitation of mammalian central neurons by acidic amino acids, Brain Research, 41 (1972)
283-301.
191 3 CURTlS, D. R., AND JOHNSTON, G. A. R., Amino acid transmitters in the mammalian central nervous system, Ergebn. Physiol., 69 (1974) 97-188. 4 FELlX, D., AND K(ONZLE, H., lontophoretic and autoradiographic studies on the role of proline in nervous transmission, Pfliigers Arch. ges. Physiol., 350 (1974) 135-144. 5 FLOREY, E., Neurotransmitters and modulators in the animal kingdom, Fed. Proc., 26 (1967) 1164-1178. 6 JOHNSON, J. L., Glutamic acid as a synaptic transmitter in the nervous system. A review, Brain Research, 37 (1972) 1-19. 7 KRNJEVI6, K., Chemical nature of synaptic transmission in vertebrates, Physiol. Rev., 54 (1974) 418 540. 8 MCLENNAN, H., Actions of excitatory amino acids and their antagonism, Neuropharmacology~ 13 (1974) 449-454. 9 McLENNAN, H., HUFFMAN, R. D., AND MARSHALL, K. C., Patterns of excitation of thalamic neurons by amino acids and by acetylcholine, Nature (Lond.), 219 (1968) 387-388. 10 MCLENNAN, H., MARSHALL, K. C., AND HUFF~aAN, R. D., The antagonism of glutamate action at central neurones, Experientia (Basel), 27 (1971) 1116. 11 VAN HARREWLO, A., AND F~FKOVX, E., EffEcts of amino acids on the isolated chicken retina, and on its response to glutamate stimulation, J. Neurochem., 20 (1973) 947-962. 12 ZARZECKL P., BLUM, P., AND SOMJEN, G., Interaction of L-proline and glutamate upon neurons in the central nervous system of the cat, Fourth Annual Meeting, Society for Neuroscience, (1974) 494 (abstract).
187
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Microiontophoretic studies of the effects of L-proline on neurons in the mammalian central nervous system
PETER ZARZECKI*, PAUL S. BLUM**, GARY E. CORDINGLEY AND GEORGE G. SOMJEN Department of Physiology and Pharmacology, Duke University, Durham, N.C. 277/0 (U.S.A.)
(Accepted February 11th, 1975)
In studies aimed at deciding the function of glutamate in synaptic transmission 3,5-v it would be helpful to have available a selective pharmacological antagonist to support the notion of glutamate-mediated synaptic excitation. Several derivatives of the stimulant amino acids have been proposed as specific glutamate antagonists, but clear evidence for their effect is lacking v. Our attention was called to e-proline since Van Harreveld and Fitkov/O 1 have reported that it reversibly and apparently competitively prevents the glutamate-induced increase of the transparency of the chick retina. The experiments described here were designed to discover whether proline is able to block excitation of central nervous system neurons by glutamate. Particular attention was paid to regional differences in the effects. We found variable effects ranging from no effect at all, to proline reversibly blocking in some cases and potentiating in others the glutamate-induced activity of single neurons. While our work was carried out, Felix and Ktinzle 4 demonstrated a depressant action of proline upon neurons of the cerebellum. Some of our results have already been reported in abstract form 12. We have applied proline from microiontophoretic electrodes in attempts to modify glutamate-induced excitation of neurons in three areas of the CNS. Proline solutions (0.5-2.0 M) were usually adjusted to a pH of 7.5-9.0. Some difficulty was encountered in passing prolonged currents from proline-containing barrels, even with barrels of low resistance (less than 50 M[]). It was sometimes observed that electrodes able to carry up to 150 nA for 15-30 rain through the proline barrel into saline, would only allow the passage of 5-10 nA into brain. To determine the effect of proline, a continuous record of the firing frequency of single neurons was made. With each neuron the response to a standard application of glutamate (1.0 M, pH 7.3, 5-25 sec, 10-100 hA) or aspartate (I.0 M, pH 8.2) was recorded before, during and after a * Present address: The Rockefeller University, New York, N.Y., U.S.A. ** Present address: Columbia University College of Physicians and Surgeons, Dept. of Neurology, New York, N.Y., U.S.A.
188 A. Proline-,10 T
u~
15]
~o E
g a J
B
g
g o
o
g og
o
go
(
I rain. Proline -~30
T
~ 5 o/ .;'~ .' , ' ~
.
,;T
~
,,
J:
,~
~.~
,
,
,
,I
,,.~ ,~ /~, / . -
E
#
2 rain.
I
C.
T
P~oline-,40
F
'
~'~
2 rnin.
Fig. 1. The antagonistic effect of iontophoretically applied L-proline upon glutamate-induced excitation of neurons in: A, the cerebral cortex; B, the spinal cord; and its lack of effect in C, the cuneate nucleus. The records are ratemeter outputs displayed on an ink-writing polygraph. Vertical deflections of the pen correspond to the number of impulses occurring within successive time periods of predetermined length. The periods of drug application are indicated by horizontal lines, and the currents used are given in nA. The cortical neuron illustrated in A was stimulated by alternate
8 sec applications of 40 nA currents of glutamate (g) and aspartate (a). The spinal neuron of B was stimulated by repeated applications of 35 nA of glutamate; cuneate neurons in C by repeated iontophoresis of 40 nA of glutamate. Gap in tracing in A is of 0.5 rain duration.
period of proline application. Control currents from NaC1 solutions (1.0 M) had no effect comparable to those of proline or glutamate. These tests were carried out in the cerebral cortex, the spinal cord and the cuneate nucleus of cats. In animals to be used for the study of cerebral cortical neurons, the cortex was exposed under ether anesthesia. Electrical recordings were made after discontinuation of the ether, and locally anesthetizing wound edges with lidocaine. Electrocorticograms dominated by slow waves and sleep spindles demonstrated the effectiveness of the local anesthesia. Spinal cord neurons were studied in unanesthetized decapitate preparations and in intact animals under barbiturate anesthesia. Cuneate nucleus neurons were tested in cats with forebrains isolated by rostropontine transection, or under chtoralose anesthesia. Neuromuscular paralysis was maintained with gallamine. The effect of proline was determined on 18 neurons of the cerebral cortex which were stimulated by the application of glutamate. Our criterion for accepting an effect
189
A, _
,P,~o], ~0
E
,*.t
0
~
CI-,2O
La,("d.~
~
~
~-/
~.,~ ~
~
~
'
B,
Pr-30
C1"30 Pr- ~,0
Pr"_.20 Pr-lO i
Pr.~-5
Pr-_.O P~'-_-5
Pr-O
!
Imin.
Fig. 2. Examples of: A, the potentiation of glutamate-induced excitation by proline; and B, independent proline-induced excitation of spinal interneurons. Polygraph records of ratemeter outputs as in Fig. 1. A: the response to repeated application of 65 nA of glutamate is potentiated during proline iontophoresis. Similar current carried by chloride ion (CI , 20) had no effect, B: the excitatory responses of another neuron to varying doses of proline. Simply removing the retaining current (Pr-, 0) allowed the diffusional release of sufficient proline to cause excitation. A retaining current of 5 nA (Pr-, --5) was adequate to prevent this diffusional release. Also shown is the negative current control (CI , 30). Seven minutes of recording are deleted at gap in tracing in B.
as being induced by proline was a reversible change of firing rate of at least 50 ~o of the pretreatment control level. N o effect was registered if this criterion was not achieved in 5-12 rain of drug application. Proline applied by currents o f 5-65 nA (electrode tip negative) reversibly prevented or reduced the excitation of 9 cells (50 ~o). This proline-induced antagonism of glutamate had a latency from several seconds to 3 min on different cells and was repeatable u p o n individual neurons. N o t included in these numbers, are cells which were apparently lost before the reversibility o f the effect could be determined and cases where the proline-filled barrel failed to deriver prolonged current. Fig. 1A illustrates the antagonistic effect of proline against excitation by both glutamate and aspartate. Equal degrees of antagonism were seen whenever both excitant amino acids were tested. The onset of the effect had a latency varying from a few seconds to 3 rain and the effect persisted for 10-170 sec following cessation o f iontophoresis. Proline did not usually alter the firing o f 'spontaneously' active cells, but a slight enhancement of u n p r o v o k e d activity was seen twice, and a small depression also twice. Spike amplitudes did not change during proline application.
190 In the cerebral cortex, the excitation of cells by glutamate was never enhanced by proline. By contrast in the spinal cord we found several examples where proline potentiated tile effect of glutamate. Fig. 2A illustrates one such case, and also demonstrates that current carried by CI ~ had no comparable effect. In this case, ~iae effect of proline was purely one of potentiating glutamate, for proline alone did not excite this cell. In the case illustrated in Fig. 2B, proline applied by itself had an excitant action. The very great sensitivity of this cell to stimulation by proline is noteworthy. Of 24 interneurons in the spinal cord, proline antagonized the effect of glutamate in 5 cases (Fig. 1 B); potentiated glutamate in 6; excited 2 cells but did not influence their glutamate-evoked firing; had no effect by itself and did not interact with glutamate in 11. Proline excited 3 additional cells upon which its interaction with glutamate could not be tested for technical reasons. Unlike the cerebral cortex and the spinal cord, in the cuneate nucleus proline failed to influence the activity of 24 neurons, including both interneurons and relay cells. Identification of the two types was by criteria similar to those established by Anderson et al. 1. An example of this lack of effect is seen in Fig. lC. Neither the firing evoked by stimulation of the superficial radial nerve, nor spontaneous or glutamate-evoked activity were altered during application of proline up to 40 nA tbr 12 min. Glutamate diethyl ester applied in the same manner, and with similar current intensities, had the depressant effect on glutamate-induced excitation which was already described by others 2,1°. The effectiveness of the diethyl ester indicates that drug-induced inhibition was not hindered in these experiments by technical failure. These observations suggest that proline is an antagonist of glutamate in selected areas of the central nervous system of cats. The differences in effects on cells found in different parts of the CNS, and indeed on different cells within the same tissue, may be related to a selective affinity of this compound for specific subsynaptic sites. This claim has relevance in view of McLennan's 8,9 proposal that there may be two types of receptors for excitant amino acids, One unspecific (affected by any excitant amino acid and found on most neurons) and the other specifically activated by glutamate and perhaps identical with the excitatory synaptic receptor. Proline is clearly not a universal blocker of the effects of glutamate; however, proline in lhct may be an antagonist at sites where glutamate acts as a synaptic transmitter but not where it acts as a non-specific stimulant. Felix and Kfinzle 4 have suggested that naturally occurring proline may have a physiological role in some parts of the mammalian central nervous system. Our results are compatible with this proposal, although not directly supporting it. Supported by grants of the U.S.P.H;S., NS 05330 and NS 10507.
] ANDERSON,P., ECCLES, J. C., SCHM1DT,R. F., AND YOKOTA,T., Identification of relay cells and interneurons in the cuneate nucleus, J. Neurophysio/., 27 (1964) 1080-1095. 2 CURTIS,D. R., DUGGAN,A. W., FELIX,D., JOHNSTON,G. A. R., TEB~OS,A. K., AND WATKINS, J. C., Excitation of mammalian central neurons by acidic amino acids, Brain Research, 41 (1972)
283-301.
191 3 CURTlS, D. R., AND JOHNSTON, G. A. R., Amino acid transmitters in the mammalian central nervous system, Ergebn. Physiol., 69 (1974) 97-188. 4 FELlX, D., AND K(ONZLE, H., lontophoretic and autoradiographic studies on the role of proline in nervous transmission, Pfliigers Arch. ges. Physiol., 350 (1974) 135-144. 5 FLOREY, E., Neurotransmitters and modulators in the animal kingdom, Fed. Proc., 26 (1967) 1164-1178. 6 JOHNSON, J. L., Glutamic acid as a synaptic transmitter in the nervous system. A review, Brain Research, 37 (1972) 1-19. 7 KRNJEVI6, K., Chemical nature of synaptic transmission in vertebrates, Physiol. Rev., 54 (1974) 418 540. 8 MCLENNAN, H., Actions of excitatory amino acids and their antagonism, Neuropharmacology~ 13 (1974) 449-454. 9 McLENNAN, H., HUFFMAN, R. D., AND MARSHALL, K. C., Patterns of excitation of thalamic neurons by amino acids and by acetylcholine, Nature (Lond.), 219 (1968) 387-388. 10 MCLENNAN, H., MARSHALL, K. C., AND HUFF~aAN, R. D., The antagonism of glutamate action at central neurones, Experientia (Basel), 27 (1971) 1116. 11 VAN HARREWLO, A., AND F~FKOVX, E., EffEcts of amino acids on the isolated chicken retina, and on its response to glutamate stimulation, J. Neurochem., 20 (1973) 947-962. 12 ZARZECKL P., BLUM, P., AND SOMJEN, G., Interaction of L-proline and glutamate upon neurons in the central nervous system of the cat, Fourth Annual Meeting, Society for Neuroscience, (1974) 494 (abstract).
