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N-methyl bicuculline and primary afferent depolarization
iv~u~oLocx 38, 525428 (1973)
EXPERIMENTAL
RESEARCH N-Methyl R. A. Neurology
Bicuculline G. E.
DAVIDOPF,
Service, University
and
NOTE
Primary S. A.
SILVEY,
Afferent KOBETZ,
Depolarization AND
H. M.
Veterans Administration Hospital, aped Department of Miami Sclzool of Medicilke, Miami, Florida
SPIRA
I
of Neurolo:gy, 33152
September 29, 1972
Received
It has been demonstrated that bicuculline, a convulsant alkaloid, blocks the action of gamma-aminobutyric acid (GABA) at various sites in the central and peripheral nervous systems of vertebrates (14). This drug is consequently a useful experimental tool despite the difficulties inherent in its low solubility in water. Pong and Graham (5) may have obviated this problem, however, by synthesizing a water-soluble quaternary derivative, N-methyl bicuculline. Before the new compotmd can be experimentally substituted for bicuculline, it is necessary to know whether its pharmacological properties are similar to the naturally occurring alkaloid. In answer to this question we offer the following experiments which explore the effects of water-soluble N-methyl bicuculline on the GABA-mediated depolarization of primary afferent terminals in the isolated frog spinal cord. The experimental trials of the new compound were performed on precisely the same preparation that had been employed in our laboratory to test bicuculline (3, 4). Lumbar spinal cords of Rarln pities were removed after laminectomy, hemisected, placed together with attached dorsal (DR) and ventral (VR) roots in a small (< 1 ml) temperature-controlled bath, and perfused with oxygenated Ringer solution. Roots were led through Vaseline-sealed slits in thin plastic partitions, covered with mineral oil, and placed on pairs of silver-silver chloride electrodes for stimulation and for a-c recording (3). The temperature of the preparation was maintained at 1.5 C. 1 The
authors
thank
hIr.
R. Adair
for
able 525
Copyright All rights
0 1973 by Academic Press, of reproduction in any form
Inc. reserved.
technical
assistance.
526
DAVIDOFP
Al
ET
Cl
61 DR.DRP/VR-DRP
.4L.
DR.DRP/VR-DRP
DR-VRP
FIG. 1. Action of bicuculline and N-methyl-bicuculline on the DRP and on polysynaptic reflex. A and B, superimposed oscilloscope sweeps of DR-DRP potential) and of VR-DRP. Al, control record obtained in Ringer solution. A5 after addition of N-methyl-bicuculline (2 X lo-’ M) to perfusate. B1, same as ferent preparation. Bz, after perfusion with bicuculline (2 X IO4 M) for C1, VR response to supramaximal DR stimulation. C,, same response obtained after adding N-methyl bicuculline (lo-’ M) to Ringer. Vertical calibration: /LV; B, 50 pv ; C, 200 cv.
the VR (larger 12 min AI, dif12 min. 30 min A, 100
N-Methyl bicuculline was synthesized by reacting methyl iodide and bicuculline in acetone for 12 hr. After precipitation, washing with an.hydrous ether, and lyophilization, a fluffy white powder was obtained. Thin-layer chromatography on silica gel using two solvent systems (n-butanol-acetic acid-Hz0 4 : 1 : 1; chloroform-methyl alcohol 1 : 1) revealed that the precipitate was homogeneous. The electrotonically conducted dorsal root potential (DRP) can be produced in the amphibian by a volley in either the DR of an adjacent spinal segment (DR-DRP) or in the VR of the samesegment (VR-DRP) (Fig. 1Ar and Br). Addition of N-methyl bicuculline to the perfusate rapidly decreasedor abolished DRPs. Figure 1As illustrates that N-methyl bicuculline (2 X 1W5 M) reduced the amplitude of the DR-DRP by 50% and that of the VR-DRP by 95% in 12 min. Bicuculline at an identical concentration affected DRP similarly (Fig. 1Bs). A concentration of Nmethyl bicuculline of 5 x 10msM or greater decreased the DR-DRP. These effects were partially reversible upon prolonged washing with Ringer solution. Since the DRP has been used extensively as an indirect measure of the depolarization of primary afferent terminals known as primary afferent depolarization (PAD), N-methyl bicuculline can thus be said to interfere with the production of PAD. In addition to these effects, Nmethyl bicuculline increased the amplitude of the VR polysynaptic potential and reflex (DR-VRP, Fig. 1C). Thus, because the chemical enhanced orthodromic reflect activity, it is clear that the DRP-blocking
527
N-METHYL-BICUCULLINE CONTROL Al
DH STIM
2
+ GABA
N-METHYL
3
+ VI STIM
BICUCULLINE
FIG. 2. Recording of antidromic compound action potential from the distal end of the DR produced by constant stimulation of DR terminals through a low resistance (4 M NaCI) glass micropipette inserted into the dorsal horn (DH). Al, control response. Addition of GABA (lo-% M) to the Ringer solution (A,) or a conditioning VR volley delivered 80 msec earlier (Aa) augmented DR terminal excitability. B, application of N-methyl-bicuculline (10e4 nr, 30 min) abolished the VR-produced PAD (B3) and the 6ABA-produced depolarization ( Bz). Five oscilloscope sweeps superimposed in each record.
of N-methyl bicuculline could not have been caused by a general depressantaction. Using Wall’s technique (6), intraspinal measurements of the excitability of DR primary afferent terminals were made. These measurements provide a direct demonstration of the PAD evoked by a VR volley (Fig. ZA,) and of the deplorization of DR terminals produced by GABA (3,4) (‘Fig. 2Az). Perfusion with N-methyl bicuculline markedly reduced increased terminal excitability whether elicited by a VR volley (Fig. 2B3) or by the application of GABA (Fig. 2Bz). In other experiments, the PAD initiated by a DR volley was similarly reduced by this compound. N-Methyl bicuculline, therefore, appears to block PAD and to antagonize the action of GABA on DR terminals as does the naturally occurring alkaloid bicuculline (3, 4). The greater solubility of the former compound confers a considerable advantage as an experimental tool and warrants further investigation of its action on neural tissue,
actions
REFERENCES D. R,, A. W. DUGCAN, D. FELIX, and G. A. R. JOHNSTON, 1971. BicuculT Iine, an antagonist of GABA and synaptic inhibition in the spinal cord of the cat, Brain Res. 32: 69-96,
I, CURTIS,
528
4. 5. 6.
ET
AL.
D. R., A. W. DUGCAN, D. FELIX, G. A. R. JOHNSTON, and H. MCLENNAN. 1971. Antagonism between bicuculline and GABA in the cat brain. Brain Res. 33: 57-73. DAVIDOFF, R. A. 1972. Gamma-aminobutyric acid antagonism and presynaptic inhibition in the frog spinal cord. Science 175: 331-333. DAVIDOFF, R. A. 1972. The effects of bicuculline on the isolated spinal cord of the frog. Exp. Neural. 35: 179-193. PONC, S. F., and L. T. GRAHAM, JR. 1972. N-Methyl bicuculline, a convulsant more potent than bicuculline. Brain Res. 42: 486-490. WALL, P. D. 1958. Excitability changes in afferent fibre terminations and their relation to slow potentials. J. Physiol. London 142: l-21.
2. CURTIS,
3.
DAVIDOFF
EXPERIMENTAL
RESEARCH N-Methyl R. A. Neurology
Bicuculline G. E.
DAVIDOPF,
Service, University
and
NOTE
Primary S. A.
SILVEY,
Afferent KOBETZ,
Depolarization AND
H. M.
Veterans Administration Hospital, aped Department of Miami Sclzool of Medicilke, Miami, Florida
SPIRA
I
of Neurolo:gy, 33152
September 29, 1972
Received
It has been demonstrated that bicuculline, a convulsant alkaloid, blocks the action of gamma-aminobutyric acid (GABA) at various sites in the central and peripheral nervous systems of vertebrates (14). This drug is consequently a useful experimental tool despite the difficulties inherent in its low solubility in water. Pong and Graham (5) may have obviated this problem, however, by synthesizing a water-soluble quaternary derivative, N-methyl bicuculline. Before the new compotmd can be experimentally substituted for bicuculline, it is necessary to know whether its pharmacological properties are similar to the naturally occurring alkaloid. In answer to this question we offer the following experiments which explore the effects of water-soluble N-methyl bicuculline on the GABA-mediated depolarization of primary afferent terminals in the isolated frog spinal cord. The experimental trials of the new compound were performed on precisely the same preparation that had been employed in our laboratory to test bicuculline (3, 4). Lumbar spinal cords of Rarln pities were removed after laminectomy, hemisected, placed together with attached dorsal (DR) and ventral (VR) roots in a small (< 1 ml) temperature-controlled bath, and perfused with oxygenated Ringer solution. Roots were led through Vaseline-sealed slits in thin plastic partitions, covered with mineral oil, and placed on pairs of silver-silver chloride electrodes for stimulation and for a-c recording (3). The temperature of the preparation was maintained at 1.5 C. 1 The
authors
thank
hIr.
R. Adair
for
able 525
Copyright All rights
0 1973 by Academic Press, of reproduction in any form
Inc. reserved.
technical
assistance.
526
DAVIDOFP
Al
ET
Cl
61 DR.DRP/VR-DRP
.4L.
DR.DRP/VR-DRP
DR-VRP
FIG. 1. Action of bicuculline and N-methyl-bicuculline on the DRP and on polysynaptic reflex. A and B, superimposed oscilloscope sweeps of DR-DRP potential) and of VR-DRP. Al, control record obtained in Ringer solution. A5 after addition of N-methyl-bicuculline (2 X lo-’ M) to perfusate. B1, same as ferent preparation. Bz, after perfusion with bicuculline (2 X IO4 M) for C1, VR response to supramaximal DR stimulation. C,, same response obtained after adding N-methyl bicuculline (lo-’ M) to Ringer. Vertical calibration: /LV; B, 50 pv ; C, 200 cv.
the VR (larger 12 min AI, dif12 min. 30 min A, 100
N-Methyl bicuculline was synthesized by reacting methyl iodide and bicuculline in acetone for 12 hr. After precipitation, washing with an.hydrous ether, and lyophilization, a fluffy white powder was obtained. Thin-layer chromatography on silica gel using two solvent systems (n-butanol-acetic acid-Hz0 4 : 1 : 1; chloroform-methyl alcohol 1 : 1) revealed that the precipitate was homogeneous. The electrotonically conducted dorsal root potential (DRP) can be produced in the amphibian by a volley in either the DR of an adjacent spinal segment (DR-DRP) or in the VR of the samesegment (VR-DRP) (Fig. 1Ar and Br). Addition of N-methyl bicuculline to the perfusate rapidly decreasedor abolished DRPs. Figure 1As illustrates that N-methyl bicuculline (2 X 1W5 M) reduced the amplitude of the DR-DRP by 50% and that of the VR-DRP by 95% in 12 min. Bicuculline at an identical concentration affected DRP similarly (Fig. 1Bs). A concentration of Nmethyl bicuculline of 5 x 10msM or greater decreased the DR-DRP. These effects were partially reversible upon prolonged washing with Ringer solution. Since the DRP has been used extensively as an indirect measure of the depolarization of primary afferent terminals known as primary afferent depolarization (PAD), N-methyl bicuculline can thus be said to interfere with the production of PAD. In addition to these effects, Nmethyl bicuculline increased the amplitude of the VR polysynaptic potential and reflex (DR-VRP, Fig. 1C). Thus, because the chemical enhanced orthodromic reflect activity, it is clear that the DRP-blocking
527
N-METHYL-BICUCULLINE CONTROL Al
DH STIM
2
+ GABA
N-METHYL
3
+ VI STIM
BICUCULLINE
FIG. 2. Recording of antidromic compound action potential from the distal end of the DR produced by constant stimulation of DR terminals through a low resistance (4 M NaCI) glass micropipette inserted into the dorsal horn (DH). Al, control response. Addition of GABA (lo-% M) to the Ringer solution (A,) or a conditioning VR volley delivered 80 msec earlier (Aa) augmented DR terminal excitability. B, application of N-methyl-bicuculline (10e4 nr, 30 min) abolished the VR-produced PAD (B3) and the 6ABA-produced depolarization ( Bz). Five oscilloscope sweeps superimposed in each record.
of N-methyl bicuculline could not have been caused by a general depressantaction. Using Wall’s technique (6), intraspinal measurements of the excitability of DR primary afferent terminals were made. These measurements provide a direct demonstration of the PAD evoked by a VR volley (Fig. ZA,) and of the deplorization of DR terminals produced by GABA (3,4) (‘Fig. 2Az). Perfusion with N-methyl bicuculline markedly reduced increased terminal excitability whether elicited by a VR volley (Fig. 2B3) or by the application of GABA (Fig. 2Bz). In other experiments, the PAD initiated by a DR volley was similarly reduced by this compound. N-Methyl bicuculline, therefore, appears to block PAD and to antagonize the action of GABA on DR terminals as does the naturally occurring alkaloid bicuculline (3, 4). The greater solubility of the former compound confers a considerable advantage as an experimental tool and warrants further investigation of its action on neural tissue,
actions
REFERENCES D. R,, A. W. DUGCAN, D. FELIX, and G. A. R. JOHNSTON, 1971. BicuculT Iine, an antagonist of GABA and synaptic inhibition in the spinal cord of the cat, Brain Res. 32: 69-96,
I, CURTIS,
528
4. 5. 6.
ET
AL.
D. R., A. W. DUGCAN, D. FELIX, G. A. R. JOHNSTON, and H. MCLENNAN. 1971. Antagonism between bicuculline and GABA in the cat brain. Brain Res. 33: 57-73. DAVIDOFF, R. A. 1972. Gamma-aminobutyric acid antagonism and presynaptic inhibition in the frog spinal cord. Science 175: 331-333. DAVIDOFF, R. A. 1972. The effects of bicuculline on the isolated spinal cord of the frog. Exp. Neural. 35: 179-193. PONC, S. F., and L. T. GRAHAM, JR. 1972. N-Methyl bicuculline, a convulsant more potent than bicuculline. Brain Res. 42: 486-490. WALL, P. D. 1958. Excitability changes in afferent fibre terminations and their relation to slow potentials. J. Physiol. London 142: l-21.
2. CURTIS,
3.
DAVIDOFF