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Angiotensin II-induced depression of purkinje cell firing and possible modulatory action on GABA responses
Neuroscience Research, 1 (1984) 369-372
369
Elsevier Scientific Publishers Ireland Ltd . NSR 00034
Short Communications
Angiotensin II-Induced Depression of Purkinje Cell Firing and Possible Modulatory Action on GABA Responses Pavich Tongroach, Sanay Sanguanrungsirikul, Boonyong Tantisira and Pongsak Kunluan Department of Physiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10500 (Thailand)
Received April 23rd, 1984 ; Accepted May 31st, 1984) Key words : angiotensin II - depressant action - Purkinje cell - modulation - GABA - bicuculline
SUMMARY Effects of octapeptide angiotensin II (All) were tested on cortical neurons of rat's cerebellum by means of microiontophoresis . It was observed that AII consistently depressed spontaneous firing of Purkinje cell, whereas other unidentified neurons were unaffected. When tested against responses of Purkinje cell to depressant putative neurotransmitters, namely, GABA, glycine, taurine, 5-hydroxytryptamine and noradrenaline, it was observed that AII specifically enhanced depressant action of GABA, while the responses to other substances were unaffected . Both AII-induced depression of cell firing and the AII-induced enhancement of GABA depression were antagonized by a specific GABA antagonist, bicuculline methochloride . We therefore suggest that All exerts an inhibitory action on Purkinje cells through its modulatory action on bicuculfne-sensitive GABA receptors .
An angiotensin II (All) afferent system has been demonstrated in the cerebellum by means of anatomical and biochemical methods . Thus, in the cerebellar tissue, radioimmunoassay shows significant quantity of All', whereas receptor binding study also suggests the presence of AII-specific binding sites' . In addition, immunohistochemistry demonstrates the presence of All-containing fibers coursing in the cerebellar white matter and diverging within the granular layer to terminate densely surrounding the Purkinje cell somata' . These findings suggest a transmitter role of All in the cerebellum . Therefore, the present experiments are aimed at demonstrating the action of this octapeptide on principal neurons of the cerebellum, i .e . the Purkinje cell, as well as on Correspondence : P. Tongroach, Department of Physiology, Faculty of Pharmaceutical Sciences, Bang-
kok 10500, Thailand . 0168-0102/84/$03 .00 © 1984 Elsevier Scientific Publishers Ireland Ltd .
370 other unidentified cortical neurons, by means of the microiontophoretic technique . The results suggest that this peptide exerts an inhibitory action on Purkinje cell, presumably through modulatory enhancement of the cells' response to GABA . Male albino rats were anaesthetized with urethane (1 .4 g/kg, i .p .), mounted in a Narishige stereotaxic frame, and the skull covering the cerebellum removed . Recording of extracellular spike potentials was made from cerebellar cortical neurons via the center barrel, 4 M NaCl filled, of a seven-barrel microelectrode (tip diameter 5-8 µm) . The outer barrels of the electrode contained either of the following solutions to be ejected to the neurons by a standard microiontophoretic method' : angiotensin II (AII, Calbiochem, 0 .001 M, pH 4 .5), angiotensin II antagonist, Sar'-Ala$-angiotensin, (saralasin, Calbiochem, 0 .001 M, pH 4 .5), y-aminobutyric acid (GABA, 0 .2 M, pH 3 .5), glycineHCl (Gly, 0 .2 M, pH 3 .5), taurine (Tau, 0 .2 M, pH 3 .5), 5-hydroxytryptamine creatine phosphate complex (5-HT, 0 .2 M, pH 4), noradrenaline bitartrate (NA, 0 .2 M, pH 4), (+ )-bicuculline methochloride (BMC, 0 .005 M in 0 .165 M NaCl, pH 3 .5) . Retaining currents (10-40 nA) were routinely applied when the drugs were not ejected . Total currents flowing through the electrode were nullified all the time by means of an automatic current balancing device through the sixth barrel of the electrode containing 2 M NaCl . Microiontophoretic application was timed precisely using a multichannel time programmer . Spike potentials were amplified by a standard set of high input impedance amplifiers and monitored on a digital memory oscilloscope (Nihon Kodhen, VC-10) . They were selected by a window slicer, counted electronically and the counts recorded on a curvilinear pen recorder for presentation of neuronal firing rates . Assessment was made of the neuronal activities before, during and after iontophoretic applications of the test substances . Two groups of cortical neurons were tested in this study . First, Purkinje cells were identified by their high firing rates and by the characteristic pattern of simple and complex spike discharges' . Eighty-five of these cells were recorded randomly in the vermis . Second, non-Purkinje cells were also sampled in the vicinity of the Purkinje cells . Iontophoretically applied AII consistently produced a dose-dependent depression of spontaneous firing of 80 Purkinje cells (94% of total) tested (Fig . 1), whereas nonPurkinje cells (6 cells tested) were non-responsive to the peptide . The responses were rapid in onset and recovery, and were antagonized by the angiotensin II antagonist, saralasin . Subsequently, further tests were made in an attempt to explore if there would be any interaction between AII and putative inhibitory transmitters reportedly active in the cerebellum, namely, GABA, Gly, Tau, NA and 5-HT . When continuous ejection of AII was superposed on pulsatile application of these substances, it was consistently observed that GABA-induced depression of Purkinje cell firing was selectively enhanced (18 neurones tested), whereas the responses to the other control agbnists were unaffected (number of cells tested for Gly, NA, 5-HT, and Tau respectively were 12, 6, 5 and 5) . Fig . 2 shows examples of such results obtained with two Purkinje cells, tested with GABA and Gly, and GABA, Gly and NA, respectively. This enhancement was
371
A1
20
40
60
80
100 20S .
Fig. 1 . Effects of iontophoretic application of angiotensin II (All) on spontaneous firing of a cerebellar Purkinje cell . The record shows that All produced depression of the cell firing in a dose-dependent manner . In this and the subsequent figure, the vertical axis represents firing rate in spikes per second . Horizontal bars underneath the record indicate the period of iontophoretic application, while the figure under each bar denotes iontophoretic current in nanoamperes (nA) . Time scale of 20 s is shown under the record .
A ANGIOTENSIN 11 50 nA
0
40
20
0
WAg" o
-
o
o
-
o
-
o
B
20S.
40
20
0
= m GABA:GLr C3 .NA 10 40 30
M
o
m
0 a M
o=
m
0
M
o=
m
o
m
n
m
Fig . 2 . Effects of continuous application of angiotensin II (All) on depressant actions of (A) y-aminobutyric acid (GABA) and glycine (GLY) ; and (B) GABA, GLY and noradrenaline (NA) ; on two Purkinje cells (A and B). All selectively potentiated the depressant action of GABA without having any appreciable effects on the actions of other control depressants, namely, GLY and NA . The peptide also failed to produce potentiation of two other control depressants, i .e. taurine and 5-hydroxytryptamine (results are not shown here) .
3 72 progressive with time of All application, and was dependent on the amount of iontophoretic current of AII . Thus, complete cessation of cell firing was usually induced from the originally submaximal dose of GABA after either prolonged application or application with higher current of AII . Further observation showed that the effects of AII, both on spontaneous firing and on GABA-induced depression of Purkinje cell firing, were antagonized by an AII antagonist, saralasin, and a specific GABA antagonist, BMC. In view of biochemical' ,' and anatomical' observations which demonstrate the presence of AII in the cerebellum, the possibility arises that this peptide may function as a neurotransmitter in this brain area . The findings in the present study that Purkinje cells, .but not other unidentified cortical neurons, consistently respond to iontophoretic application of AII confirm the presence of AII receptor, and suggest further that these receptors may be localized on the Purkinje cell membrane, or at least, on neuronal elements functionally connecting to the Purkinje cells . Such results are consistent with immunohistochemical data which elucidates an AII-containing afferent system to terminate as a dense collection of fibres surrounding Purkinje cell somata' . Although the present results suggest that AII may exert its inhibitory effects on Purkinje cells, the fmdings that this peptide specifically enhances inhibitory action of GABA give rise to the possibility that synaptic action of this peptide, if any, may be modulatory on GABA actions . This possibility is further supported by the results that both depressant action of AII and AII-induced enhancement of GABA action were antagonized by a specific GABA antagonist, BMC, indicating that inhibition seen with AII application may be mediated through BMC-sensitive GABA receptors . The actual mode of synaptic function as well as the mechanism of action of this peptide is the matter of further investigation . The authors wish to express their sincere thanks to Prof. Masao Ito for his support and encouragement. This work was supported by Rachadapisake Sompote (Royal Silver Jubilee Memorial) Grant, B .E . 2525, from Chulalongkorn University . We are also indebted to the Kajima Foundation, the Japan Neuroscience Society, Nihon Kohden Corp ., Narishige Scientific Lab ., and Takahashi Co ., for their generous gifts of instruments used in this investigation .
REFERENCES I Changaris, D.G ., Severs, W .B . and Keil, L.C ., Localization of angiotensin in rat brain, J. Histochem. Cytochem ., 26 (1978) 593-607 . 2 Eccles, J.C., Ito, M . and Szentfigothai, J., The Cerebellum as a Neuronal Machine, Springer-Verlag, Berlin, Heidelberg and New York, 1967 . 3 Geller, H .M. and Woodward, D .J., An improved constant current source for micro-iontophoretic drug application studies, Electroenceph . din . Neurophysiol., 33 (1972) 430-432 . 4 Sirett, N.E ., Bray, J.J. and Hubbard, J .I., Localization of immunoreactive angiotensin in the hippocampus and striatum of rat brain ., Brain Res., 217 (1981) 405-411 . 5 Sirett, N.E ., McLean, A .S., Bray, J.J. and Hubbards, J.I., Distribution of angiotensin II receptors in rat brain, Brain Res., 122 (1977) 299-312 .
369
Elsevier Scientific Publishers Ireland Ltd . NSR 00034
Short Communications
Angiotensin II-Induced Depression of Purkinje Cell Firing and Possible Modulatory Action on GABA Responses Pavich Tongroach, Sanay Sanguanrungsirikul, Boonyong Tantisira and Pongsak Kunluan Department of Physiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10500 (Thailand)
Received April 23rd, 1984 ; Accepted May 31st, 1984) Key words : angiotensin II - depressant action - Purkinje cell - modulation - GABA - bicuculline
SUMMARY Effects of octapeptide angiotensin II (All) were tested on cortical neurons of rat's cerebellum by means of microiontophoresis . It was observed that AII consistently depressed spontaneous firing of Purkinje cell, whereas other unidentified neurons were unaffected. When tested against responses of Purkinje cell to depressant putative neurotransmitters, namely, GABA, glycine, taurine, 5-hydroxytryptamine and noradrenaline, it was observed that AII specifically enhanced depressant action of GABA, while the responses to other substances were unaffected . Both AII-induced depression of cell firing and the AII-induced enhancement of GABA depression were antagonized by a specific GABA antagonist, bicuculline methochloride . We therefore suggest that All exerts an inhibitory action on Purkinje cells through its modulatory action on bicuculfne-sensitive GABA receptors .
An angiotensin II (All) afferent system has been demonstrated in the cerebellum by means of anatomical and biochemical methods . Thus, in the cerebellar tissue, radioimmunoassay shows significant quantity of All', whereas receptor binding study also suggests the presence of AII-specific binding sites' . In addition, immunohistochemistry demonstrates the presence of All-containing fibers coursing in the cerebellar white matter and diverging within the granular layer to terminate densely surrounding the Purkinje cell somata' . These findings suggest a transmitter role of All in the cerebellum . Therefore, the present experiments are aimed at demonstrating the action of this octapeptide on principal neurons of the cerebellum, i .e . the Purkinje cell, as well as on Correspondence : P. Tongroach, Department of Physiology, Faculty of Pharmaceutical Sciences, Bang-
kok 10500, Thailand . 0168-0102/84/$03 .00 © 1984 Elsevier Scientific Publishers Ireland Ltd .
370 other unidentified cortical neurons, by means of the microiontophoretic technique . The results suggest that this peptide exerts an inhibitory action on Purkinje cell, presumably through modulatory enhancement of the cells' response to GABA . Male albino rats were anaesthetized with urethane (1 .4 g/kg, i .p .), mounted in a Narishige stereotaxic frame, and the skull covering the cerebellum removed . Recording of extracellular spike potentials was made from cerebellar cortical neurons via the center barrel, 4 M NaCl filled, of a seven-barrel microelectrode (tip diameter 5-8 µm) . The outer barrels of the electrode contained either of the following solutions to be ejected to the neurons by a standard microiontophoretic method' : angiotensin II (AII, Calbiochem, 0 .001 M, pH 4 .5), angiotensin II antagonist, Sar'-Ala$-angiotensin, (saralasin, Calbiochem, 0 .001 M, pH 4 .5), y-aminobutyric acid (GABA, 0 .2 M, pH 3 .5), glycineHCl (Gly, 0 .2 M, pH 3 .5), taurine (Tau, 0 .2 M, pH 3 .5), 5-hydroxytryptamine creatine phosphate complex (5-HT, 0 .2 M, pH 4), noradrenaline bitartrate (NA, 0 .2 M, pH 4), (+ )-bicuculline methochloride (BMC, 0 .005 M in 0 .165 M NaCl, pH 3 .5) . Retaining currents (10-40 nA) were routinely applied when the drugs were not ejected . Total currents flowing through the electrode were nullified all the time by means of an automatic current balancing device through the sixth barrel of the electrode containing 2 M NaCl . Microiontophoretic application was timed precisely using a multichannel time programmer . Spike potentials were amplified by a standard set of high input impedance amplifiers and monitored on a digital memory oscilloscope (Nihon Kodhen, VC-10) . They were selected by a window slicer, counted electronically and the counts recorded on a curvilinear pen recorder for presentation of neuronal firing rates . Assessment was made of the neuronal activities before, during and after iontophoretic applications of the test substances . Two groups of cortical neurons were tested in this study . First, Purkinje cells were identified by their high firing rates and by the characteristic pattern of simple and complex spike discharges' . Eighty-five of these cells were recorded randomly in the vermis . Second, non-Purkinje cells were also sampled in the vicinity of the Purkinje cells . Iontophoretically applied AII consistently produced a dose-dependent depression of spontaneous firing of 80 Purkinje cells (94% of total) tested (Fig . 1), whereas nonPurkinje cells (6 cells tested) were non-responsive to the peptide . The responses were rapid in onset and recovery, and were antagonized by the angiotensin II antagonist, saralasin . Subsequently, further tests were made in an attempt to explore if there would be any interaction between AII and putative inhibitory transmitters reportedly active in the cerebellum, namely, GABA, Gly, Tau, NA and 5-HT . When continuous ejection of AII was superposed on pulsatile application of these substances, it was consistently observed that GABA-induced depression of Purkinje cell firing was selectively enhanced (18 neurones tested), whereas the responses to the other control agbnists were unaffected (number of cells tested for Gly, NA, 5-HT, and Tau respectively were 12, 6, 5 and 5) . Fig . 2 shows examples of such results obtained with two Purkinje cells, tested with GABA and Gly, and GABA, Gly and NA, respectively. This enhancement was
371
A1
20
40
60
80
100 20S .
Fig. 1 . Effects of iontophoretic application of angiotensin II (All) on spontaneous firing of a cerebellar Purkinje cell . The record shows that All produced depression of the cell firing in a dose-dependent manner . In this and the subsequent figure, the vertical axis represents firing rate in spikes per second . Horizontal bars underneath the record indicate the period of iontophoretic application, while the figure under each bar denotes iontophoretic current in nanoamperes (nA) . Time scale of 20 s is shown under the record .
A ANGIOTENSIN 11 50 nA
0
40
20
0
WAg" o
-
o
o
-
o
-
o
B
20S.
40
20
0
= m GABA:GLr C3 .NA 10 40 30
M
o
m
0 a M
o=
m
0
M
o=
m
o
m
n
m
Fig . 2 . Effects of continuous application of angiotensin II (All) on depressant actions of (A) y-aminobutyric acid (GABA) and glycine (GLY) ; and (B) GABA, GLY and noradrenaline (NA) ; on two Purkinje cells (A and B). All selectively potentiated the depressant action of GABA without having any appreciable effects on the actions of other control depressants, namely, GLY and NA . The peptide also failed to produce potentiation of two other control depressants, i .e. taurine and 5-hydroxytryptamine (results are not shown here) .
3 72 progressive with time of All application, and was dependent on the amount of iontophoretic current of AII . Thus, complete cessation of cell firing was usually induced from the originally submaximal dose of GABA after either prolonged application or application with higher current of AII . Further observation showed that the effects of AII, both on spontaneous firing and on GABA-induced depression of Purkinje cell firing, were antagonized by an AII antagonist, saralasin, and a specific GABA antagonist, BMC. In view of biochemical' ,' and anatomical' observations which demonstrate the presence of AII in the cerebellum, the possibility arises that this peptide may function as a neurotransmitter in this brain area . The findings in the present study that Purkinje cells, .but not other unidentified cortical neurons, consistently respond to iontophoretic application of AII confirm the presence of AII receptor, and suggest further that these receptors may be localized on the Purkinje cell membrane, or at least, on neuronal elements functionally connecting to the Purkinje cells . Such results are consistent with immunohistochemical data which elucidates an AII-containing afferent system to terminate as a dense collection of fibres surrounding Purkinje cell somata' . Although the present results suggest that AII may exert its inhibitory effects on Purkinje cells, the fmdings that this peptide specifically enhances inhibitory action of GABA give rise to the possibility that synaptic action of this peptide, if any, may be modulatory on GABA actions . This possibility is further supported by the results that both depressant action of AII and AII-induced enhancement of GABA action were antagonized by a specific GABA antagonist, BMC, indicating that inhibition seen with AII application may be mediated through BMC-sensitive GABA receptors . The actual mode of synaptic function as well as the mechanism of action of this peptide is the matter of further investigation . The authors wish to express their sincere thanks to Prof. Masao Ito for his support and encouragement. This work was supported by Rachadapisake Sompote (Royal Silver Jubilee Memorial) Grant, B .E . 2525, from Chulalongkorn University . We are also indebted to the Kajima Foundation, the Japan Neuroscience Society, Nihon Kohden Corp ., Narishige Scientific Lab ., and Takahashi Co ., for their generous gifts of instruments used in this investigation .
REFERENCES I Changaris, D.G ., Severs, W .B . and Keil, L.C ., Localization of angiotensin in rat brain, J. Histochem. Cytochem ., 26 (1978) 593-607 . 2 Eccles, J.C., Ito, M . and Szentfigothai, J., The Cerebellum as a Neuronal Machine, Springer-Verlag, Berlin, Heidelberg and New York, 1967 . 3 Geller, H .M. and Woodward, D .J., An improved constant current source for micro-iontophoretic drug application studies, Electroenceph . din . Neurophysiol., 33 (1972) 430-432 . 4 Sirett, N.E ., Bray, J.J. and Hubbard, J .I., Localization of immunoreactive angiotensin in the hippocampus and striatum of rat brain ., Brain Res., 217 (1981) 405-411 . 5 Sirett, N.E ., McLean, A .S., Bray, J.J. and Hubbards, J.I., Distribution of angiotensin II receptors in rat brain, Brain Res., 122 (1977) 299-312 .