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Phaclofen antagonizes baclofen-induced suppression of visually evoked responses in the cat's striate cortex
398
Brain Research, 463 (1988) 398-402 Elsevier
BRE 23170
Phaclofen antagonizes baclofen-induced suppression of visually evoked responses in the cat's striate cortex U. Baumfalk and K. Albus Max-Planck-lnstitut fiir Biophysikalische Chemie, Abteilung Neurobiologie, G6ttingen (F. R. G.)
(Accepted 12 July 1988) Key words': Phaclofen; Baclofen-induced suppression; Visually evoked response; Striate cortex
The suppression of visually evoked responses (VER) in the cat's striate cortex by baclofen was reversibly antagonized by the phosphonic acid derivative of baclofen, phaclofen. The antagonistic effect of phaclofen was seen irrespective of whether it enhanced, suppressed or did not change VER on its own. The suppression of VER by ),-aminobutyric acid (GABA) was not affected by phaclofen. Our findings support the notion (Kerr et al., Brain Research, 405 (1987) 150-154) that phaclofen is an effective baclofen antagonist in the central nervous system of mammals. Preliminary findings indicate that in spite of its possible action on GABA Breceptors, phaclofen does not significantly alter functional properties of striate cortical neurons, in particular direction and orientation sensitivity.
It is well established that there are two classes of y-aminobutyric acid ( G A B A ) receptors in the peripheral and central nervous systems: G A B A A and G A B A B 3. Inhibitory mechanisms mediated via G A B A receptors have been shown to contribute to functional properties of many neurons in the striate cortex of cats: microelectrophoretic application of the G A B A A antagonist bicuculline near single neurons may diminish orientation and direction sensitivity and alter substantially the spatial organization of receptive fields 15. Although a high density of G A B A B receptor binding sites has been demonstrated in the mammalian neocortex 9, and although the G A B A B agonist, baclofen (4-amino-3-(p-chlorophenyl)butyric acid) 2 suppresses neuronal responses in neocortical neurons l'6'7, the physiological significance of G A B A ~ receptor sites has been less clear. A major step towards understanding the functional importance of bicuculline insensitive G A B A receptor sites has been the recent demonstration that fl-(pchlorophenyl)-3-aminopropylphosphonic acid (phaclofen) reversibly antagonizes the effects of baclofen in the guinea pig's ileum and colon and in the cat's spinal cord 11. To assess the possible contribution of
G A B A B receptors to functions of the visual cortex we have investigated the effects of phaclofen on visually evoked responses of single striate neurons in the cat, and on the baclofen induced suppression of these responses. In addition to phaclofen, other compounds known to interfere with baclofen induced inhibition, such as barium (Ba2+) s and 5-aminovaleric acid (5-AVA) 12 were tested. The experiments were performed on 16 adult cats. The animals were anesthetized with Ketanest (15 mg/kg, i.m.) and R o m p u n (2 mg/kg, i.m.), followed by a continuous infusion of 2 mg/kg/h pentobarbitone in Ringer's solution. Muscular relaxation was maintained with gallamine triethiodide (Flaxedil), and the animals were artificially respired with a gas mixture of N20/O2/CO 2 (67%/30%/3%). Refractive errors of the eyes were corrected with plastic lenses which also protected the corneae. The heart rate, blood pressure, body temperature and expiratory CO 2 were continuously monitored and held at physiological levels. Extracellular recordings were obtained from a glass-coated tungsten wire fixed parallel to a 7-barrelied pipette. The distance between the tip of the electrode and the tip of the pipette averaged 30 and
Correspondence: U. Baumfalk, Max-Planck-Institut for Biophysikalische Chemie, Am Fassberg, D-3400 G6ttingen, F.R.G.
0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
399 70/~m. The pipettes were filled with the following drug solutions: G A B A (Sigma) 0.5 M in aqua dest., pH 3.5; baclofen (racemic form, Ciba-Geigy) 25 mM in 150 mM NaCI, pH 3; phaclofen (Tocris Neuramin), about 60 mM in 75 mM NaC1, pH 3.5; bicuculline methiodide (BMI) racemic form (Pierce) or (+)enantiomer (Sigma, further preparation according to ref. 14) 8.5 mM, in citrate buffer pH 3.5, 5-AVA (Sigma) 25 mM or 0.5 M in aqua dest., pH 3.5; BaC12 (Sigma) 25 mM in aqua dest., pH 3.5. Retaining currents of 15 nA were applied to all barrels. Current balancing was performed via a barrel filled with Ringer's solution. Some electrode tracks were reconstructed from electrolytic lesions. Visual stimulation was performed by projecting light bars onto a translucent screen 0.57 cm in front of the animal's eyes by a light projector operated by means of a computercontrolled stimulation program. To avoid saturation of visually evoked responses, intensity and movement velocity of an optimally oriented bar were adjusted to yield submaximal responses. The stimulus was moved back and forth across the cell's receptive field for the dominant eye. The effects of phaclofen on spontaneous and visually evoked activity were tested in 35 neurons. Evoked responses were either facilitated (9 cells), suppressed (10 cells) or not affected (10 cells). These effects of phaclofen seemed to be independent from ejection current, which averaged 88 nA in the cells facilitated, 95 nA in the cells suppressed, and 95 nA in the cells not affected by phaclofen. In a further 5 neurons evoked responses were suppressed at ejection currents between 40 nA and 100 nA and became normal or were even enhanced with higher ejection currents. When responses became facilitated the increase in response amplitude was generally less than 40% of the controls (average 22%), and usually the spontaneous activity did not change. Spontaneous activity increased only in 3 neurons in which the evoked response was enhanced by more than 60%. Two of these neurons reliably responded to visual stimulation only under phaclofen; in a third case facilitation disappeared during later periods of application. The strength of the suppression varied between neurons and seemed to be independent from the ejection cur rent. At 200 nA phaclofen completely suppressed evoked responses in one cell and reduced response
amplitude by only 80%, 30% and 10%, respectively, in 3 other cells. The difference between the effects of phaclofen on single striate cortical neurons could indicate differences in functional importance and/or location of receptor sites mediating the effects of baclofen. These effects were therefore quantified separately for the cells showing no phaclofen effects (NE), for the cells facilitated (FE), and for the cells suppressed (SE) by phaclofen. In the FE-group on average 16 nA baclofen suppressed evoked responses by about 83%; in
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Fig. 1. Responses of a standard complex cell (layer 5) to visual stimulation before, during and after microiontophoretic application of baclofen (BAC), GABA and phaclofen (PHAC). Each point represents the cell responses averaged over 5 sweeps of a light bar moving in the preferred direction across the receptive field. Movement was always perpendicular to bar orientation. The horizontal bars indicate the duration of the drug application and the numbers besides these bars indicate the ejection current (nA) used for the corresponding drugs.
400 the SE-group the respective values were 22 n A and 80%, and in the N E - g r o u p 37 n A and 44%, respectively. The average application time of baclofen for yielding maximal effects was about 10 min in the NEgroup, and about 5 min in both F E - g r o u p and SEgroup. This difference in time is due to the fact that the ejection of baclofen was started with a very low current (5 n A ) , which was increased in a stepwise manner until consistent effects were observed. Recovery times were the same in the 3 groups. These results show that phaclofen was ineffective in those neurons having a 'high d o s e - w e a k response' relationship for baclofen. The results of the interaction between baclofen and phaclofen were unequivocal. In 17 of 21 neurons tested the baclofen-induced inhibition was reversibly antagonized by phaclofen. C o m p l e t e antagonism was seen in 11 neurons (Figs. 1-3): in 5 of these 11 neurons the baclofen-induced inhibition was actually
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Fig. 3. Responses of a standard complex cell (layer 6) to visual stimulation before, during and after microiontophoretic application of baclofen (BAC), GABA and phaclofen (PHAC). The transient suppression of the responses by phaclofen was stronger with lower ejection currents (not shown here).
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Fig. 2. Responses of a special complex cell (layer 5) to visua! stimulation before, during and after application of baclofen (BAC) and phaclofen (PHAC). The evoked responses of the cell were slightly suppressed with higher ejection currents of phaclofen.
reversed and responses were enhanced under phaclofen (Fig. 1); in 4 neurons phaclofen reduced the baclofen induced inhibition by 4 0 - 7 0 % , and in the two remaining cases a slight reduction in inhibition was c o m b i n e d with a delay in onset, and a decrease in the duration of the baclofen action. Phaclofen applied with ejection currents reducing baclofen induced suppression did not affect the G A B A - i n d u c e d inhibition of e v o k e d responses (Figs. 1, 3), and this was found for 6 neurons tested. The antagonistic effect of phaclofen became stronger with increase in ejection current (Figs. 1, 2). In the case shown in Fig. 1,80 n A phaclofen did not alter the baciofen-induced inhibition which however was blocked with 200 n A phaclofen. The relationship between ejection current and antagonistic action was also d e m o n s t r a t e d by the fact that the average ejection current was higher in cases with complete antagonism (125 nA;
401 range 50 nA-250 nA) than in cases with partial antagonism (80 nA; range 20 nA-250 nA). The antagonistic effect of phaclofen was found to be independent on whether phaclofen preceded or followed this application of phaclofen (3 neurons tested; Fig. 3). The antagonistic effects on the baclofen-induced inhibition were seen irrespective of whether or not phaclofen changed evoked responses on its own (Fig. 2). Even in a neuron in which it suppressed evoked responses by 80%, phaclofen blocked the inhibition elicited by baclofen but not that induced by G A B A . The baclofen-induced suppression was antagonized by phaclofen for a wide range of cell types, i.e. in neurons having simple, complex and hypercomplex receptive fields as well as in neurons responding only to a moving stimulus. For 4 neurons inhibition by baclofen was not antagonized by phaclofen. In one of these cases the ejection current was most probably too low (30 nA). In the 3 other neurons however, ejection currents were in the range (100 nA-170 nA) where one would expect antagonistic actions (see above). Interestingly, these neurons were those most sensitive to baclofen in our whole sample, their evoked responses being strongly inhibited by ejection currents of 8 nA or less. The ineffectiveness of phaclofen in these cases might therefore be due to a competitive disadvantage caused by low concentration at the receptor site rather than to an absence of antagonistic property. 5-AVA was tested in 30 neurons. When ejected from a 0.5 M solution (3-16 nA, mean 8 nA) it suppressed the evoked responses of all 6 neurons tested. When ejected as a 25 mM solution (24 neurons; ejection currents: 12-75 nA, mean 44 nA) evoked responses were either suppressed (n = 7), facilitated (n = 5) or not changed (n = 12). The AVA-induced suppression was readily antagonized with BMI, and with continuing application of BMI cell responses became strongly enhanced. The interaction between 5AVA and baclofen was tested in 20 neurons, and the baclofen-induced inhibition was reversibly antagonized in only 3 of them. When the 5-AVA-induced suppression had been counteracted with BMI subsequent application of baclofen either inhibited or did not change evoked responses. It could not be decided definitely, however, whether the 'ineffectiveness' of baclofen in the latter cases was caused by an antagonistic action of 5-AVA or by the strong facilitation
seen with prolonged application of BMI. Ba 2+ (4 neurons; currents: 20-50 nA, mean 37 nA) either reduced baclofen induced suppression, or had no effect; it also partially or completely blocked G A B A induced inhibition in these cases. Phaclofen did not systematically change the directional selectivity of the striate cortical neurons. Of the 34 neurons tested, 20 were directionally selective, which means that their response to movement of a light stimulus into one direction (preferred response) was more than two times stronger than the response into the opposite direction (non-preferred response). Only two of these 20 neurons changed the degree of their directional selectivity under phaclofen. In one case directional selectivity increased due to a suppression, and in another cell it was diminished due to an enhancement of the non-preferred response. In a few neurons also the orientation selectivity was studied and significant changes were not observed under phaclofen. One may tentatively suggest therefore, that baclofen receptor sites, which might be in part identical with G A B A B receptors, are involved in regulating the general excitability of a neuron rather than to contribute to specific functional properties. Our findings demonstrate that phaclofen antagonizes the baclofen suppression of visually evoked responses of striate cortical neurons. A competitive mechanism is suggested by the fact that the strength of the antagonism increases with ejection current. In addition the antagonistic effect is independent on whether phaclofen precedes or follows the application of baciofen. Inhibition of evoked responses by G A B A , which was effectively antagonized by bicuculline, was not reduced by phaclofen. The discussion of possible sites of the antagonistic action of phaclofen has to consider the sites of action of baclofen. In neocortical neurons baclofen has been shown to increase postsynaptic potassium conductance and to depress postsynaptic potentials by reducing the release of excitatory amino acids, i.e. having both pre- and postsynaptic effects (for a review see ref. 9). Our findings that suppression by baclofen was antagonized completely in most neurons tested also suggest both pre- and postsynaptic sites of action for phaclofen. This point will certainly be clarified only when intracellular recording experiments are performed. Interestingly, in neurons of rat
402 frontal cortex G A B A applied in the vicinity of the cell soma does not induce baclofen-like hyperpolarizatior~s in the presence of bicuculline1°. This finding has been presented as evidence against a participation of G A B A a receptors in mediating suppression by baclofen. O n the other hand bicuculline-resistant hyperpolarizations were evoked in hippocampal neurons when G A B A was applied to the dendritic field 13. Therefore it is suggested that baclofen acts at more peripheral parts of the neuron. O n the basis of our observation that the effects of phaclofen alone were somehow correlated with the effectiveness of baclofen it is assumed that phaclofen interacts with the same receptor site as does baclofen. Phaclofen did not change evoked responses in cells with a 'high d o s e - w e a k response' relationship for baclofen. It is noteworthy that 6 of these 10 neurons were complex cells of layers 5 and 6. Therefore, the ineffectiveness of phaclofen and a low effectiveness of baclofen in these neurons could be explained by a relatively long distance between ejection site and more distal parts of the dendrites where baclofen should exert its main effects on neuronal excitability13. A possible participation of GABAB receptors in mediating suppression by baclofen in deeper layer neurons of striate cortex receives additional support from observations made
1 Baumfalk, U. and Albus, K., Baclofen inhibits the spontaneous and visually evoked responses of neurones in the striate cortex of the cat, Neurosci. Lett., 75 (1987) 187-192. 2 Bowery, N.G., Hill, D.R., Hudson, A.L., Doble, A.L., Middlemiss, A., Shaw, J. and Turnbull, M., (-)-Baclofen decrease neurotransmitter release in the mammalian CNS by an action at a novel GABA receptor, Nature (Lond.), 283 (1980) 92-94. 3 Bowery, N.G., Price, G.W., Hudson, A.L., Hill, D.R., Wilkin, G.P. and Turnbull, M.J., GABA receptor multiplicity. Visualization of different receptor types in the mammalian CNS, Neuropharmacology, 23 (1984) 219-23l. 4 Collins, G.G.S., Anson, J. and Kelly, E.P., Baclofen: effects on evoked field potentials and amino acid neurotransmitter release in the rat olfactory cortex slice, Brain Research, 238 (1982) 371-383. 5 Curtis, D.R., GABAergic transmission in the mammalian central nervous system. In P. Krogsgaard-Larsen, J. Scheel-Kruger and H. Kofod (Eds.), GABA-Neurotransmitters - - Alfred Benzon Symposium 12, Munksgaard, Copenhagen, 1979, pp. 17-27. 6 Curtis, D.R., Game, S.J.A., Johnston, J.A.R. and McCulloch, R.M., Central effects of fl-(p-chlorophenyl)-y-aminobutyric acid, Brain Research, 70 (1974) 493-499. 7 Davies, J. and Watkins, J.C., The action of fl-phenylGABA derivatives on neurones of the cat cerebral cortex, Brain Research, 7[) (1974) 5[]1-5(15. 8 G~ihwiler, B.H. and Brown, D.A., GABAFreceptor-activated K+ current in voltage-clamped CA 3pyramidal cells in
in an earlier investigation1. Here it was found that most of the cells in which bicuculline is able to only partially antagonize suppression of evoked responses by G A B A are located in layers 5 and 6. A 'low d o s e - s t r o n g response' relationship for baclofen was seen in neurons facilitated by phaclofen. In these cells drug spread might have been directed to the dendrites rather than to the cell body; here baclofen would significantly reduce the amount of excitatory transmitter released and phaclofen, by contrast, would counteract a reduction in release of transmitter mediated by intrinsic G A B A possibly via G A B A B receptors. It is still unclear whether the suppression of evoked responses by phaclofen is caused by an increase in GABA-ergic inhibition secondary to an increase in the release of excitatory amino acid transmitter 4, since we did not test bicuculline on these neurons. A local anesthetic type of action 11 does not seem to play an important role because from the 10 neurons suppressed by phaclofen the amplitude of the extracellularly recorded action potential was reduced in only one case.
We thank Drs. Rossi and K. Scheibli from CibaGeigy for providing us with baclofen.
hippocampal cultures, Proc. Acad. Sci. U.S.A., 82 (1985) 1558-1562. 9 Gehlert, D.R., Yamamura, H.I. and Wamsley, J.K., yAminobutyric acidu receptors in the rat brain: quantitative autoradiographic localization using [3H](-)-baclofen, Neurosci. Lett., 56 (1985) 183-188. 10 Howe, J.R., SutoL B. and Zieglg~insberger,W., Baclofen reduces postsynaptic potentials of rat cortical neurones by an action other than its hyperpolarizing action, J. Physiol. (Lond.), 384 (1987) 539-569. 11 Kerr, D.I.B., Ong., J., Prager, B.D. and Curtis, D.R., Phaclofen: a peripheral and central baclofen antagonist, Brain Research, 4(}5(1987) 150-154. 12 Muhyaddin, M., Roberts, P.J. and Woodruff, G.N., Presynaptic y-aminobutyric acid receptors in the rat anococcygeus muscle and their antagonism by 5-aminovaleric acid, Br. J. Pharmacol., 77 (1982) 163-168. 13 Newberry, N.R. and Nicoll, R.A., Comparison of the action of baclofen with y-aminobutyric acid on rat hippocampal cells in vitro, J. Physiol. (Lond.), 360 (1985) 161-185. 14 Pong, S.F. and Graham. Jr., L.T., A simple preparation of bicuculline methiodide, a water-soluble GABA antagonist, Brain Research, 58 (1973) 266-267. 15 SiIlito, A.M., Functional considerations of the operation of GABAergic inhibitory processes in the visual cortex. In E.G. Jones and A. Peters (Eds.), Cerebral Cortex, Vol. 2, Functional Properties o1"Cortical Cells, Plenum, New York, 1984, pp. 91 117.
Brain Research, 463 (1988) 398-402 Elsevier
BRE 23170
Phaclofen antagonizes baclofen-induced suppression of visually evoked responses in the cat's striate cortex U. Baumfalk and K. Albus Max-Planck-lnstitut fiir Biophysikalische Chemie, Abteilung Neurobiologie, G6ttingen (F. R. G.)
(Accepted 12 July 1988) Key words': Phaclofen; Baclofen-induced suppression; Visually evoked response; Striate cortex
The suppression of visually evoked responses (VER) in the cat's striate cortex by baclofen was reversibly antagonized by the phosphonic acid derivative of baclofen, phaclofen. The antagonistic effect of phaclofen was seen irrespective of whether it enhanced, suppressed or did not change VER on its own. The suppression of VER by ),-aminobutyric acid (GABA) was not affected by phaclofen. Our findings support the notion (Kerr et al., Brain Research, 405 (1987) 150-154) that phaclofen is an effective baclofen antagonist in the central nervous system of mammals. Preliminary findings indicate that in spite of its possible action on GABA Breceptors, phaclofen does not significantly alter functional properties of striate cortical neurons, in particular direction and orientation sensitivity.
It is well established that there are two classes of y-aminobutyric acid ( G A B A ) receptors in the peripheral and central nervous systems: G A B A A and G A B A B 3. Inhibitory mechanisms mediated via G A B A receptors have been shown to contribute to functional properties of many neurons in the striate cortex of cats: microelectrophoretic application of the G A B A A antagonist bicuculline near single neurons may diminish orientation and direction sensitivity and alter substantially the spatial organization of receptive fields 15. Although a high density of G A B A B receptor binding sites has been demonstrated in the mammalian neocortex 9, and although the G A B A B agonist, baclofen (4-amino-3-(p-chlorophenyl)butyric acid) 2 suppresses neuronal responses in neocortical neurons l'6'7, the physiological significance of G A B A ~ receptor sites has been less clear. A major step towards understanding the functional importance of bicuculline insensitive G A B A receptor sites has been the recent demonstration that fl-(pchlorophenyl)-3-aminopropylphosphonic acid (phaclofen) reversibly antagonizes the effects of baclofen in the guinea pig's ileum and colon and in the cat's spinal cord 11. To assess the possible contribution of
G A B A B receptors to functions of the visual cortex we have investigated the effects of phaclofen on visually evoked responses of single striate neurons in the cat, and on the baclofen induced suppression of these responses. In addition to phaclofen, other compounds known to interfere with baclofen induced inhibition, such as barium (Ba2+) s and 5-aminovaleric acid (5-AVA) 12 were tested. The experiments were performed on 16 adult cats. The animals were anesthetized with Ketanest (15 mg/kg, i.m.) and R o m p u n (2 mg/kg, i.m.), followed by a continuous infusion of 2 mg/kg/h pentobarbitone in Ringer's solution. Muscular relaxation was maintained with gallamine triethiodide (Flaxedil), and the animals were artificially respired with a gas mixture of N20/O2/CO 2 (67%/30%/3%). Refractive errors of the eyes were corrected with plastic lenses which also protected the corneae. The heart rate, blood pressure, body temperature and expiratory CO 2 were continuously monitored and held at physiological levels. Extracellular recordings were obtained from a glass-coated tungsten wire fixed parallel to a 7-barrelied pipette. The distance between the tip of the electrode and the tip of the pipette averaged 30 and
Correspondence: U. Baumfalk, Max-Planck-Institut for Biophysikalische Chemie, Am Fassberg, D-3400 G6ttingen, F.R.G.
0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
399 70/~m. The pipettes were filled with the following drug solutions: G A B A (Sigma) 0.5 M in aqua dest., pH 3.5; baclofen (racemic form, Ciba-Geigy) 25 mM in 150 mM NaCI, pH 3; phaclofen (Tocris Neuramin), about 60 mM in 75 mM NaC1, pH 3.5; bicuculline methiodide (BMI) racemic form (Pierce) or (+)enantiomer (Sigma, further preparation according to ref. 14) 8.5 mM, in citrate buffer pH 3.5, 5-AVA (Sigma) 25 mM or 0.5 M in aqua dest., pH 3.5; BaC12 (Sigma) 25 mM in aqua dest., pH 3.5. Retaining currents of 15 nA were applied to all barrels. Current balancing was performed via a barrel filled with Ringer's solution. Some electrode tracks were reconstructed from electrolytic lesions. Visual stimulation was performed by projecting light bars onto a translucent screen 0.57 cm in front of the animal's eyes by a light projector operated by means of a computercontrolled stimulation program. To avoid saturation of visually evoked responses, intensity and movement velocity of an optimally oriented bar were adjusted to yield submaximal responses. The stimulus was moved back and forth across the cell's receptive field for the dominant eye. The effects of phaclofen on spontaneous and visually evoked activity were tested in 35 neurons. Evoked responses were either facilitated (9 cells), suppressed (10 cells) or not affected (10 cells). These effects of phaclofen seemed to be independent from ejection current, which averaged 88 nA in the cells facilitated, 95 nA in the cells suppressed, and 95 nA in the cells not affected by phaclofen. In a further 5 neurons evoked responses were suppressed at ejection currents between 40 nA and 100 nA and became normal or were even enhanced with higher ejection currents. When responses became facilitated the increase in response amplitude was generally less than 40% of the controls (average 22%), and usually the spontaneous activity did not change. Spontaneous activity increased only in 3 neurons in which the evoked response was enhanced by more than 60%. Two of these neurons reliably responded to visual stimulation only under phaclofen; in a third case facilitation disappeared during later periods of application. The strength of the suppression varied between neurons and seemed to be independent from the ejection cur rent. At 200 nA phaclofen completely suppressed evoked responses in one cell and reduced response
amplitude by only 80%, 30% and 10%, respectively, in 3 other cells. The difference between the effects of phaclofen on single striate cortical neurons could indicate differences in functional importance and/or location of receptor sites mediating the effects of baclofen. These effects were therefore quantified separately for the cells showing no phaclofen effects (NE), for the cells facilitated (FE), and for the cells suppressed (SE) by phaclofen. In the FE-group on average 16 nA baclofen suppressed evoked responses by about 83%; in
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Fig. 1. Responses of a standard complex cell (layer 5) to visual stimulation before, during and after microiontophoretic application of baclofen (BAC), GABA and phaclofen (PHAC). Each point represents the cell responses averaged over 5 sweeps of a light bar moving in the preferred direction across the receptive field. Movement was always perpendicular to bar orientation. The horizontal bars indicate the duration of the drug application and the numbers besides these bars indicate the ejection current (nA) used for the corresponding drugs.
400 the SE-group the respective values were 22 n A and 80%, and in the N E - g r o u p 37 n A and 44%, respectively. The average application time of baclofen for yielding maximal effects was about 10 min in the NEgroup, and about 5 min in both F E - g r o u p and SEgroup. This difference in time is due to the fact that the ejection of baclofen was started with a very low current (5 n A ) , which was increased in a stepwise manner until consistent effects were observed. Recovery times were the same in the 3 groups. These results show that phaclofen was ineffective in those neurons having a 'high d o s e - w e a k response' relationship for baclofen. The results of the interaction between baclofen and phaclofen were unequivocal. In 17 of 21 neurons tested the baclofen-induced inhibition was reversibly antagonized by phaclofen. C o m p l e t e antagonism was seen in 11 neurons (Figs. 1-3): in 5 of these 11 neurons the baclofen-induced inhibition was actually
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Fig. 3. Responses of a standard complex cell (layer 6) to visual stimulation before, during and after microiontophoretic application of baclofen (BAC), GABA and phaclofen (PHAC). The transient suppression of the responses by phaclofen was stronger with lower ejection currents (not shown here).
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Fig. 2. Responses of a special complex cell (layer 5) to visua! stimulation before, during and after application of baclofen (BAC) and phaclofen (PHAC). The evoked responses of the cell were slightly suppressed with higher ejection currents of phaclofen.
reversed and responses were enhanced under phaclofen (Fig. 1); in 4 neurons phaclofen reduced the baclofen induced inhibition by 4 0 - 7 0 % , and in the two remaining cases a slight reduction in inhibition was c o m b i n e d with a delay in onset, and a decrease in the duration of the baclofen action. Phaclofen applied with ejection currents reducing baclofen induced suppression did not affect the G A B A - i n d u c e d inhibition of e v o k e d responses (Figs. 1, 3), and this was found for 6 neurons tested. The antagonistic effect of phaclofen became stronger with increase in ejection current (Figs. 1, 2). In the case shown in Fig. 1,80 n A phaclofen did not alter the baciofen-induced inhibition which however was blocked with 200 n A phaclofen. The relationship between ejection current and antagonistic action was also d e m o n s t r a t e d by the fact that the average ejection current was higher in cases with complete antagonism (125 nA;
401 range 50 nA-250 nA) than in cases with partial antagonism (80 nA; range 20 nA-250 nA). The antagonistic effect of phaclofen was found to be independent on whether phaclofen preceded or followed this application of phaclofen (3 neurons tested; Fig. 3). The antagonistic effects on the baclofen-induced inhibition were seen irrespective of whether or not phaclofen changed evoked responses on its own (Fig. 2). Even in a neuron in which it suppressed evoked responses by 80%, phaclofen blocked the inhibition elicited by baclofen but not that induced by G A B A . The baclofen-induced suppression was antagonized by phaclofen for a wide range of cell types, i.e. in neurons having simple, complex and hypercomplex receptive fields as well as in neurons responding only to a moving stimulus. For 4 neurons inhibition by baclofen was not antagonized by phaclofen. In one of these cases the ejection current was most probably too low (30 nA). In the 3 other neurons however, ejection currents were in the range (100 nA-170 nA) where one would expect antagonistic actions (see above). Interestingly, these neurons were those most sensitive to baclofen in our whole sample, their evoked responses being strongly inhibited by ejection currents of 8 nA or less. The ineffectiveness of phaclofen in these cases might therefore be due to a competitive disadvantage caused by low concentration at the receptor site rather than to an absence of antagonistic property. 5-AVA was tested in 30 neurons. When ejected from a 0.5 M solution (3-16 nA, mean 8 nA) it suppressed the evoked responses of all 6 neurons tested. When ejected as a 25 mM solution (24 neurons; ejection currents: 12-75 nA, mean 44 nA) evoked responses were either suppressed (n = 7), facilitated (n = 5) or not changed (n = 12). The AVA-induced suppression was readily antagonized with BMI, and with continuing application of BMI cell responses became strongly enhanced. The interaction between 5AVA and baclofen was tested in 20 neurons, and the baclofen-induced inhibition was reversibly antagonized in only 3 of them. When the 5-AVA-induced suppression had been counteracted with BMI subsequent application of baclofen either inhibited or did not change evoked responses. It could not be decided definitely, however, whether the 'ineffectiveness' of baclofen in the latter cases was caused by an antagonistic action of 5-AVA or by the strong facilitation
seen with prolonged application of BMI. Ba 2+ (4 neurons; currents: 20-50 nA, mean 37 nA) either reduced baclofen induced suppression, or had no effect; it also partially or completely blocked G A B A induced inhibition in these cases. Phaclofen did not systematically change the directional selectivity of the striate cortical neurons. Of the 34 neurons tested, 20 were directionally selective, which means that their response to movement of a light stimulus into one direction (preferred response) was more than two times stronger than the response into the opposite direction (non-preferred response). Only two of these 20 neurons changed the degree of their directional selectivity under phaclofen. In one case directional selectivity increased due to a suppression, and in another cell it was diminished due to an enhancement of the non-preferred response. In a few neurons also the orientation selectivity was studied and significant changes were not observed under phaclofen. One may tentatively suggest therefore, that baclofen receptor sites, which might be in part identical with G A B A B receptors, are involved in regulating the general excitability of a neuron rather than to contribute to specific functional properties. Our findings demonstrate that phaclofen antagonizes the baclofen suppression of visually evoked responses of striate cortical neurons. A competitive mechanism is suggested by the fact that the strength of the antagonism increases with ejection current. In addition the antagonistic effect is independent on whether phaclofen precedes or follows the application of baciofen. Inhibition of evoked responses by G A B A , which was effectively antagonized by bicuculline, was not reduced by phaclofen. The discussion of possible sites of the antagonistic action of phaclofen has to consider the sites of action of baclofen. In neocortical neurons baclofen has been shown to increase postsynaptic potassium conductance and to depress postsynaptic potentials by reducing the release of excitatory amino acids, i.e. having both pre- and postsynaptic effects (for a review see ref. 9). Our findings that suppression by baclofen was antagonized completely in most neurons tested also suggest both pre- and postsynaptic sites of action for phaclofen. This point will certainly be clarified only when intracellular recording experiments are performed. Interestingly, in neurons of rat
402 frontal cortex G A B A applied in the vicinity of the cell soma does not induce baclofen-like hyperpolarizatior~s in the presence of bicuculline1°. This finding has been presented as evidence against a participation of G A B A a receptors in mediating suppression by baclofen. O n the other hand bicuculline-resistant hyperpolarizations were evoked in hippocampal neurons when G A B A was applied to the dendritic field 13. Therefore it is suggested that baclofen acts at more peripheral parts of the neuron. O n the basis of our observation that the effects of phaclofen alone were somehow correlated with the effectiveness of baclofen it is assumed that phaclofen interacts with the same receptor site as does baclofen. Phaclofen did not change evoked responses in cells with a 'high d o s e - w e a k response' relationship for baclofen. It is noteworthy that 6 of these 10 neurons were complex cells of layers 5 and 6. Therefore, the ineffectiveness of phaclofen and a low effectiveness of baclofen in these neurons could be explained by a relatively long distance between ejection site and more distal parts of the dendrites where baclofen should exert its main effects on neuronal excitability13. A possible participation of GABAB receptors in mediating suppression by baclofen in deeper layer neurons of striate cortex receives additional support from observations made
1 Baumfalk, U. and Albus, K., Baclofen inhibits the spontaneous and visually evoked responses of neurones in the striate cortex of the cat, Neurosci. Lett., 75 (1987) 187-192. 2 Bowery, N.G., Hill, D.R., Hudson, A.L., Doble, A.L., Middlemiss, A., Shaw, J. and Turnbull, M., (-)-Baclofen decrease neurotransmitter release in the mammalian CNS by an action at a novel GABA receptor, Nature (Lond.), 283 (1980) 92-94. 3 Bowery, N.G., Price, G.W., Hudson, A.L., Hill, D.R., Wilkin, G.P. and Turnbull, M.J., GABA receptor multiplicity. Visualization of different receptor types in the mammalian CNS, Neuropharmacology, 23 (1984) 219-23l. 4 Collins, G.G.S., Anson, J. and Kelly, E.P., Baclofen: effects on evoked field potentials and amino acid neurotransmitter release in the rat olfactory cortex slice, Brain Research, 238 (1982) 371-383. 5 Curtis, D.R., GABAergic transmission in the mammalian central nervous system. In P. Krogsgaard-Larsen, J. Scheel-Kruger and H. Kofod (Eds.), GABA-Neurotransmitters - - Alfred Benzon Symposium 12, Munksgaard, Copenhagen, 1979, pp. 17-27. 6 Curtis, D.R., Game, S.J.A., Johnston, J.A.R. and McCulloch, R.M., Central effects of fl-(p-chlorophenyl)-y-aminobutyric acid, Brain Research, 70 (1974) 493-499. 7 Davies, J. and Watkins, J.C., The action of fl-phenylGABA derivatives on neurones of the cat cerebral cortex, Brain Research, 7[) (1974) 5[]1-5(15. 8 G~ihwiler, B.H. and Brown, D.A., GABAFreceptor-activated K+ current in voltage-clamped CA 3pyramidal cells in
in an earlier investigation1. Here it was found that most of the cells in which bicuculline is able to only partially antagonize suppression of evoked responses by G A B A are located in layers 5 and 6. A 'low d o s e - s t r o n g response' relationship for baclofen was seen in neurons facilitated by phaclofen. In these cells drug spread might have been directed to the dendrites rather than to the cell body; here baclofen would significantly reduce the amount of excitatory transmitter released and phaclofen, by contrast, would counteract a reduction in release of transmitter mediated by intrinsic G A B A possibly via G A B A B receptors. It is still unclear whether the suppression of evoked responses by phaclofen is caused by an increase in GABA-ergic inhibition secondary to an increase in the release of excitatory amino acid transmitter 4, since we did not test bicuculline on these neurons. A local anesthetic type of action 11 does not seem to play an important role because from the 10 neurons suppressed by phaclofen the amplitude of the extracellularly recorded action potential was reduced in only one case.
We thank Drs. Rossi and K. Scheibli from CibaGeigy for providing us with baclofen.
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