Journal of Physiology (1990), 422, pp. 433-446 With 5 figures Printed in Great Britain
433
ON THE PRESYNAPTIC ACTION OF BACLOFEN AT INHIBITORY SYNAPSES BETWEEN CULTURED RAT HIPPOCAMPAL NEURONES
BY NEIL L. HARRISON* From the Laboratory of Neurophysiology, NINDS, National Institutes of Health, Bethesda, MD 20892, USA
(Received 12 June 1989) SUMMARY
1. (-)Baclofen reduces inhibitory postsynaptic potentials (IPSPs) and the associated synaptic currents (IPSCs) at inhibitory GABAergic synapses between cultured rat hippocampal neurones. The reversal potential for the IPSC is unaltered. 2. The effect of (-)baclofen is concentration dependent; the EC50 for (-)baclofen is approximately 5 ,M. 3. Statistical analyses of the amplitude fluctuations of the IPSC in the presence of (-)baclofen suggested a presynaptic location for the depression of synaptic transmission by (-)baclofen. In control experiments, lowering extracellular Ca2+ produced similar effects. (-)Baclofen has no detectable postsynaptic actions in these cultured neurones. 4. Phaclofen (0-2-05 mM) increases IPSC amplitude but does not significantly block the depressant effect of (-)baclofen on synaptic transmission. 5. The effect of (-)baclofen is not blocked by pertussis toxin pre-treatment. 6. It is concluded that (-)baclofen acts presynaptically to reduce the release of GABA. The mechanism by which release is reduced may involve a phaclofeninsensitive GABAB receptor. INTRODUCTION
The inhibitory neurotransmitter y-aminobutyric acid (GABA) binds to and activates two classes of receptor on neuronal membranes: GABAA and GABAB receptors (Bowery, Hill, Hudson, Doble, Middlemiss, Shaw & Turnbull, 1980; Hill & Bowery, 1981; Bowery, Hudson & Price, 1987). The GABAA receptor mediates fast IPSPs in supraspinal regions of the CNS, but not the slower 'late' IPSP (Newberry & Nicoll, 1984 a). The antispastic agent baclofen has no action at GABAA receptors but binds to GABAB receptors (Hill & Bowery, 1981). Binding of baclofen to GABAB receptors is modulated by GTP (Hill, Bowery & Hudson, 1984; Karbon & Enna, 1984; Wojcik & Neff, 1984; Asano, Ui & Ogasawara, 1985), suggesting that certain actions of baclofen are mediated via interactions with GTP-binding proteins ('G proteins'; Gilman, 1986). * Present address: Department of Anesthesia and Critical Care, The University of Chicago, Chicago, IL 60637, USA.
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Baclofen increases K+ conductance and thereby hyperpolarizes hippocampal pyramidal cells (Newberry & Nicoll, 1984b; Gahwiler & Brown, 1985) and other mature CNS neurones (Gallagher, Stevens & Shinnick-Gallagher, 1984; Stevens, Gallagher & Shinnick-Gallagher, 1985; Howe, Sutor & Zieglgainsberger, 1987; Christie & North, 1988; Colmers & Williams, 1988; Connors, Malenka & Silva, 1988; Lacey, Mercuri & North, 1988). Recent evidence suggests that GABAB receptor activation mediates 'late' IPSPs in the hippocampus (Dutar & Nicoll, 1988 a), thalamus (Soltesz, Haby, Leresche & Crunelli, 1988) and dorsolateral septal nucleus (Hasuo & Gallagher, 1988), since both the late IPSP and the response to baclofen are blocked by the GABAB receptor antagonist, phaclofen (Kerr, Ong, Prager, Gynther & Curits, 1987). It also appears that both the increase in K+ conductance stimulated by baclofen and the 'late'IPSP involve a G protein, since both are abolished by pre-incubation of the tissue with pertussis toxin (Andrade, Malenka & Nicoll, 1986; Thalmann, 1988), which inactivates certain G proteins by catalysing ADP ribosylation of the protein (Ui, 1984). In addition, baclofen decreases voltage-dependent Ca2+ current in sensory neurone cell bodies (Dunlap, 1981; Desarmenien, Feltz, Occhipinti, Santangelo & Schlichter, 1984; Deisz & Lux, 1985; Dolphin & Scott, 1986). These effects are similarly prevented by preincubation of the tissue with pertussis toxin (Holz, Rane & Dunlap, 1986). Baclofen depresses both excitatory and inhibitory synaptic transmission in the rat hippocampus (Ault & Nadler, 1982; Peet & McLennan, 1986) and neocortex (Howe et al. 1987) by a presumed presynaptic mechanism. In an earlier report from this laboratory, it was suggested that presynaptic GABAB receptors might be present on the terminals of GABAergic neurones (Harrison, Lange & Barker, 1988). The present study describes in detail the actions of baclofen on GABA-mediated inhibitory synaptic transmission between cultured rat hippocampal neurones at the level of a single inhibitory synapse. In addition, the effects of phaclofen and pre-treatment with pertussis toxin were also investigated, in order to determine whether the processes mediating the synaptic depressant actions of baclofen are similar to those mediating the increase in K+ conductance and decrease of Ca2+ current. A preliminary report of certain aspects of this work has appeared (Harrison, 1988). METHODS
Cell culture Neurones from the hippocampi of day 19-20 embryonic rats were dissociated and maintained in culture for 2-5 weeks by methods similar to those previously described, using enzymatic treatment with papain (Huettner & Baughmann, 1986), followed by mechanical trituration. Donor mother rats were killed by exposure to a rapidly rising concentration of CO2 and then cervically dislocated. Embryonic rats were removed and rapidly decapitated, the brains removed and the hippocampi dissected out. Neurones were plated at 50-100000 cells ml-' onto confluent cortical astrocytes (Forsythe & Westbrook, 1988). The maintenance medium contained minimal Eagle's medium supplemented with 2 mM-glutamine and 5% horse serum (Hyclone; Logan, UT, USA).
Electrophysiological recordings
Recordings were made at room temperature (19-24 °C), on the stage of an inverted phasecontrast microscope, from neurones with cell bodies between 10 and 20 ,um in diameter. The wholecell variation of the patch-clamp technique was used to record membrane currents or potentials (Hamill, Marty, Neher, Sakmann & Sigworth, 1981) via two List EPC-7 amplifiers. Pipette-
BACLOFEN EFFECTS ON GABAERGIC SYNAPSES
435
membrane seals were 2-20 GQ; pipette-to-bath resistances prior to seal formation were 3-5 MQ. The patch pipettes were filled with an 'intracellular' solution containing (in mM): potassium gluconate, 145; K2ATP, 5; MgCl2, 2; CaCl2, 041; EGTA, 1L1; HEPES, 5; titrated to pH 7-2 with KOH. The osmolarity was adjusted to 315 mosm. In some experiments, this medium was supplemented with 0-2 mM-NaGTP. The extracellular medium contained (in mM): NaCl, 125; KCl, 5; MgCl2, 8; CaCl2, 4; D-glucose, 6; HEPES, 10; titrated to pH 7-4 with NaOH. The high level of Mg2+ in the extracellular medium was associated with a low level of spontaneous synaptic activity (Segal & Barker, 1984). The liquid-junction potential between the two solutions was negated by placing the reference electrode in a side compartment containing the 'intracellular' medium. The two compartments were connected with a bridge; an agar-saline 'plug' prevented mixing of the solutions, but ensured low (kQ) electrical resistance (Barker & Harrison, 1988). The postsynaptic events recorded were evoked by direct injection of depolarizing current via the presynaptic patch pipette (1 nA, 2-5 ms, 0-1 Hz), sufficient to trigger an action potential in the presynaptic neurone (Barker & Harrison, 1988). (-)Baclofen and GABA were applied by pressure from micropipettes (tip diameter 2-3 ,um) placed within 5-10 ,sm of the postsynaptic cell body. Data acquisition and analysis Pipette current or voltage was sampled and digitized at rates between 1 and 5 kHz, using a Data Translation analog-to-digital converter (12 bit, +5 V range). Digitized data were stored for off-line analysis on a PDP 11-23 computer. Signals were low-pass filtered at half the sampling rate. Decay time constants, TIPSC, were estimated for individual synaptic currents using a least-squares fitting routine. Synaptic latency was determined as the time elapsed between the peak of the presynaptic somatic action potential and the onset of the postsynaptic signal. Where mean values of variables are given, these are quoted as mean + standard deviation. Calculation of peak synaptic chord conductance The IPSC reversal potential, EIPSC, was obtained by linear interpolation from the current-voltage relation for peak IPSC amplitude, I, for each target cell. The driving force for Cl- ion movement, VD, was then expressed for a given holding potential, VH, as VD = VH -EIPSc. The peak synaptic conductance was then defined as gIPSC = I/(VH-EIPSC)-
Statistical analysis of amplitude fluctuations Statistical analysis of IPSC amplitude fluctuations were performed in order to determine whether the sites of action of various agents were pre- or postsynaptic. The ratio r = 12/s2, where r is mean experimental IPSC amplitude and 52 the experimental variance about the mean, was calculated for long stretches of IPSC amplitude measurements (20-200) obtained under control, drug and recovery conditions. Without assuming any particular model for release of transmitter, it is generally true that under experimental conditions that reduce output of transmitter, the mean declines in the absence of a large change in variance, and r is therefore significantly reduced, while during purely postsynaptic manipulations, both r and 52 decline so that the ratio r is relatively unaffected (e.g. Nelson, Marshal, Pun, Christian, Sherriff, Macdonald & Neale, 1983). The effect of the agents was expressed in terms of the ratio of means M = I2/'1 and R = r2/r1 where r, is the mean square-to-variance ratio under control conditions and r2 is the ratio in the presence of the agent. Values of both M and R were collected from several experiments of this type and a onetailed t test was employed to determine whether these quantities were significantly different from 1. Chemicals and reagents Baclofen isomers were from Ciba-Geigy. Phaclofen was obtained from Tocris Chemicals. Pertussis toxin was obtained from List Biologicals. RESULTS
Effects of baclofen on IPSPs IPSPs were elicited in postsynaptic elements by electrically evoking action potentials in the presynaptic neurone (stimuli 2-5 ms, 1 nA, 01 Hz) and were
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identified as monosynaptic GABA-mediated IPSPs by virtue of their short latency (1-3 ms) reversal potential (-60 to -70 mV) and sensitivity to the GABAA receptQr antagonist bicuculline (10-20 ,UM). (-)Baclofen had a consistent depressant action on inhibitory synaptic transmission (Fig. IA). In nine experiments, application of (-)baclofen from a pipette containing 10 ,lM reduced IPSP amplitude by 46% (mean±s.D.: 46+8%). A Postsynaptic neurone -50 mV
0mV
'
10 Control
(-) Baclofen
mVB
Recovery 1 ml
50 mV
-60 mV
v
Presynaptic neurone
(-) Rai-!lnfQn
mv
IJiiiiILLYLUILL
C Control
[II I ILI Jj _X- _ -_ 110
Rp-invarv
I
10.2 nA 25 ms
\I/ j10.l1nA los
_____ Baclofen (5PM)
Fig. 1. Baclofen reduces the amplitude of monosynaptic IPSPs and IPSCs. A, (-)baclofen reduces IPSP amplitude at synapses between cultured rat hippocampal neurones. The postsynaptic neurone was maintained at -50 mV by constant depolarizing current injection, while the presynaptic neurone was held near to its resting membrane potential at -60 mV. Each presynaptic action potential was elicited by a 4 ms, 1 nA current pulse. Local application of 5 ,lM-(-)baclofen from a micropipette placed near to the postsynaptic cell body results in a decrease of 50 % in IPSP amplitude. Full recovery is obtained after terminating the application of (-)baclofen. B, when the postsynaptic cell was held at -40 mV in voltage clamp, outwardly directed, exponentially decaying synaptic currents were evoked; I, postsynaptic current, V, presynaptic membrane potential. These evoked inhibitory postsynaptic currents (IPSCs) were depressed by application of (-)baclofen. The time course of the effect of (-)baclofen on the synaptic current is illustrated by the pen record. The very small outward current seen on application of baclofen was not associated with a conductance increase and is an artifact associated with the pressure application technique. C, individual IPSPs are illustrated to show the effect of (-)baclofen on the amplitude and decay of the IPSC.
Effect of baclofen on IPSCs When the postsynaptic cell was held at -40 mV in voltage clamp, outwardly directed, exponentially decaying synaptic currents were evoked. These evoked inhibitory postsynaptic currents (IPSCs) were also depressed in amplitude by application of (-)baclofen. On termination of the application of baclofen, IPSCs recovered slowly to control amplitudes (Fig. 1B). Individual IPSCs recorded at -40
J~ ~ ~ ~10mV
BACLOFEN EFFECTS ON GABAERGIC SYNAPSES GABA I
GABA
IUVtIIIJII'I1
A
437
150 pA
A
Fig. 2. GABA reduces the amplitude of inhibitory postsynaptic currents. Local application of a brief pulse (top) of 50 /sM-GABA from a micropipette placed near to the postsynaptic cell body results in a rapidly developing and decaying outward current and a dramatic decrease in IPSC amplitude (middle record - postsynaptic membrane current). The application of GABA (arrow-heads) also causes a small hyperpolarization of the presynaptic membrane potential (lower record), but this does not prevent action potential generation. Full recovery is obtained after terminating the application of GABA. Note that the decrease of IPSC amplitude outlasts the time course of the rapid outward current response. A
1200 . X 800 -X 400
a
-40 -20 -80 -60 -100 E~~~~~~~~~~~ U 400 (mV) Membrane potential g n 9! 20 50. n
B
gm**
1'0 .
-100
|
-20 -40 -60 -80 Membrane potential (mV)
Fig. 3. Baclofen does not alter reversal potential for the IPSC. A, in the presence of 5/SM(- )baclofen, IPSC amplitude was decreased at all potentials, but the reversal potential for the IPSC, EIS was unaltered (EIPSC = -62 mV). B, the synaptic chord conductance 9IPS increased with depolarization in control conditions and in the presence of (- )baclofen. Thus the decrease in synaptic transmission was independent of postsynaptic membrane potential. E, control; *, (- )baclofen.
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mV were substantially reduced in amplitude (Fig. 1 C). The response to baclofen was observed at virtually all inhibitory synapses; in sixty-five experiments in which IPSCs were recorded and (-)baclofen applied at concentrations > 1 ,UM, IPSC amplitude was reversibly reduced in sixty-three.
Effect of GABA on IPSCs When the postsynaptic cell was held at -40 mV in voltage clamp, application of brief pulses of GABA elicited rapid outwardly directed currents that decayed rapidly on terminating the pulse and were sensitive to bicuculline. In addition, GABA application also reduced the amplitude of evoked IPSCs. In many cases (e.g. Fig. 2) the IPSCs were clearly depressed for some time after the complete decay of the rapid GABAA receptor-mediated responses.
Baclofen does not alter EIPSC The IPSC recorded under these conditions reverses in polarity between -60 and -80 mV and shows outward rectification. Application of 10 /M-(-)baclofen consistently caused a large decrease in the amplitude of the IPSC, but did not alter the reversal potential for the IPSC (Fig. 3A). In the presence of (-)baclofen, synaptic chord conductance gIPsc was reduced uniformly over a large range of membrane potential, and still increased significantly with postsynaptic depolarization (Fig. 3B).
Effects of baclofen on IPSC kinetics Under control conditions, the synaptic latency (time interval between the peak of the presynaptic somatic action potential and the beginning of the postsynaptic current) is between 2 and 3 ms. The synaptic latency was unaltered when the peak IPSC amplitude was decreased by (-)baclofen (Fig. 4A). The decay time constant for the IPSC (TrpSC) was somewhat voltage dependent in most recordings, increasing about 2-fold between -110 and -40 mV (Fig. 4B). In the presence of (-)baclofen, T,pSCwas still voltage dependent and showed only a small non-significant decrease relative to control recordings. Concentration dependence of the baclofen effect The results from experiments with varying concentrations of (-)baclofen were pooled and a dose-response curve constructed for the synaptic depressant effect (Fig. 5). The IC50 for (-)baclofen was estimated as 6 /M (pipette concentration).
Postsynaptic actions of baclofen (-)Baclofen (10-100 ,tM) had no consistent or significant effect on the resting membrane potential, input resistance or spike threshold of pre- or postsynaptic neurones. In addition, (-)baclofen did not reduce postsynaptic currents evoked by exogenous GABA (Harrison et al. 1988). Statistical analysis of amplitude fluctuations Under 'control' conditions of 4 mM-Ca2" and 8 mM-Mg2+, IPSC amplitudes showed evidence of random fluctuation during data collection periods of 20-200
BACLOFEN EFFECTS ON GABAERGIC SYNAPSES
439
stimuli at 005 Hz. Longer collection periods sometimes revealed evidence for a systematic decline in IPSC amplitude with time, which was probably presynaptic in origin, since the pipette solutions employed here are effective in stabilizing postsynaptic responses to exogenous GABA over much longer time periods (Harrison & Lambert, 1989). For this reason, most data collection epochs contained fifty events, the longest being 200 events. A
L -
Control
0.2 nA 5 rms
Post Pre
50 mV v
L (-) Baclofen
Post Pre
V
B 40
-E30 _ 20
i
*
D
*
10 0
-100 -80 -60 -40 -20 Membrane potential (mV) Fig. 4. Baclofen does not alter synaptic latency or decay time of the IPSC. Traces marked 'Pre' illustrate membrane potential, V, recorded in the presynaptic cell body. Traces marked 'Post' illustrate membrane current, I, recorded under voltage clamp at -40 mV in the postsynaptic neurone. A, under control conditions, synaptic latency was between 2 and 3 ms. The synaptic latency, L, was unaltered (2-2 ms) when the peak IPSC amplitude was decreased by (-)baclofen. B, the decay time constant for the IPSC was somewhat voltage dependent in most recordings, increasing about 2-fold between -110 and -40 mV. In the presence of (-)baclofen, tIPSC was still voltage dependent and showed only a small decrease relative to control recordings. L, control; *, (-)baclofen.
Experiments with (-)baclofen were performed with a concentration of 10 ftM(-)baclofen, which reduced mean IPSC amplitude by about 70% (Table 1). In contrast to the uniform large decline in the mean, the variance was often not reduced in the presence of (-)baclofen, or reduced to a much lesser extent than was the mean.
N. L. HARRISON The ratio r was always greatly reduced by (-)baclofen, so that in a population of four synapses studied, both M and R were significantly < 1 (Table 2). Qualitatively similar results were obtained in experiments in which 'low Ca2+' solutions were applied (Table 2). This coincidental reduction in I and P/82 is consistent with a presynaptic site of action for both (-)baclofen and low Ca2+ solutions.
440
100. 0
80
c
0 0) ._
CU
Q CL C,) n
-7
-5 -6 log [(-) baclofen]
-4
Fig. 5. The effect of (-)baclofen is concentration dependent. The effect of (-)baclofen on IPSC amplitude is concentration related. Percentage reduction in IPSC amplitude is plotted against log [(-)baclofen]. The results from three to fifteen experiments at various concentrations of (-)baclofen were pooled and a dose-response curve constructed for the synaptic depressant effect. The EC50 for (-)baclofen was estimated to be approximately 6 ,UM (pipette concentration). TABLE 1. Examples taken from individual experiments of the effects of (-)baclofen and low Ca2+ medium and changes in postsynaptic membrane potential on the mean and variance of IPSC amplitude, and on the ratio P/S2 1 M 82 R P/S2 83 18 292-2 155-9 a Control 0 119 9-87 0 318 249-3 49-6 (-)Baclofen 1 441-1 51-78 151 b Control 7 61 0-147 0-205 126 3 31-0 Low Ca2+ 559-1 130-9 270 5 c Control 1-064 0-643 50 9 594-1 174-1 -50 mV 0-353 0-777 21-0 434.7 95.5 -60 mV M = I2/Pl, the ratio of means and R = r2/r (see Methods). a, b and c denote different experiments.
The effects of changes in postsynaptic membrane potential on mean and variance of IPSC amplitude were also studied. Changing the postsynaptic membrane potential from -40 to -50 mV reduced the mean IPSC amplitude by an average of 44 % and altering the postsynaptic membrane potential to -60 mV reduced the mean by 73 % (Table 1). In contrast to the results with (-)baclofen and low Ca2 , the variance was also reduced at these holding potentials. There was also no significant change in R
BACLOFEN EFFECTS ON GABAERGIC SYNAPSES
441
when the postsynaptic membrane potential was held at -80 mV, which was more negative than the reversal potential for the IPSC. In four experiments, M was significantly < 1, but R was not significantly different from 1 (Table 2), indicating that the ratio P/S2 was unaltered by the change in postsynaptic membrane potential. TABLE 2. The effects of baclofen and low Ca2+ medium and changes in postsynaptic membrane potential on the mean IPSC amplitude and on /82 M R 0-14 + 0.04* 0-28 + 0 05 (-)Baclofen Low Ca2+ 0.19+0.10* 0-26±0-05 -50 mV 0-56+0-13 1-37+0.63n.s. -60 mV 0 27 + 0 06 0-81 + 033n.s. Data from several experiments were pooled and expressed in terms ofM, the ratio of means, and R, the ratio of 1/82 values (see Methods). * Indicates a significant departure from 1 0 at the level
P
433
ON THE PRESYNAPTIC ACTION OF BACLOFEN AT INHIBITORY SYNAPSES BETWEEN CULTURED RAT HIPPOCAMPAL NEURONES
BY NEIL L. HARRISON* From the Laboratory of Neurophysiology, NINDS, National Institutes of Health, Bethesda, MD 20892, USA
(Received 12 June 1989) SUMMARY
1. (-)Baclofen reduces inhibitory postsynaptic potentials (IPSPs) and the associated synaptic currents (IPSCs) at inhibitory GABAergic synapses between cultured rat hippocampal neurones. The reversal potential for the IPSC is unaltered. 2. The effect of (-)baclofen is concentration dependent; the EC50 for (-)baclofen is approximately 5 ,M. 3. Statistical analyses of the amplitude fluctuations of the IPSC in the presence of (-)baclofen suggested a presynaptic location for the depression of synaptic transmission by (-)baclofen. In control experiments, lowering extracellular Ca2+ produced similar effects. (-)Baclofen has no detectable postsynaptic actions in these cultured neurones. 4. Phaclofen (0-2-05 mM) increases IPSC amplitude but does not significantly block the depressant effect of (-)baclofen on synaptic transmission. 5. The effect of (-)baclofen is not blocked by pertussis toxin pre-treatment. 6. It is concluded that (-)baclofen acts presynaptically to reduce the release of GABA. The mechanism by which release is reduced may involve a phaclofeninsensitive GABAB receptor. INTRODUCTION
The inhibitory neurotransmitter y-aminobutyric acid (GABA) binds to and activates two classes of receptor on neuronal membranes: GABAA and GABAB receptors (Bowery, Hill, Hudson, Doble, Middlemiss, Shaw & Turnbull, 1980; Hill & Bowery, 1981; Bowery, Hudson & Price, 1987). The GABAA receptor mediates fast IPSPs in supraspinal regions of the CNS, but not the slower 'late' IPSP (Newberry & Nicoll, 1984 a). The antispastic agent baclofen has no action at GABAA receptors but binds to GABAB receptors (Hill & Bowery, 1981). Binding of baclofen to GABAB receptors is modulated by GTP (Hill, Bowery & Hudson, 1984; Karbon & Enna, 1984; Wojcik & Neff, 1984; Asano, Ui & Ogasawara, 1985), suggesting that certain actions of baclofen are mediated via interactions with GTP-binding proteins ('G proteins'; Gilman, 1986). * Present address: Department of Anesthesia and Critical Care, The University of Chicago, Chicago, IL 60637, USA.
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Baclofen increases K+ conductance and thereby hyperpolarizes hippocampal pyramidal cells (Newberry & Nicoll, 1984b; Gahwiler & Brown, 1985) and other mature CNS neurones (Gallagher, Stevens & Shinnick-Gallagher, 1984; Stevens, Gallagher & Shinnick-Gallagher, 1985; Howe, Sutor & Zieglgainsberger, 1987; Christie & North, 1988; Colmers & Williams, 1988; Connors, Malenka & Silva, 1988; Lacey, Mercuri & North, 1988). Recent evidence suggests that GABAB receptor activation mediates 'late' IPSPs in the hippocampus (Dutar & Nicoll, 1988 a), thalamus (Soltesz, Haby, Leresche & Crunelli, 1988) and dorsolateral septal nucleus (Hasuo & Gallagher, 1988), since both the late IPSP and the response to baclofen are blocked by the GABAB receptor antagonist, phaclofen (Kerr, Ong, Prager, Gynther & Curits, 1987). It also appears that both the increase in K+ conductance stimulated by baclofen and the 'late'IPSP involve a G protein, since both are abolished by pre-incubation of the tissue with pertussis toxin (Andrade, Malenka & Nicoll, 1986; Thalmann, 1988), which inactivates certain G proteins by catalysing ADP ribosylation of the protein (Ui, 1984). In addition, baclofen decreases voltage-dependent Ca2+ current in sensory neurone cell bodies (Dunlap, 1981; Desarmenien, Feltz, Occhipinti, Santangelo & Schlichter, 1984; Deisz & Lux, 1985; Dolphin & Scott, 1986). These effects are similarly prevented by preincubation of the tissue with pertussis toxin (Holz, Rane & Dunlap, 1986). Baclofen depresses both excitatory and inhibitory synaptic transmission in the rat hippocampus (Ault & Nadler, 1982; Peet & McLennan, 1986) and neocortex (Howe et al. 1987) by a presumed presynaptic mechanism. In an earlier report from this laboratory, it was suggested that presynaptic GABAB receptors might be present on the terminals of GABAergic neurones (Harrison, Lange & Barker, 1988). The present study describes in detail the actions of baclofen on GABA-mediated inhibitory synaptic transmission between cultured rat hippocampal neurones at the level of a single inhibitory synapse. In addition, the effects of phaclofen and pre-treatment with pertussis toxin were also investigated, in order to determine whether the processes mediating the synaptic depressant actions of baclofen are similar to those mediating the increase in K+ conductance and decrease of Ca2+ current. A preliminary report of certain aspects of this work has appeared (Harrison, 1988). METHODS
Cell culture Neurones from the hippocampi of day 19-20 embryonic rats were dissociated and maintained in culture for 2-5 weeks by methods similar to those previously described, using enzymatic treatment with papain (Huettner & Baughmann, 1986), followed by mechanical trituration. Donor mother rats were killed by exposure to a rapidly rising concentration of CO2 and then cervically dislocated. Embryonic rats were removed and rapidly decapitated, the brains removed and the hippocampi dissected out. Neurones were plated at 50-100000 cells ml-' onto confluent cortical astrocytes (Forsythe & Westbrook, 1988). The maintenance medium contained minimal Eagle's medium supplemented with 2 mM-glutamine and 5% horse serum (Hyclone; Logan, UT, USA).
Electrophysiological recordings
Recordings were made at room temperature (19-24 °C), on the stage of an inverted phasecontrast microscope, from neurones with cell bodies between 10 and 20 ,um in diameter. The wholecell variation of the patch-clamp technique was used to record membrane currents or potentials (Hamill, Marty, Neher, Sakmann & Sigworth, 1981) via two List EPC-7 amplifiers. Pipette-
BACLOFEN EFFECTS ON GABAERGIC SYNAPSES
435
membrane seals were 2-20 GQ; pipette-to-bath resistances prior to seal formation were 3-5 MQ. The patch pipettes were filled with an 'intracellular' solution containing (in mM): potassium gluconate, 145; K2ATP, 5; MgCl2, 2; CaCl2, 041; EGTA, 1L1; HEPES, 5; titrated to pH 7-2 with KOH. The osmolarity was adjusted to 315 mosm. In some experiments, this medium was supplemented with 0-2 mM-NaGTP. The extracellular medium contained (in mM): NaCl, 125; KCl, 5; MgCl2, 8; CaCl2, 4; D-glucose, 6; HEPES, 10; titrated to pH 7-4 with NaOH. The high level of Mg2+ in the extracellular medium was associated with a low level of spontaneous synaptic activity (Segal & Barker, 1984). The liquid-junction potential between the two solutions was negated by placing the reference electrode in a side compartment containing the 'intracellular' medium. The two compartments were connected with a bridge; an agar-saline 'plug' prevented mixing of the solutions, but ensured low (kQ) electrical resistance (Barker & Harrison, 1988). The postsynaptic events recorded were evoked by direct injection of depolarizing current via the presynaptic patch pipette (1 nA, 2-5 ms, 0-1 Hz), sufficient to trigger an action potential in the presynaptic neurone (Barker & Harrison, 1988). (-)Baclofen and GABA were applied by pressure from micropipettes (tip diameter 2-3 ,um) placed within 5-10 ,sm of the postsynaptic cell body. Data acquisition and analysis Pipette current or voltage was sampled and digitized at rates between 1 and 5 kHz, using a Data Translation analog-to-digital converter (12 bit, +5 V range). Digitized data were stored for off-line analysis on a PDP 11-23 computer. Signals were low-pass filtered at half the sampling rate. Decay time constants, TIPSC, were estimated for individual synaptic currents using a least-squares fitting routine. Synaptic latency was determined as the time elapsed between the peak of the presynaptic somatic action potential and the onset of the postsynaptic signal. Where mean values of variables are given, these are quoted as mean + standard deviation. Calculation of peak synaptic chord conductance The IPSC reversal potential, EIPSC, was obtained by linear interpolation from the current-voltage relation for peak IPSC amplitude, I, for each target cell. The driving force for Cl- ion movement, VD, was then expressed for a given holding potential, VH, as VD = VH -EIPSc. The peak synaptic conductance was then defined as gIPSC = I/(VH-EIPSC)-
Statistical analysis of amplitude fluctuations Statistical analysis of IPSC amplitude fluctuations were performed in order to determine whether the sites of action of various agents were pre- or postsynaptic. The ratio r = 12/s2, where r is mean experimental IPSC amplitude and 52 the experimental variance about the mean, was calculated for long stretches of IPSC amplitude measurements (20-200) obtained under control, drug and recovery conditions. Without assuming any particular model for release of transmitter, it is generally true that under experimental conditions that reduce output of transmitter, the mean declines in the absence of a large change in variance, and r is therefore significantly reduced, while during purely postsynaptic manipulations, both r and 52 decline so that the ratio r is relatively unaffected (e.g. Nelson, Marshal, Pun, Christian, Sherriff, Macdonald & Neale, 1983). The effect of the agents was expressed in terms of the ratio of means M = I2/'1 and R = r2/r1 where r, is the mean square-to-variance ratio under control conditions and r2 is the ratio in the presence of the agent. Values of both M and R were collected from several experiments of this type and a onetailed t test was employed to determine whether these quantities were significantly different from 1. Chemicals and reagents Baclofen isomers were from Ciba-Geigy. Phaclofen was obtained from Tocris Chemicals. Pertussis toxin was obtained from List Biologicals. RESULTS
Effects of baclofen on IPSPs IPSPs were elicited in postsynaptic elements by electrically evoking action potentials in the presynaptic neurone (stimuli 2-5 ms, 1 nA, 01 Hz) and were
436
N. L. HARRISON
identified as monosynaptic GABA-mediated IPSPs by virtue of their short latency (1-3 ms) reversal potential (-60 to -70 mV) and sensitivity to the GABAA receptQr antagonist bicuculline (10-20 ,UM). (-)Baclofen had a consistent depressant action on inhibitory synaptic transmission (Fig. IA). In nine experiments, application of (-)baclofen from a pipette containing 10 ,lM reduced IPSP amplitude by 46% (mean±s.D.: 46+8%). A Postsynaptic neurone -50 mV
0mV
'
10 Control
(-) Baclofen
mVB
Recovery 1 ml
50 mV
-60 mV
v
Presynaptic neurone
(-) Rai-!lnfQn
mv
IJiiiiILLYLUILL
C Control
[II I ILI Jj _X- _ -_ 110
Rp-invarv
I
10.2 nA 25 ms
\I/ j10.l1nA los
_____ Baclofen (5PM)
Fig. 1. Baclofen reduces the amplitude of monosynaptic IPSPs and IPSCs. A, (-)baclofen reduces IPSP amplitude at synapses between cultured rat hippocampal neurones. The postsynaptic neurone was maintained at -50 mV by constant depolarizing current injection, while the presynaptic neurone was held near to its resting membrane potential at -60 mV. Each presynaptic action potential was elicited by a 4 ms, 1 nA current pulse. Local application of 5 ,lM-(-)baclofen from a micropipette placed near to the postsynaptic cell body results in a decrease of 50 % in IPSP amplitude. Full recovery is obtained after terminating the application of (-)baclofen. B, when the postsynaptic cell was held at -40 mV in voltage clamp, outwardly directed, exponentially decaying synaptic currents were evoked; I, postsynaptic current, V, presynaptic membrane potential. These evoked inhibitory postsynaptic currents (IPSCs) were depressed by application of (-)baclofen. The time course of the effect of (-)baclofen on the synaptic current is illustrated by the pen record. The very small outward current seen on application of baclofen was not associated with a conductance increase and is an artifact associated with the pressure application technique. C, individual IPSPs are illustrated to show the effect of (-)baclofen on the amplitude and decay of the IPSC.
Effect of baclofen on IPSCs When the postsynaptic cell was held at -40 mV in voltage clamp, outwardly directed, exponentially decaying synaptic currents were evoked. These evoked inhibitory postsynaptic currents (IPSCs) were also depressed in amplitude by application of (-)baclofen. On termination of the application of baclofen, IPSCs recovered slowly to control amplitudes (Fig. 1B). Individual IPSCs recorded at -40
J~ ~ ~ ~10mV
BACLOFEN EFFECTS ON GABAERGIC SYNAPSES GABA I
GABA
IUVtIIIJII'I1
A
437
150 pA
A
Fig. 2. GABA reduces the amplitude of inhibitory postsynaptic currents. Local application of a brief pulse (top) of 50 /sM-GABA from a micropipette placed near to the postsynaptic cell body results in a rapidly developing and decaying outward current and a dramatic decrease in IPSC amplitude (middle record - postsynaptic membrane current). The application of GABA (arrow-heads) also causes a small hyperpolarization of the presynaptic membrane potential (lower record), but this does not prevent action potential generation. Full recovery is obtained after terminating the application of GABA. Note that the decrease of IPSC amplitude outlasts the time course of the rapid outward current response. A
1200 . X 800 -X 400
a
-40 -20 -80 -60 -100 E~~~~~~~~~~~ U 400 (mV) Membrane potential g n 9! 20 50. n
B
gm**
1'0 .
-100
|
-20 -40 -60 -80 Membrane potential (mV)
Fig. 3. Baclofen does not alter reversal potential for the IPSC. A, in the presence of 5/SM(- )baclofen, IPSC amplitude was decreased at all potentials, but the reversal potential for the IPSC, EIS was unaltered (EIPSC = -62 mV). B, the synaptic chord conductance 9IPS increased with depolarization in control conditions and in the presence of (- )baclofen. Thus the decrease in synaptic transmission was independent of postsynaptic membrane potential. E, control; *, (- )baclofen.
438
N. L. HARRISON
mV were substantially reduced in amplitude (Fig. 1 C). The response to baclofen was observed at virtually all inhibitory synapses; in sixty-five experiments in which IPSCs were recorded and (-)baclofen applied at concentrations > 1 ,UM, IPSC amplitude was reversibly reduced in sixty-three.
Effect of GABA on IPSCs When the postsynaptic cell was held at -40 mV in voltage clamp, application of brief pulses of GABA elicited rapid outwardly directed currents that decayed rapidly on terminating the pulse and were sensitive to bicuculline. In addition, GABA application also reduced the amplitude of evoked IPSCs. In many cases (e.g. Fig. 2) the IPSCs were clearly depressed for some time after the complete decay of the rapid GABAA receptor-mediated responses.
Baclofen does not alter EIPSC The IPSC recorded under these conditions reverses in polarity between -60 and -80 mV and shows outward rectification. Application of 10 /M-(-)baclofen consistently caused a large decrease in the amplitude of the IPSC, but did not alter the reversal potential for the IPSC (Fig. 3A). In the presence of (-)baclofen, synaptic chord conductance gIPsc was reduced uniformly over a large range of membrane potential, and still increased significantly with postsynaptic depolarization (Fig. 3B).
Effects of baclofen on IPSC kinetics Under control conditions, the synaptic latency (time interval between the peak of the presynaptic somatic action potential and the beginning of the postsynaptic current) is between 2 and 3 ms. The synaptic latency was unaltered when the peak IPSC amplitude was decreased by (-)baclofen (Fig. 4A). The decay time constant for the IPSC (TrpSC) was somewhat voltage dependent in most recordings, increasing about 2-fold between -110 and -40 mV (Fig. 4B). In the presence of (-)baclofen, T,pSCwas still voltage dependent and showed only a small non-significant decrease relative to control recordings. Concentration dependence of the baclofen effect The results from experiments with varying concentrations of (-)baclofen were pooled and a dose-response curve constructed for the synaptic depressant effect (Fig. 5). The IC50 for (-)baclofen was estimated as 6 /M (pipette concentration).
Postsynaptic actions of baclofen (-)Baclofen (10-100 ,tM) had no consistent or significant effect on the resting membrane potential, input resistance or spike threshold of pre- or postsynaptic neurones. In addition, (-)baclofen did not reduce postsynaptic currents evoked by exogenous GABA (Harrison et al. 1988). Statistical analysis of amplitude fluctuations Under 'control' conditions of 4 mM-Ca2" and 8 mM-Mg2+, IPSC amplitudes showed evidence of random fluctuation during data collection periods of 20-200
BACLOFEN EFFECTS ON GABAERGIC SYNAPSES
439
stimuli at 005 Hz. Longer collection periods sometimes revealed evidence for a systematic decline in IPSC amplitude with time, which was probably presynaptic in origin, since the pipette solutions employed here are effective in stabilizing postsynaptic responses to exogenous GABA over much longer time periods (Harrison & Lambert, 1989). For this reason, most data collection epochs contained fifty events, the longest being 200 events. A
L -
Control
0.2 nA 5 rms
Post Pre
50 mV v
L (-) Baclofen
Post Pre
V
B 40
-E30 _ 20
i
*
D
*
10 0
-100 -80 -60 -40 -20 Membrane potential (mV) Fig. 4. Baclofen does not alter synaptic latency or decay time of the IPSC. Traces marked 'Pre' illustrate membrane potential, V, recorded in the presynaptic cell body. Traces marked 'Post' illustrate membrane current, I, recorded under voltage clamp at -40 mV in the postsynaptic neurone. A, under control conditions, synaptic latency was between 2 and 3 ms. The synaptic latency, L, was unaltered (2-2 ms) when the peak IPSC amplitude was decreased by (-)baclofen. B, the decay time constant for the IPSC was somewhat voltage dependent in most recordings, increasing about 2-fold between -110 and -40 mV. In the presence of (-)baclofen, tIPSC was still voltage dependent and showed only a small decrease relative to control recordings. L, control; *, (-)baclofen.
Experiments with (-)baclofen were performed with a concentration of 10 ftM(-)baclofen, which reduced mean IPSC amplitude by about 70% (Table 1). In contrast to the uniform large decline in the mean, the variance was often not reduced in the presence of (-)baclofen, or reduced to a much lesser extent than was the mean.
N. L. HARRISON The ratio r was always greatly reduced by (-)baclofen, so that in a population of four synapses studied, both M and R were significantly < 1 (Table 2). Qualitatively similar results were obtained in experiments in which 'low Ca2+' solutions were applied (Table 2). This coincidental reduction in I and P/82 is consistent with a presynaptic site of action for both (-)baclofen and low Ca2+ solutions.
440
100. 0
80
c
0 0) ._
CU
Q CL C,) n
-7
-5 -6 log [(-) baclofen]
-4
Fig. 5. The effect of (-)baclofen is concentration dependent. The effect of (-)baclofen on IPSC amplitude is concentration related. Percentage reduction in IPSC amplitude is plotted against log [(-)baclofen]. The results from three to fifteen experiments at various concentrations of (-)baclofen were pooled and a dose-response curve constructed for the synaptic depressant effect. The EC50 for (-)baclofen was estimated to be approximately 6 ,UM (pipette concentration). TABLE 1. Examples taken from individual experiments of the effects of (-)baclofen and low Ca2+ medium and changes in postsynaptic membrane potential on the mean and variance of IPSC amplitude, and on the ratio P/S2 1 M 82 R P/S2 83 18 292-2 155-9 a Control 0 119 9-87 0 318 249-3 49-6 (-)Baclofen 1 441-1 51-78 151 b Control 7 61 0-147 0-205 126 3 31-0 Low Ca2+ 559-1 130-9 270 5 c Control 1-064 0-643 50 9 594-1 174-1 -50 mV 0-353 0-777 21-0 434.7 95.5 -60 mV M = I2/Pl, the ratio of means and R = r2/r (see Methods). a, b and c denote different experiments.
The effects of changes in postsynaptic membrane potential on mean and variance of IPSC amplitude were also studied. Changing the postsynaptic membrane potential from -40 to -50 mV reduced the mean IPSC amplitude by an average of 44 % and altering the postsynaptic membrane potential to -60 mV reduced the mean by 73 % (Table 1). In contrast to the results with (-)baclofen and low Ca2 , the variance was also reduced at these holding potentials. There was also no significant change in R
BACLOFEN EFFECTS ON GABAERGIC SYNAPSES
441
when the postsynaptic membrane potential was held at -80 mV, which was more negative than the reversal potential for the IPSC. In four experiments, M was significantly < 1, but R was not significantly different from 1 (Table 2), indicating that the ratio P/S2 was unaltered by the change in postsynaptic membrane potential. TABLE 2. The effects of baclofen and low Ca2+ medium and changes in postsynaptic membrane potential on the mean IPSC amplitude and on /82 M R 0-14 + 0.04* 0-28 + 0 05 (-)Baclofen Low Ca2+ 0.19+0.10* 0-26±0-05 -50 mV 0-56+0-13 1-37+0.63n.s. -60 mV 0 27 + 0 06 0-81 + 033n.s. Data from several experiments were pooled and expressed in terms ofM, the ratio of means, and R, the ratio of 1/82 values (see Methods). * Indicates a significant departure from 1 0 at the level
P
