Y-AMINOBUTYRIC ACID UPTAKE and the TERMINATION of INHIBITORY SYNAPTIC POTENTIALS in the RAT HIPPOCAMPAL SLICE by RAYMOND DINGLEDINE and STEPHEN J

Home , Arecaidine, Isonipecotic acid, Nipecotic acid

J. Phy8iol. (1985), 366, pp. 387-409 387 With 10 text-figures Printed in Great Britain

y-AMINOBUTYRIC ACID UPTAKE AND THE TERMINATION OF INHIBITORY SYNAPTIC POTENTIALS IN THE RAT HIPPOCAMPAL SLICE BY RAYMOND DINGLEDINE AND STEPHEN J. KORN* From the Department of Pharmacology, University of North Carolina, Chapel Hill, NC 27514, U.S.A.

(Received 15 March 1984)

SUMMARY 1. Intracellular recordings were made from CAI pyramidal cells in the rat hippocampal slice to study the processes that influence the time course of inhibitory post-synaptic potentials (i.p.s.p.s) mediated by y-aminobutyric acid (GABA), and conductance changes evoked by ionophoretically applied GABA. 2. The GABA-uptake inhibitors, nipecotic acid and cis-4-OH-nipecotic acid (1 mM), greatly prolonged conductance increases associated with both hyperpolarizing and depolarizing responses to ionophoretically applied GABA. In contrast to their effects on GABA-evoked conductances, uptake inhibitors only slightly prolonged antidromically evoked i.p.s.p.s. Their primary effect occurred after the i.p.s.p. had decayed to 5-30 % of its peak. 4-OH-isonipecotic acid, a nipecotic acid analogue that does not inhibit GABA uptake, did not prolong i.p.s.p.s or ionophoretically evoked conductance changes. 3. Sodium pentobarbitone (100 #SM), a drug that prolongs the open time of GABA-activated chloride channels, potentiated both i.p.s.p.s and responses to ionophoretically applied GABA. Whereas pentobarbitone also prolonged i.p.s.p.s, it did not prolong responses to ionophoretically applied GABA. The prolongation of i.p.s.p.s by pentobarbitone occurred equally in both the early and late phases of the i.p.s.p., in contrast to the effects of GABA-uptake inhibitors. 4. I.p.s.p.s did not usually decay exponentially. The observation that uptake inhibitors prolonged the late but not the early decay phase of the i.p.s.p., together with the previous finding that the conductance change persists for the duration of the i.p.s.p., indicate that GABA is present in the synapse throughout much of the i.p.s.p. These data suggest that diffusion of GABA out of the synapse, a non- exponential process, is an important determinant of the i.p.s.p. decay time course. 5. Increasing the extracellular potassium concentration from 3-5 to 8-5 mm resulted in spontaneously occurring, synchronous burst firing of pyramidal cells. Cis-4- OH-nipecotic acid significantly reduced the number and amplitude ofextracellularly recorded population spikes within each burst. * Completed in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Pharmacology. Present address: Laboratory of Preclinical Studies, National Institute on Alcohol Abuse and Alcoholism' 12501 Washington Avenue, Rockville, MD 20852, U.S.A. 13-2 388 R. DINGLEDINE AND S. J. KORN 6. We conclude that diffusion, channel open time and GABA uptake all influence the time course of GABA-mediated i.p.s.p.s. The time course of a single, brief i.p.s.p. is determined predominantly by post-synaptic channel kinetics and diffusion of GABA out of the synapse, whereas the inhibition produced by prolonged synaptic bursts or relatively long application of exogenous GABA can be markedly influenced by GABA uptake. 7. A kinetic model is presented in the Appendix that qualitatively describes how diffusion, channel kinetics and cellular re-uptake may act to influence the time course of post-synaptic potentials.

INTRODUCTION The mechanisms that regulate the time course of.post-synaptic potentials in the central nervous system are not well understood. Two general classes of mechanism are likely to contribute to the termination of neurotransmitter action: first, the removal of neurotransmitter from the synaptic cleft by diffusion, cellular re-uptake or enzymic degradation, and second, the kinetics of the molecular events underlying the post-synaptic response. Acetylcholine is removed from cholinergic synapses very rapidly by hydrolysis (Koelle, 1970; Magleby & Stevens, 1972). Consequently, at the neuromuscular junction, transmitter degradation and the kinetics ofchannel activation are important influences on the decay rate of cholinergic post-synaptic currents (Magleby & Stephens, 1972; Hartzell, Kuffler & Yoshikami, 1975; Neher & Sakmann, 1976). Other neurotransmitters, including the catecholamines and amino acid neurotransmitters, are removed from the extracellular space by cellular re-uptake (Balcar & Johnston, 1973; Iversen, 1975; Iversen & Kelly, 1975). It has been hypothesized that re-uptake of neurotransmitter terminates the synaptic potentials mediated by these neurotransmitters (e.g. Iversen, 1975). Indeed, noradrenergic inhibitory post-synaptic potentials (i.p.s.p.s) in the rat locus coeruleus are greatly prolonged by the uptake inhibitor desipramine (Egan, Henderson, North & Williams, 1983). On the other hand, studies on the role of uptake in y-aminobutyric-acid-(GABA)-mediated inhibition in mammalian brain have produced conflicting results. Although neuronal responses to ionophoretically applied GABAare markedlyprolongedby GABA-uptake inhibitors (Curtis, Game & Lodge, 1976; Brown & Galvan, 1977; Lodge, Johnston, Curtis & Brand, 1977; Brown, Collins & Galvan, 1980; Alger & Nicoll, 1982b), experiments designed to determine the influence of uptake on inhibition mediated by synaptically released GABA have produced predominantly negative results (Curtis et al. 1976; Lodge et al. 1977; Brown & Scholfield, 1984; but see Matthews, McCafferty & Setler, 1981). The studies presented in this paper were undertaken to examine directly the influence of uptake on the time course of GABA-mediated i.p.s.p.s. The results indicate that cellular re-uptake of GABA plays a role in the termination of single hippocampal i.p.s.p.s only during the late decay phase. Diffusion of GABA out of the synapse and channel open time appear to be the primary determinants of the i.p.s.p. decay rate. However, uptake may assume greater importance during high- frequency activity of GABAergic interneurones. In the Appendix, a simple kinetic GABA UPTAKE AND I.P.S.P.S 389 model is presented that describes the circumstances under which slow processes such as uptake, or faster processes such as channel kinetics, would influence post-synaptic potentials. With this model, the dramatically different effects of uptake inhibition on ionophoretic GABA responses, i.p.s.p.s and epileptiform bursts can be explained. Accounts of this work have been presented to the Society for Neuroscience (Korn & Dingledine,1982, 1983; Korn, Chamberlin & Dingledine, 1984).

METHODS Slice preparation and bathing medium Rat hippocampal slices were prepared as described previously (Dingledine, 1981). Briefly, male Sprague-Dawley rats (125-200 g) were anaesthetized with ether. Hippocampi were removed and cut nearly transversely into 400 #um thick slices, which were collected in cold bathing medium and then transferred to the recording chamber. Slices were perfused from below with warm (33-36 'C) bathing medium at a rate of 05 ml/min and exposed to a warm, humidified stream of 95% 02/5% CO2 above. Bathing medium consisted of (in mM): NaCl, 130; KCI, 3-5; NaH2PO4, 1-25; NaHCO3, 24; CaCl2. 2H20, 1.5; MgSO4 7H20, 1P5; glucose, 10; bubbled with 95% 02/5% CO2 to a pH of 7-4. High-potassium medium (8-5 mM-K+) was prepared by adding 5 mM-KCI to the normal bathing medium. In many experiments, regenerative sodium currents were eliminated by perfusion with 1 uM-tetrodotoxin (TTX), or by including 50-200 mM-QX-314, a quaternary lidocaine derivative (Dahlbom, Misiorny & Truant, 1965), in the recording pipette. Neither TTX nor QX-314 had any effect on the resting input resistance or the GABA-evoked conductance change over the course of an experiment. As reported by Connors & Prince (1982), cells tended to slowly depolarize when exposed to QX-314 for more than 1 h. Recording, stimulation and ionophoresi8 Intracellular recordings from CAl pyramidal cells were made with micropipettes (35-160 MO) filled with 3 M-potassium acetate or chloride or 2 M-potassium methylsulphate plus 10 mM-potassium acetate. When i.p.s.p.s were studied, the membrane potential was held relatively constant by slight adjustments of the holding current. In some experiments, cells were depolarized by injection of steady positive current into the cell through the recording electrode. This facilitated the study of i.p.s.p.s by moving the membrane potential away from the i.p.s.p. reversal potential. Trains of hyperpolarizing current pulses (0-25-0-5 nA) were passed into the cell through the recording electrode to monitor input resistance. Whenever current was injected into the cell, the bridge balance was checked frequently and adjusted when necessary. Extracellular recordings (e.g. Fig. 8) were made from the pyramidal cell layer in region CA2/CA3, with pipettes (20-60 MO) filled with 0 9 % NaCl (w/v). Orthodromically activated i.p.s.p.s were produced by stimulation through monopolar, sharpened tungsten electrodes placed in the stratum radiatum. Antidromic activation of recurrent i.p.s.p.s was achieved by an electrode placed in the alveus on the subicular side of the cell under study. In some experiments lesions were made through the stratum oriens and stratum pyramidale, between the stimulating electrode and the recorded cell, to minimize contamination of antidromic i.p.s.p.s by excitatory post-synaptic potentials (e.p.s.p.s) (Dingledine & Langoen, 1980). The purity of antidromic i.p.s.p.s was judged on two criteria: (1) the absence of extracellular, orthodromically evoked population spikes upon 5-10 Hz stimulation in the alveus and (2) the absence of a visible e.p.s.p. in intracellular records of stimulus-evoked i.p.s.p.s (cf. Fig. 3). Orthodromic and antidromic stimuli (100 ,us, 5-400 1sA) were delivered at frequencies of 0-03-0-25 Hz. Single-barrel micropipettes, having tip diameters of 1-2 ,um and resistances of 30-100 MCI, were used for focal ionophoretic application of GABA (1 M, pH 4-65 or 0 5 M, pH 4 0) into pyramidal cell somatic and dendritic regions. Pipettes were assumed not to leak agonist excessively ifthe input resistance of the cell remained constant when the pipette was placed into the slice near the cell under study, with no ejection or backing current applied. 390 R. DINGLEDINE AND S. J. KORN

Data collection Data were collected on a four-channel chart recorder and a DEC PDP 11/03 computer, which digitized at rates of up to 10 kHz and displayed the wave forms for on- and off-line analysis. All time-course analyses were performed by computer on digitized wave forms. All intracellular data are averages of four to ten synaptic potentials or two to four ionophoretic responses. Spontaneous pyramidal cell bursts were collected on FM tape and played back for manual or computer analysis. Drug8 Drugs were applied by perfusion at 0-3-1-0 mm (uptake inhibitors) or 100 /M (pentobarbitone). Cis-4-OH-nipecotic acid and 4-OH-isonipecotic acid were supplied by Dr Povl Krogsgaard-Larsen. QX-314 was provided by Dr Bertil Takman ofAstra Pharmaceuticals. All other drugs and chemicals were purchased from Sigma.

RESULTS Useful intracellular recordings were obtained from forty-two pyramidal cells, each from a different animal. The mean resting membrane potential of this sample was -69+ 1'3 mV (S.E. of mean, n = 31) and the mean input resistance was 35 + 1'3 MCI (n = 39). Ionophoretic GABA responses Nipecotic acid and cis-4-OH-nipecotic acid (CIS), at a concentration of 1 mM, inhibit GABA uptake into both neurones and glia (Schousboe, Thorbek, Hertz & Krogsgaard-Larsen, 1979). When tested on hippocampal pyramidal cells, nipecotic acid prolonged and potentiated GABA-evoked conductances associated with both depolarizing and hyperpolarizing responses (Fig. 1 A-C). CIS, the most potent inhibitor of glial uptake available (Schousboe et al. 1979), prolonged GABA-evoked responses to a greater degree than nipecotic acid (Fig. 1 D and E, Table 1). Effects of CIS were evident after 5-20 min of perfusion; peak effects were reached approximately 10 min after onset. Effects ofnipecotic acid usually required 20 min for onset and 40 min to reach peak. Neither nipecotic acid nor CIS had any consistent effects on membrane potential or input resistance. 4-OH-isonipecotic acid, a nipecotic acid analogue that does not block GABA uptake at a concentration of 1 mm (Schousboe et al: 1979), did not prolong the time course of ionophoretic GABA responses (Fig. 4C and D). Pentobarbitone is considered to prolong and potentiate GABA-mediated post- synaptic currents by increasing the open time of GABA-activated chloride channels (Study & Barker, 1981). Perfusion with 100 /iM-pentobarbitone enhanced the magnitude of the conductance increase evoked by ionophoretically applied GABA in four out of five cells but had negligible effects on the slope of the decay or the over-all time course of the GABA-evoked response (Fig. 2, Table 1). Subsequent exposure to 1 mM-CIS prolonged the GABA-evoked response as usual (Fig. 2). This concentration of pentobarbitone is sufficient to prolong and potentiate hippocampal i.p.s.p.s (see below; Alger & Nicoll, 1982a). GABA UPTAKE AND I.P.S.P.S 391

Control BCotlo Nipecotic X

0 30- acid 0-10 N2ipecotic acid

5 0

. 0*30 1 2 3 4 GABA 1 S Time (s) C1 C Control C2 * Nipecotic acid

i s D1 1 00I E Control o i Control CIS E

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|;9|1 ~~~ V 0-03 1 t I- 3 s : 2 4 6 8 10 1 2 14 Time (s) Fig. 1. Effect of uptake inhibitors on GABA-evoked conductance changes. Results from three cells are illustrated. In A, C and D, traces representative of the GABA-evoked responses before and after drug treatment are illustrated. The decrease in input resistance evoked by GABA is plotted semilogarithmically in B and E. A-C, in two different experiments, slices were perfused with 1 mM-nipecotic acid for 40 min (A 2 and C2). In A and B, GABA was applied by ionophoresis near the dendrites (160 nA ejected for 90 ms, denoted by the filled square); in C the ionophoretic pipette was placed in the cell layer (40 ms, 160 nA; filled square). D and E, in a third experiment, slices were perfused with 1 mM-cis-4-OH-nipecotic acid (CIS) for 34 min (GABA ejected for 500 ms, 135 nA; filled bar). Note the action potential in trace D2 that occurred during the peak GABA-evoked depolarization. Cells A and Cwere recorded in the presence of 1 ,M-TTX with micropipettes filled with potassium acetate. Cell D was recorded with a KCl-filled micropipette, with no sodium channel blocker present.

Shape of i.p.s.p. decay Hippocampal i.p.s.p.s typically did not decay exponentially. When plotted semilogarithmically, i.p.s.p.s either curved down (Figs. 5 and 6) or up (Figs. 3-7), the latter especially after perfusion with uptake inhibitors (see below). Consequently, the decay could not be described appropriately by an exponential time constant. Instead, we expressed the time course of i.p.s.p.s as decay times from 90 to 30 % of peak (early 392 R. DINGLEDINE AND S. J. KORN

TABLE 1. Prolongation of GABA-mediated conductances Increase in Drug n* decay timet (%) Nipecotic acid 5 164+231 CIS 6 263 ± 33§ Pentobarbitone 4 118+12 * Cells were included only if exposed to a drug for a sufficient time to be nearly maximally affected. t Expressed as percentage of control decay time (control = 100 %) measured from 90 to 10 % of peak conductance change following an ionophoretic dose of GABA. Values represent mean +S.E. of mean. I Significantly different from control (P < 0 025) by Student's t test. § Significantly different from control (P < 0 005) and nipecotic acid (P < 0 025) by Student's t test.

Al Control 5 B - U 8 2s

1-00 Control 0 A2 PBPB n PB +CIS o 0) 30-

A3~~~~~~~~~~~~~~~~(0)

0 1 2 3 Time (s) Fig. 2. Comparison between effects of pentobarbitone and CIS on the ionophoretic GABA response. GABA was ionophoresed (70 ms, 100 nA; filled bars) every 30 s. The slice was perfused with 100 /SM-pentobarbitone (PB) for 31 min (A2) and then with pentobarbitone plus 1 mM-CIS for 25 min (A 3). The recording electrode contained potassium methylsulphate plus 50 mM-QX-314. phase) and from 30 to 10 % of peak (late phase). As will be shown, these two phases were differentially affected by uptake inhibition. I.p.8.p.8 Pyramidal cell i.p.s.p.s were evoked by antidromic or orthodromic stimulation for a period of 10-40 min in control bathing medium to monitor their stability and reproducibility. After this period, families of i.p.s.p.s were generated by varying the intensity of the stimulus current from 5 to 400 ,csA. Perfusion for 15-40 min with CIS GABA UPTAKE AND I.P.S.P.S 393 (0 3-1 mM) or nipecotic acid (1 mM) prolonged both antidromic and orthodromic i.p.s.p.s in a similar, characteristic manner. Antidromic i.p.S.p.8. Fig. 3 demonstrates the typical effects of CIS on recurrent i.p.s.p.s elicited by antidromic activation of pyramidal cell axons. The late phase of the i.p.s.p., typically after it had decayed to about 30 % of its peak, was prolonged. This prolongation occurred over a full range of stimulus intensities (Fig. 3C and D).

120- A 30° A C 0 80-

CISg40 Control 0 5 Cu mV stimuli ovaynitniebeoenaCIS 0 0 200 mns 8 1 00 250 a C*200 0 0~~~~~~~

~~oio~ :1:: 50100150 20050 000 0 100 00

membrane potential was held constant with slight adjustments of injected current. A, traces (averages of ten sweeps) of i.p.s.p.s evoked by 30 1sA stimuli. B, semilog plot of the time course of i.p.s.p.s in A, normalized for their peaks. C and D, decay times of an entire family of i.p.s.p.s before (circles) and after (squares) CIS, measured from 90-30% (C) and 30-10% (D) of peak. Note the different scales on the ordinates. The larger effect of CIS was on the later part of the i.p.s.p. decay. The recording electrode contained potassium methylsulphate and 50 mM-QX-314.

The decay times measured from 90 to 30 % of peak (early phase), and from 30 to 10% of peak (late phase), are plotted in Fig. 30 and D. The early phase of the i.p.s.p. decay, which occurred from about 40 to 100 ms after the antidromic stimulus, was relatively little affected by CIS, increasing in duration by up to 30 ms over the range of stimulus intensities employed (Fig. 30). In contrast, the late phase of the decay, which occurred between 100 and 300 ms after the stimulus, was more profoundly affected by CIS, increasing in duration by up to 150 ms (Fig. 3D). As can be seen clearly in the traces in Fig. 3A and the plot in Fig. 3B (see also Fig. 7A and B), the primary effect of CIS on antidromic i.p.s.p.s was to maiainin the hyperpolarization associated with the i.p.s.p. at 10% of the peak for hundreds of milliseconds. 394 R. DINGLEDINE AND S. J. KORN CIS did not affect the time to peak of the i.p.s.p. Often, particularly at higher stimulus intensities, the peak amplitude of the i.p.s.p. at a given stimulus intensity was decreased by CIS (cf. Fig. 7). The maximum i.p.s.p. size obtainable also was usually decreased by CIS; in the cell illustrated in Fig. 3, the maximum was reduced from 12 to 9 mV. The decreased magnitude of the i.p.s.p., which could be reversed upon return to control bathing medium, was a characteristic effect of all uptake inhibitors tested. However, this effect may have been unrelated to inhibition

A B 1 00-

4 -HRecverx isonipecoticAj'c acid C .2 0*10

0.031 u~~~~~~~~01 00mV i:ooW 2000 300 C Control Time (is) D oto 25S S 0 0.30- 4 OH-isonipecotic a mVo 10 Cc o ISOn a- :9 0.03 0.01 isonipecotic and 351mi Cfeeuntontrol0 mdu1 rcvr)2 h 3 nu ~~acid Time (s) Fig. 4. Lack of effect of 4-OH-isonipecotic acid on i.p.s.p.s and GABA-evoked conductances. Antidromic i.p.s.p.s were evoked aiternateiy with ionophoretic GABA responses. A, i.p.s.p.s are illustrated from control, after 31 min perfusion with 1 mm-4-OH- isonipecotic acid and 35 mm after return to control medium (recovery). The input resistances in these three conditions were 45 MCI in control, 52 MCI in the latter two conditions. B, the i.p.s.p.s in A were digitized and are plotted semilogarithmically, normalized for their peaks. C, responses to ionophoretically applied GABA (200 ins, 80 nA, filled bar) in control and 30 min into perfusion with 4-OR-isonipecotic acid. D, semilogarithmic plot of control (circles) and drug-treated (squares) GABA-evoked responses illustrated in C. The recording pipette contained potassium methylsulphate and 50 mM-QX-314. of uptake since 4-OH-isonipecotic acid, a structural analogue of CIS that has no activity as an uptake inhibitor (Schousboe et al. 1979), also reversibly decreased the magnitude of i.p.s.p.s (Fig. 4A). 4-OH-isonipecotic acid did not prolong i.p.s.p.s, however, consistent with its lack of effect on GABA uptake (Fig. 4A and B). Orthodromic i.p.s.p.s. The prolongation by CIS of orthodromically evoked i.p.s.p.s is illustrated in Fig. 5. As with antidromic i.p.s.p.s, the peak amplitudes of orthodromic i.p.s.p.s were usually decreased. Nonetheless, i.p.s.p.s matched for amplitude or stimulus intensity were prolonged by CIS. Orthodromic i.p.s.p.s result from both an early, fast chloride conductance, similar to that which produces an GABA UPTAKE AND I.P.S.P.S 395

A 100

0) a' 0 30 Control 0 C 0 4- 0*10- (U 10 mV 0-03- 100 ms

B 1 00 le

" 030- Cis-4-OH -nipecotate 0 0 i) 0*10- U. 0 031- 0 100 200 300 400 Time (Ms)

C 0

CU U-

I 1.Vr.*. ci 0 \00 800., - 1i2 0.1 1- -'l;. 200 ms 0 400 800 1200 1600 Time (ms)

Fig. 5. Effects of CIS on orthodromic i.p.s.p.s. Data from two experiments are illustrated. A and B, families of orthodromic i.p.s.p.s were evoked by stimulation of pyramidal cell afferents in the stratum radiatum before (A) and after (B) perfusion with 1 mM-CIS for 24 min. Each trace on the left is an average of four sweeps. The i.p.s.p.s were digitized and plotted semilogarithmically in the graphs on the right. Stimuli ranged from 100 1sA (trace 1) to 400 ,uA (trace 6) in control and 180 ,gA (trace 1) to 400 ,sA (trace 4) in CIS. Note that stimuli of equal intensity evoked i.p.s.p.s of smaller magnitude after perfusion with CIS. C, in another experiment, orthodromic i.p.s.p.s were evoked by a 100 ,zA stimulus before and after perfusion with 1 mm-CIS for 50 min. The traces on the left are averages of ten sweeps and are plotted with their peaks normalized on the right (clustered dots). The more dispersed dots in the plot on the right, which are from an i.p.s.p. recorded 2 min later at a slower digitizing rate, illustrate the later phase of the i.p.s.p. The break in the decay at about 75 ms represents the transition from the early chloride-dependent to the late potassium-dependent phase of the i.p.s.p. The recording electrode in both experiments contained potassium methylsulphate and 50 mM-QX-314. 396 R. DINGLEDINE AND S. J. KORN antidromic i.p.s.p., and a late, slow conductance thought to involve potassium (Thalmann & Ayala, 1980; Nicoll & Alger, 1981). The separation of these two components can be seen clearly in Fig. 5C. Both the slow, late hyperpolarization as well as the fast, early hyperpolarization were prolonged by CIS. Consequently, in three cells examined, the effect ofCIS was larger for orthodromic than for antidromic i.p.S.p.S.

A B 1 00

A f&ir0 30 X~ Nipecotic acid 0 W 5 *,~~~; 0-10

100 Ms 003 0 50 100 150 200 250 Time (ins) CD 250- 250 Nipecotic acid E 200- Nipecotic acid / o 200-0 a 150- 150- 0 o ~~~~~~~~~~~~~~~~0 o 100 2 100 Control 1 500 00 Control E 50 0 0- 0 5 10 1 5 20 25 0 20 40 60 80 100 L.p.s.p. peak (mV) Stimulus intensity (MA) Fig. 6. Effect of nipecotic acid on orthodromic i.p.s.p.s. A, orthodromic i.p.s.p.s (averages of six sweeps) evoked by 20 /tsA stimuli before and after (arrowhead) perfusion with 1 mM-nipecotic acid for 40 min. B, semilogarithmic plot of digitized i.p.s.p.s normalized to their peaks. C and D, time from peak to 10 % of peak plotted ver8u8 i.p.s.p. size (C) or stimulis intensity (D) for the entire family. The recording electrode contained potassium methylsulphate and 50 mM-QX-314.

Perfusion with nipecotic acid (1 mM) usually reduced the amplitude of i.p.s.p.s to such a great extent that time-course analysis was impossible. However, when i.p.s.p.s survived perfusion with nipecotic acid for 30-40 min, they were prolonged regardless of peak amplitude or stimulus intensity, albeit to a lesser extent than after perfusion with CIS (Fig. 6). As with CIS, the primary effect of nipecotic acid was to prolong the late phase (between 5 and 10 % of peak) of the i.p.s.p. (Fig. 6A and B). With prolonged washing the effects of CIS on both antidromic and orthodromic i.p.s.p.s were reversible. One example of this is demonstrated in Fig. 7, in which the i.p.s.p. labelled 'wash' in Fig. 7 D was evoked 40 min after return to control bathing medium. Pentobarbitone. A comparison between the effects of CIS and pentobarbitone on i.p.s.p.s, together with the results discussed above (Figs. 1 and 2), indicates that these two drugs prolonged i.p.s.p.s by different mechanisms. This comparison is illustrated GABA UPTAKE AND I.P.S.P.S 397

A Cis-4- 0H - nipecotate C Pentobarbitone

~Cis 5 mV 200 ms B D 100 1.00-

(U Ci0:30 CIs 0.30 PB o ~ - 0 ~ 0 010 Control' -,. 010 CU ~Wash- LL~~~~~~~~~~~~~~L 0-03 0-031 0 100 200 300 400 0 100 200 300 400 Time (ms) Time (ins) Fig. 7. Comparison between the effects of CIS and pentobarbitone on antidromic i.p.s.p.s. A and B, antidromic i.p.s.p.s (average of ten analogue sweeps) were evoked by a 40 #A stimulus before and after 43 min perfusion with 1 mM-CIS. C and D, after 45 min of perfusion with CIS, the bathing medium was switched back to control for 62 min (the trace labelled 'wash' was recorded 40 min later). The slice was then perfused with 100 /M- pentobarbitone for 16 min (PB). The recording electrode contained potassium methylsulphate plus 50 mM-QX-314. in Fig. 7, where the effects of CIS and pentobarbitone were compared on i.p.s.p.s recorded from the same pyramidal cell. CIS produced its typical effect, a prolongation of the late phase with little effect on the first 80 ms of the i.p.s.p. (Fig. 7A and B). After 40 min of wash-out, the i.p.s.p. recovered to its control time course (Fig. 7C and D). Subsequent perfusion with 100 /LM-sodium pentobarbitone then prolonged the i.p.s.p., but in contrast to the actions of CIS, pentobarbitone potentiated and prolonged the i.p.s.p. over its entire extent (Fig. 7C and D). In further contrast to CIS, pentobarbitone also increased the latency to the i.p.s.p. peak. Attenuation of pyramidal cell bur8t firing by inhibitors of GABA uptake Spontaneous pyramidal cell burst firing was produced by raising the extracellular potassium concentration from 3-5 to 8&5 mM (extracellular calcium concentration = 1P5 mM). Spontaneous bursts of greater than ten population spikes could be recorded by extracellular electrodes placed in the cell layer of hippocampal region CA2/CA3. Such bursts occurred regularly (approximately 1/s), and were followed by a quiet period which could be associated with a large negative d.c. shift in the extracellular potential (Fig. 8A). Bursts were also elicited in high extracellular potassium concentration by orthodromic stimuli delivered to the stratum radiatum of hippocampal region CA3 at a frequency of 01 Hz. Perfusion with 1 mM-CIS 398 R. DINGLEDINE AND S. J. KORN

A 1 * Control A2rr1rr'rr~* CIS A 3 Recovery n(2I~~~~~~~~~r ~~45 mVmnV 1000 ms 40 mns

8 1 C 90- 80| Control ED 70 cis - 82 cis ~60- 'EL 50- O 40- ~~~~~0 B3 Recovery 20- 10- 0- Spike amplitude (mV) Fig. 8. Attenuation of spontaneous epileptiform burst firing by CIS. Pyramidal cell burst firing was produced by raising the potassium concentration of bathing medium to 8-5 mM and recorded with an extracellular electrode placed in the cell layer of region CA2/CA3. A, 4 s continuous record of bursting in control medium with a high potassium concentration (A 1), after 20 min perfusion with CIS (A 2) and 15 min after return to control (A 3). The bursts marked by asterisks in A are shown on an expanded time scale in B to illustrate individual population spikes within a burst. C, amplitude histogram of the number of population spikes in twenty bursts is plotted. Each bin on the abscissa is 0 5 mV wide; spikes smaller than 0 5 mV could not be accurately distinguished from noise. Bursts were selected for inclusion in the histogram from 10 min of data collected on magnetic tape under each condition. dramatically reduced the size and number of population spikes within both spontaneous (Fig. 8B and C, Table 2) and stimulus-evoked bursts. The greatest effect of CIS was a profound reduction in the number of large spikes in a burst (Fig. 8C, Table 2). Concomitant with the decrease in size and number of pyramidal cell spikes, the magnitude of the post-burst, negative d.c. shift decreased. Despite the dramatic reduction by CIS of the burst intensity, the duration of each burst was not markedly shortened (Fig. 8B). The effects of CIS were reversible upon reurn to drug-free medium (Fig. 8A 3 and B3, Table 2). Similar results were obtained in seven slices. In contrast, in seven slices perfused with 1 mM-4-OH-nipecotic acid, the inactive GABA UPTAKE AND I.P.S.P.S 399

TABLE 2. Attenuation of pyramidal cell burst firing by CIS Average spike size Condition Spikes/burst (mV) Bursts/s Control 18&8+0-56 2-1 +007 (370) 1±0+0 04 CIS 73+073 1-3±0-08 (149) 1P5±0.05 Wash 13-5+0-99 2-4+0-18 (135) 1V2+0-03 In each row, the number and amplitude of population spikes within twenty spontaneous bursts (ten for wash) were measured manually. The number of spikes included in the analysis is shown in parentheses. Spikes smaller than 0 5 mV could not be accurately distinguished from noise and therefore were not measured. Bursts/s were determined from nine consecutive 10 s intervals. Values represent mean + S.E. of mean. compound, no effect on burst intensity was observed. The frequency of spontaneous bursting was not consistently changed by CIS, although it was increased in the experiment illustrated in Fig. 8.

DISCUSSION Termination of GABAergic i.p.s.p.s The principal conclusion of this study is that cellular re-uptake ofGABA, diffusion of GABA away from its receptors and GABA-activated channel kinetics all influence the duration ofi.p.s.p.s, each being important during different phases. GABA-uptake inhibitors prolonged i.p.s.p.s late in their decay, whereas increasing the channel open time with pentobarbitone potentiated and prolonged i.p.s.p.s over their entire extent. Despite the apparently small influence of uptake on single i.p.s.p.s, epileptiform bursts in high extracellular potassium concentration were markedly attenuated by GABA-uptake inhibitors. Role of uptake. Nipecotic acid and CIS prolonged GABA-evoked conductance changes in pyramidal cells, consistent with findings in other preparations (Curtis et al. 1976; Brown & Galvan, 1977; Brown et al. 1980; Alger & Nicoll, 1982b). Several lines of evidence indicate that the major site of action of these compounds was on the GABA-uptake system. Uptake inhibitors did not alter pyramidal cell resting membrane properties, an analogue ofuptake inhibitors that is inactive in biochemical studies (4-OH-isonipecotic acid) did not prolong GABA-evoked responses, and the effect of the uptake inhibitors was qualitatively different from that of pentobarbitone. In contrast to the marked prolongation of conductance changes evoked by ionophoretically applied GABA, GABA-uptake inhibitors prolonged i.p.s.p.s only slightly. We favour the explanation that the uptake process is too slow to appreciably clear the synaptic cleft of GABA during the i.p.s.p. Ionophoretic responses, being of much longer duration than i.p.s.p.s, should be more susceptible to slow removal processes such as uptake. The finding that the effect of uptake inhibition was largely limited to the slowly falling late phase of the i.p.s.p. is in agreement with this conclusion. A model of the diffusion of GABA and GABA-receptor activation is presented in the Appendix to support this interpretation. An alternative explanation for the different effectiveness of CIS on i.p.s.p.s and ionophoretic GABA responses is worth considering. It is possible that CIS inhibited 400 R. DINGLEDINE AND S. J. KORN glial but not neuronal uptake. It might be predicted, basedon geometric considerations, that glial uptake would be a more important influence on ionophoretic GABA responses whereas neuronal uptake would primarily influence synaptic responses. However, in biochemical experiments 1 mM-CIS inhibited neuronal as well as glial uptake (Schousboe et al. 1979). More importantly, the demonstration that nipecotic acid, a presumed neuronal uptake blocker, also had only a slight effect on i.p.s.p.s, argues against this explanation being solely responsible for the findings. Role of channel lifetime. Pentobarbitone is considered to increase the mean open time of GABA-activated chloride channels (Study & Barker, 1981; Segal & Barker, 1984). Given this, the marked potentiation and prolongation of i.p.s.p.s by pentobarbitone suggests that channel closure limits both the magnitude and duration of i.p.s.p.s. This is expected since the rate of chloride channel closure (mean open time of 10-20 ms by noise analysis; Barker, McBurney & MacDonald, 1982; Segal & Barker, 1984) approximates the decay rate of i.p.s.p.s, which peak in 15-20 ms and decay over 100-200 ms. In contrast to its effects on i.p.s.p.s, pentobarbitone did not prolong conductance changes evoked by ionophoretically applied GABA. This is presumably due to the slow rate of change of these responses compared with the rate of channel closure (see Appendix). Role ofdiffusion. Several observations support the conclusion that GABA is present within the synaptic cleft throughout much of the i.p.s.p. and therefore, that the rate of diffusion of GABA away from its receptors is an important determinant of the i.p.s.p. time course. First, both antidromic and orthodromic i.p.s.p.s are too long to be accounted for by the time constant of the membrane (10-25 ms; Brown, Fricke & Perkel, 1981). Indeed, the conductance increase associated with the antidromic i.p.s.p. lasts as long as the i.p.s.p. itself (Dingledine & Langmoen, 1980). Further, both GABA-uptake inhibitors and pentobarbitone prolonged the late phase of i.p.s.p.s, which would be expected only if GABA were still present in the synapse and activating receptors late in the i.p.s.p. Finally, i.p.s.p.s typically decayed non- exponentially, especially after inhibition ofthe uptake process, as would be predicted for a diffusion-limited process (see Appendix). The rate of diffusion of GABA out of the cleft may be particularly important during the early decay phase of the i.p.s.p., when removal by uptake is insignificant, yet the concentration of GABA within the cleft is presumably declining. Ultimately, uptake is a process that influences post-synaptic events by modifying neurotransmitter diffusion to and from its receptors. Being a slow process, the effectiveness of uptake would depend on neurotransmitter being in the extracellular space for a relatively long time. Consequently, uptake would be expected to have its most profound influence during relatively long synaptic events (e.g. Egan et al. 1983) or on the late phases of a faster synaptic potential. Differences between antidromic and orthodromic i.p.s.p.s Uptake inhibitors prolonged orthodromic i.p.s.p.s more than antidromic i.p.s.p.s, a finding that may be explained in two ways. First, whereas antidromic i.p.s.p.s result primarily from an increased chloride conductance, orthodromic i.p.s.p.s appear to contain at least two ionic components: the early chloride conductance which is activated by GABA-A receptors and a late, slower potassium conductance (Thalmann GABA UPTAKE AND I.P.S.P.S 401 & Ayala, 1980; Nicoll & Alger, 1981), which may be activated by GABA-B receptors (Newberry & Nicoll, 1984a). If GABA-receptor activation proves to be responsible for the late hyperpolarization evoked by orthodromic stimulation (cf. Blaxter, Davies, Carlen & Gurevich, 1984; Newberry & Nicoll, 1984b), the prolongation ofthis late conductance by uptake inhibition would be predicted. An alternative explanation relies on spatial differences between i.p.s.p.s evoked antidromically and orthodromically. Antidromic i.p.s.p.s are primarily somatic whereas orthodromic i.p.s.p.s have a more prominent dendritic component (Alger & Nicoll, 1982a). Accordingly, the differential effects of uptake inhibition may be a result of differences in (1) the duration of presynaptic release of GABA at somatic and dendritic nerve terminals; (2) the spatial distribution of uptake sites near the activated GABAergic synapses, or (3) the properties of the GABA receptor-ionophore complexes activated by the two types of stimuli. Significance of uptake in the termination of i.p.s.p.s Although the functional significance of prolonged i.p.s.p.s following uptake inhibition has not been directly assessed, some pertinent information is available. A major inhibitory action of recurrent i.p.s.p.s appears to be the hyperpolarization, since shunting of dendritic e.p.s.p.s occurred only during the brief rising phase of the i.p.s.p. (Dingledine & Langmoen, 1980). After inhibition of GABA uptake, the additional hyperpolarization during the late phase of the i.p.s.p. would presumably enhance inhibition by moving the cell farther from its firing threshold. Indeed it is clear that under certain conditions, synaptic inhibition of cell firing can be potentiated and prolonged by GABA-uptake inhibitors. For example, epileptiform burst firing of pyramidal neurones in the presence of raised extracellular potassium was markedly suppressed by CIS (Fig. 8), but not by the inactive isomer, 4-OH-isonipecotic acid. Rovira, Ben-Ari & Cherubini (1984) demonstrated in the hippocampus in 8itU that potentiation of pyramidal cell population spikes by high-frequency stimulation, a phenomenon that may be due to an impairment of GABAergic inhibition (Ben-Ari, Krnjevic & Reinhardt, 1979), is strikingly reduced by ionophoresis of the uptake inhibitors, nipecotic acid and guvacine. Also, several uptake inhibitors had a protective effect against seizures in mice (Croucher, Meldrum & Krogsgaard-Larsen, 1983). In contrast, Curtis and co-workers (Curtis et al. 1976; Lodge et al. 1977) were unable to demonstrate potentiation of GABA-mediated inhibition produced by stimulation of GABAergic pathways in cat cerebellum or spinal cord. Thus, whereas GABA uptake has relatively little influence on single i.p.s.p.s, it may have an important influence under physiological conditions that involve prolonged, high-frequency activation of interneurones. Indeed, the frequency-dependent effectiveness ofuptake inhibitors may provide a basis for selective interference with the high-frequency discharges associated with epileptiform activity. If so this would be an instance in which the selectivity of drug action was based not on chemical recognition but on differences in duration of normal i.p.s.p.s and pathological seizure discharges. 402 R. DINGLEDINE AND S. J. KORN

Application to other post-synaptic potentials The arguments used to explain our experimental results were derived from a general kinetic scheme (see Appendix) and should therefore apply to other systems, with other neurotransmitters. Noradrenaline and excitatory amino-acid neurotransmitters are removed from the extracellular space by uptake systems similar to that for GABA (Balcar & Johnston, 1973; Iversen, 1975). Egan et al. (1983) demonstrated that noradrenergic i.p.s.p.s in the rat locus coeruleus, which had a duration of 2 s or more, were very much prolonged by the uptake inhibitor desipramine. Assuming similar rate constants for uptake of GABA and catecholamines, the prolongation of this very long i.p.s.p. is consistent with our results. Similarly, Bell & Grabsch (1976) demonstrated that prolonged (> 25 s) chronotropic responses of rat heart to noradrenaline released in 5 s stimulus trains were prolonged by uptake inhibition. Whetheruptake terminatesthe action ofexcitatory amino acids remains unresolved, and may depend on the properties ofthe synapse under study. The results ofthe model presented in the Appendix suggest that very fast synaptic events, lasting several milliseconds, would be uninfluenced by a slow uptake process. This prediction is supported by observations of fast excitatory junctional currents at the locust neuromuscular junction (Clark, Gration & Underwood, 1980). Crawford & McBurney (1977) suggested, however, that the prolongation by aspartate of junctional currents at the crab neuromuscular junction could be due to inhibition of transmitter uptake. These post-synaptic currents also lasted only a few milliseconds; consequently, this interpretation rested on the assumption that the rate constant for neurotransmitter uptake in the crab preparation was 1000-2000/s. It will be important to determine whether transmitter uptake processes can proceed this rapidly because, if so, even the time course of fast synaptic potentials could then be controlled by uptake.

APPENDIX Influence of uptake and channel kinetic8 on the decay of post-synaptic potentials A simple kinetic model was developed on a PDP 11/23 computer to help explain the following three major observations of this study. First, uptake inhibitors prolonged the late phase more than the early phase of i.p.s.p.s (Fig. 3). Secondly, uptake inhibition had a relatively mild effect on i.p.s.p.s compared with responses to ionophoretically applied GABA (compare Figs. 3 and 6 to Fig. 1) or compared with epileptiform bursts in the hippocampus (Fig. 8). Thirdly, pentobarbitone significantly prolonged i.p.s.p.s but not responses to ionophoretically applied GABA (compare Fig. 7C and D to Fig. 2). In brief, the model demonstrates that the effect of inhibiting uptake or increasing mean channel open time is highly dependent on the time course of the measured response, and that the results listed above can be accounted for by this dependence.

Description of the model The model was designed to simulate the diffusion of GABA from a point source (ionophoretic pipette or nerve terminal) to its receptor and the subsequent activation of receptor-linked channels (Fig. 9). GABA UPTAKE AND I.P.S.P.S 403 Calculations were done in two stages. The first stage used an equation developed by Nicholson & Phillips (1981) for diffusion ofmolecules in the brain (their eqn. A 14), which solves for the concentration of molecules (G(t)) appearing at a point receptor (R) at time t, following the release of G0 molecules from a point source some specified distance away. G(t) is dependent on the rate and duration ofrelease ofC0, the diffusion

GA GX G'

G GG G G G G G G G GGGGGG G G G G GeG@\ S G GG

Diffusion |Go T G+R G R G Rl Kd

Fig. 9. Scheme on which kinetic model of GABA-mediated neurotransmission is based. The physical situation in the top half of the Figure was modelled by the reaction mechanism below. In this scheme, GABA (Go) is released from a point source (ionophoretic pipette in upper left) and diffuses to its receptor (R) on a pyramidal cell (P). GABA-uptake sites, which are distributed homogeneously throughout the extracellular space, remove GABA (0') at a rate determined by the first-order rate constant, Ku. The binding ofGABA to its receptor is described by simple Michaelis-Menten kinetics, and is governed by the dissociation constant, Kd. After GABA binds to its receptor (GR), a fraction of the receptors undergo a conformational change that permits the flow of chloride ions (Cl-) through open channels in the post-synaptic membrane. This conformational change is represented in the equation by the transition from OR to GR*, and is governed by the rate constants a and a. If the ionophoretic pipette were moved very close to the post-synaptic membrane, it would correspond to a presynaptic nerve terminal. distance between source and receptor, and the rate of irreversible, first-order removal of transmitter (C') from the extracellular space, as governed by the uptake rate constant Ku. In the Nicholson-Phillips equation we used their values for the extracellular volume fraction (0-21) and the diffusion coefficient as modified by tortuosity (0-32 x 10-5 cm2/S). Kd is the dissociation constant for binding of GABA to its receptors. Conventional mass action principles are assumed for the binding reaction, and it is further presumed that the binding ofG to R is very fast with respect to the rate of change of the GABA concentration and the subsequent conformational change. Under these conditions binding is always at equilibrium, and it follows that the fractional saturation of receptors at any time is given by GR G GRmax G+ Kd where GRmax = the maximum possible number of occupied receptors. 404 R. DINGLEDINE AND S. J. KORN In the second stage of the model this concentration profile, G(t), is converted to a post-synaptic response, GR*(t). Open channels (GR*) reflect a conformational change governed by the rate constants for channel opening (fi) and closing (az). The assumption that there are no spare receptors allows the fractional response to be given by GR*/GRmax. We solved for GR*(t) because our experimental measurement, the GABA-evoked conductance change or i.p.s.p., should be directly proportional to the number of open channels at any given time. The equation describing the rate of change in the number of open channels over time is: dGR* FGi da - i(AGR) - a7(GR*) = (AGRmax - GR*) [G +KJ a(GR*). (1) This equation was solved by iterative evaluation of the following equation:

GR*(t) = GR*(t-1) +,lAt(GRmax-GR*(t-1)) [G(t) - tAt(GR*(t-1)). (2) GR*(t -1) is the number of open channels calculated from the previous iteration; GR*(t) is the newly calculated number of open channels. The initial boundary condition was that, at time t = 0, GR(t) = GR*(t) = 0. Time was then incremented by At, and first GR(t), then GR*(t), were re-calculated. The value of At must be sufficiently small to ensure that dGR*/dt approximates 0 between iterations (Crank, 1975). The appropriate value was empirically determined by systematic evaluation of eqn. (2) at successively smaller values of At, until the calculated values of GR*(t) converged (see Crank, 1975, chapter 8.4). Typically, At ranged from 1 ,ss (for fast responses) to 100 pss (for slow responses). Although this model is undoubtedly over-simplified, it contains the elements that are necessary and sufficient to examine qualitatively the influence on the post-synaptic conductance of the three processes of immediate interest. Additional complexities such as a multistep binding reaction, saturable uptake, interaction of GABA with a population of spatially distributed receptors, and more complicated kinetic mechanisms of channel activation, could provide realism but would introduce extraneous variables that would be unwarranted without additional information. The critical difference between this model and that of Magleby & Stevens (1972), which described cholinergic end-plate currents, is in the influence of the driving function (GR(t)) on the decay rate of GR*. Magleby & Stevens (1972) were able to consider situations derived from one of two simplifying assumptions: that the transmitter concentration rose and fell either very quickly, as was postulated for nerve-released acetylcholine, or very slowly, as was later demonstrated for ionophoretically released acetylcholine (Dionne & Stevens, 1975). Our model was designed to examine the intermediate condition, in which the time course of the transmitter concentration change approximated that of the synaptic potential. Unlike the equations of Magleby & Stevens (1972), the resulting equation had no explicit solution but had to be solved numerically. Effect ofuptake inhibition or change in channel lifetime on responses ofdifferent duration The model was used to examine the circumstances under which inhibition ofGABA uptake (decreased Ku) or prolongation ofmean channel open time (decreased a) would GABA UPTAKE AND I.P.S.P S 4405 be expected to influence the time course of GABA-evoked responses. We used a value of 06/s for the uptake rate constant (Ku). This value was calculated from a simple model of the uptake process and measurements of the initial uptake rate into hippocampal neurones in primary culture (D. Hoch & R. Dingledine, in preparation). The value of the channel-closing rate constant (a), 100/s, is compatible with that determined from fluctuation analysis of GABA-mediated currents in cultured mouse spinal neurones (Barker et al. 1982) and rat hippocampal neurones (Segal & Barker, 1984). Fig. 10 (upper panels) illustrates the effects of inhibiting uptake or prolonging channel open time on quickly falling responses, similar in duration to i.p.s.p.s, and on gradually declining responses, similar in duration to conductance changes evoked by ionophoretically applied GABA. Complete inhibition of the uptake process (AU) had negligible effects on the concentration of GABA at the receptor following the brief pulse (Fig. 10A 1). Consequently, the time course of the conductance change following the brief pulse was only slightly affected (Fig. 1OA 2). The effect that was produced was limited to a slight prolongation of the late phase of the response. In contrast, the amount and duration of GABA appearing at the receptor following the long pulse was markedly increased by inhibition of GABA uptake (Fig. lOB 1). In particular, the tail of the GABA concentration profile, between 2 and 5 s, was prolonged. Consequently, the resulting response was both potentiated and greatly prolonged (Fig. lOB 2). These differences can be explained simply by a consideration of the relative values of the uptake time constant and the duration of the response; in 200 ms, uptake with a first-order rate constant of 06/s could sequester only 11 % of the GABA whereas in 4 s it could remove 91 % of the GABA. A similar argument demonstrates why the early phase of the brief response was unaffected by uptake. In order to influence the brief response to a similar extent as the long response, an uptake rate constant of 50/s was required. When the rate constant for channel closing was decreased from 100/s to 33/s (increasing mean open time from 10 to 30 ms), the amplitudes of both brief (Fig. IOA 2) and long (Fig. lOB 2) responses were potentiated. This simply reflects an increase in the percentage open time for any given fractional occupation of the receptors. However, the decay rate of only the brief response was decreased. These results are consistent with our data and can be explained as follows. During ionophoretic GABA responses the rate of change of the macroscopic conductance is very slow relative to the channel closing rate. It is therefore unaffected by moderate changes in the closing rate constant. As the duration of the conductance change becomes shorter, in the range of an i.p.s.p., the rate of change of the macroscopic conductance becomes comparable to the rate of channel closing, and the mean channel open time assumes a more prominent role in controlling the decay rate. To illustrate further the importance of response duration on the effectiveness of uptake inhibitors and pentobarbitone, we generated families ofcurves similar to those in Fig. 1OA and B. The control decay times, measured from 90 to 10 % ofpeak, ranged from 30 ms to 3 s. This was accomplished by adjusting the duration of GABA release from 0 5 to 50 ms and the distance between GABA source and receptor from 0 5 to 50 gsm. By far the more important variable in these calculations was the distance between source and receptors; the decay time was much more sensitive to the 406 R. DINGLEDINE AND S. J. KORN

0 50 A 1 Brief pulse B 1 Long pulse 0040- 00304 <0-20 ,A XA 010c-,AU ~ ~ ~ 0100

G050- A2 82 C 0230 0 W0.10 E AUC eraeutk C~~~

0.0 02 04 06 00 10 20 30 40 50 Time (s) Time (s)

W 5s C Decrease uptake E

3 Increase channel open time

0) U. 0.03 0.10 030 1.00 3.00 Control decay time (90-10%) (s) Fig. 10. Calculated effect ofchanging uptake rate and channel open time on GABA-evoked responses of different time courses. A and B, the kinetic model described in Fig. 9 was used to produce GABA concentration profiles (A 1 and B 1) and the resultant conductance changes (A2, B2) that were either brief and rapidly decaying (A), to simulate the time course of i.p.s.p.s, or longer and more slowly falling (B), to simulate the time course of a response to ionophoretically applied GABA. The GABA concentration profiles at the receptor (A 1 and B 1) are plotted as a unitless ratio to the Kd for the binding reaction. The calculated conductance changes are plotted on a semilogarithmic scale as a fraction of the maximal possible conductance change (with saturating GABA concentration). The effect of changing the uptake rate constant from 06/s to 0-0/s is shown by the curves labelled AU. The effect of increasing the mean channel open time from 10 to 30 ms (decreasing a from 100/s to 33/s) is shown by the curves labelled Aa. In each case, the control curves (Ku = 0 6/s, a = 100/s) are labelled C. In A, GABA was released in a 30 ms pulse from a distance of 4 lam; in B, GABA was released in a 200 ms pulse from a distance of 20 jam. In all cases, the channel-opening rate constant, fi, equalled 100/s. The graph in C summarizes the results of thirteen sets of calculations such as those shown in A and B. The time course of the GABA response was varied systematically by changing the diffusion distance and the amount and duration of release. For each condition, the fold increase in decay time (1 = no change) measured from 90 to 10% of peak response was calculated following a change in the uptake rate constant (circles) or channel-closing rate constant (squares), and plotted as a function of the control decay time in seconds (abscissa). Ku was changed from 0-6/s to 0-0/s; a was changed from 100/s to 33/s. GABA UPTAKE AND I.P.S.P.S 407 diffusion distance than the duration of the pulse. The response prolongation due to inhibition of uptake (circles) or increase in channel mean open time (squares) was calculated from each curve and plotted in Fig. 10C. The data in this graph are qualitatively unchanged by the choice of rate constants; the curves merely shift horizontally. The effectiveness ofuptake inhibition was strictly dependent on the rate of decline of the response. Quickly falling responses were unaffected by uptake, but as the response decay time became comparable to the uptake time constant (1-65 s), elimination ofuptake markedly lengthened the decay time ofthe response. Conversely, increasing the channel open time prolonged only the decay time of responses the duration of which was of the same order of magnitude as the channel open time. The slight prolongation of the longer responses by this manipulation was due predominantly to a broadening of the peak. Interestingly, peak broadening by pentobarbitone was a feature of both our data (Fig. 2) and those of Higashi & Nishi (1982).

We thank Dr Povl Krogsgaard-Larsen for generous supplies of several GABA-receptor agonists and GABA uptake inhibitors, Dr Bertil Takman of Astra Pharmaceuticals for supplying us with QX-314, Dr Barry Pallotta for valuable discussions during the course of this project and Ms Sandi Batchelor for excellent secretarial assistance. This work was supported by a grant from the Society of Sigma Xi to S. Korn and by N.I.H. grant NINCDS 17771.

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