SYNAPSE 11~249-258 (1992)

GABAB Receptor-MediatedInhibitory Postsynaptic Potentials Evoked by Electrical Stimulation and by Glutamate Stimulation of Interneurons in Stratum Lacunosum-Moleculare in Hippocampal CAI Pyramidal Cells In Vitro -

SYLVAIN WILLIAMS AND JEAN-CLAUDE LACAILLE Centre de Recherche en Sciences Neurologiques et Departement de Physiologie, Universite de Montreal, Montdal, Quebec, Canada H3C 357

KEY WORDS

Interneurons, GABA, Intracellular, Slices

ABSTRACT

Following micropressure application of glutamate (500 p M ) in stratum lacunosum-moleculare (L-MI, inhibitory postsynaptic potentials (glut-IPSPs) were recorded in CA1 pyramidal cells. These glut-IPSPs were blocked by tetrodotoxin (1pM) and, thus, were probably generated by the activation of local interneurons. The effects of pharmacological antagonists on glut-IPSPs and on electrically-evoked early and late IPSPs were assessed in the same cells during the same application of the antagonist. Local application of the GABA, antagonist 2-OH saclofen (1-4 mM) reduced both glutIPSPs and late IPSPs but not early IPSPs. In contrast, the GABAB antagonist phaclofen (20 mM) reduced late IPSPs but not early IPSPs or glut-IPSPs. Barium (1mM),a blocker of some K’ channels, diminished late IPSPs but not early IPSPs or glut-IPSPs. Early IPSPs were blocked by the GABAA antagonists bicuculline and picrotoxin but late IPSPs and glut-IPSPs were not. Repetitive electrical stimulation depressed early and late IPSPs as well as glut-IPSPs, suggesting that interneurons activated with glutamate were also stimulated electrically. Thus, interneurons in str. lacunosum-moleculare appear to inhibit pyramidal cells via a GABAB receptor-mediated IPSP. The discrepancy in the pharmacological profile of the GABAB glut-IPSPs and of the GABAB late IPSPs may suggest the presence of two GABABmechanisms in CA1 pyramidal cells. 0 1992 Wiley-Liss, Inc.

(Alger and Nicoll, 1982a,b; Andersen et al., 1964a,b; Ben-Ari et al., 19811, and 2) a slower “late” IPSP resultIn Ammon’s horn (CA1 and CA3) of the hippocampus, ing from the activation of GABAB receptors and the 80 to 95% of local circuit cells (interneurons) appear opening of potassium channels (Alger, 1984; Dutar and immunoreactive to gamma-aminobutyric acid (GABA) Nicoll, 1988a,b; Knowles et al., 1984). or to its synthesizing enzyme glutamic acid decarboxIt has been proposed that in the CA1 region, the early ylase (Woodson et al., 1989). Although interneurons and late IPSPs may implicate different types of intercomprise only about 10% of the total hippocampal cell neurons releasing GABA on separate populations of population (Olbrich and Braak, 1985), their inhibitory GABA receptors located on pyramidal cells (Alger and input is crucial in maintaining proper excitatory levels 1988; Lacaille et al., Nicoll, 1982a,b; Kunkel et al., in pyramidal cells (Traub et al., 1987). GABAergic in1989; Segal, 1990). Previous studies have shown that hibitory postsynaptic potentials (IPSPs)are evoked folGABAA early IPSPs were evoked when pyramidal cells lowing electrical stimulation of hippocampal afferent pathways (Alger, 1984; Alger and Nicoll, 1982a,b; were antidromically activated, suggesting the involveKnowles et al., 1984). These IPSPs recorded intracellu- ment of recurrent inhibitory interneurons (Alger and larly in pyramidal cells reveal a biphasic response: 1)a rapid “early” IPSP mediated by the activation of Received August 27,1991; accepted in revised form November 22,1991 GABAAreceptors and the opening of chloride channels INTRODUCTION

0 1992 WILEY-LISS. INC.

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Nicoll, 1982a,b). Interneurons responsible for mediating this IPSP have been described using paired recordings. Interneurons located in, or near, stratum (str.) pyramidale (basket cells; Knowles and Schwartzkroin, 1981) and near the str. oriens-alveus junction (vertical cells; Lacaille et al., 1987) were excited by pyramidal cell collaterals and in turn inhibited pyramidal cells, thus producing recurrent inhibition, They may therefore mediate the GABAA early IPSP. The late IPSP appears to be mediated by feedforward interneurons making inhibitory synapses on pyramidal cell dendrites (Alger and Nicoll, 198213). One such interneuron is located at the border of str. radiatum and lacunosummoleculare (L-M) (Kawaguchi and Hama, 1987, 1988; Kunkel et al., 1988; Lacaille and Schwartzkroin, 1988a,b). Intracellular activation of these cells inhibited pyramidal cells solely in a feedforward fashion, producing IPSPs with slow time course. The hypothesis that interneurons located in L-M underlie the electrically-evoked late IPSP was tested by locally activating these interneurons with applications of glutamate in this layer and by recording the IPSPs evoked in pyramidal cells (glut-IPSPs) (Williams and Lacaille, 1990). These experiments revealed that the glut-IPSPs were different from both the early and the late IPSPs. The null potential of the glut-IPSPs was similar to the equilibrium potential of the early IPSP, but the glut-IPSPs were insensitive to bath application of both the GABAA antagonist bicuculline and the GABAS antagonist phaclofen. However, in recent experiments, these glut-IPSPs were antagonized by local application of a different GABAB receptor antagonist, 2-OH-saclofen. The present series of experiments were therefore undertaken to further examine the pharmacological profile of these glut-IPSPs and their relation to the electrically-evokedIPSPs. Preliminary reports of this work have appeared in abstract form (Williams and Lacaille, 1991a,b).

MATERIALS AND METHODS Slices and intracellular recordings Hippocampal slices were obtained as described previously (Lacaille, 1991; Lacaille and Schwartzkroin, 1988a,b; Lacaille and Williams, 1990; Williams and Lacaille, 1990) from male Sprague-Dawley rats (Charles River Laboratories) weighing 150-225 gm. Briefly, the animals were anesthetized with ether, and decapitated. The brain was quickly removed from the skull and placed in ice-cold, oxygenated, artificial cerebrospinal fluid (ACSF; see below). The hippocampus was dissected free and slices were cut transversely (thickness 400-450 pm) using a McIlwain tissue chopper. Slices were placed on a nylon net in a gadfluid interface chamber and perfused with ACSF of the following composition (in mM): NaC1, 124; KC1, 5; NaH,PO,, 1.25; MgSO,, 2; CaCl,, 2; NaHCO,, 26; dextrose, 10. ACSF was saturated with 95% 0,/5% CO,

and slices were continuously perfused at 1 mllmin. Slices and ACSF were maintained at a temperature of 35 * 0.5%. The upper surface of the slices was exposed to a warmed, humidified gas mixture (95%0,/5% CO,). Micropipettes were pulled from fibre-filled capillary tubing of borosilicate glass using a Brown-Flaming micropipette puller (Sutter Instr. P-80). Micropipettes (40-75 MR) were filled with 4M potassium acetate and 0.01 M KC1. Intracellular recordings were obtained from pyramidal cells of the CA1 region using conventional recording techniques. Intracellular responses were recorded with intracellular recording amplifiers (Neuro Data IR-283) equipped with an active bridge circuit for current injection, displayed on a digital storage oscilloscope (Gould 1604) and stored in digitized format using a video cassette recorder (Neuro Corder DR 886) for later retrieval and analysis. Intracellular recordings were considered acceptable if neurons had stable membrane potentials without holding current. Bridge balance was carefully monitored throughout the experiments and adjusted when necessary. Intracellular responses were digitized using a microcomputer (Everex 3000) with data acquisition boards (Axon Instr. TL-1-125) and analyzed using pClamp (Axon Instr.). Resting membrane potential (RMP) was measured after withdrawal from the cell. Action potential amplitude was measured from RMP, and cellular input resistance (R1J from the maximum voltage change during a 100 ms, -0.5 nA current pulse.

IPSPs Glutamate (500 pM; l-glutamic acid monosodium salt, Sigma) was dissolved in saline (0.9%). A fine tipped micropipette was filled with glutamate and the tip was gently broken to yield a drop of 30-50 pm diameter when micropressure (10-200 ms, 30 psi) was applied to the back of the pipette. The glutamate-containing pipette was lowered into str. L-M of the CA1 region 200-400 pm lateral t o the intracellular recording electrode. The glutamate pipette was moved into the tissue in 10 pm steps until glutamate ejection elicited a hyperpolarization in pyramidal cells. In order to compare the glutamate-evoked responses to the electrically-evoked IPSPs, orthodromic synaptic responses were elicited by stimulating the Schaffer collaterals with constant current (100400 FA, 50 ps) delivered via a stimulus isolation unit (WPI A3601 using a monopolar resin-coated tungsten microelectrode placed in str. radiatum, near the str. lacunosum-moleculare border. The measurements of the latency to peak of the IPSPs were taken from the onset of glutamate or electrical stimulation to the peak amplitude of the IPSP. The IPSP decay was measured from the peak amplitude to the return to pre-stimulation membrane potential. Glut-IPSPs and electrically evoked early and 1ateIPSPs were always recorded in the same cells. Also, the efficacy of pharmacological antagonists were tested on glut-IPSPs and on

GABA, IPSPs EVOKED FROM L-M INTERNEURONS

A

251

B

I

A GLUTAMATE

TTX

+

-58

GLUTAMATE BrnV

120 Ins

80 rns

Fig. 1. 'ITX block of glutamate- and electrically-evoked IPSPs. A: Glutamate application (pulse, 30 ms, 30 psi) to str. L-M elicited an IPSP (triangle) in control medium. Perfusion of the slice with TTX (I p M ,12 min) blocked the glutamate IPSP (bottom trace). B: In the same cell, electrical stimulation elicited a n early (circle) and a late (square) IPSP in control medium (-58 mV). In the presence of "x,electrically evoked responses were also blocked. In this and following figures, action potentials are truncated.

electrically-evoked early and late IPSPs during the same application of the antagonist.

t-test was used for statistical comparisons of differences between means.

Materials Solutions of (-) bicuculline methiodide (BIC; 100 pM, Sigma), picrotoxin (PIC; 1.6 mM, Sigma), phaclofen (PHAC; 20 mM, RBI), 2-OH-saclofen (1, 2, and 4 mM, RBI), and barium chloride (1 mM, Sigma) were prepared in saline (0.9%)and kept frozen. Pharmacological agents were applied locally, resulting in more rapid drug application and effects, and facilitating the monitoring of response recovery. The day of the experiment a pipette was filled with the proper drug solution, its tip broken back to yield a drop with a diameter of approximately 100 pm when pressure (100-200 ms, 30 psi) was applied. The drug filled pipette was placed into the dendritic area of the pyramidal cell being recorded from, perpendicular to the cell layer and approximately 100 pm into str. radiatum. For the picrotoxin experiments, it was diluted in ACSF t o its final concentration (10-20 p M ) and applied to the bath via a three-way valve. Application of GABA, antagonists was considered effective when the early IPSP was blocked and electrical stimulation produced 2 or more spikes. In some experiments synaptic transmission was blocked by perfusing the slices with ACSF containing tetrodotoxin (TTX; 1p M , Sigma). Results are expressed as mean z S.D., unless otherwise indicated. Student's

RESULTS Glutamate-evoked IPSPs Intracellular recordings were obtained from 193 neurons. Membrane properties of the cells sampled were 85.3 ? 13.4 mV (n = 193) for action potential amplitude, -63.3 2 8.3 mV (n = 178) for RMP and 29.4 & 10.6 M a for Ri, (n = 191). In 88 cells, hyperpolarizations were elicited following the application of glutamate to the L-M layer (glut-IPSPs). In 17 cells, glutIPSPs could not be elicited after several glutamate applications during multiple descents in this layer. Recordings from 88 cells were lost before more than one descent with the glutamate pipette could be completed and a glut-IPSP found. Representative examples of hyperpolarizing responses evoked by glutamate application to L-M and by electrical stimulation of str. radiatum are shown in Figure 1, along with their block by TTX. In this cell, glutamate applied in L-M, in normal ACSF, elicited an IPSP with an amplitude of - 3.6 mV at a peak latency of 168 ms. Electrical stimulation evoked an excitatory postsynaptic potential (EPSP) followed by an early IPSP of -11.3 mV (25 ms latency) and a late IPSP of -6.4 mV (130 ms latency). After perfusion of the slice for 12 min with ACSF containing TTX (1pM), the glu-

S. WILLIAMS AND J.-C. LACAILLE

252 A

CONTROL

B

2

A GLUTAMATE

- OH - SACLOFEN ___

7 n A

L

160 ms

120 ms

C

WASH

D

CONTROL 2

- OH - QACLOFEN

7 n A

160 ms

O

i

120ms

Fig. 2. Effects of 2-OH-saclofen on IPSPs. A: Glutamate-evoked IPSP (top trace, triangle) and electrically-evoked (bottom trace, arrow) early IPSP (circle) and late IPSP (square) were elicited in control medium. B: Thirty seconds following the local application of 2-OHsaclofen (2 mM) the glut-IPSP and the late IPSP were largely reduced.

The early IPSP was only slightly affected. C: After 3 min, a partial recovery of the responses was observed. D: Overlay of traces of A (control) and of B (immediately following the application of %OHsaclofen), showing the antagonism of the late IPSP (square)and of the glut-IPSP (triangle) by 2-OH-saclofen.

60% of control, respectively, and the amplitude of the glut-IPSP had recovered to 46% of control. In all cells tested, 2-OH-saclofen significantly reduced the late IPSP (46.3 -r- 18.8%of control, n = 7;P< 0.001) and the glut-IPSP (47.3 -t 17.2% of control, n = 9;P< 0.005). The early IPSP was not significantly altered (89.8 10.8%of control, n = 7;P> 0.05). In 2 of these cells, the effects of 2-OH-saclofen were tested only on the glutIPSP. In the remaining 7 cells, the sensitivity of glutIPSPs and of electrically-evoked early and late IPSPs were assessed in the same cell during the same application of 2-OH-saclofen.The effects of 2-OH-saclofenwere reversible with the amplitude of the early, late and glutamate IPSPs recovering to 94%, 78%, and 93% of control, respectively (n = 7). All three concentrations of Effects of GABA, antagonists 2-OH-saclofen tested were as effective in antagonizing The efficacy of the GABAB antagonist 2-OH-saclofen glut-IPSPs and late IPSPs (1mM, n = 1, amplitude of in blocking the IPSPs evoked by glutamate and by elec- glut-IPSP and late IPSP: 53% and 55% of control, retrical stimulation was first examined. An example of spectively; 2 mM, n = 5: 50%and 48% of control, respecthe effect of locally applied 2-OH-saclofen (2 mM) is tively; and 4 mM, n = 1:23% and 31%of control, respecshown in Figure 2. Following the application of 2-OH- tively). Different effects were observed with the GA13AB ansaclofen, the amplitude of the glut-IPSP was reduced t o 23% of control. In the same cell, during the same appli- tagonist phaclofen, as illustrated in Figure 3. Following cation of 2-OH-saclofen, the amplitude of the late IPSP the application of phaclofen (20 mM), the amplitude of was also diminished (31%of control). In this cell, the the late IPSP was diminished to 56% of control. But, in amplitude of the early IPSP was slightly attenuated the same cell and during the same application of (88%of control). Three minutes later, the amplitude of phaclofen, the amplitude of the early IPSP and of the the early and late IPSPs had recovered t o 102% and glut-IPSP were not reduced (116% and 95% of control,

tamate and electrically evoked synaptic potentials were abolished completely. Similar results were found in all 3 cells tested with TTX. Overall, the electrophysiological characteristics of the glut-IPSPs were similar to those reported in a previous study (Williams and Lacaille, 1990). In a sample of 20 cells, the mean amplitude of the glut-IPSP was -2.0 -+ 0.5 mV (mean V, -58.0 ? 5.3 mV). The mean latency of the peak amplitude was 127.2 2 48.6 ms and the mean decay for the glut-IPSP was 319.5 2 146.7 ms. In 14 cells examined, the null potential of the glut-IPSP was -69.6 i- 4.5 mV. In 13 of 14 cells, the glut-IPSPs displayed little or no response reversal at negative membrane potentials ( - 75 mV to - 100 mV).

*

GABAB IPSPSEVOKED FROM L-M INTERNEURONS

A

B

CONTROL

PHACLOFEN

253 C

CONTROL PHACLOFEN

-65

A GLUTAMATE

A

A

LLL

‘ 0

0

Fig. 3. Selective antagonism of the late IPSP by phaclofen. A: Glutamate-evoked IPSP (top trace, triangle) and electrically evoked (arrow, bottom trace) early (circle) and late (square) IPSPs elicited in control ACSF. B: Following the local application of phaclofen (20 mM), the amplitude of the late IPSP was reduced but the amplitude of the

respectively). In 4 cells tested, phaclofen significantly reduced the amplitude of the late IPSP to 47 2 15.5%of control (P< 0.05). The amplitude of the early IPSP and of the glut-IPSP, recorded in the same cells and during the same applications of phaclofen, remained unchanged (109 k 11.4% and 101 r 17.8% of control, respectively; P> 0.05).

early IPSP and of the glut-IPSP were not. C: Traces before (A) and after (B) application of phaclofen are overlaid to show the selective reduction of the late IPSP (square) but not of the early or glutamate IPSP by phaclofen.

Effects of GABAA antagonists The characteristics of the early, late and glutamateIPSPs were compared in two groups of cells in normal or in picrotoxin containing medium. In 6 cells perfused with picrotoxin (10-20 pM) in which the early IPSP was blocked, the mean amplitude of the glut-IPSPs was -2.7 k 1.1 mV (mean Vm-55.5 & 8.4 mV). Their mean latency to peak was 232.7 t 35.2 ms and they decayed Effects of barium in 524.3 i 246.2 ms. In normal medium and in picroSince barium has been shown to block certain K + toxin, the mean peak latency of the glut-IPSPs were channels, including those linked to GABAB receptors statistically different (P< O.OOl>,whereas the ampli(Gahwiler and Brown, 1985; Knowles et al., 1984; Newtude and decay were not. The measures of equilibrium berry and Nicoll, 1985),the sensitivity of the early, late, potential of glut-IPSPs elicited in the presence of picroand glutamate IPSPs to this ion was assessed. As toxin were also compared to those obtained in control shown in Figure 4 for a representative cell, locally apmedium. In normal medium, the mean equilibrium poplied barium reduced the amplitude of the late IPSP to tential of the glut-IPSP was -69.6 2 4.5 mV (n=14), 64% of control. However, in the same cell and during whereas in the presence of picrotoxin, the mean equilibthe same application of Ba2+,the amplitude of the glutrium potential was -68.5 2 5.3 mV (n=4). The differIPSP and of the early IPSP were augmented (175% and ence between these means was not statistically signifi116% of control, respectively). In this cell, 16 min fol- cant (P> 0.05). The effectiveness of local application of lowing the application of barium, the amplitude of the the GABA, antagonist bicuculline (100 pM) in blocking early, late and glutamate IPSPs had recovered to 123%, glutamate- and electrically evoked IPSPs was also ex90%, and 92% of control, respectively. In 4 cells tested, amined in two cells. The early IPSP was blocked combarium significantly reduced the late IPSP to 64.2 2 pletely after the application of bicuculline, but in the 8.8% of control (P< 0.01). The peak amplitude of the same cells, and during the same bicuculline applicaearly and glutamate IPSPs, recorded in the same cells, tion, the mean peak amplitude of the glut-IPSP was were slightly increased during the same application of 122% of control. The late IPSP appeared potentiated barium, although not significantly (112.5 2 7.6% and but it may have been augmented by additional intrinsic 116.5 2 37.6%, respectively; P> 0.05). In these cells, conductances activated by the burst of action potentials the amplitude of the early, late, and glutamate IPSPs (i.e. slow after-hyperpolarizations). recovered to 109.5 17.6%, 84.7 13.1%, and 87.0 Frequency dependent depression 6.5% of control, respectively. The mean recovery period Repetitive stimulation of afferent fibres has been was 12.0 ? 6.3 min after barium application. In one of these 4 cells, a late portion of the glut-IPSP appeared shown to depress the early and late GAJ3Aergic IPSPs blocked in a similar manner to the late IPSP. In all (Ben-Ari et al., 1981; Thompson, 1989). To test the hypyramidal cells tested with local application of Ba2+,a pothesis that both electrical and glutamate stimulation depolarization of their resting membrane potential and activated overlapping populations of interneurons, the a widening of their action potential were observed (data amplitude of the glut-IPSPs was examined before and not shown), in addition to a reduction of the late IPSP. after frequency-dependent depression of the early and

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S. WILLIAMS AND J.-C. LACAILLE

254 A

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Fig. 4. Effects of barium on IPSPs. A Glutamate-evokedIPSP (top trace, triangle) and electrically evoked (arrow, bottom trace) early IPSP (circle)and late IPSPs (square)were elicited in control ACSF. B: Seven minutes after the local application of barium (1 mM), the late IPSP was reduced but the glut-IPSP and the early IPSP were aug-

mented. C: Sixteen minutes after applicationof barium, the amplitude of the responses had recovered to near their control values. D: Superimposed traces before (A) and after (B) application of barium, to show the selective reduction of the late IPSP (square)VB. the small augmentation of the early IPSP and of the glut-IPSP by Ba".

late IPSPs. Electrical and glutamate stimulations were given as shown in Figure 5. In this cell, before repetitive stimulation, glutamate evoked an IPSP with a peak amplitude of -2.7 mV. Electrical stimulation elicited an early IPSP of -6.5 mV and a late IPSP of -4.2 mV. The electrical stimulation was then repeated at a frequency of 3 Hz for 10 sec. At the 30th stimulation, the IPSPs were greatly depressed (Fig. 5) with the early IPSP now being depolarizing (+ 5.6 mV) and the amplitude of the late IPSP reduced to -0.2 mV. Glutamate applied 2 sec after the last electrical stimulation did not elicit a glut-IPSP. Two minutes after the end of repetitive stimulation, both the glutamate- and the electrically evoked IPSPs had recovered to control amplitude. Although in this particular cell the membrane potential hyperpolarized slightly (to -68 mV) during the train of stimulation, it was still far enough from the equilibrium potential to evoke early and glut-IPSPs in the control period. In 6 cells tested, before repetitive electrical stimulation, the mean amplitude was -8.8 2 1.5 mV for the early IPSP and -5.8 i 1.4 mV for the late

IPSP (mean V, -59.5 L 2.9 mV). The mean amplitude of the glutamate IPSP was -2.4 & 0.7 mV (mean V, -59.3 ? 3.7). Following repetitive stimulation, the mean amplitude of the early and the late IPSPs were depressed to +1.3 ? 1.9 mV and -2.0 5 1.5 mV, respectively (mean V, -60.3 5 6.9 mV). The mean amplitude of the glutamate evoked IPSP also was reduced to -0.5 L 0.8 mV (mean V, -59.8 ? 5.7 mV). The differences in mean amplitude, before and after repetitive stimulation, of the early, late and glutamate IPSPs were statistically significant (P< 0.01). DISCUSSION The main finding of the present study was that IPSPs evoked by glutamate stimulation of interneurons in str. lacunosum-moleculare, like the electrically elicited late IPSPs, appeared mediated by GABA, receptors. However, the pharmacological profile of both types of IPSPs seemed different, suggesting a dissociation between these responses (Table I).

GABAS IPSPS EVOKED FROM L-M INTERNEURONS

255

A STIMULATION GLUTAMATE

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Fig. 5. Frequency-dependent depression of the early, late, and glutamate IPSPs. A: Stimulation paradigm with glutamate and electrical stimulation given before and after repetitive electrical stimulation. The stimulation events numbered 1-6 elicited the corresponding responses depicted in B-E. B: In the control period (traces 1 and 2), a glutamate IPSP (triangle) and an early (circle) and late (square) IPSP were elicited. C: After repetitive electrical stimulation (3 Hz for 10 seconds), the 30th stimulation (trace 3) elicited a reversed early IPSP

and a reduced late IPSP. Glutamate applied 2 sec after the end of the stimulation train did not elicit an IPSP (trace 4). D: Following a recovery period of 2 min, the glutamate IPSP (trace 5)and the electrically evoked early and late IPSPs (trace 6) recovered to control levels. E: Traces in the control period and following the stimulus train are superimposed for the glut-IPSP (1 and 4) and the electrically evoked IPSPs (2 and 3), showing the depression ofboth the glutamate- and the electrically evoked IPSPs following repetitive electrical stimulation.

Glutamate-IPSPs The amplitude, time course, null potential, lack of response reversal, and TTX sensitivity of the L-M evoked glut-IPSPs in the present study were similar to the previously reported properties of glut-IPSPs similarly evoked from L-M stimulation (Williams and Lacaille, 1990). These glut-IPSPs probably resulted from the activation of local GABAergic interneurons in str. L-M. The insensitivity of the glut-IPSPs to local application of bicuculline in the present experiments confirmed previous evidence of little involvement of GABAA receptors in these glutamate responses

(Williams and Lacaille, 1990). We extended this confirmation by showing that glut-IPSPs evoked during perfusion with picrotoxin have a similar amplitude, decay, equilibrium potential and lack of reversal potential than glut-IPSPs evoked in normal ACSF. Their longer peak latency may be due to disinhibition of inhibitory interneurons in picrotoxin (Lacaille, 1991; Misgeld and Frotscher, 1986) resulting in greater interneuron activation by glutamate. The insensitivity of these glutIPSPs to GABAa antagonists is in contrast to the sensitivity of IPSPs evoked by glutamate stimulation in str. pyramidale and at the str. orienslalveus border which

S. WILLIAMS AND J.-C. LACAILLE

256

TABLE I. Properties of glutamate- and electrically evoked IPSPs (mean amplitude f S.D. in % of control) Treatment

TTX 2-OH-saclofen Phaclofen

Ba2+

Early IPSP (n)

Late IPSP (n)

glut-IPSP

0% (3) 90 f 11% (7) 109 f 11% (4) 112 f 8%

0% (3) 46 f 19% (7) 47 k 16% (4) 64 ?c 9% (4) Increased (2) Increased

0%

(4)

Bicuculline

0%

Picrotoxin

0%'

(2)

Repetitive electrical stimulation

(6) Reversed (6)

(4

(3) 47 f 17% (9) 101 f 18% (4) 116 f 38% (4)

122 f 11% (2) 135 f 55%'

(6)

(6)

34 f 26%

21 f 33% (6)

(6)

~~

'Percentage of mean response from cells in normal ACSF.

are blocked by GABAAbut not by GABAB antagonists (Madison and Nicoll, 1988; Samulack and Lacaille, 1991).

GABAB receptor-mediated IPSPs The GABAB antagonist 2-OH-saclofen is an effective blocker of GABAB mediated responses such as the late IPSP (Davies et al., 1990; Lambert et al., 1989). In the present study, local application of 2-OH-saclofen decreased the late IPSP. At the same time, the IPSP elicited by glutamate application to str. L-M was also reduced similarly. Thus, these results suggest that glut-IPSPs are mediated via GABAB receptors. However, discrepancies were observed in the pharmacological profile of glut-IPSPs and late IPSPs when other blockers of GABAB responses were used. Firstly, local application of the GABA, antagonist phaclofen significantly reduced the late IPSP, as previously reported by others (Dutar and Nicoll, 1988a,b; Malouf et al., 1990). But in the same cells and during the same application, phaclofen had no effect on the glut-IPSP, confirming results obtained previously with bath application of this antagonist (Williams and Lacaille, 1990). Secondly, local application of Ba2+,a blocker of certain K' channels including some linked to GABA, receptors (Gahwiler and Brown, 1985; Newberry and Nicoll, 1985) and the late IPSP (Alger, 1984; Knowles et al., 1984), decreased the late IPSP in all cells tested. However, in the same cells and during the same application of Ba2+, it did not reduce the glut-IPSP, but rather increased it slightly. These results with GABAB antagonists suggest that glut-IPSPs and late IPSPs, although both mediated by GABAB receptors, may involve different mechanisms. As shown in Table I, glut-IPSPs were not totally antagonized by 2-OHsaclofen. Thus, glut-IPSPs may have possibly been due to a substance other than GABA. However, in our experiments, both glut-IPSPs and late IPSPs were blocked to a similar extent by 2-OH-saclofen (approx-

imately 50%). Therefore, the residual portion of the IPSPs most probably resulted from a certain proportion of GABABreceptors not exposed to the antagonist since 2-OH-saclofenwas applied locally in str. radiatum. Possible mechanisms Our observation of a dissociation between GABAB responses are not unique. There have been several reports suggesting the existence of phaclofen-resistant GABAB receptor subtypes (for review see Sivilotti and Nistri, 1991). Firstly, in the hippocampus, 2-OHsaclofen antagonized both the pre- and postsynaptic GABAB receptors, but phaclofen blocked only postsynaptic and not presynaptic receptors (Davies et al., 1990; Dutar and Nicoll, 1988b; Harrison, 1990; Harrison et al., 1988; Wang and Dun, 1990; but see also Seabrook et al., 1990). Secondly, in cortex, baclofen may bind to two GABABreceptor sites that phaclofen, but not 2-OHsaclofen, can discriminate (Al-Dahan et al., 1990). Thirdly, baclofen has been shown to activate a phaclofen-insensitive GAEiAB receptor that promotes receptor-mediated CAMP accumulation in cortex (Scherer et al., 1988). Finally, in the periphery, actions of baclofen were mediated by both phaclofen-sensitive and phaclofen-resistant GABAB receptors (Blandizzi et al., 1991). Recent studies using hippocampal microcultures have also reported IPSPs similar to the glutIPSPs of the present study. With paired recordings from inhibitory and excitatory cells, certain proportions of slow IPSPs elicited in excitatory cells (presumed pyramidal cells) were insensitive to both bicuculline and phaclofen (Segal and Furshpan, 1990). There have also been several indications of an heterogeneity of conductances mediating GABAB responses. For example, baclofen-activated, GABAB hyperpolarizations displayed inward rectification in CA3 pyramidal cells, but GABA-activated, GABAB responses did not (Inoue et al., 1985; Ogata et al., 1987). Furthermore, the K+ channel blocker 4-aminopyridine (Storm, 1990) antagonized the response mediated by baclofen but did not affect the GABA-activated, GABAB response (Inoue et al., 1985; Ogata et al., 1987).Finally, it was recently reported that, in contrast to the postsynaptic actions of baclofen which were blocked by Ba2+, its presynaptic effects were insensitive to Ba2+ (Lambert et al., 1991). The difference in pharmacological profile of glutIPSPs and electrically evoked late IPSPs observed in the present study may have been pharmacological artefacts due to the low potency and/or poor tissue penetration of phaclofen. However, two lines of evidence suggest otherwise. First, in the case of the antagonist phaclofen, it was not effective in reducing the glut-IPSP despite the fact that it was effective in antagonizing the late IPSP during the same application in the same cell. Similar results have also been obtained with bath application of phaclofen (Williams and Lacaille, 1990).

GABA, IPSPS EVOKED FROM L-M INTERNEURONS

Thus, it is unlikely that the inefficacy of phaclofen in blocking the glut-IPSP may have been due to poor penetration. Second, Ba2+ which blocks the potassium channels linked to GABAB receptors (Gahwiler and Brown, 1985; Knowles et al., 1984; Newberry and Nicoll, 1985), was also effective in reducing the late IPSP, while a t the same time it was ineffective in blocking the glut-IPSP in the same cell. Taken together these results suggest that the different pharmacological profile of glut-IPSPs and late IPSPs may be due to the presence of different GABAB receptor mechanisms in CA1 pyramidal cells.

257

have unique intrinsic properties and connectivity (Lacaille et al., 1989). This interneuron can alternate from regular spiking to bursting upon membrane hyperpolarization (Lacaille and Schwartzkroin, 1988a,b), apparently as a result of a prominent low-threshold Ca2+ conductance (Fraser and MacVicar, 1991). The connectivity of stellate cells is also distinct since their axon terminates in both the CA1 region and the dentate gyms (Kunkel et al., 1988; Lacaille and Schwartzkroin, 1988a). Because of these unique intrinsic properties and connectivity, stellate interneurons may play a pivotal role in the modulation of hippocampal activity. The present study has begun to provide some evidence about the mechanisms of this modulation of activity which appear to involve GABAB receptors. The exact nature of the GABAB receptor-mediated mechanisms remains unclear. Finally, our results support the hypothesis (Lacaille et al., 1989) that the stellate cells of str. lacunosum-moleculare may be implicated in the GABAB-mediatedlate IPSP in CA1 pyramidal cells.

Repetitive stimulation Although the results of experiments with antagonists of GMABresponses suggested differences between late IPSPs and glut-IPSPs, the results with repetitive electrical stimulation suggested that interneurons stimulated with glutamate may also have been involved in the electrically evoked IPSP. Repetitive electrical stimulation depresses GABAergic IPSPs via a number of ACKNOWLEDGMENTS mechanisms involving both pre- and post-synaptic factors (Ben-Ari et al., 1981; McCarren and Alger, 1985; This research was supported by a grant from the Thompson, 1989; Davies et al. 1990). In the present Medical Research Council of Canada (J.-C.L.). J.-C.L. is study, following repeated electrical stimulation, the a Scholar from Fonds de la recherche en sante du QueIPSPs evoked by glutamate were always reduced in bec and a member of the Groupe de recherche sur le parallel to the early and late IPSPs. Since the depres- systeme nerveux central (FCAR) and of the Reseau de sion is due in large part to postsynaptic factors (Ben-Ari recherche en sante mentale FRSQ-Universite de Monet al., 1981; McCarren and Alger, 1985; Thompson, treal. S.W. was supported by a studentship from the 1989) and since the reduction in glut-IPSPs and electri- Savoy Foundation. The authors wish to thank C. Gaucally evoked IPSPs were always in parallel, these re- thier and D. Cyr for graphic and photographic assissults suggest that the interneurons stimulated with tance, H. Dussault for secretarial assistance and Dr. glutamate may also have been activated by electrical Donald D. Samulack for constructive comments on earstimulation. Therefore, the glut-IPSP may be a compo- lier versions of this manuscript. nent of the electrically evoked IPSP. Since both IPSPs were reduced by 2-OH-saclofen,but a portion of the late REFERENCES IPSP was phaclofen-sensitive and the glut-IPSP was Al-Dahan, MI., Tehran, M.H.J., and Thalmann, R.H. (1990) Effects of phaclofen-insensitive, the late component of the electri2-hydroxy-saclofen, a n antagonist of GABA, action, upon the binding of baclofen and other receptor ligands in rat cerebrum. Brain cally evoked IPSP may therefore consist of two GABAB Res., 526:308-312. subcomponents. If this would be the case, Ba2+ and Alger, B.E. (1984) Characteristics of a slow hyperpolarizing synaptic potential in rat hippocampal pyramidal cells in vitro. J. Neurophysphaclofen should be less effective in blocking the late iol., 52:892-910. IPSP than 2-OH-saclofen. Our experiments were incon- Alger, B.E., and Nicoll, R.A. (1982a) Feedforward dendritic inhibition clusive on this matter, since 2-OH-saclofen produced a in rat hippocampal pyramidal cells studied in vitro. J. Physiol. (Lond.), 328:105-123. greater reduction of the late IPSP than Ba2+but was as Alger, B.E., and Nicoll, R.A. (1982b) Pharmacological evidence for two effective as phaclofen. More definitive experiments kinds of GABA receptors on rat hippocampal pyramidal cells studied in vitro. J . Physiol. (Lond.), 328:125-141. with bath application of antagonists are needed to clarAndersen, P., Eccles, J.C., and Loyning, Y. (1964a) Location of ify this issue. postsynaptic inhibitory synapses on hippocampal pyramids. J. NeuFunctional implications The characteristics of the IPSPs elicited in pyramidal cells by glutamate applications to str. L-M resembled those of IPSPs elicited from stellate interneurons in paired recording studies (Lacaille and Schwartzkroin, 1988b; Williams and Lacaille, 1990). Thus, the IPSPs evoked with glutamate in the present study may have originated principally from stimulation of stellate interneurons of str. L-M. Stellate interneurons appear to

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