J. Physiol. (1984), 351, pp. 327-342 327With 7 text-figuresPrinted in Great Britain

THE REVERSAL POTENTIAL OF EXCITATORY AMINO ACID ACTIONON GRANULE CELLS OF THE RAT DENTATE GYRUS

BY VINCENZO CRUNELLI, SUSAN FORDA AND JOHN S. KELLYFrom the Department of Pharmacology, St George's Hospital Medical School,

Cranmer Terrace, London SW17 ORE

(Received 22 August 1983)

SUMMARY

1. The responses of granule cells to glutamate, aspartate, N-methyl-D-aspartate(NMDA), quisqualate and kainate applied by ionophoresis on to their dendrites inthe middle molecular layer of the dentate gyrus were studied with intracellularelectrodes using an in vitro hippocampal slice preparation. On passive depolarization75% of the granule cells displayed anomalous rectification, which persisted in thepresence of TTX and TEA but was eliminated by Co2+ or the intracellular injectionof Cs+.

2. Short ionophoretic applications of all the excitatory amino acids evokeddose-dependent depolarizations that were highly localized: movement of the iono-phoretic electrode by as little as 10 j#m could substantially change the size of theresponse. The depolarizations evoked by glutamate, aspartate, quisqualate andkainate were unaffected by TTX and Co2+. The depolarization evoked by NMDA wasunaffected by TTX but markedly reduced by Co2+.

3. Following intracellular injection of Cs+, neurones could be depolarized to+ 30 mV and the depolarizations produced by glutamate, quisqualate, NMDA andkainate reversed. The reversal potentials (E) were Eglutamate - 56+ 0 4 mV;ENMDA: 1P8+ 1 9 mV; Equisqualate: -39+ 1.9 mV; Ekainate: -4-6+ 2-0 mV. Theexcitatory post-synaptic potential (e.p.s.p.) evoked by stimulation of the medialperforant path could also be reversed and Ee.p s p was -5-5+ 1 1 mV.

4. The 6 mV difference between ENMDA and the equilibrium potential for the otherexogenously applied excitatory amino acids and the statistically significant differencebetween ENMDA and Ee.p.s.p. (P < 0O005; d.f.: 7) is consistent with our earlierhypothesis that both the transmitter released by the medial perforant path andexogenously applied glutamate are unlikely to interact with NMDA receptors.

INTRODUCTION

The granule cells of the dentate gyrus receive their major excitatory input fromperforant path fibres which originate in the entorhinal cortex (Lorente de No, 1934;L0mo, 1971; Steward, 1976). In a recent paper (Crunelli, Forda & Kelly, 1983a), wepresented evidence from the use of the specific antagonist y-D-glutamylglycine thatexcitatory amino acid receptors of the quisqualate/kainate type mediate the

V. CRUNELLI, S. FORDA AND J. S. KELLY

excitation evoked by stimulation of the medial perforant path, and argued that thesefindings offer strong support for the view that glutamate may be the transmitter(White, Nadler, Hamberger, Cotman & Cummins, 1977; Storm-Mathisen & Iversen,1979; Wheal & Miller, 1980). In a further attempt to identify the endogenoustransmitter and the receptors which mediate excitation at this synapse we havestudied the action of glutamate and its more potent agonists N-methyl-D-aspartate(NMDA), quisqualate and kainate, on the granule cell dendrites by comparing theirreversal level (E) with that of the endogenous transmitter.Although there have been a number of attempts to determine the E for the

depolarization of glutamate and other excitatory amino acids in mammalianneurones (MacDonald & Poreitis, 1982), no attempt has been made to compare allthese values with that of the excitatory post-synaptic potential (e.p.s.p.) on the samecell. This is not surprising, since the link between depolarization and the change inmembrane input resistance (Rm) is poorly understood and there are conflictingreports as to the nature of the Rm changes induced by glutamate and its agonists(Ziegelgansberger & Puil, 1973; Engberg, Flatman & Lambert, 1979; Puil, 1981;Dodd, Dingledine & Kelly, 1981; Assaf, Crunelli & Kelly, 1981; Hablitz & Langmoen,1982).In this study, in an attempt to overcome some of the problems which arise from

the disparate location ofthe conductances evoked by the endogenous and exogenouslyapplied transmitters (Johnston & Brown, 1983) we have applied all four amino acidsby micro-ionophoresis from the same multibarrelled pipette on to a patch of highglutamate sensitivity close to a region of the dendrites shown in previous studies invivo to be associated with the peak of the synaptic current (Jefferys, 1979).Furthermore, in the presence of intracellular Cs+ (Johnston, Hablitz & Wilson, 1980),we have been able to inject sufficient intracellular depolarizing current to reverse thevoltage response to the application of glutamate, NMDA, quisqualate and kainateand the e.p.s.p. evoked by stimulation of the medial perforant path and to comparetheir null potentials in the same cell.

METHODS

Hippocampal slices (400 ,um thick) were prepared from decapitated rats (Charles River, 200 g)using a McIlwain tissue chopper. The slices were maintained and subsequently used for electro-physiological recording as previously described (Assaf et al. 1981). Briefly, they were initially keptin an incubation chamber and 1 h before the beginning of each experiment, slices were transferredto the recording chamber and perfused with a warmed (36-0+05 00) continuously oxygenated (95%02, 5% C02) medium consisting of (mM): NaCl, 134; KC1, 5; KH2PO4, 1P25; Mg2SO4, 2; CaCl2, 2;NaHCO3, 16; and glucose, 10.

Intracellular electrodes were made from omega glass tubing ('Kwick-fil', Clark ElectromedicalInstruments) and were filled with 1 M-K acetate (80-120 MCI) or 1 M-CsCl (30-60 MQ). Intracellularelectrodes filled with 2 M-CsCl or Cs2SO4 led to immediate depolarization of the cells to -5 mVand a complete loss in Rm, indicating that the cells were injured. They could not therefore be usedto impale granule cells. Cs+ were injected into the cell by the passage of 800 ms depolarizing currentpulses at 1 Hz. A precision electrometer (M-707, WP Instruments) was used to measure potentialsand to inject current through the single recording micro-electrode. The resting membrane potentialof the cell was measured from the change in potential seen on withdrawal of the electrode fromthe cell. Plots of the voltage-current relationships were constructed from measurements of thesteady-state voltage responses to families of hyperpolarizing and depolarizing pulses of current

328

DEPOLARIZATION OF GRANULE CELLS BY AMINO ACIDS

(100-120 ms in duration) injected through the recording micro-electrode. Throughout experimentsconcerning reversal potentials the bridge balance was more carefully monitored by one experimenteron an oscilloscope. The time constant was calculated from voltage transients produced bysmall amplitude hyperpolarizing pulses of current which lay within the linear portion of thevoltage-current plot. The initial 60 ms of each voltage transient were digitized at 2-5 kHz usinga PDP 11/23 (Crunelli, Forda, Brooks, Wilson, Wise & Kelly, 1983c), normalized with respect tothe plateau voltage and the logarithm plotted against time. The time constant was then obtainedby linear regression. Glass micropipettes (3-4 jam tip) filled with 1 M-NaCl were used as stimulatingelectrodes (15-50 #ss, 1-0 V, 0-5-1 Hz) and positioned along the afferent fibres of the medialperforant path in the middle third of the molecular layer (McNaughton, 1980), 1-2 mm away fromthe recorded cell. TTX (10-5 M) in normal medium and C02+ (5-15 mM) in low Ca2+ medium(134 mM-NaCl, 5 mM-KCl, 2 mM-MgCl2, 0-2 mM-CaCl2, 16 mM-NaHCO3 and 10 mM-glucose) wereapplied to the surface of the slice near the impaled cell by droplet application using a Hamiltonmicrosyringe.Drugs were ejected ionophoretically from an independently mounted six-barrelled micropipette

(6-9,um tip) positioned along the dendritic tree of the impaled neurone in the molecular layer ofthe dentate gyrus at the same level as the stimulating electrode. After impalement the tip of theionophoretic electrode was slowly advanced into the slice in 5-10um steps until a fast risingdepolarization could be observed in response to a short (100-700 ms) application ofglutamate. lono-phoretic barrels contained a selection of the following drugs: L-glutamate (1 M; pH 8), L-aspartate(1 M; pH 8), NMDA (20 mm in 150 mM-NaCl; pH 8), quisqualate (20 mm in 150 mM-NaCl; pH 8),kainate (20 mm in 150 mM-NaCl; pH 8), and NaCl (1 M; pH 8). Retaining currents of 1-5 nA wereapplied to the individual barrels when necessary. At the end of each impalement, the effect ofionophoretic application of the drugs was re-tested to evaluate any electrical coupling between theionophoretic pipette and the recording electrode. Results were stored on a Racal FM 4D taperecorder and later analysed with a PDP 11/23 computer (Cambridge Electronic Design Ltd.)(Crunelli, Forda, Kelly & Wise, 1983b).

RESULTS

Experiments comparing the actions of glutamate with those of the agonists were

performed only on those granule cells (Crunelli et al. 1983a) (n = 78) whose restingmembrane potential (mean + S.E. of mean: -62 + 0-2 mV; range 46-80) remainedstable throughout the course of the experiment. The time constant of these cells was10-4 + 0-4 ms (mean +S.E. of mean; range 8-414-0) and the input resistance45+0 3 MQ (mean ±s.E. of mean; range 20-78).

Membrane properties of granule cellsIn thirteen out of eighteen granule cells tested, a steady passive depolarization of

the cell produced by the injection of current caused a 25% increase in the amplitudeof the voltage deflexion evoked by constant pulses of hyperpolarizing current. On anumber of cells voltage-current plots showed this apparent increase in Rm to persistin the presence of TTX (10-5 M, droplet application) (Fig. 1 A). Indeed, the regionof apparent increased Rm became more obvious, since the cells no longer fired anda greater range of depolarizing current could be used to explore the region of interest(range -70 to -40 mV) which in control medium began just subthreshold to the levelrequired for initiating action potentials. This concentration ofTTX had no effect onthe resting membrane potential, time constant or Rm (Table 1). Even large pulses(1 nA) of depolarizing current evoked only a passive depolarization, and there wasno sign of regenerative activity.

In the presence and absence ofTTX, the addition of Co2+ (5-15 mM) to the surface

329

TTX

-20

_ 1 0*

:@

-120 mV

Control

-30

-1 .*

-140 mV

Control

-20

-1

1 nA

-120 mV

Co2+

1 nA

-130 mV

TTX +Co2*

1 nA

-120 mV

Fig. 1. Voltage-current plots from three different granule cells in normal medium and inthe presence of TTX and Co2+. In the control and Co2+-containing medium depolarizingpulses of greater amplitude than shown on the plots evoked action potentials and couldno longer be used to determine membrane input resistance. In A, the apparent increasein input resistance at membrane potentials between -60 and -40 mV, the restingmembrane potential (-60 mV) and input resistance (42 MCI) were unaffected by TTX.In B, in control solutions, a region of apparent increase in input resistance can be clearlyobserved between -70 and -50 mV. In the presence of Co2+ (10 mM), the cell depolarizedby 10 mV, the resting membrane input resistance increased from 52 to 80 MCI and theanomalous rectification disappeared. In C, the anomalous rectification present between-60 and -45 mV was abolished following the application of TTX and Co2+. However,in this particular cell C02+ did not cause any significant depolarization. The restingmembrane input resistance increased from 49 to 82 MCI. The membrane potential of-63 mV was unchanged.

330 V. CRUNELLI, S. FORDA AND J. S. KELLY

Control

1 nA

A

-201

-1 .:

-40

-11

-1 o...-0{0.

C

-20

-1*Ie

1 nA

1 nA

-120 mV

DEPOLARIZATION OF GRANULE CELLS BY AMINO ACIDS 331

of the slice usually caused a depolarization of up to 10 mV, abolition of anomalousrectification, a 50% increase in Rm and an increase in the time constant (Figs. 1 B,2A and Table 1). Thus these results raise the possibility that, in granule cells, aCa2+-dependent K+ current is activated at potentials close to the resting potential,as previously suggested by Bernardo & Prince (1981) in CA1 cells. Fig. 2A also showsthat Co2+ abolished the sag often seen to follow the initial phase of the electrotonicpotential evoked by large pulses of hyperpolarizing current in normal media.Although the addition of TEA to the slice (10 mm, drop application) increased the

TABLE 1. Membrane potential, input resistance and time constant of granule cells in the presenceof TTX and Co2+

Membrane Input resistance (MCI) Time constant (ms)Cell potentialno. (mV) Control TTX c02+ Control TTX Co2+

1 64 54 56 10 1 9.42 59 46 45 12-8 12-23 60 53 59 11-3 9 44 66 26 22 10-2 9-85 61 67 66 10-4 9 56 70 52 55 8-4 8-17 80 55 90 10 0 - 21-08 60 42 60 9-79 60 43 75 9 0 211010 80 42 64 10-3 - 15-511 63 42 68 8-7 - 18-312 60 36 49 14-0 15-0

width of the action potentials, anomalous rectification in the depolarizing directionremained unchanged.The apparent increase in Rm was also reduced or abolished by the injection of Cs+

into the cell. Typically Cs+ increased the Rm and decreased the resting membranepotential by approximately 30 mV, and action potentials evoked by pulses ofdepolarizing current were of prolonged duration (80 ms) and reduced amplitude(50 mV). These effects were partially reversed when the cell was repolarized by theinjection of current. At -50 mV, in fact, the action potential was 40 ms wide and70 mV in amplitude. Cells successfully injected with Cs+ could be depolarized up to+ 30 mV and in eight out of seventeen cells all the outward rectification seen withK acetate electrodes at membrane potentials positive to -20 mV was blocked (Fig.2Bi). In the remaining nine cells some outward rectification still occurred, causinga 20-40% decrease in Rm (Fig. 2B2) as opposed to 60% observed in controlexperiments.

Response pattern of the excitatory amino acidsThe responses to short ionophoretic applications of glutamate and aspartate were

very similar and consisted of a rapid, dose-dependent depolarization which could beaccompanied either by no change in Rm, a decrease in Rm, or an increase followedby a decrease. These changes in Rm appeared to be dependent on the rate and

332 V. CRUNELLI, S. FORDA AND J. S. KELL Y

magnitude of depolarization, which in turn was critically dependent on the positionof the ionophoretic electrode in the slice (Fig. 3). As the depolarization evoked bythe same ionophoretic pulse of current increased in amplitude, the Rm changed froman increase or no change, to an increase followed by a decrease. As the pipetteapproached a point of maximum sensitivity only a decrease in Rm was seen. At thispoint the latency to the onset of depolarization could be as short as 10 ms and themaximum rate of depolarization approached 0 05 mV/ms. However, on occasion themaximum rate of rise could reach 0 07 mV/ms (Fig. 3).

Al A2

I_

701 70E * ***O,00

50- 501E .0.

Cr. ~~~ ~ ~ ~ ~ ~ ~ ~ ~~S*30 30 040 -20 0 20 -40 -20 0 20

Membrane potential (mV)Fig. 2. Effect ofC02+ and Cs+ on membrane properties ofgranule cells. A shows electrotonicpotentials (upper traces) produced by the injection ofhyperpolarizing current pulses (lowertraces) in control medium (Al) and after the application of a droplet of low Ca2+ mediumcontaining 5 mM-Co2+ (see Methods) to the surface of the slice (A2). Note how C02+depolarizes the cell by 10 mV and increases the time constant and the Rm. Co2+ alsoabolished the sag clearly seen in the largest electrotonic potential in the control medium.Calibrations bars equal: 20 mV, 2 nA and 20 ms. B1 and B2 show the change in resistanceover a range of membrane potentials in two different cells injected with Cs+. In Bi Cs+was able to completely abolish outward rectification while B2 is an example of a granulecell in which injection of Cs+ was only able to reduce the outward rectification, leavinga 30% decrease in Rm between membrane potentials of -20 to + 20 mV.

Responses to NMDA were considerably different from those described above. Lowdoses produced a bursting pattern of firing (never seen with glutamate or the otheragonists) and high doses resulted in a huge depolarization of up to 45 mV from whichthe cell could take several minutes to recover. Although NMDA produced the samepattern of changes in Rm as those described above for glutamate, the apparentincrease in Rm associated with NMDA applications was often more pronounced. Themaximum rate of depolarization, however, was somewhat slower (0-01 mV/ms)although the latency to onset was of the same magnitude (10 ms).The depolarizing response to quisqualate was similar to glutamate, although in any

given cell the position of the ionophoretic electrode at which the maximum rate ofdepolarization was obtained with quisqualate was several micrometres away from the

DEPOLARIZATION OF GRANULE CELLS BY AMINO ACIDS

position which produced the maximum rate of depolarization for glutamate. Theresponse to quisqualate was usually accompanied by a decrease in Rm, but rarely anincrease was observed. The latency of the onset of the depolarization was about 15 msand the maximum rate of depolarization 0 05 mV/ms, thus similar to the valuesobtained for glutamate.

Kainate produced a marked depolarization (up to 40 mV) followed by a recoverywhich, in any given cell, was always slower than that for the other agonists (Robinson& Deadwyler, 1981). These features made it particularly difficult to select the

A B

Glutamate

AA a A A A la A A A

Quisqualate

A A A A

NMDA

Fig. 3. Differential sensitivity of the same granule cell to glutamate and NMDA. In A thevoltage records show the response to all three amino acids when the position of the pipettewas at an optimum for glutamate sensitivity and in B when the pipette was at an optimumfor NMDA sensitivity. In A the maximum mean rate of depolarization for glutamate was0107 mV/ms compared with 0-01 mV/ms and 0-006 mV/ms for quisqualate and NMDArespectively. In B the maximum rates of depolarization were: glutamate 0104 mV/mis,quisqualate 0-05 mV/ms and NMDA 0-01 mV/ms. Triangles indicate 50 ms and 70 msionophoretic applications of glutamate, quisqualate and NMDA respectively. The iono-phoretic currents were: glutamate 103 nA, quisqualate 130 nA, NMDA 100 nA. Calibrationbars equal 10 mV and 4 s.

appropriate ionophoretic parameters that would produce a substantial depolarizationfollowed by reasonably fast recovery. Kainate usually evoked a decrease in Rm.Rarely, however, the depolarization was accompanied by no change in Rm.

In four cells manual voltage clamp was used to show that the depolarizationproduced by glutamate, NMDA and quisqualate was accompanied by an apparentincrease in Rm in the absence of a change in voltage (Fig. 4).

Effect of TTX and Co2+ ions on the depolarization evoked by amino acid8

When all the regenerative activity was suppressed by the droplet addition ofTTXon fifteen cells in fifteen different slices, glutamate (n = 13), NMDA (n = 15),quisqualate (n = 7) and kainate (n = 4) continued to be equally effective in depolar-izing granule cells and are thus unlikely to exert their effects via excitatoryinterneurones. Indeed on occasion, TTX appeared to enhance both the depolarizations

333

V. CRUNELLI, S. FORDA AND J. S. KELLY

A B

NMDA

Glutamate

Quisqualate

Fig. 4. The effect of glutamate, NMDA and quisqualate on membrane input resistance.In A the upper traces of each pair are voltage recordings showing the depolarization andchanges in input resistance evoked by the ionophoretic application of NMDA (700 ms),glutamate (2 s) and quisqualate (2 s). The lower traces show the injection ofconstant pulsesof hyperpolarizing current used to test the input resistance of the cell. B shows how thevoltage response was negated by the passage of d.c. current through the intracellularelectrode. During the application of glutamate and NMDA the 2nd and 3rd pulses of thevoltage trace are clearly increased in amplitude even though the voltage is held constant.However, during quisqualate application the increase in size of the voltage pulse was verymuch smaller. Black bars above the voltage records indicate the onset and offset ofthe ionophoretic applications of excitatory amino acids, and the amplitude of actionpotentials evoked by drug-induced depolarizations has been attenuated by the computerplotting sub-routine. Drug ejection currents were: NMDA, 252 nA; glutamate, 496 nA;and quisqualate, 359 nA. Calibration bars equal 20 mV, 0.5 nA, 12 s (A) and 4 s (B).

and the increases in Rm. Presumably, in the absence of cell firing, indirect activationof restorative K+ currents was reduced. In the presence of TTX, the pattern ofchanges in Rm produced by the excitatory amino acids remained unaltered.In the presence ofCo2+ the depolarizing responses to glutamate (n = 11), aspartate

(n = 3) and quisqualate (n = 3) persisted but the apparent increases in Rm seen inthe absence ofCo2+ were abolished. In seven out ofeight cells (in eight different slices)a droplet of Co2+ rapidly (1 min) abolished the responses to NMDA. However, in thecontinuing presence of Co2+, the responses to NMDA slowly (3 min) recovered,although the apparent increases in Rm associated with control responses to NMDAwere abolished. Thus our finding, that the responses to NMDA but not those to

334

DEPOLARIZATION OF GRANULE CELLS BY AMINO ACIDS

glutamate, aspartate, quisqualate and kainate are blocked by Co2+, is in keeping withthe hypothesis that Co2+ may be a competitive antagonist of NMDA (Ault, Evans,Francis, Oakes & Watkins, 1980). Recently, in hippocampal pyramidal neurones, thedepolarization produced by NMDA has been shown to be completely blocked by Co2+(Dingledine, 1983).These experiments also showed the after-hyperpolarization (a.h.p.) in granule cells

following glutamate-, aspartate- and occasionally NMDA- or quisqualate-induceddepolarization to be abolished both by the external application of Co2+ and theinternal injection of Cs+. The magnitude of the a.h.p. was dependent on the extentto which the cell was depolarized and was unaffected by the application of TTX orby the use of 1 M-KCl electrodes. The a.h.p. produced by glutamate reversed at- 75.1 + 1-04 mV (mean+S.E. of mean) and on the same five cells this was identicalto the reversal potential of the a.h.p. which followed a similar depolarizationproduced by the injection of a pulse (100 ms) of depolarizing current into the cell,- 75-2 + 0-5 mV (mean+ S.E. of mean). Our observations in granule cells on the a.h.p.are therefore similar to those reported for CA1 cells (Nicoll & Alger, 1981) and supporttheir conclusion that the a.h.p. depends on a Ca2+ influx. The reversal potential forthe a.h.p. determined in this study also supports the suggestion that the a.h.p. is dueto a Ca2+-activated increase in K+ conductance (Nicoll & Alger, 1981; Hotson &Prince, 1980; Brown & Griffiths, 1983).

The reversal potential of the excitatory amino-acid-evoked depolarizationThe reversal potential for glutamate, NMDA, quisqualate and kainate was

determined using CsCl electrodes and recording the responses to the application ofexcitatory amino acids over a range of membrane potentials from -60 mV to+ 30 mV. During depolarization of the cell by current the responses to the excitatoryamino acids decreased in amplitude and at a membrane potential of about -5 mV,inverted and changed in sign. Reversals were always symmetrical and no biphasicreversals were observed (Fig. 5). The E calculated by linear regression analysis (Fig.7) were: Egiutamate: -56+0-4 mV (mean+s.E. of mean) (n = 11); ENMDA:1 8 + 1 9 mV (n = 10); Equisquaate:-3-9± 1-9 mV (n = 4) and Ekainate:-46 + 2-0 mV(n = 4). Statistical analysis showed no significant differences between the E ofglutamate, quisqualate and kainate. The reversal level of NMDA, however, wassignificantly different from that of glutamate (paired t test; t: - 3-15, d.f.: 10,P < 0 025).

The reversal potential of the e.p.s.p.Under the same experimental conditions using CsCl electrodes the e.p.s.p. evoked

by stimulation of the medial perforant path reversed at- 59+±11 mV (mean +S.E.of mean) (n = 14) (Figs. 6 and 7). In the only cell successfully impaled with a1 M-Cs2So4-filled electrode Egiutamate was -5*1 mV and Ee.p.s.p. was -6-8 mV.Thalmann & Ayala (1982) have reported that the e.p.s.p. evoked by the perforantpath may be followed by a y-aminobutyric acid mediated inhibitory post-synapticpotential (i.p.s.p.) and a late picrotoxin-insensitive hyperpolarization. In our handsthe reversal of the e.p.s.p. was symmetrical in all the cells. However, similar resultsto those ofThalmann & Ayala (1982) were obtained by replacing the glass stimulating

335

V. CRUNELLI, S. FORDA AND J. S. KELLY

Glutamate Quisqualate

M 8 wM 10

M 14 2 0

-7 -7

-18 Ti -24

NMDA Kainate

2 2

rll-4 0

-11 -7

-16 -19

Fig. 5. Reversal of the response to glutamate, quisqualate, NMDA and kainate in aCs+-loaded granule cell. Typical records of each amino-acid-evoked response are shownat four different membrane potentials. The reversal levels calculated by regression analysiswere Eiutamate: 5-4 mV; Equisquaiate: - 73 mV; ENMDA: -4-4 mV and Ekainate:- 8-6 mV (Table 2, cell 4). Downward deflexions of the trace are the voltage response ofthe cell to pulses ofhyperpolarizing current that were kept constant during each individualdrug application. The mean input resistance of the cell was 62 MC. lonophoreticapplications were: glutamate: 405 nA, 4 s; quisqualate: 130 nA, 3 s; NMDA: 393 nA, 1 8;kainate 98 nA, 600 ms. Calibration bars equal 10 mV and 10 s.

electrode by a tungsten electrode. Using these electrodes to produce a wider focus

of stimulation, the e.p.s.p. reversed at - 19-4+ 1-8 mV (mean +s.E. of mean) (n = 8)in control solutions and - 6-3 mV in the presence of bicuculline (10-5 M, dropapplication). Brown & Johnston (1983) have recently obtained a similar reversal levelfor the current responsible for the mossy fibres excitation of CA3 pyramidal cells,using CsCl electrodes and bicuculline to block contamination of an i.p.s.p. This isanother synapse where a substance structurally related to the excitatory amino acidshas been suggested as the endogenous transmitter (Storm-Mathisen & Iversen, 1979).Table 2 shows results from all the cells in which at least two or more reversal

336

DEPOLARIZATION OF GRANULE CELLS BY AMINO ACIDS

23

4

-8 9eL

-25

337

-35 --II

Fig. 6. Reversal of the e.p.s.p. evoked by stimulation of the medial perforant path in agranule cell loaded with Cs+. Typical voltage records show the e.p.s.p. at five differentmembrane potentials. In this cell, which is the same cell as shown in Fig. 5 (Table 2, cell4), the reversal level calculated by regression analysis was -9-8 mV. The arrow marksstimulation of the medial perforant path and calibration bars equal 10 mV and 20 ins.

TABLE 2. The reversal potential of excitatory amino acids and of the e.p.s.p. evoked by stimulationof the medial perforant path

E.p.s.p. Glutamate

-6-0 -8-2-5-5 -6-7-4-2 -2-0-9-8 -5-4

-2-0-6-5

-7-5-7.0-5-5-4-3-8-0-6-7

NMDA

-0.9

-4.42-2

-4-3-5-0

3.711-08-45.9

Quisqualate

-7-00.1

-73

-1-4

Kainate

-7-5-5.0

-8-61-0

*b Mean

+ S.E.

of mean

-6-9+0-6 1-8+2-2

a Only neurones in which two or more reversal potentials were measured on the same cell arereported in this Table. Each value represents the reversal potential (in mV) in Cs'-injected granulecells calculated by linear regression analysis from plots similar to that shown in Fig. 9.

b Refers only to those eight cells in which Eepp p and ENMDA were measured in the same cell.* t -4-38; d.f. 7; P < 01005 (paired t test).

Cellno.

123456789101112

I

V. CRUNELLI, S. FORDA AND J. S. KELLY

201 mV

10.

-10'

20

10

-10

K\VMr

15 -60(mV)

mV

-.-~~~~~~~~~~~~~~~~

15

Fig. 7. The reversal potential of the e.p.s.p. and depolarizations evoked by glutamate,NMDA and kainate in a Cs+-loaded neurone. The graphs show the relationship betweenthe synaptic event or amino-acid-evoked response (ordinate) and the membrane potential(abscissa). The regression line intercepts the ordinate at - 6-0 mV for the e.p.s.p. (r = 0 99),-8'2 mV for glutamate (r = 0O98), -0-9 mV for NMDA (r = 0-98) and -7-5 mV forkainate (r = 0-97) (Table 2, cell 1).

potentials could be measured. Statistical analysis (paired t test) showed Ee.p s.p. andENMDA to be significantly different (P < 0'005) on eight cells in which both valueswere determined. There was no significant differences between Ee.p.s.p. and the E ofthe other excitants.

DISCUSSION

The passive membrane properties of granule cellsIn this study the resting membrane potential, Rm and time constant of the granule

cells are similar to those reported by other workers from in vitro preparations ofgranule cells (Barnes & McNaughton, 1980; Brown, Fricke & Perkel, 1981). However,voltage-current curves determined using families of current pulses, and passivedepolarization of the membrane potential to levels immediately subthreshold for theinitiation of action potentials, revealed anomalous rectification not seen by earlierworkers (Barnes & McNaughton, 1980). The anomalous rectification was insensitiveto TTX and blocked by Co2+, thus similar to that seen in motoneurones by Barrett,Barrett & Crill (1980) and in CA1 neurones by Halliwell & Adams (1982), but different

E.p.s.p. Glutamate

VM _

-60(mV)

201mV

-30

10.

-30

NMDA

v 't .-15

-10*

Kainate201mV

Vm-60(mV)

-30

10l

VMram p-

-60(mV)

-30 15

-10l

~~~~~~ -A m. --me l

338

0

0

DEPOLARIZATION OF GRANULE CELLS BY AMINO ACIDS

from that observed by Hotson, Prince & Schwartzkroin (1979) in CA1 cells, in whichthe anomalous rectification appeared to be sensitive to both TTX and Ca2+ channelblockers.

Effect of excitatory amino acidsThe depolarization of the granule cells produced by glutamate, aspartate, NMDA,

quisqualate and kainate is consistent with the well-documented excitatory action ofthese amino acids on vertebrate neurones (Engberg, Flatman & Lambert, 1978;Engberg et al. 1979; Constanti, Connor, Galvan & Nistri, 1980; Puil, 1981; Robinson& Deadwyler, 1981). In the present work the latency to onset and the comparativerates of depolarization of glutamate, quisqualate and NMDA suggest that in themiddle third of the molecular layer there is either a greater density of glutamate andquisqualate receptors or receptors which possess a higher affinity for glutamate andquisqualate than for NMDA. It is possible that the greater sensitivity of thepost-synaptic membrane to glutamate may be related to the innervation of this regionof the dendrites by the terminal branches of the medial perforant pathway (Steward,1976).Glutamate, aspartate, NMDA and quisqualate all produced, although to differing

degrees, an apparent increase in Rm that was unaffected by TTX but blocked by Co2+.Although a rise in Rm associated with drug-induced depolarizations may be explainedas a receptor-activated reduction in membrane conductance (Krnjevic, Pumain &Renaud, 1971), it is impossible to rule out that the same voltage-dependentmechanisms seen during anomalous rectification are responsible for the apparentincreases in Rm seen during the amino-acid-evoked depolarization and that no specificreceptor-activated phenomenon is involved. However, based on voltage-clampexperiments on cultured spinal neurones, MacDonald, Poreitis & Wojtowicz (1982)have suggested that the action of aspartate causes an increase in a voltage-dependentNa+ current. Unfortunately our experiments do not allow us to distinguish betweenthese two possibilities. It is however, worth noting that on the same cell the apparentincrease in Rm evoked by NMDA is greater than that evoked by glutamate. Thissuggests that either NMDA is acting at a different dendritic site or there is a greaterdegree of receptor-specific activation of a voltage-dependent conductance.

The reversal level of the excitatory amino acid and the e.p.s.p.In vertebrates the direct comparison ofthe reversal potentials for glutamate-evoked

responses and the e.p.s.p. has been reported previously (Engberg et al. 1979; Mathews& Wickelgren, 1979). In this study we have been able to eliminate the contributionof the voltage-sensitive rectifying properties of the cells by the intracellular injectionof Cs+ (Hablitz & Langmoen, 1982). Cs+ have been shown to block K+ channels(Bezanilla & Armstrong, 1972) and Cs+ has been used to eliminate the outward K+current in molluscan neurones (Tillotson, 1979) and the K+ component of the inwardcurrent in CA3 hippocampal neurones (Johnston et al. 1980).Using Cs+ the e.p.s.p. appears to be a relatively pure event. The depolarizing and

hyperpolarizing envelopes of the e.p.s.p. were mirror images of each other, andcontamination of the e.p.s.p. during the construction of voltage-current curves isunlikely. In addition, in our earlier paper (Crunelli et al. 1983a) the decay time of

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the e.p.s.p. evoked under the same experimental conditions was shown not to changeduring specific post-synaptic antagonism of the e.p.s.p. by y-D-glutamylglycine. Theaverage values reported for Ee.p s.p. in this paper are 6-16 mV more positive thanthe value we estimated in our earlier paper by extrapolation from voltage-currentrelationships (Crunelli et al. 1983 a) and presumably results from the use ofintracellularCs+ and the subsequent blockade ofa K+-mediated conductance initiated by the entryof Ca2+ during both the e.p.s.p. and passive depolarization.The reversals ofthe amino-acid-evoked potentials were symmetrical and unlike the

biphasic responses to glutamate which have been reported in cultured spinal neurones(Wojtowicz, Gysen & MacDonald, 1981). It should be emphasized that the Edetermined in this paper are not the true equilibrium potentials for the e.p.s.p.,glutamate, NMDA, quisqualate or kainate and could be distorted by the disparatelocation of the depolarization evoked by the amino acids and the e.p.s.p.s on thedendritic tree of the granule cells and the changes in membrane potential evoked bycurrents applied through an electrode impaled in the soma. However, by comparingthe reversal levels and the maximum rates of depolarization on the same cells someof these differences have been negated. Although we are not yet in the position tovoltage clamp these cells or to make a detailed analysis of the way in which thedendritic events are attenuated by recording from the soma, the fact that we canrecord the quantal nature ofthe fluctuations at the peak ofthe e.p.s.p. and differentialrates of rise ofamino-acid-induced depolarizations are two features ofour work whichwould lead Johnston & Brown (1983) to suggest that the errors associated withelectronic disparity are small compared with errors due to other factors. The otherfactors they mention such as changes in ionic currents during large depolarizations,base-line noise, field potentials superimposed on the intracellular response and thepresence of unblocked voltage-dependent conductances are also minimized by ourexperimental paradigm.

Since ENMDA is statistically different from Ee.p.s.p. and Eglutamate and 6 mV morepositive than Ekainate and Equisqualate our results support the hypothesis (Crunelliet al. 1983a) that the excitatory transmitter of the medial perforant path interactswith receptors shown to interact with exogenously administered glutamate, quisqua-late and kainate but not NMDA. Furthermore, the differential sensitivity of theseamino acids to the presence ofCo2+ and the block ofglutamate-induced depolarizationsby y-D-glutamylglycine but not 2-amino-5-phosphonovaleric acid (Crunelli, Forda,Collingridge & Kelly, 1982) provide additional evidence for the view that glutamatereceptors are quite distinct from those operated by NMDA in the middle molecularlayer of the dentate gyrus, and thus different from those in the spinal cord whereglutamate acts on all three receptor types (Watkins & Evans, 1981).

The authors are grateful to John Wise for developing the computer programs essential for thecompletion of this study, Mrs P. Harper and Mrs A. Keable for typing the manuscript, S. Prestwichand M. J. S. Kelly for preparing the illustrations, the Wellcome Trust and the MRC (GrantG8219655N) for financial support. S. Forda is in receipt of an SERC/ICI Case Award.

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