Modulation of decay kinetics and frequency of GABAA receptor-mediated spontaneous inhibitory postsynaptic currents in hippocampal neurons

Neuroscience Vol. 49, No. 1, pp. 13-32, 1992

0306-4522/92$5.00+ 0.00 PergamonPress Ltd © 1992IBRO

Printed in Great Britain

MODULATION OF DECAY KINETICS A N D FREQUENCY OF GABA A RECEPTOR-MEDIATED SPONTANEOUS INHIBITORY POSTSYNAPTIC CURRENTS IN HIPPOCAMPAL NEURONS T. S. OTIS and I. MODY* Department of Neurology and Neurological Sciences, M016, Stanford University School of Medicine, Stanford, CA 94305, U.S.A. Abstract--Inhibitorypostsynaptic currents mediated by spontaneous activation of GABA~receptors were studied using whole-cell voltage-clamp recordings in granule cells of the adult rat (postnatal day 60+) dentate gyrus in 400-#m-thick coronal half-brain slices maintained at 34-35°C. The average amplitude of spontaneous inhibitory postsynaptic currents remained constant during a given recording period (i.e. no rundown was noted). The spontaneous cmTentshad an average conductance between 200-400 pS, were mediated by CI- flux through GABA, receptor/channels since they reversed at the CI- equilibrium potential and were blocked by bicuculline or picrotoxin. Their mono-exponential decay time-constants (range: 4.2-7.2 ms) were prolonged by midazolam and pentobarbital in a dose-dependent manner. The effect of midazolam was reversed by the benzodiazepine receptor antagonist flumazenil (RO 15-1788) which, by itself, had no effect on the decay time-constant. The decay time-constant was also dependent on membrane voltage and on temperature. A 132-mV change in membrane potential produced an e-fold prolongation of the decay while the Ql0 (between 22-37°C) of the decay rate was 2.1. Within a given neuron, the frequency of spontaneous GABAergic events was remarkably constant over long time-periods, though the mean frequency among different cells showed large variability. Spontaneous miniature inhibitory postsynaptic currents also persisted under experimental conditions such as the presence of extracellular tetrodotoxin (1 #M), Cd2+ (200/zM) or lowered extracellular Ca2+/elevated Mg:+, which effectively abolished all stimulus-evoked GABAergic neurotransmission. The frequency of tetrodotoxinresistant miniature events was increased by elevating extracellular K + concentration and was diminished by the GABABreceptor agonist (-)baclofen only at a dose (50 #M) which was an order of magnitude larger than that required to depress stimulus-evoked responses. These findings are consistent with different mechanisms being responsible for the spontaneous and stimulus-evoked release of GABA from interneuron terminals and also identify pre- and postsynaptic modulatory factors of the endogenous, action-potential-independent, GABAergic neurotransmission as being important determinants of the excitability level of mammalian CNS neurons.

In various preparations, the study of the amplitude of unitary evoked and spontaneous synaptic events has yielded valuable information about the quantal nature of transmission at a particular synapse.25,39,52,65 In the mammalian CNS, spontaneously released GABA from terminals of inhibitory interneurons produces a considerable tonic inhibitory activity~'~7'28'4°'53which persists in the absence of action potential firing. 17'23'24'56'62'7°'72's2We will refer to spontaneous inhibitory postsynaptic currents recorded in *To whom correspondence should be addressed, Abbreviations: ACSF, artificial cerebrospinal fluid; BAPTA, 1,2-bis(O-aminophenoxylethane-N,N,N',N'-tetraacefic

acid; CNQX 6-cyano-7-uitroquinoxaline-2,3-dione; DAP5, D-2-amino-5-phosphonovalericacid; E., activation energy; EGTA, ethyleneglycolbis(aminoethylether)tetra-acetate; HEPES, N-2-hydroxyethylpiperazine-N'2-ethanesulfonic acid; IEI, inter-event interval; eIPSCs, evoked inhibitory postsynaptic currents; slPSCs, spontaneous inhibitory postsynaptic currents; mlPSCs, miniature inhibitory postsynaptic currents; mepps, miniature endplate potentials; P, postnatal day; %, decay-time constant; T~, rise-time constant; TTX, tetrodotoxin, 13

the presence of action potential activity as sIPSCs while the miniature spontaneous events which occur independently of action potential firing will be referred to as mlPSCs. Traditionally, the sIPSCs have been studied at room temperature in Cl--filled hippocampal CAI pyramidal cells voltage-clamped with single sharp microelectrodes/7~8,7° More recently, the higher signal-to-noise ratio and the possibility for intracellular diffusion provided by the patch-clamp technique in brain slicess'33 allows a more rigorous investigation of the modulation of amplitude and decay kinetics of both slPCs and mIPSCs. This technique also enables the study of factors involved in the control of synaptic GABA release. 24'56'6°'62's2 Modulation of GABAA receptor function underlies the mechanism of action of several clinically employed drugs such as benzodiazepines, barbiturates, anticonvulsants and anesthetics, t°'4L~'st'67'6s's4Recent work has clearly demonstrated the vast molecular heterogeneity of GABA, receptors in different brain regions42"59'7s raising the possibility of region-specific drug effects. In this context, it is critical to determine the effect of these agents on inhibitory events evoked

14

T.S. OTIS and I. MODV

by endogenously released G A B A in various parts of the mammalian brain. By using the neurons as delicate biosensors, slPSCs provide an extremely powerful means of looking at both postsynaptic modulation of G A B A receptors and G A B A release processes in specific brain regions, On the postsynaptic side, the modulation of G A B A A receptor/channels is not restricted to the extracellular surface of the cell membrane. Putative intraneuronal modulatory sites identified by molecular biological and biochemical studies have been inferred from electrophysiological recordings using whole-cell dialysis in acutely dissociated or cultured mammalian neurons. In such recordings, GABAA receptor-mediated currents have been reported to be modulated by protein kinase C 79 and also to rundown in the absence of intracellular high-energy phosphates or as a result of an impaired intraneuronal calcium homeostasis.16'32,37'83 Furthermore, the kinetics of GABAA receptor/channels can be modified by the transmembrane voltage. 31'87'88 This mechanism is believed to enhance the efficacy of G A B A e r g i c inhibition by preventing excessive activation of voltage- and neurotransmitter-gated ion channels during depolarization. On the presynaptic side, our understanding of neurotransmitter release relies mainly on what is known about the control of A C h release at the neuromuscular j u n c t i o n Y '38,39,43 At this synapse, the frequency of miniature endplate potentials (mepps) can be modulated by osmotic pressure, extracellular Ca 2+ and Mg 2+ concentrations or polarization of the presynaptic endings. 39 In general, the control of the frequency of mepps is thought to depend on conditions of the presynaptic membrane, while their amplitude receptor/channel aptic membrane. neurotransmitter

modulation is a function of the kinetics as well as of the postsyn38 Yet the control of spontaneous release at synapses of the mam-

malian CNS is less clear. The study of spontaneous miniature and quantal evoked postsynaptic currents has enormously benefited from the application of the whole-cell recording method to mammalian brain slice preparations. 5.6,24,44,49,56.6°,62,82 Recordings in the thin slice preparation used in most wholecell recording studies show very few, if any, spontaneous postsynaptic events in the absence of an osmotic challenge, 6,24 thereby curtailing a detailed investigation currents, The aim of some of the factors known

of

the

frequency

of

spontaneous

the present study was to investigate above postsynaptic and presynaptic to modulate G A B A A receptor kinetics

and neurotransmitter release mechanisms in 400-/~mthick hippocampal slices maintained at 34-35°C. In this preparation the frequency of GABAA receptormediated spontaneous events is quite high and persists at high rates in the presence of tetrodotoxin (TTX). 56'6° Part of the results presented here have been published as abstracts. 55,6~

EXPERIMENTAL PROCEDURES Slice preparation Recordings were made from granule cells of the dorsal dentate gyrus in coronal half-brain slices (400/~m thickJ obtained from adult [postnatal day (P) 60 + ; 200-400 g] and young (P 15) Wistar rats. Briefly, following Na-pentobarbital anesthesia (60 mg/kg, i.p.), animals were decapitated, the brain quickly dissected and immersed for l -2 min in cold (4°C) artificial cerebro-spinal fluid (ACSF) solution. The brain was glued, frontal side down, to a brass platform with cyanoacrylate cement, and coronal whole-brain slices were prepared with a Vibratome (Lancer Series 1000). The slices were then hemisected and stored oxygenated at 32'C in a storage chamber until individually transferred to the recording chamber at the start of an experiment. Recordings were performed in a modified "Oslo-type" recording chamber (Nadler, Vancouver BC, Canada), with the slices usually immobilized by thin nylon netting (bridal veil) and two small platinum weights. These served to minimize movement during recording, and to break surface tension, allowing a 50-20-/~m film of ACSF to cover the slice. During experiments, slices were maintained at 34-35°C by a heating bath/water pump (Haake). This was calibrated, and its temperature setting was never more than 2°C higher than that of the medium in contact with the tissue, throughout a temperature range of 23-37°C. For experiments in which the temperature was changed, data were not collected for at least 5 min after re-setting the bath temperature. The ACSF contained (in mM): 126 NaCI, 2.5 KCI, 2 CaCI2, 2 MgC12, 26 NaHCO~ 1.25 NaH2PO4, and 10 glucose, continuously bubbled with 95%02/5%C02 (pH 7.35 + 0.05). To obtain e-fold and e2-fold increases in [K÷]0 without changing the C1 equilibrium potential, additional KC1 was substituted for equimolar amounts of NaCI to give final KCI concentrations of 6.8 and 18.6 mM. respectively. Drugs were added to the ACSF at concentrations indicated, and were.bath-applied by manually switching a Teflon rotary valve (Rheodyne).

Whole-cell recordings Whole-cell voltage-clamp recordings were obtained using borosilicate glass capillaries with an inner filament (KG-35, 1.5mm o.d., Garner Glass) pulled to 2-2.5/~m outer tip diameters (0.54).8pm diameter of the lumen) using a two-stage vertical Narishige PP'83 puller. Intracellular solutions varied according to experimental conditions, with patch pipettes containing either (in mM): 140 CsCI (or KCI), I0 HEPES, 2 MgC12 or 135 Cs-gluconate (or K-gtuconate), 10 HEPES, 5 CsC1 (or KC1), and 2 MgC12. All solutions were titrated to pH 7.2 with CsOH (or KOH). Additional pipette contents will be noted in the figure legends and included: 5mM N-(2,6-dimethylphenylcarbamoylmethyl)triethylammonium bromide (QX-314), 11 mM 1,2bis(O-aminophenoxylethane)-N,N,N',N'-tetraacetic acid (BAPTA)/I mM CaC12 (particularly for cells held at potentials more depolarized than - 5 0 m V for substantial periods of time), or a high-energy support system (4 mM Mg-ATP, 20 mM phosphocreatine and 50 U/ml creatine phosphokinase26). Total osmolality for all intraceltular solutions ranged from 255 to 285 mosm as measured on a Wescor 5500vapor pressureosmometer. Recordings in young (P 15) granule cells were made with the above-mentioned CsC1based solution or with the following intracellular solution 24 (in mM): 140 N-methyl-n-glucamine, 125 HC1, 2 CaCl2, 10 EGTA, 2 ATP, 1 MgCI2, 10 HEPES. Most recordings were made with an Axopatch-lD amplifier (Axon Instruments, Burlingame, CA) with > 80% series resistance compensation. For recordings in young slices a List EPC-7 patch-clamp amplifier has also been used. Access resistances typically ranged from 5-20 Mf~. To eliminate the possible contribution of access resistance to the filtering of the responses, some recordings were done

Decay kinetics and frequency of IPSCs using an Axoclamp-2A amplifier operated in the discontinuous voltage-clamp mode with a switching frequency of 12-15 kHz. As described in detail previously, +2'82recordings were obtained by lowering patch electrodes into the granule cell layer, while monitoring voltage responses to 100-pA current pulses and applying suction to form > C ~ seals. In previous studies employing identical methods 8" neurons were filled with Biocytin and immunostained, yielding only granule cells. In addition, the membrane properties of the neurons in this study (RN: 200-350 Mf~, RMP with Cs-filled pipettes: - 55 to - 70 mV) are consistent with those ohtained from morphologically identified granule cells, Event detection

All experiments were digitized (44 kHz) with a pulse-code modulated Neurocorder 484 (Neuro Data) and were stored on VHS videotapes. Following the experiments, recordings were played back, filtered (d.c. to 1-3 kHz, eight pole Bessel) and re-digitized (6-25 kHz) using an event detection program (SCAN or CDR; Strathclyde Electrophysiology Software, courtesy of Dr J. Dempster) running on Intel 80286/80287 or 80386/80387 based IBM/AT compatible computers equipped with Data Translation DT-2821 A/D converters, For detection of individual events, a trigger level was set at approximately three standard deviations above/below the baseline noise. As the noise ranged between 5-10 pA peak-to-peak in most recordings, the trigger level was typically set at 8-13 pA level. A time-criterion was also specified for the frequency/amplitude analyses. That is, events had to remain above/below the trigger level for 0.75-1 ms to be detected. As a test of the capabilities of the program, we ran square voltage pulses of variable duration (1-10 ms) separated by different intervals (2-200ms) through the event detector. The method could accurately record and measure successive events separated by intervals as short as 4 ms. A running baseline (2 ms) also allowed the exact determination of individual amplitudes in compound events. Each waveform which satisfied the trigger criteria was labeled as an event by the software. We routinely scrolled through the detected waveforms and rejected any noise which spuriously met trigger specifications. In addition, this type of analysis allowed us to eliminate multiple detections during large sIPSCs whose waveform often remained within the trigger criterion window and caused retriggering, Three types of systematic errors could be made by our event-detection procedure. The first arises when the smallest events are below the limit of trigger detection. This problem was very unlikely given that frequency measurements were always made under conditions in which E o - I/~ > 55 mV, where the unitary mlPSC size has been reported ~,56.6z7° to range between 15 and 20pA and our peak-to-peak noise never exceeded 10 pA. The second error may be introduced by measuring amplitudes of smaller events followed immediately (within 3 ms)by larger amplitude events. Under these conditions, the peak amplitude within the detection window would incorrectly be measured from the larger, secondary event. In a sample of three neurons this occurred in no more than 2% ofdetected IPSCs (mean 1.2%). Finally, IPSCs in this preparation result from GABA release from a number of independent presynaptic endings. In cases where two IPSCs generated from different presynaptic terminals occurred within I ms of each other, the two events would incorrectly be labeled as one. We have no way of estimating what percentage of the recorded spontaneous events fall into this category, but it is likely to be a small fraction of the total based on the rapid rise-time of the events. 62'82 Given the above concerns, we suggest our measurements to be lower limits of the actual frequencies, All frequency/amplitude measurements listed in Table 1 were obtained from continuous 30-120-s periods containing at least 300 events (except some 18.6 mM [K + ]o experiments

15

in which 10-s periods were adequate to collect more than 1000 events). Analysis o f decay kinetics

Miniature endplate potentials or currents can be best described by the difference of two exponential functions, one related to the rising phase, the other to the decaying phase of the waveform 7s [Eqn (1)]. For double-exponential decay kinetics (e.g. Fig. 3) the equation becomes a difference between a decaying double-exponential function and a rising single exponential function [Eqn (2)]. The kinetics will be determined on single or averaged waveforms. We have successfully used the non-linear least-squares Simplex fitting algorithm tS,Ss to fit the following equations to the digitized events: l ( t ) = A t x (e -t/. . . . e -t/,,), I(t)

=

(1)

(A t x e -t/,D~ + A2 × e -t/+o2)

- (A t + A2) x e-t/", whereI(t) isthelPSCasafuncti°n°ftime,

(2)

AtandA2are

amplitude scaling factors (pA), and ¢Dl, ¢D2 and z, are the two decay- and rise-time constants respectively (ms). These equations gave excellent fits both to the rising and decaying phases of individual IPSCs, as shown in Figs 3 and 5. The xD of IPSCs was determined by two other methods as well. The first was the fluctuation analysis3°'75 of the membrane current trace over a period of 30-60 s which included hundreds of sIPSCs. Following a fast Fourier transform and the fit of a Lorentzian function to the spectral density, the half-power (cut-off) frequency (fc) is related to the mean zD by the formula: zD = I/2n~. The fluctuation analysis provides an additional advantage by allowing the determination of the baseline noise variance and of the mean slPSC conductance independently of any arbitrarily set detection limits for spontaneous currents and without having to average several hundred individual events. The slope of the linear relationship between the mean current and the vailance can be used to calculate the average conductance of the events. The second approach, which was used more frequently, was to fit a single- or a double-exponential function to the decaying phase of individual or averaged sIPSCs using the Levenberg-Marquardt algorithm t3 (e.g. Figs 3, 4). The function had the following form: l ( t ) A 1 × e -t/'°' + A: x e -'/'°2, (3) =

where I(t) is the current as a function of time (t), A t and A2 are amplitude constants (A2 = 0 for single exponentials), and ~ot and zD2 are the two decay-time constants. In practice this was done by fitting the above doubleexponential function to hundreds of single individual events in which no other spontaneous events could be detected during the decaying phase. The two decay time-constants were then examined. If a single exponential was the better fit, the fitting routine usually converged to two ¢vs of comparable values or to a zm and a very large ( > 1000 ms) zo2 coupled with a very small A 2. The individual events with clearly double-exponential decay components were marked and used for averaging (e.g. Fig. 3B). Monoexponential fits were repeated then for the remaining spontaneous IPSCs (e.g. Fig. 3A). In the case of adult neurons maintained at physiological temperatures where our previous studies ~'+2 have indicated the consistently mono-exponential decay kinetics of IPSCs, we have resorted to the above method or to simply fitting single-exponentials. In the latter case, the accuracy of the individual fits was determined in 10-20 randomly selected events. The digitized points of the decay phase of each of these events were numbered. Two traces were generated for each event from the even- and odd-numbered data-points. Both traces of a given individual event were fitted separately m to a single exponential function with a Simplex based minimization

16

T.S. OTIs and !. MODY

routine, tS'Ss If fits to both records yielded comparable (within the S.EM.) Tos, then the mono-exponential fit was considered accurate, Of the three methods used to quantify decay kinetics, the fitting of rising and decaying exponentials was preferred, because it described rise- and decay-time constants concurrently,75but for the analysis of decay kinetics, single- or double-exponential fitting was more routinely used.

Cumulative probability distributions To avoid bias by the subjective binning of events into histograms, most amplitude and inter-event interval distributions were also plotted as cumulative probability distributions. These probability plots have the advantage that two or more normal distributions can be readily distinguished graphically.86 In such plots normal curves were approximated by the logistic equation. 4 The curves were generated by the Simplex fitting~5"58of two or more equations in which all parameters (ratio of curves, means, and standard deviations) were allowed to vary independently until the least sum of residuals squared was established. Most cumulative probability plots of amplitudes could be approximated by a single sigmoidal curve (a single normal distribution) or by the sum of two sigmoidal curves (sum of two normal distributions, e.g. Fig. 12B).

Materials All chemicalswere purchased from Sigma except 6-cyano7-nitroquinoxaline-2,3-dione (CNQX, Tocris Neuramin), D-2-amino-5-phosphonovaleric acid (D-AP5, Cambridge Research Biochemicals and Tocris Neuramin), TTX (Calbiochem), N-methyl-D-glucamine(Fluka) and QX-314 (generous gift of Astra Pharmaceuticals), RO-15788 and midazolam (Roche).

the amplitude of slPSCs was constant over long recording periods ( > 1 h) when patch electrodes were filled with standard intracellular solutions. We have also recorded slPSCs with an intracellular support system26'45 (Mg-ATP, creatine phosphokinase and creatine phosphate), which by itself had no effect on the mean amplitude of slPSCs and, as such, had no obvious advantages over standard electrode filling solutions. The % of slPSCs was also steady during long recording periods if no partial re-sealing of the patch electrode's opening occurred. The partial re-sealing of the cell membrane within the electrode lumen was found to be the most critical factor in long-term recordings. The resultant increase in access resistance markedly slowed down the rise-time and % of slPSCs. This problem was more likely to occur during the initial 15-20min of whole-cellrecordings and in some cases could be ameliorated or reversed by applying small positive pressure to the electrode. Cells were discarded from the present study if the access resistance at the end of the recording was > 25 Mf~ which, in our experience, was the upper limit for avoiding significant access resistance artifacts.

Reversal potential of evoked and spontaneous inhibitory postsynaptic currents

We have compared the slPSCs to synaptic currents evoked by stimulation of GABAergic fibers or interneurons. These experiments were done in the absence RESULTS of TTX with stimulating electrodes positioned in the hilus of the dentate gyrus. Consistency of spontaneous inhibitory postsynaptic In the presence of the excitatory amino acid antagcurrent amplitudes over time onists D-AP5 (40#M) and CNQX (10#M), large Whole-cell patch-clamp recordings provide an synaptic currents with reversal potentials determined ideal opportunity for intracetlular diffusion of nerve by the EcE could be evoked by stimulating in the hilus cells. 5°'64 Since there is assumed to be a gradual just underneath the granule cell layer. These prediffusion of the cell's interior, time-dependent de- sumably monosynaptic~8'19 GABAA currents were ctines in the amplitude of ionic currents recorded in completely abolished by bicuculline methiodide the whole-cell configuration have been taken as evi- (10 p M), as were the spontaneous IPSCs (not shown). dence for the loss of the intracellular constituents A comparison of the I / V relationships for these two necessary for the maintenance of full voltage- or types of currents is displayed in Fig. I. Figure 1A neurotransmitter-gated ion channel activity. 16'26'45 shows evoked GABAergic currents (elPSCs) Given their small molecular weight, high-energy recorded over a range of holding potentials, while phosphates are the most likely candidates to rapidly Fig. 1B displays average slPSCs recorded from the diffuse out of the cytoplasmu and the consequent same cell at identical membrane potentials. The dilution would be responsible for the wash-out of shapes of both I / V curves are linear between - 7 0 ionic currents. 26'45Accordingly, the gradual rundown and + 50 mV, but note that in this granule cell the of channel activity in whole-cell recordings can be slope conductance of the evoked IPSC is ten times prevented by including a high-energy phosphate re- larger than the average slPSC slope conductance. In generating system26 into the patch electrode which a sample of four other cells, depending on the continuously refurbishes the intracellular constitu- stimulus, the IPSC conductance was found to be ents necessary for full functional expression ofchan10-25 times larger than the average slPSC conducnel activity, tance (487.7 +42.3 pS; mean + S.E.M.; n = 5). To ensure that the observed effects on amplitude Finally, in all Cl--loaded neurons the elPSCs and and frequency of slPSCs were not due to a gradual slPSCs had a single reversal potential ( - 2 . 7 +_ rundown phenomenon, we examined the possibility 0.4 mV; n = 5), i.e. there was no evidence for the of time-dependent changes in the GABA^ receptor- existence of a different CI- reversal potential in the mediated slPSCs. With small cell-to-cell variability, soma vs dendrites of granule cells.

Decay kinetics and frequency of IPSCs A

17

e eo T

slPSC:pA)

|

I

/o

-is-i0-i~--i"-,--,¢l 2 s

B

~-40]

go

VM(mY)

140pA Fig. 1. Current-voltage (I/V) plots of the elPSC and sIPS(; are linear and reverse at EQ. A granule cell recorded with 140 mM CsCI inside of the pipette was claml~:l at different holding potentials, and both elPSCs (A) and slPSCs (B) were collected. The I/V plot in (2 shows an approximately ten times larger evoked conductance (slope conductance = 12.4 nS), which is otherwise identical in shape to the sIPSC (slope conductance = 1.48 nS) I/V relationship. The slight rectification evident for the slPSCs at I'M= - 2 5 mV to +25 mV is probably due to the inability of resolving very small spontaneous currents by the triggering protocol when the I / i s close to Eo.

Decay kinetics and the effect o f GABA, receptor modulators The decay kinetics of spontaneous synaptic currents generally reflect the kinetics of the single receptor channels activated by the respective neurotransmitter. 24'3°'~ Miniature endplate potentials or

currents can be best described by the difference of two exponential functions, one related to the rising phase the other to the decaying phase of the waveform 6s (see Experimental Procedures). The equations gave excellent fits to the timecourses of individual or averaged sIPSCs, as shown in Fig. 2. Less than 10% of slPSCs recorded at room temperature in young (P < 21) animals 24 had doubleexponential decay kinetics (Fig. 2, bottom trace and Fig. 3B). In a sample of five neurons of 15-day-old animals we have found predominantly mono-exponential decay kinetics of slPSCs (mean ~D= 21.8 _+ 2.7 ms; Fig. 3A). The results from one such neuron are shown in Fig. 3. In three of five neurons, a small fraction ( < 5 % ) of slPSCs, mono-exponential % longer than 4 0 m s were also found (Fig. 3A). In all five neurons, the slow exponential decay component of the double-exponential decays fitted to the average of five to twenty slPSCs was always less than 30 ms (26.4 + 1.3 ms; Fig. 3B, bottom panel). Based on the consistency of the 10-90% rise times of averaged events (Fig. 3, bottom panels) it is unlikely that electrotonic filtering was responsible for converting biexponential decay kinetics into single exponentials, The slPSCs recorded at physiological temperatures in adult preparations showed a mono-exponential decay (Figs 2, 4). In over 100 neurons held at m e m b r a n e potentials near rest ( - 60 to - 80 mV) the decay fitted to the average of 100-1500 slPSCs

ranged between 4.7-7.2ms. ~,62,s2 To eliminate the possible contribution of the access resistance in interfering with the time-course of slPSCs, the wholecell recording in Fig. 4 has been obtained using a PND 70+. 350 C

i ~t'~ ..

1~

"~ ~ t"

~ "'

'"

'OR

=

0.47 ms

xo(1)= 4.28ms

J 5 pA 10 ms PND 1 5, 220 C '."'.-"~J~ .

~i/" I/'

.

.. .~ . . . . . ~' , ,~. . 0 : , ~ . .. " '¢0(1)" 1.75 ms xo(2)-17.79 ms

V I t 0 pA 10 ms Fig. 2. The method for estimating the kinetics of slPSCs. Top panel: slPSC recorded in a dentate gyrus granule cell from an adult preparation [postnatal day (PND) 70+] at 35°C in the presence of I pM TTX. Bottom panel: slPSC recorded from a postnatal day 15 rat using identical conditions (except for the thickness of slices and lack of cleaning procedure) to those d~cribed by Edwards et al.u The solid lines indicate the fits obtained through a non-linear Simplex method using equations (1) and (2) (see Experimental Procedures) for single-and double-exponential decays, respectively. The rise (T,) and decay (To) time-constants are indicated. Both neurons were held a t - 5 0 m V .

18

T.S. OTIS and 1. MODY

switching single-electrode voltage clamp (switching frequency of 14.7 kHz). The sIPSCs recorded under these conditions were noisier but had comparable decay kinetics (mean zD = 5.3 + 1.7 ms, n = 3 neurons) to those recorded using continuous voltageclamp on the Axoclamp-2A 56'6zs2 or conventional patch-clamp amplifiers. At membrane potentials close to the resting potentials of granule cells ( - 6 0 to - 8 0 mV), the above range of mono-exponential % of slPSCs was also confirmed by another method. Fluctuation analysis of a membrane current trace comprising hundreds of sIPSCs 3°'75 is shown in Fig. 5A in a sample neuron. The half-power (cut-off) frequency (.~) was 32.7 Hz corresponding to a mean r. of 4.87 ms. The variance of the baseline noise (Fig. 5B) and the mean conductance of sIPSCs could be determined independently of any arbitrarily set detection limits and without

averaging individual events. This method of determination of mean sIPSC conductance resulted in cornparable values (mean zD: 5.7 _+ 1.9 ms, n = 5 neurons) to those obtained from averaging individual slPSCs, as shown in Fig. 5C. If the r[, of sIPSCs reflects the closing kinetics of GABA~ receptor channels, then pharmacological agents which increase G A B A A channel mean open time or burst duration are expected to prolong the rD of sIPSCs. The prolongation of rD of GABAergic currents by some anesthetics in voltage-clamped hippocampal neurons has already been shown. :s,56 We have investigated the effect of the benzodiazepine receptor agonist midazolam and antagonist flumazenil (RO 15-1788) on the zo of sIPSCs. Midazolam (5-10/~M) produced a marked prolongation of zo (234-t-21% of control, n = 5 neurons, at 10 # M midazolam) but not of v, (Fig. 6A, Fig. 7 insert).

A.

B.

40

2O

30

22.5 ± 6.4 ms (88.7%)

15

t-

¢-.

;

=

>

20

¢

d z

6 . 8 % of total

xot 2.4 ± 0.7 ms

10

o 10

42.1 ± 2.8 ms 4.5%)

0

Z

5

0 0

10

20

30 ~o (ms)

40

S W

50

~

m

27.9 + 12.2 ms

~ 10

20

30 ro (ms)

40

~ ........................................... ,o,,o.oo, 20 ms

.11 20 ms

Fig. 3. Decay kinetics of sIPSC in a young (P 15) granule cell recorded at 22°C. (A) Top panel: histogram of 314 (93.2% of total 337) sIPSC zDs (bin width = 1.67 ms) fitted with the sum of two Gaussian distributions with the indicated means and S.D. The percentages of total r~s included in each distribution are indicated in parentheses, Bottom panel: two randomly selected averages (10 slPSCs each) of different amplitudes showing comparable mono-exponential %s in the same neuron. The values of the two zDs are indicated from top to bottom for the small and large amplitude average responses, respectively. Exponential fits are indicated by solid lines. The 10-90% rise-times were 0.70 and 0.65 ms, respectively. (B) Top panel: histogram of the fast and slow %s for the 23 slPSC (6.8% of total 337) in the same neuron which showed clear double-exponential decay kinetics (bin width = 1.67 ms). Two separate single Gaussian distributions were used to fit the distributions of Zol and zD2, respectively (means and S.D.s are indicated). Bottom panel: two sample averages (five slPSCs each) of different amplitudes showing the double-exponential decay kinetics in the same neuron. The values for the ,Ds are indicated from top to bottom for the smaller and larger amplitude average responses. Exponential fits are indicated by solid lines. The 10-90% rise-times for the two averaged records were 0.75 and 0.65 ms, respectively. Holding potential was - 7 0 mV and the electrode was filled with (in mM): 140 CsCI, I0 HEPES, 2 MgCI2, 5 QX-314. Individual events were filtered at 3 kHz and digitized at 20 kHz.

50

Decay kinetics and frequency of IPSCs This effect was antagonized by the benzodiazepine receptor antagonist flumazenil ( R O 15-1788; 10/~M) and could be overcome by increasing the dose of midazolam in the presence of flumazenil (Fig. 6B). Perfusion of flumazenil (10 p M) alone had no significant effect on % of the slPSCs (109 _+ 17% of control, n =4). The amplitude of histograms of m I P S C s in a n o t h e r granule cell before and during the perfusion of 10/~ M midazolam are shown in Fig. 7. The average amplitude of the events following perfusion of the benzodiazepine receptor agonist is similar to that recorded under control conditions. C o m p a r a b l e results were obtained in three other neurons. The frequency of the events was not altered by midazolam. Pentobarbital prolonged % in a dose-dependent m a n n e r (Fig. 8). To ascertain that the pentobarbital effect reached its steady-state, the dose-response curve was obtained by incubating the slices for at least 30 min in a given concentration of pentobarbital before the recordings were undertaken. Each point

19

represents the average slPSC % from five granule cells recorded from at least two separate slices incubated at the given concentration of the drug. A concentration of 10 p M pentobarbital significantly increased %, and at 250/~M, the zo was increased ten-fold c o m p a r e d with the control. With concentrations of pentobarbital greater than 250/~M, individual sIPSCs could not be completely resolved since they

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Fig. 4. Decay kinetics of sIPSCs in an adult (P 90 +) granule cell recorded at 35°C. The neuron was voltage-clamped using a discontinuous voltage-clamp amplifier (Axoclamp2A) with a switching frequency of 14.7 kHz to avoid access resistance artifacts. Top panel: histogram of 150 sIPSC zDs (bin width =0.33 ms) fitted with a Gaussian distribution with the indicated mean and S.D. Bottom panel: three randomly chosen averages (composed of 5-10 sIPSCs each) of different amplitudes showing similar mono-exponential %s in the same neuron. The values of the three rDs are shown from top to bottom for the smallest to largest amplitude average responses. The exponential fits are indicated by solid lines. The 10-90% rise times of the averaged records ranged between 0.34--0.40ms. Holding potential was - 6 5 m V and the electrode was filled with (in raM): I40 CsC1, 10 HEPES, 2 MgC12, 5 QX-314. Events were filtered at 3 kHz and digitized at 25 kHz.

-so

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Fig. 5. Fluctuation analysis used to estimate the decay-time constant and average conductance of slPSCs. (A) Lorentzian fit to the spectral density function of a 60-s segment of current noise in which slPSCs were present (V h ---60mV). The data were filtered at 3 kHz (8 pole Butterworth), digitized at 12.5 kHz and were separated into traces containing no sIPSCs (background, open circles) and traces with several sIPSCs (filled circles). The frequency at which the spectral density is half maximal (f~) is 32.7 Hz corresponding to a slPSC zo of 4.8 ms. (B) The average variance of the background noise was 12.63 pA 2and was not related to the mean current. (C) The average variance of the current traces containing sIPSCs was 169.11pA 2 and an average slPSC conductance (GszPSC) of 388 pS could be calculated from the slope of the linear relationship between mean current and variance.

20

T.S. OTIS and !. MODY

did not decay to baseline before merging with subsequent events,

~ 40

In nine cells in which slPSCs were recorded over a wide range of holding potentials, we observed a slower rate o f decay (increased "CD) o f individual IPSCs as the membrane was depolarized. One such cell is depicted in Fig. 9A, B. In Fig. 9A, average traces of 10-15 events were normalized and superimposed (outward currents are inverted for clarity), illustrating that while the rate of rise appears to be less dependent on voltage, the rate of decay changes as a function of VM. Figure 9B is a plot of the same data, with mono-exponential decay-time constants best-fit to the above current traces at their respective membrane potentials. As calculated from

~> m

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slPSC Amplitude (pA) Fig. 7. Lack of effect of midazolam on the amplitude distribution of mIPSCs. The indicated number of events were collected during 180-s periods in control ACSF and in the presence of I0 #M midazolam, respectively. Amplitude histograms were constructed with a bin width of I pA (shaded bars: midazolam). Midazolam failed to alter the mean amplitude of mlPSCs (indicated with + S.D.) while prolonging the zo (inset shows the average of each 175 individual mlPSCs recorded during the two experimental conditions). The frequency of the mIPSCS was unaltered by midazolam (control: 5.58Hz, midazolam: 5.38 Hz). The lower number of individual events depicted during the 180-s period in the presence of midazolam can be attributed to the 30% more rejection rate compared with control conditions which resulted from the emergence of several secondary events before the individual mlPSCs could decay to baseline. The intracellular solution consisted of (in mM): 130 Cs-gluconate, 10 HEPES, 2 MgC12 and 5 QX-314, and the holding potential was + 5 mV.

10ms

B 35 I.tMMidazolam + t 0 I.tMRO 1 5-1 788

5(l:R=0.71;XD=14.18ms) i.tMMidazolam + t 0 p.M RO 15-1788

j 5 pA 10 ms Fig. 6. The benzodiazepine receptor agonist midazolam increases the decay-time constant of slPSCs, an effect which is antagonized by flumazenil (RO 15-1788). In the upper panel, the average of 10 slPSCs collected in ACSF containing 10#M CNQX and 40/~M D-AP5 is shown in comparison to an average slPSC following application of 5 #M midazolam to the bath. % increased from 8.28 to 16.31 ms. This effect was completely antagonized by perfusion of 10 # M flumazenil (RO 15-1788). Average slPSCs collected later from the sam= neuron shown above demonstrate that flumazenil (RO 15-1788) restores the decay-time constant to its control value of 7.95 ms. This antagonism can be further overcome by increasing the concentration (35/~ M) of midazolam in the bath which restored % to 14.18 ms, suggesting a competitive interaction with flumazenil (RO 15-1788). Note that the treatments altered relatively little the rise-time (zR) of the averaged slPSC. The continuous lines are Simplex least-squares fits using equation (1) (see Experimental Procedures). The rise-(%) and decay-(z,) time constants are indicated. The electrode contained (in raM): 130 Cs-gluconate, 10 HEPES, 2 MgC12 and 5 QX-314, and the holding potential was +6mV.

the regression line, an e-fold increase in ~o would result from depolarizing the membrane by 147 mV. Figure 9C shows a regression line fitted to data obtained in seven cells, each with different intracellular and extracellular recording conditions (see legend). F r o m these data an e-fold increase in % was calculated to result from a depolarization of 132.3 mV at 34°C. This voltage-dependence of r~ is essentially similar to that found in hippocampal C A 1 neurons recorded at r o o m temperature with sharp microelectrodes. ~7

Effect of temperature on decay kinetics Temperature is known to affect the rise- and decay-rate of miniature synaptic events ]7'69 and most previous studies on the sIPSCs of hippocampal neurons in the slice preparation were done at room temperature. 24"7°We wanted to determine if the higher and presumably more physiological temperature (34-35°C) during our recordings was responsible for the significantly faster % obtained in our study. Average IPSC traces obtained from three different granule cells held at - 5 5 mV, each over a partial but overlapping range of temperatures, are shown in Fig. 10. As was found with membrane voltage, the rate of rise of the events was less sensitive to the experimental variable (in this case, temperature). This can be seen in Fig. 10A, along with the monoexponential decays fitted to each average trace.

Decay kinetics and frequency of IPSCs Figure 10B is an Arrhenius plot of the same data fitted by linear regression with error bars representing the S.D. of %s fit to individual events. The line has the form y = 20.1 - 6.8 x; where y is ln(z~-~) and x is 103/T. F r o m this equation, the Q l0 of the decay rate (1/zD) was calculated to be 2.1 and the activation energy (Ea) for the reaction which controls the decay of IPSCs was calculated as 56.5 kJ/mol. The Ql0 is less than previously estimated with sharp microelectrode recordings, ~7 but the Ea value for the decay of IPSCs is in good agreement with the 69.3 kJ/mol obtained for the miniature endplate current decay reaction. 69

21

rents are shown at faster sweep-speed in Fig. l l B . Note the different amplitudes, and the similar decays of the currents. In Fig. 11C, average traces of these three different "types" of currents have been normalized to the same scale to show the nearly identical decay rates. In the absence of excitatory synaptic transmission, electrical stimulation of interneurons can elicit currents with similar mono-exponential

A "~

f

Kinetics o f evoked and spontaneous events can be identical A granule cell voltage-clamped at a holding potential of + 2 0 m Y , with 4 0 # M D-AP5 and 1 0 p M C N Q X in the bath to block excitatory synaptic transmission, is shown in Figs 11 and 12. The stimulating electrode was placed < 30 # m away immediately underneath the granule cell layer. The recording electrode contained 4 m M C I - and the resulting CIgradient caused outward currents at this holding potential ( + 20 mV). Spontaneously generated, large amplitude (75-200 pA) outward currents were evident, occurring at regular intervals, but intermingled with considerably smaller amplitude (20-60 pA), randomly occurring events which were presumably a mixture of slPSCs and mlPSCs (Fig. 11A). In the same cell, currents evoked by stimulation just below the granule cell layer, presumably mediated by direct excitation of GABAergic interneurons 19 close to the granule cell body, 2,t~,76were often greater than 300 pA (not shown in Fig. 1 I A). Three representative cur-

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lPontobarbitall(M) Fig. 8. Pentobarbital increases the % of sIPSCs in a dose-dependent manner. Each point on the curve represents the average slPSC % recorded from five granule cells + S.E.M. Slices were incubated in the given concentration of pentobarbital for over 20 min to obtain steady-state drug effects. Decay-time constants (%) were derived from mono-exponential functions fitted to the decaying phase of averaged sIPSCs using the Levenberg-Marquardt algorithm. With concentrations of pentobarbital above 250#M, the timeconstants still increased, but quantitative measurement of % was impractical due to the overlap of successive individual sIPSCs.

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Fig. 9. The slPSCs decay-time constant (%) increases as a function of transmembrane voltage. (A) Averages of 10-30 sIPSCs recorded at different holding potentials were normalized and superimposed (outward currents were inverted). Total sweep length is 51.2 ms. Note that the rate of rise of each trar~e is identical despite the different rates of decay. (B) The values of % calculated from the same traces were plotted on a logarithmic scale as a function of the holding potential. The regression line has a slope of 2.96 x 10-3 (s/V) and yields a ro of 10.97 ms at V, = 0 mV. (C) The slope of the relationship between % and membrane voltage was independent of the intracellular fill. A regression line has been fitted to a scatter plot of seven cells recorded under a variety of different recording conditions. The best-fit regression line has the form log(%)=10.00328 x II. + 0.930, and predicts an e-fold increase in % for every 132.3mV increase in V.. Each symbol represents a different cell with an intracellular fill (in raM), as follows. Filled squares, circles and diamonds: 140 CsC1, 10 HEPES, 2 MgCI2 (with extracellular TTX and phaclofen); filled inverse triangles: 110 Cs-gluconate, 5 CsC1, 1 BAIrrA, 1.1 CaCI2, 10 HEPES, 2 MgCI2, 5 QX-314 (no extracelhilar TTX); open diamonds: CsC1 140, 11 BAPTA, 1.1 CaCI2, 10 HEPES, 2 MgCI2, 5 QX-314 (no extracellular TTX); open circles: 140 CsCI, 10 HEPES, 2 MgCI2 (with extracellular "FIX); open triangles: 130 Cs-gluconate, 5 CsCI, 10 HEPES, 2 MgC12 (with extracellular TTX).

22

T.S. OT1Sand I. MODY

A

~o=18.9ms;21oC B ~

v

vD ¢~1~ co , -~ , , 6

3.2 (a4)

3.3

3.4

(ao)

(21)

r " x~03 (K'~) (o e)

Fig. 10. The decay rate of individual sIPSCs is accelerated by increasing the temperature. (A) Average sIPSC traces recorded at the same holding potential, but at different temperatures from three granule cells. Mono-exponential decays have been fit to each trace, and are superimposed (bold lines). While the activation of the channel appears to have little temperature sensitivity indicated by the comparable rates of rise, the decay rates of the average sIPSCs increase with increasing temperature. Total sweep length is 51.2 ms. (B) Arrhenius plot of the averaged data, with log(l/To) plotted as a function of lIT. Error bars are S.D. from 10-30 different sIPSC averages recorded in three different neurons. The line yields a Q~0 for the decay rate (1/To) of 2.1.

decay rates to the spontaneous currents, though stimulation in the molecular layer often elicits currents that decay more slowly than the sIPSCs. 6° The amplitude distribution of all sIPSCs recorded from this granule cell, depicted in Fig, 12A, clearly showed evidence for at least two normally distributed populations. Under the assumption that a rhythmic discharge of action potentials in the presynaptic neuron gave rise to the larger amplitude events, we chose to separate the regularly occurring, but spontaneously generated larger amplitude currents from the smaller randomly occurring sIPSCs. This was done in order to test whether the inter-event interval (IEI) distributions for the two types of events would also show differences. An arbitrary level of 75 pA was selected. Currents with amplitudes larger than 75 pA were classified as "regular", and were distinguished from the "random" events smaller than 75pA. Evoked currents were not included in this analysis. The histograms resulting from this classification are shown in Fig. 12A, C. As mentioned above, the two

distinct populations apparent in the amplitude histogram were best described by the sum of two normal distributions yielding means of 52 pA and 150 pA, for the random and regular populations, respectively. The amplitude data were also displayed as a cumulative probability distribution (Fig. 12B) and showed that at least two populations of slPSCs, each described by Gaussian distributions, were evident. A clear inflection separates the sum of two sigmoidal curves obtained without the bias of binning or an arbitrary separation of amplitudes. The appropriateness of the 75 pA cut-off was further demonstrated by the histogram of IEIs. All intervals between two consecutive random events and between successive regular events could be measured separately. Accordingly, Fig. 12C demonstrates two distinct populations of events: one is best-fit by an exponential probability density function with a mean of IEI of 129ms, and corresponds to a mixture of slPSCs and mlPSCs, while the other appears to be normally distributed around a mean of 140 ms. The cumulative probability plot of IEI was fitted by the sum of an exponential and a normal distribution. The point where the two curves describing the cumulative interval distributions became distinguishable is clearly illustrated in Fig. 12D. Granule cells, without stimulating electrodes placed in close proximity on the hilar side, had skewed sIPSC amplitude distributions and purely exponential probability density functions of IEIs (e.g. Fig. 13) characteristic of random events. Serendipitous recordings such as the one illustrated above can, however, provide some insight into the relationship between sIPSCs and action potential mediated depolarization of GABAergic terminals. Thus, action potential-dependent or stimulus-evoked GABAergic synaptic transmission may operate independently of random, spontaneous GABA release, 6° despite kinetically indistinguishable postsynaptic actions.

Spontaneous inhibitory postsynaptic current Jrequency in dentate gyrus granule cells With the C1- reversal potential set at either 0 mV (by including 140 mM C I inside the recording pipette), or at - 55 mV (by using pipettes filled with only 9 mM CI ), holding potentials ranging from 55 to 75 mV away from Ecl provided sufficient driving force such that sIPSCs were evident immediately after breakthrough 62 (n > 10 cells). Consistent with previously reported findings,62 in the majority of analysed cells, the probability density functions for IEIs were mono-exponential. The average frequency of stPSCs in granule cells recorded under different conditions varied considerably. Measurements from 25 cells in a histogram format are displayed in Fig. 13A. All neurons were recorded in ACSF containing 1/~M TTX. In this sample, the average IEI was 221.4 ms, with a standard deviation of 146.5 ms. This corresponds to an average frequency of 4.5 Hz. Perhaps more importantly, within a given cell, the rate

Decay kinetics and frequency of IPSCs of spontaneous events was relatively constant over long recording periods even in the absence of TTX. This is shown in Fig. 13B, where the frequency just after breakthrough was approximately 19.6 Hz (mean IEI = 51.2 ms) and was unaltered at 35 rain or at l h after breakthrough. Variations in the mean are bound to occur simply due td the relatively short (30 s) sampling periods.

Blocking transmembrane Ca 2+ currents reduces but does not abolish spontaneous G.4BAergic activity It has been suggested that the reduction in transmembrane Ca 2+ currents with the inorganic Ca 2+ channel antagonist Cd 2+ is not sufficient to block sIPSPs recorded in CA1 pyramidal cells. 17 Relatedly, a class of miniature endplate potentials at the neuromuscular junction persists in the absence of extracellular calcium. 25 Presumably both evoked

23

IPSCs and TTX-sensitive sIPSCs depend on transmembrane Ca 2+ currents, but the release mechanisms underlying TTX-resistant mIPSCs may be less dependent on Ca 2+ entry through Ca 2+ channels of the presynaptic terminal. This was tested in two ways, by either bath application of low Ca2+/high Mga+ ACSF, or in separate experiments by perfusion of 200 taM CdCl2 (a concentration of Cd 2+ sufficient to completely abolish voltage-dependent Ca 2+ currents in the postsynaptic neurons, n = 4). These recordings were performed in the absence of TTX, so that evoked IPSCs could be monitored to gauge the effectiveness of presynaptic Ca 2+ channel blockade. Such an experiment for the high Mg 2+ condition is shown in Fig. 14A. Peak amplitude of the evoked response is plotted vs time, demonstrating that the reduced extracellular Ca 2+, coupled with the high concentration of Mg 2+, gradually abolishes the evoked IPSC. Representative traces of evoked

A

0.5 s B

C Evok.

.

Regular

_

_ ....

Iso,^

30 ms

Fig. 11. Regularly and randomly occurring sIPSCs and directly evoked IPSCs differed in amplitude, but not in decay rate in a dentate gyrus granule cell. The whole-cell patch pipette contained (in mM): 135 Cs-gluconate, l0 HEPES and 2 MgC12. All traces depict postsynaptic currents recorded from a granule cell held at +20mV, continuously perfused in ACSF containing 10/~M CNQX and 40/aM D-APS. Evoked IPSCs were collected by stimulating just underneath the granule cell layer, on the hilar side. (A) A slow-sweep speed recording shows the large variation in amplitudes, and the regularity of the larger amplitude events (> 75 pA). (B) Three "types" of IPSCs are depicted, with regular and random events arbitrarily distinguished on the basis of their amplitudes (see text for details). (C) Averages of 2-20 traces were normalized and aligned by their rate of rise. Mono-exponential decays fitted to the average evoked IPSC, regular sIPSC and random sIPSC are comparable.

24

T.S. OTis and I. MODY

responses t a k e n before a n d after M g 2~ perfusion are s u p e r i m p o s e d in Fig. 14B. C u m u l a t i v e probability distributions detail a reduction in the m e a n amplitude (Fig. 14C) a n d the m e a n IEI (Fig. 14D). In three cells, the average of the ratios o f e x p e r i m e n t a l / c o n t r o l m e a n amplitudes was 0.82_+0.12 ( m e a n ± S . E . M . ) while the average o f the ratios of m e a n frequencies was 0.93 ± 0.15. This was in the range t h a t would be expected if action potential input to the terminals were abolished. 61 Two experiments with perfusion of 200 # M extracellular CdCl2 resulted in m e a n ratios of 0.87 a n d 0.86 for average amplitudes a n d frequencies, respectively. Experiments were also c o n d u c t e d in a n extracellular solution c o n t a i n i n g 4 m M M g 2~ and 0 m M Ca2+/l m M E G T A . In four granule cells,

A 150

exposure to this Ca2+-free solution abolished evoked postsynaptic currents within t 0 m i n while m l P S C s persisted. Five minutes after evoked IPSCs could n o longer be elicited, the m e a n experimental/control ratio was 0.88 (average amplitudes were reduced from 36.6±4.3pA to 2 9 . 3 _ + 3 . 6 p A , n = 4 ) while the same ratio for the frequencies was 0.67 (average frequency was reduced from 13.0 ± 3.4 Hz to 8.2 ± 2.1 Hz, n = 4). Some slices (n = 5) were incub a t e d in the 0 m M Ca2+/1 m M E G T A A C S F for over 2 h. T h o u g h recordings were difficult to o b t a i n u n d e r these conditions, two granule cells showed n o r m a l input resistances a n d sodium spikes upon depolarization, but no s p o n t a n e o u s events could be detected.

B Random s l P S C s

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Fig. 12. Histograms and cumulative probability plots of amplitudes and inter-event intervals (lEIs) show at least two populations of slPSCs. The data are from the same cell as in Fig. 2, excluding the evoked responses. (A) Based on the amplitude histograms, an arbitrary separation level of 75 pA was chosen. Events were then classified as "regular" (i>75 pA) or "random" (<75 pA) in order to generate the frequency distributions. The amplitudes of all spontaneous (i.e. non-evoked) events were obtained from a 2-min recording period. The Gaussian distributions (solid lines) of the form: [(N x BW)/(a x ~/2;t)] x e -~x-~)2/~2 (where N is the total number of events, BW is bin width, tr is the standard deviation and /~ is the mean) were superimposed on the histogram, one describing the population of random slPSCs. with a mean ~ ) = 52 pA (shaded bars), and the other fitted to the larger, regular events with a mean ~ ) of 150 pA (open bars). (B) The same data are plotted as amplitude, in ascending order, vs cumulative probability. It is clear that the curve is the sum of two sigmoidal (normal) cumulative probability distributions. The fits to the sum of two logistic equations which approximate the sigmoidat normal curves' are shown, indicating the fractional contribution of each type of event to the global distribution. (C) Using the same amplitude discrimination criteria, the range of intervals between two consecutive events of either type is displayed in histogram format. Two entirely different distributions can be distinguished: the regular events (open bars) are best described by a Gaussian distribution function (fitted line with mean = 140 ms), while the random events (shaded bars) are best described by an exponential probability density function (fitted line) of the form: (N × BW/T) × e-t/r, where T is the mean inter-event interval (129 ms). (D)The cumulative probability plot of inter-event intervals was fitted with the sum of an exponential (1 e -e/r) and a sigmoidal (normal) distribution and implies two separable populations with the indicated percentage contributions to the global distribution.

Decay kinetics and frequency of IPSCs A

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Inter-Event

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Interval (ms)

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Fig. 13. The frequency of sIPSCs is relatively constant over long periods of time within a #oven cell, but varies widely between granule cells. (A) The histogram of average IEI obtained in 25 granule cells recorded in separate slices in the presence 1 #M TI'X demonstrates the large cell-to-cell variability of IEIs. Each average was calculated from IEIs measured during a 2-min period shortly after breakthrough. (B) A cumulative probability plot shows the constancy of IEIs in a #oven neuron measured over 30-s segments at three different time-points during a recording in the absence of TTX. Average IEIs measured at 1 min, 3 5 imn and l h following breakthrough were 51.2 ms, 40.8 ms and 47.1 ms, respectively. The cumulative probability describing the IEIs measured at 34.5-35 min is plotted for every tenth point (dots) for clarity and has been fitted with a mono-exponential equation of the form P = 1 - e -'/r', where P is the cumulative probability, t is the IEI and T is the mean IEI (40.8 ms).

25

of GABAergic neurons or that of their terminals while recording postsynaptic currents in the granule cells with the membrane potential clamped at -- 55 mV. This was accomplished by elevating [K + ]o e- and e2-fold (from 2.5 m M to 6 . 8 m M and to 18.6raM, respectively), or by bath application of 5 0 # M ( - ) b a c l o f e n . Baclofen was used because of its activity as a G A B A , agonist, ~t with the aim of determining how presynaptic G A B A B receptors feed back 19'2°'34'6° on TTX-resistant spontaneous G A B A release. All experiments were performed in 1 # M TTX, previously determined to block all evoked synaptic transmission and action potential activity. Presynaptic modulation of G A B A release was evident despite the blockade of voltage-dependent sodium currents. This was best demonstrated by perfusion of medium containing an e2-fold increase in [K+]o (from 2.5 m M to 18.6 mM). A n experiment in which a complete wash was obtained is displayed in Fig. 15. Despite the large inward shift in the holding current during the high [K+]o perfusion, individual events could still be easily identified (Fig. 15A). The cumulative probability plots show that while there was little change in mIPSC amplitude during the

Presynaptic manipulations of action potential independent GABA release We have attempted to alter the frequency or amplitude of mIPSCs by altering the m e m b r a n e potential

elevation of [K + ]o, the frequency of mIPSCs increased dramatically (Fig. 15B, C). This experiment showed a reversible 88% decrease in the mean IEI. The mean percentage decrease in 10 experiments was 40% compared to the average control IEI, as summarized in Table 1. This change was significant as measured by the Wilcoxon paired-sample signed rank test (P < 0.025). We observed a decrease in the average amplitude of events after the wash of 18.6 m M [K + ]o. As expected, with a smaller elevation of [K + ]o, the decrease in average IEI was less. Table 1 shows no significant decrease (9%) in the average IEI when [K + ]o was increased e-fold (from 2.5 m M to 6.8 mM). Perfusion of the GABA~ receptor agonist baclofen (50 # M ) increased the average IEI and reduced the average amplitude. The baclofen effects persisted in all cases following washout of the drug, even in experiments where more than 25 min were allowed

Table 1. Presynaptic modulation of miniature inhibitory postsynaptic currents Treatment

n

Control (ms)

Experimental (ms)

Ratio (exp./con.)

6.8 mM [K + ]o 18.6raM 0K+]o 50 #M (-)baclofen

10 10 10

184.7 + 71.8 243.2+ 182.1 200.1 + 128.6

167.4 + 94.5 121.1 + 102.5 277.1 + 155.2

0.92 _+0.31 0.59+0.47* 1.58 + 0.72*

Effect of an e-fold, or of an e2-fold increase in extracellular K + concentration ([K + ]o) or of the GABA~ receptor agonist (-)baclofen on the inter-event intervals of granule cell mIPSCs. All recordings were done at holding potentials of - 6 0 to --80mV in C1--loaded neurons in the presence of I gM TTX. The data were collected at least l0 min following the beginning of the experimental condition. Values are mean + S.D. The asterisks indicate that the average ratios are significantly different from 1.0 (P < 0.025; Wilcoxon paired-sample signed rank test). Note that mean ratios #oven in the Ratio column were calculated from individual experimental/control ratios in each of the l0 cells and are different from the ratio of the average values #oven by dividing the Experimental and Control columns.

26

1. S. OTIS and I. MoDY

for the wash. Table 1 shows that the average increase (59%) in the mean IEI produced by 50/~ M baclofen was significant (P < 0.025, Wilcoxon paired-sample signed rank test). Lower concentrations of baclofen (1-10/~M) were ineffective in altering the amplitude or frequency of sIPSCs or mIPSCs. 6° DISCUSSION

Intraeellular diffusion and spontaneous inhibitory postsynaptic current amplitudes Most of our recordings were performed without a high-energy phosphate support system or exogenous Ca 2+ chelators added to the intracellular solution, U n d e r these conditions, the lack of significant timedependent declines in the amplitude of GABAA receptor-mediated sIPSCs during whole-cell recordings lasting in some cases in excess of 2 h is in marked contrast to the fast rundown of CI currents evoked by applications of exogenous G A B A in acutely dissociated hippocampal ~6'83 or cultured neurons? 2 There are several possible explanations for this discrepancy, Firstly, the intracellular environment of neurons recorded in the whole-cell mode in our slice preparations may not be sufficiently perturbed. This is A

6 rnMMg2*,0.SmMca~ ,

n.

B -':-

;..

400

unlikely since recordings of voltage-dependent Ca 2 currents, which are known to rundown in the absence of intracellular high-energy phosphates. 2~ sho~ a gradual time-dependent decrease over about 30-40 min in our preparation when recorded without the support system. Furthermore, in a preparation similar to ours, maintained at room temperature, the possibility of induction of long-term potentiation diminished significantly after about 20 min of wholecell recording presumably due to the diffusion of an intracellular factor. 49 Assuming that in our recordings there was adequate diffusion of the cells' cytoplasmic constituents, alternative explanations must be explored to account for the independence of sIPSCs from intracellular high-energy phosphates. It is possible that the requirement of high-energy phosphates in acutely dissociated and cultured neurons is not due to the necessity of maintaining a receptor in a phosphorylated state, but rather serves to fulfil an indirectly related requirement, such as the cells' need for maintenance of a normal Ca" + homeostasis. As this homeostasis is impaired, secondary effects such as the decline of G A B A A receptormediated currents may result. ~6'37~83 Given this, our findings suggest that the more intact (i.e. less

6 mM M02*

•

300

•

•

o "o

• 200

E i <1oo 0

• •• . . . . . . I

2

3

4

•

~,

'•

*' * • • • • * • *

5

6

7

100pA ' 8

30ms

9

Time (min.)

C

~. 0.6 oJ > _~

i~

D

6.raMMg=2+,0.3 mMCa2.

Control

E 0.2

~- 0.6 ® // m "5 E 0.2

0 0.0

0 0.0

/

/~,-:~Control ean 8 8 8 p

. )

:3

a2.

//

(Meafi = 67,7 ms)

"1

0

25

50

75 100 125 150 175 200 Amplitude (pA)

0

50

100 150 200 250 300 Inter-Event Interval (ms)

350

Fig. 14. Evoked synaptic transmission is blocked upon perfasion of 6mM Mg2+/0.3 mM Ca 2+, while slPSCs remain. (A) Perfusion of a high Mg2+/low Ca2+-containing extracellular solution for the time marked by the horizontal arrow at the top of the graph resulted in the complete blockade of evoked compound PSCs. (B) Two evoked responses (averages of two sweeps each), each taken at the times marked by the arrowheads in A. It is evident even from these average traces that large amplitude sIPSCs persist in the absence of evoked release. (C, D) The reductions in the mean amplitude (39%) and increase in IEI (56%) are shown in cumulative probability distributions. The intracetlular fill was (in raM): 140 CsCI, 10 HEPES, 2 MgC1z and 5 QX-314. The neuron was voltage clamped at - 6 0 mV for the course of the experiment.

Decay kinetics and frequency of IPSCs traumatized) neurons of the slice preparation maintained at body temperature may be equipped with more efficient intracellular Ca 2+ regulatory mechanisms which do not easily rundown during whole-cell recordings. This hypothesis is supported by the findings that inclusion of extracellular Ca 2+ chelators (e.g. BAPTA) into the intracellular solution did not alter the amplitude or % of sIPSCs. ~ Alternatively, the concentration of G A B A applied to the receptors may also be an important factor in the rundown of the responses. 33 Here we studied very small spontaneous currents which are presumably activated by different concentrations of G A B A in the synaptic cleft than the exogenously applied agonists employed for producing rundown in previous studies. 14'1~'32'37's3 It was beyond the scope of the present study to rigorously test this hypothesis, but we are in the process of addressing this question by studying the possible rundown of large, evoked G A B A , currents as well as the time-dependent

27

changes in responses evoked by short applications of exogenous GABAA receptor agonists during wholecell recordings. Lastly, the neurons recorded from in the present study were at a different stage of maturation than the acutely dissociated neurons (usually taken from animals younger than 21 days of age) or the embryonically isolated cultured neurons. Given the possible changes in GABA^ receptor channels during development, 29'47 it remains to be determined whether immature GABA^ receptor channels are subject to different modulatory mechanisms than their adult counterparts.

Decaykinetics The prolongation of sIPSCs at lower than body temperatures most likely reflects the slower kinetics of GABA^ receptor channels. As recently shown for inhibitory synapses on young granule cells,~ we have also found some sIPSCs with double-exponential

A I II llIM K ,

Neme 18 mM K*

JiN~ Ni

B

C Wash

18.6 mM K* ( M ~ ? . 7 eu)

1.0

.

I

/ / , '~DIIltroI

oio=t o.ol / I! . 0

2s

0> O.4H / / (Melm-64.0m)

H /i

. . . . . . . . . so 7s 100 1is Amplitude (pA)

~o 1so

0.0

: o

1

1

/

loo 2oo soo Inter-Event Interval (ms)

Fig. 15. An e2-fold increase in extracellular potassium concentration (from 2.5 to 18.6mM) reversibly increases the frequency of sIPSCs. (A) During the time indicated by the horizontal bar on the chart recording (lower panel), ACSF containing an additional 16.1 mM KC1 (substituted for NaCI) was perfused (both control and high [K+]o solutions contained 1/JM TTX). Faster sweeps depicted above highlight the massive, yet reversible, increases in frequency and net inward current associated with the perfusion. Note that events can still be well discriminated despite the 250 pA increase in holding current. (B) Cumulative probability of sIPSC amplitudes. The elevated [K+]o caused a slight (12%) increase in the range of amplitudes of sIPSCs, but initiated a significant decline (32%) in average amplitude that persisted through 15 min of wash. (C) The average IEI for this cell declined by 88%, and returned to within 20% of control level following wash. NSC 49/I--B

400

28

T.S. OTIs and I. MODY

decay kinetics in young neurons at room temperature. However, in our preparation the single exponential decay kinetics (with a % in the range of 22-25 ms) predominated while long ( ~ 54 ms)exponential components as reported by Edwards et al. 24 were not found in the averaged records. Different kinetics of single- and double-liganded G A B A , receptor/ channels 14 have been proposed to account for the double-exponential decay kinetics of spontaneous and evoked IPSCs in granule cells on postnatal days 15-21. A possible explanation for the discrepancy between our findings and those of Edwards et al. 24 is that the "cleaning" procedure, 23'24 routinely used in the thin slice preparation, may have altered the kinetics of the subsynaptic GABAA receptor/channels by removal of some active synapses from the soma or by altering G A B A reuptake mechanisms into neurons and glial cells. The slightly faster mono-exponential decay kinetics of slPSCs in adult vs young neurons at room temperatures (Figs 3, 10) may result from the developmental changes in the GABAA receptor/channel ~j, c~2, 0~447 and 7229 mRNA expression beyond the third postnatal week. Indeed, developmental changes in the function of muscle acetylcholine receptors resulting in faster closing rates of the channel have been described and attributed to the different molecular forms of the receptor. 54 Although the anatomical organization of the GABAergic system in the dentate gyrus appears to be complete by the third postnatal week 71'77 it is possible that functional maturation of inhibition continues past the anatomical development, The tD of slPSCs should reflect kinetic properties of G A B A A receptor channels z4 and is consistently altered by membrane voltage, temperature and drugs known to modulate the mean open-time of G A B A , receptor channels. The prolongation of slPSCs upon membrane depolarization ~v,6z is consistent with the increased probability of opening of GABAA receptor channels at positive membrane potentials 8788 and the rectification of these channels in dentate gyrus granule cells of enzyme-treated slice preparations. 3t It is also of considerable interest how the prolongation of slPSCs by drugs matches the pharmacological profile of GABAA receptors. One of the observations in the present study indicates that the benzodiazepine antagonist flumazenil (RO 15-1788) had no effect by itself on the z~ of slSPCs whereas it reversed the prolongation produced by midazolam, a benzodiazepine agonist. Our data do not support the co-release of an endogenous flumazenil-sensitive benzodiazepine site agonist together with GABA from the terminals of dentate gyrus inhibitory interneurons. The results are also not consistent with the possibility that sufficiently high ambient levels of an endogenous benzodiazepine liberated by non-GABAergic terminals alters slPSC kinetics. Stimulus-evoked IPSCs, however, may be different in this regard. 4° The lack of effect of the benzodiazepine receptor agonist on the average amplitude of mlPSCs is

intriguing. Benzodiazepines are known to increase the affinity of the receptor for G A B A by increasing the association rate of G A B A with the receptor. This usually results in larger whole-cell responses to exogenously applied agonists (e.g. Refs 80, 85). At the channel level, benzodiazepines are believed to enhance the probability of opening in long duration bursts; 51'85 similar effects result from increasing the concentration of GABA. 85 Thus, the increased z,, paralleled by an unchanged mean amplitude of mlPSCs in the presence of midazolam raises the possibility that subsynaptic G A B A Areceptors may be already saturated by the amount of GABA present in the synaptic cleft following action potential independent release. This situation would be analogous to the high G A B A concentration/GABA~ receptors model proposed in simulation studies. ~4 Frequency currents

o f spontaneous

inhibitory postsynaptic

As interneurons of the dentate gyrus are known to have high rates of spontaneous spiking, 57'7~ in most experiments the contribution of action potential activity to presynaptic terminal depolarization was abolished by including TTX in the extracellular recording solutions. The frequencies of mlPSCs measured in this study (1.6-25 Hz) are one to two orders of magnitude larger than the 2 10/min (0.033q3.166 Hz)reported previously24 at this synapse in young animals. This may be explained by a functional maturation of GABAergic inhibition similar to that seen in the rat neocortex where prior to P7. in spite of numerous GABAergic neurons and large postsynaptic G A B A responses, slPSC activity is absent and develops gradually throughout the following two weeks of postnatal life (J. J. LoTurco, personal communication). Granule cells of the dentate gyrus, however, are still mitotic during this time, and it is likely that an increase in GABAergic circuitry continues past P21. The increased frequency of slPSCs during ontogeny may be correlated with an increased number of GABAergic presynaptic terminals, but this is not supported by anatomical evidence. 7~77 The increase in inhibitory input must then reflect functional development of the GABAergic system. Furthermore, in our experiments the high slPSC frequency observed in 400-/~m-thick slices regardless of the age of the preparation suggests that the "cleaning" procedure together with the use of thin ( < 300/~m) slices23'24 may have unintentionally but effectively removed some functional GABAergic terminals from the granule cells. Most probability density functions of IEI distributions recorded in this study were mono-exponential. 62'82 Our observation regarding the relative constancy of slPSC frequency over long periods of time is an important characteristic of the GABA release mechanism underlying generation of slPSCs. It makes it rather unlikely that slPSCs are generated by damaged interneuron terminals from which

Decay kinetics and frequency of IPSCs G A B A would gradually "leak out". If interneurons were deteriorating over the course of an experiment, one would expect to see a systematic change in the frequency of sIPSCs with time. Moreover, if the procedure of preparing slices were damaging the tissue, and sIPSCs resulted merely from the trauma, recordings made just after cutting the slices should have had higher rates of slPSCs than those performed several hours later. The variation in frequency was unchanged throughout the life of a group of slices, and n o systematic trends in frequency were seen during a day's experiments. The constancy of IEI in a given cell raises the possibility that a tonic inhibition some of which is independent of action potential activity, much like any intrinsic inhibitory conductance, may play an important role in depressing or selectively filtering the excitation impinging upon granule cells,

Does the release of GABA responsiblefor the miniature inhibitory postsynaptic currents depend on presynaptie Ca 2+ entryP Our findings cannot unambiguously resolve the question whether or not the action potential-independent mlPSCs are generated by a Ca2+-independent release of GABA. However, the G A B A release mechanism responsible for the generation of mIPSCs appears to be different than the purely Ca2+-indepe ndent G A B A release described in the retina. TM The occurrence of discrete mIPSCs with fast rise-times points to a vesicular release mechanism. Clearly, extracellular Ca 2+ need not be present for the mIPSCs to occur, which raises the following possibilities. The resting level of Ca 2+ in the interneurons or in their terminals may always be above the threshold level required for spontaneous G A B A release. In this case, lowering extracellular Ca 2+ concentrations, and even perfusing 0 mM Ca2+/1 mM E G T A solutions cannot sufficiently reduce the intracellular free Ca 2+ concentration. Alternatively, Ca 2+ may not be the limiting factor for neurotransmitter release, but rather may act in a more permissive role, as a cofactor, postulated in the calcium-voltage hypothesis of neurotransmitter release. 36 In this scheme, if the Ca 2+ affinity of the release process is lower than the resting intracellular Ca 2+ concentration, transmembrane voltage fluctuations will precipitate G A B A release. Finally, our findings may be explained by fluctuations in intraterminal Ca :+ levels perhaps mediated by oscillatory Ca 2+ release from i n t r a c e l l u l a r s t o r e s . 7 Such a mechanism has been proposed at the neuromuscular junction based on the periodic changes in mepp frequency. 63 Further support, albeit indirect, regarding this hypothesis comes from our experiments in which slices were incubated in 0 mM Ca2÷/1 mM EGTA for longer than 2 h. The absence of mlPSCs under these conditions implies that the release mechanism may be dependent on a C a 2+ pool that can be depleted during a long incu-

29

bation with Ca2+-free/EGTA containing extracellular medium.

Presynaptic manipulations Increased transmitter release is commonly observed in the presence of elevated [K+]o at the neuromuscular junction. 2L43In our experiments, since action potentials are not contributing to the excitation of the synaptic terminals, the increased mIPSC frequency during elevated [K+]o must stem from a direct depolarization of synaptic terminals. An e-fold increase in [K + ]o will result in a shift by EK by 23 mV, while an e:-fold increase depolarizes EK by 46 mV. These presynaptic terminal depolarizations will almost surely lead to an increase in free intraterminal Ca 2+. The shift in EK, however, is not the only depolarizing influence in the terminals. Increased release of transmitter onto presynaptic receptors localized on the terminals themselves, as well as voltage-dependent conductances intrinsic to the interneurons' synaptic terminals, could affect the rate of spontaneous G A B A release. The increase in mIPSC frequency may involve a combination of increased C a 2+ influx, as well as second messenger mediated Ca 2+ release from intracellular stores located near the G A B A release sites. These possibilities need to be examined in future experiments. The reduction of mlPSC frequency in response to baclofen occurred at a much higher concentration of GABAA agonist than expected. Monosynapticaily activated G A B A release in response to a large electrical stimulus will feed back and activate presynaptic GABAB autoreceptors, ~9'6° and also depresses large monosynaptically evoked IPSCs in granule cells.6° In addition, presynaptic inhibition can result from exogenous application of low concentrations (1 # M) of the GABAB receptor agonist baclofen. 6° Why then are the concentrations of baclofen needed to reduce the frequency of mlPSCs (i.e. action potential-independent spontaneous G A B A release) an order of magnitude higher? The pharmacology of GABA~ receptors is not well characterized, but at least two conductances have been reported to be coupled via second messengers to GABA- and baclofen-activated receptors. It has been implied that a G-protein coupled activation of K + conductance is responsible for the paired-pulse depression of monosynaptic IPSPs described in the CA1 region. 19 G A B A , receptors have also been shown to be negatively coupled to voltagedependent Ca 2+ conductances, however (for review, see Refs 9, 12), and GABA~ receptor activation reduces Ca 2+ entry into neurons generated by repetitire stimulation in the hippocampus. 35 Our obserration that considerably higher concentrations of baclofen are required for the depression of TTXinsensitive mIPSC frequency as compared with depression of stimulus-evoked G A B A release may have two explanations. First, the receptors or presynaptic terminals involved in the paired-pulse depression of stimulus evoked IPSCs may be spatially or

30

T.S. OTIS and I. Moov

functionally distinct from those involved in the baclofen-mediated m l P S C depression. Second, activation of a given G A B A B receptor may have differential effects on action potential-independent release and evoked release of G A B A . 6° CONCLUSIONS Our present findings complemented by previous results l'17,22,24,2s'4°'s6,62,7°'s2 provide evidence for a substantial tonic inhibitory activity in the mammalian C N S mediated by GABA^ receptors. This inhibition is reduced but not abolished upon blocking all action potential activity and thus may serve as an important continuous filtering/shunting mechanism in the CNS. Inputs which are spatially and temporally dissynchronous with this inhibitory activity will be able to exert their full influence on the principal neurons. Altering this inhibitory activity by exogenous (anxiolitics, anesthetics,

antiepileptics) or endogenous modulators (e.g. norepinephrine, 22 serotonin 69a) may result in dramatic shifts in neuronal excitability of entire nerve cell aggregates. Clearly, some endogenous modulatory systems could have an effect solely restricted to interneurons. 27 By studying this tonic inhibitory activity during plastic alterations in nervous system function, which involves the loss of GABAergic neurons, 3'67 significant insight could be gained into both normal and pathological functioning of the major inhibitory neurotransmitter system of the mammalian brain. Acknowledgements--This work was supported in part by NIH grants NS-12151, NS-27528 and a Klingenstein Fellowship in the Neurosciences to I.M.T.S.O. is a Howard Hughes Predoctoral Fellow. We would like to thank Drs J Huguenard, G. K6hr, J. J. LoTurco, and K. J. Staley for valuable discussions; Dr J. Dempster for providing the Strathclyde Electrophysiology Software; and J. T. Palmer for expert technical assistance.

REFERENCES

1. Alger B. E. and Nicoll R. A. (1980) Spontaneous inhibitory postsynaptic potentials in hippocampus: mechanism for tonic inhibition. Brain Res. 20, t95-200. 2. Amaral D. G. (1978) A Golgi study of cell types in the hilar region of the hippocampus in the rat. J. comp. Neurol. 182, 851-904. 3. Arai H., Emson P. C., Mountjoy C. Q., Carrasco L. H. and Heizmann C. W. (1987) Loss of parvalbumin-immunoreactive neurones from cortex in Alzheimer-type dementia. Brain Res. 418, 164-169. 4. Barlow R. (1990) Cumulative frequency curves in population analysis. Trends pharmac. Sci. 11, 404-406. 5. Bekkers J. and Stevens C. (1988) NMDA and non-NMDA receptors are co-localized at individual excitatory synapses in cultured rat hippocampus. Nature 341, 230-233. 6. Bekkers J., Richerson G. and Stevens C. (1990) Origin of variability in quantal size in cultured hippocampal neurons and hippocampal slices. Proc. natn. Acad. Sci. U.S.A. 87, 5359-5362. 7. Berridge M. J. (1990) Calcium oscillations. J. biol. Chem. 265, 9583-9586. 8. Blanton M. G., LoTurco J. J. and Kriegstein A. R. (1989) Whole cell recording from neurons in slices of reptilian and mammalian cerebral cortex. J. Neurosci. Meth. 30, 203--210. 9. Bormann J. (1988) Electrophysiology of GABA A and GABA Dreceptor subtypes. Trends Neurosci. 11, 112 116. 10. Bowery N. G. (1983) Actions and Interactions o f GABA and Benzodiazepines. Raven Press, New York. 11. Bowery N. G., Hill D. R., Hudson A. L., Doble A., Middlemiss D. N., Shaw I. and TurnbuU M. (t980) (-)-Bactofen decreases neurotransmitter release in mammalian CNS by an action at a novel GABA receptor. Nature 283, 92-94. 12. Bowery N. (1989) GABA B receptors and their significance in mammalian pharmacology. Trends pharmac. Sci. 10, 401-407. 13. Brown K. M. and Dennis J. S. (1972) Derivative free analogs of the Levenberg-Marquardt algorithms for non-linear least squares approximation. Numer. Math. lg, 289-297. 14. Busch C. and Sakmann B. (1990) Synaptic transmission in hippocampal neurons: numerical reconstruction of quantal IPSCs. Cold Spring Harbor Symp. quant. Biol. 55, 69-80. 15. Caceci M. S. and Cacheris W. P. (1984) Fitting curves to data: the simplex algorithm is the answer. Byte 9, 340-362. 16. Chen Q. X., SteLzer A., Kay A. R. and Wong R. K. S. (1989) GABA^ receptor function is regulated by phosphorylation in acutely dissociated guinea-pig hippocampal neurones. J. Physiol. 420, 207-221. 17. Collingridge G. L., Gage P. W. and Robertson B. (1984) Inhibitory post-synaptic currents in rat hippocampal CA1 neurones. J. Physiol. 356, 551-564. 18. Davies S. N. and Collingridge G. L. (1989) Role of excitatory amino acid receptors in synaptic transmission in area CAI of rat hippocampus. Proc. R. Soc. B. 236, 373-384. 19. Davies C. H., Davies S. N. and Collingridge G. L. (1990) Paired-pulse depression of monosynaptic GABA-mediated inhibitory postsynaptic responses in rat hippocampas. J. Physiol. 424, 513-531. 20. Deisz R. A. and Prince D. A. (1989) Frequency-dependent depression of inhibition in guinea-pig neocortex in vitro by GABA B receptor feed-back on GABA release. J. PhysioL 412, 513-542. 21. Del Castillo J. and Katz B. (1954) Changes in endplate activity produced by presynaptic polarization. J. Physiol. 124, 586-604. 22. Doze V. A., Cohen G. A. and Madison D. V. (1991) Synaptic localization of adrenergic disinhibition in the rat hippocau~pus. Neuron 6, 889-900. 23. Edwards F. A., Konnerth A., Sakmann B. and Takahashi T. (1989) A thin slice preparation for patch clamp recordings from neurones of the mammalian central nervous system. Pflfigers Arch. 414, 600-612. 24. Edwards F. A., Konnerth A. and Sakmann B. (1990) Quantat analysis of inhibitory synaptic transmission in the dentate gyrus of rat hippocampal slices: a patch-clamp study. J. Physiol. 430, 213-249. 25. Fatt P. and Katz B. (1952) Spontaneous subthreshold activity at motor nerve endings. J. Physiol. 117, 109-128.

Decay kinetics and frequency of IPSCs

31

26. Forscher P. and Oxford G. S. (1985) Modulation of calcium channels by norcpinephrine in internally dialyzed avian sensory neurons. J. gen. Physiol. 85, 743-763. 27. Freund T. F. and Antal M. (1988) GABA-containing neurons in the septum control inhibitory interneurons in the hippocampus. Nature 336, 170-173. 28. Gage P. W. and Robertson B. (1985) Prolongation of inhibitory postsynaptic currents by pentobarbitone, halothane and ketamine in CA1 pyramidal cells in rat hippocampus. Br. J. Pharmac. 85, 675-681. 29. Gambrana C., Bcattie C. E., Rodriguez Z. R. and Siegel R. E. (1991) Region-specific expression of messenger RNAs encoding GABA A receptor subunits in the rat brain. Neuroscience 45, 423-432. 30. Gardner D. and Stevens C. (1980) Rate-limiting step of inhibitory post-synaptic current decay in Aplysia buccal ganglia. J. Physiol. 304, 145-164. 31. Gray R. and Johnston D. (1985) Rectification of singie GABA-gated chloride channels in adult hippocampal neurons. J. Neurophysiol. 54, 134-141. 32. Gyenes M., Farrant M. and Farb D. H. (1988) "Run-down" of GABA Areceptor function during whole-cell recording: a possible role for phosphorylation. Molec. Pharmac. 34, 719-723. 33. Gyenes M., Gibbs T. T. and Farb D. H. (1991) ATP-dependent run down of GABA^ receptor function during whole-cell recording is stimulated by pcntobarbital. Soc. Neurasci. Abstr. 17, 1523. 34. Harrison N. L., Lange G. D. and Barker J. L. (1988) (-)Baclofen activates presynaptic GABA B receptors on GABAergic inhibitory neurons from embryonic rat hippocampus. Neurasci. Lett. ~ , 105-109. 35. Heinemann U., Hamon B. and Konnerth A. (1984) GABA and baclofen reduce changes in extracellular free calcium in area CA1 of rat hippocampal slices. Neurosci. Lett. 47, 295-300. 36. Hochner B., Parnas H. and Parnas I. (1989) Membrane depolarization evokes neurotransmitter release in the absence of calcium entry. Nature,342, 433-435. 37. Inoue M., Oomura Y., Yakushiji T. and Akaike N. (1986) Intracellular calcium ions decrease the affinity of the GABA receptor. Nature 324, 156-158. 38. Katz B. (1962) The transmission of impulses from nerve to muscle, and the subcellular unit of synaptic action. Proc. R. Soc. Lond. B. 155, 455-479. 39. Katz B. (1969) The Release o f Neurotransmitter Substances. Thomas, Springfield, IL. 40. King G. L., Knox J. J. and Dingledine R. (1985) Reduction of inhibition by a benzodiazcpine antagonist, Rol 5-1788, in the rat hippocampal slice. Neuroscience 15, 371-378. 41. Krogsgaard-Larsen P., Scheel-Kruger J. and Kofod H. (1979) GABA-Neurotransmitters. Munkgaard Press, Copenhagen. 42. Levitan E. S., Schoficld P. R., Burt D. R., Rhce L. M., Wisden W., Kohler M., Fujita N., Rodriguez H. F., Stepbenson A., Darlinson M. G., Barnard E. A. and Seeburg P. H. (1988) Structural and functional basis for GABA A receptor heterogeneity. Nature 335, 76-79. 43. Lilcy A. W. (1956) The effectsof prcsynaptic polarization on the spontaneous activityat the mammalian neuromuscular junction. ]. Physiol. 134, 427-433. 44. LoTurco J. J., Mody I. and Kriegstcin A. R. 0990) Differential activation of glutamate receptors by spontaneously released transmitter in slices of ncocortcx. Neurosei. Lett. 114, 265-271. 45. MacDonald J. F., Mody I. and Salter M. W. (1989) Regulation of N-methyl-D-aspartate receptors revealed by intracellular dialysis of routine neurones in culture. J. Physiol. 414, 17-34. 46. MacDonald R. L., Rogers C. J. and Twyman R. E. (1989) Barbiturate regulation of kinetic properties of the GABAA receptor channel of mouse spinal neurones in culture. J. Physiol. 417, 483-500. 47. MacLennan A. J., Brecha N., Khrcstchatisky M., Stcrnini C., Tillakaratne N. J. K., Chiang M.-Y., Anderson K., Lai M. and Tobin A. J. (1991) Independent cellularand ontogenic expression of mRNAs encoding three ~ polypeptides of the rat GABA^ receptor. Neuroscience 43, 369-380. 48. Maglcby K. L. and Stevens C. (1972) A quantitative description of end-plate currents. J. Physiol. 223, 173-197. 49. Malinow R. and Tsien R. W. (1990) Presynaptic enhancement shown by whole-cell recordings of long-term potentiation in hippocampal slices.Nature 346, 177-190. 50. Marry A. and Neher E. 0983) Tight-seal whole-cell recordings. In Single-Channel Recording (cds Sakmann B. and Neher E.), pp. 107-122. Plenum Press, New York. 51. Mathers D. A. (1987) The GABA^ receptor: new insights from single channel recording. Synapse I, 96-101. 52. Mattcson D. R., Kriebel M. E. and Llados F. (1981) A statisticalmodel indicates that miniature end-plate potentials and unitary evoked end-plate potentials are composed of subunits. J. theor. Biol. 90, 337-363. 53. Miles R. and Wong R. K. S. 0984) Unitary inhibitory synaptic potentials in the guinea-pig hippocampus in vitro. J. Physiol. 356, 97-113. 54. Mishina M., Takai T., Imoto K., Noda M., Takahashi T., Numa S., Methfessel C. and Sakmann B. (1986) Molecular distinction between fetal and adult forms of muscle acetylcholine receptor. Nature 321, 406-411. 55. Mody I. and Otis T. S. (1991) The postsynaptic actions of spontaneously released GABA. Soc. Neurosci. Abstr. 17, 526. 56. Mody I., Tanelian D. L. and MacIver M. B. (1991) Halothane enhances tonic neuronal inhibition by elevating intracellular calcium. Brain Res. 538, 319-323. 57. Mfiller W. and Misgeld U. (1990) Inhibitory role of dentate hilus neurons in guinea pig hippocampal slice. J. Neurophysiol. 64, 46-56. 58. Nedler J. A. and Mead R. (1965) A simplex method for function minimization. Comput. J. 7, 308-313. 59. Olsen R. W. and Tobin A. J. (1990) Molecular biology of GABA^ receptors. Fedn Am. Soc. exp. Biol. J. 4, 1469-1480. 60. Otis T. S. and Mody I. (1992) Differential activation of GABA^ and GABA, receptors by spontaneously released transmitter. J. Neurophysiol. 67, 227-235. 61. Otis T. S., Staley K. J. and Mody I. (1990) Characteristics of action potential independent spontaneous inhibitory currents. Soc. Neurosci. Abstr. 16, 166. 62. Otis T. S., Staley K. J. and Mody I. (1991) Perpetual inhibitory activity in mammalian brain slices generated by spontaneous GABA release. Brain Res. 545, 142-150. 63. Pawson P. A. and Grinnell A. D. (1989) Oscillation period of mepp frequency at frog neuromuscular junctions is inversely correlated with release efficacy and independent of acute Ca 2+ loading. Proc. R. Soc. Lond. B 237, 489-499.

32

T.S. OTIS and 1. MODY

64. Pusch M. and Neher E. (1988) Rates of diffusional exchange between small cells and a measuring patch pipette, l"fliiger.~ Arch. 411, 204-211. 65. Redman S. (1990) Quantal analysis of synaptic potentials in neurons of the central nervous system. Physiol. Rev. 70, 165 198. 66. Ribak C. E. and Seress L. (1983) Five types of basket cell in the hippocampal dentate gyrus: a combined Golgi and electron microscopic study. J. Neurocytol. 12, 577~i97. 67. Robert E. (1986) Failure of GABAergic inhibition: a key to local and global seizures. In Basic Mechanisms of Epilepsies: Molecular and Cellular Approaches (eds Delgado-Escueta A. V., Ward A. A., Woodbury D. M. and Porter R. J.), pp. 319-341. Advances in Neurology, Vol. 44. Raven Press, New York. 68. Roberts E., Chase T. N. and Tower D. B. (1976) GABA in Nervous System Function. Raven Press, New York. 69. Robertson B. and Wann K. T. (1984) The effect of temperature on the growth and decay times of miniature end-plate currents in the mouse diaphragm. Brain Res. 29~, 346 349. 69a. Ropert N. and Guy N. (1991) Serotonin facilitates GABAergic transmission in the CAI region of rat hippocampus in vitro. J. Physiol. 441, 121-136. 70. Ropert N., Miles R. and Korn H. (t 990) Characteristics of miniature inhibitory postsynaptic currents in CA 1 pyramidal neurones of rat hippocampus. J. Physiol. 428, 707-722. 71. Rozenberg F., Robain O., Jardin L. and Ben-Ari Y. (1989) Distribution of GABAergic neurons in late fetal and early postnatal rat hippocampus. Devl Brain Res. 50, 177 187. 72. Sakmann B., Edwards F. A., Konnerth A. and Takahashi T. (1989) Patch clamp techniques used for studying synaptic transmission in slices of mammalian brain. Quart. Jl exp. Psychol. 74, 1107-1118. 73. Scharfman H. E., Kunkel D. D. and Schwartzkroin P. A. (1990) Synaptic connections of dentate granule cells and hilar neurons: results of paired intracellular recordings and intracellular horseradish peroxidase injections. Neuroscience 37, 693-707. 74. Schwartz E. A. (1987) Depolarization without calcium can release y-aminobutyric acid from a retinal neuron. Science 238, 350 355. 75. Segal J. R., Ceccarelli B., Fesce R. and Hurblut W. P. (1985) Miniature endplate potential frequency and amplitude determined by an extension of Campbell's theorem. Biophys. J. 47, 183-202. 76. Seress L. and Ribak C. E. (1983) GABAergic cells in the dentate gyrus appear to be local circuit and projection neurons. Expl Brain Res. 50, 173-182. 77. Seress L. and Ribak C. E. (1988) The development of GABAergic neurons in the rat hippocampal formation. An immunocytochemical study. Devl Brain Res. 44, 197 209. 78. Shivers B. D., Killisch I., Sprengel R., Sontheimer H., K6hler M., Schofield P. R. and Seeburg P. H. (1989) Two novel GABA A receptor subunits exist in distinct neuronal subpoputations. Neuron 3, 327-337. 79. Sigel E. and Baur R. (1988) Activation of protein kinase C differentially modulates neuronal Na t, Ca ~ and 7-aminobutyrate type A channels. Proc. natn. Acad. Sci. U.S.A. 85, 6192-6196. 80. Sigel E. and Baur R. (1988) Allosteric modulation by benzodiazepine receptor ligands of the GABA^ receptor channel expressed in Xenopus oocytes. J. Neurosci. 8, 289-295. 81. Simon W. (1986) Mathematical Techniques for Biology and Medicine. Dover Publications, New York. 82. Staley K. J. and Mody I. (1991) Integrity of perforant path fibers and the frequency of action potential independent excitatory and inhibitory synaptic events in dentate gyrus granule cells. Synapse 9, 219-224. 82a. Staley K. J., Otis T. S. and Mody I. (1992) Membrane properties of dentate gyrus granule cells: comparison of sharp microelectrode and whole-cell recordings. J. Neurophysiol. 67 (in press). 83. Stelzer A., Kay A. R. and Wong R. K. S. (1988) GABAA-receptor function in hippocampal cells is maintained by phosphorylation factors. Science 241, 339-341. 84. Tallman J. F., Paul S. M., Skolnick P. and Gallager D. W. (1980) Receptors for the age of anxiety: pharmacology of the benzodiazepines. Science 207, 274-281. 85. Twyman R. E., Rogers C. J. and Macdonald R. L. (1989) Differential regulation of y-aminobutyric acid receptor channels by diazepam and phenobarbital. Ann. Neurol. 25, 213 220. 86. Van der Kloot W. (1989) Statistical and graphical methods for testing the hypothesis that quanta are made up of subunits. J. Neurosci. Meth. 27, 81-89. 87. Weiss D. S. (1988) Membrane potential modulates the activation of GABA-gated channels. J. Neurophysiol. 59, 514-527. 88. Weiss D. S., Barnes E. M. Jr and Hablitz J. J. (1988) Whole-cell and single channel recordings of GABA-gated currents in cultured chick cerebral neurons. J. Neurophysiol. 59, 495-513. (Accepted 10 March 1992)