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Journal of Physiology (1992), 445, pp. 261-276 With 7 figures Printed in Great Britain

EXCITATORY AND INHIBITORY SYNAPTIC CURRENTS AND RECEPTORS IN RAT MEDIAL SEPTAL NEURONES

BY R. SCHNEGGENBURGER, J. LOPEZ-BARNEO* AND A. KONNERTHt From the Max-Planck Institut fur biophysikalische Chemie, D-3400 Gittingen, Germany and the * Departamento de Fisiologia y Biof'sica, Facultad de Medicina, Universidad de Sevilla, 41009 Sevilla, Spain

(Received 12 February 1991) SUMMARY

1. A thin-slice preparation was used to study the postsynaptic potentials and the underlying currents of visually identified rat medial septal (MS) neurones under tight-seal voltage- and current-clamp conditions. 2. Upon stimulation of the afferent fibres, all MS neurones exhibited a sequence of excitatory-inhibitory postsynaptic potentials (EPSP-IPSP). Under voltage clamp, with potassium glutamate as internal solution and at negative holding potentials (Vh), this synaptic pattern appeared as an initial inward current followed by a longer lasting outward current. 3. The inward postsynaptic current was completely abolished by 5 ftM-6-cyano-7nitroquinoxaline-2,3-dione (CNQX) whereas the outward current disappeared in the presence of 10 /tM-bicuculline. Thus the major excitatory and inhibitory synaptic inputs were identified as being due to activation of quisqualate/kainate glutamatergic and y-aminobutyric acid (GABAA) receptors, respectively. 4. At positive Vh a CNQX-resistant component of the excitatory postsynaptic current (EPSC) was revealed. This component was slower than the one mediated by the quisqualate receptor and was abolished by 3-3(2-carboxypiperazine-4-yl)propyl1-phosphonate (CPP), indicating that N-methyl-D-aspartate (NMDA) receptors are involved in excitatory synaptic transmission in MS cells. The existence of the two main subtypes (NMDA and non-NMDA) of glutamatergic receptors in MS neurones was also confirmed by the responses of the neurones to bath application of the different agonists (glutamate, quisqualate, kainate and NMDA). 5. The CNQX-sensitive EPSC had a reversal potential near 0 mV. The fast rise time ( 0 7 ms) indicates a somatic location of the excitatory synapses. The relaxation kinetics of the fast EPSC were fitted by a single exponential function with a time constant of 1 13 + 0-1 ms. This parameter was independent of Vh. Fast EPSCs were blocked by CNQX in a dose-dependent manner (dissociation constant, KD = 0 2 /M). 6. Inhibitory postsynaptic currents (IPSCs) were studied in symmetrical chloride solutions after blockade of the excitatory receptors. The current-voltage relation -

t To whom correspondence should be addressed. MS 9146

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was linear and reversed at 0 mV. The IPSCs had a fast rise time and their decay was best fitted by the sum of two exponentials with time constant of approximately 20 and 50 ms (Vh = -60 mV). The IPSCs were abolished by bicuculline (KD = 1 tM), a selective antagonist of GABAA receptors. As expected, bath application of GABA produced large whole-cell currents. 7. In many cells, in addition to the usual EPSP-IPSP sequence, failures of either the EPSP or the IPSP were frequently observed during the experimental protocol. These results are consistent with a feed-forward type of inhibition in the large MS neurones. In addition, the pattern and kinetic properties of these synaptic inputs seem to determine the rhythmical firing typical of MS cells. INTRODUCTION

It is well established that the medial septum/diagonal band-hippocampal projection, one of the major basal forebrain cholinergic pathways, participates in the generation of the electroencephalogram 'theta' rhythm (Green & Arduini, 1954; Petsche, Stumpf & Gogolak, 1962; Apostol & Creutzfeldt, 1974; Dutar, Lamour & Jobert, 1985; Bland & Bland, 1986). This slow rhythmical activity, necessary for the normal mnemonic functions of the hippocampus, has gained clinical interest in recent years because in some mammalian species its impairment produces a syndrome similar to pre-senile dementia in humans (Gray & McNaughton, 1983; Buzsaki, Leung & Vanderwolf, 1984), and a loss of basal forebrain cholinergic neurones is associated with Alzheimer's disease (Whitehouse, Price, Struble, Clark, Coyle & DeLong, 1982; Sarter, Schneider & Stephens, 1988). Nevertheless, the mechanisms underlying the generation of the 'theta' rhythm are not well known. Spontaneous neuronal discharges recorded from the medial septum (MS) of alert animals have not been observed in septal slices (Segal, 1986; Alvarez de Toledo & Lopez-Barneo, 1988). However, guinea-pig MS neurones studied in vitro by intracellular recordings have membrane ionic conductances that on depolarization make them capable of generating bursts of action potentials; thus it has been proposed that the rhythmical bursting activity of MS neurones could be the result of the intrinsic membrane properties accentuated by their synaptic inputs (Alvarez de Toledo & Lopez-Barneo, 1988). Interestingly, it has been suggested that the 'theta' rhythm requires recurrent hippocampal synaptic modulation of MS neuronal activity (McLennan & Miller, 1974a) and/or asynchronous synaptic activity by brain stem afferents terminating in the MS area (for a recent review see Stewart & Fox, 1990). Although it is known that MS neurones receive, either directly or indirectly via the lateral septum, afferents from the hippocampus and other brain regions, the physiology of their synapses is practically unknown. Field potential studies have shown that MS neurones receive strong excitation through the fornix (McLennan & Miller, 1974a). In addition, there is morphological evidence indicating the existence of y-aminobutyric acid (GABA)-ergic synapses in the large MS cells (Onte'niente, Geffard, Campistron & Calas, 1987) and physiological studies show that application of GABA into the MS inhibits the spontaneous bursting discharges (McLennan & Miller, 1974b; Lamour, Dutar & Jobert, 1984; Dutar, Rascol & Lamour, 1989) and hyperpolarizes MS neurones (Segal, 1986). The aim of the present work was to study the pattern and basic properties of the

SYNASPTIC CLURRENTS IN MEDIAL SEPTUMl26M 263

postsynaptic currents of MS neurones upon stimulation of the afferent fibres and to identify the synaptic receptors involved. These questions were addressed by recording synaptic currents in voltage-clamped rat MS neurones. We used a thin-slice preparation that allows patch clamping of visually identified neurones (Edwards, Konnerth, Sakmann & Takahashi, 1989).

Fig. 1. Appearance of a medial septal neurone injected with Lucifer Yellow and location of (dye-filled) patch pipette and stimulating electrode. Note the absence of prominent dendrites. Calibration bar = 20 ,um.

METHODS

Young (14-20-day-old) Wistar rats were decapitated and a piece of the brain containing the septal region was rapidly isolated and placed in ice-cold saline solution. Thin septal coronal slices, 10(-150 tm thick, were cut with a vibratome and kept at 33 °C until use in saline solution bubbled with 95 % 02-5 % CO2. During the experimental procedure a slice was transferred to the recording chamber where it was continuously superfused with the standard solution. The procedures for cleaning and patch clamping of individual neurones were the same as previously described (Edwards et al., 1989). All experiments were performed using the whole-cell variant of the patchclamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981) with fire-polished borosilicate patch pipettes of 2-4 AMQ coated with Sylgard resin (General Electric Company, NY, USA). Ionic currents were recorded with an EPC-7 patch-clamp amplifier (List Electronics, Germany). Signals were stored on videotape using a PCM/VCR device (Instrutech, NY, USA). For later analysis, signals were low-pass filtered to 5 kHz with an 8-pole Bessel filter (Frequency Devices, MA, USA), digitized, and stored in a VME-bus computer system. A glass pipette with 5-10 ,um tip diameter filled with external solution was gently applied to the slice for focal electrical stimulation with square pulses of 100-300 Its duration and 2-10 V in amplitude. In most experiments the stimulating electrode was placed within the fibre system along the mid-line of the medial septum,

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at a distance of 50-100 ,um from the recording position. The data presented here results from stable recordings (more than 45 min) in thirty-one MS neurones. These neurones of the medial septum-diagonal band complex were identified by their location and relatively large size (15-25 ,um diameter). The location and morphology of the cells studied are the same as those of cholinergic MS neurones identified in rats of the same age (Armstrong, Bruce, Hersh & Gage, 1987). The arrangement for recording and stimulation, as well as the appearance of a neurone injected with Lucifer Yellow are illustrated in Fig. 1. The external solution was (mM) 125 NaCl, 2-5 KCl, 2 CaCl2, 1 MgCl2, 1 NaH2PO4, 26 NaHCO3, 10 glucose. This solution was continuously bubbled with 95 % 02-5% CO2 to increase its oxygen content and to maintain a pH of 7-4. The standard internal solution contained (mM) 140 CsCl, 4 Na-ATP, 2 MgCl2, 10 EGTA, 10 HEPES, with pH 7-3. In most experiments Cs' was used as the major intracellular cation in order to block outward current through K+ channels and to facilitate the measurement of synaptic currents. However, in some experiments we also employed an internal solution with potassium glutamate, the composition of which is given in the figure legends. Experiments were performed at room temperature (approximately 22°C). The dendrite membrane of neurones subjected to whole-cell patch clamp sometimes escapes voltage control; thus, synaptic currents generated in regions remote from the soma show distorted kinetics (see recent reports by Hestrin, Nicoll, Perkel & Sah, 1990 and Llano, Marty, Armstrong & Konnerth, 1991). However, we think that in our preparation this problem is minimal because the dendritic arbour is relatively small (see Fig. 1), and because the synapses are most likely located in regions which are electrically close to the cell body. Apart from other evidence discussed later, an indication of good space and voltage clamp was that capacity transients evoked by 10 mV hyperpolarizing square pulses from the holding potential of -70 mV had an exponential decay and could be perfectly cancelled by a single time-constant voltage command signal. Application of drugs was done by changing the control perfusion solution in the bath to a solution containing the desired compound. A complete exchange of the external solution could be achieved in less than 30 s. The following drugs were used in the present experiments; tetrodQtoxin (TTX), GABA, bicuculline, glutamate, quisqualate, kainate, N-methyl-D-aspartate (NMDA) (all from Sigma, Germany). The glutamatergic antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 3(2-carboxypiperazine-4-yl)propyl1-phosphonate (CPP) were purchased from Tocris Neuramin Ltd, Bristol, UK. RESULTS

Identification of the major synaptic inputs In current-clamp recordings from MS neurones, single shock stimulation of the afferent fibres elicited a typical pattern of synaptic potentials consisting of a relatively fast excitatory postsynaptic potential (EPSP) followed by an inhibitory postsynaptic potential(IPSP) lasting more than 100 ms (Fig. 2A). When the EPSP exceeded a certain threshold, the cells generated action potentials of large amplitude followed by long lasting hyperpolarizations a feature previously noted in intracellular recordings from MS neurones (Segal, 1986; Alvarez de Toledo & LopezBarneo, 1988). This EPSP-IPSP sequence could be observed in all cells (n 7) at the normal resting potential (near -55 mV) dialysed with the potassium glutamate solution. The EPSP amplitude was enhanced on membrane hyperpolarization and decreased in amplitude on membrane depolarization. Similar shifts in the membrane potential had the opposite effect on IPSPs (Fig. 2B). All synaptic responses were abolished when TTX (1 #m) was added to the external solution which indicates that propagated action potentials were required to trigger transmitter release (not =

shown). The ionic currents underlying the two synaptic potentials could be clearly recorded in the whole-cell voltage-clamp configuration as illustrated in Fig. 3A. The top trace (a) is the current elicited by a single electrical stimulus in a cell dialysed with the potassium glutamate solution. At this holding potential (-40 mV) the stimulus

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evoked an initial fast inward current of about 100 pA of amplitude, that generated the EPSP, followed by a smaller but longer lasting outward current, responsible for the slower IPSP observed under current clamp. The outward current was eliminated by 10 uM-bicuculline (trace b), a competitive antagonist of the GABAA receptor, B

A

-40 mV

-55 mV-

20 ms

5 mV

50 ms

Fig. 2. Current-clamp recordings in MS neurones after stimulation of afferent fibres. A, at resting membrane potential (-55 mV) stimulation (250 /us, 4-3 V) evokes a sequence of excitatory-inhibitory synaptic potentials. Large EPSPs can reach threshold for action potential generation. B, superimposed sweeps of the EPSP-IPSP sequence recorded at different membrane potentials. Membrane potential changes were obtained by DC current injection into the cell, and the values are given next to each trace. Cells were filled with a modified internal solution containing 135 mM-potassium glutamate instead of CsCl (see Methods).

whereas the inward current disappeared after application of 5 jtM-CNQX, an agent that inhibits glutamate receptors of the non-NMDA type (trace c; Honore, Davies, Drejer, Fletcher, Jacobsen, Lodge & Nielsen, 1988). The effects of bicuculline and CNQX were completely reversible as shown by the bottom trace (d). Thus the EPSP and IPSP elicited in resting MS neurones by stimulation of neighbouring fibres are due, respectively, to the activation of non-NMDA glutamate and GABAA receptors. However, a significant percentage of NMDA receptors also exists in MS cells. The synaptic current generated by activation of these receptors could be revealed by blocking the bicuculline-sensitive inhibitory current component and holding the membrane potential at + 40 mV to relieve the block of NMDA receptor channels by external Mg2+ (Fig. 3B, trace a; Nowak, Bregestovski, Ascher, Herbet & Prochiantz, 1984; Mayer, Westbrook & Guthrie, 1984). At this membrane potential the excitatory postsynaptic current (EPSC) was outward and had two clear components: a fast component of large amplitude followed by a slow component of smaller size. Trace b shows that the fast EPSC was blocked by CNQX revealing the slow

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component of the EPSC in isolation. The slow EPSC was reversibly blocked by CPP, a potent antagonist of NMDA receptors (Davies, Evans, Herrling, Jones, Olverman, Poork & Watkins, 1986). Excitatory postsynaptic currents Figure 4A shows superimposed EPSCs on an expanded time scale. Each trace is the average of twenty consecutive responses recorded at holding potentials between B

Control

A

v a

Control

.V a

Bic (10 pM) b

CNQX (5 pM) b

Bic (10 pM), CNQX (5 pM)

CNQX (5 um), CPP (5 gm)

c c

Wash-out of CPP

Wash-out

d _,I

d 100

pA

5

20

ms

pA

50 ms

Fig. 3. Major synaptic current components in MS neurones recorded under whole-cell voltage clamp. A, synaptic currents recorded at a holding potential (V') of -40 mV in the control external solution (a), and after addition of 10 IaM-bicuculline (Bic) (b) or 10 ,ambicuculline plus 5 ,uM-CNQX (c). The effects of these pharmacological agents were completely reversible (d). Each trace is the average of six consecutive recordings. The arrow-head marks the onset of the stimulation artifacts, which have been removed. Internal solution as in Fig. 2. B, synaptic currents recorded at a Vh of + 40 mV in the control external solution (a) and after addition of 5 jtM-CNQX (b) or 5 ,uM-CNQX plus 5 ,am-CPP (c). Wash-out of CPP is shown in d. Each trace is the average of six consecutive sweeps. The external solution contained, in all cases, 10,uM-bicuculline. The internal solution contained CsCl (see Methods).

-80 and + 40 mV. At negative membrane potentials large inward currents, of about 300 pA at -80 mV, are recorded in isolation due to activation of the CNQX-sensitive fast EPSC. With negative holding potentials the contribution of the NMDA

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component is almost negligible. At positive membrane potentials the current is outward and the fast component is followed by the slower NMDA component. The peak current (measured at the time indicated by the dotted line) as a function of the membrane potential is plotted in Fig. 4B. Here, as in most experiments, internal Cs' B

A

/ (pA)

Vh (mV)

100 pA 2 ms

D

C

10 T 0

/norm 0,5 100

5

.

+

pA

ms

o.o 0.01

1

100

[CNQX] (pM) Fig. 4. Properties of fast excitatory postsynaptic currents (EPSCs). A, excitatory currents recorded at various , (from -80 to +40 mV). Each trace is the average of twenty consecutive sweeps. The vertical dotted line marks the peak current amplitude. Arrowheads indicate the onset of stimulation. B, plot of the peak current amplitude as a function of V,. EPSC reversed at a membrane potential near 0 mV. C, time course of a single EPSC recorded at a VF of -60 mV. The rise time to half-maximal amplitude is indicated by the vertical dotted lines. The horizontal dashed line indicates halfamplitude. The decay of the EPSC was fitted by a single exponential function which is superimposed on the current trace. D, dose-response curve of the blockade of EPSCs by bath application of CNQX. Normalized peak currents (Inorm) recorded from three cells at a Vl7 of -60 mV are plotted as a function of CNQX concentration. The KD value of the fitted curve is 0-2 /LM. All experiments were done in the presence of 10 /aM-bicuculline.

was used instead of K+ to block voltage-dependent K+ channels and thus to favour an accurate measurement of synaptic currents. The reversal potential of the fast EPSC was near 0 mV, which indicates that the channels involved in the generation of the current were equally permeable to Na+ and Cs+ ions. In the presence of CNQX,

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the peak current-voltage relation of the slow NMDA component had, as in other central neurones (Hestrin et al. 1990; Keller, Konnerth & Yaari, 1991), a strong rectification at negative voltages (not shown). The kinetic properties of the fast EPSC are illustrated by the single trace of Fig. 4C, which was recorded at the holding potential of -60 mV. Stimulation is marked B Quisqualate (5 gM)

A Glutamate (50 gM)

2

250 pA

pA

1 min

1 min

D NMDA (50!uM)

C Kainate (20pM)

10 pA 1 min

250 pA 1 min

Fig. 5. Membrane currents induced by application of different glutamatergic agonists. A, 50 /LM-glutamate; B, 5 uM-quisqualate; C, 20 /M-kainate; D, 50 ,M-NMDA. All drugs were applied for 1 min at holding potentials of + 40 and -60 mV (upper and lower traces, respectively). Arrows indicate the start of agonist application.

by an arrow, and rise time to half-amplitude by two vertical dotted lines. The current has a fast rising phase followed by complete relaxation to the holding current level in 5 or 6 ms. The decay is well fitted by a single exponential which is superimposed on the current trace. At this membrane potential the average time to half-amplitude and the decay time constant were, respectively, 0 45 + 0-08 (equivalent to a 10-90% rise time of approximately 0-7 ms) and 1 13 + 0-11 ms (mean+ S.D., n = 6). No voltage dependence of the two kinetic parameters was

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observed (not shown). The dose-dependent inhibitory action of CNQX on the fast EPSC is shown by the normalized plot of Fig. 4D with data obtained from three cells. Large concentrations (10 AM) of CNQX completely eliminated the synaptic current with a KD value of 0-2 aM. The various glutamatergic receptor types responsible for synaptic transmission in MS neurones were identified by measuring the current induced in cells briefly exposed to bath application of the different agonists. A summary of the electrical responses is shown in Fig. 5. Each agonist was tested at two different holding potentials (-60 and + 40 mV), and the instant of application, which lasted for 1 min, is indicated by the vertical arrows. Glutamate, probably one of the physiological transmitters, produced a current much smaller at -60 than at + 40 mV indicating that it preferentially activated NMDA receptors. Among the three selective agonists tested, quisqualate elicited the most powerful response followed in potency by kainate and NMDA. At + 40 mV, a membrane potential at which NMDA channels are not blocked by Mg2+, NMDA (50 JLM) elicited an outward current of almost 0 4 nA, again demonstrating the existence of a significant proportion of NMDA glutamatergic receptors in MS cells.

Inhibitory postsynaptic currents Inhibitory postsynaptic currents (IPSCs) were studied in isolation after blockade of glutamatergic synapses with CNQX and CPP. Superimposed currents elicited by several constant consecutive stimuli (200 ,us and 6 2 V) are shown in Fig. 6A (top panel). Since we employed symmetrical chloride concentrations these currents are inward at -60 mV. The traces also illustrate the fluctuation in current amplitude, which was seen in all cells tested (n = 14). This phenomenon was not studied in detail but, as reported by others (Edwards, Konnerth & Sakmann, 1990), it probably represents the all-or-none nature of transmission at single synaptic boutons where the number of postsynaptic channels determines the size of quantal events. The bottom trace, an average of twelve single consecutive sweeps, illustrates that the IPSC has a fast rise time and a decay that can be fitted by the sum of two exponentials with time constants of approximately 20 and 50 ms at -60 mV. Figure 6 C shows a plot of average peak IPSC amplitude as a function of the membrane potential. In our ionic conditions (symmetrical Cl-) the current-voltage curve was linear and the reversal potential was at 0 mV. As expected for highly Cl-permeable channels, changing Cl- gradients shifted the reversal potential of the IPSP (Fig. 2) or the IPSC (Fig. 3). Bicuculline produced a dose-dependent attenuation of the inhibitory currents (Fig. 6B) with complete elimination at 10 tM (KD 1/tM). These results identify the receptors mediating inhibitory synapses at MS neurones as belonging to the GABAA type. In fact, transient bath application of GABA (20,aM, Fig. 6D) produced large whole-cell currents whose amplitude and direction were determined by the electrochemical driving force for chloride ions.

Variability in the synaptic inputs Although upon synaptic activation all MS neurones displayed the EPSP-IPSP sequence illustrated in Fig. 2, complete failures of either the excitatory or the inhibitory input were frequently observed. This phenomenon is illustrated in Fig. 7A

R. SCHNEGGENB URGER AND) OTHERS

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by voltage recordings obtained from a neurone under current clamp and dialysed with the potassium glutamate solution. Records a-c, respectively, show responses consisting of an EPSP, an IPSP, or the more usual EPSP-IPSP pattern. The last response (c) is quite similar to the sum of trace a and b (record d). Although we could A

B

~~300 |y

/~~~~~~-

I(PA) 100 lOOpA

50

C

0.1

ms-

1

10

[Bicuculline] (PM) D

/(PA) 150 Vh (MV) -80 -40

40

+40 mV -60 mV

-150

250 pA 1 min

Fig. 6. Properties of inhibitory postsynaptic currents (IPSCs). A, upper panel, four consecutive IPSCs recorded at a E~of -60 mV' are superimposed to illustrate the variability in postsynaptic current amplitude. Lower panel. the decay of IPSCs (average of twelve consecutive recordings at -60 mV) is fitted by the sum of two single exponentials (continuous lines). The double exponiential function (dots) superimposes perfectly on the decay phase of the current trace. B. blockade of IPSC by bicuculline. Means+ ±S.D. (n = 10. at each bicuculline concentration) of IPSC peak current amplitude are plotted as a function of bicuculline concentration. The K, value for the fitted curve is 1 aIm. All IPSCs were recorded in the presence of 5 /am-CNQX and 5 /4am-CPP. C, plot of the IPSC peak current versus Vh. Current-voltage relation is linear and the reversal potential is at 0 mV. D, membrane currents generated by bath application of GABA (20 /Im) at Vh of + 40 and - 60 mV. Application of the drug lasted 40 s and the onset is indicated by

arrows.

not assess whether failures occurred because the afferent fibres failed to generate action potentials, the fact that sometimes only EPSPs or only IPSPs occurred

suggests that excitation and inhibition of MS neurones are mediated by different fibres. A simple synaptic circuit that could account for our observations is depicted in Fig. 7B. The large septo-hippocampal MS neurones most likely receive direct glutamatergic excitatory fibres, whereas inhibition probably involves local GABAergic interneurones. These ideas are in agreement with previous work in which, based on field potential analysis, it was suggested that MS neurones are

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directly excited by fornix afferents and that inhibition observed after fimbrial stimulation is mediated by interneurones (McLennan & Miller, 1974a). A more recent immunocytochemical study (Onteniente et al. 1987) has confirmed the existence of small GABAergic interneurones in the MS region. B

A a

b

C

10 mV d

50 ms

Fig. 7. Afferent stimulation of the large MS neurones. A. recording in a MS cell of an EPSP (a), an IPSP (b) or the usual EPSP-IPSP sequence (c). The computer summation of traces a and b are indicated in d. B, local synaptic circuitry of the medial septum with a GABAergic interneurone (0) synapsing onto a large MS neurone. DISCUSSION

Although the large neurones of the MS-diagonal band complex have special neurophysiological (and perhaps pathophysiological) relevance, the nature and physiology of their afferent synapses were unknown. Our results indicate that these cells generate strong excitatory and inhibitory postsynaptic currents in response to stimulation of the afferent fibres. In neurones from young animals we could reliably record the postsynaptic currents underlying the synaptic potentials and study some of their pharmacological and biophysical properties. Excitatory current-s and receptors Synaptic excitation of MS neurones mainly involves activation of non-NMDA receptors but an appreciable NMDA component, that becomes significant on membrane depolarization, also exists. Similar types of excitatory receptors are known to participate in synapses of other central neurones (Blake, Brown & Collingaridgye, 1988; Gallagher & Hasuo, 1989; Hestrin et at. 1990; Konnerth, Keller & Lev-Tov, 1990; Keller et at. 1991). Among the different glutamatergic receptor agonists tested, quisqualate was the most powerful in depolarizing MS cells. Glutamate, which could be the physiological transmitter, had, however, an effect very similar to that of NMDA, with strong rectification at negative membrane potentials due to blockade of the channels by extracellular Mg"~ (Mayer et at. 1984; Nowak et at. 1984).

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The time course of the fast and slow components of the stimulus-evoked EPSCs could be compared in isolation by using selective antagonists. The kinetics of the NMDA EPSC were not studied in detail but they were clearly slower than those of the fast, CNQX-sensitive, EPSC. Similar differences in the kinetic properties have been reported for the two EPSC components in other central neurones (Konnerth TABLE 1. Kinetics of quisqualate/kainate EPSCs recorded in patch-clamped central neurones* Time constant Age (days) Temperature (°C) of decay (ms) Reference Neuronal type 3-7 + 14 Konnerth et al. 1990 5-9 22-24 Spinal cord motoneurones Hestrin et al. 1990 67 + 13 20-28 Room temperature Pyramidal cells of hippocampus 5 1 + 2-8 Sah et al. 1990 20-28 Room temperature Hippocampal interneurones Llano et al. 1991 8-22 20-22 Cerebellar Purkinje cells 8-3 + 1 6 Parallel fibre 6-4 + 1 1 Climbing fibre 21-24 Keller et al. 1991 30-90 10-21 Granule cells of hippocampus 11 +01 This paper 14-20 20-22 Medial septal neurones * All experiments were performed on dialysed neurones from rat brain slices using the whole-cell configuration of the patch-clamp technique. Time constant values are given as means+ S.D.

et al. 1990; Sah, Hestrin & Nicoll, 1990; Keller et al. 1991). The fast EPSC, mediated by the quisqualate/kainate glutamatergic receptor type, had a rise time of approximately 0 7 ms and an exponential decay with a time constant of 141 ms. These kinetic parameters are probably dependent on the intrinsic biophysical properties of the receptor-ionic channel complex but, as discussed in detail previously (Hestrin et al. 1990; Llano et al. 1991), they can be influenced by the electrotonic structure of the neurone if the synapses are electrically remote from the soma (see also Johnston & Brown, 1983). The compact morphology of MS cells, the reversal potential of the EPSC at 0 mV (similar to the reversal potential measured for single glutamate-activated channels in membrane patches), and the fast kinetics of the synaptic currents altogether suggest that in our preparation space and voltage clamp were reasonably good. The rate of desensitization of the postsynaptic receptors has been postulated to account for the time course of decay of the synaptic signal (Trussell & Fischbach, 1989), and in fact, our decay time constant measurement is similar to the mean channel lifetime ( 1 ms) of the smallconductance channels activated by glutamate (Cull-Candy & Usowickz, 1989). The kinetic parameters of the CNQX-sensitive fast EPSC recorded in MS neurones are faster than those measured in any other dialysed central neurone so far studied with the patch-clamp technique. To facilitate comparison, decay time constants and some of the relevant experimental variables in different preparations are summarized in Table 1. Decay time constants of less than 1 ms have been found for the somatic excitatory synapses between primary afferents and cat spinal cord motoneurones (0 3 ms at 37 °C using a single-microelectrode voltage clamp; Finkel & Redman, 1983). Differences in the kinetic properties of non-NMDA EPSCs could reflect a %

273 SYNAPTIC CURRENTS IN MEDIAL SEPTUM variability in the molecular properties of the receptor-ionic channel complexes found in various neuronal types but, as discussed above, they may also depend on the proximity of the synapses with respect to the soma. The fast kinetics and the relativet small dispersion of the average time constant measured in MS neurones (see Table 1) suggest that the excitatory synapses are close to the cell body. This conclusion is in accord with recent immunocytochemical work (Onteniente et al. 1987) where it was found that several types of non-GABAergic synaptic boutons are located around the soma of the large MS neurones.

Inhibitory currents All MS neurones studied displayed stimulus-evoked IPSCs that were responsible for the generation of the long lasting IPSPs observed under current clamp. IPSPs and IPSCs of MS neurones resemble inhibitory synaptic signals recorded from other central neurones in their sensitivity to bicuculline and in their dependence on the Clgradient, which indicates that they were due to activation of the GABAA receptor type (Collingridge, Gage & Robertson, 1984; Gallagher & Hasuo, 1989; Edwards et al. 1990). The existence of GABAergic synapses in the medial septum was previously suggested by morphological studies demonstrating the existence of GABA immunoreactive boutons terminating on the soma of the large MS cells (Onteniente et al. 1987). In addition, it was known that local application of GABA hyperpolarizes MS neurones (Segal, 1986) and inhibits the spontaneous discharges of MS cells recorded in vivo (McLennan & Miller, 1974b; Lamour et al. 1984). The IPSCs of MS neurones had a fast rising phase comparable to the activation time of IPSCs observed in hippocampal CAI (Collingridge et al. 1984) and dentate gyrus (Edwards et al. 1990) neurones. In adult rat CAl neurones the decay of the EPSC follows a single exponential time course that at -58 mV has a time constant of 5 ms (25 °C; Collingridge et al. 1984). In contrast, in dentate gyrus granule cells from young (17-21-day-old) rats, the decay is fitted by two exponentials with time constants of 2 and 54 ms (-50 mV membrane potential and 21-23 °C; Edwards et al. 1990). Interestingly, the decay of IPSCs in MS neurones, although requiring double exponential fit, was much slower (time constants of 20 and 50 ms at -60 mV). Time constant values similar to our own have been observed in stimulus-evoked GABAergic IPSCs recorded in intermediate lobe cells of the hypophysis (Schneggenburger & Konnerth, 1990). These kinetic differences are likely to be due to variability in the subunit composition of the GABAA receptors among the various cell types rather than to distortion of the current signals by the electrotonic structure of the cells. In fact, it has been reported that GABAA-receptor channel subtypes with different conductance and gating properties can be assembled by a combination of different GABAA-receptor subunits (Verdoorn, Draguhn, Ymer, Seeburg & Sakmann, 1990).

Implications of the synaptic currents for MS function The kinetic properties of the EPSCs and IPSCs observed in the medial septum suggest that they may play an important part in the normal physiological activity of MS neurones. The burst firing typical of MS cells upon synaptic bombardment is probably favoured by the particularly fast kinetics of the quisqualate/kainate

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receptors which mediate most of the excitatory synaptic current generated at the resting potential of the cells (around -55 mV; Alvarez de Toledo & Lopez-Barneo, 1988). Both the long lasting IPSPs and the pronounced after-hyperpolarization, typical of the action potentials of MS neurones (Alvarez de Toledo & Lopez-Barneo, 1988), surely contribute to determine the duration of silent periods and therefore influence the rhythmicity of the cells. The NMDA receptors, mainly significant during the depolarized state of the cells, could be involved in long term modifications of the excitability of MS neurones, which is an aspect not studied yet. Glutamatergic and GABAergic synaptic receptors may also be involved in several pathophysiological conditions. Glutamate toxicity has been suggested to be responsible for neuronal death in Alzheimer's disease and injection into basal forebrain cholinergic nuclei of benzodiazepine receptor antagonists (which interact with the GABAA receptorchannel complex) has been assayed as therapeutical strategy in animal models of senile dementia (for recent reviews see Mayer & Westbrook, 1987; Sarter et al. 1988). The authors are grateful to Mrs F. Friedlein for technical help and to Dr Robert Chow for comments on the manuscript. This research was supported by the Deutsche Forschungs-

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