A Comparison of Spontaneous EPSCs in Layer II and Layer IV-V Neurons of the Rat Entorhinal Cortex In Vitro

Transcription

1 JOURNALOF NEUROPHYSIOLOGY Vol. 76, No. 2, August Printed in U.S.A. A Comparison of Spontaneous EPSCs in Layer II and Layer IV-V Neurons of the Rat Entorhinal Cortex In Vitro NICOLA BERRETTA AND ROLAND S. G. JONES Department of Pharmacology, University of Oxford, Oxford OX1 3QT, United Kingdom SUMMARY AND CONCLUSIONS 1. We have compared the characteristics of spontaneous excitatory postsynaptic currents (sepscs) in neurons of layer IV-V and layer II of the rat entorhinal cortex (EC) using whole cell voltageclamp recordings in a slice preparation. 2. The frequency of sepscs was similar in the two layers, but the events in layer IV-V had a larger mean amplitude, faster rise time, and were faster to decay. The difference in amplitude could be attributed to the presence of a population of larger events in the layer IV-V neurons that were not present in layer II. 3. Electrotonic length was greater in layer II neurons, suggesting that the difference in kinetics of the sepscs may be explained partly by electrotonic attenuation. 4. The frequency of sepscs in both layers was reduced by tetrodotoxin (TTX) to a similar extent ( 15-20%). However, the amplitude distribution was unchanged in layer II, whereas in layer IV-V TTX abolished most of the larger amplitude sepscs cyano-7-nitroquinoxaline-2,3-dione or 6-nitro-7-sulphamoylbenzo ( f ) -quinoxaline-2,3-dione, abolished most of the sepscs in neurons of both layers. However, even at negative holding potentials, a population of slower time-course sepscs remained in the presence of these antagonists. 6. The slow sepscs were more frequent in layer IV-V but had similar characteristics in both layers, being increased in amplitude at more positive holding potentials or in Mg*+ -free medium, and blocked by 2-amino-5phosphonovalerate (AP5 ). 7. AP5 alone (i.e., without addition of a-amino-3-hydroxy-5- methyl-4-isoxazolepropionic acid antagonists) reduced the peak amplitude and decay phase of sepscs in layer IV-V neurons but appeared to have little effect on amplitude and only a weak effect on decay phase in layer II. 8. Thus both layer IV-V and layer II neurons of the EC suffer continuous spontaneous excitation. However, layer IV-V neurons exhibit larger amplitude sepscs, probably mediated by release of multiple quanta of neurotransmitter. In addition, although both types of neurons display spontaneous excitation mediated by N- methyl-d-aspartate receptors, this component appears more pronounced in the deeper layers. INTRODUCTION Any scheme of hippocampal function must take into account the control of its activity exerted by the entorhinal cortex (EC). The EC receives converging inputs from primary sensory and higher-order association neocortical areas. In turn, it provides the major source of afferent input to the hippocampal formation. There is a topography to this projection such that layer II of the EC provides input mainly to the dentate gyrus, whereas layer III projects primarily to CA1 and CA3. There also may be a projection from layer IV to CAl. A large part of hippocampal output is directed back to the EC, primarily layer V, which redistributes infor- mation to the neocortex (Swanson et al. 1987; Witter 1993; Witter et al. 1989). Increasing attention is being paid to the role of the EC as a dynamic processor of synaptic activity entering and leaving the hippocampus. This role depends, to a large extent, on synaptic interactions within the EC as well as the properties of the different populations of constituent neurons. It has been suggested that the dynamic balance between inhibition and excitation in the neurons of the deep layer of the EC may differ from that in the superficial layers (Jones 1993 ), and this may be important in determining the treatment and destination of information entering and exiting the EC. The pivotal role of the EC is emphasized by considering dysfunction as well as function. For example, the EC suffers some of the most severe degenerative changes associated with Alzheimer s disease (Braak and Braak 1991). These changes effectively isolate the hippocampus from neocortical inputs and may be the basis of the learning and memory deficits associated with the disease. Evidence is also beginning to point to the EC as a primary site at which temporal lobe seizures may originate, propagate, and reverberate (e.g., Ben-Ari et al. 1981; Du et al. 1993; Geddes et al. 1990; Jones 1988; Jones and Heinemann 1988; Jones and Lambert 1990a,b; Rutecki et al. 1989; Spencer and Spencer 1994). On the basis of studies in a slice preparation of rat EC, we have suggested that the deeper layers (IV-V) may be particularly susceptible to epileptogenesis and may be the site of seizure initiation (Jones 1993; Jones and Heinemann 1988; Jones and Lambert 1990a). In contrast, synchronized epileptiform activity in layer II appears less severe (Jones 1994; Jones and Lambert 1990a), and these observations have prompted the view that deep layers may be seizure sensitive whereas superficial layers may be relatively seizure resistant (Jones 1993 ). A primary aim of this laboratory is to determine the properties of the neurons and the networks in these two layers that may impart these relative sensitivities to epileptogenesis. The recent use of whole cell patch-clamp recordings in cortical slices has revealed that most neurons suffer a perpetual bombardment of spontaneously released transmitters, both excitatory (glutamate) and inhibitory [ y-aminobutyric acid (GABA)] (e.g., LoTurco et al. 1990; Otis et al. 1991). It has been suggested that the balance between spontaneous inhibition and excitation may be an important factor in determining excitability in cortical networks and that disturbances in this balance could contribute to synchronized epileptiform discharges (Otis et al. 1991). We have embarked on an analysis of spontaneous synaptic activity in EC neurons and are making a comparison between neurons of layer /96 $5.00 Copyright The American Physiological Society 1089

2 N. BERRETTA AND R. S. G. JONES II and layers IV-V to determine if the properties of this activity could reflect their susceptibilities to epileptogenesis. The present study describes the physiological and pharmacological characteristics of spontaneous, glutamate-dependent synaptic excitation. Some of these results have been presented in abstract form (Berretta and Jones 1995a-c). METHODS Experiments were conducted on combined slices of EC and hippocampus from adult male Wistar rats ( g). Animals were decapitated after being anesthetized with an intramuscular injection of ketamine, and the brain was removed and placed in artificial cerebrospinal fluid (ACSF) chilled to 4OC. Entorhinalhippocampal slices (450~pm thick) were cut as described previously (Jones and Heinemann 1988 ) using a vibroslice ( Campden Instruments) and then transferred directly to the recording chamber where they were maintained at the interface between ACSF (34 t 0.2 C; 1 ml/min) and moist carbogen gas (95% 02-5% CO*). The ACSF contained (in n&i) 126 NaCl, 3 KCl, 24 NaHC03, 1.25 NaH,P04, 2 MgS04, 2 CaCl,, and 10 D-glucose. This was bubbled continuously with carbogen and had a ph of at the operating temperature. Whole cell recordings were obtained from neurons in layer IV- V and layer II of the EC, using the blind-patch technique (Blanton et al. 1989). Electrodes were pulled from borosilicate glass ( 1.2 mm OD, 0.69 mm ID; CEI) using a Narishige PP-83 electrode puller. They had resistances of 4-6 MSt and were filled with (in mm) 130 Cs-methansulphonate, 5 N-2-hydroxyethylpiperazine- N -2-ethanesulfonic acid, 0.5 ethylene glycol-bis (P-aminoethyl ether) -N,N, N,N -tetraaceit acid, 1 MgCl,, 1 NaCl, 0.34 CaC&, and 5 QX-3 14 (buffered to ph 7.3 with CsOH). Recording electrodes were placed under visual control in layer II or layer IV/V of the EC. A positive pressure was applied to the electrode before it touched the slice, then it was advanced slowly into the tissue until it approached a neuron, as revealed by a small increase in electrode resistance. A cell-attached configuration was obtained by releasing the pressure and applying a light suction to the electrode until a gigaohm seal (>4 Ga) was obtained. Whole cell access was achieved by gentle suction until the patch ruptured. Unless otherwise stated, neurons were voltage clamped at -60 mv. Access resistance (Rs) was estimated from the formula: Rs = Vt/Ic, where Vt was a test voltage pulse, and Zc was the peak amplitude of the capacitative transient induced by Vt after an accurate compensation of the pipette capacitance in the cell-attached configuration. Rs in both layers ranged between 10 and 36 MS2 (24.4 t 0.7 Mfi, mean t SE, y2 = 69). Series resistance compensation was not used, to maintain the highest possible signal-to-noise ratio but cells where Rs changed by > 15% were discarded. The mean input resistance was t 15.5 MO (~1 = 26) in layer II neurons and MS2 (n = 34) in layer IV/V. Currents were recorded with an Axopatch 200A amplifier (Axon Instruments), filtered at 2 khz, digitized at 48 khz, and recorded on a DAT recorder (SONY, Digital DTC ES). Acquisition and analysis of spontaneous events was performed off-line using the Strathclyde Electrophysiology Software, WCP V 1.1 (courtesy of J. Dempster, University of Strathclyde, UK). Spontaneous excitatory postsynaptic currents (sepscs) were sampled at 12.5 khz [ 3.3 khz for isolated N-methyl-D-aspartate ( NMDA) -receptor-mediated sepscs] and captured using a threshold-crossing detector set above the noise level. Events that did not show a typical EPSC waveform were rejected manually. Peak amplitude, interval between events, rise time (time from 10 to 90% of the peak amplitude), decay time constant (calculated by least square fittings of the experimental record), and T-50% (decay time from peak to 50% of peak amplitude) of the sepscs were determined. Synaptic responses also were evoked using a bipolar platinum stimulating electrode placed on the surface of the slice to activate intracortical synaptic pathways. Evoked responses were analyzed with the pclamp software package (Axon Instruments). For statistical analysis, sepscs measurements were expressed as cumulative probability distributions and compared using the Kolmogorov-Smimov nonparametric test (Van der Kloot 199 1). In some experiments, sepsc amplitude distribution has been plotted as a cumulative mean amplitude distribution. To obtain this plot, the amplitudes were sorted in ascending order and the mean was calculated progressively. This plot is particularly suitable for showing graphically when a distribution has been altered as a result of a change in larger sized events while smaller events have not been affected (Van der Kloot 199 1). Numerical values in the text are expressed as mean 2 SE. Estimates of membrane electrotonic length (L) were obtained by measuring the slow ( 71 ) and the fast ( 72) time constants of the current transient induced by a negative voltage step ( 10 mv, 150 ms) and substituting these into the equation, L = (r/2) (9~~ - T*y2(T1-72y2 (Jackson 1992; Rall 1969; Soltesz and Mody 1994). The presence of active, voltage-dependent membrane conductances can clearly influence length constants estimated in this way (Major et al. 1994). Layer II, but not layer V cells often showed a pronounced sag in the membrane response to negative voltage steps, probably as a result of activation of the inwardly rectifying conductance Zh (see Jones 1994). However, this sag disappeared within lo- 15 min of commencing recording, and care was taken to make measurement of membrane time constants at a time when the sag was no longer present. We cannot rule out complications induced by the presence of other active conductances, although the inclusion of Cs2 in the electrodes should obviate these to some extent. Drugs used were 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 6-nitro-7-sulphamoylbenzo ( f ) -quinoxaline-2,3-dione (NBQX), D- or D,L-2-amino-5-phosphonovaleric acid (AP5 ), bicuculline methochloride (Tocris Cookson), and tetrodotoxin (TTX; Sigma), and all were applied by bath perfusion. All salts used for the preparation of ACSF were from BDH, whereas salts used for electrode filling solution were from Sigma, apart from QX 314, which was from Alomone Labs. RESULTS Whole cell recordings from layer IV-V (~1 = 39) and layer 11 ( n = 30) neurons, at a holding potential of -60 mv, invariably revealed spontaneous synaptic activity, detectable as spontaneously occurring inward currents. These events were abolished by glutamate receptor antagonists (see below) but were insensitive to the GABA* receptor antagonist bicuculline, suggesting that they were sepscs resulting from the release of glutamate from presynaptic terminals. sepscs occurred in neurons of the two layers at similar frequencies ( 1.44 t 0.09 Hz, n = 39 in layer IV-V and 1.42 t 0.08 Hz, n = 30 in layer II). In layer IV-V there was clear evidence that sepscs could occasionally occur in clusters, in a burst-like fashion. Such a pattern did not occur in all neurons, but can be seen in the upper record of Fig. 1 AI. In layer II neurons burst-like patterns of activity were much less evident (Fig. 1 BI ). Very occasionally there may have been a tendency for events to group in this way, but this effect was much less obvious than in the deep cells. Amplitude of sepscs The frequency distribution of the amplitudes of the sepscs were represented by histograms with a single peak, skewed toward larger amplitude events. The mean amplitude

3 ANEOUS EPSCS IN THE ENTORHINAL CORTEX I I I 500 msec 20pA FIG. 1. Al : spontaneous excitatory postsynaptic currents (sepscs) recorded in a layer IV-V neuron. Six traces are consecutive recordings of spontaneous activity. Inward currents largely occurred in a random fashion although there were occasional bursts of events (top). Frequency distribution of peak amplitudes of sepscs from this layer IV-V neuron (A2) is described by a histogram with a characteristic skewed shape and a long tail of large amplitude sepscs. Bl: sepscs shown recorded in a layer II neuron, and although frequency distribution shown in B2 still had a skewed shape, tail of larger amplitude events was not present. Note slower kinetics of sepscs in layer II I I of the sepscs in layer IV-V was slightly greater than that in layer II (15.67 t 0.52 pa, y1 = 34 and t 0.36 pa, n = 26, respectively) and recordings from neurons in the deeper layers were characterized by the presence of larger amplitude sepscs than superficial neurons. Thus in layer IV-V neurons, sepsc amplitude ranged up to -200 pa, whereas in layer II neurons, they rarely exceeded 50 pa (Fig. 1, A2 and B2). It was clear that in layer IV-V relatively few events occurred in the upper ranges of amplitude, and this is reflected in the similarities between the mean amplitudes for the two layers. However, the mean amplitudes of sepscs in the two layers, compared with a t-test, were not significantly different. Such a simple comparison for the entire populations of cells recorded is not appropriate because amplitude will vary as a function of access resistance. For this reason, to compare the properties of sepscs in more detail, we selected seven cells in layer IV-V and nine cells in layer II on the basis of similar and relatively low ( <20 MS2) access resistances ( and 16.3 t 1.1 MO, respectively). In addition, a nonparametric statistical analy- sis was used to examine differences in amplitude distribution (see below). sepscs (150) were collected from each neuron and pooled as cumulative probability distributions. Figure 2Al shows that sepsc amplitude in layer IV-V neurons was significantly larger than in layer II (P < 0.001). When the same sepscs were plotted as cumulative mean amplitude distributions (Fig. 2A2), it was apparent that the difference mainly was due to the presence of the larger amplitude events in layer IV-V neurons ( >40 PA), whereas the amplitude distributions of the smaller sepscs (<40 PA) in the two layers did not greatly diverge. Kinetics of sepscs sepscs in the whole population of neurons recorded in layer IV-V had a mean rise time of 1.87 t 0.12 ms and a T-50% of 5.68 t 0.34 ms (n = 34). In layer II neurons, sepscs had a rise time of 2.52 t 0.12 ms and a T-50% of 8.02 t 0.44 ms (n = 26). These kinetic parameters were

4 -111--m--. 1 l l ) N. BERRETTA AND R. S. G. JONES C IO rise time (msec) T-50% (msec) D 3.0 * 40 l FIG. 2. A : cumulative probability distributions shown of peak amplitude ( f ) and cumulative mean amplitude distributions (2) of sepscs recorded from 7 layer IV-V neurons (-) and 9 layer II neurons (- - - ) selected on basis of similar access resistance (see text). Events ( 150) were collected from each neuron and pooled. Difference between 2 distributions in I was significant (P < 0.001). Cumulative mean amplitude distribution 2 clearly shows presence of larger amplitude sepscs in layer IV-V. B: cumulative probability distributions shown of rise time ( I) and T-50% (2) of sepscs recorded from layer IV-V neurons (-) and layer II neurons ( ) shown in A. Separation of curves clearly suggests that events in layer IV-V neurons are faster to peak (P < 0.001) and faster to decay (P < 0.001) than events in layer II. These differences are further illustrated in C where averaged sepscs of a layer II (top) and a layer IV-V neuron are shown. Decay phase of sepscs could be fitted with a single exponential function ( L): average estimates shown of electrotonic length of neurons in A and B and reveals that layer II neurons are less electronically compact than those in layer IV-V. Difference was significant (P = 0.02). c E \f-,-- : 10 msec 5 PA 2 Q) 0.6.g 1.80 I Layer IV-V Layer II compared more quantitatively in the two populations of neurons, which were selected on the basis of similar access resistances (see above) because, again, differences in the series resistance might contribute to differences in kinetics (Spruston et al. 1993, 1994). This analysis clearly has confirmed differences in kinetics. The cumulative distribution analysis indicated that sepscs in, the selected layer IV-V neurons had significantly faster rise and decay times (P < 0.001, both plots; Fig. 2B). Averaged sepscs from two neurons with a similar access resistances ( 12 and 11 MO, in layer II and layer IV-V, respectively) illustrate these differences (Fig. 2C). The averaged rise and decay time constants in layer II were 1.68 and 6.21 ms, whereas in the layer IV-V neuron they were 0.80 and 3.27 ms. A difference in the electrotonic properties of deep and superficial neurons, perhaps related to dendritic organization, could account for a difference in the kinetics of the synaptic responses (Spruston et al. 1993, 1994; see also Soltesz and Mody 1994). We have estimated the electrotonic length (L) of the subpopulation of neurons used for comparison of the sepsc waveform (see Jackson 1992; Soltesz and Mody 1994). Both populations of neurons were poorly electrotonitally compact, with a L value of 2.48 t 0.29 in layer II and 1.57 t 0.15 in layer IV-V neurons (Fig. 2 0). Electrotonic length in layer II neurons was significantly greater (P = 0.02, t-test unpaired data). Accepting the constraints imposed by estimates of length constants, these results may suggest that a different degree of dendritic filtering could be a factor in the slower time course of the sepscs in layer II. Dendritic filtering also could account for the differences in amplitude distribution of sepscs, so we have attempted to correlate rise time and T-50% with peak amplitude in the

5 ANEOUS EPSCS IN THE ENTORHINAL CORTEX 1093 same neurons. A lack of correlation in these parameters has been assumed to indicate that electrotonic attenuation is not a major cause of peak amplitude variability (Arancio et al. 1994; McBain and Dingledine 1992; Raastad et al. 1992; Rall 1969; but see Spruston et al ). It was clear that larger events were often among those with faster kinetics. However, the reverse was not true, i.e., that small events tended to have slow kinetics. Therefore, in the overall population of sepscs, we found no significant correlation between amplitude and kinetics (r in all cells, for all plots, data not shown). Thus although differences in dendritic filtering and/or location of synaptic boutons on the dendritic tree might be partly responsible for the differences in amplitude, it is likely that additional factors might determine the presence of larger amplitude sepscs in layer IV-V. Efect of l7x on sepscs Spontaneous synaptic events recorded in TTX are considered to reflect activity-independent release of single quanta from individual release sites ( Arancio et al. 1994; Jonas et al. 1993)) and a lack of change in the sepsc amplitude distribution in TTX may indicate that activity-dependent sepscs are mediated by the release of single quanta of neurotransmitter from presynaptic terminals (Arancio et al. 1994). Therefore we have determined the effect of TTX on sepscs in the EC. The effectiveness of TTX was confirmed by the complete block of evoked synaptic responses. Care was taken to ensure that the access resistance was unaltered throughout perfusion with TTX. TTX (0.5 PM) reduced the frequency of sepscs in both layers by 1520%, indicating that most of the sepscs were miniature events. The frequency in layer IV-V was reduced from 2.17 t 0.33 to 1.80? 0.37 Hz (n = 5) and in layer II from 1.61 t 0.17 to Hz (n = 5). However, the effect on the amplitude distribution of sepscs was not the same in the two layers. Figure 3 shows pooled cumulative distributions of sepsc amplitude and interval in layer IV- V( n = 5; Fig. 3, left) and layer II (n = 5) neurons (Fig. 3, right; 150 events per neuron), before and after perfusion of TTX. In layer IV-V, both the interval and amplitude cumulative probability distributions were changed significantly (P < 0.001, both plots). In contrast, in layer II, there was a significant difference only for the interval distribution (P < ), whereas the cumulative amplitude distribution was unaltered (P > 0.9; Fig. 3A). Figure 3B also shows that the change in the amplitude distribution in layer IV-V neurons was mainly due to loss of larger amplitude sepscs. When the same amplitude measurements plotted in Fig. 3A were pooled as cumulative mean amplitude distributions (Fig. 3C), it was clear that TTX caused a loss of larger amplitude events in layer IV-V neurons, whereas the mean amplitude distribution in layer II neurons was unaffected. However, as shown in Fig. 4, even in the presence of TTX, layer IV-V neurons still displayed larger amplitude sepscs. Therefore, the difference between the two layers cannot be explained solely on the basis of activity-dependent sepscs in layer IV-V. Pharmacology of sepscs Evoked and sepscs in CA1 and CA3 of the hippocampus exhibit both a-amino-3-hydroxy-5-methyl-4-isoxazolepropi- onic acid ( AMPA) / kainate and NMDA-receptor-mediated components, suggesting a colocalization of the two glutamate receptors at the same synapses (Bekkers and Stevens 1989; Hestrin et al. 1990; McBain and Dingledine 1992). Evoked synaptic responses in layer IV-V and layer II neurons in the EC also exhibit AMPA/kainate- and NMDAmediated components. However, the NMDA component appears more pronounced in layer IV-V neurons (Jones 1987, 1994; Jones and Heinemann 1988). We now have examined the role of AMPA/kainate and NMDA receptors in mediating sepscs. Again, in all studies access resistance was monitored throughout to ensure that it remained stable. Perfusion with the AMPA-receptor antagonists, CNQX or NBQX ( 10 PM), abolished fast sepscs in both layer IV- V (n = 11) and layer II (n = 7) neurons (Vh -60 mv). However, under these conditions, it was still possible to detect occasional slower time-course spontaneous events (see Figs. 5-7). The time course of these events is clearly shown in Fig. 5A, where averaged sepscs from a layer IV- V neuron are shown in the presence and the absence of CNQX ( 10 PM). Rise time and decay time constants in control conditions were 0.9 and 5.5 ms, respectively, whereas for the events recorded in the presence of CNQX, the same parameters were 6.8 and 26.5 ms. That these slower time-course sepscs were synaptic events was supported by the similarity of their kinetics to evoked synaptic responses recorded at the same time. Figure 5B shows evoked and spontaneous EPSCs, scaled in amplitude, and superimposed at the time of the first negative deflection. The low frequency of the slow sepscs ( see below), made a statistical comparison of the events in layer IV-V and layer II neurons difficult. However, the events in the two layers did not reveal any dramatic differences in their amplitude and decay time constant, whereas the rise time appeared to be somewhat faster in layer IV-V than layer II neurons (see Table 1). Moreover, the interval between slow sepscs in layer IV-V neurons was less than half that observed in layer II neurons, suggesting a higher incidence of these events in layer IV-V than in layer II neurons. Perfusion with Mg *+ -free ACSF (in the presence of CNQX or NBQX) resulted in a marked increase in frequency of the slow sepscs. The interval between these events decreased from _ to 2.86? 1.09 s in layer IV-V (n = 3), and from t to 2.90 t 1.01 s in layer II (n = 2). There was also an increase in amplitude and some larger events were recorded clearly under these conditions (Fig. 6). All these slow time-course events were abolished by perfusion with the NMDA-receptor antagonist D,L- AP5 (100,uM) (n = 6; Fig. 6A). The slow sepscs increased in amplitude when the neuron was voltage clamped at -40 mv compared with -70 mv and reversed in polarity when recorded at positive holding potentials (n = 7; Fig. 7). This voltage dependence is characteristic of that expected for NMDA-receptor-mediated responses (Hestrin et al. 1990). Evoked synaptic responses, recorded from the same neuron showed similar voltage-dependent behavior (Fig. 7). Thus the effects of Mg*+ removal, AP5 and depolarization clearly suggest that the slow events were pure NMDA-receptor-mediated sepscs. Finally, in three neurons (n = 2 layer IV-V and n = 1 layer II) sepscs also could be observed in the presence of NBQX plus TTX, so, the NMDA-receptormediated sepscs were, at least in part, activity-independent.

6 l l ) 1094 N. BERRETTA AND R. S. G. JONES Layer IV-V Layer II % 'a :.- Y OII intewal (set) interval (set) FIG. 3. A : solid lines show cumulative probability distributions of peak amplitude (top) and interval (bottom) of sepscs recorded from 5 layer IV-V neurons (left) and 5 layer II neurons (right). Distribution shown ( ) after addition of tetrodotoxin (TTX; 0.5 PM). In layer IV-V neurons, leftward shift in amplitude distribution indicates a reduction in peak amplitude whereas rightward shift in interval curve is indicative of a reduced frequency. Changes in both parameters were highly significant (P < 0.001). In layer II neurons only, frequency of events was reduced in TTX (P < 0.001) and there was no change in peak amplitude. Curves were derived from 150 events collected from each neuron and pooled. B: cumulative mean amplitude distributions derived from layer IV-V neurons (left) and layer II neurons (right) shown above. Perfusion with TTX ( l abolished larger events in layer IV-V neurons, whereas distribution of smaller events essentially was unaltered IO We have tried to determine whether the NMDA-receptormediated sepscs were spontaneous events occurring independently of the fast sepscs or whether they were residual components of sepscs that would have displayed previously both AMPA/kainate and NMDA components. These experiments have determined the effect of AP5 on spontaneous synaptic activity without addition of AMPA receptor antagonists. Membrane potential was held at -40 mv to maximize the contribution of NMDA receptors to the synaptic response. A relatively low concentration of bicuculline ( l- 2 PM) allowed us to inhibit substantially the spontaneous inhibitory activity that was apparent at -40 mv without inducing spontaneous epileptiform discharges. Under these conditions, D-APS (50 PM) caused a reduction in the peak amplitude and the T-50% in the sepscs of layer IV-V neurons (n = 5)) indicating that a NMDA component contributed to the peak and the late phase of the sepscs. Figure 8A shows an example from one neuron. There was a leftward shift of the cumulative peak amplitude and T-50% distribution induced by D-AP5 (P < 0.002, both plots; Fig. 8Al). The averaged sepsc from the same neuron (Fig. 8A2) illustrates the AP5-sensitive component in the peak and the late phase. Similar results have been obtained in the presence of TTX (0.5 PM, y2 = 3). In contrast, no significant change was observed after perfusion with D-AP5 in layer II neurons (n = 4). The graphs in Fig. 8Bl show that there was no change in the cumulative peak amplitude distribution (P > 0.8) and only a slight leftward shift in the cumulative T- 50% distribution. The latter was not significant (P > 0.4). The averaged sepscs (Fig. 8 B2) clearly show that there was very little difference in peak amplitude or decay phase after perfusion with D-APS. DISCUSSION Neurons in both superficial and deep layers of the rat EC show continuous spontaneous excitatory synaptic activity, indicating a tonic excitation by glutamate. This activity was only partially affected by perfusion of TTX and therefore is supported largely by miniature events. Occasionally, sepscs in layer IV-V were observed to occur in bursts. However, these bursts did not show any regular pattern and could not be reliably detected throughout the same experiment. Therefore, analysis of changes in their occurrence after pharmacological manipulations was difficult to study. The distribution of sepsc amplitude in both layers could be represented by histograms with a single peak, skewed toward larger amplitude. Such variability in sepsc amplitude has been observed in other areas of the CNS (Arancio et al. 1994; Edwards et al. 1990; Jonas et al. 1993; Livsey and Vicini 1992). One possible explanation for this variability is that sepscs do not all originate from the same site on the dendritic tree and are differently filtered by the cable properties of the neuron, before reaching the soma. This is conceivable for EC neurons, given their poor electrotonic compactness. However, neither rise time nor T-50% were

7 l l ) l l ) ANEOUS EPSCS IN THE ENTORHINAL CORTEX 1095 A A control $. ; 4 msec 40 msec CNQX ms B NBQX (10 PM) Evoked EPSC Spontaneous EPSC v I d0 :5 FIG. 4. A : cumulative probability distributions of sepsc amplitude of a layer IV-V neuron (Zefi) and a layer II neuron (right) before (-) and after ( ) perfusion with TTX (0.5 PM; 250 events included in each plot). Perfusion with TTX reduced amplitude of sepscs in layer V (P < 0.001) but was without effect in layer II (P > 0.9). B: curves shown for layer II and layer IV-V (layer II control omitted for clarity) in a single plot. Control condition (-) and TTX (- - - ) shown for layer IV-V neuron as is TTX ( l for layer II neuron. Difference between amplitude distributions of sepscs in layer IV-V and layer II obtained in presence of TTX was still highly significant (P < 0.001). correlated significantly with peak amplitude, as might be expected if synaptic location was the main source of sepsc variability (Arancio et al. 1994; McBain and Dingledine 1992; Raastad et al. 1992). The lack of correlation was not due to the presence of activity-dependent sepscs, because even for miniature events recorded in TTX, rise time or T- 50% were not correlated to the peak amplitude. Moreover, a similar broad, skewed shape also has been reported for miniature EPSCs originating from single synaptic boutons (Liu and Tsien 1995). These recent findings suggest that the heterogeneity of sepsc amplitude may be related to intrinsic variability at individual synapses. Different characteristics of the sepscs in layer IV-V and layer II neurons sepscs in layer IV-V exhibited faster kinetics than in layer II. The greater electrotonic length of superficial neurons strongly suggests that differences in dendritic filtering might be a factor underlying the different kinetics. In the dentate gyrus, similar differences in the kinetics of spontaneous inhibitory postsynaptic currents and in electrotonic lengths have been found between hilar neurons and granule cells, and a difference in the electrotonic cable structure of the neurons has been suggested to be partly responsible (Soltesz and Mody 1994). However, it should be noted that msec 25 msec FIG. 5. All traces are averaged records of sepscs recorded in layer IV- V neurons at a holding potential of -60 mv. A: records show sepscs before (left) and after (right) addition of 6-cyano-7-nitroquinoxaline-2,3- dione (CNQX, 10 PM). Events remaining in presence of a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) antagonist had a slower rise time and decay time compared with those recorded in control artificial cerebrospinal fliud (ACSF) medium. This clearly is illustrated when traces are expanded and superimposed on the same gain and time base (lower line). Decay phase of both events was fitted with a single exponential function ( l. B : in presence of 6-nitro-7-sulphamoylbenzo ( f ) -quinoxaline-2,3-dione (NBQX, 10 PM) and bicuculline ( 10 PM) residual sepscs had remarkably similar kinetics to response elicited by electrical stimulation. Two responses are shown (top), and these are shown scaled in amplitude and superimposed in lower line. receptor channel properties also appear to be a factor in determining the shape of AMPA receptor-mediated postsynaptic currents (Sarantis et al. 1993). Many AMPA receptor subunits with different functional properties have been cloned so the possibility exists that the different kinetics of sepscs in deep and superficial neurons of the EC could reflect the presence of different populations of AMPA-recep- TABLE 1. Characteristics of CNQX-resistant sepscs in layer IV-V and layer II Layer IV-V Layer II Peak, pa Rise time, ms t 1.75 T-50%, ms Interval, s CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; sepsc, spontaneous excitatory postsynaptic current.

8 1096 N. BERRETTA AND R. S. G. JONES A Control CNQX Mg++ free D,L-APS 200 msec 20pA FIG. 6. A : selected records of sepscs in a layer IV-V neuron. Perfusion with CNQX (10 PM) abolished most events but small, slow time-course sepscs still could be detected (top right). Perfusion with a Mg*+ -free medium increased frequency of these events and larger events also now could be detected. All these events in Mg *+ -free medium were abolished on addition of D,L-2-amino+phosphonovalerate (D,L-APS ). B : frequency distribution of peak amplitudes of sepscs of a layer IV-V neuron recorded in CNQX ( 10 PM). Bin size was 1 pa and sepscs for both histograms were collected during a recording period of 3 min. Filled columns show events recorded in presence of 2 mm Mg*+. Removal of Mg*+ (clear columns) clearly increased number of events, and much larger events now were apparent. tor subunits (Hestrin 1993; Livsey and Vicini 1992; Livsey et al. 1993; McBain and Dingledine 1993). Another interesting finding was the presence of larger amplitude sepscs in deep layer neurons. The effect of TTX strongly suggested that differences in the activity-dependent spontaneous release might be the most important factor responsible for the presence of larger amplitude sepscs in layer IV-V neurons. The amplitude distribution of sepscs was unaffected by TTX in layer II neurons, whereas in layer IV-V neurons, the larger amplitude sepscs were abolished (see Fig. 3). These data suggest that activity-dependent and -independent sepscs in layer II are mediated by the release of single quanta of glutamate, whereas in layer IV-V, activity-dependent sepscs are mediated mainly by the release of multiple quanta. It is unlikely that this activity-dependent spontaneous activity is associated with the release of more than one synaptic vesicle from a single release site, because the release of a single quantum should almost saturate subsynaptic receptors (Tang et al. 1994). Therefore the most likely explanation is the presence of more release sites on layer IV-V neurons through branching of afferent axons. Thus the difference in sepsc amplitude distributions between layer IV-V and layer II is probably due mainly to a difference in activity-dependent presynaptic release. However, larger amplitude sepscs in layer IV-V neurons cannot be explained only in terms of release of multiple quanta, because the cumulative distributions obtained in TTX still indicated the presence of larger amplitude sepscs in the deep neurons (see Fig. 4). NMDA and AMPA component of the sepscs Most of the spontaneous excitatory synaptic activity in both layer II and IV-V resulted from activation of AMPA receptors. However, discrete spontaneous events were still detected in the presence of AMPA receptor antagonists. A number of characteristics indicated that these were synaptic events due to the activation of NMDA receptors: a slow rise

9 Vh -70 mv ANEOUS EPSCS IN THE ENTORHINAL CORTEX Vh -40 mv Vh +40 mv sepscs # 200 msec llopa Evoked EPSCs FIG. 7. Records were selected (not consecutive) from a recording of sepscs in a layer IV-V neuron in presence of NBQX ( 10 PM) and bicuculline ( 10 ym). Those on Zej? show occasional small, slow sepscs and also EPSC evoked by electrical stimulation recorded at a holding potential of -70 mv. Holding at a potential of -40 mv (center) resulted in an increase in amplitude of sepscs, and this was paralleled by a similar change in evoked response. Finally, at positive holding potential of 40 mv (right), both spontaneous and evoked events reversed in polarity. 50 msec 20 pa time and decay (Hestrin et al. 1990) and a similarity in time course to evoked synaptic responses recorded at the same time; an increase in amplitude with depolarization and reversal at positive holding potentials (Hestrin et al. 1990; McBain and Dingledine 1992) ; an increase in amplitude and frequency in Mg2+ -free ACSF (Hestrin et al. 1990) (although the latter effect could be related to a facilitation of presynaptic release of glutamate) ; amplitude distributions in Mg2+ -free ACSF still represented by single peak histograms, skewed toward larger amplitude events (Fig. 6B); and abolition by an NMDA-receptor antagonist. That EC neurons recorded in slices may display discrete sepscs purely mediated by NMDA receptors is of particular interest. In cultures of rat hippocampus, Bekkers and Stevens ( 1989) recorded NMDA-receptor-mediated sepscs in ACSF containing no added magnesium. NMDA-receptormediated spontaneous activity has been detected in slice preparations, but only as current fluctuations in CA1 pyramids (Sah et al. 1989) and neocortical neurons (LoTurco et al. 1990). In CA3 neurons (McBain and Dingledine 1992) and in the superior colliculus (Hestrin 1992)) NMDA-receptor-mediated sepscs were detected only at positive holding potentials, whereas EC neurons could display such events at negative holding potentials and in the presence of 2.0 mm Mg2+. This could indicate a particularly powerful NMDAreceptor-mediated spontaneous excitation in EC neurons. However, it could be argued that current fluctuations re- corded in the hippocampus and neocortex (LoTurco et al. 1990; Sah et al. 1989) are composed of many discrete spontaneous events that merge and cannot be detected separately, and that in the EC these events can be detected because they occur less frequently. We believe this is unlikely, because APS-sensitive current fluctuations could be detected in the hippocampus and neocortex only at depolarized holding potentials or in the absence of Mg2+. Pure NMDA-receptor-mediated sepscs appear to occur more frequently in layer IV-V than in layer II neurons. It is difficult to rule out the possibility that the lower incidence of these discrete events is due to detection problems caused by the presence of smaller amplitude slow sepscs in layer II neurons. In any case, considering differences in either frequency or in amplitude, this may indicate a more pronounced contribution of NMDA receptors to the spontaneous synaptic activity in the deep layers. A greater contribution of NMDA receptors to the synaptic transmission in layer IV-V also has been suggested for evoked responses (Jones 1987, 1994; Jones and Heinemann 1988). The effect of AP5 seemed to confirm the presence of a stronger contribution from NMDA receptors to sepscs in layer IV-V. sepscs in layer II showed little change in peak amplitude or T-50% in AP5, but in layer IV-V there was a reduction in both parameters. Similar results were observed for miniature events recorded in TTX, ruling out the possibility that the release of multiple quanta may be responsible for this pro-

10 N. BERRETTA AND R. S. G. JONES Peak (PA) b 1 0 ;o io 4 0 T-50% (msec) Peak (PA) T-50% (msec) AP5 IOmsec FIG. 8. All experiments were performed without addition of AMPA antagonists. A : cumulative probability distributions of peak amplitude (left) and T-50% (right) of sepscs in a layer IV-V neuron, before (-) and after ( ) perfusion with D-APS (50 PM; 250 events selected for each plot). Leftward shift of both curves shows that blockade of N-methyl- D-aspartate receptors results in a reduction in both peak amplitude (P < 0.002) and decay phase (P < 0.001) of sepscs. These effects are illustrated clearly by averaged records in 2. B: representative results in a layer II neuron. In contrast to layer IV-V, there was little change in peak amplitude and only a small effect on decay of sepscs after perfusion with D-APS. nounced NMDA component. It is conceivable that poor clamping in the dendrites could result in an overestimation of the NMDA receptor component due to local depolarization. However, these experiments were conducted in a region of the current/voltage relationship for NMDA responses (holding potential -40 mv) that should not result in increase in these responses if dendritic depolarization did occur. Nevertheless, even considering the possibility that the absolute measurements of the NMDA component were overestimated, the greater electrotonic length of layer II neurons might result in greater space-clamp errors. Therefore, the difference in the contribution of NMDA receptors to the sepscs would have been even more evident. The larger NMDA component of sepsc peak amplitude in layer IV-V also could be a reason why larger amplitude sepscs were recorded in this layer. For example, local depolarizations due to poorly clamped dendritic synaptic currents could have resulted in larger amplitude sepscs through an enhanced contribution of NMDA receptors. However, if this hypothesis were true, larger amplitude sepscs would be expected to show a greater T-50% as an indication of a more pronounced contribution of NMDA receptors. On the contrary, larger amplitude sepscs often were characterized by faster kinetics than small sepscs. In the hippocampus, it has been suggested previously that NMDA and AMPA receptors may be largely colocalized (Bekkers and Stevens 1989). However, it has been demonstrated recently that the majority of synapses in CA1 pyramidal neurons have only NMDA receptors (Liao et al ). We cannot yet say whether NMDA and AMPA receptors may be colocalized at synapses in the EC. This point could be answered by determining whether the pure NMDA receptor-mediated sepscs occurred independently of the fast events, when AMPA receptors were not blocked. The fast, CNQX-sensitive events could occur with a frequency of 52-3 Hz. Therefore it was very difficult to pick out the few slow events that would be interspersed between the fast sepscs, to find them uncontaminated by the fast sepscs, and to be confident that such events were the same slow sepscs seen when AMPA receptors were blocked. We are fairly certain that such events did occur in pharmacologically untreated slices (see lower trace in Fig. IA1 ) and this may well suggest discrete NMDA and AMPA synapses in EC neurons. However, the relative rarity of these events precluded any meaningful frequency analysis. Concluding remarks We now have shown that neurons in the EC undergo continuous excitement by spontaneously released glutamate,

11 ANEOUS EPSCS IN THE ENTORHINAL CORTEX 1099 and it is conceivable that this could help establish the overall level of excitability in the synaptic networks of the EC. There is good evidence to suggest that the deeper layers of the EC may be the site of initiation of epileptiform discharges (Jones and Lambert 1990a,b), and a number of observations in the present experiments indicate that spontaneous synaptic activity might have a greater effect on the excitability of deeper neurons of EC than on superficial neurons: the presence of bursts of events; the presence of a population of large, activity-dependent sepscs; a greater contribution of NMDA receptors to spontaneous activity; and more frequent pure NMDA receptor-mediated sepscs. These differences could contribute to a greater susceptibility of neurons in the deeper layers to epileptogenesis, although this remains a speculative possibility at present. We thank J. Dempster (University of Strathclyde, UK) for providing the Strathclyde Electrophysiology Software for acquisition and analysis of data and C. Stoub for providing the program for the Kolmogorov-Smirnov test. This work was supported by The Wellcome Trust and The Royal Society. Present address of N. Berretta: S.I.S.S.A.-I.S.A.S., International School for Advanced Studies, Via Beirut 4, Trieste, Italy. Address for reprint requests: University Department of Pharmacology, Mansfield Road, Oxford OX1 3QT UK. Received 12 September 1995; accepted in final form 4 March REFERENCES ARANCIO, O., KORN, H., GULYAS, A., FREUND, T., AND MILES, R. Excitatory synaptic connections onto rat hippocampal inhibitory cells may involve a single transmitter release site. J. Physiol. Lund. 48 1: , BEKKERS, J. M. AND STEVENS, C. F. NMDA and non-nmda receptors are co-localized at individual synapses in cultured rat hippocampus. Nature Lond. 341: , BEN-ARY, Y., TREMBLAY, E., RICHE, D., GHILINI, G. AND NAQUET, R. Electrographic, clinical and pathological alterations following systemic administration of kainic acid, bicuculline or pentetrazole: metabolic mapping using the 2-deoxyglucose method with special reference to the pathology of epilepsy. Neuroscience 6: , BERRETTA, N. AND JONES, R. S. G. Spontaneous AMPA/kainate and NMDA mediated synaptic currents in layer V neurons of the rat entorhinal cortex in vitro (Abstract). J. Physiol. Lond. 483: 61P, 1995a. BERRETTA, N. AND JONES, R. S. G. A comparison of spontaneous, glutamate mediated synaptic currents in neurons of layer II and layer V of the rat entorhinal cortex in vitro (Abstract). J. Physiol. Lond. 487: 63P, 1995b. BERRETTA, N. AND JONES, R. S. G. Differences in the contribution of NMDA-receptors to spontaneous excitation in neurons in layer II and layer V of the rat entorhinal cortex in vitro (Abstract). J. Physiol. Lond. 487: 62P, 1995~. BLANTON, M. G., LOTURCO, J. J., AND KRIEGSTEIN, A. R. Whole-cell recording from neurons in slices of reptilian and mammalian cerebral cortex. J. Neurosci. Methods 30: , BRAAK, H. AND BRAAK, E. Neuropathological staging of Alzheimer-related changes. Acta Neuropathol. Berlin 82: , Du, F., WHETSELL, W. O., ABOU-KHALIL, B., BLUMENKOPF, B., LOTHMAN, E. W., AND SCHWARCZ, R. Preferential neuronal loss in layer III of the entorhinal cortex in patients with temporal lobe epilepsy. Epilepsy Res. 16: , EDWARDS, F. A., KONNORETH, A., AND SAKMANN, B. Quanta1 analysis of inhibitory synaptic transmission in the dentate gyrus of rat hippocampal slices: a patch-clamp study. J. Physiol. Lond. 430: , GEDDES, J. W., CAHAN, L. D., COOPER, S. M., KIM, R. C., CHOI, B. H., AND COTMAN, C. W. Altered distribution of excitatory amino acid receptors in temporal lobe epilepsy. Exp. Neural. 108: , HESTRIN, S., NICOLL, R. A., PERKEL, D. J., AND SAH, P. Analysis of excitatory synaptic action in pyramidal cells using whole-cell recording from rat hippocampal slices. J. Physiol. Lond. 422: , HESTRIN, S. Developmental regulation of NMDA receptor-mediated synaptic currents at a central synapse. Nature Lond. 357: , HESTRIN, S. Different glutamate receptor channels mediate fast excitatory synaptic currents in inhibitory and excitatory cortical neurons. Neuron 11: , JACKSON, M. B. Cable analysis with the whole-cell patch clamp. Theory * and experiment. Biophys. J. 61: , JONAS, P., MAJOR, G. AND SAKMANN, B. Quanta1 components of unitary EPSCs at the mossy fibre synapse on CA3 pyramidal cells of rat hippocampus. J. Physiol. Land. 472: , JONES, R. S. G. Complex synaptic responses of entorhinal cortical cells in the rat to subicular stimulation in vitro: demonstration of an NMDA receptor-mediated component. Neurosci. Lett. 8 1: , JONES, R. S. G. Epileptiform events induced by GABA-antagonists in entorhinal cortical cells in vitro are partly mediated by N-methyl-D-aspartate receptors. Brain Res. 457: , JONES, R. S. G. Entorhinal-hippocampal connections: a speculative view of their function. Trends Neurosci. 16: 58-64, JONES, R. S. G. Synaptic and intrinsic properties of neurons of origin of the perforant path in layer II of the rat entorhinal cortex in vitro. Hippocampus 4: , JONES, R. S. G. AND HEINEMANN, U. Synaptic and intrinsic responses of medial entorhinal cortical cells in normal and magnesium-free medium in vitro. J. Neurophysiol. 59: , JONES, R. S. G. AND LAMBERT, J. D. C. Synchronous discharges in the rat entorhinal cortex in vitro: site of initiation and the role of excitatory amino acid receptors. Neuroscience 34: ,199Oa. JONES, R. S. G. AND LAMBERT, J. D. C. The role of excitatory amino acid receptors in the propagation of epileptiform discharges from the entorhinal cortex to the dentate gyrus in vitro. Exp. Bruin Res. 80: , 1990b. LIAO, D., HESSELER, N. A., AND MALINOW, R. Activation of postsynaptically silent synapses during pairing-induced LTP in CA1 region of hippocampal slice. Nature Lond. 375: , LIU, G. AND TSIEN, R. W. Properties of synaptic transmission at single hippocampal synaptic boutons. Nature Lond. 375: , LIVSEY, C. T., COSTA, E., AND VICINI, S. Glutamate-activated currents in outside-out patches from spiny versus aspiny hilar neurons of rat hippocampal slices. J.Neurosci. 13: , LIVSEY, C. T. AND VICINI, S. Slower spontaneous excitatory postsynaptic currents in spiny versus aspiny hilar neurons. Neuron 8: , LOTURCO, J. J., MODY, I., AND KRIEGSTEIN, A. R. Differential activation of glutamate receptors by spontaneously released transmitter in slices of neocortex. Neurosci. Lett. 114: , MAJOR, G., LARKMAN, A. U., JONAS, P., SAKMANN, B., AND JACK, J. J. B. Detailed passive cable models of whole-cell recorded CA3 pyramidal neurons in rat hippocampal slices. J. Neurosci. 14: , MCBAIN, C. J. AND DINGLEDINE, R. Dual-component miniature excitatory synaptic currents in rat hippocampal CA3 pyramidal neurons. J. Neurophysiol. 68: 16-27, MCBAIN, C. J. AND DINGLEDINE, R. Heterogeneity of synaptic glutamate receptors on CA3 stratum radiatum interneurons of rat hippocampus. J. Physiol. Lond. 462: , OTIS, T. S., STALEY, K. J., AND MODY, I. Perpetual inhibitory activity in mammalian brain slices generated by spontaneous GABA release. Bruin Res. 545: , RAASTAD, M., STORM, J. F., AND ANDERSEN, P. Putative single quantum and single fibre excitatory postsynaptic currents show similar amplitude range and variability in rat hippocampal slices. Eur. J. Neurosci. 4: , RALL, W. Time constants and electrotonic length of membrane cylinders and neurons. Biophys. J. 9: , RUTECKI, P. A., GROSSMAN, R. G., ARMSTRONG, D., AND IRISH-L EWEN, S. Electrophysiological connections between the hippocampus and entorhinal cortex in patients with complex partial seizures. J. Neurosurg. 70: , SAH, P., HESTFUN, S., AND NICOLL, R. A. Tonic activation of NMDA receptors by ambient glutamate enhances excitability of neurons. Science Wash. DC 246: , SARANTIS, M., BALLERINI, L., MILLER, B., SILVER, R. A., EDWARDS, M., AND ATTWELL, D. Glutamate uptake from the synaptic cleft does not shape the decay of the non-nmda component of the synaptic current. Neuron 11: , SOLTESZ, I. AND MODY, I. Patch-clamp recordings reveal powerful GA- BAergic inhibition in dentate hilar neurons. J. Neurosci. 14: , SPENCER, S. S. AND SPENCER, D. D. Entorhinal-hippocampal interactions in medial temporal lobe epilepsy. Epilepsia 35: , 1994.