Temporal overlap of excitatory and inhibitory afferent input in guinea-pig CA1 pyramidal cells

Brain slices

Transverse hippocampal slices (Yamamoto, 1972; Schwartzkroin, 1975) were obtained from adult guinea-pigs (Hartley, from Harlan Sprague-Dawley, Inc., Indianapolis, IN, USA; 150-200 g). Guinea-pigs were anaesthetized by inhalation of halothane (2-bromo-2-chloro-1, 1, 1-trifluoroethane) before decapitation with an animal guillotine. After removal of the brain and isolation of the hippocampus, slices of 450 μm thickness were cut on a McIlwain tissue chopper. The procedures conformed with the guidelines of the Institutional Animal Care and Use Committee (protocol no. 9808069). Slices were superfused in an interface recording chamber (Fine Science Tools, Belmont, CA, USA) with a solution saturated with 95 % O₂ and 5 % CO₂ (temperature, 30-32°C) of the following composition (mM): 118 NaCl, 3 KCl, 25 NaHCO₃, 1·2 NaH₂PO₄, 1·2 MgCl₂, 1·7 CaCl₂ and 11 D-glucose.

Electrophysiological recordings

Data aquisition and analysis

Evoked responses were elicited by stimulation of stratum radiatum Schaffer collateral-commissural fibres through a pair of insulated tungsten bipolar electrodes (stimulation range 15-800 μA). Voltage signals of dual recordings were recorded and amplified with an Axoprobe-1A (Axon Instruments). Voltage and current signals of single recordings recorded in both dSEVC and current clamp were amplified by Axoclamp (Axon Instriments). Data were sampled at rates ranging from 2·5 to 10 kHz (-3 dB) and filtered with a cut-off frequency of 1·5 kHz. The voltage and current signals were fed into separate channels of an A/D converter (Digidata 1200, Axon Instruments) digitized, stored and analysed off-line using pCLAMP6 software from Axon Instruments on a Pentium PC. Statistical significance was accepted at the P < 0·05 level in various tests.

Drugs

With the exception of intracellularly applied biocytin (Sigma), all drugs, bicuculline (Bic), picrotoxin (PTX), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), D-aminophosphonovalerate (D-AP5) and saclofen, were applied by bath perfusion. CNQX, D-AP5 and saclofen were purchased from Tocris Cookson, Inc. (Ballwin, MO, USA); all other drugs were from Sigma.

Abbreviations

t_(i) is the poststimulation latency to the event specified by the subscript. For example, t_{(P,monoIPSP,onset)} denotes the latency between stimulation artifact and onset of the stimulation-evoked monosynaptic IPSP in a pyramidal (P) cell.

Δt_(i-j) is the time lag between two events. For example, Δt_{(I,AP,peak-P,IPSP,onset)} denotes the latency between peak of an AP in an interneuron (I) and onset of the unitary IPSP in a synaptically coupled pyramidal cell.

g_GABAA is the GABA_A receptor-mediated conductance. AHP denotes afterhyperpolarization; V_m is membrane potential, V_thres is AP threshold potential, and V_rest is resting membrane potential.

Monosynaptic inhibition

Comparison of the kinetics of orthodromic excitation and monosynaptic inhibition in the same cells was achieved by the following protocol: the orthodromic response was recorded first. The same stimulation (with respect to site and intensity) was then applied following application of CNQX, D-AP5 and saclofen (Fig 2, Fig 3 and Fig 4; Table 1). The resulting pharmacologically isolated inhibitory responses (monosynaptic IPSP, IPSC and conductance change (g_GABAA)) were in all cases completely blocked by bicuculline (100 μM, not shown).

Temporal comparison of monosynaptic IPSP/IPSC and EPSP/EPSC

In nine dendritic and seven somatic preparations, recordings were carried out in both current clamp and dSEVC (Fig 2, Fig 3 and Fig 4; Table 1A).

Kinetics of IPSCs were faster than IPSPs (Table 1A; Fig. 2D). Mean differences between t_(IPSP,peak) and t_(IPSC,peak) were 5·7 ± 0·7 ms in dendritic (n= 9) and 5·2 ± 0·5 ms in somatic (n= 7) recordings. In contrast to IPSP/IPSCs, no significant differences in kinetics were observed between EPSPs and EPSCs: mean differences between t_(EPSP,peak) and t_(EPSC,peak) were only 0·8 ± 0·2 ms in dendritic (n= 9) and 0·7 ± 0·3 ms in somatic (n= 7) recordings.

The latencies to onsets of all responses - orthodromic EPSP/EPSCs, monosynaptic IPSP/IPSCs - were statistically identical in both groups of recordings, somata and dendrites (ANOVA) (Table 1A). With the exception of latencies to onset, however, kinetics of IPSP/IPSCs were considerably slower than those of EPSP/EPSCs. For example, rise times (10-90 %) and latencies to peak of the monosynaptic IPSP, were on average more than twice as long as those of the orthodromic EPSP (Table 1A and B, Fig. 2B). Mean differences between t_(EPSP,peak) and t_(IPSP,peak) were 8·0 ± 1·1 ms in dendritic and 10·6 ± 1·6 ms in somatic recordings. Temporal overlap between EPSP and IPSP was modest (Fig. 2B). Due to the faster kinetics of IPSCs, mean differences between t_(EPSC,peak) and t_(IPSC,peak) were somewhat smaller: 4·3 ± 0·7 ms in dendritic (n= 9) and 7·1 ± 0·9 ms in somatic recordings (n= 7). However, temporal overlap between EPSC and IPSC was still rather small (Fig. 2C). The same conclusion was reached when comparing EPSCs and IPSCs that were measured in somatic whole-cell recordings. Latencies to t_(EPSC,peak) (5·8 ± 1·2 ms; n= 5) and to t_(IPSC,peak) (12·5 ± 1·5 ms; n= 5) and mean differences (6·8 ± 1·2 ms) were not statistically different when comparing the respective values obtained from sharp-electrode and whole-cell recordings (P > 0·05, ANOVA).

Components of monosynaptic inhibition

In a second approach, latency to onset of the evoked monosynaptic IPSP was obtained through the evaluation of its components: (a) antidromic AP in an interneuron (Fig. 5) and (b) unitary IPSP (Fig. 6). The values of latency to onset of the monosynaptic IPSP were similar in the two modes of measurements, i.e. via summation of components (Fig 5 and Fig 6) and directly during pharmacological isolation (Fig 2, Fig 3 and Fig 4, Table 1): the ranges of IPSP onsets were 1-3·9 ms (summation of components) and 1·7-3·9 ms (during isolation); the means were 2·6 ms (summation of components) and 2·8 ms (during isolation).

Feedforward inhibition

Dual recordings of pyramidal cells and stratum pyramidale interneurons (n= 17) were carried out to determine the latencies to onset of feedforward IPSPs (Fig. 7). As shown in Fig. 7A, the onset of the feedforward IPSP was determined by the sum of components: (i) latency to peak of orthodromic APs in interneurons (t_{(I,ortho-AP,peak)}) (Fig. 7Aa), and (ii) latency to onset of the unitary IPSP in the pyramidal cell (Δt_{(I,AP,peak-P,IPSP,onset)}) (Fig. 7Ab). A 1 ms constant was generally substituted for Δt_(I,AP,peak-_{P,IPSP,onset)} in non-coupled pairs (see Fig. 7A).

The temporal overlap between feedforward inhibition and orthodromic EPSP in pyramidal cells was measured by projecting the latency of the feedforward IPSP onto the orthodromic EPSP elicited in the simultaneously recorded pyramidal cell (Fig. 7B). Onsets of the feedforward IPSP occurred in 13 of 17 dual recordings during the EPSP rising phase. Temporal overlap was quantified by a scale defined by onset (assigned value ‘1′) and peak of the pyramidal cell EPSP (assigned value ’0′) (see legend to Fig. 7). By this measure, temporal overlap ranged from 0·75 (i.e. feedforward inhibition was present during 75 % of the EPSP rising phase) to -0·21 (i.e. feedforward inhibition started during the early decay phase of the EPSP). On average (all interneurons taken together, n= 17), the onset of the feedforward inhibitory response coincided with the last quarter of the EPSP rising phase (factor 0·255). Slightly faster onsets of feedforward inhibition were mediated by axo-axonic cells (mean overlap, 0·43 ± 0·22) compared with those of bistratified cells (mean overlap, 0·20 ± 0·1) and basket cells (mean overlap, 0·23 ± 0·1).

In Fig. 7C, the values of feedfoward IPSP onset (measured in the interneurons of the 17 pairs shown in Fig. 7B), were projected onto a standard EPSP (2·8 ms latency to onset and 7·8 ms latency to peak: these values were the average from 39 dendritic and 24 somatic pyramidal cell recordings). Comparing Fig. 7B and C shows that the distribution of feedforward IPSP onsets was similar. Using the standard EPSP (as in Fig. 7C), a measure of feedforward inhibition can be obtained from single interneuron recordings (e.g. from the basket cell shown in Fig. 9 and recordings of Lucifer Yellow-stained interneurons from a previous study (Stelzer et al. 1994). The calculated values of temporal overlap feedforward IPSP-orthodromic EPSP based on all interneuron recordings (n= 37; shown in Fig. 7D) confirm the results of the smaller stratum pyramidale interneuron population (n= 17; shown in Fig. 7C): in 31 of 37 comparisons of stratum pyramidale interneuron-mediated IPSPs, feedforward inhibition occurred during the rising phase of the average pyramidal cell EPSP.

A very similar distribution was seen for IPSPs mediated by stratum lacunosum-moleculare (L-M) interneurons: in 10 of 14 recordings, L-M-mediated IPSPs coincided with the EPSP rising phase, and 4 of 14 with the early decay phase of the averaged EPSP.

IPSP onsets obtained from alveus-oriens (A-O) interneuron recordings were significantly slower (Fig. 7D). In 6 of 7 A-O interneurons, IPSP onsets coincided with the decay phase of the EPSP, and in only 1 of 7 with the EPSP rising phase. Furthermore, the mean of temporal overlap between A-O cell-mediated IPSPs and the EPSP (-0·24 ± 0·16) was significantly different from that of pyramidale interneurons (0·30 ± 0·05; P < 0·002, ANOVA) and L-M interneurons (0·24 ± 0·09; P < 0·01). The late onset of A-O cell-mediated IPSPs is probably due to the fact that A-O interneuron activation was implemented via recurrent excitation through CA1 pyramidal cell APs (Blasco-Ibanez & Freund, 1995; Maccaferri & McBain, 1995). This notion is supported by the delayed onset of the stimulation-evoked EPSP in A-O interneurons (6·4 ± 1·3 ms, mean ±s.e.m., n= 7). In comparison, mean onset of orthodromic EPSPs in stratum pyramidale interneurons was 2·7 ± 0·3 ms (n= 28; Table 3B).

Differential excitability of interneurons and pyramidal cells

The protocol of comparing time courses of feedforward inhibition and EPSP required the concomitant elicitation of synaptic APs in the interneuron, but subthreshold EPSPs in the pyramidal cell (Fig. 7A). This condition was met in a large range of stimulation intensities due to differential excitability of interneurons and pyramidal cells (Scharfman, 1991). For example, in the paired recording shown in Fig. 8A, synaptic spikes in the axo-axonic cell were elicited at 20 μA, whereas > 500 μA was required for synaptic spikes in the concomitantly recorded pyramidal cell dendrite. In addition, double spike responses in the interneuron were accompanied by subthreshold EPSP responses in the pyramidal cell over a large range of stimulation intensities (Fig. 8Ac). In Fig. 8B, probabilities of synaptic spikes were compared in a basket cell and four pyramidal cells (3 dendrites and 1 soma) that were recorded consecutively during the same interneuron recording. The basket cell responded with synaptic APs at any intensity (with 100 % probability at intensities > 300 μA). In contrast, none of the three concomitantly recorded pyramidal cell dendrites exhibited spike responses at any of the selected stimulation intensities. In the pyramidal cell soma, 10-20 % of synaptic APs were observed at intensities of 500 and 700 μA, respectively.

It seems that in CA1 pyramidal cells - similar to neocortical pyramidal cells (Ling & Benardo, 1998) - the magnitude of inhibitory responses reaches an upper limit at stimulation intensities at which excitation is far below its maximum. In the dual recordings shown in Fig. 8A and B, interneurons responded in all recordings with synaptic spikes at the stimulation intensities that elicited half-maximal EPSPs in the pyramidal cells (at 300 μA in Fig. 8A; marked by arrows in Fig. 8B b). With stimulation intensities adjusted to generate about half-maximal EPSPs in the pyramidal cells (e.g. in the experiments shown in Fig. 7), synaptic interneuron APs were elicited in 57 of 62 pairs (in 39 of 57 cases with a 100 % probability, in 18 cases with probabilities between 34 and 90 %). Taken together, these data show that maximal efficacy of feedforward inhibition is reached by stimulation intensities that generate only about 50 % of maximal EPSPs in pyramidal cells.

Temporal overlap of feedforward inhibition and EPSP: role of stimulation intensity

As shown in Fig. 8A (at 20 μA), synaptic interneuron APs could be elicited at stimulation intensities that were below the threshold for eliciting pyramidal cell EPSPs. This was observed in 16 of 24 combinations of interneuron-pyramidal cell recordings at which such low intensities were applied. These data demonstrate that the weakest excitatory input into CA1 is accompanied by large activation of the feedforward inhibitory pathway. What is the temporal overlap between feedforward inhibition and evoked excitation for such weak excitatory input?

Activation kinetics changed with increasing stimulation intensity in both interneurons (Lacaille, 1991) (Fig. 9E) and pyramidal cells (Fig. 10E). As illustrated in Fig. 9 in a single interneuron recording, the decrease in t_(I,AP,peak) (Fig. 9D) was accompanied by a parallel increase in the slope of the orthodromic EPSP (Fig. 9C and F). However, the changes of interneuron kinetics do not generally result in a change of temporal overlap between feedforward inhibition and evoked excitation in the pyramidal cell. This is illustrated in Fig. 10. There was a close correlation between latencies to peaks of interneuron APs (t_(I,AP,peak); Fig. 10B) and latencies to EPSP peak in pyramidal cells (Fig. 10A). As a result, the difference of t_{(P,EPSP,peak)} and t_(I,AP,peak) (Fig. 10C) and temporal overlap of feedforward inhibition and orthodromic excitation (Fig. 10F) were similar in the entire range of stimulation. Latencies to onset were not generally affected by stimulation intensity (Fig 9E and 10E). These data show that the high degree of temporal overlap between feedforward inhibition and excitation - measured at half-maximal EPSPs (Fig. 7) - is also seen at lower stimulation intensities and smaller EPSPs.

Recurrent inhibition

The time course of the recurrent inhibitory response was evaluated via measurements of its underlying components (Fig. 11): (1) latency to peak of synaptic pyramidal cell AP (t_(P,AP,peak); Fig. 11Aa, P); (2) latency between pyramidal cell AP peak and unitary AP peak in a coupled interneuron (Δt_(P,AP,peak-_{I,unitaryAP,peak)}; Fig 11Ab and 12A and B); (3) latency between interneuron AP peak and onset of unitary IPSP in the pyramidal cell (Δt_(I,AP,peak-_{P,mono-IPSP,onset)}; Fig. 11Ac).

The results of this approach can be summarized as follows (see below for details). First, the earliest onsets of the recurrent IPSP occur several milliseconds (> 3 ms) after the peak of the orthodromic EPSP in pyramidal cells. Thus recurrent inhibition is not a factor in the control of the EPSP rising phase. Second, recurrent inhibitory responses are prominently expressed at the time of the peak of the orthodromic population IPSP: in the recordings of Fig 11, Fig 12 and Fig 13, earliest onsets of recurrent inhibition occurred at poststimulation latency t= 11 ms (see Fig. 11B); the peak of the population IPSP was measured at t= 26 ms. Third, a widespread range of recurrent IPSP onsets (in the pyramidal cell shown in Fig 11, Fig 12 and Fig 13 from 11 to 99·8 ms; see Fig. 11B) were observed.

Measurements of all three components could be obtained from a mutually coupled bistratified cell and pyramidal cell apical dendrite (Fig 11, Fig 12 and Fig 13). In this pair, strong coupling was observed between the pyramidal cell and the interneuron: pyramidal cell APs elicited large unitary EPSP without failure (in many cases suprathreshold) in the interneuron (Fig. 12). In reverse, the interneuron was (albeit weakly) coupled to the recorded dendrite (Fig. 13). The onsets of mono- and feedforward IPSPs in relation to the pyramidal cell EPSP in this pair (Fig. 11B) were in the general range of respective onsets seen in other cells (see Fig. 2 and Fig. 7).

Latencies to peak of pyramidal cell AP

In pyramidal cells, the latencies to peaks of synaptic APs (component a, Fig. 11A) coincided roughly with the latencies to their EPSP peaks (e.g. Fig. 4B; Table 1B for single recordings; Fig 8A and 11A for dual recordings). In the pyramidal cell of the dual recording shown in Fig 11, Fig 12 and Fig 13, mean latency to synaptic AP peak was 9·8 ± 0·3 ms (n= 27; range 8·6-11·4 ms; Fig 11Aa and Fig 12D); mean latency to EPSP peak was 9·4 ± 1·1 ms (n= 12, range 5·6-12·0 ms). Since the onset of recurrent inhibition starts several milliseconds after the EPSP peak (i.e. the delay is the sum of components b and c, see below), it is clear that recurrent inhibitory influences are not present during the slope or peak of the orthodromic EPSP.

Latencies of recurrent disynaptic IPSPs

Summation of all three components of recurrent inhibition

The shortest latency to onset of the recurrent (trisynaptic) IPSP (sum of respective minima of components a-c) was 11·0 ms. The longest latency to onset (sum of respective maxima of components a-c) was 99·8 ms (Fig. 11B).

Monosynaptic inhibition

Since the monosynaptic inhibitory response is elicited by direct electrical stimulation of interneuron processes (Fig. 5) and is therefore not observed in situ, the question arises as to why such detailed examination of the kinetics was carried out (Figs 2-5; Table 1). First, the orthodromic response of CA1 pyramidal cells is a frequently used experimental measure of excitability. The presented data (Figs 2-4; Table 1) show that - with monosynaptic inhibition being an integral component - GABA_A-mediated influences are present before the onset of the orthodromic EPSP in dendrites. Second, the monosynaptic inhibitory response could be isolated pharmacologically (Davies et al. 1990). It is inferred that the main finding regarding the kinetics of monosynaptic inhibition (g_GABAA faster than IPSCs and IPSPs) applies also to feedforward and feedback inhibitory responses. Third, the similarity of latencies to IPSPs measured directly (Figs 2-4; Table 1) and indirectly via summation of components (Figs 5 and 6) demonstrated the feasibility of the indirect approach via summation of components. The latter was the sole approach in the evaluation of feedforward (Fig. 7) and feedback inhibition (Fig. 11).

Feedforward inhibition

The kinetics of interneuron activation reported here are in agreement with those reported previously: overall latency to disynaptic IPSPs (Miles, 1990b), poststimulation intervals to interneuron AP peaks (Lacaille, 1991; Scharfman, 1991), and differences in latency between interneuron and pyramidal cell firing (Ashwood et al. 1984). Kinetics of dendritic unitary IPSPs, however, were found to be faster in cases of strongly coupled cells (see Table 2) than reported previously (Miles, 1990b; Buhl et al. 1994a.

Data in Fig. 7B -D show that fastest feedforward inhibitory responses cover 75 % of the EPSP rising phase. There is convincing evidence that onsets of feedforward inhibitory responses are even faster than illustrated in Fig. 7. First, as inferred from the analysis of monosynaptic inhibition (see Fig. 3B, Table 1B), onsets of g_GABAA could be detected before the corresponding IPSPs. Second, the interneurons used in the dual recordings shown in Fig. 7 represent a selection of non-spontaneously firing cells (with a higher V_rest and thus somewhat longer latencies to synaptic spikes). Third, onsets of feedforward IPSPs (Fig. 7) were calculated by substituting a 1 ms latency for the onset of the unitary dendritic IPSP. However, latencies to onsets of unitary IPSP could be as short as 0·2 ms (Fig. 6D). Therefore - by taking variability of onsets into account - fast onsets of feedforward IPSPs could precede those shown in Fig. 7 by 0·8 ms. Using 0·2 ms instead of 1 ms as latency to unitary IPSP (shown in Fig. 8A, see arrows at 300 and 500 μA), complete temporal overlap with the orthodromic pyramidal cell EPSP was observed.

The high degree of temporal overlap between feedforward inhibition and orthodromic excitation (Figs 7 and 8A) is a consequence of considerably shorter latencies of interneuron APs (Fig. 12D) (Ashwood et al. 1984). Steeper EPSP slopes of the interneuron at a given intensity (Table 3A) indicate that the differences in activation kinetics and excitability in the two cell types were linked. Cellular properties of interneurons such as higher input resistance (Table 3A) or different AMPA receptor subunit expression (McBain & Dingledine, 1993; Jonas et al. 1994) could be responsible.

Recurrent inhibition

Results show that recurrent inhibitory responses have no impact on the rising phase and peak of the orthodromic EPSP in CA1 pyramidal cells: earliest onsets of recurrent IPSPs occur several milliseconds after the EPSP peak (Table 1B; Fig. 11B). Moreover, orthodromic activation of the recurrent inhibitory pathway is the exception. First, elicitation of orthodromic APs in pyramidal cells occurs in general only at very high stimulation intensities (Fig. 8). In addition, orthodromic activation of recurrent inhibition is most probably confined to interneurons that are not connected in a feedforward fashion. In interneurons that are connected in a feedforward manner, the AHP of the synaptic AP prevents recurrent unitary APs (Fig. 12Da). The second of synaptic double spikes is more likely to be generated by feedforward excitation (cf. Fig. 8A at 700 μA, and Fig. 12Db).

The importance of recurrent inhibition in CA1 may lie - in analogy to the CA3 region (Miles & Wong, 1986, 1987) - in containing recurrent excitation (Deuchars & Thomson, 1996; Ali et al. 1998). The average latencies of recurrent IPSPs (8·3 ms) in the dual recording (Figs 11-13) were somewhat longer than described for disynaptic recurrent IPSPs in CA3 (Miles, 1990a) and CA1 (Sik et al. 1995). Possible reasons are as follows. First, interneuron V_rest was somewhat higher than average due to the interneuron selection criteria of lack of spontaneous firing. Second, the value of the onset of the unitary IPSP (about 2 ms) was longer due to weak inhibitory coupling (see Table 2). Third, recurrent activation kinetics of bistratified cells could have been slower in comparison with basket cells (Ali et al. 1998).

A suprising observation in the mutually coupled pair of cells shown in Figs 11-13 was the occurrence of delayed unitary APs riding on plateau EPSPs in interneurons (Fig. 12). The combination of the slow and fast unitary interneuron APs (Fig. 12C) - even faster values have been reported (Miles, 1990a; Sik et al. 1995) - generated a wide range of latencies (Figs 11B and 12C). This could be a mechanism in the observed long-lasting presence of GABA_A-mediated synaptic influences following afferent stimulation (see Fig. 1Aa). The prolonged presence of inhibition conceivably enhances the efficacy of containing recurrent excitation. However, delayed unitary interneuron APs were obtained in a single pair of cells and the general occurrence of this phenomenon remains to be established.

Inhibitory conductances

Although IPSP/IPSCs and g_GABAA were evoked by the same stimulus (Figs 2-4), they may not represent the same event. It was shown in motoneurons that the inhibitory action was considerably faster than indicated by the IPSP (Araki et al. 1960). Our kinetic measurements of g_GABAA suggest that GABA_A-mediated inhibition may also be considerably faster than indicated by the IPSC (Fig. 3D; Table 1). Current measurements were used in previous studies for assessing the effects of inhibitory shunting (Edwards, 1990; Staley & Mody, 1992). While efficacy of shunting can be measured by the time integral of the current waveform, which is subject to much less distortion than the amplitude (Jack et al. 1975; Carnevale & Johnston, 1982), measurements of charge transfer to soma do not decrease errors of kinetics that are associated with current measurements. In contrast to IPSP/IPSCs that are derived from spatially diverse sources containing axial and membrane components, inhibitory conductance changes occur in the immediate vicinity of the synapse (Araki et al. 1960; Andersen et al. 1980; Koch et al. 1983).

Based on the temporal interaction of EPSCs and IPSCs, it could have been concluded that synaptic inhibition may primarily affect later parts of the orthodromic EPSP/EPSC such as the delayed NMDA component (Staley & Mody, 1992). Based on the kinetics of g_GABAA (Table 1; Fig. 4), however, it can be concluded - at least in CA1 - that the orthodromic EPSP/EPSC rising phase is tightly controlled by GABA_A-mediated inhibition.

How will activity-dependent modification of inhibition change the orthodromic EPSP/EPSC?

A consequence of the shown complete temporal overlap of afferent excitation and inhibition in CA1 pyramidal cells (Figs 4 and 8) is that any modification of GABA_A-mediated inhibition will affect the slope and peak of the EPSP. In CA1, the net effect of LTP-inducing tetanization on synaptic inhibition is an EPSP potentiation due to long-term disinhibition caused by impairment of GABA_A receptor function. The evidence is as follows. First, barring effects on GABA release mechanisms, the main action of tetanization on monosynaptic inhibition is impairment of GABA_A receptor function (Stelzer et al. 1994). More importantly, a long-term reduction of monosynaptic inhibitory responses by tetanization was demonstrated (Stelzer et al. 1994). Second, impairment of GABA_A receptor function is also the main modification of the efficacy of feedforward inhibitory responses. At the stimulation intensities that are typically used for controls in LTP studies (e.g. 50 % of pyramidal cell EPSP amplitudes, arguably higher in field potential studies), feedforward inhibitory responses are close to their upper limit (see Fig. 8A and B). The maximal expression of feedforward inhibition during control occludes a potential tetanization-induced increase of interneuron excitability. An enhancement of recurrent inhibition caused by pyramidal cell LTP has no impact on the pyramidal cell EPSP rising phase or peak due to lack of temporal overlap (Fig. 11B). In summary, the presented data of temporal interaction of afferent excitation and inhibition in CA1 pyramidal cells support the previous postulate of disinhibition as LTP mechanism (Stelzer et al. 1994; Wang & Stelzer, 1996).