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. 1999 Mar 15;515(Pt 3):757–767. doi: 10.1111/j.1469-7793.1999.757ab.x

Long-term suppression of synaptic transmission by tetanization of a single pyramidal cell in the mouse hippocampus in vitro

Yevgenij Yanovsky 1, Helmut L Haas 1
PMCID: PMC2269175  PMID: 10066902

Abstract

  1. The consequences of stimulating a single pyramidal cell in the CA1 area of the hippocampus for synaptic transmission in the stratum radiatum were investigated.

  2. Tetanic activation of single pyramids caused by depolarizing current injection, but not an equal number of distributed action potentials, reduced excitatory transmission by 20%, with a delayed onset, for more than 1 h.

  3. EPSPs in the tetanized pyramidal cells were increased for equally long periods but this was not the cause of the field EPSP reduction. Spontaneous somatic IPSPs were not affected; evoked IPSPs were decreased in the tetanized cell.

  4. Paired pulse facilitation of the field EPSPs was unchanged.

  5. The field EPSP reduction was markedly diminished by a knife cut along the base of pyramidal cells in CA1.

  6. The addition of antagonists of GABA, NMDA and metabotropic glutamate receptors blocked or diminished the field EPSP slope reduction evoked by intracellular stimulation.

  7. Simultaneous recordings revealed long-lasting excitations of interneurons located in the outer oriens layer as a result of single pyramid tetanization.

  8. Intense firing of small numbers of pyramidal cells can thus persistently inhibit mass transmission through the hippocampus. This effect involves activation of interneurons by glutamate receptors.

Pyramidal cells in the CA1 area of the rodent hippocampus target with their recurrent collaterals both neighbouring pyramidal cells and GABAergic interneurons. Local collaterals and consequently the excitatory interactions among CA1 pyramidal cells are relatively sparse (Amaral & Witter, 1989; Amaral et al. 1991; Radpour & Thomson, 1992). The incidence of observing connections between pairs of pyramidal cells in CA1 was approximately 1 in 100 tests (Deuchars & Thomson, 1996). Eleven per cent of the hippocampal neurons display GABA-like-immunoreactivity; the vast majority of these cells are located within the regions containing apical and basal dendrites of pyramids (Woodson et al. 1989).

Spike generation in a small number of pyramidal neurons can suppress further discharges from those cells and their neighbours through recurrent GABAergic inhibition (Knowles & Schwartzkroin, 1981; Arai et al. 1995). The interneurons involved in such action include the following. (1) Basket cells receiving afferent and recurrent input (Frotscher, 1985; Frotscher, 1989; Freund & Buzsaki, 1996). One basket cell may innervate 500–1600 postsynaptic neurons in a 400 μm slice (Miles et al. 1996). In vivo (Sik et al. 1995) a single basket cell may be connected with 1500–2500 pyramidal cells. (2) Interneurons located close to the alveus, which contain somatostatin, sending their axons to stratum lacunosum-moleculare (McBain et al. 1994; Sik et al. 1995). These interneurons are primarily involved in feedback circuits (Freund & Buzsaki, 1996). (3) Bistratified and horizontal trilaminar cells (Buhl et al. 1994; Sik et al. 1995), located within or near the stratum pyramidale or at the stratum oriens-alveus border. A single bistratified neuron may innervate approximately 2500 pyramidal cells (Sik et al. 1995). The laminar distribution of their dendritic trees enables them to receive input from commissural- associational fibres and from local recurrent collaterals. (4) Another distinct type of interneuron occurring at the stratum oriens-alveus border (Sik et al. 1994) projects across subfield boundaries but one-quarter of the axon endings are located in CA1. These cells are also likely to be driven primarily by the local collaterals of CA1 pyramidal cells.

Direct activation of interneurons at the alveus-oriens border by glutamate or vasoactive intestinal peptide (VIP) causes a long-lasting suppression of field EPSPs (Yanovsky et al. 1997). IPSPs evoked by alveus stimulation are not potentiated after high-frequency stimulation of the afferent fibres in stratum radiatum (Haas & Rose, 1982), but tetanic stimulation of the alveus leads to an NMDA-dependent enhancement of IPSPs recorded in pyramids (Grunze et al. 1996). Several types of hippocampal interneurons have been tested for the occurrence of long-term potentiation (LTP); Maccaferri & McBain (1996) found LTP only as a result of passive propagation from pyramidal cells, while others have described LTP on interneurons which receive recurrent collaterals from the pyramids (Ouardouz & Lacaille, 1995) or a persistent enhancement of IPSPs evoked by alveus stimulation (Grunze et al. 1996). We have now examined the consequences of tetanic activation of single pyramidal cells in the CA1 region and present evidence for a long-term reduction of excitatory transmission in a large area of stratum radiatum which is mediated by a long-lasting activation of interneurons in the oriens region.

METHODS

Seventy-two male mice (3 months old) of the NMRI strain (Tierversuchs-anstalt der Heinrich-Heine-Universität (TVA) Düsseldorf, Germany) were stunned and rapidly decapitated, and the brains were quickly removed and placed into ice-cold Krebs- Ringer solution. The experiments were performed in accordance with German law and permitted by the Bezirksregierung Düsseldorf. Slices from the dorsolateral hippocampus, 500 μm thick, were cut parallel to the dorsal surface of the brain using a vibroslice. After preincubation for at least 1 h the slices were transferred to a custom-built recording chamber, submerged in oxygenated medium (95 % O2 and 5 % CO2) at 32°C and perfused at 1 ml min−1 with the following solution (mM): 124.0 NaCl, 3.7 KCl, 2.0 CaCl2, 1.3 MgSO4, 26.0 NaHCO3, 1.24 NaH2PO4 and 10 glucose (pH 7.4).

Glass micropipettes for intracellular recording were filled with 4 M potassium acetate (pH 7.4). Biocytin (Sigma, 1 %) was sometimes included in the pipette solution (1 M potassium acetate) to allow reconstruction of the recorded neuron using a camera lucida. Signals were fed to a high-impedance DC amplifier (Axoclamp-2A), continuously recorded on tape, simultaneously digitized and displayed on a computer monitor using the pCLAMP software package (Axon Instruments). Potentials were continuously plotted on a chart recorder. Membrane properties of neurons were determined during steady state conditions more than 30 min after the penetration. Resting membrane potential was determined as the potential change upon withdrawal of the electrode from the cell.

Glass micropipettes filled with perfusion medium were used for extracellular recordings of field potentials (3–5 MΩ). They were placed in stratum radiatum for field EPSP recording. Signals were amplified, recorded on tape and digitized for computer analysis. The amplitude and slope of EPSPs were measured. Mean values and standard errors of the mean are given in the figures. Non-parametrical statistics were used for determination of significance (Wilcoxon test for two independent samples). Bipolar electrodes were placed at the stratum radiatum to stimulate Schaffer collaterals and commissural fibres at 0.1 Hz; constant current pulses had a duration of 0.1 ms. The interval between depolarizing pulse and Schaffer collateral-commissural stimulation was 5 s. In some experiments synaptic activation was entirely absent during the phase of intracellular tetanization; there was no pairing of intracellular and fibre stimulation. Micropipettes for extracellular recording of activity of CA1 interneurons in the stratum oriens were filled with 4 M NaCl (10–20 MΩ). The spontaneous firing of interneurons was continuously recorded by a ratemeter. Latency and number of spikes in response to Schaffer collateral stimulation were measured.

Drugs

Bicuculline methiodide (a GABAA antagonist) was from Sigma, Deisenhofen, Germany; CGP 55 845A (a GABAB antagonist) was from Ciba-Geigy, Basel, Switzerland. In experiments where both these antagonists were bath applied the Mg2+ concentration was increased from 2 to 6 mM to prevent burst firing. (RS)-α-Methyl-4-carboxyphenylglycine (MCPG; a metabotropic glutamate receptor antagonist) and DL-2-amino-5-phosphopentanoic acid (DL-AP5; an NMDA antagonist) were from Tocris Cookson, Bristol, UK.

RESULTS

The results presented here are based on recordings from 119 CA1 pyramids and 12 interneurons. Pyramidal cells received an intracellular stimulation by positive current injection (500 pA, 500 ms, 10 times at 0.1 Hz, Fig. 1C) while the field EPSP in stratum radiatum was continuously monitored (Fig. 1E). Nine of the neurons were recorded in stratum oriens, filled with biocytin and reconstructed by drawing a camera lucida picture. Seven cells of this group were identified as displaced pyramidal cells; two were apparently dendritic recordings as the cell body was located in the pyramidal layer. Although a minimal difference in electrophysiological properties between misplaced and regular pyramids was found, there was no difference in the effect of intracellular stimulation on excitatory transmission in stratum radiatum. Therefore the two groups are considered together. The mean electrophysiological properties for all pyramidal cells were as follows: resting potential, −70.3 ± 0.4 mV; action potential amplitude, 87.1 ± 1.4 mV; input resistance, 78.4 ± 3.2 MΩ; time constant, 12.3 ± 0.8 ms; spontaneous firing rate, if present, 0.53 ± 0.08 s−1. Only eight cells showed spontaneous activity. One of these is illustrated in Fig. 1. The stimulation of Schaffer collaterals- commissural fibres was weak, usually subthreshold for action potentials in control conditions. The latency of EPSPs was 3.8 ± 0.1 ms, and their amplitude 8.4 ± 0.6 mV. Positive current injections (500 ms, 0.5 nA) evoked trains of 13.6 ± 1.2 spikes.

Figure 1. Intracellular recording from a pyramidal cell and consequences of its tetanization on field potentials.

Figure 1

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A, spontaneous action potentials. B, response to afferent stimulation. C, response to strong depolarizing pulse (+500 pA, 500 ms). Ten such pulses were used for intracellular stimulation (tetanization). D, hyperpolarizing pulses, 100–500 pA. E, change in field EPSP slope following intracellular stimulation (10 times); left, diagram; right, averaged field EPSPs at points 1 and 2. F, scheme of experimental situation. al, alveus; so, stratum oriens; sp, stratum pyramidale; sr, stratum radiatum; sl-m, stratum lacunosum-moleculare.

Suppression of field EPSPs

Ten positive current injections at an interval of 10 s caused a slow decline of the concurrently monitored field EPSP in 38 of 46 experiments. Figure 2A illustrates the amplitudes of all field EPSPs in an individual experiment. The decline seemed to occur in two phases, one with a duration of about 20 min and the second with a much longer duration of 1–2 h or more. In most experiments (n = 42) full recovery of field EPSPs from the reduction induced by intracellular tetanization was not obtained during a 60 min observation period (Fig. 2B and C). The average decline of field EPSPs in response to intracellular pyramid activation was by 24.3 ± 4.4 % (P < 0.01, n = 31, Fig. 2C). In only four experiments the field EPSP reduction waned within 1 h; one of these is illustrated in Fig. 1E. It is possible that only the first of two apparent phases of the effect was present in these cases. When instead of pyramid tetanization by 500 ms depolarizing pulses a smaller permanent depolarization causing a similar number of spikes within the same time period of 100 s was given in four experiments no change occurred in EPSP slopes (0.25 ± 0.04 versus 0.24 ± 0.04 mV ms−1). In five experiments where pyramid stimulation consisted of a protocol imitating the theta rhythm (a short pulse eliciting 3 action potentials at 5 Hz, 40 times) no changes were seen in the field EPSPs (0.27 ± 0.04 versus 0.25 ± 0.03 mV ms−1, difference not significant).

Figure 2. Intracellular stimulation of single CA1 pyramidal cells suppresses synaptic transmission in stratum radiatum.

Figure 2

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A, stimulation of single CA1 pyramidal cell (10 times, 500 ms, +500 pA pulses at 0.1 Hz) decreases field EPSPs in stratum radiatum. Each point represents the amplitude of a field EPSP elicited every 10 s. The diagram illustrates every EPSP between −5 and +5 min; after 5 min the time base changes and only every 10th EPSP is given. Inset shows 1 of the 10 responses to intracellular stimulation given during the time indicated by shaded area. Note slow onset of the effect on field EPSPs. B, another single experiment featuring averaged EPSP slopes normalized to the mean of control. C, the mean of 31 experiments; D, the mean of 7 experiments in which both the intracellular EPSP (intEPSP) in the pyramidal cell and the field EPSP (fEPSP) in stratum radiatum were measured. Tetanization at vertical arrows, time 0.

In a further series of seven experiments double field recordings were made in the stratum radiatum of CA1, one at the level of the intracellularly recorded pyramid and one at a distance of 300 μm; this distance represents about half the extent of CA1 in the mouse hippocampal slice. The remote field EPSP displayed a longer latency (1–1.5 ms) and a slightly slower slope but no difference from the close recording site in the reduction induced by intracellular pyramid stimulation (Fig. 3). At 30 min after intracellular tetanization the EPSPs were reduced to 79.8 ± 3.3 % for the close and 85.5 ± 2.1 % for the remote recording site. This difference is not significant indicating the involvement of neurons with widespread axonal projections in the stratum radiatum.

Figure 3. Equal EPSP slope reduction after single pyramid tetanization recorded at two different sites in stratum radiatum.

Figure 3

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The close recording site was at the level of the tetanized pyramid, indicated by filled circles in the diagram below; the remote (300 μm) recording site is represented by open circles. Mean of 7 experiments.

Paired pulse facilitation

In four experiments paired pulses were employed for synaptic activation in order to gain an indication on the locus of the depressant effect. Three different stimulation intensities allowed a comparison of EPSP pairs with the first (unconditioned) EPSPs displaying similar slopes before and after induction of the depression. The facilitation ratios were the same before (1.25 ± 0.02) and after (1.26 ± 0.2) the intracellular tetanization indicating a postsynaptic locus of action at the pyramidal dendrites.

Long-term potentiation of intracellularly measured EPSPs

Along with the reduction of the field EPSPs the slope of intracellularly registered EPSPs in the tetanized pyramid increased by 34.1 ± 8.4 % (n = 7, Fig. 2D). This was not the reason for the field EPSP inhibition as the intracellular EPSPs remained mostly subthreshold for action potential generation. Thus the altered response of this pyramidal cell has no consequence for the activation of interneurons through our test stimulus.

GABAergic inhibition

Sixteen experiments were performed in the absence of GABAergic inhibition. First, both antagonists of GABAA and GABAB actions were added to the medium: bicuculline (12 μM) and CGP 55 845A (0.6 μM). This treatment abolished the suppression of the field EPSP slope evoked by intracellular tetanization of a single pyramidal cell (n = 8, Fig. 4A). Some EPSP slope reduction was present after 50–60 min. Thus the EPSP suppression depends on GABAergic inhibition at least during the first three-quarters of an hour after intracellular pyramid tetanization. The degree of EPSP slope suppression was smaller in a medium containing only the GABAB blocker CGP 55 845A (0.6 μM, Fig. 4B, n = 8). The maximal field EPSP inhibition was by 11.6 % at 45 min after the intracellular tetanus as compared with 21.6 ± 4.0 % at the same time point in control conditions. The difference between the suppression in control and in the presence of the GABAB blocker was significant for the time from 10 to 55 min (P < 0.1). The suppression of field EPSPs evoked by intracellular tetanization of a single pyramidal cell thus involves both types of GABA receptors.

Figure 4. Block of EPSP slope reduction by GABA antagonists.

Figure 4

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A, a mixture of both GABAA and GABAB antagonists blocks this reduction completely for 40 min and markedly reduces it after that time. B, the GABAB antagonist alone blocks about half of the EPSP reduction. Bicuculline (GABAA antagonist, 12 μM) and CGP 55 845A (GABAB antagonist, 0.6 μM) were used. Control curve in this and following figures is the same as in Fig. 2C.

Horizontal GABAergic interneurons at the alveus-oriens border region express somatostatin (Freund & Buzsaki, 1996). Therefore the possibility that somatostatin may be involved in the suppression of field EPSPs was tested with local applications of this peptide in six slices. A small reduction of EPSP slope was noted which did not reach significance.

Lesion of recurrent pathway

A likely path for the observed EPSP depression after single pyramid tetanization is the recurrent activation of interneurons through pyramidal axon collaterals. We tested this in slices where a knife cut had severed the pyramidal axons just below the pyramidal layer in stratum oriens (Fig. 5). In all eight experiments the first phase of EPSP depression was absent, but a small late depression seemed to persist (to 91.8 ± 5.0 %, not significant).

Figure 5. Lesion of recurrent pathway.

Figure 5

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A, a cut along the base of CA1 pyramids prevents the first phase of field EPSP depression following the intracellular tetanization. B, time course of changes in field EPSP slopes in intact slices (•) and in slices with the illustrated cut in stratum oriens (^).

Spontaneous IPSPs in pyramidal cells

Spontaneous IPSPs in pyramidal cells (n = 6) were recorded with chloride-filled electrodes as positive potentials. The intracellular stimulation of the pyramid changed neither the frequency nor the amplitude of spontaneous IPSPs (6.4 ± 0.9 counts per second (c.p.s.) in control conditions and 6.6 ± 0.9 c.p.s. 25 min after intracellular stimulation). The IPSP amplitudes were divided into three groups: from 1 to 2 mV, from 2 to 4 mV and more than 4 mV. For the ‘small’ events the frequency was 4.1 ± 0.9 c.p.s. in control and 4.3 ± 0.4 c.p.s. at 30 min after intracellular stimulation; for ‘medium’ IPSPs: 1.4 ± 0.4 and 1.8 ± 0.7 c.p.s. at 30 min; and for ‘large’ IPSPs: 0.7 ± 0.2 and 0.9 ± 0.4 c.p.s., respectively.

Evoked IPSPs in the tetanized pyramidal cell

The amplitudes of IPSPs evoked by Schaffer collateral- commisural fibre stimulation were measured in pyramidal cells (n = 8) before and after intracellular tetanization. Measurements were taken at −62 mV, 45 ms (early IPSP) and 190 ms (late IPSP) following afferent stimulation during 60 min after intracellular tetanization of the recorded pyramid. We found a decrease of both IPSP types: the early IPSP by 39.6 ± 5.9 % and the late IPSP by 28.4 ± 7.8 % at 30 min after intracellular stimulation.

Involvement of NMDA receptors

A further series of experiments tested for the involvement of NMDA receptors in this plastic process. No changes in field EPSP slope were found after intracellular tetanization in the presense of 50 μM of the NMDA receptor blocker dl-AP5 (n = 7, Fig. 6). The phenomenon is thus entirely NMDA dependent.

Figure 6. EPSP slope reduction is NMDA receptor dependent.

Figure 6

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dl-AP5 (NMDA antagonist, 50 μM, n = 7) abolished the EPSP reduction caused by pyramid tetanization.

Involvement of metabotropic glutamate receptors

The metabotropic glutamate receptor antagonist (RS)-α-methyl-4-carboxyphenylglycine (MCPG) at a concentration of 500 μM decreased the slope of the field EPSP in stratum radiatum by 29.5 ± 7.6 %. Full recovery occurred in about 30 min and was followed by a small rebound excitation of 5.8 ± 3.9 % (Fig. 7B). This EPSP depression was the same in control conditions or when paired with the intracellular tetanization: the maximal early decrease following tetanization was by 26 ± 4.2 % in the presence of MCPG (Fig. 7A). After washout of the MCPG a small EPSP slope reduction was apparent in experiments with tetanization (by 13.3 ± 8.1 %). Thus MCPG blocked the first phase of EPSP depression evoked by tetanization but reduced the second phase (Fig. 7C); the decrease without MCPG was 17 ± 3.6 % at the same time point, the 35th minute after tetanization.

Figure 7. Action of the metabotropic glutamate receptor antagonist MCPG on the slope of the field EPSPs in stratum radiatum.

Figure 7

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A, with single pyramid tetanization at arrow. B, without tetanization, action of MCPG alone. C, difference between A and B, revealing the effect of single pyramid tetanization without the inhibitory effect of MCPG (500 μM).

Simultaneous recordings from pyramidal cells and interneurons

We recorded the firing of 12 interneurons located in the outward half of stratum oriens together with an intracellular registration from a pyramidal cell. All of the interneurons displayed spontaneous brief single action potentials and responded to Schaffer collateral stimulation with a latency that was different from that of the pyramidal cell and the population field. Displaced pyramidal cells, also recorded in this region, had no spontaneous activity, responded to afferent stimulation synchronously with the intracellularly recorded pyramidal cells, and had broader action potentials.

The mean firing frequency of the 12 interneurons was 34.1 ± 12.4 action potentials per minute (c.p.m.). According to their response latency to Schaffer collateral stimulation they fell into two categories: nine neurons discharged after the pyramidal cells, seven of them with a single spike. Two responded with one to two action potentials. The mean latency in this group was 9.6 ± 0.3 ms. The second group of three interneurons responded to afferent stimulation with a group of spikes (from 3 to 12) with the first spike before or during the discharge of the simultaneously recorded pyramidal cells; the mean latency was 4.7 ± 0.5 ms. In the simultaneously recorded pyramidal cells the mean latencies were as follows: EPSP, 3.0 ± 0.15 ms (n = 12); action potential, 5.9 ± 0.3 ms (n = 9); population spike, 5.6 ± 0.2 ms (n = 4).

Changes in spontaneous and evoked activity of interneurons after tetanization of pyramidal cell

Tetanization of a simultaneously recorded pyramidal cell led to an increase in spontaneous activity in 8 of 12 interneurons. The mean frequency during 10 min of control before the tetanization was 34.1 ± 12.4 c.p.m.; during the sequential 10 min periods after tetanization it was 52.1 ± 22.1, 63.6 ± 23.4, 62.5 ± 27.3, 143.4 ± 72.4, 83.5 ± 28.4 and (from 50 to 60 min) 64.7 ± 36.5 c.p.m. (Fig. 8A). The larger increase between 20 and 40 min was due to the appearance of bursts in three cells.

Figure 8. Changes in spontaneous and evoked firing of oriens interneurons after single pyramid tetanization.

Figure 8

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A, firing rates averaged over 10 min in 8 interneurons; tetanization of pyramid at arrow; cpm, counts per minute. B, left, control response to afferent stimulation in interneuron (top, extracellular record) and pyramid (below); simultaneous recording before tetanization. Right, interneuron responses 10 and 60 min after tetanization of the pyramidal cell. All pictures are superpositions of five traces. First spike in the interneuron records occurs before the pyramidal action potential.

Intracellular stimulation of the pyramid led to a decreased latency, by 9.8 ± 2.3 % (P < 0.1), of the first spike in the response of seven interneurons to Schaffer collateral stimulation. In 2 of the 3 interneurons with a discharge latency less than that in pyramidal cells a slowly rising increase in the number of spikes in the response to afferent stimulation occurred after intracellular pyramid tetanization. The number of spikes increased from 3.6 spikes min−1 in control to 5.5 spikes min−1 at 30 min, 6.4 spikes min−1 at 60 min, and 4.5 spikes min−1 at 90 min after tetanization (Fig. 8B) in one of these interneurons. The second one displayed an increase from 11.3 spikes min−1 in control (mean of 10 trials) to 24.5 spikes min−1 at 15 min after intracellular stimulation. This level remained unchanged during the further registration period of 55 min. Thus, intracellular tetanization of one pyramid led to a long-lasting increase in spontaneous or evoked activity in most of the interneurons studied.

DISCUSSION

Intense intracellular stimulation of single CA1 pyramidal cells leads to a long-lasting reduction of excitatory synaptic transmission from afferents in stratum radiatum to the apical dendrites of pyramids. The effect is likely to be caused by recurrently activated interneurons and seems to occur postsynaptically on apical dendrites through a long-lasting change in activity of these cells. It is completely blocked by NMDA receptor antagonism and reduced by metabotropic glutamate receptor (mGluR) antagonism.

This effect of single pyramidal cell stimulation was observed in an extended region along at least half of CA1 in hippocampal slices of the mouse. Two network properties may be responsible: extended recurrent collaterals of pyramids and widespread axonal arborization of the affected interneurons (reviewed by Freund & Buzsaki, 1996). Thus strong firing of single (or a few) pyramidal cells has a profound effect on synaptic transmission in a large surrounding area. This action is likely to result from a long-lasting increase in firing of interneurons recurrently activated from a single pyramid.

Action potentials evoked by current injection in the cell soma can passively or actively invade the dendritic tree (Wong & Stewart, 1992; Stuart & Sakmann, 1994) and open voltage-sensitive Ca2+ channels (Markram & Sakmann, 1994; Magee et al. 1995). This can explain the long-term potentiation of intracellularly registered EPSPs evoked by afferent stimulation in tetanized pyramids, which was also previously described in slices from rats (Kullmann et al. 1992; Kuhnt et al. 1994). This effect is, however, not responsible for the field EPSP suppression reported here as the intracellularly registered EPSPs remained subthreshold for firing action potentials in the tetanized neuron. The increase of the intracellularly registered EPSP in the tetanized cell was presumably counteracted by the general EPSP decrease documented with the field recording and would have been even larger without the latter effect.

The field EPSP suppression depends on intact pyramidal axons, and therefore the most likely path is via activation of recurrent fibres to interneurons. The spontaneous firing of basket cells, as judged from the observation of spontaneous IPSPs, is not changed as a result of pyramid tetanization and most, if not all, spontaneous IPSPs recorded from CA1 pyramidal cells in vitro are of perisomatic origin (Miles et al. 1996). Evoked IPSPs in the tetanized cell were indeed slightly reduced. The interneurons responsible for the somatic IPSPs are probably not the same population that mediates the effect on field EPSPs; on the other hand the intracellular stimulation may have changed the postsynaptic responsiveness - as it did for the EPSP in the opposite direction (Pitler & Alger, 1994). Inhibition of the neuronal population projecting to pyramidal somata by another population of interneurons activated by intracellular tetanization provides a disinhibitory mechanism (McMahon & Kauer, 1997) for which a morphological substrate exists (Cobb et al. 1997; Freund & Gulyas, 1997).

We show here an increase in spontaneous and evoked activity of interneurons located in stratum oriens of CA1 as a result of pyramid tetanization. The passive propagation of LTP from pyramidal cells (Maccaferri & McBain, 1996) is not responsible for the described changes in activity of interneurons as the one pyramidal cell tetanized in our experiments remained mostly below firing threshold. The population of pyramidal cells located more closely to the stimulation area are more likely to fire action potentials in response to Schaffer collateral-commissural stimulation. They could activate interneurons at synapses located near those originating from the tetanized pyramidal cell, a scene for heterosynaptic LTP. Moreover, most CA1 interneurons are aspinous (Freund & Buzsaki, 1996), and aspinous interneurons usually lack synaptic specificity of LTP (Cowan et al. 1998). Heterosynaptic LTP has been shown to be NMDA dependent (Clark & Collingridge, 1996; Murphy et al. 1997) and could be, through long-lasting interneuron activation, the cause of our NMDA-dependent suppression of field EPSPs.

We have seen three kinds of changes in activity of the two types of oriens interneurons encountered, responding before or after the pyramidal cell firing: (1) an increase in the frequency of spontaneous firing was observed in both groups; (2) a reduction in response latency occurred only in cells firing after the pyramids; and (3) an increase in the number of spikes in response to afferent stimulation occurred only in the two cells which responded monosynaptically to afferent stimulation. A direct activation of these cells is possible but the observed latencies make this unlikely. A response to afferent stimulation after the population spike indicates the lack of direct input from Schaffer collaterals - a property of ‘horizontal’ cells. Cells responding with short latency, monosynaptically, are likely to be ‘vertical’ interneurons (Lacaille et al. 1987; Freund & Buzsaki, 1996; Maccaferri & McBain, 1996). Thus, conventional LTP and a long-lasting increase in spontaneous firing occurred only on putative vertical cells while spontaneous firing increases and latency reduction were observed in putative horizontal cells which show, interestingly, a prominent temporal summation of EPSPs (Ali & Thomson, 1997). The tonic rather than the phasic changes in interneuron activity are likely to be the major reason for the observed reduction in the field EPSP.

Tetanic stimulation of stratum oriens in conjunction with postsynaptic depolarization increases EPSPs in local interneurons. This form of LTP is prevented by bath application of NMDA and mGluR antagonists (Ouardouz & Lacaille, 1995). The spontaneous firing of interneurons also increases, NMDA dependently, as a result of afferent tetanization (Stelzer et al. 1994; Poncer & Miles, 1995) and a high NMDA sensitivity of IPSP potentiation following antidromic tetanization of pyramidal cells was observed by Grunze et al. (1996). The long-lasting depression of transmission described in our report is, like these different forms of interneuron plasticity, dependent on NMDA receptor and mGluR activation. A combined role of metabotropic and NMDA receptors has been put forward by several authors (Musgrave et al. 1993; Ben-Ari & Aniksztejn, 1995). Such a mechanism on interneurons would be in line with our finding that MCPG and dl-AP5 each reduced or blocked the effect of intracellular tetanization.

Several immunocytochemical studies show a striking staining of horizontally running dendrites at the stratum oriens-alveus border (Martin et al. 1992; Gorcs et al. 1993). Baude et al. (1993) provided direct evidence that all mGluR1α-positive neurons in this location are also immunoreactive for somatostatin, the marker of horizontal interneurons projecting to the stratum lacunosum-moleculare. These cells respond to ACPD, the metabotropic glutamate agonist, with strong depolarization and a dramatic increase of firing for a long period of time (McBain et al. 1994). Local application of t-ACPD in the alveus region causes a persistent reduction of excitatory transmission in stratum radiatum (Yanovsky et al. 1997).

Our data favour the involvement of interneurons located in the outer oriens regions but do not exclude contributions by interneurons with vertically oriented dendrites located elsewhere which are innervated by pyramidal axons. The discharges of these interneurons evoked the inhibition of excitatory transmission in the stratum radiatum by activating both GABAA and GABAB receptors. Each type of receptor contributed about half of the effect.

The slow rise of the field EPSP suppression may depend on slow intracellular processes leading to the plasticity; similar time courses and phases have recently been described as a result of cyclic AMP-mediated long-term increases in excitability (Selbach et al. 1997). Ca2+ entry induced by depolarizing pulses increases the sensitivity of cerebellar Purkinje cells to GABA and induces a retrograde inhibition of presynaptic terminals. This may be achieved by a diffusible messenger (Llano et al. 1991). A similar mechanism could be at work in the hippocampus, especially for the late phase of EPSP depression in our experiments, which is not removed by severing the pyramidal axons and metabotropic glutamate receptor block. A depolarization-induced suppression of GABAergic inhibition in pyramidal cells has been described by Pitler & Alger (1994); this phenomenon appears and wanes within seconds after depolarization and is presumably unrelated to our finding of a long-lasting reduction of evoked IPSPs in the tetanized pyramid.

Discharge of single interneurons can modify the phase of intrinsic oscillations in the target pyramidal cells or prevent their firing altogether (Cobb et al. 1995; Miles et al. 1996). Conversely, a discharging pyramidal cell may induce firing in many of its target interneurons (Gulyas et al. 1993). Because a single pyramidal cell innervates hundreds of interneurons and one interneuron in turn can innervate 1000–3000 pyramidal cells (Buhl et al. 1994; Li et al. 1994; Sik et al. 1995), discharging a single pyramidal neuron is indeed expected to affect tens of thousands of other pyramidal cells (Freund & Buzsaki, 1996). Even with the smaller number of involved neurons in the restricted circuitry of a slice the wide ranging and long-lasting effects described here reflect the intricate interaction of the network. Combined activation of metabotropic and ionotropic receptors may trigger oscillatory responses in interneurons of basal dendritic layers (Carmant et al. 1997). Such behaviour was indeed observed on two of our recorded cells in the region of basal pyramidal dendrites after tetanization of one pyramid (Fig. 8B).

Intense discharges of pyramidal cells occur under physiological and pathophysiological conditions. Sharp waves (Buzsaki, 1986) or epileptiform discharges would be expected to induce a long-lasting reduction of excitatory transmission. In particular our results suggest that a single or a few repetitively discharging pyramids can dampen mass transmission in the region. The functional role of this long-lasting inhibition may include a suppression of further input from CA3 and entorhinal cortex following an intense discharge of small populations of pyramidal neurons, for instance during sharp waves and γ-oscillations (Traub et al. 1996). Demarcation of place fields in the CA1 area of exploring rats can occur within 5 min (Tanila et al. 1997), and better focusing of place cells requires at least 10 to 30 min (Wilson & McNaughton, 1993), time courses which are similar to the ones observed for the phenomenon presented in our report. A tetanized pyramid displays LTP while the input to the region is suppressed: a mechanism perfectly suitable for the improvement of spatial selectivity which occurs during learning in a new environment.

Acknowledgments

This work was supported by Human Frontier Science Program ‘Network cooperativity and memory formation in the hippocampus’. We are grateful to Petra Schwarz for biocytin preparations.

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