Stress-facilitated LTD induces output plasticity through synchronized-spikes and spontaneous unitary discharges in the CA1 region of the hippocampus

Neuroscience Research 49 (2004) 229–239

Stress-facilitated LTD induces output plasticity through synchronized-spikes and spontaneous unitary discharges in the CA1 region of the hippocampus Jun Cao a,b , Nanhui Chen c , Tianle Xu d , Lin Xu a,b,∗ a

Laboratory of Learning and Memory, Kunming Institute of Zoology, The Chinese Academy of Sciences, Kunming 650223, Yunnan, PR China b Graduate School of the Chinese Academy of Sciences, Beijing 100039, PR China c Center of Brain and Mind, Kunming Institute of Zoology, The Chinese Academy of Sciences, Kunming 650223, PR China d Laboratory of Synaptic Physiology, Institute of Neuroscience, The Chinese Academy of Sciences, Shanghai 200031, PR China Received 12 November 2003; accepted 1 March 2004 Available online 24 April 2004

Abstract Long-term potentiation (LTP) and long-term depression (LTD) of the excitatory synaptic inputs plasticity in the hippocampus is believed to underlie certain types of learning and memory. Especially, stressful experiences, well known to produce long-lasting strong memories of the event themselves, enable LTD by low frequency stimulation (LFS, 3 Hz) but block LTP induction by high frequency stimulation (HFS, 200 Hz). However, it is unknown whether stress-affected synaptic plasticity has an impact on the output plasticity. Thus, we have simultaneously studied the effects of stress on synaptic plasticity and neuronal output in the hippocampal CA1 region of anesthetized Wistar rats. Our results revealed that stress increased basal power spectrum of the evoked synchronized-spikes and enabled LTD induction by LFS. The induction of stress-facilitated LTD but not LFS induced persistent decreases of the power spectrum of the synchronized-spikes and the frequency of the spontaneous unitary discharges; However, HFS induced LTP in non-stressed animals and increased the power spectrum of the synchronized-spikes, without affecting the frequency of the spontaneous unitary discharges, but HFS failed to induce LTP in stressed animals without affecting the power spectrum of the synchronized-spikes and the frequency of the spontaneous unitary discharges. These observations that stress-facilitated LTD induces the output plasticity through the synchronized-spikes and spontaneous unitary discharges suggest that these types of stress-related plasticity may play significant roles in distribution, amplification and integration of encoded information to other brain structures under stressful conditions. © 2004 Elsevier Ireland Ltd and The Japan Neuroscience Society. All rights reserved. Keywords: Long-term depression (LTD); Long-term potentiation (LTP); Spontaneous unitary discharges; Evoked synchronized-spikes; Power spectrum; Stress; Hippocampus

1. Introduction The hippocampal network is involved in the acquisition, consolidation and retrieval phase of hippocampus-dependent memory (Tang et al., 1999; Martin et al., 2000; Shimizu et al., 2000; Nakazawa et al., 2002). The hippocampus has a time-limited role in the permanent storage of memory (Reed and Squire, 1998; Kapur and Brooks, 1999; Debiec et al., 2002; Manns et al., 2003) and the formation and storage of memory depend on the interactions between the hippocam∗ Corresponding author. Tel.: +86-871-5195889/5195402; fax: +86-871-5191823. E-mail address: [email protected] (L. Xu).

pus and its sub-adjacent areas or neocortex (Fries et al., 2003; Silva, 2003; Wittenberg et al., 2002; Squire et al., 2001; Frankland et al., 2001; Bontempi et al., 1999). Therefore, the pyramidal cells of the CA1 region, the major output of the whole hippocampal networks, is considerably important in transferring encoded information to its sub-adjacent areas such as the subiculum or neocortex. At present, such studies on the relationship between the field EPSP, synaptic inputs, and population spike, outputs of the postsynaptic pyramidal neurons, have been reported as E–S coupling (Abraham et al., 1985; Bernard and Wheal, 1995; Fujii et al., 1999; Daoudal et al., 2002; Staff and Spruston, 2003). Furthermore, two different results related to synaptic plasticity and spontaneous unitary discharges have been reported in

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the hippocampus under non-stressed conditions (Deadwyler et al., 1976; Kimura and Pavlides, 2000). Spontaneous spikes are usually regarded as noise, unrelated to neural information processing. Yet, it has been shown in the sensory system that cell spontaneous activity signifies the functional connectivity and its reorganization for information processing (Decharms and Merzenich, 1996; Dinse et al., 1993; Johnson and Alloway, 1996). Moreover, cell spontaneous activity itself is functionally significant in ongoing network dynamics that has a major influence on sensory processing in its specific interactions with the activity evoked by sensory inputs (Arieli et al., 1995, 1996). It is well known that firing rate of the hippocampal CA1 pyramidal cells depends on the spatial location of the rats, which suggests that contextual environment information related to exploration behavior is encoded by cell activity (Thompson and Best, 1990; Wiener, 1996; Zhou et al., 1999; Hollup et al., 2001); on the other hand, the hippocampal plasticity of synaptic input within the network, long-term potentiation (LTP) and long-term depression (LTD), is believed to be the mechanisms underlying certain types of learning and memory (Bliss and Collingridge, 1993; Malenka and Nicoll, 1999; Martin et al., 2000). Generally, a great number of studies is involved in the mechanisms and modulation of synaptic plasticity and its roles in learning and memory (Kim et al., 1996; Izaki and Arita, 1996; Xu et al., 1997, 1998a,b; Soderling and Derkach, 2000; Wang et al., 2003; Malenka, 2003; Lee et al., 2003; Okada et al., 2003; Xiong et al., 2003, 2004; Yang et al., 2004). It has been a common observation that LTD is most easily generated in 1–3 weeks rats (Dudek and Bear, 1993; Wagner and Alger, 1995; Wasling et al., 2002). However, with development, the induction of LTD is very difficult in non-stressed adult animals (5–10 weeks) and facilitated by behavioral stress (Kim et al., 1996; Xu et al., 1997, 1998b), but LTP can be induced in non-stressed adult animals and blocked by stress (Diamond et al., 1992; McEwen, 1999; Shors et al., 1989). Thus, stress itself provides a strong behavioral manipulation for the study of LTD in adult animals. Therefore, to understand the effects of LTP and LTD on the output of postsynaptic neurons in the CA1 region of the hippocampus, we simultaneously and separately recorded long-term synaptic plasticity, synaptic input, in the stratum radiatum, and the spontaneous unitary discharges and the evoked synchronized-spikes of postsynaptic CA1 cells, neuronal output, in anesthetized adult animals under stressed and non-stressed conditions.

2. Materials and methods 2.1. Animals Experiments were carried out on male Wistar rats (inbred strain, Animal House Center, Kunming General Hospital, Kunming), weighed from 200 to 250 g (8–10 weeks). Ani-

mals were group-housed with free access to water and food in an established animal house having a 12 h light:12 h darkness cycle and a thermoregulated environment. The animal care and experimental protocol were approved by the Yunnan Health Department, China. 2.2. Stress and non-stress protocol Behavioral stress was evoked by elevated platform stress as described previously (Xu et al., 1997, 1998b; Yang et al., 2003, 2004; Xiong et al., 2003, 2004). The elevated platform stress was recently suggested as depression/anxiety animal model, indicating that this type of mild stress formed permanent experience memory (Rocher et al., 2004). The animals were anesthetized immediately after the stress procedure. Non-stressed animals were carefully taken out of their home cage and anesthetized immediately. 2.3. Electrophysiology Both LTP and LTD experiments were carried out under pentobarbitone sodium (50–60 mg/kg, i.p.) anesthesia and core temperature was maintained at 37 ± 0.5 ◦ C. Recordings of field EPSP were made from the CA1 stratum radiatum of the hippocampus in response to ipsilateral stimulation of the Schaffer collateral/commissural pathway using techniques similar to those described (Xu et al., 1998b; Wei et al., 2002). Two stainless steel screws (1.5 mm diameter) were inserted into the skull through a drill hole without piercing the dura. One served as a ground electrode (7 mm posterior to bregma and 5 mm left of the midline), the other served as the reference electrode (8 mm anterior to bregma and 1 mm left of the midline). The field EPSP recording and stimulating electrodes were made by gluing together a pair of twisted Teflon-coated 90% platinum/10% iridium wires (50 ␮m inner diameter, 75 ␮m outer diameter; WPI, USA). Recordings of synchronized-spikes/spontaneous unitary discharges were made from CA1 pyramidal cell bodies of the hippocampus using techniques similar to those described (Chen et al., 2001). The recording and reference electrodes for synchronized-spikes/spontaneous unitary discharges were made by the same wires and glued together. However, the spike recording electrode is made ∼0.2 mm longer than the reference electrode to avoid the possible interference from the field EPSP and to have a high signal/noise ratio recordings of single unit. Finally, the field EPSP recording electrodes and spike recording/reference electrodes were glued together again. The field EPSP recording electrode was made ∼0.2 mm longer than spike recording/reference electrodes in order to simultaneously record the dendrites and the pyramidal cell body respectively. Such a technique was different from the techniques in previous studies of E–S coupling or spontaneous unitary discharges in dentate gyrus (Deadwyler et al., 1976; Abraham and Bliss, 1985; Kimura and Pavlides, 2000). The advantage of the technique was that the field EPSP and synchronized-spikes/spontaneous

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unitary discharges were recorded simultaneously and separately. Electrode implantation sites were identified by using stereotaxic coordinates. The recording electrode was inserted 3.5 mm posterior to bregma and 2.5 mm right of the midline and the stimulating was inserted 4.2 mm posterior to bregma and 3.5 right of the midline. The optimal depth of the electrode in the stratum radiatum and the pyramidal cell bodies of the CA1 region of the dorsal hippocampus was determined by using electrophysiological criteria. All the electrodes were verified by postmortem (Fig. 1A).

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In most of the implantations, the recording electrode for synchronized-spikes/spontaneous unitary discharges was then optimized as indicated by fast synchronized-spikes (without exception, the latencies of most spikes is <10 ms as the response to the field EPSP; Fig. 2B) and high signal/noise ratio for spontaneous unitary discharges. Experiments test EPSP were evoked at a frequency of 0.033 Hz (30 s interval) and at a stimulus intensity adjusted to give an EPSP amplitude of 50% of maximum. There were no differences on the stimulus required to evoke 50% of maximum EPSP between stressed and non-stressed an-

Fig. 1. Localization of electrodes and isolation of single units in the CA1 region of the hippocampus. (A) Electrodes were localized in the stratum radiatum (the synaptic input of EPSPs), the Schaffer collateral/commissural pathway and the postsynaptic pyramidal cells (the output of evoked spikes/single-units). Stimulus (St), the field EPSP (EPSPs), synchronized-spikes/spontaneous unitary discharges (evoked spikes/single-units) and reference (the reference electrode for single unit) electrodes were shown. (B) Single units of pyramidal cells (width >300 ␮s) were discriminated by both wave width (␮s) and amplitude (␮V) (Spike Histogram Extension, Chart Software, PowerLab). One single unit was identified as a cluster.

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chronously recorded at 40 kHz (Scope Software, Powlab, ADInstruments, Australia). Spontaneous unitary discharges were continuously recorded by tape recorder for future off-line analysis (RMG-5304; Nihonkoholen Corporation, Tokyo, Japan). Single units were filtered (300 Hz–3 kHz), discriminated (Fig. 1B) and analyzed by Spike Histogram Extension (Chart Software, PowerLab, ADInstruments, Australia). Pyramidal cells were separated from interneurons by independent criteria, including spike duration (Skaggs et al., 1996) low discharge frequency of pyramidal cells (Buzsaki et al., 1983). Stimulus artifact and evoked spikes could be easily excluded out in single unit analysis by their frequency (0.033 Hz). However, Single unit analysis or the measures for population spikes was not appropriate for the synchronized-spikes. Thus, the power spectrum of the synchronized-spikes was measured after fast Fourier transformation of sweeps of 50 ms duration (thin lines in Fig. 2B). 2.4. Statistical analysis

Fig. 2. Simultaneously recordings of the field EPSP, synchronized-spikes and spontaneous unitary discharges in the CA1 region of the hippocampus. (A) One representative trace of spontaneous unitary discharges (Chart Software, PowerLab) was shown from inhibition to firing after a stimulus. Stimulus artifact (AF) determined the time of the onset of field EPSP and evoked synchronized-spikes. Synchronized-spikes: evoked spikes; inhibition period of spontaneous unitary discharge: inhibition; spontaneous unitary discharges: spontaneous spikes. Calibration bars: horizontal = 40 ms; vertical = 8 ␮V. (B) Pure synchronized-spikes (thin line) and the field EPSP (thick line) were simultaneously shown (Scope Software, PowerLab) and exactly matched in time. The synchronized-spikes (calibration bars: horizontal = 1 ms; vertical = 2 ␮V) were discharged constantly at the rising phase of the field EPSP (calibration bars: horizontal = 1 ms; vertical = 2 mV; the stimulus artifact in the field EPSP recording was very tiny here due to low pass filter (<3 kHz). (C) The effects of HFS and LFS on the inhibition time of the spontaneous unitary discharges. No significant difference was shown before/after HFS (490.1 ± 52.1 ms for baseline vs. 518.3 ± 63.4 ms after HFS, F(1,7) = 1.24, P > 0.05) and before/after LFS (525.8 ± 70.2 ms for baseline vs. 553.9 ± 60.5 ms after LFS, F(1,7) = 1.82, P > 0.05).

imals. In LTD experiment, After 40 min stable baseline recording, a low frequency stimulation (LFS) consisted of 900 pulses at 3 Hz was delivered to the Schaffer collaterals/commissural pathway in stressed and non-stressed animals. In LTP experiment, a high frequency stimulation (HFS) consisted of ten trains of 20 stimuli at 200 Hz with 2 s intertrain interval was given in non-stressed and stressed animals. The synchronized-spikes and field EPSP were syn-

Statistical comparisons were made by using least significant difference of repeated one-way ANOVA. Significance level was set at P < 0.05. Data from field EPSP recordings in LTP and LTD experiments were expressed as mean ± S.E.M.% of baseline EPSP amplitude. Data from spontaneous unitary discharges recordings were expressed as both mean ± S.E.M.% of baseline firing rate and averaged actual firing rate (Hz). Data from the evoked synchronized-spikes recordings were shown as power spectrum (mV/ms).

3. Results 3.1. The field EPSP, the evoked synchronized-spikes and the spontaneous unitary discharges The field EPSP and the evoked synchronized-spikes were recorded simultaneously by stimulating the Schaffer collateral/commissural pathway (Figs. 1A and 2B) at an interval of 30 s but the spontaneous unitary discharges were recorded continuously by tape recorder (Figs. 1A and 2A) and discriminated (Fig. 1B). The unitary discharges of the postsynaptic neurons were synchronized at the rising phase of the field EPSP with a latency of ∼3–10 ms (∼1 ms delay after the rising of the field EPSP; Fig. 2B), indicating that the evoked synchronized-spikes were driven directly by the field EPSP. The finding suggested that the evoked synchronized-spikes, outputs of the field EPSP, might be a driving source of postsynaptic depolarization, which is required for induction of synaptic plasticity, presumably driven by other inputs or back propagation of postsynaptic action potentials (Bi and Poo, 2001). Thus, the synchronized-spikes might contribute to the induction of synaptic plasticity. Simultaneously recording the spontaneous unitary discharges showed that there was a period of inhibition

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for ∼500 ms immediately following the field EPSP and evoked synchronized-spikes (Fig. 2A), which might be due to GABAergic system-mediated feed forward inhibition (Ashwood et al., 1984), indicating that the spontaneous unitary discharges were also determined by the integration of synaptic excitation. The inhibition time of the spontaneous unitary discharges during baseline period was primarily similar to that after HFS (F(1,7) = 1.24, P > 0.05; Fig. 2C), indicating that HFS did not altered the inhibition period of spontaneous firing. Similarly, there was no difference in inhibition period before and after LFS (F(1,7) = 1.82, P > 0.05; Fig. 2C), indicating that LFS did not influence the inhibition period of the spontaneous firing. Interestingly, ∼500 ms (∼2 Hz) is close to 1 Hz (1000 ms) or 3 Hz (333 ms) which are widely used for the induction of LTD and far from 100 Hz (10 ms) or 200 Hz (5 ms) which are widely used for the induction of LTP. 3.2. The effects of LTD and LFS on the spontaneous unitary discharges of postsynaptic pyramidal cells After 40 min stable baseline of field EPSP and evoked synchronized-spikes/spontaneous unitary discharges recordings, a reliable LTD of the field EPSP amplitude in stressed group was induced by LFS (n = 6, 80.9 ± 0.9% of baseline 60 min after LFS, F(1,4) = 5213, P < 0.05 compared with baseline; Fig. 3A). Correspondingly, the relative frequencies of the spontaneous unitary discharges for postsynaptic hippocampal CA1 pyramidal cells (18 single units) persistently decreased with the induction of LTD by LFS (n = 18; 41.4 ± 9.5% of baseline 60 min after LFS, F(1,16) = 294, P < 0.05 compared with baseline; Fig. 3B). Similarly, the distribution of the actual discharges from 15 single units (3 single units excluded) before LFS and after LTD was far from and below 45◦ diagonal line (Fig. 4A). These results indicated that LTD of synaptic input could induce the changes of postsynaptic neuron. In contrast, LTD in the CA1 stratum radiatum of the hippocampus was not induced by LFS in na¨ıve (non-stressed) group (n = 3, 101.7 ± 3.8% of baseline 60 min after LFS, F(1,1) = 0.52, P > 0.05 compared with baseline; Fig. 3C). Under this condition, the averaged relative frequencies of the spontaneous unitary discharges for postsynaptic pyramidal cells (9 single units discriminated by Spike Histogram Software from three rats; Fig. 1B) were not altered by LFS protocol (n = 9, 103.9 ± 14% of baseline 60 min after LFS, F(1,7) = 0.20, P > 0.05 compared with baseline; Fig. 3D); further analysis in Fig. 4B showed that the distribution of actual frequency from all 9 single units before and after LFS was close to 45◦ diagonal line. These findings indicated that LFS protocol was unable to induce LTD under non-stressed condition and did not significantly change the spontaneous firing rate of single units. Taken together, stress could facilitate LTD induction by LFS (n = 6, 80.9 ± 0.9% of baseline 60 min after LFS in stressed group, F(1,4) = 5213, P < 0.05 compared

Fig. 3. Persistent decrease of the spontaneous unitary discharges was associated with stress-facilitated LTD. (A) LTD of the field EPSP amplitude was induced by LFS (3 Hz, bar) in the stressed animals (n = 6, 80.9 ± 0.9% of baseline after LFS, F(1,4) = 5213, P < 0.05 compared with baseline). (B) The averaged relative discharges from 18 single units were strikingly decreased when LTD was induced under stressful condition (n = 18, 41.4 ± 9.5% of baseline 60 min after LFS, F(1,16) = 294, P < 0.05 compared with baseline). Each point was the averaged value over 10 min. (C) LTD of the field EPSP amplitude was not induced by LFS (3 Hz, bar) in the non-stressed animals (n = 3, 101.7 ± 3.8% of baseline 60 min after LFS, F(1,1) = 0.52, P > 0.05 compared with baseline). (D) The averaged relative discharges from 9 single units were not significantly changed when no LTD was induced in non-stressed animals (n = 9, 103.9±14% of baseline 60 min after LFS, F(1,7) = 0.20, P > 0.05 compared with baseline). Each point was the averaged value over 10 min. (Insets) Representative traces of the field EPSP at the time indicated by the numbers on the graph. Horizontal bar = 5 ms; vertical bar = 1.5 mV.

with baseline; F(1,7) = 4710, P < 0.05 compared with non-stressed group; Fig. 3A and C) and stress-facilitated LTD further induced the decrease of spontaneous unitary discharges(n = 18; 41.4 ± 9.5% of baseline 60 min after LFS, F(1,16) = 294, P < 0.05 compared with baseline; F(1,25) = 271, P < 0.05 compared with non-stressed group; Fig. 3B and D). Thus, the plasticity of decreased synaptic input, in the CA1 stratum radiatum but not LFS protocol that significantly induced persistent suppression of the spontaneous output of the postsynaptic neurons.

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Fig. 4. Comparison of the averaged actual frequency distribution before and after LFS under stressed and non-stressed condition. Frequency of each single unit before LFS was shown as X-axis; and that after LFS or after LTD was shown as Y-axis. One single unit was shown as each point. (A) Discharge frequency from 18 single units in stressed animals was lower than 2 Hz. To match with the baseline discharge frequency in non-stressed animals, single units (higher than 1 Hz) were excluded from this analysis. Most of the single units (15 single units) were far from the 45◦ diagonal line and close to X-axis, indicating that LTD induction decreased their discharge frequency. (B) Baseline discharge frequency from nine pyramidal cells in non-stressed animals was lower than 1 Hz over 40 min. Nine points (single units) before and after LFS in na¨ıve (non-stressed) group were close to the 45◦ diagonal line, indicating that LFS did not change their discharge frequency.

3.3. The effects of LTP and HFS on spontaneous unitary discharges on the postsynaptic pyramidal cells LTP of the field EPSP amplitude was not induced by HFS in stressed group (n = 3, 98.9 ± 2.5% of baseline 60 min after HFS, F(1,1) = 1.54, P > 0.05 compared with baseline; Fig. 5A). Also, the relative frequencies of the spontaneous unitary discharges for postsynaptic neurons (9 single units) in this group were not significantly changed after HFS (n = 9, 105.8 ± 12% of baseline 60 min after HFS, F(1,7) = 2.70, P > 0.05 compared with baseline; Fig. 5B). Similarly, the distribution of actual frequency from 9 single

Fig. 5. No changes of the spontaneous unitary discharges were associated with LTP. (A) LTP was not induced by HFS (200 Hz, arrow) in stressed animals (n = 3, 98.9 ± 2.5% of baseline 60 min after HFS, F(1,1) = 1.54, P > 0.05 compared with baseline). (B) The averaged relative discharges from 9 single units was not significantly changed when no LTP was induced (n = 9, 105.8 ± 12% of baseline 60 min after HFS, F(1,7) = 2.70, P > 0.05 compared with baseline). Each point was the averaged value over 10 min. (C) LTP was induced by HFS (200 Hz, arrow) in the non-stressed animals (n = 6, 126.1±3.5% of baseline 60 min after HFS, F(1,4) = 7047, P < 0.05 compared with baseline). (D) The averaged relative discharges was calculated from 16 single units discriminated by Spike Histogram Software and expressed as the mean ± S.E.M.% of baseline frequency. Each point was the averaged value over 10 min. LTP induction did not enhance the averaged relative discharges (n = 16, 96.6±11.9% of baseline 60 min after HFS, F(1,14) = 2.85, P > 0.05 compared with baseline). (Insets) Representative traces of the field EPSP at the time indicated by the numbers on the graph. Horizontal bar = 10 ms; vertical bar = 1 mV.

units before HFS and after HFS were close to the 45◦ diagonal line (Fig. 6A), which indicated that actual frequencies remain unchanged after HFS. These findings revealed that HFS protocol, which was unable to induced LTP in the CA1 stratum radiatum under stressed condition, did not change the discharge frequency of single units. However, in non-stressed group, a reliable LTP of the field EPSP amplitude was induced by HFS (n = 6, 126.1 ± 3.5% of baseline 60 min after HFS, F(1,4) = 7047, P < 0.05 compared with baseline; F(1,7) = 5477, P < 0.05 compared with

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to the 45◦ diagonal line (Fig. 6B), similar to that in stressed group (Fig. 6A). These findings revealed that LTP of synaptic input did not cause significant change of the spontaneous output in postsynaptic pyramidal neurons in the CA1 region of the hippocampus. 3.4. The effects of stress on the baseline evoked synchronized-spikes of the postsynaptic pyramidal cells

Fig. 6. Comparison of the averaged actual frequency distribution before and after HFS under stressed and non-stressed condition. Frequency of each single unit before HFS was shown as X-axis; and that after LTP and HFS was shown as Y-axis. One single unit was shown as each point. (A) Discharge frequency from 9 single units in stressed animals was lower than 1 Hz. The 9 single units were close to the 45◦ diagonal line, indicating that HFS did not influence their discharge frequency. (B) Baseline discharge frequency from 16 pyramidal cells in the non-stressed animals was lower than 1 Hz over 40 min. All the single units were close to the 45◦ diagonal line, indicating that LTP induction did not change their discharge frequency (16 single units).

stressed group; Fig. 5A and C), indicating that LTP could be induced by HFS under normal condition and blocked by stress. Surprisingly, after the induction of LTP, the averaged relative discharges of the spontaneous unitary discharges (16 single units) remained primarily unchanged (n = 16; 96.6 ± 11.9% of baseline 60 min after HFS, F(1,14) = 2.85, P > 0.05 compared with baseline; F(1,23) = 3.76, P > 0.05 compared with stressed group; Fig. 5D and B), different from previous findings in dentate gyrus of the hippocampus (Deadwyler et al., 1976; Kimura and Pavlides, 2000). In addition, the distribution about the actual discharges of 16 single units before HFS and after LTP in this group was close

Interestingly, in LTD and LTP experiments, the power spectrum of the synchronized-spikes during 40 min baseline period in stressed animals was significantly larger than that in non-stressed animals (12.3 ± 0.22 mV/ms for baseline from stressed animals versus 6.5 ± 0.3 mV/ms for baseline from non-stressed animals in LTD experiments; F(1,7) = 431, # P < 0.05; Fig. 7A; 11.8 ± 0.5 mV/ms for baseline from stressed animals versus 6.6 ± 0.1 mV/ms for baseline from non-stressed animals in LTP experiments; F(1,7) = 395, # P < 0.05; Fig. 7B) while there were no significant differences in stimulus intensity/field EPSP test between stressed and non-stressed animals (F(1,16) = 0.42; P > 0.05; Fig. 7C) in the hippocampus, indicating that behavioral stress increased the baseline synchronized-spikes without affecting basal synaptic transmission. The changes of hippocampal basal synaptic efficacy during/after stress experience was not found (Xu et al., 1997; Alfarez et al., 2002; Yang et al., 2004) except a recent report in VTA (Saal et al., 2003) but other changes such as theta rhythm in the hippocampus associated with stress was documented (Simonov and Rusalova, 1980; Yamamoto, 1998). Thus, our findings, which stress persistently increased the basal synchronized-spikes, suggested that stress might lay down traces in the hippocampus by the synchronized-spikes but not the field EPSP, and that the increased basal synchronizedspikes might be one of stress-induced plasticity to facilitate the induction of LTD in adult animals. 3.5. The effects of LTD/LFS and LTP/HFS of the evoked synchronized-spikes of the postsynaptic pyramidal cells In LTD experiments, after 40 min stable baseline of field EPSP and evoked synchronized-spikes, the power spectrum of the synchronized-spikes after LFS was not significantly changed (n = 3, 6.8 ± 0.4 mV/ms after LFS versus 6.5 ± 0.3 mV/ms for baseline; F(1,1) = 1.72, P > 0.05; Fig. 7A; right) when there was no LTD induced by LFS in non-stressed animals (Fig. 3C). However, the power spectrum of the evoked synchronized-spikes after LFS was significantly decreased (n = 6, 8.9 ± 0.1 mV/ms after LTD induction versus 12.3 ± 0.22 mV/ms for baseline; F(1,4) = 165, ∗ P < 0.05; Fig. 7A; left) following the induction of LTD by LFS in stressed rats (Fig. 3A), similar to previous E–S depression studies (Daoudal et al., 2002). The comparison of the results between stressed and non-stressed animals revealed that the decrease of the evoked synchronized-spikes output was also accom-

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Fig. 7. LTD/LFS and LTP/HFS had different effects on the evoked synchronized-spikes under stressed and non-stressed condition. (A) The synchronized-spikes in LTD experiments. During 40 min baseline period the power spectrum of the synchronized-spikes of stressed animals was significantly larger than that of non-stressed animals (12.3 ± 0.22 mV/ms from six stressed animals vs. 6.5 ± 0.3 mV/ms from three non-stressed animals; F(1,7) = 431, # P < 0.05). Furthermore, in stressed animals the power spectrum was significantly decreased (n = 6, 12.3 ± 0.22 mV/ms for baseline; 8.9 ± 0.1 mV/ms after LTD induction; F(1,4) = 165, ∗ P < 0.05 compared with baseline; left) after the induction of LTD by LFS. However, in na¨ıve (non-stressed) animals, there was no difference in the power spectrum (n = 3, 6.5 ± 0.3 mV/ms for baseline; 6.8 ± 0.4 mV/ms after LFS; F(1,1) = 1.72, P > 0.05 compared with baseline; right). In addition, the decreased synchronized-spikes after LTD in stressed animals was still higher than those in the non-stressed animals (8.9 ± 0.1 mV/ms after LTD induction from stressed animals vs. 6.8 ± 0.4 mV/ms after LFS from non-stressed group; F(1,7) = 110, $ P < 0.05; Fig. 7A). (B) The synchronized-spikes in LTP experiments. During 40 min baseline period the power spectrum of the synchronized-spikes of stressed animals was significantly larger than that of non-stressed animals in LTP experiments (11.8±0.5 mV/ms from stressed animals vs. 6.6±0.1 mV/ms from non-stressed animals; F(1,7) = 395, # P < 0.05). In addition, in non-stressed animals, the power spectrum was significantly increased (n = 6, 6.6 ± 0.1 mV/ms for baseline; 9.0 ± 0.2 mV/ms after LTP induction; F(1,4) = 153, ∗ P < 0.05 compared with baseline; right) after the induction of LTP by HFS. However, in stressed animals the power spectrum remained unchanged following HFS (n = 3, 11.8 ± 0.5 mV/ms for baseline; 11.9 ± 0.6 mV/ms after HFS; F(1,1) = 0.06, P > 0.05 compared with baseline; left). Furthermore, the unchanged synchronized-spikes after HFS in stressed animals was still higher than those in the non-stressed animals (11.9 ± 0.6 mV/ms after HFS from stressed group vs. 9.0 ± 0.2 mV/ms after LTP induction from non-stressed animals; F(1,7) = 102, $ P < 0.05; Fig. 7B). (C) Input/output test before HFS (LTP) and LFS (LTD). No differences were shown between stressed and non-stressed animals during 40 min baseline period (F(1,16) = 0.42, P > 0.05). The stimulus intensity (V; not the actual stimulus intensity, controlled by the outputs of Scope Software to a stimulus isolator).

panied by stress-facilitated LTD of synaptic input but not LFS. However, in LTP experiments, the power spectrum of the synchronized-spikes after HFS remained unchanged (n = 3, 11.9 ± 0.6 mV/ms after HFS versus 11.8 ± 0.5 mV/ms for baseline; F(1,1) = 0.06, P > 0.05; Fig. 7B; left) when there was no LTP of synaptic input following HFS in stressed animals (Fig. 5A), indicating that HFS protocol, which was unable to induce LTP of synaptic input under stressed animals, did not change the evoked output of postsynaptic neuron. However, the power spectrum of the synchronized-spikes in non-stressed animals after HFS was significantly increased (n = 6, 9.0 ± 0.2 mV/ms after LTP versus 6.6 ± 0.1 mV/ms for baseline; F(1,4) = 153, ∗ P < 0.05; Fig. 7B; right) by induction of LTP (Fig. 5C), similar to previous findings in EPSP/population spike coupling studies (Abraham et al., 1985; Bernard and Wheal, 1995). The different changes of the spectrum following HFS under non-stressed and stressed animals revealed that the output through evoked synchronized-spikes was increased by LTP of synaptic input but not by HFS protocol. In addition, the decreased synchronized-spikes after LTD and the unchanged synchronized-spikes after HFS in stressed animals was still higher than those in the non-

stressed animals (8.9 ± 0.1 mV/ms after LTD induction from stressed animals versus 6.8 ± 0.4 mV/ms after LFS from non-stressed group; F(1,7) = 110, $ P < 0.05; Fig. 7A; 11.9 ± 0.6 mV/ms after HFS from stressed group versus 9.0 ± 0.2 mV/ms after LTP induction from non-stressed animals; F(1,7) = 102, $ P < 0.05; Fig. 7B). The findings revealed that behavioral stress, which could increase the basal synchronized-spikes, had the effect on the synchronizedspikes following LFS/HFS.

4. Discussion We recorded the field EPSP from stratum radiatum while simultaneously monitored the outputs of postsynaptic neurons by synchronized-spikes and spontaneous unitary discharges in the CA1 pyramidal cells of the dorsal hippocampus (Fig. 1A) following LTD and LTP. Such a technique was different from the techniques in previous studies of E–S coupling or spontaneous unitary discharges in dentate gyrus (Deadwyler et al., 1976; Abraham and Bliss, 1985; Kimura and Pavlides, 2000). We tried to address the question primarily unknown: Does stress-related synaptic plasticity have an impact on the output of postsynaptic neurons?

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Our results first demonstrated that the spontaneous unitary discharges (Fig. 3B) were persistently decreased with stress-facilitated LTD (Fig. 3A) and remained unchanged (Fig. 3D) with no LTD (Fig. 3C), indicating that the naturalistic outputs of the CA1 region of the hippocampus to its sub-adjacent areas or neocortex were due to stress-facilitated LTD but not due to LFS. The LTD could not be so specific as LTP and might spread to other synapses although it is input-specific (Xu et al., 1997, 1998b), which means synaptic plasticity generates at one set of stimulus-activated synapses but not in other synapses on the same cell. Thus, if the population of depressed synapses was large, the spontaneous unitary discharges of postsynaptic neurons could be decreased with the LTD. The second explanation is that the LTD of the excitatory–excitatory synapses could reduce the input of postsynaptic pyramidal cells. Meanwhile, the input of inhibitory–inhibitory synapses might be increased by stimulation (Melanie et al., 2003) and then prevented the output of postsynaptic neurons. Thus, the increased inputs of inhibitory–inhibitory synapses and decreased input of excitatory–excitatory synapses resulted in large decrease of the output of postsynaptic neurons. Anyway, the output plasticity of postsynaptic neurons in the CA1 region of the hippocampus make it possible that long-term synaptic plasticity may induce further plasticity in the efferent projection areas at least in present stressed condition and thus may contribute to the permanent storage of the information of stress events. Surprisingly, on the other hand, the spontaneous unitary discharges of the postsynaptic CA1 cells (Fig. 5D) did not increase with LTP and also remained unchanged when there was no LTP following HFS. LTP is highly input-specific in the CA1 region of the hippocampus (Malenka and Nicoll, 1999), which means synaptic plasticity generates at one set of stimulus-activated synapses but not in other synapses on the same cell. However, the spontaneous unitary discharges could be driven by the synaptic excitation from all the synapses on the same cell. If the population of potentiated synapses was small, the increase of naturalistic outputs could be too small to be detected with LTP. Alterative explanation is that although LTP of excitatory–excitatory synapses could increase the input of postsynaptic neurons, the input of inhibitory–inhibitory synapses might be largely enhanced either (Melanie et al., 2003). Thus, the integration of synaptic excitation between excitatory–excitatory and inhibitory–inhibitory synapses could balance the overall excitability of pyramidal cell. The present findings of the spontaneous unitary discharges are different from the previous reports which may be due to the different sub-region of the hippocampus (Deadwyler et al., 1976; Kimura and Pavlides, 2000). Namely, here the CA1 region of the hippocampus was examined and the dentate gyrus of the hippocampus was done in the previous reports (Deadwyler et al., 1976; Kimura and Pavlides, 2000). However, in the present study the firing patterns (such as complex spikes) of the spontaneous unitary discharges in LTP and LTD experiments were

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not analyzed. Further investigations are needed to understand whether different forms of synaptic plasticity may induce different patterns of the unitary discharges rather than frequencies. The present study also examined the effects of LTD and LTP on the evoked synchronized-spikes of the CA1 region of the hippocampus. The results demonstrated that the synchronized-spikes were persistently decreased (Fig. 7A) after stress-facilitated LTD and remain unchanged (Fig. 7A) when no LTD was induced in non-stressed animals, indicating that the evoked outputs of the CA1 region of the hippocampus to its sub-adjacent areas or neocortex were also decreased following LTD. However, the synchronizedspikes (Fig. 7B) were increased following LTP and remained unchanged when there was no LTP, clearly revealed that the evoked output of postsynaptic neurons were enhanced with LTP. At present, decrease/increase of the synchronizedspikes after LTD/LTP are in agreement with the findings of previous studies that LTD/LTP are associated with population spike depression/potentiation (E–S coupling) (Abraham et al., 1985; Fujii et al., 1999; Daoudal et al., 2002; Staff and Spruston, 2003). In summary, the different effects of LTD and LTP on the output (the synchronized-spikes and the spontaneous unitary discharges) were observed respectively in the hippocampus. The results in the LTD experiments clearly suggested that the enhanced basal synchronized-spikes, long-term decrease of the spontaneous and evoked synchronized-spikes were the novel types of plasticity induced by stress experience or by stress-facilitated synaptic plasticity, implicating their possible roles in distribution, amplification and integration of encoded information to other brain structures.

Acknowledgements This work was supported by grants from The Chinese Academy of Sciences (KSCX2-SW-04) and The National Natural Science Foundation of China (39870280, 39925011, 39930080) and The National Basic Research Program (G1999054000) to L.X.

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