Sequence dependence of post-tetanic potentiation after sequential heterosynaptic stimulation in the rat auditory cortex

Authors


Corresponding author K. Shibuki: Department of Neurophysiology, Brain Research Institute, Niigata University, Asahi-machi, Niigata 951-8585, Japan. Email: shibuki@bri.niigata-u.ac.jp

Abstract

  • To investigate the mechanisms for the coding stimulus sequence in the auditory cortex (AC), post-tetanic potentiation (PTP) was recorded after sequentially combined heterosynaptic stimulation was applied in rat AC slices.

  • Brief tetanic stimulation (TS) was applied at two sites on AC slices at intervals of 0.5–10 s. PTP of field potentials was induced by the earlier TS, rather than the later TS. PTP was followed by sequence-dependent long-term potentiation (LTP).

  • Using Ca2+ imaging in the slices loaded with rhod-2, a Ca2+ indicator, a sequence-dependent distribution of PTP was found in AC slices.

  • The sequence-dependent PTP in excitatory postsynaptic potentials (EPSPs) was observed in supragranular pyramidal neurons.

  • The sequence dependence of PTP was not significantly affected by 1 μm bicuculline, an antagonist of GABAA receptors, or 100 μm 2-hydroxysaclofen, an antagonist of GABAB receptors.

  • Depolarization and firing recorded in pyramidal neurons during the later TS were less vigorous than when the slices were incubated in the control medium. However, this suppression of the responses during the later TS was not observed in the presence of 50 μm atropine, an antagonist of muscarinic receptors.

  • PTP was induced by the earlier and later TS in the presence of 50 μm atropine, so that the sequence dependence of PTP was abolished. Pirenzepine (50 μm), an antagonist of muscarinic M1 receptors, but not methoctramine (30 μm), an antagonist of M2 receptors, eliminated the sequence dependence of PTP.

  • These findings suggest that the sequence dependence of PTP in AC might have a role in the temporal processing of auditory information on the scale of seconds.

Perception of auditory stimuli requires temporal information processing, although its mechanisms are not fully understood. Temporal information is converted into spatial information in the neural networks (Buonomano & Merzenich, 1995). In birds, sounds received by both ears with an interaural time difference less than 100 μs are mediated through delay-line circuits, and cause coincidental occurrence of EPSPs and the resulting generation of a spike at particular neurons in the brainstem (Moiseff & Konishi, 1981). Processing on the scale of several hundreds of milliseconds can be achieved by slow synaptic potentials in AC (Buonomano, 2000). However, little is known regarding the cellular mechanisms underlying the auditory information processing on the scale of seconds.

The neural networks in AC are suitable for the induction of LTP (Kudoh & Shibuki, 1997). AC of adult animals exhibits receptive field plasticity (Edeline & Weinberger, 1993; Ohl & Scheich, 1997) and plasticity of sound representation (Recanzone et al. 1993; Kilgard & Merzenich, 1998a). The sound discrimination ability of rats is enhanced by long-lasting sound exposure, and this enhancement is blocked by pressure injection of an antagonist of NMDA receptors into AC (Sakai et al. 1999). However, these reports do not exclude the possibility that synaptic plasticity might have a role other than a modulatory one in AC.

Various aspects of synaptic plasticity are sensitive to the temporal structure of synaptic inputs, so that synaptic plasticity may be intimately related to temporal information processing. The efficacy of the induction of LTP in the hippocampus is influenced not only by the number or average frequency of TS used for the induction of LTP, but also by the temporal structure of the TS (Tsukada et al. 1994). The correlated activities of pre- and postsynaptic neurons induce LTP or long-term depression (LTD) according to the temporal relationship of the pre- and postsynaptic activities within a time window of tens of milliseconds (Markram et al. 1997; Bi & Poo, 1999; Feldman, 2000). The responses to the temporally combined test stimuli are differentially potentiated by LTP (Buonomano et al. 1997). AC is required for discriminating complex sound patterns (Kelly & Whitfield, 1971; Cranford et al. 1976). Therefore, temporal information processing of auditory stimuli in the order of seconds, which is required for discriminating complex sound patterns, may be processed in AC, and the mechanism may be related to the sensitivity of synaptic plasticity to temporal structures of synaptic inputs. In the present study, therefore, we investigated the sequence-dependent properties of PTP induced by sequentially combined heterosynaptic stimulation in AC slices. Since the involvement of polysynaptic circuits in the induction of LTP in AC is known (Kudoh & Shibuki, 1997), we discuss the possible roles of polysynaptic circuits in the sequence-dependent properties of PTP.

METHODS

Slice preparations

The experiments in this study were performed according to the guidelines of Niigata University, and had the approval of the Ethics Committee of Niigata University. Slice preparations were obtained from Wistar rats of both sexes (4-7 weeks old). The rats were deeply anaesthetized with ether and except for the nose, were immersed in ice-cold water, for 3 min to reduce the brain temperature. They were decapitated and a block of brain tissue including AC was dissected out. The location of AC was determined to be that of area 41 of the temporal cortex (Krieg, 1946). Frontal slices (400 μm thick) of AC were prepared from the block in an ice-cold medium using a microslicer (DTK-1500, Dosaka, Osaka, Japan). The composition of the medium was (mm): NaCl, 124; KCl, 5; NaH2PO4, 1.24; MgSO4, 1.3; CaCl2, 2.4; NaHCO3, 26; and glucose 10. This medium was bubbled with 95 % O2-5 % CO2 for at least 2 h before use. After incubation at room temperature (21-23 °C) for more than 1 h, the slices were transferred to a small recording chamber (about 0.3 ml in volume), in which the slices were kept submerged. The recording chamber was maintained at room temperature, and was continuously perfused with the oxygenated medium at the flow rate of 1 ml min−1.

Field potential recording

The location of AC in slices was confirmed under a binocular microscope by a faint band at the position of layer IV. This band is likely to reflect the well-developed thalamo-cortical axons in layer IV. We used pieces of Teflon-coated platinum wire (metal diameter, 50 μm) as stimulating electrodes. The cut end of the wire was placed at two different places (SA and SB, Fig. 1a) on the border between the white matter and layer VI in the slices. The relative location of SA and SB in the dorso-ventral (or medio-lateral) axis was randomly determined in each slice. The distance between SA and SB was 350 μm, unless otherwise specified. This value was determined as the maximal distance for evoking PTP of field potentials recorded in supragranular layers in the middle of SA and SB. Biphasic current pulses (duration of each phase of pulses: 200 μs) were used to reduce stimulus artefacts. These pulses were applied through the electrodes placed at SA or SB as test stimuli. The field potentials in the slices were recorded through a metal electrode in supragranular layers in the middle of SA and SB. This metal electrode was made from a piece of silver wire (200 μm in diameter), which was electrolytically polished in 10 % KNO3 solution and insulated with polyvinyl chloride, except for the part within 60 μm from the tip. Signals were amplified 10 times with a handmade electronic circuit using an operational amplifier (OPA128LM, Burr-Brown, Tucson, AZ, USA), and were passed through a band-pass filter between 0.2 Hz and 10 kHz. The output was stored in a computer (PC-9801BA2, NEC, Tokyo, Japan) via an analog-digital converter board (ADXM-98A, Canopus, Kobe, Japan) for further analysis. We developed the basic programs used for the recording and analysis using the software library supplied by Canopus. The amplitudes of the trans-synaptic components in the field potentials were measured from the peak amplitudes of the second negativity in the traces. We used the measurement of the peak amplitudes instead of the initial slopes, since they were less affected by the preceding antidromic or presynaptic activities than the initial slopes. Only when the amplitudes of the trans-synaptic field potentials elicited by test stimuli at the intensity of 200 μA were larger than 1.0 mV were the slices used for PTP recording. The current intensities of the test stimuli (50-200 μA) were adjusted to ensure that half-maximal responses of trans-synaptic field potentials (amplitude: about 1 mV) were elicited. Responses were elicited by alternate stimulation of SA and SB at intervals of 30 s, and two consecutive traces were averaged. To induce PTP, sequential TS with 30 pulses at 100 Hz was applied to SA and SB in this order. The interval between TS at each pathway was 1 s (Fig. 1B), unless otherwise specified. The sequential TS was applied to the slices twice at an interval of 1 min for inducing clear PTP. Simultaneous TS of SA and SB or TS of SA only was also used for the induction of PTP. The amplitudes of the field potentials were normalized by the averaged value in five consecutive traces immediately before TS. The magnitude of PTP was estimated as the relative amplitude 10 min after cessation of TS.

Figure 1.

PTP in AC slices

A, schematic drawing of the recording electrode and two stimulated sites (SA and SB). B, timing of sequential TS applied at SA and SB at intervals of T (0-30 s). C, PTPA (•) and PTPB (○) in the field potentials (FP) induced by simultaneous TS of SA and SB. Insets show the field potentials in response to SA and SB stimulation recorded before and after TS (*). D, PTPA and delayed PTPB induced by TS of SA only. E, PTPA without accompanying PTPB induced by sequential TS of SA and SB at intervals of 1 s. F, amplitudes of PTPA and PTPB induced under various stimulus conditions. Vertical bars indicate s.e.m.

Ca2+ imaging

For visualizing the distribution of PTP in AC, we used Ca2+ imaging. Rhod-2 was used as a Ca2+ indicator, since it has a low affinity (Kd= 1.0 μm) for Ca2+ (Minta et al. 1989), and does not prevent the induction of PTP in AC (Seki et al. 1999). For loading with rhod-2, slices were incubated in the medium containing a 10 μm concentration of the tetraacetoxymethyl ester of rhod-2 (rhod-2 AM), which was solubilized with 0.1 % dimethyl sulfoxide and 0.1 % cremophore EL (polyoxyethylated castor oil), at room temperature for 1 h. Slices were rinsed with the normal medium for at least 30 min before Ca2+ imaging. A slice loaded with rhod-2 AM was transferred into a recording chamber custom-made for Ca2+ recording and was placed on an inverted epifluorescence microscope (TMD-300, Nikon, Tokyo, Japan). This chamber has a membrane filter (H100A, Advantec, Tokyo, Japan). This filter, made of hydrophilic polytetrafluoroethylene (PTFE), was permeable to the oxygenated perfusing medium, and became transparent in the medium. A slice was placed on the filter and kept submerged during recording. The use of this chamber and an inverted epifluorescence microscope enabled observations of the Ca2+ signal in 400 μm thick slices using a × 4 objective lens without the interference of electrodes. The fluorescent Ca2+ image was recorded with an excitation wavelength of 546 ± 10 nm and an emission wavelength > 590 nm using a cooled CCD camera system (ARGUS HiSCA, Hamamatsu Photonics, Japan). No marked autofluorescence of the membrane filters or slices was detected under this condition. The images (30 × 30 pixels) were sequentially recorded at intervals of 5.3 ms. Data of 30 consecutive images were divided by the first image pixel by pixel for normalization, and the changes in fluorescence intensity (ΔF/F) were estimated. For demonstrating the spatial distribution of PTP (Fig. 4), the changes (ΔF/F) elicited by single-pulse stimulation of SA or SB were observed. To improve the quality of images, images taken 15.9, 21.2 or 26.5 ms after the stimulation 1-4 min before TS (total 12 images) were averaged using the public domain NIH Image software (written by Wayne Rasband at the US National Institute of Health and available from the Internet). The image obtained before TS was compared with the averaged image taken 7-10 min after TS, and PTP of the Ca2+ signal was estimated from the difference. For demonstrating neural activities during TS (Fig. 6), the fluorescence changes (ΔF/F) elicited by sequential TS were observed at the end of the earlier and later TS.

Figure 4.

PTP visualized using Ca2+ imaging in AC slices

A and B, fluorescence changes (ΔF/F) elicited by SA stimulation before (A) and after (B) sequential TS in AC slices loaded with rhod-2. C, PTPA measured as the difference in ΔF/F (B - A). D and E, fluorescence changes elicited by SB stimulation before (D) and after (E) sequential TS. F, PTPB measured as the difference in ΔF/F (E - D). The pseudocolour scale of fluorescence changes (ΔF/F) is applicable to A-F. G, spatial distribution of PTPAF/F > 6 %, red) and PTPBF/F > 6 %, blue) superimposed on the grey image of the slice. R (*) shows the recording site of the field potentials. H, traces showing PTPA but no PTPB in the field potentials recorded in the same slice as that shown in A-G. I, spatial distribution of PTP in a different slice.

Figure 6.

Influence of GABAergic modulation on the sequence dependence of PTP in AC slices

A, depolarization and firing during sequential TS (intersite distance, 350 μm; time interval, 1 s) before and after application of 1 μm bicuculline. Two traces recorded in the same neurone are superimposed. The resting membrane potentials are shown with a dashed line. B, depolarization and firing during sequential TS (intersite distance, 350 μm; time interval, 1 s) before and after application of 100 μm 2-hydroxysaclofen. C, sequence-dependent PTP induced by sequential TS (intersite distance, 350 μm; time interval, 1 s) in the presence of 1 μm bicuculline. D, sequence-dependent PTP induced in the presence of 100 μm 2-hydroxysaclofen.

Patch recording from pyramidal neurons

PTP of EPSPs was recorded in supragranular pyramidal neurons in AC slices using a perforated and blind slice patch technique, as described previously (Kudoh & Shibuki, 1997). A glass micropipette was filled with a medium containing (mm): K2SO4, 92; KCl, 31; Hepes, 10; MgCl2, 1 and adjusted to pH 7.4 with KOH. Amphotericin B dissolved in dimethyl sulfoxide (10 mg ml−1) was added to the patch medium (final concentration, 50 μg ml−1). The tip was filled with an amphotericin B-free medium to facilitate the sealing between the pipette and cell membrane. The resistance of the recording electrode was 11-13 MΩ. This electrode was inserted into supragranular layers, and a positive pressure of about 10-30 mmHg was applied in the pipette during insertion. After a sudden increase in the access resistance of the electrode, the pressure on the electrode was removed. In successful cases, the access resistance and resting membrane potential continuously decreased for about 10 min before these parameters stabilized. The intracellular potentials were amplified 10 times with an amplifier for intracellular recording (MEZ-8300, Nihon Kohden, Tokyo, Japan), and the obtained signals were processed by the same system as that used for field potential recordings. Neurons showing a resting membrane potential more negative than -60 mV and a membrane resistance larger than 30 MΩ were selected. Pyramidal neurons were identified in terms of an antidromic spike elicited by white matter stimulation at an intensity not larger than 500 μA (Kudoh & Shibuki, 1996). EPSPs were elicited by the stimulation of SA or SB. PTP was measured with respect to the rising slopes of EPSPs. The intensity of the test stimuli (50-200 μA) was adjusted so that the rising slope was about 1 mV ms−1. PTP was induced by the same stimulus parameters as those used for field potential recording.

Whole-cell recording was also used in the experiments in which PTP was induced in pyramidal neurons by pairing depolarizing current injection with stimulation of SA and SB. A glass micropipette was filled with a solution containing (mm): potassium gluconate, 130; KCl, 10; Hepes, 10; EGTA, 0.5; MgCl2, 1; Na-ATP, 4; Na-GTP, 1; sucrose, 16, with pH adjusted to 7.2 with KOH. This electrode was inserted into the supragranular layers, and a positive pressure of 10-30 mmHg was applied in the pipette during insertion. After a sudden increase in the access resistance of the electrode, negative pressure was applied to obtain whole-cell and current-clamp recordings. To induce PTP, a depolarizing current (1 nA, 100 ms) was injected into a pyramidal neuron with simultaneous stimulation of SA and SB at 50 ms after the initiation of the current injection, and this paired stimulation was repeated 100 times at 1 Hz.

Drugs

Bicuculline, 2-hydroxysaclofen and carbachol were purchased from Sigma, and atropine, pirenzepine, methoctramine and linopirdine were from RBI (Natick, USA). These drugs were applied to the slices by adding them to the perfusing medium. Rhod-2 AM and cremophore EL used for Ca2+ imaging were obtained from Dojindo Laboratories (Kumamoto, Japan) and Sigma, respectively. Amphotericin B used for perforated patch recording was purchased from Wako (Tokyo, Japan).

RESULTS

Sequence dependence of PTP in AC slices

To induce PTP, we applied TS at two different sites located 350 μm apart (SA and SB, Fig. 1a). PTP of field potentials in response to the single pulse test stimulation applied at SA (PTPA) or SB (PTPB) was recorded in supragranular layers in the middle of SA and SB. These sites were stimulated with TS of 30 pulses at 100 Hz with or without intervals (Fig. 1B). Simultaneous TS of SA and SB induced both PTPA and PTPB (Fig. 1C and F). TS of SA only induced PTPA and weak PTPB (Fig. 1D and F). However, sequential TS of SA and SB at intervals of 1 s induced PTPA but almost no PTPB (Fig. 1E and F). The amplitude of PTP induced by the earlier TS (PTPA) was always larger than that of PTPB regardless of the relative location of SA and SB in the slices. The difference was statistically different after sequential TS (P < 0.003, Wilcoxon signed rank test), but not after simultaneous TS of SA and SB (Fig. 1F). The amplitude of PTPA induced by TS of SA only was usually larger than that of PTPB although the difference in Fig. 1F was not statistically significant (P > 0.05).

To characterize the sequence dependence of PTP, we varied the stimulus conditions for the induction of PTP. The sequence dependence of PTP was clearly observed at intervals of 1 s. We varied the intervals between the initiation of TS of SA and TS of SB. A significant difference in the amplitudes between PTPA and PTPB was observed after sequential TS at intervals of 0.5-10 s (Fig. 2a). However, no significant difference between PTPA and PTPB was observed at 15 and 30 s. The intersite distance between SA and SB was changed from 350 μm to 150 μm. Although the amplitude of PTPA was slightly larger than that of PTPB, the sequence dependence of PTP observed for this reduced spatial separation was not clearly found even at the intervals of 1 s (Fig. 2B). These results indicate that sequentially combined heterosynaptic inputs, stimulated separately at a distance of 350 μm between SA and SB and at time intervals of 0.5-10 s, induce sequence-dependent PTP.

Figure 2.

Stimulus conditions required for the sequence dependence of PTP

A, difference in PTP (PTPA - PTPB) following simultaneous or sequential TS at intervals of 0.5-30 s. Asterisks show significant differences between PTPA and PTPB (P < 0.005-0.03, Wilcoxon signed rank test). B, PTPA (•) and PTPB (○) induced by sequential TS of SA and SB at intersite distance of 150 μm (time interval, 1 s). C, PTPA and PTPB observed for 1 h after sequential TS of SA and SB (intersite distance, 350 μm; time interval, 1 s). Insets show sample traces. D, sequence-dependent PTP induced by sequential TS with 15 pulses at 50 Hz (intersite distance, 350 μm; time interval, 1 s). E, sequence-dependent PTP induced by sequential TS with 30 pulses at 100 Hz at intervals of 1 s, which was applied at SA and SB 350 μm apart. Insets show a schematic drawing of the experiment and sample traces. In this experiment, SA and SB were located in supragranular layers, although they were on the border between layer VI and the white matter in other experiments in this figure. F, PTPA and PTPB induced by the first sequential TS of SA and SB (intersite distance, 150 μm; time interval, 1 s) followed by the second sequential TS in the reversed order. The second TS was applied to the slices 10 min after the first TS. Insets show the traces recorded immediately before the first TS (a and a'), the second TS (b and b') and 10 min after the second TS (c and c').

PTP is usually followed by LTP in AC slices (Kudoh & Shibuki, 1996, 1997). We observed the sequence-dependent changes in the field potentials for 1 h after cessation of sequential TS (intersite distance, 350 μm; time interval, 1 s). The difference between PTPA and PTPB was maintained for more than 1 h in all of the four slices tested (Fig. 2C). However, the amplitude of field potentials gradually declined, probably because we used TS of minimal intensity to demonstrate the sequence-specific properties of PTP. Therefore, we focused on PTP in the initial 10 min after cessation of sequential TS in the rest of the experiments.

We changed the stimulus parameters of TS to estimate the minimal stimulus frequency required for the significant difference in the amplitude between PTPA and PTPB. Sequential TS with 15 pulses at 50 Hz was applied to SA and SB (intersite distance, 350 μm; time interval, 1 s). This stimulation induced PTPA and a smaller PTPB (PTPA, 149 ± 3 %; PTPB, 123 ± 3 %; mean ±s.e.m.; n = 9; Fig. 2D), and the difference in the amplitudes between PTPA and PTPB was significant (P < 0.01, Wilcoxon signed rank test).

LTP and PTP in supragranular layers can be induced in the horizontal pathways to supragranular layers (Hirsch & Gilbert, 1993; Seki et al. 1999). We tested the sequence dependence of PTP induced by TS applied through a pair of stimulating electrodes placed 350 μm apart in supragranular layers (time interval, 1 s). The field potentials were recorded midway between SA and SB (Fig. 2E inset). Sequential TS of SA and SB induced PTPA (166 ± 4 %, n = 8) but not PTPB (96 ± 5 %), and the difference was statistically significant (P < 0.02, Mann-Whitney U test).

To confirm the equivalence of TS applied at SA and SB placed on the border between layer VI and the white matter, we tested the effect of sequential TS in the reversed order after sequence-dependent PTP was induced (intersite distance, 350 μm; time interval, 1 s). The first TS (SA followed by SB) induced PTPA and almost no PTPB, while the second TS in the reversed order evoked PTPB and no further PTPA (Fig. 2F). Thus, PTPA and PTPB of similar amplitude were induced by the first and second TS, respectively, indicating the equivalence of TS applied at SA and SB.

Independence of the stimulated pathways

We tested whether independent pathways were stimulated at SA and SB placed on the border between layer VI and the white matter. The amplitude of postsynaptic potentials evoked by the stimulation at SA, SB, or SA plus SB was measured at the intersite distances of 350 and 150 μm (Fig. 3a). The sum of the responses evoked separately by the stimulation at SA and at SB was almost equal to or slightly smaller than that evoked by the simultaneous stimulation at SA and SB at the intersite distances of 350 and 150 μm (Fig. 3B). The independence of the pathways was also tested after sequence-dependent PTP was induced at the intersite distance of 350 μm. The increase in the amplitude of the field potentials evoked by the stimulation at SA plus SB (0.52 ± 0.11 mV, n = 5) was almost equal to the increase in the potentials evoked by the stimulation at SA (0.53 ± 0.09 mV), while almost no increase was observed in the potentials evoked by the stimulation at SB (0.08 ± 0.03 mV).

Figure 3.

Independence of the pathways stimulated at SA and SB

A, field potentials elicited by the stimulation at SA, SB and SA plus SB at the intersite distance (d) of 350 μm and 150 μm. B, amplitudes of postsynaptic potentials elicited by the stimulation at SA, SB and SA plus SB. The sum of the amplitudes of the potentials elicited by the stimulation at SA and SB are also shown. C, paired pulse facilitation at intervals of 50 ms. The stimulation at SA or SB was followed by the stimulation at the same site, or the stimulation at SA was followed by the stimulation at SB. The intersite distance (d) was 350 or 150 μm. D, paired pulse facilitation measured as the amplitude of the second postsynaptic potentials normalized by the amplitude of the first responses.

We also tested the independence of pathways using paired pulse facilitation. Although the two pulses applied at the same sites (SA or SB) at an interval of 50 ms produced clear facilitation in the second response, no facilitation was observed when the stimulation at SA was followed by the stimulation at SB at the intersite distance of 350 μm (Fig. 3C). At the intersite distance of 150 μm, some facilitation was observed, but it was significantly smaller than that after paired pulse stimulation of the same sites (P < 0.05, Wilcoxon signed rank test). These findings suggest that the two pathways stimulated at SA and SB were independent of each other at the intersite distance of 350 μm, while parts of the stimulated pathways might overlap at the intersite distance of 150 μm.

Spatial distribution of sequence-dependent PTP

Previously, we demonstrated PTP and LTP using Ca2+ imaging in AC slices loaded with rhod-2 (Seki et al. 1999). We used this technique to demonstrate the spatial distribution of PTP induced by TS with the stimulus parameters used for field potential recordings (SA and SB, on the border between layer VI and the white matter; intersite distance, 350 μm; time interval, 1 s). The fluorescence changes (ΔF/F) reflecting [Ca2+]i increase were elicited by test stimulation with a single pulse applied at SA or SB (Fig. 4a and D). The stimulus intensity was adjusted so that the extent of the fluorescence increases in response to the stimulation at SA or SB were similar. The fluorescence changes were elicited again by test stimulation with a single pulse applied at SA or SB after sequential TS (Fig. 4B and E). The distribution of the fluorescence increase 15.9-26.5 ms after test stimulation was enlarged after 7-10 min TS. However, the extent of the enlargement in the response to SA stimulation was larger than that to SB stimulation (Fig. 4B and E). Since the fluorescence changes (ΔF/F) varied almost linearly with the field potentials (Seki et al. 1999), PTPA and PTPB were measured as the difference in ΔF/F in response to test stimulation before and after sequential TS (Fig. 4C and F). PTPA and PTPB exhibited no overlapping distribution, and PTPA but not PTPB was induced in the supragranular layers between SA and SB (Fig. 4G). This finding was confirmed by the field potential recording in the same slice (Fig. 4H). The predominant distribution of PTPA was also observed in another slice (Fig. 4I), indicating that the sequence but not the relative position of the stimulation determined the distribution of PTP. Of the eight slices loaded with rhod-2 and showing PTPA with no PTPB in the field potentials, the predominant distribution of PTPA was observed in the Ca2+ imaging in six slices, while PTP was not clear from the Ca2+ imaging in the other two slices.

Sequence-dependent PTP recorded in pyramidal neurons

The field potentials in AC are mainly attributed to the activities of pyramidal neurons (Kudoh & Shibuki, 1996). Therefore, we investigated sequence-dependent PTP in intracellularly recorded pyramidal neurons, which were identified as neurons exhibiting antidromic firing in response to white matter stimulation (Kudoh & Shibuki, 1996). The resting membrane potentials, membrane resistance and series resistance in these neurons were -73 to -60 mV, 32-48 and 36-58 MΩ, respectively. We measured the rising slope of EPSPs, since this parameter was not affected by 1 μm bicuculline (data not shown) and was not expected to be contaminated by inhibitory postsynaptic or action potentials. The slope of EPSPs in response to SA stimulation was made equivalent to that in response to SB stimulation by adjusting the stimulus intensity. After sequential TS, the EPSPs in response to SA stimulation exhibited potentiation (Fig. 5a). However, the changes in EPSPs were variable from cell to cell. Therefore, they were defined as PTP only when all the responses recorded in the five traces 6-10 min after TS were larger than any of the five control responses recorded before TS (corresponding to a statistically significant difference of P < 0.005, Mann-Whitney U test). PTPA was observed in 18 of 33 cells recorded. In contrast, PTPB was observed in only one of the 18 cells showing PTPA and no cells showed PTPB only (Fig. 5B). The effects of simultaneous TS of SA plus SB or TS of SA only were also tested. After simultaneous TS of SA plus SB, both PTPA and PTPB were observed in only two of 29 cells tested (Fig. 5C). Others showed PTPA only (n = 12), PTPB only (n = 11) or no PTP at all (n = 4). After TS of SA only, PTPA was observed in 13 of 24 cells tested, and PTPB was observed in six cells (Fig. 5D). The main effects of sequential TS was to suppress the induction of PTPB, since the number of cells showing PTPB after sequential TS (n = 1/33) was much smaller than that after TS of SA only (n = 6/24) or simultaneous TS of SA and SB (n = 13/29). These findings obtained from intracellular recordings accord with those obtained from the field potential recordings as a whole. Therefore, it is strongly suggested that sequence-dependent PTP in field potentials is attributed to that in pyramidal neurons, at least in part.

Figure 5.

PTP in pyramidal neurons

A, PTPA induced by sequential TS in 18 of the 33 cells tested and simultaneously recorded responses to SB stimulation. Insets show sample traces before and after (*) TS. B, relationship between the amplitudes of PTPA and PTPB in each pyramidal neuron after sequential TS. In B, C, D and F, neurons with PTPA only (•), PTPB only (○), both PTPA and PTPB (▪) and no PTP (×) are shown. C, relationship between the amplitudes of PTPA and PTPB in each pyramidal neuron after simultaneous TS of SA and SB. D, relationship between the amplitudes of PTPA and PTPB in each pyramidal neuron after TS of SA only. E, PTPA and PTPB induced by pairing depolarizing current injection with simultaneous stimulation of SA and SB. F, relationship between the amplitudes of PTPA and PTPB in each pyramidal neuron after being induced by pairing depolarizing current injection with stimulation of SA and SB. Insets show the responses elicited by the stimulation only (left), current injection only (middle) and stimulation paired with current injection (right). In A-D, perforated patch technique was used, and PTP was induced by the same TS protocol as that used in the field potential recordings. In E and F, whole-cell patch technique was used, and PTP was induced by the paired stimulation.

Of the 29 cells tested after simultaneous TS of SA plus SB, 23 cells exhibited either PTPA only or PTPB only (Fig. 5C), indicating that most synapses could not exhibit both PTPA and PTPB. However, both pathways stimulated at SA and SB might never exhibit PTP to the same neuron. To test this possibility, we induced PTP by pairing current injection (1 nA, 100 ms) with simultaneous stimulation of SA and SB at 50 ms after the initiation of the current injection (Fig. 5E). In this experiment, the stimulation of SA and SB, which was unable to fire a neuron by itself, elicited a spike during current injection (Fig. 5E insets). This paired stimulation was repeated 100 times at 1 Hz. Although the amplitudes of PTP induced by this paired stimulation were slightly smaller than those induced by TS, they were less variable from cell to cell. Of the 15 cells tested, 14 cells exhibited both PTPA and PTPB, indicating that most pyramidal neurons could have both types of synapses exhibiting PTPA and PTPB.

Chemical modulation underlying the sequence dependence of PTP

The characteristic distribution of PTPA and PTPB in AC after sequential TS requires non-linear interactions between the inputs activated by the stimulation of SA and SB. A simple explanation of the sequence dependence is that the ability of the later TS to induce PTPB might be inhibited by the earlier TS via GABAergic modulation. The extent of inhibition was estimated by comparing the depolarization and firing in pyramidal neurons during the earlier TS and the later TS. In all of the 20 neurons tested, the extent of the first depolarization and firing during the earlier TS was larger than that of the second responses during the later TS (Fig. 6a). The difference was slightly diminished in the presence of 1 μm bicuculline (Fig. 6a), an antagonist of GABAA receptors. However, 100 μm 2-hydroxysaclofen (Fig. 6B), an antagonist of GABAB receptors, was not effective (Fig. 6B). We also induced PTP by sequential TS in the presence of antagonists for GABA receptors (Fig. 6C and D). The difference in PTP (PTPA - PTPB) induced in the presence of 1 μm bicuculline was 60 ± 6 % (n = 7), and that in the presence of 100 μm 2-hydroxysaclofen was 74 ± 16 % (n = 6). These values are not significantly different (Mann-Whitney U test) from that recorded in normal medium (78 ± 13 %, n = 11), suggesting that GABA receptors do not have an important role in the sequence dependence of PTP.

Cholinergic modulation is important for the induction of neural plasticity in the neocortex in vivo, and the receptive field plasticity in AC is blocked by atropine, a muscarinic receptor antagonist (Metherate & Weinberger, 1989). Therefore, we tested the effect of atropine. Application of 50 μm atropine enlarged the amplitude of field potentials by 157 ± 5 % (n = 9). These changes were cancelled by adjusting the intensity of test stimuli. We tested the effect of 50 μm atropine on the extent of inhibition of the depolarization and firing in pyramidal neurons during the later TS (Fig. 7a and B). The responses during the later TS were similar to those during the earlier TS in the presence of atropine (n = 8). Although the amplitude of the depolarization measured 20 ms after the later TS was significantly smaller than that of the first depolarization in control medium (P < 0.03, Wilcoxon signed rank test), it was increased in the presence of 50 μm atropine, and a difference between the amplitudes of the first and second depolarizations was not observed (Fig. 7C). The amplitude of the second depolarizations was not clearly affected by 1 μm bicuculline and 100 μm 2-hydroxysaclofen (Fig. 7C), although the amplitude of the first depolarization was increased in the presence of bicuculline.

Figure 7.

Effects of atropine on the depolarization and firing of neurons during sequential TS

A, depolarization and firing during sequential TS (intersite distance, 350 μm; time interval, 1 s). B, depolarization and firing in the presence of 50 μm atropine. The traces in A and B were recorded in the same neuron. C, amplitudes of depolarization measured 20 ms after cessation of TS at SA or SB in control medium, in the presence of 1 μm bicuculline, 100 μm hydroxysaclofen and 50 μm atropine. Differences between the amplitudes of the first and second responses are also shown.

We tested the effect of atropine on the induction of sequence-dependent PTP in AC slices. Both PTPA and PTPB were induced by sequential TS in the presence of 50 μm atropine. The amplitudes of PTPA and PTPB were 148 ± 8 % (n = 9) and 146 ± 6 %, respectively (Fig. 8a). The difference (PTPA - PTPB) was 2 ± 5 % (Fig. 8F), and was significantly smaller than the control value recorded in the absence of atropine (78 ± 13 %, n = 11, P < 0.0003, Mann-Whitney U test).

Figure 8.

Cholinergic modulation of the sequence dependence of PTP in AC slices

A, PTPA and PTPB induced by sequential TS in the presence of 50 μm atropine. Insets show sample traces. B, sequence-dependent PTP induced in the presence of 10 μm carbachol plus 50 μm atropine. C, PTPA and PTPB induced in the presence of 50 μm pirenzepine. D and E, sequence-dependent PTP induced in the presence of 30 μm methoctramine (D), and 50 μm linopirdine plus 50 μm atropine (E). F, PTPA - PTPB shown in A-E.

Since the application of atropine alone was sufficient to enlarge the amplitude of the field potentials, acetylcholine is expected to be continuously released in the slices. If so, the sustained release of acetylcholine may be sufficient to maintain the sequence dependence of PTP. To test this possibility, we applied 10 μm carbachol, an agonist of cholinergic receptors, in the presence of atropine. The changes in the field potentials elicited by 50 μm atropine were cancelled by carbachol at this dose. In the presence of this cocktail, PTPA but not PTPB was induced by sequential TS (Fig. 8B and F), suggesting that the sustained release of acetylcholine was sufficient to maintain the sequence dependence of PTP.

Muscarinic receptors are divided into several subtypes, some of which are known to affect the induction of PTP and LTP (Abe et al. 1994; Auerbach & Segal, 1996; Shimoshige et al. 1997). To identify the subtype of muscarinic receptors related to the sequence dependence of PTP in AC slices, we induced sequence-dependent PTP in the presence of pirenzepine, an antagonist of M1 receptors (Maclagan & Faulkner, 1989), and methoctramine, an antagonist of M2 receptors (Van Charldorp et al. 1988). Pirenzepine (50 μm) slightly reduced the amplitude of PTPA and increased that of PTPB so that the sequence dependence of PTP was completely abolished (Fig. 8C and F). These effects were similar to those of atropine. In contrast, 30 μm methoctramine exhibited almost no effect on the induction of sequence-dependent PTP (Fig. 8D and F), suggesting that the cholinergic modulation of the sequence dependence of PTP was exerted mainly via M1 but not via M2 receptors.

Activation of M1 receptors inhibits M-currents (Marrion, 1997). Linopirdine, an inhibitor of M-currents (Lamas et al. 1997), enhances neurotransmitter release including acetylcholine (Provan & Miyamoto, 1994), and can substitute cholinergic inputs to facilitate learning and plasticity (Brioni et al. 1993). Therefore, we tested the sequence dependence of PTP in the presence of atropine and linopirdine. The sequence dependence of PTP was clearly observed in the presence of this cocktail (Fig. 8E and F), suggesting that the effect of cholinergic modulation on sequence-dependent PTP was mediated via M1 receptors and the subsequent inhibition of M-currents.

DISCUSSION

Sequence dependence of PTP in AC and temporal information processing

In the present study, the sequence dependence of PTP in AC slices was observed in the field potentials, Ca2+ signals and EPSPs in pyramidal neurons. The time differences required for the sequence dependence were in the order of seconds. Slow synaptic potentials mediated by GABAB receptors are used for temporal information processing (Buonomano, 2000). However, the sequence dependence of PTP in the present study was observed after heterosynaptic stimulation at time differences much longer than the duration of synaptic potentials mediated by GABA. Antagonists of GABAA and GABAB receptors did not significantly affect the sequence dependence of PTP in the present study (Fig. 6). Combined pre- and postsynaptic activities elicit synaptic potentiation and depression depending on the temporal relationship of the two within the time windows of tens of milliseconds (Markram et al. 1997; Bi & Poo, 1999; Feldman, 2000). However, the time differences required for the sequence dependence of PTP in the present study were clearly outside this time window. Therefore, it is possible that the sequence dependence of PTP in AC has a unique role in temporal information processing in the order of seconds.

Role of polysynaptic excitation in the induction of PTP

The importance of polysynaptic excitation during the induction of LTP in AC has been demonstrated in our previous study (Kudoh & Shibuki, 1997). In the present study, sequence-dependent PTP was induced by TS applied through a pair of stimulating electrodes placed 350 μm apart in supragranular layers (Fig. 2E). Therefore, the synapses between supragranular pyramidal neurons connected with horizontal axon collaterals are probably involved in the sequence-dependent PTP. Some findings in the present study are difficult to understand without assuming the involvement and dynamic properties of polysynaptic pathways during the induction of PTP. First, TS of SA induced not only PTPA but also weak PTPB at the intersite interval of 350 μm (Fig. 1D and Fig. 5D), although the pathways activated by the stimulation at SA and SB were independent of each other at this intersite distance (Fig. 4). This discrepancy is explained by assuming that TS of SA elicited the polysynaptic activation of many neurons, which were not directly stimulated by the single pulse stimulation at SA (Fig. 9a). It is possible that some pathways activated by single pulse stimulation at SB but not at SA exhibit PTP (PTPB) after this polysynaptic activation. Second, in the majority of neurons tested simultaneous TS of SA and SB did not induce both PTPA and PTPB (Fig. 5C), although simultaneous stimulation of SA and SB paired with depolarizing current injection induced both PTPA and PTPB (Fig. 5F). Although this discrepancy is difficult to understand, it might be explained by assuming unidirectional propagation of polysynaptic excitation in a local circuit during TS, because of the refractoriness of neural activities. If a particular neuron is involved in a local circuit of rightward-dominant propagation during TS, PTPA is expected to be more easily induced in this neuron than PTPB (Fig. 9B). In contrast, a neuron involved in a local circuit of leftward-dominant propagation will exhibit PTPB, but almost no PTPA. Sequential TS of SA and SB produced PTPA in only half of the neurons (Fig. 5a). These neurons might be involved in local circuits of leftward-dominant propagation, while others could be part of local circuits with the opposite direction of propagation.

Figure 9.

Role of polysynaptic excitation in the induction of PTP

A, PTPA and weak PTPB induced after TS of SA only. Weak PTPB was probably induced by spreading polysynaptic excitation. The schematic drawing of three pyramidal neurones connected to each other with horizontal axon collaterals is shown. These neurons can be stimulated antidromically via their axons. B, PTPA and no PTPB induced in a neuron (middle) by simultaneous TS of SA and SB. PTPB was not induced in this neuron, probably because the input stimulated at SB could activate the right neuron only during the refractory period of the middle neuron. C, PTPA and no PTPB induced by sequential TS in a neuron (middle). This neurone could not respond to the later TS of SB with depolarization and firing sufficient to induce PTPB. D, PTPA and PTPB induced by sequential TS in a neuron (middle) in the presence of atropine. This neurone could respond to the later TS of SB with depolarization and firing sufficient to induce PTPB.

Role of cholinergic inputs in the sequence dependence of PTP

Cholinergic modulation has been reported to be important in the induction of neural plasticity in sensory cortices in vivo. The plasticity of ocular dominance columns in the visual cortex is facilitated by cholinergic modulation (Bröcher et al. 1992). Whisker stimulation paired with acetylcholine applied iontophoretically produced long-lasting modification of neural responses in the somatosensory cortex, and the expression of this modification requires the presence of exogenous acetylcholine (Shulz et al. 2000). The receptive field plasticity in AC is facilitated by cholinergic modulation (Metherate & Weinberger, 1989). The combined presentation of sound stimuli and electrical stimulation of the basal forebrain produces plasticity of the receptive field or the sound representation in AC (Bakin & Weinberger, 1996; Kilgard & Merzenich, 1998a). The cholinergic inputs are involved in the changes in the temporal response properties of AC neurons induced by training (Kilgard & Merzenich, 1998b). All these findings strongly suggest an important role of cholinergic inputs in the sensory cortices, including AC.

The induction of synaptic potentiation is not simply facilitated or blocked under the influence of cholinergic modulation. Paired pulse stimulation can induce LTD in response to the second pulse in the presence of cholinergic agonists (Kirkwood et al. 1999). Oscillatory field potentials in hippocampal slices are generated by the application of cholinergic agonists, and the stimulation locked with the particular phase of the oscillatory field potentials can induce potentiation or depression depending on the phase (Huerta & Lisman, 1995). In the present study, the sequence dependence of PTP, but not the induction of PTP, was blocked by atropine (Fig. 8a). Bath application of carbachol was able to rescue the sequence dependence of PTP blocked by atropine (Fig. 8B), and therefore, tonic cholinergic modulation may be sufficient for the sequence dependence of PTP in AC.

The involvement of M1 receptors in the sequence dependence of PTP in AC is suggested from the pharmacological experiments in the present study (Fig. 8C and D). M1 receptors are coupled with a number of secondary messenger systems (Loffelholz, 1996), and the inhibition of M-currents by the activation of M1 receptors is one of the possible mechanisms of cholinergic influence on the sequence dependence of PTP. Linopirdine, known as a cognitive enhancer (Fontana et al. 1994) and an inhibitor of M-currents (Lamas et al. 1997), can substitute cholinergic inputs to facilitate learning and plasticity (Brioni et al. 1993). The disappearance of the sequence dependence of PTP in the presence of atropine was rescued by linopirdine. Enhancement of neurotransmitter release including acetylcholine (Provan & Miyamoto, 1994) may be the underlying mechanism for the facilitatory effect of linopirdine for the sequence dependence of PTP. M-currents are recorded in pyramidal neurons (Munakata & Akaike, 1993). Activation of M1 receptors and subsequent suppression of M-current probably have profound effects on the excitability of pyramidal neurons. The resulting changes in polysynaptic activities during the later TS, as shown in Fig. 7a, may be required for the sequence dependence of PTP in AC.

Mechanisms underlying the sequence dependence of PTP

The sequence dependence of PTP in the present study is probably attributed to a novel mechanism of heterosynaptic interactions. Simple summation of PTP elicited by TS of SA only and TS of SB only cannot explain the sequence dependence of PTP induced by sequential TS, since TS of SA (or SB) only evoked both PTPA and PTPB. Therefore, non-linear heterosynaptic interaction is required for the sequence dependence of PTP. Heterosynaptic depression is attributed to several mechanisms, a part of which may be mediated by muscarinic receptors (Kozhemyakin & Kleschevnikov, 1994). Therefore, the present findings might be explained by heterosynaptic depression. It is reported that sequential TS applied at intervals of minutes induces potentiation in response to the earlier TS followed by its depotentiation after the later TS (Stäubli & Chun, 1996; Doyére et al. 1997). The result, obtained after sequential TS, was potentiation in response to the later TS in these studies. In the present study, sequential TS applied at intervals of seconds induced potentiation in response to the earlier but not later TS. This discrepancy can be attributed to the differences in the induction protocol or in the circuits. Although we tried to demonstrate sequence-dependent PTP in the slices obtained from the visual cortex, the sequence dependence of PTP was not clearly demonstrated (data not shown). Therefore, the induction of sequence-dependent PTP in AC might be dependent on the properties of AC circuits, which are expected to be specialized for auditory information processing.

In this study, atropine blocked the sequence dependence of PTP. It also blocked the inhibition of the second depolarization and firing during the later TS. Therefore, it is natural to speculate that the diminished responses during the later TS in control medium caused the inability to exhibit PTPB after sequential TS of SA and SB. During the later TS at SB, presynaptic activities are not always accompanied by the firing of their postsynaptic cells, and this situation does not favour the induction of PTPB. Associative LTD is induced in hippocampal neurons when presynaptic activities are preceded by the firing of their postsynaptic cells (Debanne et al. 1994). The conditions required for the induction of associative LTD might also be satisfied in our experiments after sequential TS, since the presynaptic activities activated by TS of SB were preceded by the firing of their postsynaptic cells during the earlier TS (Fig. 9C). Although the earlier TS of SA only could produce weak PTPB, this weak PTPB would be cancelled by the subsequent depression induced by the later TS, and no apparent changes might be produced by sequential TS in the pathways activated by the stimulation at SB after all. However, in the presence of atropine, PTPB was induced, probably because polysynaptic excitation during the later TS was not inhibited (Fig. 9D). The characteristic features of PTP demonstrated in the present study can be explained only by assuming the involvement of polysynaptic activities. Although we have no direct evidence supporting this hypothesis, such properties of polysynaptic activities in local circuits, if any, might play an important part in information processing in AC.

Acknowledgements

We thank H. Nawa and R. Hishida for reading the manuscript, and Y. Tamura and N. Taga for technical assistance. This work was supported by grants from the Japanese Government, CREST and Toyota RIKEN.

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