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NEUROSCIENCE |
1 Department of Information Physiology, National Institute for Physiological Sciences, Okazaki 444-8787, Japan
2 School of Life Sciences, The Graduate University for Advanced Studies (SOKENDAI), Okazaki 444-8787, Japan
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Abstract |
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(Received 30 May 2006;
accepted after revision 8 June 2006;
first published online 15 June 2006)
Corresponding author M. Miyata: Department of Information Physiology, National Institute for Physiological Sciences, Myodaiji, Okazaki 444-8787, Japan. Email: mmiyata{at}nips.ac.jp
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Introduction |
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Corticothalamic projections, in association with primary sensory inputs, coordinate firing responses of thalamic neurons in sensory processing in vivo (Krupa et al. 1999; Fanselow et al. 2001; Temereanca & Simons, 2004). Since both types of synapses are glutamatergic, glutamate receptors are a key molecule in sensory-driven firing responses of thalamic neurons (Salt & Eaton, 1989, 1991; Sillito et al. 1990; Eaton & Salt, 1996). In in vivo experiments, it has been reported that distinct types of glutamate receptors temporally integrate the firing responses of VB neurons to sensory stimulation. Non-NMDA receptors are responsible for the initial firing responses induced by somatosensory stimulation, but NMDA receptors are involved in delayed firing responses of VB neurons (Salt & Eaton, 1989). However, it remains unknown how these glutamate receptors are associated with corticothalamic and/or lemniscal synapses during the firing of VB neurons.
Corticothalamic synapses are distinguished from the primary sensory synapses by their properties, kinetics of synaptic transmissions and short-term plasticity in vitro (McCormick & von Krosigk, 1992; Castro-Alamancos, 2002; Blitz & Regehr, 2003), but the composition of glutamate receptors in both corticothalamic and lemniscal synapses remains unclear. We therefore compared glutamate receptor composition, temporal properties of synaptic transmission, and their impacts on respective firing responses of corticothalamic and lemniscal synapses in the mouse VB. Our results demonstrated that prominent NMDA receptors (NMDARs) in corticothalamic synapses play a crucial role in determining the temporal signature of firing responses of VB neurons to corticothalamic stimuli, whereas firing responses to lemniscal stimuli are mainly regulated by AMPA receptors (AMPARs).
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Methods |
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VB neurons were visualized under a microscope (BX50WI; Olympus) with an infrared differential interference contrast video system (C2400-79H; Hamamatsu Photonics, Hamamatsu, Japan). Whole-cell recordings were made with 2–4.5 M
recording pipettes containing (mM): 120 caesium methane-sulphonate, 10 Hepes, 1 EGTA, 2 MgCl2, 0.1 CaCl2, 20 NaCl, 5 QX314, 2 ATP-Na2, 0.5 GTP-Na (pH 7.3 with CsOH, 298–310 mosmol l–1) for voltage-clamp recordings. In the case of current-clamp and loose-patch recordings, the internal solution contained (mM): 120 potassium gluconate, 10 Hepes, 1.0 EGTA, 2 MgCl2, 0.1 CaCl2, 10 NaCl, 2 Na2ATP, 0.5 Na2GTP (pH 7.3 with KOH, 295–310 mosmol l–1).
An EPC9 patch-clamp amplifier (HEKA, Lambrecht, Germany) was used for voltage-clamp whole-cell recordings. EPSCs were mainly recorded at holding potentials of –70 mV with filtering at 2–10 kHz and digitized at 50 kHz. Series resistances were monitored on-line and the uncompensated series resistance was typically less than 9 M. A series resistance compensation of 75% was used during recordings from VB neurons. Liquid-junction potentials between the internal solutions and ACSF were corrected. The chord conductance mediated by non-NMDARs was measured by the peak amplitudes of non-NMDAR-mediated EPSCs at holding potentials from –90 to 0 mV. The conductance mediated by NMDARs was measured by amplitude of NMDAR-mediated currents at holding potentials between +40 and +5 mV. For current-clamp recordings, Axoclamp 2B (Molecular Devices, Sunnyvale, CA, USA) or EPC9 was used. We recorded from VB neurons at approximately their resting membrane potentials (see Results). When a cell became depolarized within 5 mV during a recording, we applied a small negative current through the pipette to hold the potential at around –70 mV. Loose-patch recording was performed as previously described (Kondo & Marty, 1998). Synaptic responses were evoked using a concentric electrode (tip diameter, 25 µm; Inter Medical, Nagoya, Japan) placed on the internal capsule for recording corticothalamic responses and on the medial lemniscus for recording lemniscal responses as reported previously (Castro-Alamancos & Calcagnotto, 2001; Castro-Alamancos, 2002). The stimulus consisted of a 100 µs-duration bipolar pulse of constant current steps (< 100 µA) using a biphasic isolator (BAK Electronics, Oxford, UK). The stimulus was delivered at 0.05 Hz. Data acquisition was performed using the PULSE program (HEKA, version 8.54). Pulse Fit (HEKA, version 8.54) and Igor Pro (Wavemetrics, Lake Oswego, OR, USA) were used to analyse the data. The statistical significance was determined using a t test (unpaired or paired) and ANOVA depending on the experimental design.
Drugs
DL-2-amino-5-phosphonopentanoic acid (DL-AP5), 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulphonamide disodium salt (NBQX), 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX), 7-(hydroxyimino)cyclo-propa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt) (RS)-
-methyl-4-carboxyphenylglycine (MCPG), 2-(4-benzylpiperidino)-1-(4-hydroxyphenyl)-1-propanol hemitartrate (ifenprodil hemitartrate), SYM 2206 and SYM 2081 were purchased from Tocris Cookson (Avonmouth, UK). GYKI 53655 (Sigma-Aldrich) was gifted from Drs. H. Kamiya and T. Momiyama.
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Results |
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Whole-cell recordings were taken from 241 VB neurons. We recorded corticothalamic EPSCs evoked by the stimulation of the internal capsule in horizontal brain slices. We also recorded lemniscal EPSCs evoked by the stimulation of lemniscal fibres in the same slices. In 16% of the recorded VB neurons, both corticothalamic and lemniscal EPSCs were recorded from the same VB neuron (Fig. 1). We distinguished the two synaptic responses by their characteristics. First, corticothalamic EPSCs increased monotonically with increasing stimulus intensity, indicating that they were composed of a large number of small unitary events (Fig. 1A). In contrast, lemniscal EPSCs in the vast majority of the neurons (27 out of 30 neurons) showed all-or-none responses (Fig. 1B). A few neurons (3 out of 30 neurons) displayed two steps of amplitudes with increasing stimulus intensity. The threshold stimulation resulted in a unitary response that always had the same amplitude. Thus, lemniscal EPSCs were composed of one or two large unitary events. Second, the two types of synaptic responses were different in short-term plasticity. Corticothalamic EPSCs showed the paired-pulse facilitation when the same stimulus was delivered twice with a short interval (Fig. 1A). At the 50 ms interval, the amplitude of the second EPSC, on average, was 195.1 ± 18.2% (mean ± S.E.M., n = 20) greater than that of the first EPSC amplitude in corticothalamic synapses (Fig. 1C). The facilitation was observed up to the 200 ms intervals, although the facilitated amplitude was smaller toward longer intervals. In contrast, all lemniscal EPSCs led to the paired-pulse depression (Fig. 1B). At the 50 ms interval, the amplitude of the second EPSC was reduced to 71.7 ± 9.0% (n = 20) of that of the first EPSC in lemniscal synapses (Fig. 1D). The amplitude remained depressed at still longer intervals up to 1000 ms. These synaptic properties were consistent with those found in a previous study that examined EPSPs (Castro-Alamancos, 2002).
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We next investigated whether there is any difference in the composition of NMDAR-mediated and non-NMDAR-mediated components between corticothalamic and lemniscal EPSCs. Non-NMDAR-mediated (i.e. AMPAR- and kainate receptor-mediated) EPSCs were recorded at a membrane potential of –90 mV (Fig. 2A). The stimulus strength for corticothalamic synapses was adjusted to evoke EPSCs with a peak amplitude of 
200 pA (206.0 ± 21.5 pA, mean ±
S.D., n
= 10). The stimulus strength for lemniscal synapses was fixed at the suprathreshold level that produced a unitary response. The peak amplitude of the unitary response was 1420.1 ± 432.1 pA (n
= 10). The peak conductances of non-NMDARs were 2.3 ± 0.2 nS (mean ±
S.D., n
= 8) and 15.7 ± 4.5 nS (n
= 8) in corticothalamic and lemniscal synapses, respectively. Corticothalamic EPSCs showed large rise-time and decay-time constants in comparison with lemniscal EPSCs. NMDAR-mediated EPSCs were evoked at the same stimulus strength in the presence of NBQX (50 µM), a non-NMDAR antagonist, at a holding potential of +40 mV to relieve the voltage-dependent Mg2+ block of the NMDAR channel (Mayer et al. 1984; Nowak et al. 1984) (Fig. 2A). The peak amplitudes of NMDAR-mediated EPSCs were 395.4 ± 99.9 pA (mean ±
S.D.) and 853.6 ± 221.4 pA in corticothalamic and lemniscal synapses, respectively. The NMDAR-mediated conductances were 10.8 ± 2.2 nS (mean ±
S.D.) and 22.3 ± 7.3 nS in corticothalamic and lemniscal synapses, respectively. The NMDAR-mediated EPSCs were completely abolished by DL-AP5, an NMDAR antagonist, at 100 µM (data not shown). These results indicated that both corticothalamic and lemniscal EPSCs consisted of NMDAR- and non-NMDAR-mediated currents. The peak current ratio of NMDAR-mediated EPSCs at +40 mV to non-NMDAR-mediated EPSCs at –90 mV was significantly larger in corticothalamic synapses (Fig. 2B and 191.9 ± 31.0%, mean ±
S.D., n
= 10) than in lemniscal synapses (Fig. 2B and 60.1 ± 16.2%, P < 0.01, unpaired t test, n
= 10). The ratios of NMDAR-mediated conductance to non-NMDAR-mediated conductance were 470.0 ± 72.5% (mean ±
S.D.) and 142.0 ± 25.6% in the corticothalamic and lemniscal synapses, respectively, indicating that the relative contribution of the NMDAR-mediated component was significantly greater in corticothalamic synapses than that in lemniscal synapses (P < 0.01, unpaired t test).
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To further investigate the properties of the non-NMDAR-mediated component in the two types of synapses, we recorded corticothalamic and lemniscal EPSCs in the presence of 100 µM DL-AP5. We confirmed that non-NMDAR-mediated currents were completely blocked by the AMPA and kainate receptor antagonists NBQX (50 µM; Fig. 4A and B) and CNQX (50 µM, n = 5). The basic properties of non-NMDAR-mediated corticothalamic and lemniscal EPSCs are summarized in Table 1. The 10–90% rise time of corticothalamic EPSCs was 1.97 ± 0.61 ms (mean ± S.D., n = 20), which was significantly longer than that of lemniscal EPSCs (0.93 ± 0.25 ms, P < 0.01, unpaired t test, n = 16). The longer rise time may result from more distal locations of corticothalamic synapses than lemniscal synapses (Jones & Powell, 1969; Bourassa et al. 1995; Zhang & Deschenes, 1997), or from asynchronous glutamate release from corticothalamic fibres.
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1: 5.14 ± 1.29 ms; 88.4 ± 5.7%) and slow (2: 87.9 ± 14.5 ms) time constants, whereas the decay of lemniscal EPSCs was fitted with a single exponential (4.1 ± 2.1 ms), suggesting the difference in the composition of non-NMDARs between corticothalamic and lemniscal synapses. Since AMPA and kainate receptors have fast and slow gating kinetics, respectively (Lerma et al. 2001), we examined the kainate receptor-mediated component of corticothalamic EPSCs. The change in the amplitude of corticothalamic EPSCs was investigated after the application of GYKI 53655 (50 µM) and SYM 2206 (100 µM), both of which are selective AMPAR antagonists (Pelletier et al. 1996; Bleakman et al. 1996; Li et al. 1999). In the presence of DL-AP5, corticothalamic EPSCs were partially blocked by the AMPAR antagonists, but a resistant component remained (Fig. 4A and C; GYKI 53655: 13.9 ± 5.1%, mean ±
S.D., n
= 12; SYM 2206: 15.9 ± 6.5%, n
= 12). The decay of resistant currents was best fitted with a single exponential (Fig. 4E; 1: 59.6 ± 23.8 ms). The resistant component was completely blocked by 50 µM NBQX (n
= 10; Fig. 4A) and/or 20 µM SYM 2081, a kainate receptor antagonist (Jones et al. 1997; DeVries, 2000; Cho et al. 2003), indicating that the component was mediated by kainate receptors. In contrast, a unitary lemniscal EPSC was completely blocked by GYKI 53655 (50 µM; Fig. 4B and D) and SYM 2206 (100 µM). In addition to GYKI 53655 or SYM 2206, the subsequent application of NBQX did not change the EPSC amplitude in lemniscal synapses (Fig. 4F). These results indicated that corticothalamic synapses contained a significant amount of kainate receptor-mediated EPSCs. In contrast, no kainate receptor-mediated currents were detected and AMPAR-mediated currents were dominant in lemniscal EPSCs. Postsynaptic summation of corticothalamic and lemniscal EPSCs induced by repetitive stimulation and their contribution by glutamate receptors
Due to the different composition of NMDA, kainate and AMPA receptors between corticothalamic and lemniscal synapses and the fact that NMDA and kainate receptors have slower kinetics than those of AMPARs, we next examined how these differences would affect the postsynaptic summation of EPSCs by repetitive stimulation. We first stimulated corticothalamic or lemniscal fibres using 5-pulse trains at 1–50 Hz and compared compound EPSCs before and after the bath application of 100 µM DL-AP5. In this experiment, holding potentials were kept at –60 mV in order to observe NMDAR-mediated currents. Compound EPSCs in corticothalamic synapses decreased after the application of DL-AP5 in a frequency-dependent manner (Fig. 5A). Percentage charges of NMDAR components of compound EPSCs were 32.8 ± 4.93 and 13.5 ± 8.6% (mean ± S.E.M., n = 5) at 50 and 10 Hz, in corticothalamic synapses (Fig. 5C). On the other hand, percentage charges of NMDAR components of compound EPSCs in lemniscal synapses were much smaller than those in corticothalamic synapses (Fig. 5B and D, and 4.93 ± 2.2 and 2.86 ± 1.14 at 50 and 10 Hz, respectively, n = 5).
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In lemniscal synapses, postsynaptic summations of EPSCs were not significantly different before and after the bath application of DL-AP5. At 50 Hz, for example, the fourth to the first EPSCres ratio was 178.2 ± 13.5% without DL-AP5, and 174 .0 ± 15.1% (n
= 10) in the presence of the drug. Overall these values were much smaller than the corresponding ones of corticothalamic EPSCs (Fig. 5F, open circles). No summation was observed during low-frequency (\#8804;
10 Hz) repetitive stimulation in lemniscal EPSCs either with or without DL-AP5 (Fig. 5B and F).
Since corticothalamic EPSCs contained a significant kainate receptor-mediated component (Fig. 4), which also has slow kinetics, we next investigated how kainate receptor-mediated currents contribute to the summation of EPSCs during the repetitive stimulation. In the presence of DL-AP5 (100 µM), GYKI 53655- and SYM 2206-resistant currents that were evoked in response to the corticothalamic repetitive stimulation were measured (Fig. 6A). The kainate receptor-mediated currents also displayed the summation in a frequency-dependent manner. The percentage of kainate receptor-mediated charges of the total EPSC charge increased progressively in a frequency-dependent manner, being 13.8 ± 2.7% (mean ± S.E.M., n = 10) at 50 Hz, 10.2 ± 2.4% at 20 Hz, and 2.1 ± 0.85% at 5 Hz (Fig. 6C).
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In contrast to corticothalamic EPSCs, lemniscal EPSCs were almost completely abolished by the bath application of GYKI 53655 (50 µM) and/or SYM 2206 (100 µM) in the presence of DL-AP5, even when the repetitive stimulation was applied at 50 Hz (Fig. 6B). An additional application of NBQX (98.8 ± 2.0%, n = 8) and/or MCPG (101.2 ± 2.1%, n = 10) did not cause any significant change in the charge at various frequencies (P > 0.05, paired t test).
Contrasting temporal properties of firing responses in the two types of synapses
During the repetitive stimulation, the postsynaptic summation was significantly greater at high frequencies than at lower frequencies in corticothalamic synapses, but not in lemniscal synapses. The question at hand was how these different synaptic properties were transformed into different temporal firing patterns of VB neurons. To answer this question, we recorded firing responses (spikes), in the loose patch mode, from VB neurons in response to a 10-pulse train stimulation delivered to corticothalamic and lemniscal fibres. For corticothalamic fibres, we first increased the stimulation intensity gradually until the threshold for spike generation was reached in the 9th or 10th stimulus in the train at 10 Hz, the frequency at which the postsynaptic summation started to appear (Fig. 5E), and then applied the stimulation of the same intensity at 40 Hz, the frequency at which the summation was evidently larger (Fig. 5E). In the case of lemniscal fibres, we confirmed the appearance of spikes in an all-or-none fashion at the threshold of stimulus intensity and fixed the stimulus intensity at the suprathreshold level, and then applied the pulse train at 20 Hz.
The firing responses to these stimulation protocols allowed us to distinguish between corticothalamic and lemniscal synapses. To quantify the difference between the two types of synapses, we measured the success rate for spike generation in each set of 30 trials. The stimulation of corticothalamic fibres induced a late-persistent firing pattern (Fig. 7A, upper trace). The probability for the spike generation was the lowest for the first three stimuli and increased from the fourth stimulus on (Fig. 7B, filled circles; success rates: 0.0 ± 0.0% (mean ± S.E.M.) for the first stimulus, 91.6 ± 2.8% for the 9th stimulus, n = 15). In contrast, the stimulation of lemniscal fibres resulted in an onset-transient firing pattern (Fig. 7A, lower trace). The probability for the spike generation was the highest at the onset of the pulse train and decreased rapidly in subsequent stimuli (Fig. 7B, open circles; success rates: 100.0 ± 0.0% for the first stimulus, 0.0 ± 0.0% for the 9th stimulus, n = 15).
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To elucidate different contributions to firing responses by AMPA, kainate and NMDA receptors in corticothalamic and lemniscal synapses, we recorded VB neurons (including a subset of neurons described in the preceding section, which were repatched) with and without the presence of respective receptor antagonists in a whole-cell current-clamp configuration. The resting membrane potential of these VB neurons was –67.9 ± 3.9 mV (n
= 30) and the input resistance was 193.2 ± 52.3 M (n
= 30). We applied the same stimulation protocols to corticothalamic and lemniscal fibres as in the loose-patch experiment. In the control experiment, the stimulation to corticothalamic fibres induced EPSPs, which displayed the temporal summation and evoked action potentials between the fourth and tenth stimuli (late-persistent firing; Fig. 8A). The probability for the generation of action potentials was the lowest after the first three stimuli and increased between the fourth to tenth stimuli (success rates: 0.0 ± 0.0%, mean ±
S.E.M., for the first stimulus, 86.2 ± 5.6% for the 9th stimulus, n
= 34; Fig. 8C, open circles). In contrast, after the stimulation to lemniscal fibres, action potentials appeared at the onset of each pulse train (onset-transient firing) and EPSPs weakened drastically in the course of the stimulation (Fig. 8B). The probability for the generation of action potentials was the highest at the onset of the stimulation and decreased rapidly in subsequent stimuli (Fig. 8D, open circles; success rates: 85.0 ± 3.8% for the first stimulus, 0.0 ± 0.0% for the 9th stimulus, n
= 12).
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Next, we bath-applied SYM 2801 (20 µM) to determine the contribution of kainate receptors to the late-persistent firing. After 10 min application of SYM 2081, the spike generation was attenuated but the late-persistent pattern was maintained (Fig. 8A, middle traces). The mean success rates for spike generation were 5.00 ± 4.33% for the 5th stimulus (significantly lower than the corresponding value in the control experiment, P < 0.01, n = 10) and 87.5 ± 6.25% for the 9th stimulus (Fig. 8C, filled diamonds). The result from this experiment suggested that kainate receptors, similar to AMPARs, were only partially involved in the late-persistent firing.
Finally, we examined the contribution of NMDARs to the late-persistent firing. The bath application of DL-AP5 (100 µM) disturbed the stability of the late-persistent firing and resulted in a marked decrease in the probability of the spike generation in all stimuli in the pulse train (Fig. 8A, right traces). The mean success rates for spike generation were 8.33 ± 6.45% for the 4th stimulus and 7.78 ± 3.75% for the 9th stimulus, both of which were significantly lower than those in the control experiment (P < 0.01, one-way repeated ANOVA, n = 10; Fig. 8C, filled triangles). Taken together, these data demonstrated that while AMPA, kainate and NMDA receptors were all necessary for the spike generation in response to the corticothalamic stimulation, NMDARs were the determinant contributor to the late-persistent firing response.
In contrast to corticothalamic synapses, the bath application of 100 µM DL-AP5 did not significantly affect the probability of spike generation in lemniscal responses, with the success rates for spike generation being 79.3 ± 8.8% for the first stimulus and 0.0 ± 0.0% for the 9th stimulus (P > 0.05, in comparison with the corresponding values in the control experiment, n = 5; Fig. 8B and D, filled triangles). The bath application of GYKI 53655 (50 µM), on the other hand, completely blocked lemniscal EPSPs and spike generation throughout the pulse train (Fig. 8D, filled circles; success rates for spike generation: 0.0 ± 0.0% for the first stimulus; 0.0 ± 0.0% for the 9th stimulus, n = 7). This result indicated that AMPARs were essential for the onset-transient firing response in lemniscal synapses.
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Discussion |
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Larger ratio of NMDAR component to non-NMDAR component in corticothalamic EPSCs than in lemniscal EPSCs
NMDARs are responsible for somatosensory-driven responses in VB neurons in vivo (Salt, 1986; Salt & Eaton, 1989), yet their synaptic origin in VB neurons remains undetermined. Primary sensory synapses activate NMDARs in the lateral geniculate nucleus (Scharfman et al. 1990), but the involvement of NMDARs has not been definitively demonstrated in lemniscal synapses. NMDARs are also activated by the corticothalamic stimulation both in vivo (Deschenes & Hu, 1990; Eaton & Salt, 1996) and in vitro (Scharfman et al. 1990; Kao & Coulter, 1997; von Krosigk et al. 1999). One of our primary findings was that the relative contribution of the NMDAR-mediated component, in comparison with non-NMDAR-mediated component, was much greater in corticothalamic EPSCs than in lemniscal EPSCs, suggesting that NMDARs in corticothalamic synapses play a principal role in evoking somatosensory-driven responses of VB neurons. Functional NMDARs are a heterometric complex composed of NR1 and NR2 subunits. It has been reported that NR2B-containing NMDAR-mediated currents shows a similar I–V relationship to NR2A-containing NMDAR-mediated currents, but has a longer decay time course (Monyer et al. 1994). We found that NR2B-mediated currents existed only in corticothalamic synapses, but not in lemniscal synapses, and the decay-time constant was much longer in corticothalamic synapses than that in lemniscal synapses even under ifenprodil. Presumably, it is because the electrotonic length from the soma to the site of corticothalamic synapses is larger than that from the soma to the site of lemniscal synapses.
Existence of kainate receptor component in corticothalamic EPSCs but not in lemniscal EPSCs
In the present study, we used two AMPAR-selective antagonists, SYM 2206 and GYKI 53655, the latter being the most potent antagonist available. GYKI 53655- and SYM 2206-resistant currents in corticothalamic synapses were completely blocked by NBQX and/or SYM 2081, indicating that they were kainate receptor-mediated currents. SYM 2081 is commonly used as an antagonist of kainate receptors because SYM 2081 rapidly desensitizes kainate receptor-mediated responses (Jones et al. 1997; DeVries, 2000; Cho et al. 2003). In contrast, kainate receptor-mediated currents were not detected in lemniscal synapses, a finding consistent with the results reported in a previous study (Binns et al. 2003).
It has been shown that kainate receptors are expressed in the mouse VB. Among five subunits of kainate receptors, GluR5, 6, 7, and KA2 exist in the rodent VB (Bahn et al. 1994). Furthermore, GluR5/6/7 immunolabelling in mouse VB neurons is seen at the postsynaptic sites of corticothalamic synapses and corticothalamic synapses are much more heavily labelled with the anti-GluR5/6/7 complex antibody than lemniscal synapses (Bolea et al. 2001). Thus, our electrophysiological results are supported by these anatomical findings. However, Bolea and colleagues reported no kainate receptor-mediated currents in corticothalamic EPSCs. This discrepancy may be attributed to the differences in experimental conditions between the two studies. They recorded EPSCs at a lower temperature (22–25°C), which is known to reduce kainate receptor-mediated currents (Kidd & Isaac, 2001). In addition, they used a different AMPA receptor antagonist, GYKI 52466, which has been shown to reduce kainate receptor-mediated currents by 20–30% (Paternain et al. 1995).
Relation to developmental changes of glutamate receptors in the two types of synapses
It has been revealed in developmental studies that major changes in the composition of glutamate receptors have already occurred before and around postnatal day 12 (P12) in the rodent thalamus. At corticothalamic synapses, for example, NMDAR-mediated currents are dominant during the first 12 postnatal days. However, after P12, other than the presence of the NMDAR component, there is also a substantial increase in non-NMDAR-mediated currents (Golshani et al. 1998). The expression of NR2B in the mouse thalamus is already high at birth, which remains at about the same level through the postnatal development, and starts to decline at around P7. In the meantime, the NR2A expression starts to appear and increases gradually during the next 2–3 weeks to reach the adult level. It is also reported that mRNA of kainate receptors is only weakly detected in the adult rat thalamus, but strongly expressed during the early postnatal development until around P12 (Bahn et al. 1994). In the mouse VB, the immunoreactivity of kainate receptors is detectable at least at P15–P19 (Bolea et al. 2001). However, it is still unclear whether these developmental changes occur simultaneously in both corticothalamic and lemniscal synapses. Our results were obtained from juvenile animals (P12–P17). Thus, our observation that corticothalamic and lemniscal synapses were composed of different components of AMPA, NMDA and kainate receptors reflected the distinct composition of glutamate receptors in the two types of synapses after their major changes in the postnatal development. It is unknown at the moment if the same composition is maintained in the adult.
Firing responses in corticothalamic and lemniscal synapses and their regulations by different composition of glutamate receptors
It is proposed that firing responses evoked by the synaptic stimulation with a train of pulses can be generally defined by such elements as membrane time constants, kinetics of EPSCs and short-term plastic properties (Zucker, 1989). The present study showed that the postsynaptic temporal summation of corticothalamic EPSCs and in part the short-term facilitation of synaptic transmission led to the late-persistent firing pattern. On the other hand, lemniscal EPSCs, which exhibited fast kinetics of EPSCs, and the short-term depression of synaptic transmission, resulted in the onset-transient firing pattern. The location of synaptic contacts is thought to contribute to the temporal summation of EPSCs and/or EPSPs in response to the repetitive stimulation. As corticothalamic and lemniscal synapses are located at the distal and proximal portion of the dendrite, respectively (Jones & Powell, 1969; Bourassa et al. 1995; Zhang & Deschenes, 1997), corticothalamic EPSCs, in comparison with lemniscal synapses, would be more strongly attenuated and exhibit slower kinetics when synaptic currents are measured at the soma (Bloomfield & Sherman, 1989; Hausser & Roth, 1997; Destexhe et al. 1998; Destexhe, 2000). Another determinant for the kinetics of EPSCs and/or EPSPs is the composition of glutamate receptors. The large NMDAR component in corticothalamic synapses induced marked postsynaptic summation at the high frequency stimulation because NMDARs have slower kinetics than AMPARs. Especially in current-clamp condition, activated NMDARs, which were relieved from the voltage-dependent Mg2+ block, led to large postsynaptic summation of EPSPs and finally reached the threshold to generate action potentials as the late-persistent firing response.
Kainate receptors with slow kinetics may also play a certain role on the firing response. Although kainate receptors in corticothalamic synapses were a minor contributor to the late-persistent firing, kainate receptor-mediated EPSCs showed the frequency-dependent summation because of the slow kinetics of kainate receptors, as have been reported for several types of neurons (Mulle et al. 1998; Bureau et al. 2000; Kidd & Isaac, 2001). This summation would lead to the generation of tonic depolarization in response to the repetitive stimulation in a frequency-dependent manner. Thus, in association with the AMPAR activation, the kainate receptor-mediated depolarization also helps to relieve the voltage-dependent Mg2+ block of NMDARs, which were essential for the late-persistent firing response (Fig. 7C). In addition, kainate receptors in corticothalamic synapses may also contribute to transmitting the average cortical activities (Frerking & Ohliger-Frerking, 2002). It is well known that depolarization switches the firing pattern from burst mode to tonic mode by activating low-threshold T-type calcium channels (Destexhe et al. 1998; Williams & Stuart, 2000; Zhuravleva et al. 2001). Taken together, we propose that prominent NMDA and kainate receptors with slow kinetics in corticothalamic synapses contribute to generate a prolonged depolarization and a fast switch between the burst and tonic modes, depending on the cortical activity. This would enable a more rapid response than those generated by conventional neuromodulators such as mGluRs, which operate on a time scale of a few hundreds of milliseconds (McCormick & von Krosigk, 1992).
On the contrary, the large AMPAR-component in lemniscal synapses displayed EPSCs with fast kinetics that did not induce a visible postsynaptic summation. It has been reported that NMDARs trigger additional action potentials with longer latencies and act as a modulator, whereas AMPARs evoke precisely timed action potentials with short latencies and detect the timing information (Blitz & Regehr, 2003). These different functions of glutamate receptors may be responsible for the differences in the temporal features of firing responses in the two synapses in vivo. For example, corticothalamic synapses facilitate and prolong firing responses of VB neurons to a whisker movement with a long latency (Krupa et al. 1999; Temereanca & Simons, 2004). Lemniscal inputs, in contrast, generate spikes in VB neurons with a short latency (Krupa et al. 1999; Fanselow et al. 2001).
Functional implications of corticothalamic and lemniscal synapses
As shown in the present study, lemniscal synaptic properties were transformed into the onset-transient firing in response to the stimulation with a train of pulses, because most of recorded cells were rather hyperpolarized like in the burst mode (Sherman & Guillery, 1998). In another sense, VB neurons did not faithfully transfer lemniscal inputs, even if repetitive inputs reached VB neurons in the burst mode. Castro-Alamancos (2002) reported that a VB neuron that was tonically depolarized closer to its firing threshold (tonic mode) is able to overcome the disturbance of transfer and relay lemniscal sensory information faithfully in response to high-frequency inputs. Therefore, the relay of sensory information is warranted when lemniscal inputs and depolarization coincide. We suggest that NMDAR-mediated currents may play the role of generating or maintaining the depolarized state in vivo.
Taken together, our results strongly suggest that the properties of corticothalamic synapses are useful in amplifying or gating lemniscal sensory information, depending on cortical activity. A large temporal summation of corticothalamic EPSCs was observed at high frequency including gamma frequency (20–50 Hz), which is considered to engage the cortex and thalamus during the arousal state (Steriade, 1993). This gain system in corticothalamic synapses may underlie the sensory processing (Murphy et al. 2000; Sillito & Jones, 2002; Miyata et al. 2003; Jung et al. 2004), spatiotemporal definition of receptive fields (Temereanca & Simons, 2004), and switching the firing modes during the arousal state (Guillery & Sherman, 2002). In sum, this type of facilitation can provide an elegant stimulus-specific transfer of somatosensory information through lemniscal synapses, depending on cortical activity.
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, n
= 10). The current amplitude was normalized to the amplitude at +40 mV.
: application of DL-AP5, n
= 10; 
: co-application of DL-AP5 and GYKI 53655, n
= 10; 
: application of SYM 2081, n
= 10). Error bars indicate the mean ±
S.E.M.D, probabilities for the spike generation for lemniscal synapses (