Impaired Feedforward Inhibition of the Thalamocortical Projection in Epileptic Ca²⁺ Channel Mutant Mice, tottering

Thalamocortical responses in adult wt and tg mice

In the somatosensory cortex, layer IV is the main gateway for afferent fibers originating in the respective thalamic relay nuclei (for review, see Douglas and Martin, 2004). The interplay between the cerebral cortex and the thalamus can be investigated using rodent thalamocortical brain slice preparations, which are unique in including both the ventrobasal nucleus of the thalamus and the somatosensory (barrel) cortex with the functional connectivity between them (Agmon and Connors, 1991). There are two major types of morphologically different excitatory neurons in layer IV: spiny stellate cells and pyramidal cells (Simons and Woolsey, 1984). Because it was technically easier to identify pyramidal cells in the slice preparation under the DIC video microscopy, we focused on layer IV pyramidal cells in this study so that a relatively homogenous neuronal population could be studied.

Voltage-clamp recordings were made from layer IV pyramidal cells of the barrel cortex in the whole-cell patch-clamp configuration (Fig. 1A). Most of the recorded neurons were examined morphologically by including biocytin in the pipette and staining after electrophysiological recordings. EPSCs and IPSCs evoked by thalamic stimulation were measured in the presence of the NMDA receptor antagonist APV (100 μm) and compared between wt and epileptic tg mice (P21–P30) (Fig. 1B1).

• Download figure
• Open in new tab
• Download powerpoint

Figure 1.

IPSCs and EPSCs recorded in layer IV pyramidal cells in response to thalamic stimulation. A, Left, A thalamocortical brain slice preparation (cytochrome c oxidase staining). Scale bar, 1 mm. The black pointer indicates a typical position of stimulation. A barrel is indicated by a dotted circle. Recordings were made from neurons within a barrel. VB, Ventrobasal nucleus of the thalamus; IC, internal capsule. Right, The scheme of the thalamocortical circuit. B1, Synaptic currents evoked by thalamic stimulation were recorded from layer IV pyramidal cells in wt and tg mice (P21–P30). EPSCs were recorded at a holding potential of −60 mV (top traces). When the holding potential was 0 mV, IPSCs became prominent (bottom traces). B2, The outward currents at a holding potential of 0 mV were blocked by picrotoxin (50 μm) (left) and also abolished by CNQX (10 μm) (right). B3, The IPSC and EPSC traces are superimposed in an expanded timescale (the same data used in B1 for wt). The mean delay between the onset EPSC and IPSC was 1.3 ± 0.08 ms (n = 12) for wt and 1.4 ± 0.15 ms (n = 11) for tg mice. C, The relationship between the excitatory and inhibitory conductances. Each point represents data from a single neuron.

When the holding potential was −60 mV, prominent EPSCs were observed in layer IV pyramidal cells of wt and tg mice (Fig. 1B1, top traces). At the holding potential of 0 mV, EPSCs became very small and there appeared large outward currents in wt, whereas the current amplitude was much smaller in tg mice (Fig. 1B1, bottom traces). The outward currents were GABA_A receptor-mediated IPSCs, because they were completely blocked by the GABA_A receptor antagonist picrotoxin (50 μm) (Fig. 1B2). Both EPSCs and IPSCs were completely abolished by the non-NMDA receptor antagonist CNQX (10 μm) (Fig. 1B2). Because the synaptic delay between the stimulation and the onset of EPSCs was short (2.1 ± 0.12 ms; n = 12), EPSCs were mainly monosynaptic responses. In contrast, the onset of IPSCs always followed the onset of EPSCs, with the mean delay between the onsets of EPSCs and IPSCs of 1.3 ± 0.08 ms (n = 12) (Fig. 1B3). Together with the CNQX sensitivity of IPSCs, these observations suggested that IPSCs were mediated by a disynaptic response, i.e., by inhibitory interneurons excited by excitatory thalamocortical inputs, forming a circuit of feedforward inhibition. The observation that layer IV pyramidal neurons receive strong feedforward inhibition was consistent with the previous reports (Agmon and Connors, 1992; Porter et al., 2001).

Figure 1C shows the relationship between the conductance values for EPSCs and IPSCs obtained from individual cells, in which the excitatory conductance ranged from 1 to ∼8 nS in both wt and tg mice. The inhibitory conductance in wt became larger with increasing excitatory conductance, although the conductance values were quite variable. In contrast, the inhibitory conductance in tg mice was very small, even with a large excitatory conductance. The ratio of inhibitory conductance to excitatory conductance was significantly reduced in tg mice (2.34 ± 0.60 for wt, n = 12; 0.46 ± 0.06 for tg, n = 11; **p < 0.01, t test). These results demonstrated that feedforward IPSCs disynaptically evoked by thalamic stimulation were disproportionately decreased in layer IV pyramidal cells of tg mice.

Locally evoked IPSCs and EPSCs

Thalamocortical responses recorded in layer IV pyramidal cells were composed of at least two components of monosynaptic EPSCs and disynaptic IPSCs. The lags of the synaptic onsets between two types of responses were short. Pharmacological blockade of EPSCs also abolished IPSCs. These facts made it difficult to evaluate synaptic currents quantitatively, especially IPSCs. To circumvent this problem, we measured IPSCs and EPSCs evoked by local stimulation within the somatosensory cortex. The stimulating electrode was placed at ∼100 μm from the soma of the recorded pyramidal cells. IPSCs were recorded in the presence of CNQX (10 μm) and APV (100 μm), and EPSCs were recorded in the presence of bicuculline (10 μm). For measuring IPSCs, a pipette solution with a high chloride concentration was used so that IPSCs were observed as inward currents at a holding potential of −60 mV. IPSCs were blocked by bicuculline in both wt and tg mice (data not shown).

Local stimulation easily evoked large IPSCs in wt mice at P21–P30 (1079 ± 227 pA; n = 10, 1.0 mA stimulation) (Fig. 2A). In contrast, the IPSC amplitudes evoked by local stimulation were drastically reduced in tg mice at P21–P30 (145 ± 33 pA; n = 10, 1.0 mA stimulation). Statistical test using two-way repeated-measures ANOVA revealed a significant effect of the genotype (F_(1,18) = 12.288; p < 0.004) on IPSC amplitude. Additionally, there was a statistically significant interaction between stimulus intensity and genotype (F_(4,72) = 7.484; p < 0.001). Post hoc analyses using the Bonferroni’s test revealed that the IPSC amplitudes were significantly smaller in tg than wt mice at stimulus intensities ≥0.8 mA [*p < 0.05, **p < 0.01 (Fig. 2A)]. Conversely, the EPSC amplitudes evoked by local stimulation increased with increment of stimulus intensity. Although EPSC amplitude appeared smaller in tg than wt mice (554 ± 96 pA, n = 10 in wt; 368 ± 88 pA, n = 10 in tg; 1.0 mA stimulation), two-way repeated-measures ANOVA failed to show a significant effect of the genotype (F_(1,18) = 3.907; p > 0.06) (Fig. 2B).

• Download figure
• Open in new tab
• Download powerpoint

Figure 2.

Impaired IPSCs of layer IV pyramidal cells in tg mice (P21–P30). IPSCs and EPSCs were evoked by local stimulation in layer IV pyramidal cells in wt and tg mice at P21–P30. A, IPSCs were recorded in the presence of CNQX (10 μm) and APV (100 μm). The insets show IPSC traces evoked by 1.0 mA stimulation. The IPSC peak amplitude was plotted against the intensity of stimulation for wt and tg mice. The IPSC amplitude was significantly smaller in tg mice, and two-way repeated-measures ANOVA revealed a significant effect of the genotype (p < 0.004). B, EPSCs were recorded in the presence of bicuculline (10 μm). The insets show EPSC traces evoked by 1.0 mA stimulation. The EPSC amplitudes increased with increment of stimulus intensity in both wt and tg mice, and two-way repeated-measures ANOVA failed to show a significant effect of the genotype (p > 0.06). *p < 0.05 and **p < 0.01 indicate significant differences between wt and tg mice at each stimulus intensity, by multiple comparisons using Bonferroni’s test (no asterisks indicate no significant differences).

To see whether a similar synaptic impairment was observed in other primary sensory cortical areas, we also examined the primary auditory cortex. We recorded IPSCs of pyramidal cells evoked by local stimulation as used in the somatosensory cortex. In both wt (n = 10) and tg (n = 10) mice, locally evoked IPSC amplitudes increased with increments of stimulus intensity. Two-way repeated-measures ANOVA revealed no significant effect of the genotype on layer IV IPSC amplitudes in the primary auditory cortex (F_(1,18) = 0.249; p > 0.6) (supplemental Fig. 1, available at www.jneurosci.org as supplemental material).

Miniature IPSCs in layer IV pyramidal neurons

The responses evoked by local stimulation demonstrated that inhibitory synaptic transmission onto layer IV pyramidal neurons in the somatosensory cortex was drastically impaired in epileptic tg mice. The site of the impairment could be presynaptic or postsynaptic. One possibility was that neurotransmitter release from presynaptic terminals was not functional in tg mice because of the mutation of the Ca_V2.1 channel, although, in a normal condition, local stimulation should evoke neurotransmitter release at a high probability from the synaptic terminals of the thalamocortical projection. The other possibility was that postsynaptic GABA_A receptors were reduced in number or their functional properties were altered in tg mice. To examine the two possibilities, mIPSCs were recorded in the presence of CNQX, APV, and TTX (1 μm) (Fig. 3A). TTX was used to block voltage-dependent Na⁺ channel so that spontaneous firings of neuronal populations were suppressed. The mIPSC amplitude (36.6 ± 0.6 pA, n = 7 in wt; 33.0 ± 0.5 pA, n = 7 in tg) and IEI (17.8 ± 0.5 ms, n = 7 in wt; 21.2 ± 0.5 ms, n = 7 in tg) appeared similar between tg and wt mice. However, a statistical test of the cumulative probability distributions (Fig. 3B,C) showed a significant shift toward smaller amplitudes (Kolmogorov–Smirnov test, p = 0.002) and a significant shift toward longer IEIs (p = 0.001) in tg mice. These results suggested that, although there are slight alterations on the presynaptic (prolonged IEIs) or postsynaptic (reduced mIPSC amplitude) side, these changes could not account for the greatly reduced evoked IPSCs in layer IV pyramidal cells of tg mice.

• Download figure
• Open in new tab
• Download powerpoint

Figure 3.

mIPSCs in layer IV pyramidal cells in wt and tg mice (P21–P27). mIPSCs were recoded in the presence of TTX (1 μm). A, Traces of mIPSCs recorded in layer IV pyramidal cells of wt (top) and tg (bottom) mice. Holding potential −60 mV. B, C, Cumulative distributions of the mIPSC peak amplitude (B) and IEI (C) of wt and tg mice. Number of events was 2800 for each.

Number of inhibitory neurons

Another possibility to cause the markedly reduced IPSCs in layer IV was that the number of inhibitory interneurons was reduced in layer IV of tg mice. To test this possibility, distribution of inhibitory neurons was analyzed by the immunohistochemical method using polyclonal antibody against GAD (McLaughlin et al., 1975).

There was no obvious difference in the distribution of GAD-immunoreactive cells in the barrel cortex between wt and tg mice (Fig. 4). The density of GAD-immunoreactive cells in layer IV showed no statistical difference between wt mice (184 ± 26 cells/mm²; n = 5) and tg mice (192 ± 24 cells/mm²; n = 5; p > 0.05, t test). Cell bodies of large, presumable pyramidal, neurons in layer IV in both wt and tg mice were similarly surrounded by immunoreactive GABAergic terminals (Fig. 4, insets, arrows). Furthermore, tg mice grow without epilepsy until 3 weeks of age, suggesting that inhibitory neurons and their connections develop normally (see below). Putting all these results together, we concluded that the impaired IPSCs in tg mice was caused by functional impairments of neurotransmitter release from the presynaptic nerve terminals.

• Download figure
• Open in new tab
• Download powerpoint

Figure 4.

Distribution of inhibitory neurons and their axon terminals in the barrel cortex. Distribution of inhibitory neurons and their axons in the layer IV of the barrel cortex was studied by an immunohistochemical method using anti-GAD antibody. GAD-immunoreactive neurons in wt and tg mice (P21–P24). There was no obvious difference in distribution of GAD-immunoreactive neurons between wt and tg mice. Arrows point to GAD-immunoreactive neurons. Scale bars, 200 μm. Insets show that punctately stained GAD-immunoreactive axon terminals were observed around somata of large cells (indicated by the arrows). Scale bars, 50 μm.

Layer-specific impairment of IPSCs

Thalamocortical afferents form synapses not only with neuronal elements in layer IV including pyramidal cells (White and Hersch, 1981) but also with corticothalamic pyramidal cells, which are located in lower layer V and upper layer VI and project to the ventrobasal complex of the thalamus (White and Hersch, 1982; White and Keller, 1987). To test whether the impairment of IPSCs was dependent on the laminar position in the somatosensory cortex, locally evoked IPSCs and EPSCs were recorded in layer V pyramidal cells. Different from the layer IV neurons, the reduction in IPSC amplitude was not observed in layer V pyramidal cells in tg compared with that of wt mice. IPSC amplitudes with 1.0 mA stimulation were 999 ± 389 pA (n = 10) in wt and 1179 ± 312 pA (n = 10) in tg mice. Two-way repeated-measures ANOVA revealed no significant effect of the genotype (F_(1,18) = 0.236; p > 0.6) (Fig. 5A) on layer V IPSC amplitudes.

• Download figure
• Open in new tab
• Download powerpoint

Figure 5.

IPSCs recorded in layer V pyramidal cells of wt and tg mice (P21–P30). IPSCs and EPSCs evoked by local stimulation were recorded in layer V pyramidal cells of wt and tg mice. A, The IPSC peak amplitude was increased with increments of the stimulus intensity in wt and tg mice. The insets show IPSC traces by 1.0 mA stimulation. B, The EPSC amplitude in tg mice was reduced. The insets show IPSC traces by 10 mA stimulation.

Conversely, the EPSC amplitude was reduced in layer V pyramidal cells of tg mice (392 pA ± 34 pA; n = 10, 10 mA stimulation) compared with that of wt mice (615 ± 83 pA; n = 10, 10 mA stimulation). Two-way repeated-measures ANOVA showed a significant effect of the genotype (F_(1,18) = 6.100; p < 0.03) (Fig. 5B), but post hoc analyses did not show statistical differences between wt and tg mice at any stimulus intensities.

Developmental changes in IPSCs

Homozygous tg mice develop absence seizures at ∼3 weeks of age (Noebels, 1984). If the impairment of IPSCs contributes to the epileptogenesis, the IPSCs may show a related developmental change. To examine this prediction, the developmental effect on the synaptic transmission was studied by comparing nonepileptic young (P14–P16) and epileptic adult (P21–P30) tg mice.

In younger mice, the amplitudes of locally evoked IPSCs in layer IV and layer V pyramidal cells were not significantly different between wt and tg mice [layer IV, F_(1,18) = 0.351, p > 0.5 (Fig. 6A); layer V, F_(1,18) = 1.973, p > 0.1 (Fig. 6B)]. Moreover, the evoked EPSC amplitudes in layer IV and layer V were not significantly different in P14–P16 mice [layer IV, F_(1,18) = 0.653, p > 0.4 (Fig. 6C); layer V, F_(1,18) = 0.579, p > 0.4 (Fig. 6D)]. The IPSC and EPSC amplitudes evoked by maximum stimulation of P14–P16 and P21–P30 mice were summarized in Figure 6E. In tg mice, the EPSC amplitudes was smaller, but the reduction was not as remarkable as layer IV IPSC reduction at P21–P30.

• Download figure
• Open in new tab
• Download powerpoint

Figure 6.

IPSCs and EPSCs in layer IV and V pyramidal cells of younger wt and tg mice (P14–P16). A–D, The IPSC and EPSC peak amplitudes of layer IV (A, C) and V (B, D) were plotted against the intensity of stimulation for wt and tg mice at P14–P16. In both layers, the IPSC and EPSC peak amplitudes increased with increments of stimulus intensity in wt and tg mice. The insets show IPSC and EPSC traces by 1.0 mA stimulation. E, Averaged IPSC and EPSC peak amplitudes evoked by 1.0 mA stimulation in P14–P16 (left) and P21–P30 (right) mice. All data are represented as mean ± SEM for n = 10. **p < 0.01, Bonferroni’s test.

During the development period from P14–P16 to P21–P30, the IPSC amplitudes of tg mice dramatically diminished in the layer IV pyramidal cells (Fig. 6E). The IPSC amplitudes in layer V did not show such a developmental change (Fig. 6E). These results indicated that the impairment of IPSCs in layer IV was well correlated with the onset of absence seizure.

Developmental switching of Ca²⁺ channel subtypes

The previous studies demonstrated that Ca²⁺ channels involved in neurotransmitter release switch developmentally from the Ca_V2.2 (N-type) to Ca_V2.1 (P/Q-type) Ca²⁺ channels at various mammalian fast synapses (Iwasaki et al., 2000). Although the contribution of the Ca_V2.2 channel to EPSCs in layer IV pyramidal cells of the visual cortex was reported not to change developmentally (Iwasaki et al., 2000), developmental changes in Ca²⁺ channel subtypes of cortical IPSCs were not well investigated. Because the developmental switch usually occurs at ∼2 weeks of age, it was expected that the switching from Ca_V2.2 to Ca_V2.1 Ca²⁺ channel might lead to the impairment of IPSCs in tg mice.

Developmental changes in sensitivity of IPSCs to the Ca_V2.1 channel-selective blocker ω-Aga-IVA (200 nm) and the Ca_V2.2 channel-selective blocker ω-CTx-GVIA (3 μm) were studied in layer IV pyramidal cells of wt mice. At P14–P15, application of ω-Aga-IVA only weakly blocked IPSCs (Fig. 7A, left). Subsequent coapplication of ω-CTx-GVIA almost completely blocked the remaining IPSCs. Conversely, at P21–P22, the IPSCs were more strongly reduced by application of ω-Aga-IVA (Fig. 7B, left). The ω-Aga-IVA-sensitive fraction of IPSCs was increased from 20 ± 4% at P14–P15 (n = 7) to 45 ± 3% at P21–P22 (n = 7) (**p < 0.01, t test). In tg mice, however, the developmental change in sensitivity of IPSCs to ω-Aga-IVA was not as evident as in wt (25 ± 4% at P14–P15, n = 3; 18 ± 7% at P21–P22, n = 3; p = 0.43) (Fig. 7A,B, right). These results demonstrated that, whereas the Ca_V2.1 channel plays a minor role in young mice, it plays a predominant role in wt adult mice but not in tg adult mice for IPSCs in layer IV pyramidal cells of the somatosensory cortex.

• Download figure
• Open in new tab
• Download powerpoint

Figure 7.

Developmental changes in ω-Aga-IVA sensitivity of IPSCs in layer IV pyramidal cells. A, B, Time course of the peak IPSC amplitude in response to application of ω-Aga-IVA (200 nm; gray bar) and additional ω-CTx-GVIA (3 μm; white bar). The insets show IPSC traces at the time points indicated by the numbers. ω-Aga-IVA reduced the IPSC amplitude by 20% at P14–P15 (A, left). ω-Aga-IVA reduced the IPSCs amplitude by 45%, and additional ω-CTx-GVIA almost completely blocked IPSCs at P21–P22 (B, left). However, the developmental change of IPSC in ω-Aga-IVA sensitivity was not observed in tg mice (A, B, right).

Thalamocortical PSPs in wt and tg mice

We finally examined how the IPSC impairment in tg mice affects excitability in layer IV pyramidal cells using current-clamp recording (Fig. 8). Mean resting membrane potentials of layer IV pyramidal cells were similar in wt mice (−69.4 ± 1.0 mV; n = 16) and tg mice (−69.6 ± 0.8 mV; n = 14; p > 0.05). The intrinsic membrane properties were then examined using negative current injection (−100 pA, 500 ms duration). There were no significant differences between wt and tg mice in membrane input resistance (157 ± 5 MΩ, n = 16 in wt; 172 ± 9 MΩ, n = 14 in tg; p > 0.05) and membrane time constant (13.9 ± 0.8 ms, n = 16 in wt; 12.9 ± 0.9 ms, n = 14 in tg; p > 0.05) of layer IV pyramidal cells.

• Download figure
• Open in new tab
• Download powerpoint

Figure 8.

Postsynaptic potentials in layer IV pyramidal cells in response to thalamic stimulation. A, Typical responses of the thalamocortical PSPs in layer IV pyramidal cells of wt (left) and tg (right) mice, with increments of the stimulus intensity. B, Vertical expansion of the thalamocortical PSPs with the 400 μA stimulus intensity, which are identical to those in A. Note that the thalamic stimulation with 400 μA intensity evoked transient depolarization followed by long-lasting hyperpolarization in wt mice (top) but long-lasting depolarization in tg mice (bottom). The early transient depolarization is indicated by Peak, whereas the late long-lasting hyperpolarization and depolarization are indicated by arrowheads. The membrane potentials before the stimulation were set to −60 mV by current injection. Stimulus artifacts were truncated. C, Time course profiles of thalamocortical PSPs with 400 μA stimulus intensity in wt and tg mice. PSP values were calculated as membrane potentials at each time point after stimulation minus those just before stimulation. Peak amplitudes of the early transient depolarization (indicated by Peak) were searched from 2 to 7 ms after stimulation in individual preparations. The time course profiles were significantly different between wt and tg mice (p < 0.02, two-way repeated-measures ANOVA). *p < 0.05, **p < 0.01, Bonferroni’s test between genotype at each time point. D, Stimulus intensity profiles of thalamocortical PSPs at 40 ms after stimulation. The stimulus intensity profiles were significantly different between wt and tg mice (p < 0.03, two-way repeated-measures ANOVA). *p < 0.05, **p < 0.01, Bonferroni’s test between genotypes at each stimulus intensity.

We then examined differences in thalamocortical PSPs in layer IV pyramidal cells of wt and tg mice (P21–P30) (Fig. 8A,B). Thalamic stimulation with weak stimulus intensity evoked just EPSPs in layer IV pyramidal cells of both wt and tg mice (50 μA in Fig. 8A), which was reported previously in wt mice (Beierlein et al., 2003). In wt mice, stronger thalamic stimulation evoked transient (∼1 ms) depolarization, followed by long-lasting hyperpolarization in layer IV pyramidal cells (400 μA in Fig. 8A, left, B, top) because of recruitment of thalamocortical feedforward IPSCs (Agmon and Connors, 1992; Porter et al., 2001). In contrast to wt mice, we found that 400 μA stimulation evoked transient depolarization followed by small but long-lasting depolarization in layer IV pyramidal cells of tg mice (Fig. 8A, right, B, bottom).

The differences in thalamocortical PSPs were then quantified between wt and tg mice. The EPSP amplitudes with 50 μA stimulation were not different (4.11 ± 0.90 mV, n = 10 in wt; 3.69 ± 0.52 mV, n = 10 in tg; p > 0.05, t test). Amplitudes of the early transient depolarization with 400 μA stimulus intensity were also not different (2.76 ± 0.99 mV, n = 10 in wt; 2.50 ± 0.43 mV, n = 10 in tg; p > 0.05) (Fig. 8C, Peak). However, thalamic stimulation with 400 μA intensity evoked long-lasting hyperpolarization in wt (−1.91 ± 0.84 mV at 40 ms after stimulation; n = 10) (Fig. 8C) but long-lasting depolarization in tg mice (1.16 ± 0.66 mV at 40 ms after stimulation; n = 10) (Fig. 8C). Two-way repeated-measures ANOVA revealed a remarkable difference in time course profiles of the thalamocortical PSPs with 400 μA intensity (F_(1,18) = 7.679; p < 0.02) (Fig. 8C). Also, there was a significant difference in stimulus intensity profiles of the thalamocortical PSPs at 40 ms after stimulation, revealed by two-way repeated-measures ANOVA (F_(1,18) = 5.515; p < 0.03) (Fig. 8D). These results indicated that thalamic inputs preferentially induce long-lasting depolarization in layer IV pyramidal cells of tg mice.

Layer IV inhibitory synapses dysfunction in epileptic tg mice

Our principal finding of this study was a significant reduction (87%) in the amplitude of locally evoked IPSCs, but not that of EPSCs, in layer IV pyramidal cells located in the somatosensory cortex of epileptic tg mice. Similar results were obtained with the thalamocortical responses.

To identify the mechanism of the IPSC amplitude reduction, we made several experiments and obtained the following results. (1) GAD-immunoreactive inhibitory neurons were not decreased in number in layer IV of tg mice, and GAD-immunoreactive nerve terminals were well observed around presumable pyramidal cells. (2) mIPSCs could be recorded in layer IV pyramidal cells of tg mice. Although the amplitude was smaller and interevent interval was prolonged in tg mice, these changes were relatively mild. These results suggested that the postsynaptic apparatus was, at least, functional. (3) IPSCs once normally developed in younger mice, but IPSCs became impaired in later developmental stages, when the inhibitory synaptic transmission to layer IV pyramidal cells became more dependent on the Ca_V2.1 channel. All of these results suggest that significant impairment of inhibitory synaptic transmission in layer IV pyramidal neurons was not caused by failure of wiring or synapse formation but was caused primarily by dysfunction of neurotransmitter release from presynaptic terminals. Our results, however, do not exclude involvement of secondary effects of the mutation in the pathogenesis of absence seizures, because imbalance of excitation and inhibition could lead indirectly to subtle developmental rearrangements in the number or placement of synaptic inputs.

Layer-specific dysfunction of synaptic transmission in tg mice

Because the Ca_V2.1 channel is the predominant Ca²⁺ channel type in the brain, it was anticipated that some parts of the cerebral cortex were impaired in tg mice. In fact, a cortical microdialysis assay showed severely attenuated releases of both glutamate and GABA evoked by high K⁺ solution (Ayata et al., 2000). In the present study, our analyses at a finer level revealed that synaptic dysfunctions are relatively confined to specific neuronal populations. The reduction in IPSCs was prominent in layer IV pyramidal cells, but it was not observed in layer V pyramidal cells in tg mice. The EPSC amplitude in layer V pyramidal cells was also moderately reduced.

Types and functions of layer IV interneurons

The organization of neocortical circuitry is considerably complicated (for review, see Douglas and Martin, 2004). A simplified diagram shows that layer IV neurons are a main gateway receiving inputs from the thalamus, although they receive strong inputs from neurons in layer VI and other layers. Inhibitory interneurons in layers IV that are activated by the thalamocortical projections are morphologically heterogeneous in terms of their dendrites and axons (Porter et al., 2001). The inhibitory interneurons are often grouped into two types based on the patterns of the intrinsic firing, which are the fast-spiking (FS) cells and the low-threshold-spiking (LTS) cells (Gibson et al., 1999; Beierlein et al., 2000; Amitai et al., 2002). Inputs from thalamocortical relay cells are more selective for FS cells and frequently and strongly excite them (Agmon and Connors, 1991; Porter et al., 2001; Beierlein et al., 2002), whereas the thalamocortical inputs only rarely and weakly excite LTS cells (Gibson et al., 1999). Thus, FS neurons play a dominant role in feedforward inhibition of the thalamocortical projection, and this feedforward inhibition is defective in tg mice.

Development-dependent dysfunction of inhibitory synapses

Absence seizure in tg mice begins at ∼3 weeks of age (Noebels and Sidman, 1979). Inhibitory synaptic transmission in layer IV is intact in nonepileptic young tg mice but becomes impaired later. These findings imply a relationship between onset of absence epilepsy and impairment of inhibitory synaptic transmission. The Ca²⁺ channel involved in neurotransmitter release often switches developmentally from the Ca_V2.2 (N-type) channel to the Ca_V2.1 (P/Q-type) channel at various mammalian fast synapses (Iwasaki et al., 2000). In the present work, the sensitivity of inhibitory synaptic transmission to ω-Aga-IVA increased developmentally in layer IV, indicating the subtype switching. Together, we speculate that the neurological phenotype becomes overt at ∼3 weeks through the subtype switching, which makes some groups of synapses more dependent on the Ca_V2.1 channel. However, we do not think that subtype switching alone can explain such drastically impaired inhibitory synaptic transmission in the cortical layer IV. Whereas the Ca_V2.1-dependent component of the inhibitory synaptic transmission was ∼45% in adult wt mice, the IPSC amplitude in layer IV pyramidal cells was more severely reduced in tg mice (down to 13%). Other mechanisms, including various secondary effects, also must contribute to the drastic reduction.

Compensation of Ca²⁺ channel subtypes in tg mice

For the phenotypic effect of the tg mutation, two factors seem to be involved: predominance of the Ca_V2.1 channel and compensation by other subtypes. If the Ca_V2.1 channel is a trivial component, the mutation will not affect the function. If the compensation is adequate, mutational effects will not appear. Compensatory effects of other Ca²⁺ channel subtypes have been investigated in other synapses of tg mice. The hippocampal CA3–CA1 synapses are maintained because of compensatory upregulation of Ca_V2.2 (Qian and Noebels, 2000). A similar situation is observed in the neuromuscular junction (Plomp et al., 2000). Conversely, cerebellar Purkinje cells lack an ability of compensation. Our preliminary observation showed a drastic reduction in IPSC amplitude measured in deep cerebellar nucleus neurons evoked by white matter stimulation (presumably Purkinje axons) (Matsushita and Imoto, 2002). In the present work, we do not know whether the compensatory upregulation of Ca_V2.2 occurred in layer IV inhibitory neurons, but it is evident that the upregulation, if any, was insufficient.

These lines of evidence imply that different neuronal types have various capacity of compensatory upregulation of other Ca²⁺ channel subtypes in response to the Ca_V2.1 channel impairment, and such a variation may underlie selective vulnerability of certain parts of neuronal networks. Molecular determinants of the compensatory upregulation will be an interesting subject for future studies.

Functional implications

Absence epilepsy is characterized by SWDs in wide cortical areas, whose underlying mechanism involves the thalamocortical circuitry (for review, see Manning et al., 2003). To date, synaptic and intrinsic defects in the thalamocortical circuitry have been extensively studied in tg mice. In the thalamus of tg mice, there are several studies focusing on the role in the generation of the SWDs of low-voltage-activated T-type Ca²⁺ currents in thalamic relay neurons (Zhang et al., 2002; Song et al., 2004). Defects of synaptic transmission were also reported in the thalamus of tg mice (Caddick et al., 1999). In the present study, we found a significant impairment of feedforward inhibitory connection from the thalamus to the input layer of the somatosensory cortex.

The thalamocortical feedforward inhibition is an important determinant of excitability to cortical layer IV neurons. As shown in Figure 8B, single thalamic stimulation induced transient (∼1 ms) depolarization in layer IV pyramidal cells of wt mice, whereas it induced long-lasting depolarization in layer IV pyramidal cells of tg mice, because of the impairment of the feedforward inhibition. The longer depolarization in tg mice means that thalamic inputs tend to produce bursting activity in layer IV pyramidal cells. Thus, these results imply that synchronous bursts in thalamic relay cells, observed in vivo, may be readily transformed into synchronous bursts in layer IV pyramidal cells of tg mice, which could be partially reflected as SWDs in the EEG of the somatosensory cortex.

We found that IPSC impairment was observed in the somatosensory cortex but not in the primary auditory cortex (supplemental Fig. 1, available at www.jneurosci.org as supplemental material). Interestingly, a previous study reported that SWDs first originate in the somatosensory cortex and then rapidly spread to other regions of the cortex in the WAG/Rij rat model of absence seizures (Meeren et al., 2002).

In summary, we demonstrated that the effect of the tg mutation was variable depending on neuronal cell types and developmental stages, and that the feedforward inhibition was severely impaired in the somatosensory thalamocortical pathway. Because the tg phenotype is expected to arise from a complex pattern of altered transmitter release in the cerebral cortex, the thalamus, and other brain regions, further search for synaptic defects is necessary particularly in synaptic connections directly related to the thalamocortical interplay to understand the pathophysiology of absence epilepsy.