By the use of modern in vivo single-cell anatomo-electrophysiological methods, this study has unravelled novel thalamic cellular and network mechanisms underlying the generation of SWD in a well recognized genetic model of absence epilepsy. The main observations are as follows. First, in the somatosensory system, rhythmic TC and RTN AP discharges occur in synchrony during generalized SWD. Second, layer VI CT rhythmic firing usually phase-leads related thalamic, relay and reticular, discharges. Third, both discharges are a consequence of a depolarizing wave beginning by a barrage of EPSPs, which is very likely induced by layer VI CT neurones. Fourth, in TC cells displaying a hyperpolarization-activated ramp-shaped depolarization during the application of hyperpolarizing current pulses, the EPSP barrage coincides with a post-inhibitory ramp-shaped depolarization, which can trigger an apparent low-threshold Ca2+ spike. Fifth, in RTN cells the EPSP barrage can further trigger apparent voltage-dependent depolarizations. Sixth, TC and RTN neurones in both GAERS and NE rats have similar passive membrane properties, and epilepsy-related intracellular events are basically similar to those correlated with the natural 5–9 Hz rhythm.
Using in vivo paired single-cell recordings to define the time relationships between somatosensory-related cortical and thalamic regions is a challenging problem, for at least two reasons: (1) whether the two neurones of any recorded pair belong to functionally connected cortical and thalamic zones, and (2) the electrical behaviour of any single neurone is not always representative of the activity of the cellular population to which it belongs.
The absence of cisternal drainage and the performance of minute craniotomies-duratomies allow for reliable single-cell anatomo-electrophysiological investigation of living intact brain networks (see Methods). It prevents significant outflow of the cerebrospinal fluid, keeping the brain volume constant in the cranial cavity, preventing tissue slump, and avoiding swelling and oedema. These surgical conditions permit great precision recordings from intracerebral stereotaxic targets (±200 μm) and the obtaining of homogeneous cellular samples.
Although control experiments strongly indicate that my paired recordings were conducted in functionally connected thalamic and cortical regions (see Methods), the corresponding neurones were always located approximately in the same region of the somatosensory system (centred at the forepaw area) and were not systematically in register in a point-to-point manner. Moreover, the electrophysiological (spike-triggered averaging) and anatomical cellular data provide no evidence that the two neurones of any pair were in direct synaptic connection, even when they had approximately the same receptive field. In spite of this, during paired cortical and thalamic recordings, the thalamic micropipette could be moved up or down, within a range of ±350 μm, while keeping the location of the intracortical micropipette stable, without revealing significant variations in the temporal relationships between cortical and thalamic firings. However, corticothalamic cells have a more variable temporal relation with the ECoG SW complex than that shown by related thalamic neurones. That is why multi-unit recordings were used to corroborate in compelling fashion that the rhythmic firing of layer VI cells indeed precedes related thalamic AP discharges.
Relay cellular events during normal and epileptic 5–9 Hz oscillations
The intracellular recordings of the present study reveal that any TC depolarizing wave always includes an EPSP barrage, which can concur with apparent intrinsic depolarizations. The recurrent EPSP barrage might primarily be generated by CT cellular discharges. Indeed, the paired TC-CT recordings of the present study show that epilepsy-related CT firing usually phase-leads at the millisecond scale the corresponding thalamic discharges. Under the experimental conditions of this study, spontaneous or induced prethalamic (especially lemniscal) EPSPs can occur without altering the time course of the TC rhythmic activity. Furthermore, rhythmic EPSP barrages obviously occur during the earliest stage of the normal or epileptic oscillation and precede the occurrence of apparent intrinsic activities (see below). The recordings further reveal that, in the midst of the steady hyperpolarization, the intrinsic Ca2+ spike and the EPSP barrage can concur at some cycles but as a rule the barrage occurs systematically earlier than the low-threshold Ca2+ spike.
Importantly, we have identified a cellular type having the ability to generate a post-hyperpolarization ramp-shaped depolarization. It increases in amplitude with increasing hyperpolarization, resembling the depolarizing sag underlain by a H-current (McCormick & Pape, 1990; Soltesz et al. 1991). In agreement with previous in vitro (Turner et al. 1997) and in vivo (Pinault et al. 1998) observations, the present findings thus strongly suggest that the somatosensory thalamus contains two distinct TC types. Furthermore, the one that displays the sag has an average input resistance higher than the one that does not. However, because in in vivo conditions both the activities of the synaptic inputs and the content of the extracellular space are not under the experimenter's control, one may not exclude the existence of a continuum between these two apparently distinct types. Indeed, whether recorded TC cells that do not apparently exhibit a depolarizing sag in the trough of a current-induced hyperpolarization do not have functional H-channels is a question that merits further investigation. The hyperpolarization-activated cation conductance is well known to contribute at least to the determination of the resting membrane potential and to the generation of intrinsic pacemaker activities (see reviews by Pape, 1996 and Lüthi & McCormick, 1998).
The present work further demonstrates that both cellular types operate during normal and epileptic 5–9 Hz oscillations. The TC cells showing an apparent depolarizing sag have a relatively high membrane input resistance, making them more excitable than those that do not display the sag. It is shown that the post-hyperpolarization ramp-shaped depolarization usually develops shortly after the occurrence of the first few cycles of rhythmic EPSP barrages. Furthermore, this rebound depolarization coincides with such a barrage and can seemingly trigger or facilitate the triggering of an AP discharge. Assuming that the depolarizing sag reflects an H-current, it is expected to contribute to the TC oscillations by enhancing the rate of repolarization of the membrane during the summation of CT-induced EPSPs. Of course, the contribution of such intrinsic rebound depolarization depends in particular on the depth of the hyperpolarization. Knowing that the recurrent EPSP barrage does not systematically reach the AP threshold (see also Pinault et al. 1998), the hyperpolarization-activated ramp-shaped depolarization should increase the probability of reaching the firing threshold on each cycle of the rhythmic activity, probably by facilitating the generation of a low-threshold Ca2+ spike. Thereby, the post-hyperpolarization rebound depolarization ending with a low-threshold Ca2+ spike topped by an AP discharge might emerge like a resonant phenomenon.
The present study also reveals that, during the natural and epileptic 5–9 Hz oscillations, the hyperpolarizing wave that follows the depolarizing wave mainly includes a barrage of GABAA IPSPs triggered by RTN cells. This means that during such oscillations RTN cells, which are well known to shape TC activities (Kim et al. 1997), should effectively curtail the depolarizing waves through the activation of GABAA receptors. Such a powerful inhibitory mechanism would explain why TC cells do not systematically exhibit a high-frequency AP burst on each cycle of the SWD (see also Pinault et al. 1998). Thereby, RTN cells might further prevent the development in TC neurones of a NMDA-dependent depolarization similar to that identified in vivo in cat TC neurones after lesion of the RTN (Deschênes & Hu, 1990). On the other hand, RTN cells might strongly activate H-channels in their target TC neurones (Lüthi & McCormick, 1998).
The TC intracellular recordings of the present study are somehow different from those obtained in the ketamine- xylazine anaesthetized cat during SWD (Steriade & Contreras, 1995). Indeed, these latter recordings revealed two subpopulations of TC neurones, one with units discharging synchronous robust AP bursts (60 %), the other with tonically inhibited cells (40 %). In the present recording sample, most TC cells occasionally emit a robust AP burst during every cycle of the rhythmic activity (see also Pinault et al. 1998). This indicates that in the rat, large populations of TC cells fire in synchrony during every cycle of absence-related SWD. The difference observed between feline and rodent SWD-related TC activities may simply be accounted for by the fact that, in contrast to what is observed in the rat somatosensory thalamus (see Introduction), a significant number of interneurones exist in the feline thalamus (Jones, 1985). Also, it should be highlighted that feline SWD develop during ketamine- xylazine anaesthesia from sleep-related oscillations (Steriade et al. 1994; Steriade & Contreras, 1995) whereas rodent absence-related SWD principally emerge from wake-related oscillations (Semba & Komisaruk, 1984; Danober et al. 1998; Pinault et al. 2001), and similar absence seizures likewise occur in a spontaneous manner under neuroleptanalgesia (Inoue et al. 1993; Seidenbecher et al. 1998; Pinault et al. 2001).
In conclusion, during normal and epileptic 5–9 Hz oscillations most if not all of the TC neurones recorded from in this study exhibit at least a recurrent EPSP barrage-IPSP barrage sequence, which can lead to an AP discharge (Fig. 12). In relay cells that display a presumed H-current, the corresponding depolarizing wave is amplified by a post-hyperpolarization rebound depolarization, which probably includes a low-threshold Ca2+ spike. In those that do not, the depolarizing wave can trigger only such an intrinsic spike, very probably in the midst of the 5–9 Hz oscillations where cellular synchronization and hyperpolarization are maximal.
Figure 12. Summary of the reported data
Schematic diagram showing likely spatio-temporal cellular interactions within the TC system occurring during natural medium voltage 5–9 Hz oscillations in control non-epileptic rats (A) and during SWD in GAERS (B). In both NE rats and GAERS, at least two types of TC neurones coexist, of which one (TC2) is endowed with a presumed H-current. Note that thalamic, relay and reticular, discharges occur in much more synchronous and phase-locked manners during SWD than during natural 5–9 Hz oscillations. C, a mounting of SW-related extracellular CT and intracellular RTN and TC activities. From top to bottom: a SW complex (ECoG), an extracellular CT discharge, an intracellular RTN discharge, and two typical intracellular TC discharges. The second TC cell (TC2) exhibits a presumed H-current, coinciding with an EPSP barrage. The ramp-shaped depolarization, which includes a presumed Ih, can trigger a low-threshold Ca2+ spike (LTS). In RTN cells, the EPSP barrage can trigger voltage-dependent components (V-components). D, schematic drawing of the anatomical relationships between the three main elements that make up the TC system.
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Reticular cellular events during normal and epileptic 5–9 Hz oscillations
The intracellular recordings presented reveal that in RTN cells the recurrent, threshold or subthreshold, depolarizing wave includes an initial EPSP barrage, which can trigger apparent intrinsic depolarizing events. Again, the initial EPSP barrage might be generated by CT inputs. Indeed, individual EPSPs have a time course similar to individual EPSPs recorded in TC neurones during the same SW-related period. Furthermore, the paired recordings carried out show that epilepsy-related CT discharges usually occur a few milliseconds before the corresponding reticular discharges. It is worth mentioning that the recurrent EPSP barrage is often recorded during the earliest stage of the 5–9 Hz oscillations. Although the dual single-unit recordings reveal that TC and RTN rhythmic discharges occur in synchrony, especially during SWD, TC neurones may contribute to the triggering of RTN EPSP barrages, as demonstrated in a thalamic slice preparation (Bal et al. 1995).
A recent in vivo intracellular study has suggested that the recurrent depolarizing wave is mainly accounted for by a low-threshold Ca2+ potential brought about by the summation of depolarizing potentials (Slaght et al. 2002). The present intracellular recordings with QX-filled micropipettes strongly suggest that the prominent depolarization may include other components. On the basis of the known intrinsic properties of RTN cells (e.g. Mulle et al. 1986; Avanzini et al. 1989) and the plural effects of intracellularly injected QX-314 molecules (Andrade, 1991; Perkins & Wong, 1995; Talbot and Sayer, 1996), the low-threshold and high-threshold QX-resistant depolarizing components are presumably mediated in part by Ca2+ currents. The high-threshold QX-resistant depolarization may involve intrinsic and/or NMDA-dependent entries of Ca2+. Indeed, in in vitro experiments, NMDA induces membrane depolarization with sustained firing (Spreafico et al. 1988; de Curtis et al. 1989). The QX-sensitive depolarizing component may be mediated in part by a non-inactivating inward current carried by Na+, similar to the one recorded in cat RTN cells (Mulle et al. 1986).
The present study thus strongly indicates that in RTN cells, spontaneous SWD or natural 5–9 Hz oscillations are associated with rhythmic, high-frequency burst discharges, which are initiated by CT-induced EPSP barrages. These can further trigger diverse voltage-dependent conductances (Fig. 12), which might operate individually or in concert, strengthening the burst discharges and thus the GABA-mediated hyperpolarizations in target TC neurones.
CT cells play a primary role in the synchronization of relay and reticular neurones
The present recordings indicate that, during the generation of SWD, CT cells probably exert a leading role in the synchronization of their target TC and RTN neurones. During absence seizures, related TC and RTN cells display rhythmic discharges in a synchronous manner, which appear to be triggered by CT-induced EPSPs. Moreover, my paired recordings reveal that CT cells have a propensity to discharge on average 7 ms before thalamic relay and reticular neurones, whether or not the two neurones of any recording pair (CT-TC or CT-RTN) are in direct synaptic connection. In addition, as observed during single-unit and multi-unit recordings, CT cells can fire in a rhythmic manner a certain time before related TC and RTN neurones, and rhythmic EPSP barrages also occur in these neurones during the earliest stage of the normal as well as epileptic rhythmic activity. Thus, these findings raise an important question as to whether layer VI CT neurones behave in a synchronous fashion, as the synaptic pacemaker of thalamic 5–9 Hz oscillations. The answer to this question requires further studies to determine whether this pacemaker activity is either inherent to such CT cells, or the result of a network oscillation.
Corticothalamic neurones are glutamatergic, by far exceed in number their target TC and RTN neurones, and are very effective in generating larger excitatory synaptic conductances in RTN than in TC neurones (Golshani et al. 2001). Therefore, they could reliably synchronize large populations of relay neurones, directly through glutamatergic excitations and, indirectly and massively, through GABAergic inhibitions. Corticothalamic cells are thus expected to play the role of booster-drivers that excite related relay and reticular neurones in a rhythmic and synchronous fashion, thereby forcing the TC system to oscillate. The recordings of the present study indicate that this should occur in a much more powerful manner during SWD than during the natural 5–9 Hz rhythm, at least in the somatosensory system.
In addition, the present thalamic and cortical extracellular field potential recordings show that natural 5–9 Hz oscillations, which give rise to SWD (Pinault et al. 2001), can occur in layer VI several seconds before being propagated synchronously to related TC and RTN neurones. These results are in line with those obtained from multi-site cortical and thalamic field potential recordings in another well characterized genetic model of absence epilepsy, the WAG/Rij rats (Meeren et al. 2002). The hypothesis of a primary role of CT cells in the synchronization of thalamic neurones also finds support in the fact that in GAERS, ECoG signs of SWD precede the full development of electroencephalographic epileptic discharges in the thalamus (Seidenbecher et al. 1998). Furthermore, in vitro electrophysiological data suggest that CT cells might exert an essential control in the generation of paroxysmal rhythmic activities within the TC-RTN network (Bal et al. 2000; Blumenfeld & McCormick, 2000).
However, two previous studies conducted in neuroleptanalgesied GAERS (Seidenbecher et al. 1998) and WAG/Rij rats (Inoue et al. 1993), a genetic model of absence epilepsy similar to GAERS, showed that thalamic neurones fire well before cortical neurones, supporting the hypothesis of a leading role of the thalamus in the development of genetically determined SWD. The discrepancy between these results and those of the present study merits discussion. In the Inoue et al. (1993) study, the ECoG electrodes were located in the frontal cortex and there is no evidence that the thalamic unit activities were recorded at functionally related sites. In the present study, the ECoG electrodes were located in the frontoparietal (somatosensory) cortex and all thalamic unit activities were recorded in the related somatosensory thalamus. In the Seidenbecher et al. (1998) study, layer VI cells, which represent the principal cortical source of thalamic inputs (see Introduction), were not recorded from. On the other hand, it is noteworthy to emphasize that the multi-unit recordings of the present study reveal, in accordance with the results of Seidenbecher et al. (1998), that thalamic neurones have the propensity to fire a few milliseconds earlier than their target layer IV/V cells. Paired single-cell IV/V-VI recordings confirm these findings (D. Pinault, unpublished observations).
In summary, the present findings thus reveal the likely sequence of neuronal events occurring in the somatosensory system of GAERS during the development of SWD: layer VI CT neurones play a major role in the rhythmic, synchronized excitation of thalamic, relay and reticular, neurones (Fig. 12). Furthermore, to all appearances, TC neurones massively synchronize cortical neurones of middle layers.
Switch from natural 5–9 Hz oscillations to SWD in GAERS, a possible scenario
In neuroleptanalgesied rats the medium-voltage 5–9 Hz rhythm has an internal frequency that is lower (by 2-3 Hz) than that of the equivalent physiological rhythm recorded in motionless GAERS and NE rats (D. Pinault, unpublished observations). It is thus seemingly identical to an identified rodent sensorimotor 7-12 Hz rhythm, which also occurs during body immobility (Nicolelis et al. 1995; Fanselow et al. 2001). In normal rats, this rhythm begins in the cortex and then spreads to the thalamus and brainstem (Nicolelis et al. 1995). In GAERS, the natural medium-voltage oscillation gives rise to SWD (Pinault et al. 2001), which are always accompanied by whisker twitching (see also Vergnes et al. 1982; Semba & Komisaruk, 1984). This natural rhythm does not give rise to SWD in control NE rats, meaning that it is not itself sufficient for generating absence-related epileptic activity.
Understanding the factors that induce SWD from natural medium-voltage 5–9 Hz oscillations in GAERS is of crucial importance. The present study reveals that, contrary to TC and RTN neurones, CT cells do not usually discharge robust bursts of APs. In a previous study, we have shown that, during the development of SWD, RTN cells start to fire in the burst mode almost always before TC neurones (Pinault et al. 2001; see also Fig. 2). It is thus tempting to put forward that a CT induced resonance phenomenon may contribute to the switch of the natural medium-voltage 5–9 Hz oscillation into SWD in GAERS. On the basis of the present findings, the corresponding scenario might be as follows: (1) CT cells somehow launch and maintain the natural 5–9 Hz rhythm, assuring the phase-locking of threshold and subthreshold thalamic, relay and reticular, oscillations; (2) because RTN cells are endowed with powerful, synaptic and intrinsic, electroresponsive properties, they start to react – or to resonate – massively before TC neurones, exhibiting rhythmic robust high-frequency AP burst discharges (Pinault et al. 2001); (3) these RTN bursts generate GABAA-dependent IPSP barrages in their target TC cells, (4) in those having an H-current, the RTN-induced hyperpolarizing wave triggers a rebound ramp-shaped depolarization leading to an AP discharge underlaid by a low-threshold Ca2+ potential (Lüthi & McCormick, 1998). This discharge in turn reinforces reciprocal cellular interactions between the thalamic relay and reticular nuclei (see also Bal & McCormick, 1993) through the recruitment of new active RTN units, thereby strengthening the GABAA-dependent hyperpolarization in larger populations of TC neurones. These massively recruit new active units at the cortical level, in particular in layer IV/V. The cycle somehow starts again, and cellular hypersynchronization thereby would be generated.
Other possibilities for the switch of natural oscillations into SWD may exist. For instance, an in vitro study has demonstrated that layer V pyramidal neurones are able to generate synchronized oscillations in the same frequency range when NMDA receptors are activated in a tonic fashion (Silva et al. 1991). NMDA responses are considerably more important in middle and deep layers of the cerebral cortex in GAERS than in control rats (Pumain et al. 1992). Such excitatory responses, however, might result from the activity induced by hypersynchronous TC inputs. Also, recent in vivo and in vitro studies have shown that neocortical disinhibition generates synchronized 7-14 Hz oscillations, which are driven by deep cortical layers and which spread to the thalamus from layer VI (Castro-Alamancos, 2000; Castro-Alamancos & Rigas, 2002). Thus, the cause(s) switching medium-voltage 5–9 Hz oscillations into SWD in genetic models of absence epilepsy is a fundamental issue requiring additional investigation.
This study reveals novel spatio-temporal dynamics of the interactions between cortical and thalamic neurones occurring during the generation of rodent, normal and epileptic, 5–9 Hz oscillations. It strongly suggests that synchronized CT neurones represent a possible synaptic pacemaker of thalamic 5–9 Hz oscillations. Furthermore, in GAERS the TC and RTN rhythmic firings that are associated with SWD are provoked by intracellular, synaptic and intrinsic, mechanisms that are very similar to those associated with the natural medium-voltage 5–9 Hz rhythm. This suggests that, in GAERS, absence-related SWD correspond to a hypersynchronous wake-related physiological oscillation. The hypersynchronization mechanism would depend on a CT-induced resonance phenomenon.