Location and size of the cortical infarct
Focal thrombotic lesions were performed unilaterally in the somatosensory cortex using the Rose Bengal photothrombosis technique (Watson et al., 1985
; Kharlamov et al., 2003
) on P21 rats (n=20). All lesioned animals had highly consistent, well-demarcated areas of focal infarction within the right somatosensory cortex (). The area of cortical infarction appeared as a cystic, scarred area of 2 mm in diameter. A Nissl-labeled coronal section taken through the lesion () demonstrated extensive neuronal loss in the area of the infarct core that extended to the subcortical white matter without damaging the hippocampus. The infarct core was usually dislodged during tissue sectioning. In cortex adjacent to the infarct core, neither cell loss nor cytoarchitectural abnormalities were detected (data not shown). The lesion location, size and depth were highly reproducible (n=22 animals). No abnormalities were detected in the brains of control animals who received Rose Bengal i.v. injection without photostimulation (n=14) or in sham operated photostimulated rats who did not receive Rose Bengal injection (n=6). The schematic in illustrates the post-thrombotic degeneration of corticothalamic axons (black crosses) and secondary degeneration of thalamocortical relay axons (red crosses), which is complete by the end of the first week.
Location of retrograde cell death in thalamus
We first examined the effect of the cortical infarct on cell death in thalamus. The injury was first assessed with NeuN labeling which showed cell loss specifically in the ventral posterolateral (VPL) thalamic nucleus ipsilateral to the cortical infarct (n=6 animals) (). The cell loss in VPL was consistently co-localized with a strong GFAP labeling () signifying gliosis (Schmidt-Kastner et al., 1993
). The cell loss and gliosis were indistinguishable 7, 14, 42 and 60 days after the cortical infarct (data not shown), indicating that cell loss was essentially complete by the end of the first week following the infarct. Interestingly, the cell loss and gliosis were restricted to VPL and absent in the adjacent nRT or other thalamic relay nuclei (VL, VPL, VPM, Po; ). Neither cell loss nor gliosis were observed in the contralateral thalamus () (n=6 animals).
Figure 2 Cell death and gliosis in the ventral posterolateral thalamic nucleus. A–F, Horizontal thalamic 50 μm sections (every 250 μm; upper row (A) most dorsal; lower row (F) most ventral) from an injured rat with combined immmunolabeling (more ...)
Loss and disorganization of corticothalamic and thalamocortical fibers
We next examined the extent of the loss of corticothalamic and thalamocortical axons after injury as detected with Neurofilament 200 kDa (NF) labeling in four animals. As illustrated in , the parallel organization of the fibers apparent in the contralateral (unaffected) hemisphere (arrows) was lost in the hemisphere ipsilateral to the cortical infarct (n=4 animals). The loss of fibers was restricted to the VPL region and remaining fibers were disorganized (, crossed arrows).
Morphological changes in nRT
Under visual inspection, we observed a disorganized structure in terms of soma orientation. Somatic changes were first examined with parvalbumin labeling of nRT neurons. The size of the soma was similar in the injured and control cells (long axis ~25 μm). However, the fusiform shape of somata typically observed in control slices and in the contralateral side was lost in the injured side, where the cells appeared more circular (, ). In nRT adjacent to the injured VPL, the circularity of the cells was increased by 25 % (injured: 0.65 ± 0.01; n=396 cells vs contralateral: 0.52 ± 0.01; n=379 cells from 4 rats; p<0.0001). The circularity of nRT cells located ipsilateral to the injury, but >300 μm from the injured VPL, was less affected, but still 18 % larger than the corresponding contralateral nRT region (injured: 0.52 ± 0.01; n=382 cells vs 0.44 ± 0.01; n=341 homotopic contralateral cells; p<0.0001).
Furthermore, the normal parallel orientation of nRT cells in the unaffected (contralateral) side was lost in the injured side (). This cellular disorganization was quantified by measuring the standard deviation of the major axis angle of the nRT cells, which was significantly (p<0.0001) increased in the nRT adjacent to the injured VPL (30.6 ± 2.3 degrees), compared with the corresponding contralateral nRT (15.2 ± 1.4 degrees). Major axis angle of nRT cells located far (>300 μm) from the injured VPL was not different compared to that of cells from the corresponding contralateral nRT region (ipsilateral: 19.5 ± 7.3 degrees; contralateral: 23.0 ± 0.0 degrees; p>0.1).
Moreover, NF and parvalbumin were co-localized in somata and fibers on the contralateral side but not the injured side () (n=4 rats). Because parvalbumin and NF are normally expressed in somata and fibers (Majak et al., 1998
; Lee and Cleveland, 1994
; Clinton et al., 2004
; Clinton and Meador-Woodruff, 2004
), the reduced labeling on the side ipsilateral to the cortical infarct () suggests a loss or decrease in their expression after injury, which is associated with a more circular somatic shape.
Interestingly, the morphological changes described above were also found two months following the cortical infarct (data not shown), suggesting that these changes are long-lasting and do not represent a transient, perhaps pre-apoptotic phenomenon.
Dendrites and axons
The dendritic and axonal changes were assessed following a three-dimensional reconstruction of nRT neurons filled with biocytin (). The cortical infarct induced striking morphological changes in nRT neurons projecting to the injured cell-deprived thalamic areas (VPL) and in nRT cells projecting to the thalamic areas less affected by cell loss and gliosis (VPM; ). Cortical infarct resulted in (1) decreased mean dendritic length (); (2) decreased mean number of dendritic arborizations (see ); (3) decreased mean axonal length (); and (4) decreased mean number of axonal boutons in the relay thalamic nuclei. Interestingly, the number of small boutons (<0.5 μm) per cell was not significantly decreased whereas the number of big boutons (>0.5 μm) was decreased by ~93 % in the injured nRT cells compared with the controls (). It is important to note that the cortical infarct-induced morphological changes described above (decrease in the dendritic and axonal lengths, circular soma and decreased total number of synaptic boutons) were observed not only in nRT neurons projecting to the injured thalamus (VPL) () but also in those projecting to VPM (), which was not affected by cell death or gliosis (). This result suggests that the loss of boutons in the axons of nRT cells does not result from a local cell loss in the target thalamic nucleus, but rather from a pathological change in nRT cells following cortical stroke. No major differences in axon collaterals within nRT were observed (data not shown).
Morphological changes in nRT neurons following a cortical infarct.
Decreased intrinsic excitability in nRT cells
Passive membrane properties
In order to examine if the altered morphology of nRT cells was associated with a change in intrinsic excitability, we first examined the membrane V–I
relationship, which was determined by measuring membrane potential changes in response to a series of intracellular square current pulses (). The input resistance (Rin
) and membrane time constant (τm
), measured from the to responses to current steps in linear portion of the V–I
plot, were each decreased by 30 % in injured nRT neurons compared to control cells (). The membrane capacitance (Cm
) was unchanged () suggesting that the reduced Rin
did not result from an increased membrane surface but rather from an increased membrane leak conductance. Other basic electrical membrane properties includingresting membrane potential and action potentialwaveform were similar in injured and control cells (). In both cell populations, an inward rectification was observed in response to negative current pulses inducing hyperpolarizations past −95 mV (). This inward rectification was likely caused by the hyperpolarization-activated inward cationic current (Ih
) and inwardly rectifier K+
) (Abbas et al., 2006
; Rateau and Ropert, 2006
analysis (; ) confirmed a decreased excitability after injury, showing (1) a 32 % decrease in slope (see Methods); and (2) an 85 % increase in rheobase (i.e. the minimal current intensity required to trigger an action potential firing; ). Moreover, the injured cells reached a maximal firing rate of only ~60 Hz (), whereas the control cells reached a maximal firing rate of at least 80 Hz. All the alterations in intrinsic excitability described above persisted 4–6 weeks following the cortical stroke (Supplemental Figure 2
) at a time when the seizures are expected to occur (Kelly et al 2001
; Kharmalov et al. 2003
), showing that the early post-stroke alterations in nRT are a long-term phenomenon, rather then a transient alteration. These findings reinforce the potential link between these post-stroke thalamic alterations and the later epileptogenesis.
Figure 4 Effects of focal cortical infarct on electrical membrane properties of nRT neurons. A–B, Voltage responses of control (A) and injured (B) nRT thalamic neurons (top traces) to intracellular injection of positive and negative square current pulses (more ...)
Comparison of electrical membrane properties of injured and control nRT neurons.
Low-threshold calcium spike
In 97 % of nRT cells (32 out of 33) from control rats a hyperpolarization to −95 mV or more was followed by a rebound of excitation characterized by a low-threshold Ca2+
spike (LTS; Llinas and Jahnsen, 1982
; Steriade et al., 1990
) crowned by a burst of action potentials ( black traces). Interestingly, in the injured rats, the same hyperpolarizations failed to induce rebound bursts in 50 % of cells (13 cells out of 26). However, in 3 of those cells (out of 13), an LTS could be evoked by injecting a positive current pulse of +100 pA after the hyperpolarizing step (, middle panel; Lesion center
; see the protocol at the top). These results indicate that LTS was absent in ~38 % (10 cells of 26) of nRT cells ipsilateral to the cortical infarct suggesting a functionally complete loss of T-type channels in these cells. In those ipsilateral nRT cells in which an LTS could be evoked as a post-hyperpolarization rebound response, the mean maximal number of action potentials within the burst was 9.7 ± 1.1 (range, 3–14 action potentials; n=13 cells). In the control cells, by contrast, the maximal number of action potentials in a burst was significantly higher (13.7 ± 0.79; range, 6–23 action potentials, n=32 cells; p<0.01). Moreover, in the injured nRT cells which expressed LTS (n=16 cells), the LTS was less robust than in control cells (, ) and its kinetics were altered. Specifically, the LTS decayed more rapidly in the injured cells compared to the controls (). Interestingly, nRT cells located far from the thalamic injury (> 300–400 μm from the portion of nRT adjacent to VPL) expressed larger LTS responses than those nRT cells located proximal to the center of VPL injury (), despite the increased membrane leak. Such responses were less robust and decayed much faster than in control cells but slower than the LTS of nRT cells close to the VPL injury ().
Figure 5 Low-threshold calcium spikes are decreased in nRT cells after injury. A, Voltage responses (top traces) from control and injured nRT cells to intracellular injections of negative and positive square current pulses (bottom traces) from −80 mV. (more ...)
These alterations in LTS properties persisted 4–6 weeks following the cortical stroke when the seizures are expected to occur. Specifically, in the injured nRT cells (1) the maximal number of action potentials in a post-hyperpolarization rebound burst was twofold reduced (injured: 4.8 ± 2.0; range, 0–10 action potentials, n=5cells; control: 9.6 ± 1.6; range, 4–20 action potentials, n=8 cells; p<0.05); (2) the intra-burst maximal firing frequency was reduced (injured: 46.2 ± 20.3 Hz; n=5 cells; control: 101.6 ± 13.9 Hz; n=8 cells; p<0.05).
T-type calcium current is altered after injury
The altered LTS properties in nRT cells ipsilateral to cortical infarct could be due to a reduced number of T-channels and/or a change in their biophysical properties. In order to answer this question, we recorded T-currents from control and injured nRT cells with the same steady-state inactivation (SSI) protocol illustrated in (bottom).
Steady-state inactivation of T-type currents
The injured cells exhibited an abnormal voltage dependence of T-type calcium currents (, left panel), with the steady-state inactivation (SSI) curve showing a hyperpolarized shift compared with the control nRT cells (; injured V50%: 104.1 ± 1.5 mV, n = 10 cells; control V50%: −99.9 ± 1.2 mV; n = 15 cells; p<0.05). The Boltzmann function slope factor was similar in both cell groups (injured: 5.1 ± 0.7 mV, n = 10 cells; control: 5.2 ± 0.4 mV, n = 15 cells; p>0.5). Such a hyperpolarized shift in the half-maximal voltages (V50 %) for the SSI could be due to the fact that the cells have an increased leak conductance after injury (see ). However, the hyperpolarized shift of the V50% of the SSI was also observed using a CsCl internal solution that blocks leak channels and improves uniformity of voltage control (control: −84.4 ± 0.5 mV; n=3 cells; injured: −89.4 ± 1.1 mV; n=3 cells; p<0.05), which strongly suggests that the hyperpolarized shift was not due to the leak.
Decreased peak density of T-type calcium current
Interestingly, the peak T-current amplitude was decreased by ~42 % in the injured cells (57.6 ± 8.6 pA, n=18) compared with the controls (99.7 ± 14.3 pA, n=18; p<0.05; left panel). The peak current density was similarly decreased by ~44 % in the injured cells (0.9 ± 0.1 pA/pF, n=18) compared with the controls (1.6 ± 0.3 pA/pF, n=18; p<0.05; right). Thus the difference in peak amplitude cannot be accounted for by cell size.
Increased T-current inactivation rate
The weighted decay time constant (τD,W) of T-current was decreased by 26 % in the injured cells (203.8 ± 21.2 ms, n=18) compared with the controls (273.9 ± 18.9 ms, n=18 cells; p<0.05) (). The same decreases in T-current amplitude and τD,W were found with CsCl-filled recording electrodes (data not shown), indicating that these changes were independent from the recording electrode internal solution.
Loss of Cav3.3 isoform in the ipsilateral nRT following cortical infarct
The decreased amplitude and faster decay rate of T-currents as well as the hyperpolarized shift in the SSI could result from a loss of T-channels and/or a change in their isoform expression and/or location. Three different isoforms of T-channels have been identified in mammals: α1G
3.2 and α1I
3.3 (Lee et al., 1999
; McRory et al., 2001
; Murbartian et al., 2002
; Perez-Reyes, 2003
), with all expressed to some extent in nRT neurons (McKay et al., 2006
), and each with differences in their voltage-dependence and kinetic properties. Because Cav
3.3 channels are (1) widely expressed in nRT somata and dendrites (McKay et al., 2006
), (2) have slower inactivation kinetics compared to the other two isoforms (Lee et al., 1999
) and (3) have the most depolarized half-inactivation voltage (4 mV compared with Cav
3.1 and 12 mV compared with Cav
3.2 when expressed in HEK-293 cells; Lee et al., 1999
), we tested the hypothesis that this isoform was lost in nRT after cortical infarct leading to reduced current amplitudes and faster decay kinetics. Indeed, Cav
3.3 immunolabeling was dramatically decreased in nRT ipsilateral to the cortical injury () in every case (n=4 animals out of 4), particularly in nRT adjacent to the injured, cell deprived VPL, and its loss was less prominent in nRT cells far from VPL (>300 μm).
Altered burst morphology in injured nRT cells
We next examined the consequence of the decreased amplitude and decay time of T-current on maximal burst firing properties of nRT cells, as assessed by injecting a positive current pulse of same intensity (+100 pA push pulse) following hyperpolarizing current steps of variable intensity (see protocol in at the top; Porcello et al., 2003
). Following similar membrane potential hyperpolarizations, the maximal number of action potentials within the burst was reduced in nRT neurons adjacent to the injured VPL compared to the control cells (control: 14.8 ± 0.8 action potentials; range, 11–22; n=15 cells from 8 rats; injured: 8.0 ± 1.2 action potentials; range, 3–16; n=13 cells from 6 rats; p<0.0001) but not significantly reduced in nRT cells far (>300 μm) from the injury (11.1 ± 2.1 action potentials; range 3–17; n=7 cells from 4 rats; p>0.05) (). This result is in agreement with the decreased amplitude of T-current in the injured cells ().
In control cells, independent of the level of hyperpolarization, each burst showed the accelerating-decelerating spike firing pattern characteristic of a LTS-evoked burst in nRT neurons (Mulle et al., 1986
; Spreafico et al., 1988
; Avanzini et al., 1989
; Bal and McCormick, 1993
;Contreras et al., 1993
; Slaght et al., 2002
) reaching a peak value ≥180 Hz (n=15 cells from 8 control rats) (). In nRT neurons adjacent to the injured VPL ( Lesion center
) the accelerating pattern was lost in 62 % of cells, was observed only for the first inter-spike interval in 23 % of cells, and an accelerating/decelerating pattern was observed only in 15 % of cells. Moreover, in all nRT cells adjacent to VPL (n=13 cells) the peak frequency value was lower (~120 Hz) compared with the control cells (n=15, see above) ( Lesion center
In nRT neurons located ipsilateral to the cortical infarct, but far from the injury (>300 μm), the accelerating-decelerating pattern was conserved, although its kinetics were different from the control cells ( lesion far). Specifically, (1) following weak hyperpolarization the latency of the peak was delayed by ~100 ms compared to the control cells ( Control vs Lesion far); and (2) the decay time of the instantaneous firing frequency was much shorter (~50 ms) than in the control cells (>70 ms) but longer than in the nRT cells located in the center of injury (<30 ms) (). This result is in agreement with the voltage-clamp recording of the T-currents, which showed a faster decay time in the injured cells ().
Decreased excitability results from upregulation of Ikir
The decreased excitability of nRT neurons in the injured animals could be due to an increased leak current. Among the currents that could potentially contribute to the leak, the inwardly rectifying potassium (Kir) current is a good candidate (Hagiwara et al., 1978
; Bichet et al., 2003
; Rateau and Ropert, 2006
). We therefore tested the effect of Ba2+
, which blocks IKir
(Hagiwara et al., 1978
) without affecting the H-current (Ih
; Pape, 1996
), on nRT cells from control and injured animals. We examined the effect of Ba2+
on the membrane I–V
relationship of nRT cells by measuring currents generated by voltage steps from −65 to −135 mV in voltage-clamp mode (). nRT neurons from control animals expressed current responses similar to those described by previous studies (Santoro et al., 2000
; Rateau and Ropert, 2006
). As expected from the current-clamp recordings (see above), between −65 and −85 mV, which is the range of the resting membrane potential (see ), nRT cells from injured animals had a significantly lower Rin
compared with the controls (control: 382.6 ± 25.9 MΩ, n=5 cells; injured: 261.8 ± 35.2 MΩ, n = 5 cells; p<0.05; ). Bath application of 1 mM Ba2+
induced an inward current in nRT cells from both control and injured animals (). Between −65 and −85 mV, Ba2+
application increased the membrane Rin
by 33 % in the control cells and by 134 % in the injured cells. After Ba2+
application, the Rin
of control and injured nRT cells were similar (calculated in voltage range of −65 – −85 mV; control: 507.5 ± 36.2 MΩ; n=5 cells; injured: 613.6 ± 192.3; n=5 cells; p>0.5; left). The effect of Ba2+
at this potential could be due to the block of a weakly rectifying Kir-current (Bichet et al., 2003
Figure 7 Ba2+ restores the membrane input resistance but not the low-threshold calcium spike in the injured nRT cells. A–B, Current recordings in nRT cells from control (A) and injured (B) animals before and after application of 1 mM Ba2+. Two representative (more ...)
We also calculated the Rin at potentials more negative than −105 mV using the slope 2+ conductance (Rin=1/gm, where gm is the slope conductance), and found that before Ba application, membrane Rin of nRT cells from injured animals was significantly lower (p<0.05) compared with those from control animals (control: 331.6 ± 51.9 MΩ; n=5 cells; injured: 241.97 ± 5.36 MΩ; n=5 cells; p<0.05; right). However, 1 mM Ba2+ application strongly reduced I–V slope in nRT cells from both control and injured animals (), corresponding to a 44 % increase in Rin in the control cells, and, interestingly, to a much stronger increase (160 %) in the injured cells ( right). Thus, after Ba2+ application, the membrane Rin values in the hyperpolarized range for control and injured cells were similar (control: 478.3 ± 58.2 MΩ; n=5 cells; injured: 629.1 ± 147.6 MΩ; n=5 cells; p>0.1; right).
Our results indicate that after cortical injury nRT neurons express higher Kir-conductance compared to the controls at both resting potential (~−70 mV) as well as at hyperpolarized membrane potentials (<−105 mV) and that IKir contributes strongly to the leak. Because blocking IKir restored the Rin of the injured cells, it is likely that this current underlies the leak of nRT cells after injury.
did not restore the post-inhibitory LTS, even though Ba2+
is a more effective charge carrier than Ca2+
in nRT T-channels (Huguenard and Prince, 1992
) suggesting that the decrease or absence of LTS (see , ) and the hyperpolarized shift in the SSI curves of injured cells () are not due to the leak but rather a loss and/or a change in the T-type calcium channel subunit expression.
Evoked excitatory activity in nRT
Axon excitability and recruitment
In order to examine if glutamatergic synaptic transmission was altered by cortical infarct we stimulated cortical and thalamic afferents in the internal capsule () and compared the evoked excitatory post-synaptic current (EPSC) in nRT cells from control and injured animals. The intensity of stimulation required to induce an evoked EPSC at threshold (i.e. presumed activation of a single presynaptic fiber; see Methods) in nRT cells was similar in control and injured rats (control: 27.1 ± 4.8 V; n=13 cells vs injured: 22.1 ± 2.7 V; n=17 cells; p>0.5), suggesting that the excitability of the remaining glutamatergic axons projecting to nRT was not altered by the cortical infarct. Moreover, input-output relationships for evoked EPSCs were not altered in injured cells (data not shown), suggesting that axonal recruitment was not altered by the cortical infarct.
Injury decreases the amplitude of the evoked EPSC
EPSCs were evoked in nRT neurons by stimulating the internal capsule () to activate glutamatergic fibers of passage from cortex or dorsal thalamus projecting to nRT. The amplitude of the evoked EPSCs at threshold was significantly decreased in the injured nRT cells (78.9 ± 13.0 pA; n=17 cells) compared with the controls (213.7 ± 63.5 pA; n=13 cells; p<0.0001) ().
Injury slows decay of the evoked EPSC
The weighted decay time constant of internal capsule evoked EPSCs was increased by ~85 % in the injured nRT cells (2.4 ± 0.4 ms; n=17) compared with the controls (1.3 ± 0.1 ms; n=13 cells; p<0.05) (). Synaptic efficacy, as measured by area of the evoked EPSCs, showed no significant change with injury (control: 535 ± 199 fC, n=13 cells; injured: 269 ± 63 fC, n=17; p>0.05). Thus, the overall total charge of the evoked EPSCs was similar in the injured and control nRT cells (p>0.05), compensating for the decrease in amplitude. The 10–90 % rise time of evoked EPSCs was not altered in the injured animals (control: 0.5 ± 0.1 ms; n=13 cells; injured: 0.6 ± 0.1 ms; n=17 cells; p>0.4).
Injury decreases the paired-pulse ratio
Repetitive stimulation can elicit both paired-pulse facilitation (PPF) and paired-pulse depression (PPD). These phenomena can be described by the paired-pulse ratio (PPR) that is regarded as an index of presynaptic efficacy (Thomson et al., 1993
; Markram and Tsodyks 1996
; Zucker, 2002
). We examined the PPR in nRT neurons of both control and injured animals at different inter-stimulus intervals to test the hypothesis that the glutamatergic synaptic efficacy in nRT may undergo an alteration after cortical infarct. In control cells a PPF (PPR > 1) was observed in 83 % of cells at 50 Hz, in 80 % of cells at 100 Hz and in 27 % of cells at 200 Hz. However, after injury, a PPF was observed only in 50 % of cells at 50 Hz, in 42 % of cells at 100 Hz and in 9 % of cells at 200 Hz. The reduction in PPR in the injured cells was observed at all frequencies (50–200 Hz) (Supplemental Figure 1A
; see also ) but was significant only at 50 and 100 Hz (p<0.05 at 50 and 100 Hz; p>0.05 at 200 Hz; Supplemental Figure 1D
). However, even though the mean PPR was not significantly reduced at 200 Hz, its median value was lower in injured cells versus the controls (Supplemental Figure 1A
Effects of cortical injury on amplitude of successive EPSCs evoked by stimulus trains
In addition to the PPR, synaptic efficacy can be measured by the time constant of decay of amplitudes of successively evoked EPSCs during a stimulus train. Synaptic responses elicited by trains of 5 stimuli (delivered in the internal capsule, see Methods) at 1.5x threshold were measured in neurons from control and injured rats at stimulus frequencies of 50, 100 and 200 Hz (; Supplemental Figure 1
). During the train, there was a successive decline in amplitudes of successive EPSCs from the second evoked response within the train (see and Supplemental Figure 1C
). The decline in amplitude of successive EPSCs was measured, starting at the second evoked EPSC, by an exponential fit and was similar in control and injured cells for 50 Hz (control: 67.31 ± 5.93 ms, n=6 cells; injured: 88.36 ± 80.28 ms, n=10 cells; p>0.05), 100 Hz (control: 17.04 ± 5.87 ms, n=5 cells; injured: 23.76 ± 12.58 ms, n=12 cells; p>0.5) and 200 Hz stimulus trains (control: 5.88 ± 5.15 ms, n=10 cells; injured: 6.35 ± 3.35 ms, n=12 cells; p>0.5). Furthermore, the train-pulse ratio (TPR), defined as the ratio between the fifth evoked EPSC by the first, was significantly lower at 50 Hz in the injured cells, but was not significantly different from the control at 100 or 200 Hz stimulation trains (Supplemental Figure 1B,D
Effects of cortical infarct on the excitatory synaptic potentials evoked by 200 Hz stimulus trains
We next examined the membrane potential correlate of the evoked EPSCs. Thalamic relay neurons are capable firing high frequency bursts of action potentials in vitro
(Bal et al., 1995
; Huguenard and Prince, 1994) and in vivo
during normal spindle-wave activity and epileptic spike-and-wave discharges (Steriade, 2005
; Pinault, 2003
; Paz et al., 2007
). Relay neuron bursts are characterized by frequencies of ~200 Hz which induces high frequency bursts of EPSPs in the target nRT neurons (Bal et al., 1995
). In order to examine if this integrative property of nRT cells was altered after injury, we studied the excitatory post-synaptic response evoked by internal capsule stimulation with 200 Hz trains of 5 stimuli at 1.5x threshold intensity (see above). Activation of cortical/thalamic afferents to nRT neurons from control animals resulted in a short latency burst of action potentials ( left
). The number of elicited action potentials depended on the membrane potential of the cell. At depolarized membrane potentials, few action potentials were evoked, whereas at more hyperpolarized levels at which the T-current is deinactivated (Steriade et al., 1990
), the same stimulation induced a LTS ( arrow) crowned by a burst of Na+
action potentials. However, in the injured rats, the same stimulation protocol (i.e. 1.5x threshold stimulus train at 200 Hz) elicited fewer action potentials at depolarized membrane potentials ( at the top: compare right versus left panels) and neither LTS nor action potential firing were observed at more hyperpolarized levels of membrane potential (, at the bottom: compare right versus left panels).
Spontaneous synaptic activity in nRT
To determine whether the cortical infarct affected the synaptic activity of nRT GABAergic neurons, we examined spontaneous excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) in cells from injured and control animals.
Surprisingly, despite a massive loss of cortical and thalamic glutamatergic afferents to nRT (, ) and reductions in evoked EPSC (), the mean frequency of the spontaneous EPSCs was not reduced in nRT neurons of injured animals (2.4 ± 1.9 Hz; n=16 cells) compared with the controls (2.3 ± 1.4 Hz; n=20 cells; p>0.5 control vs injured cells; representative traces ). We next examined if the kinetics of the isolated spontaneous EPSCs events were altered following injury (). Based on the analysis of 1900 sEPSCs from 20 control cells and 1600 sEPSCs from 16 injured nRT cells), none of the following parameters were found to be affected by the injury: amplitude (control: 19.7 ± 0.8 pA; injured: 22.4 ± 0.8 pA; p>0.05), charge (control: 25.7 ± 2.0 fC; injured: 26.6 ± 2.0 fC; p>0.5), half-width (control: 0.84 ± 0.05 ms; injured: 0.78 ± 0.07 ms; p>0.1), 10–90 % rise time (control: 0.21 ± 0.01 ms; injured: 0.20 ± 0.01 ms; p>0.1), and decay time constant (control: 0.81 ± 0.05 ms; injured: 0.72 ± 0.08 ms; p>0.1).
Similarly, mean IPSC frequency was not different in nRT neurons from injured animals (0.78 ± 0.09 Hz; n=19 cells) compared to controls (0.99 ± 0.18 Hz; n=28 cells; p>0.3; representative traces ). IPSC kinetics were also unaffected; no significant differences were found in amplitude (control: 16.79 ± 0.77 pA; injured: 16.16 ± 1.05 pA; p>0.6), 10–90 % rise time (control: 2.30 ± 0.02 ms; injured: 2.39 ± 0.05 ms; p>0.08), half-width (control: 74.8 ± 3.99 ms; injured: 82.40 ± 5.92 ms; p>0.27), or decay time constant (control: 77.34 ± 2.94 ms; injured: 83.03 ± 4.11 ms; p>0.25; ). In addition, there were no significant differences in evoked IPSC amplitude (control: 238.77 ± 54.0 pA; injured: 266.11 ± 73.27 pA; p>0.7) or weighted decay time constant (control: 160.59 ± 21.48 ms; injured: 158.19 ± 20.61 ms; p>0.4; control: n=12 cells; injured: n=7 cells; ).