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Focal cortical injuries result in death of cortical neurons and their efferents and ultimately in death or damage of thalamocortical relay (TCR) neurons that project to the affected cortical area. Neurons of the inhibitory reticular thalamic nucleus (nRT) receive excitatory inputs from corticothalamic and thalamocortical axons and are thus denervated by such injuries, yet nRT cells generally survive these insults to a greater degree than TCR cells. nRT cells inhibit TCR cells, regulate thalamocortical transmission, and generate cerebral rhythms including those involved in thalamocortical epilepsies. The survival and reorganization of nRT following cortical injury would determine recovery of thalamocortical circuits following injury. However, the physiological properties and connectivity of the survivors remain unknown. In order to study possible alterations in nRT neurons, we used the rat photothrombosis model of cortical stroke. Using in vitro patch clamp recordings at various times following the photothrombotic injury, we show that localized strokes in the somatosensory cortex induce long-term reductions in intrinsic excitability and evoked synaptic excitation of nRT cells by the end of the first week after the injury. We find that nRT neurons in injured rats show (1) decreased membrane input resistance, (2) reduced low-threshold calcium burst responses, and (3) weaker evoked excitatory synaptic responses. Such alterations in nRT cellular excitability could lead to loss of nRT-mediated inhibition in relay nuclei, increased output of surviving TCR cells and enhanced thalamocortical excitation, which may facilitate recovery of thalamic and cortical sensory circuits. In addition, such changes could be maladaptive, leading to injury-induced epilepsy.
Neocortical injuries initiate processes of cortical cell death and orthograde and retrograde degeneration of corticothalamic and thalamocortical axons, respectively (Peacock and Combs, 1965; Matthews, 1973; Iizuka et al., 1990; Sorensen et al., 1996), as well as gliosis in affected structures (Iizuka et asl., 1990; Agarwala and Kalil, 1998). Although thalamic reticular (nRT) neurons are susceptible to excitotoxic injury following local stroke, they generally survive cortical lesions (Ross and Ebner, 1990). The functional long-term consequences of cortical stroke are predicted by the size and location of the lesion, and by the capacity of the brain to compensate and reorganize lost functions, sometimes leading to post-traumatic epilepsy by mechanisms that are poorly understood. Several studies suggest that the thalamus is involved in generation of both partial and generalized seizures (reviewed by Huguenard and Prince, 1997). Over the past decade most of the emphasis has been on the role of thalamus in diffuse corticothalamic epilepsies. Much less is known about the role of the thalamus in partial epilepsies.
The nRT, located at the crossroads of the thalamocortical system (Figure 1), receives both upstream and downstream excitatory signals (Jones, 1985) and powerfully modulates output of thalamic relay neurons (reviewed by Steriade, 2005). nRT cells produce divergent inhibitory output (Crabtree et al., 1998) that can both drive thalamocortical oscillations related to generalized absence seizures, and potentially suppress feedback of focal cortical seizures (Huguenard and Prince, 1997; Steriade, 2005). Survival and reorganization of nRT following cortical lesions is expected to play a role in recovery of thalamocortical circuits. A malfunction of the surviving GABAergic nRT neurons could profoundly affect thalamocortical activity and the results may be either favorable or disadvantageous, compensating for lesion-induced deficits while causing a deterioration of function or even post-lesional epilepsy (So et al., 1996). However, the effects of cortical infarct on the physiological properties and connectivity of the nRT survivors remain unknown. Because these questions are crucial to our understanding of the mechanisms of post-stroke thalamocortical reorganization leading to epilepsy, we used the cortical photothrombosis brain infarction rat model (Watson et al., 1985; Kelly et al., 2001; Kharlamov et al., 2003) to study the anatomical and physiological changes of nRT following well-controlled focal infarction in the somatosensory cortex. Such lesions have been shown to result in post-stroke epilepsy with seizures originating in the ipsilateral perilesional cortex and associated with motor arrest of the animal (Kelly et al., 2001; Kharlamov et al., 2003). In the present study, we first determined the precise location of cell death in thalamus following focal thrombosis in the somatosensory cortex. Furthermore, we examined the intrinsic excitability of nRT cells both central and peripheral to the region of maximal post-stroke damage. We further examined whether the mechanisms central to oscillatory activity in this nucleus were altered by determining the ability of these neurons to fire bursts of action potentials. Finally, we asked if the excitatory or inhibitory synaptic inputs to nRT neurons were altered.
All experiments were performed according to protocols approved by the Stanford Institutional Animal Care and Use Committee and every precaution was taken to minimize stress and the numberof animals used in each series of experiments.
The photothrombosis method allows for the selective placement of cortical infarcts with reproducible area, depth and location (Watson et al., 1985; Dietrich et al., 1987; Kharlamov et al., 1994, 1996; Schiene et al., 1996; Redecker et al., 2002). Photothrombosis was performed as described by Watson et al. (1985) as modified by Kharlamov et al (2003) on three week old (postnatal day 21; P21) Sprague-Dawley rats (n=25), an age at which the thalamic circuits have attained a relatively mature state (Warren and Jones, 1997). Briefly, animals were anesthetized with a mixture of ketamine (80 mg/kg) and xylazine (12 mg/kg) i.p. The scalp was incised in the midline and retracted to expose the desired area of the skull, and the periosteum removed. Rose Bengal (40 mg/kg) (Sigma, St. Louis, MO) dissolved in 0.9% saline was injected into the tail vein and a light from a 3 mm diameter fiberoptic cable connected to 200 W illuminator was focused on the skull for 20 minutes. The optical system was designed to have an emission spectrum that encompassed the in vivo absorption range of Rose Bengal (maximum absorbance at 562 nm). The light beam was centered 4.5 mm lateral and 2.5 mm caudal to Bregma to induce a thrombotic lesion in the right somatosensory cortex (Figure 1A,B) (Paxinos and Watson, 1986). The scalp was sutured and the animal allowed to recover. Buprenorphine (0.02 mg/kg) was administered as necessary following the surgery. Animals received a lethal dose of sodium pentobarbital (200 mg/kg, i.p.) 7–40 days after the cortical injury for electrophysiological recordings or 6–60 days after injury for histology. Control animals received the same injection of Rose Bengal but were not photostimulated (n=16, aged P21) or were photostimulated after vehicle (NaCl) i.v. injection without Rose Bengal (n=6, aged P21). In 3 rats the injury extended into hippocampus and striatum. These animals were excluded from the study.
Control or littermate injured Sprague Dawley rats (P28 to P35) were anesthetized with pentobarbital (200 mg/kg, i.p.) and decapitated. The brains were rapidly removed and immersed in an ice-cold (4°C) slicing solution containing (in mM): 234 sucrose, 2.5KCl, 1.25 NaH2PO4, 10 MgSO4, 0.5 CaCl2, 26 NaHCO3, and 11 glucose, equilibrated with 95% O2 and 5% CO2, pH 7.4. Horizontal thalamic slices (250–270 μm) containing the thalamic ventrobasal complex (VB) and reticular nucleus (nRT) were cut with a Lancer Series 1000 vibratome (Vibratome, St. Louis, MO). The slices were incubated, initially at 32°C for 1 h and then at room temperature, in artificial cerebrospinal fluid (ACSF) containing (in mM): 126 NaCl, 2.5 KCl, 1.25 NaH2PO4, 2 MgCl2,2 CaCl2, 26 NaHCO3, and 10 glucose, equilibrated with 95% O2 and 5% CO2, pH 7.4. In some experiments barium chloride (BaCl2, 1 mM) was added to the superfusate.
Recordings were made at room temperature (22–25 °C) using whole-cell patch-clamp methodology. Following incubation, brain slices were transferred to the recording chamber and superfused with ACSF at a flow rate of 2 ml/min. Recordings were obtained from nRTneurons visually identified using differential contrast optics with a Zeiss (Oberkochen, Germany) Axioskop microscope and an infraredvideo camera.
Recording electrodes made of borosilicate glass had a resistance of 2–4 MΩ when filled with intracellular solution. For IPSCs, the internal solution contained (in mM): 135 CsCl, 10 Hepes, 10 EGTA, 5 QX-314, 2 MgCl2. pH was adjusted to 7.3 with CsOH (290 mOsm). ECl− was estimated ~ 0 mV based on the Nernst equation. During IPSC recordings, neurons were clamped at −65 mV and events were pharmacologically isolated by bath application of the ionotropic glutamate receptor blockers 6,7-Dinitroquinoxaline-2,3-dione (DNQX, 20 μM, Ascent Scientific) and 2-Amino-5-phosphonopentanoic acid (AP-5, 100 μM, Ascent Scientific). For EPSCs and current-clamp recordings, the internal solution contained (in mM): 120 K-gluconate, 11 KCl, 1 MgCl2, 1 CaCl2, 10 Hepes, 1 EGTA. pH was adjusted to 7.4 with KOH (290 mOsm). ECl− was estimated ~ −60 mV based on the Nernst equation. Potentials were corrected for −15 mV liquid junction potential. During EPSC recordings, neurons were clamped at −80 mV and events were pharmacologically isolated by bath application of the GABAAR antagonist picrotoxin (50 μM, Tocris). In all recording conditions, access resistance was monitored and cells were included for analysis only if the series resistance was <20 M Ω and the change of resistance was <25% over the course of the experiment. In addition, adequacy of voltage clamp was demonstratedby T-current steady-state inactivation (SSIT) protocols in which peak latencies remained constant after voltage-clamp steps to −75 mV from a range of hyperpolarizing conditioning potentials (Figure 6A1–A2, dashed lines). Current amplitude from the inactivation protocol, normalized to maximum, was plotted as a function of prepulse membrane potentials, and best-fitted with a Boltzmann function: I/Imax = (A1−A2)/(1 + e(V−V50%)/k) + A2 where V50% and k represent the half-maximal voltage and Boltzmann slope factor, respectively; A1 and A2 represent initial and final I/Imax values, respectively. SSIT was used as a measure to physiologically identify nRT cells (Huguenard and Prince, 1992; Deleuze and Huguenard, 2005).
Evoked EPSCs in nRT were obtained with a concentric bipolar stimulating electrode positioned in striatum (Str.) or internal capsule (ic) (Figure 8A), activating corticothalamic and thalamocortical fibers. A minimal stimulation protocol (50 % failures and 50 % successes) was used to activate putative single thalamocortical or corticothalamic glutamatergic fibers. Single and train stimuli (5 pulses at 50–200 Hz) were applied. Evoked IPSCs were obtained with the stimulating electrode positioned in nRT ~100–200 μm from the recording electrode to activate intra-nRT axons. A minimal stimulation protocol (50 % failures and 50 % successes) was defined as threshold and evoked currents were recorded with the stimulation intensity set at 1.5X threshold (Huntsman et al., 1999) (Figure 9B4). Frequency-current (F-I) curves were obtained by applying depolarizing square current pulses from a holding potential of −80 mV, and F-I curve slope was calculated by measuring action potential (AP) frequency during the last 100 ms of 400 ms of depolarization to minimize contributions of LTS bursts.
A Digidata 1320 digitizer and pClamp9 (Molecular Devices) were used for data acquisition and analysis. Signals were amplified with an Axopatch 200A amplifier (Molecular Devices), sampled, and filtered at 10 kHz. EPSCs and IPSCs were detected and analyzed with wDetecta, a custom postsynaptic current detection program (http://huguenardlab.stanford.edu/apps/wdetecta). The amplitude of action potentials was calculated as the potential difference between their voltage threshold and the peak of the waveform. Numerical values are given as means ± SEM unless stated otherwise. Statistical box charts show: mean (central dot); median (large horizontal line); maximal and minimal values (short horizontal lines); 99 % and 1 % range (crosses); percentile 25–75 range (box); 5–95 range (whiskers). Statistical significance was assessed by performing a one-way ANOVA, t-test or Mann-Whitney Rank Sum test. Statistical analyses were performed with Sigma Stat 3.5 (Ashburn, VA) and Origin 7.0 (Microcal Software, Northampton, MA). For evoked EPSCs the peak-to-baseline decay phase of the resulting current trace was fitted by the double exponential function: I = A1e−t/τ1 + A2e−t/τ2; where A1 and A2 are the slow and fast amplitude components, and τ1 and τ2 are the slow and fast decay time constants, respectively. The weighted decay time constant (τD,W) was calculated using the equation: τD,W = (τ1A1+τ2A2)/(A1+A2).
During electrophysiological experiments, nRT cells were filled with 0.2–0.5 % biocytin (Sigma, St. Louis, MO) included in the internal solution. At the end of the recordings, whole slices were then fixed with 4% paraformaldehyde solution and processed using the standard avidin–biotin peroxidase method (Horikawa and Armstrong, 1988; Tseng et al., 1991) or immunofluorescent staining. nRT cells recorded from control and injured animals were located at the same dorsal coordinates. Biocytin-filled cells were reconstructed 3-dimensionally (Figure 3B) using a Zeiss Axioskope Microscope attached to a Neurolucida system (MBF Bioscience). Dendrites and axonal arbors were traced at 50X and putative axonal boutons were marked to allow for differentiation between small (<0.5 μm) and big boutons (>0.5 μm) (Figure 3C,D). Dendritic branching was examined by using a Sholl analysis, which measures the degree of branching as a function of distance from the soma (Figure 3B) (Sholl, 1953). Briefly, a series of concentric circles were centered on the soma at 10 μm intervals, and the number of dendrites intersecting each circle was counted (Figure 3B). Fluorescent biocytin images were obtained using a confocal microscope with a 63× oil-immersion objective. The angle and circularity (circularity=4π•area•perimeter−2) of the parvalbumin labeled nRT cells were calculated using ImageJ (Rasband, 2006). A circularity value of 1.0 indicates a perfect circle. Angle is the angle of the major axis of the cell body, which was used to quantify the variability in orientation of individual nRT cells within the nucleus.
In some cases, 6–60 days after injury, animals were anesthetized with pentobarbital (200 mg/kg ip) and perfused transcardially with saline followed by 4% paraformaldehyde (Sigma, St Louis, MO) in 0.1 M phosphate buffer (Sigma, St Louis, MO), pH 7.4. The brains were removed and post-fixed in 4% phosphate-buffered paraformaldehyde at 4 °C overnight. Digital images were taken for documentation of the position and extent of lesions (Figure 1A) and the brains were cryoprotected with 30 % sucrose buffer and frozen on dry ice. Horizontal or coronal 50 μm sections were obtained with a sliding freezing microtome (Microm, HM 400; Heidelberg) and Nissl stained for histological analysis of the cortical injury induced by Rose Bengal photothrombosis (Figure 1B). Free floating sections were pre-incubated twice with 50% alcohol for 10 minutes each time and later in 10% normal goat serum in phosphate-buffered saline, followed by incubation with the primary antibody against glial fibrillary acidic protein (GFAP; polyclonal, 1:500; Millipore, Temecula, CA) done in conjunction with neuron-specific nuclear protein (NeuN;monoclonal, 1:1000; Millipore, Temecula, CA) or with parvalbumin (PV; monoclonal, 1:1000; Sigma, St Louis Missouri) at 4 °C overnight on a shaker. Other sets of sections were incubated with Neurofilament-200 KD (NF) monoclonal antibody (Sigma, St Louis Missouri) at a concentration of 1:100 in conjunction with Parvalbumin polyclonal antibody (Millipore Temecula, CA) at a concentration of 1:1000. The T-type channel Cav3.3 polyclonal antibody (1:500) (kindly provided by Terrance P. Snutch, University of British Columbia) was combined with parvalbumin monoclonal antibody at a concentration of 1:1000 (Sigma, St Louis Missouri). The slices were rinsed in PBS and incubated with the correspondent fluorescent second antibodies (Molecular Probes, Eugene, Ore) at a concentration of 1 ug/ml. Sections were mounted on slides using Vectashield Mounting Media (Burlingame, CA) and the double immunofluorescence was assessed with a laser confocal microscope (Zeiss LSM 510).
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 (Figure 1A). 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 (Figure 1B) 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 Figure 1C 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.
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) (Figure 2B2–F2). The cell loss in VPL was consistently co-localized with a strong GFAP labeling (Figure 2D2) 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; Figure 2C2). Neither cell loss nor gliosis were observed in the contralateral thalamus (Figure 2A1–F1) (n=6 animals).
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 Figure 3A, 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 (Figure 3A, crossed arrows).
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 (Figure 3A, ,6D).6D). 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 (Figure 3A). 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 (Figure 3A) (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 (Figure 3A) 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.
The dendritic and axonal changes were assessed following a three-dimensional reconstruction of nRT neurons filled with biocytin (Figure 3C–D). 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; Figure 3C–D). Cortical infarct resulted in (1) decreased mean dendritic length (Table 1); (2) decreased mean number of dendritic arborizations (see Figure 3B); (3) decreased mean axonal length (Table 1); 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 (Table 1). 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) (Figure 3C) but also in those projecting to VPM (Figure 3D), which was not affected by cell death or gliosis (Figure 2). 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).
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 (Figure 4A,B). 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 (Figure 4C1–2,D,F1–2). The membrane capacitance (Cm) was unchanged (Figure 4F3) 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 (Table 2). In both cell populations, an inward rectification was observed in response to negative current pulses inducing hyperpolarizations past −95 mV (Figure 4D). This inward rectification was likely caused by the hyperpolarization-activated inward cationic current (Ih) and inwardly rectifier K+ current (IKir) (Abbas et al., 2006; Rateau and Ropert, 2006). F-I analysis (Figure 4E1–2; Table 2) 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; Table 2). Moreover, the injured cells reached a maximal firing rate of only ~60 Hz (Figure 4E1–2), 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.
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 (Figure 4A, C1–2 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 (Figure 5A, middle panel; Lesion center; see the protocol Figure 5B 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 (Figure 4A vs B, C2, ,6A)6A) and its kinetics were altered. Specifically, the LTS decayed more rapidly in the injured cells compared to the controls (Figure 5C). 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 (Figure 5A), 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 5C).
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).
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 Figure 6A1 (bottom).
The injured cells exhibited an abnormal voltage dependence of T-type calcium currents (Figure 6A3, left panel), with the steady-state inactivation (SSI) curve showing a hyperpolarized shift compared with the control nRT cells (Figure 6A3; 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 Figure 4). 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.
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; Figure 6B1,B2 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; Figure 6B2 right). Thus the difference in peak amplitude cannot be accounted for by cell size.
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) (Figure 6C1–2). 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.
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/Cav3.1, α1H/Cav3.2 and α1I/Cav3.3 (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 Cav3.3 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 Cav3.1 and 12 mV compared with Cav3.2 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, Cav3.3 immunolabeling was dramatically decreased in nRT ipsilateral to the cortical injury (Figure 5D) 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).
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 Figure 5B 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) (Figure 5A). This result is in agreement with the decreased amplitude of T-current in the injured cells (Figure 6B1–2).
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) (Figure 5B). In nRT neurons adjacent to the injured VPL (Figure 5B 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) (Figure 5B 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 (Figure 5B lesion far). Specifically, (1) following weak hyperpolarization the latency of the peak was delayed by ~100 ms compared to the control cells (Figure 5B 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) (Figure 5C). This result is in agreement with the voltage-clamp recording of the T-currents, which showed a faster decay time in the injured cells (Figure 6C1–2).
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 (Figure 7A,B). 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 Table 2), 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; Figure 7C). Bath application of 1 mM Ba2+ induced an inward current in nRT cells from both control and injured animals (Figure 7A,B). 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; Figure 7C left). The effect of Ba2+ at this potential could be due to the block of a weakly rectifying Kir-current (Bichet et al., 2003).
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; Figure 7C right). However, 1 mM Ba2+ application strongly reduced I–V slope in nRT cells from both control and injured animals (Figure 7A2,B2), corresponding to a 44 % increase in Rin in the control cells, and, interestingly, to a much stronger increase (160 %) in the injured cells (Figure 7C 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; Figure 7C 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.
Interestingly, Ba2+ 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 Figure 4A–C, 6A–D) and the hyperpolarized shift in the SSI curves of injured cells (Figure 6A3) are not due to the leak but rather a loss and/or a change in the T-type calcium channel subunit expression.
In order to examine if glutamatergic synaptic transmission was altered by cortical infarct we stimulated cortical and thalamic afferents in the internal capsule (Figure 8A) 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.
EPSCs were evoked in nRT neurons by stimulating the internal capsule (Figure 8A) 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) (Figure 8B1).
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) (Figure 8B2). 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).
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 Figure 8C1–3) 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).
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 (Figure 8C1; 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 Figure 8C1,C3 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).
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 (Figure 8D 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 (Figure 8D 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 (Figure 8D at the top: compare right versus left panels) and neither LTS nor action potential firing were observed at more hyperpolarized levels of membrane potential (Figure 8D, at the bottom: compare right versus left panels).
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 (Figures 1, ,2)2) and reductions in evoked EPSC (Figure 8B1), 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 Figure 9A1). We next examined if the kinetics of the isolated spontaneous EPSCs events were altered following injury (Figure 9 A2–3). 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 Figure 9B1). 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; Figure 9B2–3). 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; Figure 9B4).
The present study provides the first complete description of intrinsic and synaptic plasticity in nRT following focal cortical strokes and demonstrates how, despite compensatory changes in synaptic excitation, altered intrinsic properties result in a reduced responsiveness of nRT that persists during epileptogenesis. Briefly, in the rat photothombosis model of stroke (Watson et al., 1985; Kharlamov et al., 2003), we showed that a local infarct in the somatosensory cortex induces (1) cell death and gliosis specifically in VPL by the end of the first week; (2) morphological changes in nRT cells ipsilateral to the infarct and, in particular, a decrease in the number and proportion of large vs small boutons on axons from nRT cells within the affected (VPL) and non-affected (VPM) thalamic relay nuclei; (3) decreased intrinsic excitability of nRT cells due to an increased Kir-conductance; (4) reduced post-inhibitory rebound of excitation and a reduced ability to fire bursts of action potentials; (5) decreased expression of Cav3.3 T-type channels; (6) decreased amplitude and decay time of T-currents; (7) hyperpolarized shift in the steady-state inactivation of T-currents; (8) no change in the spontaneous synaptic excitatory and inhibitory activity; and (9) decreased paired-pulse ratio and altered synaptic efficacy. These results indicate that cortical thrombosis decreases the intrinsic excitability and reduces evoked synaptic excitatory currents in nRT neurons, which together might produce a powerful decrease in inhibitory drive onto thalamocortical relay cells and an overall increase in thalamocortical activity.
One of the main findings of this study is the decreased intrinsic excitability of nRT cells after a cortical infarct. Specifically, the 30 % decrease in membrane Rin and τm were complete by the end of the first week after the cortical infarct. We showed that the leak resulted from an increased Kir-conductance after injury. Indeed, in presence of Ba2+, which blocks IKir without affecting Ih (Pape, 1996), Rin of injured and control nRT cells were similar. In both control and injured cells, IKir-induced leak was higher at hyperpolarized membrane potentials (<−105 mV) compared to the resting potential (~−70 mV). This result was expected because IKir is normally expressed by nRT neurons and underlies ~50% of leak <−100 mV (Rateau and Ropert, 2006). Interestingly, in injured nRT cells compared to controls, Kir-conductance was increased by the same factor at <−105 mV and at ~−70 mV, suggesting that this conductance will shunt synaptic depolarizations at the resting membrane potential. The mechanisms leading to increased IKir in nRT neurons remain uknown. However, upregulation of Kir2-channels has been reported in dentate gyrus granule cells in temporal lobe epilepsy (Young et al., 2009) and was suggested to be a local adaptive mechanism that could counterbalance hyperexcitability in epileptic hippocampus.
The second key finding is the loss or reduction of LTS and the decreased amplitude and decay kinetics of T-currents after cortical infarct. Blockade of the leak channels was not able to restore the LTS, suggesting that its loss or decrease was not due to the leak, but rather to loss of T-type calcium channels. In thalamus, differences in distribution and kinetic properties of T-currents influence the nature of oscillatory output involved in both the normal sleep-wave cycle and the abnormal spike-and-wave discharges of absence epileptic seizures (Huguenard et al., 1993; Pape et al., 2004; Joksovic et al., 2006). Molecular analyses have identified three isoforms of T-type channels (Cav3.1, Cav3.2, Cav3.3) (Lee et al., 1999; McRory et al., 2001; Perez-Reyes, 2003) all expressed in nRT neurons (McKay et al., 2006). Cav3.3 channels are (1) 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. Therefore, we tested the hypothesis that this isoform was lost in nRT after injury, leading to reduced T-current amplitudes, faster decay kinetics and hyperpolarized half-inactivation voltages. Indeed, the cortical infarct led to a strong reduction in the expression of Cav3.3 in ipsilateral nRT neurons, especially those adjacent to VPL, which is damaged by cell loss and gliosis. Cav3.3 loss was less prominent in nRT cells located >300 μm from the lesion, explaining why LTS was less affected (but still abnormal) in those cells. Because the hyperpolarized shift in half-inactivation voltages of T-currents in the injured nRT cells was also observed after blockade of leak channels, it is unlikely that it resulted from the increased leakiness.
Our results suggest that the decreased T-current amplitude and decay kinetics, as well as its hyperpolarized steady-state inactivation are due to a loss of Cav3.3 isoform in nRT ipsilateral to the infarct. Thus, Cav3.3 loss, together with the increased Kir leak-conductance, is likely to affect the nature of oscillatory output of nRT onto surviving relay thalamocortical cells and alter sensory processing.
Cortical infarcts led to cell death in VPL, which could alter thalamic sensory information processing (Tokuno et al., 1992; Sela et al., 2009), and to long-lasting (≥2 months) morphological changes in the ipsilateral nRT. Specifically, the dendritic length of partially deafferented nRT cells was decreased and somata lost their characteristic fusiform shape. These changes are likely due, at least in part, to the loss of NF, an important component of cytoskeleton whose decreased expression in thalamus has been reported in various neurological disorders (Lee and Cleveland, 1994; Clinton et al., 2004; Clinton and Meador-Woodruff, 2004 http://www.nature.com/npp/journal/v29/n7/full/1300451a.html). Interestingly, there was a dramatic decrease in the number of boutons on axons emanating from nRT cells within both the damaged thalamic relay nucleus (VPL) and less affected areas of thalamus (e.g., VPM). This result suggests that the cortical infarct affects not only inhibition from nRT in the functionally related damaged thalamus but also in other thalamic relay nuclei, thus resulting in a widespread effect on thalamocortical network excitability. Interestingly, intra-nRT axons did not seem affected (data not shown), which is in agreement with the finding that intra-nRT synaptic inhibition was not altered by the cortical infarct.
Because axon excitability was unchanged, the smaller amplitude of the EPSCs evoked by threshold stimulation, presumably activating a single presynatpic axon, could result from a reduced number of release sites per axon. However, the reduced amplitude of evoked EPSCs was associated with slower decay kinetics, thus maintaining the total charge of the response and suggesting postsynaptic compensation. If slowly decaying evoked EPSCs resulted from a change in specific AMPA GluR subunit composition (Dingledine et al., 1999; Schlesinger et al., 2005), one would expect to observe longer decay in spontaneous EPSCs. One possible explanation for this discrepancy would be activation of different receptors during spontaneous and evoked EPSCs. For example, injury might alter expression of extrasynaptic receptors or glutamate transporters.
Despite a massive loss of glutamatergic afferents to nRT, the mean frequency of spontaneous EPSCs was not reduced in nRT neurons, suggesting compensatory activity in the remaining corticothalamic and thalamocortical fibers. Furthermore, PPR, which is regarded as an index of presynaptic efficacy (Thomson et al. 1993; Markram and Tsodyks 1996; Zucker, 2002), was decreased during 50–200 Hz stimulus trains, suggesting an increased probability of release after injury. This could explain, at least in part, why the frequency of spontaneous EPSCs in nRT cells was not decreased after injury. In addition to PPR, synaptic efficacy can be measured by the time constant of decay of amplitudes of successively evoked EPSCs, and by the train-pulse ratio, which were not altered significantly at 100–200 Hz, suggesting a strong compensation of synaptic efficacy after injury. Thus, the decreased evoked firing of action potentials in nRT cells after injury is unlikely to result from a decreased synaptic efficacy, but rather from a decreased intrinsic excitability due to loss of T-channels and increased Kir-conductance preventing an efficient summation of EPSPs and a sufficient depolarization to elicit a LTS.
Altogether, the results suggest that partially deafferented nRT cells are less likely to be excited and inhibit surviving thalamocortical cells, possibly leading to enhanced thalamocortical activity.
The critical finding of this study is the decreased intrinsic excitability of nRT cells as well as the loss of Cav3.3 channels leading to reduced ability to fire bursts of action potentials. The results demonstrate how, despite unchanged intra-nRT synaptic inhibition and compensatory alterations in excitatory synaptic activity, the change in intrinsic properties affects synaptic integration in nRT and most likely alters its widespread output onto relay nuclei. Future studies will be required to determine the extent to which such changes are dependent on the developmental stage of the animal. The injured thalamus may not initiate seizures (Williams, 1965), but the infarct, via its long-term effect on nRT, could affect the control of thalamocortical output and enhance the intensity of positive excitatory feedback to cortex, which might participate in post-stroke development and spread of epileptiform activity.
We thank Karina Scalise, Astra S. Bryant and Whitney McDonald for assistance with animal husbandry. This work was supported by NS06477 and NS007280.