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Inward currents induced by ischemia in rat spinal cord dorsal horn neurons
© Chen et al; licensee BioMed Central Ltd. 2007
Received: 21 March 2007
Accepted: 25 April 2007
Published: 25 April 2007
Hypoxia and ischemia occur in the spinal cord when blood vessels of the spinal cord are compressed under pathological conditions such as spinal stenosis, tumors, and traumatic spinal injury. Here by using spinal cord slice preparations and patch-clamp recordings we investigated the influence of an ischemia-simulating medium on dorsal horn neurons in deep lamina, a region that plays a significant role in sensory hypersensitivity and pathological pain. We found that the ischemia-simulating medium induced large inward currents in dorsal horn neurons recorded. The onset of the ischemia-induced inward currents was age-dependent, being onset earlier in older animals. Increases of sensory input by the stimulation of afferent fibers with electrical impulses or by capsaicin significantly speeded up the onset of the ischemia-induced inward currents. The ischemia-induced inward currents were abolished by the glutamate receptor antagonists CNQX (20 μM) and APV (50 μM). The ischemia-induced inward currents were also substantially inhibited by the glutamate transporter inhibitor TBOA (100 μM). Our results suggest that ischemia caused reversal operation of glutamate transporters, leading to the release of glutamate via glutamate transporters and the subsequent activation of glutamate receptors in the spinal dorsal horn neurons.
Glutamate is the principle neurotransmitter that mediates sensory transmission in the spinal cord dorsal horn. Under physiological conditions, glutamate is released synaptically by primary afferent fibers, descending terminals from supraspinal regions, and excitatory interneurons in the spinal cord dorsal horn . The synaptically released glutamate is rapidly taken up through glutamate transporters located at presynaptic terminals, postsynaptic cells, and on the surrounding glia cells [2–5]. These transporters keep extracellular glutamate at low levels to ensure high fidelity sensory transmission, to limit nonspecific neuronal excitation and hyperactivity, and to prevent excitatory toxicity [3, 6].
Increased glutamate concentrations in extracellular spaces can occur as a consequence of CNS tissue injury, which in turn can produce neuronal hyperactivity and secondary neuronal tissue damage due to excitatory toxicity . It has been shown that extracellular glutamate levels increased significantly in the brain following ischemic and hypoxic injury [8, 9]. In the spinal cord, ischemia and hypoxia can occur under a number of pathological conditions including traumatic spinal cord injury, tumors within the spinal cord, spinal stenosis, cardiac arrest, massive hemorrhagic shock, and surgical procedures [10–14]. These conditions often cause spinal blood vessel compression, resulting in spinal cord ischemia and hypoxia. Similar to the brain, spinal cord ischemia and hypoxia also can result in the increases of extracellular glutamate levels to cause neuronal excitatory toxicity in the spinal cord. When these pathological processes occur in the dorsal horn of the spinal cord, sensory functions may be significantly altered to result in pathological pain states.
Leak of glutamate from damaged cells and release of glutamate from synaptic sites were thought to contribute to the elevation of extracellular glutamate concentrations under pathological conditions. However, studies have suggested that a change of glutamate transporter function plays a critical role in the sustained elevation of extracellular glutamate levels during ischemia and hypoxia [9, 15]. Under physiological conditions, glutamate transporters co-transport one glutamate molecule and 3 Na+ ions into the cell to maintain the concentration gradient of micromolar extracellular glutamate against millimolar intracellular glutamate [16, 17]. This active transport function is supported by the transmembrane ion gradients established by Na+-K+ ATPase [16, 17]. Under pathological conditions, for example, during brain ischemia and hypoxia, ATP is depleted and Na+-K+ ATPase function is impaired. This subsequently results in the loss of transmembrane ion gradients and thereby reducing the driving force for the active uptake of glutamate from extracelular glutamate . In fact, studies using brain tissues suggested that the depletion of intracellular energy not only compromises glutamate uptake, but also can result in glutamate release through glutamate transporter system due to the reversal operation of the glutamate transporters .
In the present study, we tested the hypothesis that ischemic condition results in the reversal operation of glutamate transport system to cause glutamate release and subsequent excitation of sensory neurons in the spinal cord dorsal horn. The study may have implications in pathological pain states associated with ischemic and hypoxic conditions in the spinal cord dorsal horn .
In this study, we show that ischemia induced significant inward currents in dorsal horn neurons of the spinal cord and that the inward currents could be completely blocked by ionotropic glutamate receptor inhibitors as well as by glutamate transporter inhibitors. This observation is consistent with a previous study using brain slice preparations showing that glutamate transporter inhibitors blocked ischemia-induced inward currents . These findings suggest that ischemia may cause reversal operation of glutamate transporters, leading to the release of glutamate via glutamate transporters and the subsequent activation of glutamate receptors in the neurons of the spinal dorsal horn and brain. Our study, however, cannot exclude other mechanisms that may contribute to ischemia-induced membrane depolarization as shown in hippocampal neurons [18, 19].
We observed that the onset of ischemia-induced inward currents took many minutes. This delay in the onset suggests that intracellular energy depletion is a slow process under our experimental conditions and transporter reversal does not occur before intracellular energy is substantially depleted. Interestingly, we have found that the onset times of ischemia-induced currents are age-dependent, being shorter in older animals and longer in younger ones. One explanation for the age-dependence is that the rates of intracellular energy depletion may be different between younger and older animals under ischemic conditions. The onset times of ischemia-induced currents were shortened when primary afferent fibers were stimulated electrically or with the noxious stimulant capsaicin. This is mort likely due to fast energy depletion when primary afferent fibers were stimulated. In addition to the demonstration of ischemia-induced inward currents, we have also showed that inward currents evoked by exogenous glutamate were significantly larger under ischemia condition than under normal condition. This is probably because the actual concentrations of exogenous glutamate that reached the recorded neurons were higher under ischemia condition than under normal condition. This result suggest that prior to the reversal operation of glutamate transporters, glutamate uptake is severely compromised under ischemia condition. Glutamate transporters are expressed on both neuronal and glia cells in the spinal cord dorsal horn [4, 5]. To date, five subtypes of glutamate transporters have been cloned: GLAST (EAAT1), GLT-1 (EAAT2), EAAC-1 (EAAT3), EAAT4 and EAAT5 . GLAST, and GLT-1 are predominantly localized in astrocytes [2, 20, 21], while EAAC-1, EAAT4, and EAAT5 appears to be mostly neuronal [22–25]. While both neuronal and glial glutamate transporters actively participate in the uptake of extracellular glutamate [26, 27]. Glutamate-induced excitatory toxicity under ischemia conditions appears to be mainly due to the impairment of glial glutamate transporters [3, 28, 29].
Previous studies have demonstrated that TBOA blocks glutamate uptake under physiological conditions . We showed that ischemia-induced glutamate release was inhibited by glutamate transporter inhibitor TBOA in the present study. These results together suggest that TBOA bi-directionally blocks glutamate transporters. Effects of TBOA on sensory behaviors have previously been studied in both normal animals and animals with pathological pain conditions. In normal animals, intrathecal injection of TBOA was shown to induce nociceptive behaviors, such as licking, shaking, and caudally directed biting . These effects were thought due to the block of glutamate uptake by TBOA, which subsequently results in the elevation of extracellular glutamate levels to cause hyperactivity in the spinal cord dorsal horn neurons . Interestingly, under pathological pain conditions, glutamate transporter inhibitors were found to produce anti-nociceptive effects. For example, glutamate transporter inhibitors were shown to attenuate the induction of allodynia induced by PGE2, PGF2a, and NMDA , to reduced formalin-induced nociceptive responses, and to attenuate Complete Freund's adjuvant (CFA)-evoked thermal hyperalgesia [5, 33]. In addition, transient knockdown of spinal GLT-1 led to significant reduction of nociceptive behavior in the formalin model [5, 33]. It has been proposed that pathological pain conditions may cause a depletion of intracellular energy in the spinal cord dorsal horn, which subsequently reverses glutamate transporters to release glutamate and to produce hyperactivity in the spinal cord dorsal horn neurons . Therefore, by blocking glutamate release from its transporters, glutamate transporter inhibitors may execute an anti-nociceptive effect under pathological conditions . The present in vitro study supports the rationales for the use of glutamate transporter inhibitors.
Principles of laboratory animal care (NIH publication No. 86-23, revised 1985) were followed in all the experiments described in the present study. Spinal cord slice preparations and patch-clamp recordings were described in details in our previous studies . In brief, Sprague Dawley rats at the postnatal age of 4–21 days were used. Transverse spinal cord slices were prepared from lumbar enlargement of the spinal cords. The thickness of each slice was 400 μm. The spinal cord slices were maintained in a basket submerged in ~200 ml Krebs solution at 24°C. The Krebs solution contained (in mM): NaCl 117, KCl 3.6, CaCl2 2.5, MgCl2 1.2, NaH2PO4 1.2, NaHCO3 25 and glucose 11; the solution was saturated with 95 % O2 and 5% CO2 and had pH of 7.4. In each experiment, a spinal cord slice was transferred to a recording chamber and placed on the stage of an upright IR-DIC microscope. The slice was perfused at ~10 ml/min with the Krebs solution and all experiments were performed at room temperature (24°C). To induce ischemia, the slices were perfused by a ischemic bath solution, which is modified Kreb's solution that had no glucose (replaced with 7 mM sucrose) and contained 1 mM sodium cyanide , and the bath solution was deoxygenated by continuously bubbling with 100% nitrogen.
Individual neurons were identified under an IR-DIC microscope with a 40× water immersion objective. Whole cell patch-clamp recordings were made in deep lamina (lamina V) neurons with electrodes that were filled with an internal solution contained (mM): Cs2SO4 110, TEA-Cl 5, CaCl2 0.5, MgCl2 2, EGTA 5, HEPES 5, pH 7.3. The resistance of electrodes was ~5 MΩ when filled with the internal solution. The access resistance was below 30 MΩ and was not compensated. Signals was amplified and filtered at 2 kHz (Axopatch 200B) and sampled at 5 kHz using pCLAMP 7.0 (Axon Instruments). In all recordings, neurons were voltage-clamped at -30 mV. In some experiments, primary afferent fibers were stimulated by focal electrical stimulation at the dorsal root entry zone or by chemical stimulation with capsaicin. For the focal electrical stimulation, the stimulation intensity was 100 μA and the stimulation frequency was 100 Hz. For chemical stimulation, capsaicin at the concentration of 2 μM was bath applied to stimulate capsaicin-sensitive nociceptive afferent fibers. Pharmacological tests, including the tests of CNQX (20 μM), APV (50 μM), and TBOA (100 μM) were performed by applications of these compound through bath solution. Unless otherwise indicated, all recordings were performed in the presence of 20 μM bicucullin and 2 μM strychnine.
Data were presented as mean ± SEM. Student's t-tests were used for statistical analysis and significance was considered at the level of the p < 0.05.
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