Enhanced pre-synaptic glutamate release in deep-dorsal horn contributes to calcium channel alpha-2-delta-1 protein-mediated spinal sensitization and behavioral hypersensitivity
© Nguyen et al; licensee BioMed Central Ltd. 2009
Received: 05 December 2008
Accepted: 12 February 2009
Published: 12 February 2009
Nerve injury-induced expression of the spinal calcium channel alpha-2-delta-1 subunit (Cavα2δ1) has been shown to mediate behavioral hypersensitivity through a yet identified mechanism. We examined if this neuroplasticity modulates behavioral hypersensitivity by regulating spinal glutamatergic neurotransmission in injury-free transgenic mice overexpressing the Cavα2δ1 proteins in neuronal tissues. The transgenic mice exhibited hypersensitivity to mechanical stimulation (allodynia) similar to the spinal nerve ligation injury model. Intrathecally delivered antagonists for N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxyl-5-methylisoxazole-4-propionic acid (AMPA)/kainate receptors, but not for the metabotropic glutamate receptors, caused a dose-dependent allodynia reversal in the transgenic mice without changing the behavioral sensitivity in wild-type mice. This suggests that elevated spinal Cavα2δ1 mediates allodynia through a pathway involving activation of selective glutamate receptors. To determine if this is mediated by enhanced spinal neuronal excitability or pre-synaptic glutamate release in deep-dorsal horn, we examined wide-dynamic-range (WDR) neuron excitability with extracellular recording and glutamate-mediated excitatory postsynaptic currents with whole-cell patch recording in deep-dorsal horn of the Cavα2δ1 transgenic mice. Our data indicated that overexpression of Cavα2δ1 in neuronal tissues led to increased frequency, but not amplitude, of miniature excitatory post synaptic currents mediated mainly by AMPA/kainate receptors at physiological membrane potentials, and also by NMDA receptors upon depolarization, without changing the excitability of WDR neurons to high intensity stimulation. Together, these findings support a mechanism of Cavα2δ1-mediated spinal sensitization in which elevated Cavα2δ1 causes increased pre-synaptic glutamate release that leads to reduced excitation thresholds of post-synaptic dorsal horn neurons to innocuous stimuli. This spinal sensitization mechanism may mediate at least partially the neuropathic pain states derived from increased pre-synaptic Cavα2δ1 expression.
Chronic pain derived from nerve injuries, or neuropathic pain, is a common clinical problem affecting over 50 million people in the United States . Neuropathic pain may manifest as spontaneous pain, pain resulting from stimuli that are normally innocuous (allodynia), and increased pain responses to suprathreshold stimuli (hyperalgesia), all of which affect adversely the quality of patients' daily-life. Unfortunately, neuropathic pain tends to be long-term, and difficult to manage due to the poor efficacies and severe side effects associated with current conventional analgesic treatments. It appears that neuropathic pain with various etiologies can lead to similar behavioral endpoints that response differentially to pharmacological agents, suggesting that different mechanisms may underlie specific neuropathic pain states deriving from different pathological conditions. Thus, the development of target specific, safe and effective agents for neuropathic pain management relies on new insights into mechanisms of different neuropathic pain states.
Findings from previous studies have indicated that peripheral nerve injury induces a dramatic upregulation of the voltage-gated calcium channel (VGCC) α2δ1 subunit (Cavα2δ1) in sensory neurons and spinal dorsal horn [2–6], which correlates with neuropathic pain development [2, 3, 7]. Knocking down of injury-induced Cavα2δ1 upregulation in dorsal spinal cord with intrathecal antisense oligodeoxynucleotides results in allodynia reversal in spinal nerve injured rats . In addition, transgenic mice overexpressing Cavα2δ1 in neuronal tissue display tactile allodynia similar to nerve injury models that can be blocked by intrathecal gabapentin , a drug that binds to the Cavα2δ1 proteins with high affinity  and has anti-neuropathic pain properties in patients and experimental animal models [2, 3, 10–16]. Furthermore, blocking axonal transport of injury-induced Cavα2δ1 from DRG neurons to their pre-synaptic terminals in spinal dorsal horn diminishes tactile allodynia in nerve-injured animals . Together, these findings suggest that elevated Cavα2δ1 likely contributes to behavioral hypersensitivity through a pre-synaptic mechanism at the spinal cord level. This hypothesis, however, has not been tested directly.
Since elevated Cavα2δ1 in the transgenic mice has been shown to cause dorsal horn neuron hyperexcitability, behavioral hypersensitivity ; enhanced glutamate neurotransmission is an important mediator in neuropathic pain processing [17, 18]; and elevated Cavα2δ1 may modulate various calcium channels that are known to regulate spinal pre-synaptic neurotransmission [19–25], we examined if elevated Cavα2δ1 mediated deep-dorsal horn neuron excitability and behavioral hypersensitivity through activation of glutamate receptors, and if so, whether it was mediated by a pre-synaptic or post-synaptic mechanism. We tested these hypotheses in the injury-free Cavα2δ1-overexpressing transgenic mouse model showing dorsal horn neuron hyperexcitability and behavioral hypersensitivity without the influence from other nerve injury factors .
A similar tactile allodynia state is developed in spinal nerve injured mice and injury-free Cavα2δ1 transgenic mice
A spinal glutamatergic pathway mediates behavioral hypersensitivity in the Cavα2δ1-transgenic mice
Cavα2δ1-mediated spinal hyperexcitability through enhanced pre-synaptic glutamate release, but not likely through enhanced intrinsic excitability of dorsal horn neurons
We reported previously that elevated Cavα2δ1 in the TG mice, without other injury induced factors, was sufficient to cause WDR neuron hyperexcitability and behavioral hypersensitivity . Data from our previous studies have indicated that intrathecal injection of gabapentin, a drug that binds to Cavα2δ1 proteins and has anti-neuropathic pain efficacy in the SNL model [2, 3, 10–12] and patients [13–15], can reverse the behavioral hypersensitivity in the Cavα2δ1 TG mice , supporting that Cavα2δ1-mediated WDR neuron activation contributes at least partially to the behavioral hypersensitivity. Since the wind-up response, a phenomenon of progressively exaggerated WDR neuron excitability to repetitive C-fiber stimulation, is similar between the TG and WT littermates , the hyperexcitability of the TG dorsal horn neurons is not likely mediated by a facilitated state mediated by the wind-up mechanism.
While previous evidences have indicated a clear correlation between nerve injury-induced Cavα2δ1 upregulation and neuropathic pain states [3, 4, 7], pathways underlying Cavα2δ1-mediated behavioral hypersensitivity have not been identified. Since cellular changes in spinal cord and DRG after a prolonged peripheral nerve injury include altered expression of large numbers of genes [5, 6, 30], multiple factors induced by nerve injury may contribute to neuropathic hypersensitivity. In an attempt to study if upregulated Cavα2δ1 is sufficient to mediate behavioral hypersensitivity, and if so, what is the underlying mechanism, we have generated TG mice overexpressing Cavα2δ1 in neuronal tissues and compared their behavioral sensitivity to mechanical stimulation with the SNL model. Our data indicated that the TG mice have tactile allodynia similar to that observed in the SNL model. Since similar behavioral hypersensitivity and increased Cavα2δ1 expression occur in both models, and other factors such as compensatory changes in the TG mice and other injury-induced factors in the SNL model are not likely the same, our data, while do not allow the complete exclusion of other factors, strongly support that increased Cavα2δ1 proteins are sufficient in mediating tactile allodynia observed in these models.
The pharmacology profiles in allodynia reversal by the spinally delivered, selective glutamate receptor antagonists are consistent with the pharmacology profiles of allodynia reversal by these agents in the SNL model as reported by others . These data suggest that increased Cavα2δ1 mediates allodynia through a pathway related to a selective activation of AMPA/kainate and NMDA receptors. In contrast, metabotropic glutamate receptor antagonist, AP3, was without effect in allodynia reversal. This is consistent with the notion that metabotropic glutamate receptors are mainly involved in acute and persistent pain states associated with input from afferent C-fibers, but less so on tactile allodynia likely mediated by input from Aβ fibers .
Since the TG mice have elevated Cavα2δ1 in different neuronal tissues ranging from DRG in the peripheral to spinal cord and supraspinal cord locations in the central nervous system , it is possible that increased Cavα2δ1 expression in peripheral as well as central locations in the TG mice contributes to the behavioral hypersensitivity through a glutamate receptor-dependent mechanism. Therefore, the enhanced dorsal horn neuronal excitability could derive from altered pre-synaptic neurotransmission, intrinsic post-synaptic neuron excitability or descending modulation from supra-spinal locations in the TG mice. While our data do not allow the exclusion of potential contributions to tactile allodynia by activated AMPA/kainate and NMDA receptors at supra-spinal locations in the TG mice, Chaplan et al.  reported that only spinal, but not i.c.v., injection of NMDA receptor antagonists reversed injury-induced allodynia in the SNL model. This suggests that activation of a spinal glutamatergic pathway is critical in the development of neuropathic pain states post peripheral nerve injury.
Data from this study further demonstrate that, in contrast to the enhanced excitability of TG WDR neurons to natural stimuli reported previously , acutely evoked excitability of WDR neurons to C-fiber-strength electric stimulation is not altered in the TG mice. While mechanisms underlying this discrepancy are not clear, the simplest explanation is that detected WDR neuron excitability to natural stimuli derives from neuronal activation by inputs from multiple sensory fibers including A-fibers, and elevated Cavα2δ1 expression results in a higher excitability of the WDR neuron to A-fiber-, but not C-fiber-, strength stimulation (see discussion below). Thus, it is likely that increased Cavα2δ1 expression mediates tactile allodynia through a Cavα2δ1-dependent pathway by enhancing synaptic strength of A-fibers. Indeed, this hypothesis is consistent with reported findings that: (a) The basal level of Cavα2δ1 expression is relatively high in small DRG neurons, low in large DRG neurons [6, 8], and there is a dramatic increase in Cavα2δ1 expression in large DRG neurons after SNL injury  or in the Cavα2δ1 TG mice ; (b) increased Cavα2δ1 expression correlates with development of allodynia mainly mediated by large Aβ fibers, but not formalin- and carrageenan-induced acute pain states mainly mediated by nociceptive C-fiber inputs in the TG mice , and formalin- or carrageenan-induced pain models do not have increased Cavα2δ1 expression in DRG and spinal cord (data not shown); (c) increased Cavα2δ1 expression in the TG mice does not alter the receptive field sizes of WDR neurons, nor the excitability of these neurons to repetitive C-fiber stimulation (wind-up) ; (d) In the model of sciatic nerve chronic constriction injury with increased Cavα2δ1 expression , a subpopulation of dorsal horn neurons exhibits abnormal afterdischarges to mechanical and thermal stimuli, but the number of dorsal horn neurons showing wind-up phenomenon to repetitive C-fiber stimulation remains unchanged .
The increase in frequency of AMPA/kainate and NMDA receptor mediated mEPSCs in deep dorsal horn neurons from the TG mice suggests that elevated spinal Cavα2δ1 proteins lead to an increased rate of quanta release of excitatory neurotransmitter glutamate from pre-synaptic terminals at the resting state, which in turn, enhances excitability of post-synaptic dorsal horn neurons through mainly AMPA/kainate receptor activation. Since the deep dorsal horn neurons for in vitro and vivo recordings were selected from a similar dorsal horn lamina, and very few non-WDR neurons (which were excluded) were detected in this lamina during neuron sampling in vivo, it is highly likely that a similar population of WDR neurons was examined in our in vivo and in vitro recording. Our data indicate that elevated Cavα2δ1 proteins do not change the amplitudes of AMPA/kainate and NMDA receptor-mediated mEPSCs. This suggests that neither the quantum size of glutamate release nor the intrinsic excitability of post-synaptic dorsal horn neurons to glutamate receptor activation is changed by increased Cavα2δ1 expression. Direct measurements of glutamate release in deep dorsal horn and membrane excitability of WDR neurons from TG mice would further help to clarify these issues.
Nevertheless, findings from this study allow us to propose the following mechanism of Cavα2δ1-mediated tactile allodynia (Fig. 7): At the resting state under normal synaptic functions, the expression levels of Cavα2δ1 in large DRG neurons and A-fiber terminals are relatively low, so as the rate of quanta glutamate release from A-fiber terminals that maintains a normal neuronal excitation threshold to innocuous stimulation. The Cavα2δ1 expression levels in small DRG neurons and C-fiber terminals are relatively high, but may have little effect on glutamate release from C-fiber terminals at a resting state due to the lack of C-fiber inputs. This high level of basal Cavα2δ1 expression in small DRG neurons may set up a ceiling effect for the influence of Cavα2δ1 on WDR neuron excitability to C-fiber or windup stimulations (discussed below).
Mild pre-synaptic membrane depolarization in response to an action potential leads to the opening of VGCC, triggering calcium influx and a quantum release of excitatory neurotransmitter glutamate. Binding of glutamate to post-synaptic AMPA receptors at a resting state causes a conformational change of the receptors that is linked to channel opening, which is terminated by desensitization of the receptors . In addition, glutamate activation of kainate receptors at the pre-synaptic sensory neuron terminals can reduce glutamate release , and that at spinal interneurons can induce inhibitory transmitter release . This heterosynaptic regulation, which is transient due to kainate receptor desensitization, also plays a role in terminating transient activation of post-synaptic dorsal horn neurons under normal physiological conditions . Under normal physiological conditions, released glutamate is insufficient in activating post-synaptic NMDA receptors (Fig. 7A).
After peripheral nerve injuries, injury-induced Cavα2δ1 upregulation in DRG neurons, more so in large DRG neurons, leads to increased axonal transport of Cavα2δ1 proteins to central pre-synaptic terminals, which in turn causes an increased rate of basal release of glutamate through a yet identified mechanism. This "glutamate leaking" causes partial sensitization of deep dorsal horn neurons by activating mainly AMPA/kainate receptors at the resting state. These "sensitized" dorsal horn neurons therefore have lowered excitation thresholds to a subsequent release of glutamate in response to innocuous or mild nociceptive stimuli that could evoke further conformational change of the AMPA/kainate receptors and prolonged channel opening, leading to spinal hyperexcitability. In addition, signals of mild noxious stimulation could be further amplified in these sensitized dorsal horn neurons due to their low thresholds to NMDA receptor activation, leading to exaggerated painful sensations (Fig. 7B). These hyperexcitability states are sensitive to antagonists of AMPA/kainate and NMDA receptors. However, neither the partial neuron sensitization by glutamate leaking from A-fiber terminals nor the increased Cavα2δ1 expression in C-fiber terminals has any detectable effect on WDR neuron excitability to high intensity C-fiber stimulations (the ceiling effect discussed above).
The detail mechanisms regarding how elevated Cavα2δ1mediates enhanced pre-synaptic glutamate release remain elusive (Fig. 7C). Based on in vitro data indicating that Cavα2δ1 expression is required for normal membrane expression of functional VGCC [36–40], which is critical in mediating pre-synaptic neurotransmission, it is possible that elevated Cavα2δ1 leads to an enhanced VGCC expression at the pre-synaptic terminals. However, there is no direct evidence so far to support this hypothesis. Alternatively, recent findings in drosophila point to the direction that Cavα2δ plays a critical role in formation of pre-synaptic structures and active zones related to pre-synaptic neurotransmission . Indeed, it has been proposed that the ability of descending excitatory 5HT3 mediated projections to induce a state-dependency in the actions of gabapentin may also occur with the monoamine receptor interacting with pre-synaptic mechanisms at the spinal level . Thus, it is possible that increased Cavα2δ1 causes enhanced synaptic strength and/or synapse numbers by promoting formation of more active zones through a yet identified mechanism. These two possibilities may not be mutually exclusive, and deserve further investigation.
In conclusion, findings from this study support that elevated Cavα2δ1 causes spinal hypersensitivity and tactile allodynia through at least partially enhanced pre-synaptic glutamate release, which may lead to sensitization of post-synaptic dorsal horn neurons by lowering their thresholds to innocuous stimuli. This spinal sensitization may contribute to the neuropathic pain states derived from increased pre-synaptic Cavα2δ1 expression.
Transgenic mice over-expressing the Cavα2δ1 gene were generated with a transgenic vector containing the mouse brain Cavα2δ1 cDNA (Genbank accession number U73484) down-stream of a 6.5 kb murine thy-1.2 gene extending from the promoter region to the intron after exon 4 without exon 3 and its flanking introns as described previously . Thy-1 is a member of the immunoglobulin superfamily that is expressed in both neuronal and non-neuronal tissues , and deletion of exon 3 and its flanking introns has been shown to eliminate expression in non-neuronal cells . Only adult male TG mice and their WT littermates were used for the experiments. All the mice were fertile, appeared normal with respect to grooming, social interactions and feeding, and showed no signs of abnormality or any obvious motor defects, tremor, seizure, or ataxia .
All animal care and experimental procedures were performed based on protocols approved by the Institutional Animal Care Committees of the University of California, Irvine, University College London and Indiana University.
The surgical procedure of SNL was performed as described . Briefly, under isoflurane anesthesia, the left L5 spinal nerve of mice were exposed and ligated with a silk suture distal to DRG and proximal to their conjunction to form the sciatic nerve. Sham operations were performed in the same way except that spinal nerves were not ligated.
Behavioral response to mechanical stimulation was tested blindly as described before and post drug treatments . Briefly, mice were allowed to acclimatize for 1 hr in a clear plastic cage with a wire mesh bottom. The 50% paw withdrawal thresholds (PWT) to calibrated von Frey filament (Stoelting, Wood Dale, IL) stimulation were determined using a modified up-down method of Dixon . A series of filaments, starting with one that has a buckling weight of 0.41 g, were applied in consecutive sequence to the plantar surface of the left hindpaw with a pressure causing the filament to buckle. Paw lifting within 5 s was considered a positive response and led to the use of the next weaker filament. Absence of a paw lifting after 5 s led to the use of the next filament with increasing weight. The 50% paw withdrawal threshold was calculated as described before  from the resulting scores of six measurements starting from the one prior to the first positive response; or until four consecutive positive (assigned a score of 0.01 g) or five consecutive negative (assigned a score of 3 g) responses had occurred. Paw withdrawal thresholds from each hindpaw of a SNL mouse were recorded separately, or averaged paw withdrawal thresholds from both hindpaws of the WT and injury-free Cavα2δ1 TG mice were used for comparing sensitivities between injury and non-injury side in SNL mice or between injury-free WT and TG mice, respectively, with or without drug treatment.
Drug working solutions were prepared in sterile saline before intrathecal injections between lumbar regions 5–6 (5 μL/mouse) . In some cases that multiple drugs were used in the same group of animals, a three-day drug-free period was introduced between each drug to ensure that the previous drug was completely out, even though all the drug effects in this study were terminated within 24 hr post injection.
Electrophysiological recordings in dorsal horn neurons in vivo
Recordings were performed by examiners blinded to the mouse genotype. Animals were placed in a stereotaxic frame under anesthesia and the L3–L6 spinal segments were exposed with a laminectomy. Extracellular recordings were made from individual wide dynamic range neurons using parylene coated tungsten electrodes (A-M Systems, USA). Neurons were visualized, isolated, and discriminated based on their spike amplitudes and waveforms. All neurons used for recording had receptive fields over the hindpaw. Depth of the neuron was measured from the surface of the spinal cord. Spontaneous activity was quantified over a period of at least 100 s in the absence of any evoked stimulus on isolation of a neuron.
Electrical stimulation was given via two needles inserted into the receptive field and the evoked activity was characterized and quantified as follows. A train of 16 stimuli was given (2 ms pulse duration, 0.5 Hz at three-times the C-fiber threshold), and the evoked neuronal responses were quantified using online analysis (Spike 2 software, CED Cambridge UK). The evoked neuronal responses were superimposed as a post-stimulus histogram and responses identified – based on fiber conduction velocity and the resulting latency – as C-fiber-evoked (50–250 msec) action potentials. Neuronal responses occurring after the C-fiber latency band of the neuron (250–800 ms) were classed as post discharge. The 'input' (non-potentiated response) was determined as: Input = [action potentials (50–800 ms) evoked by first pulse at 3 times C-fiber threshold] × total number of pulses.
Electrophysiological recordings in dorsal horn of spinal cord slices
Recordings were performed by examiners blinded to the mouse genotype. The animals were anesthetized with ketamine-HCl (1 mg/kg, i.p.) and the spinal cord was extracted by hydraulic extrusion. Transverse slices (330 μm) were cut from the L5 lumbar enlargement using a vibratome (VT1000S; Leica, Nussloch, Germany) in an ice-cold (4°C) sucrose solution containing (in mM): 230 sucrose, 26 NaHCO3, 2.5 KCl, 1.25 NaH2PO4, 0.5 CaCl2, 10 MgSO4, 10 glucose, pH 7.4, 290–305 mOsm/L, equilibrated with 95% O2 and 5% CO2. The slices were maintained in an artificial cerebrospinal fluid (ACSF) containing (in mM): 130 NaCl, 3 KCl, 2 CaCl2, 2 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, and 10 glucose, pH 7.4, 295–305 mOsm/L. The ACSF was continuously equilibrated with 95% O2 and 5% CO2, and slices were incubated for > 1 h before recording. Recording electrodes were prepared from borosilicate glass (World Precision Instruments, Sarasota, FL) using a horizontal electrode puller (P-97; Sutter Instruments, Novato, CA). Electrodes had resistances of 2–4 MΩ when filled with an intracellular solution containing (in mM): 92 CsMeSO4, 43 CsCl, 1 MgCl2, 2 EGTA, 5 TEA, 10 HEPES and 2 Mg-ATP, pH 7.4, 295–300 mOsm/L. Oxygenated ACSF was used as bath solution, and the flow rate was adjusted to 2–3 mL/min. Depth of the neuron was measured from the surface of the spinal cord and neurons in the deep dorsal horn were visualized with an infrared-differential interference contrast (DIC) microscope (BX50WI; Olympus Optical, Tokyo, Japan) and a CCD camera. All recordings were performed at 25 ± 1°C with an Axopatch 200B amplifier (Molecular Devices, Foster City, CA). During whole-cell recordings, series resistance (8–15 MΩ) was monitored periodically, and cells with > 15% changes were excluded from the analysis. Signals were filtered at 1 kHz and digitized at a sampling rate of 5 kHz using a data-acquisition program (Axograph 4.6; Molecular Devices).
To record mEPSCs, tetrodotoxin (1 μM) and bicuculline (30 μM) were continuously applied in the bath solution. At a holding potential of -70 mV, the recorded mEPSCs were mainly mediated by AMPA/kainate receptors because application of AMPA/kainate receptor antagonist CNQX (10 μM) could reduce mEPSCs by 91.3 ± 5.3% and 93.9 ± 3.3% in WT and TG neurons (n = 5 in each group), respectively. In the presence of CNQX (10 μM), NMDA receptor-mediated mEPSCs were recorded at a holding potential of +50 mV.
Indicated significant changes were defined by the two-tailed p values as determined with unpaired Student's t test, or two-way ANOVA followed by Bonferroni post-tests.
Supported in part by NIH grants: NS40135, DE14545 (Z. D. Luo). We would like to thank Mr. Timothy Yolo for his assistant in maintaining the transgenic mice. David Nguyen was supported by a Medical Student Anesthesia Research Fellowship from the Foundation for Anesthesia Education and Research.
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