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. Author manuscript; available in PMC: 2019 May 29.
Published in final edited form as: Neurosci Lett. 2018 Apr 5;676:41–45. doi: 10.1016/j.neulet.2018.04.011

Blockade of α2-adrenergic or metabotropic glutamate receptors induces glutamate release in the locus coeruleus to activate descending inhibition in rats with chronic neuropathic hypersensitivity

Ken-ichiro Hayashida 1,2,*, Masafumi Kimuram 2, James C Eisenach 2
PMCID: PMC5960428  NIHMSID: NIHMS959354  PMID: 29627342

Abstract

Locus coeruleus (LC)-spinal noradrenergic projections are important to endogenous analgesic mechanisms and can be activated by local glutamate signaling in the LC. The current study examined the local glutamatergic, GABAergic, and noradrenergic influences on glutamate release in the LC and noradrenergic descending inhibition in rats 6 weeks after spinal nerve ligation (SNL). Intra-LC injection of the α2 adrenoceptor antagonist idazoxan or the group 2 metabotropic glutamate receptor (mGluR) antagonist (RS)-α-Methyl-4-tetrazolylphenylglycine (MTPG) increased withdrawal thresholds in SNL animals and this was reversed by the blockade of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in the LC or α2-adrenoceptors in the spinal cord, but not in normal animals. Neither blockade of GABA-A nor GABA-B receptors in the LC affected withdrawal thresholds in normal and SNL animals. Intra-LC perfusion of idazoxan increased extracellular glutamate in the LC in SNL animals but not in normal animals. Intra-LC perfusion of MTPG increased extracellular glutamate in the LC in both normal and SNL animals. These results suggest that local noradrenaline and glutamate tonically inhibit glutamate release in the LC after peripheral nerve injury and this may contribute to reduced descending inhibition in response to noxious input during chronic neuropathic pain.

Keywords: Locus coeruleus, Endogenous analgesia, Descending inhibition, Glutamate, Noradrenaline

1. Introduction

Although endogenous analgesia is regulated by several peripheral and central mechanisms [4, 5, 26], the descending locus coeruleus (LC)-spinal pathway plays a key role in many circumstances [15]. We recently demonstrated in rats with chronic neuropathic hypersensitivity that peripheral nerve injury reduces noxious stimulation-evoked glutamate release in the LC, subsequently reducing evoked LC neuronal activity and spinal noradrenaline release to impair pain-evoked endogenous analgesia [15]. These data are consistent with clinical observations that patients with established neuropathic pain have a reduced ability to physiologically recruit descending inhibition [17].

Among various neurotransmitters modulating neuronal activity in the LC, glutamate is considered as the primary excitatory regulator, acting through α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors [24]. Glutamate also inhibits its own release via presynaptic group 2/3 metabotropic glutamate receptors (mGluRs) [20]. We recently demonstrated in rat LC that the astroglial glutamate transporter-1 (GLT-1) is essential for maintaining low extracellular glutamate [14], and that peripheral nerve injury decreases expression of GLT-1 to increase basal extracellular glutamate concentrations and thereby reduces evoked glutamate release via presynaptic mGluRs [15]. In contrast to glutamate, GABA acts as the principal inhibitory neurotransmitter on LC neurons via GABA-A and GABA-B receptors [12, 23] and also inhibits evoked glutamate release via presynaptic GABA-B receptors in the LC [25]. We previously reported in rats that spinal nerve ligation (SNL) increases basal GABA release and GABA-synthesizing enzyme glutamic acid decarboxylase immunoreactivity in the LC [28], indicating increased GABA tone after nerve injury. In the LC, noradrenaline stimulates α2 adrenoceptors to auto-inhibit LC neurons [1, 12]. Noradrenaline is also known to reduce presynaptic glutamate release via stimulation of α2 adrenoceptors in the central nervous system [20]. Although peripheral nerve injury may not affect basal noradrenaline release in the LC, it increases expression of the noradrenaline-synthesizing enzyme dopamine-β-hydroxylase and of α2 adrenoceptors in the LC [2, 18], indicating increased noradrenaline signaling capacity after nerve injury.

Although influences of glutamate, GABA, and noradrenaline on LC neuronal activity are well studied, their tonic influences on pain modulation and glutamate regulation in the LC, especially those in chronic neuropathic pain condition, are still not understood. In the current study, we examined the effects in rats 6 weeks after SNL of intra-LC microinjection of the α2 adrenoceptor antagonist idazoxan, the group 2 mGluR antagonist (RS)-α-Methyl-4-tetrazolylphenylglycine (MTPG), the GABA-A receptor antagonist bicuculline, and the GABA-B receptor antagonist CGP 35348 on chronic neuropathic hypersensitivity. We also measured the effects of some of these manipulations on extracellular glutamate in the LC after SNL surgery using in vivo microdialysis.

2. Materials and methods

2.1. Animals

Male Sprague-Dawley rats (Harlan Industries, Indianapolis, IN), weighing 220–280 g, were housed under a 12-h light-dark cycle, with free access to food and water, and all studies were performed during the light portion of the cycle. All experiments were conducted at 6 weeks after SNL surgery under the approval by the Animal Care and Use Committee at Wake Forest School of Medicine (Winston–Salem, NC).

2.2. SNL surgery and LC cannula implantation

Unilateral L5 and L6 SNL was performed as described previously [13]. Briefly, under anesthesia with 2.0% isoflurane, the right L5 and L6 spinal nerves were tightly ligated using 6-0 silk suture. Five weeks after SNL surgery, LC cannula implantation was performed as described previously [7]. Briefly, under anesthesia with 2% isoflurane, a sterile stainless-steel guide cannula (CXG-8 for microdialysis, Eicom Co., Kyoto, Japan, or C315G for drugs injection, Plastic one, Roanoke, VA) was implanted into the right LC. The coordinates for placement of the tip of the guide cannula aiming at the ventral LC area were 9.8mm posterior and 1.4mm lateral to the bregma, and 6.5 mm ventral from the surface of the dura mater, according to the rat brain atlas [21]. Animals were allowed to recover from the surgery at least one week prior to the experiment. After the experiment, all animals received an intra-LC injection of methylene blue (0.5 μl) and were euthanized by an intraperitoneal injection of pentobarbital (150 mg/kg). The brain was removed and sectioned, and the placement of the cannula was verified visually. This study included data from only animals with successful LC cannula placement (170 of 182 rats).

2.3. Drugs administration and behavior test

The doses of antagonists for the current experiments were guided from previous reports [3, 8, 19, 25]. Six weeks after SNL surgery, animals were randomly assigned to drug treatment on two occasions separated by at least 4 days. For intra-LC drug administration, the GABA-A receptor antagonist bicuculline (Sigma-Aldrich Co., St. Louis, MO), the GABA-B receptor antagonist (3-Aminopropyl) (diethoxymethyl) phosphinic acid (CGP 35348; Sigma-Aldrich Co.), and the α2-adrenoceptor antagonist idazoxan hydrochloride (Tocris Bioscience, Bristol, UK) were dissolved in saline. The group 2 metabotropic glutamate receptor antagonist (RS)-α-Methyl-4-tetrazolylphenylglycine (MTPG; Tocris Bioscience) was dissolved in 0.033 M NaOH, diluted with saline to the final concentrations. Drugs (0.5 μl/rat) were injected through the LC guide cannula at a rate of 0.1 μl/min. To block spinal α2-adrenoceptors, idazoxan (30 μg/10 μl/rat) was intrathecally injected via the L5–L6 intervertebral space under brief 2% isoflurane anesthesia using a 30-gauge needle as previously described [15]. Behavioral tests were performed prior to and 30 minutes after administration. The person performing the behavioral test (M. K.) was blinded to drug and dose. Withdrawal thresholds in the hindpaw were measured with a Randall-Selitto analgesimeter (Ugo Basile, Comerio, Italy) as previously described [22]. A cutoff pressure of 250 g was used to avoid potential tissue injury. All animals were trained for 3 days with this apparatus before baseline values were measured.

2.4. Microdialysis for extracellular glutamate in the LC

Glutamate microdialysis in the LC was performed according to our previous report [25]. In rats 6 weeks after SNL surgery, anesthesia was induced with 2% isoflurane and then maintained with 1.5% isoflurane during the study. A heating blanket was used to maintain rectal temperature at 36.5° ± 0.5°C. Microdialysis probes (CX-I-8-01, outer diameter = 0.22 mm, inner diameter = 0.20 mm, length = 1 mm; Eicom Co.) were inserted into the right LC 1 h before the experiment and perfused with Ringer’s solution (1.0 μl/min). Then, after two 30-min baseline samples, idazoxan or MTPG were perfused for 90 min. Glutamate contents in the microdialysates were measured by the high-pressure liquid chromatography system with electrochemical detection (HTEC-500, Eicom Co.), as we previously reported [25]. Noradrenaline concentrations in the first and second microdialysis samples from each animal were averaged as a baseline value. Changes in noradrenaline concentrations during drug perfusion were presented as percentage of baseline.

2.5. Statistical Analysis

We determined the sample size for each experiment based on our previous experience and an a priori power analysis was not performed. Data are presented as mean ± SD. Behavioral data were analyzed by one way analysis of variance (ANOVA) followed by Tukey post hoc test and microdialysis data were analyzed by two-way repeated-measures ANOVA followed by Tukey post hoc test using Sigma Plot software (Systat Software Inc, Chicago, IL). P < 0.05 was considered significant.

3. Results

3.1. Effects of intra-LC antagonists on withdrawal thresholds

SNL resulted in hypersensitivity, as indicated by lower paw withdrawal thresholds (83 ± 10 g, n=61, p<0.001) ipsilateral to nerve injury than those of normal animals (139 ± 24 g, n=24). In SNL rats, intra-LC injection of the α2 adrenoceptor antagonist idazoxan (F2,20 = 18.87, p<0.001) or the group 2 mGluR antagonist MTPG (F2,21 = 14.35, p<0.001) increased withdrawal thresholds in the hindpaw, while the GABA-A receptor antagonist bicuculline (F2,19 = 2.57, p=0.10) or the GABA-B receptor antagonist CGP 35348 (F2,20 = 1.55, p=0.24) did not (Fig. 1A). Post hoc testing revealed that intra-LC idazoxan (30 ng, P < 0.001) or MTPG (2 μg, p < 0.001) significantly reduced hypersensitivity after SNL compared to saline or vehicle, respectively. In normal animals, none of these drugs at these doses affected withdrawal thresholds in the hindpaw (Fig. 1B). Since GABA-A and GABA-B receptor antagonists failed to affect hypersensitivity in SNL animals, we focused on the effects of intra-LC idazoxan and MTPG in the following experiments.

Fig. 1.

Fig. 1

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Effects of intra-LC injected GABA, α2 adrenergic, and mGlu receptor antagonists on mechanical withdrawal thresholds in SNL and normal rats. SNL (A) and normal animals (B) received an intra-LC injection of saline (SAL), CGP 35348 (CGP, 45, 450 ng), bicuculline (BIC, 1, 10 ng), idazoxan (IDA, 3, 30 ng), vehicle for MTPG (VEH), or MTPG (0.4, 2 μg). Withdrawal thresholds in the right hindpaw were measured prior to (baseline) and 30 min after intra-LC injection. Data (mean ± SD) are presented as change from baseline. * vs saline. # vs vehicle.

A simultaneous intra-LC injection of the AMPA receptor antagonist CNQX (0.3 μg) significantly reduced the antihypersensitivity effects from intra-LC injected idazoxan (30 ng, p=0.005, Fig. 2A) or MTPG (2 μg, p=0.002, Fig. 2B), indicating a role of glutamatergic signaling in the LC. Intrathecal idazoxan (30 μg) also significantly reduced the antihypersensitivity effects from intra-LC idazoxan (P<0.001) or MTPG (P<0.001), indicating the importance of spinal α2-adrenoceptor activation. Neither intra-LC AMPA receptor antagonist CNQX nor intrathecal idazoxan affected withdrawal thresholds in intra-LC saline injected SNL animals.

Fig. 2.

Fig. 2

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Role of LC-spinal noradrenergic pathway in antihypersensitivity from intra-LC injected idazoxan or MTPG in SNL rats. (A) SNL animals received an intra-LC injection of saline (SAL) or idazoxan (IDA, 30 ng) with or without an intra-LC CNQX (0.3 μg) or intrathecal idazoxan (IT-IDA, 30 μg). (B) SNL animals received an intra-LC injection of vehicle (VEH) or MTPG (2 μg) with or without an intra-LC CNQX or IT-IDA. Withdrawal thresholds in the right hindpaw were measured prior to (baseline) and 30 min after drug treatments. Data (mean ± SD) are presented as change from baseline. * vs saline alone (A) or vehicle alone (B). # vs intra-LC idazoxan alone (A) or MTPG alone (B).

3.2. Effects of intra-LC perfused antagonists on extracellular concentrations of glutamate

Basal glutamate concentrations in microdialysates from the right LC of SNL animals (1.9 ± 2.5 ng/30 μl, n = 45) were significantly greater than those of normal animals (0.9 ± 0.7 ng/30 μl, p = 0.02, n = 40), consistent with our previous observations [15, 25]. For idazoxan-induced glutamate release in SNL animals, there was a significant main effect of treatment (F2, 66 = 5.46, p = 0.01), but no main effect of time (F3, 66 = 2.53, p = 0.06) or the treatment X time interaction (F6, 66 = 2.00, p = 0.08) (Fig. 3A). On the other hand, in normal animals, intra-LC perfusion of idazoxan did not affect the glutamate concentration in the LC (treatment; F2, 63 = 0.88, p = 0.43, time; F3, 63 = 2.23, p = 0.09, treatment X time interaction; F6, 63 = 1.05, p = 0.40). For MTPG-induced glutamate release in SNL animals, there were significant main effects of treatment (F2, 73 = 7.40, p = 0.003), time (F3, 78 = 5.34, p = 0.002), and the treatment X time interaction (F6, 73 = 4.05, p = 0.001) (Fig. 3B). Similarly, for MTPG-induced glutamate release in normal animals, there were significant main effects of treatment (F2, 60 = 3.94, p = 0.036), time (F3, 60 = 5.04, p = 0.004), and the treatment X time interaction (F6, 60 = 2.47, p = 0.033). Post hoc testing revealed that 100 μM MTPG significantly increased LC glutamate release in both normal (P = 0.029) and SNL (P = 0.002) animals.

Fig. 3.

Fig. 3

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Effects of intra-LC perfused idazoxan and MTPG on extracellular glutamate concentrations in the LC of normal and SNL rats. In normal and SNL animals, (A) idazoxan (IDA, 10, 100 μM), (B) MTPG (10, 100 μM), or vehicle (VEH) was perfused into the right LC through the microdialysis probe for 90 min. Changes in glutamate concentrations in microdialysates are presented over time during drug perfusion as percentage of baseline. Data are presented as mean ± SD.

4. Discussion

Regulation of release of the prototypical excitatory neurotransmitter glutamate has been intensely studied in the spinal cord and brain, yet much less so in the LC. We previously demonstrated in rats with chronic neuropathic hypersensitivity that peripheral nerve injury down-regulates GLT-1 to increase basal extracellular glutamate in the LC, and thereby increases basal activity of noradrenergic neurons [15]. However, the increased basal extracellular glutamate also results in auto-inhibition of evoked glutamate release in the LC, and subsequent impaired pain-evoked endogenous analgesia in chronic neuropathic pain [15]. The current study extends these observations by showing that basal glutamate release in the LC is tonically inhibited not only by glutamate itself but also by noradrenaline in chronic neuropathic pain and removing this tonic inhibition is sufficient to activate descending inhibition and reduce hypersensitivity after nerve injury.

As the primary excitatory neurotransmitter in the LC, glutamate activates AMPA receptors to increase noradrenergic neuronal activity [24], but also activates presynaptic mGluRs to autoinhibit glutamate release from its terminals[20]. Our current and previous observations show that intra-LC mGluR blockade increases extracellular glutamate concentrations in the LC to activate descending inhibition and thereby reduces hypersensitivity in SNL rats via downstream release of noradrenaline in the spinal cord [15]. Although pharmacologic stimulation of spinal α2 adrenoceptors produces antinociception in normal rats [6], we previously demonstrated that an intra-LC administered gabapentin induces glutamate release in the LC to activate the descending noradrenergic pathway but produces no antinociception in normal rats [7, 25]. Consistently, the current study demonstrates that increased LC glutamate by intra-LC mGluR blockade does not affect mechanical withdrawal thresholds in normal rats, indicating little or no role of endogenous noradrenergic tone in antinociception in the normal condition.

Stimulation of α2 adrenoceptors hyperpolarizes the cell membrane to reduce firing of noradrenergic neurons in the LC and thereby inhibits noradrenaline release [1, 12]. Although peripheral nerve injury may not affect basal noradrenaline release in the LC, it increases the expression of α2 adrenoceptors in the LC and enhances the inhibitory effects of α2 adrenoceptor agonists on LC neuron firing [2]. Intra-LC blockade of α2 adrenoceptors induces glutamate release in the LC 6 weeks after SNL injury but not in normal LC, and produces antihypersensitivity effect in SNL rats which is inhibited by intra-LC blockade of AMPA receptors and spinal blockade of α2 adrenoceptors. These results indicate that, despite the increased basal activity [2, 15], descending noradrenergic projections in chronic neuropathic hypersensitivity are under the tonic inhibitory influence not only from auto-inhibition of noradrenergic neurons but also from inhibition of tonic glutamate release in the LC via stimulation of α2 adrenoceptors by noradrenaline.

Although the current study did not examine the basal GABA release in the LC 6 weeks after SNL, we previously reported the increase in basal LC GABA release 2 – 3 weeks after nerve injury [28], indicating increased LC GABA tone after nerve injury. As GABA inhibits LC neuronal activity via GABA-A and GABA-B receptors in normal rats [12, 23], we hypothesized that intra-LC blockade of GABA receptors would reduce hypersensitivity after nerve injury via disinhibition of noradrenergic neurons in the LC. However, in contrast to this hypothesis, the current study shows that neither intra-LC injected bicuculline (1–10 ng) nor CGP 35348 (45–450 ng) affects hypersensitivity in rats 6 weeks after SNL. Previous study in rats showed that bilateral intra-LC injection of bicuculline (200 ng/site) inhibits hypersensitivity 5 weeks after chronic constriction injury of the infraorbital nerve [11], although this dose of bicuculline can affect other brain regions via an intracerebroventricular injection [9, 10, 16, 27]. The discrepancy between the current and previous [11] results may be due to differences of bicuculline doses or site of nerve injury. It should be noted that the current dose range of intra-LC bicuculline (1 and 10 ng) was shown to block the inhibitory influence of endogenous GABA on LC neurons in rats [3]. Together, these results suggest that inhibition of tonic GABA influence on LC neuronal activity in chronic neuropathic pain may not affect hypersensitivity.

In summary, the current study demonstrates that local noradrenaline and glutamate tonically inhibit glutamate release in the LC 6 weeks after neuropathic injury and this may contribute to reduced descending inhibition in response to noxious input during chronic neuropathic pain.

Highlights.

  • LC-injected idazoxan or MTPG decreased hypersensitivity 6 weeks after nerve injury

  • LC-GABA antagonists did not affect hypersensitivity after nerve injury

  • LC-idazoxan, -MTPG, or -GABA antagonists did not affect normal nociception

  • LC-CNQX or spinal idazoxan reduced antihypersensitivity from LC-idazoxan or -MTPG

  • LC-idazoxan or -MTPG induced LC glutamate release in nerve-injured rats

Acknowledgments

This work was supported by grants DA27690 from the National Institutes of Health (Bethesda, Maryland, USA) and KAKENHI 16K08985 from the Japan Society for the Promotion of Science (Tokyo, Japan) to K. H..

Abbreviations

LC

locus coeruleus

mGluR

metabotropic glutamate receptor

GLT-1

glutamate transporter-1

SNL

spinal nerve ligation

Footnotes

Conflicts of interest: Dr. Eisenach has consulted in the past 3 years with Adynxx (San Francisco, CA, USA) unrelated to this research regarding clinical development of a product to speed recovery after surgery.

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