Keiko Takasu

Role of descending noradrenergic system and spinal α2‐adrenergic receptors in the effects of gabapentin on thermal and mechanical nociception after partial nerve injury in the mouse

1 To gain further insight into the mechanisms underlying the antihyperalgesic and antiallodynic actions of gabapentin, a chronic pain model was prepared by partially ligating the sciatic nerve in mice. The mice then received systemic or local injections of gabapentin combined with either central noradrenaline (NA) depletion by 6‐hydroxydopamine (6‐OHDA) or α‐adrenergic receptor blockade. 2 Intraperitoneally (i.p.) administered gabapentin produced antihyperalgesic and antiallodynic effects that were manifested by elevation of the withdrawal threshold to a thermal (plantar test) or mechanical (von Frey test) stimulus, respectively. 3 Similar effects were obtained in both the plantar and von Frey tests when gabapentin was injected intracerebroventricularly (i.c.v.) or intrathecally (i.t.), suggesting that it acts at both supraspinal and spinal loci. This novel supraspinal analgesic action of gabapentin was only obtained in ligated neuropathic mice, and gabapentin (i.p. and i.c.v.) did not affect acute thermal and mechanical nociception. 4 In mice in which central NA levels were depleted by 6‐OHDA, the antihyperalgesic and antiallodynic effects of i.p. and i.c.v. gabapentin were strongly suppressed. 5 The antihyperalgesic and antiallodynic effects of systemic gabapentin were reduced by both systemic and i.t. administration of yohimbine, an α2‐adrenergic receptor antagonist. By contrast, prazosin (i.p. or i.t.), an α1‐adrenergic receptor antagonist, did not alter the effects of gabapentin. 6 It was concluded that the antihyperalgesic and antiallodynic effects of gabapentin are mediated substantially by the descending noradrenergic system, resulting in the activation of spinal α2‐adrenergic receptors.

Pain relief by gabapentin and pregabalin via supraspinal mechanisms after peripheral nerve injury

The antihypersensitivity actions of gabapentin and pregabalin have been well characterized in a large number of studies, although the underlying mechanisms have yet to be defined. We have been focusing on the supraspinal structure as a possible site for their action and have demonstrated that intracerebroventricular (i.c.v.) administration of gabapentin and pregabalin indeed decreases thermal and mechanical hypersensitivity in a murine chronic pain model involving partial ligation of the sciatic nerve. This novel supraspinally mediated analgesic effect was markedly suppressed by either depletion of central noradrenaline (NA) or blockade of spinal α2‐adrenergic receptors. Moreover, i.c.v. injection of gabapentin and pregabalin increased spinal NA turnover in mice only after peripheral nerve injury. In locus coeruleus (LC) neurons in brainstem slices prepared from mice after peripheral nerve injury, gabapentin reduced the γ‐aminobutyric acid (GABA) type A receptor‐mediated inhibitory postsynaptic currents (IPSCs). Glutamate‐mediated excitatory synaptic transmission was hardly affected. Moreover, gabapentin did not reduce IPSCs in slices taken from mice given a sham operation. Although gabapentin altered neither the amplitude nor the frequency of miniature IPSCs, it reduced IPSCs together with an increase in the paired‐pulse ratio, suggesting that gabapentin acts on the presynaptic GABAergic nerve terminals in the LC. Together, the data suggest that gabapentin presynaptically reduces GABAergic synaptic transmission, thereby removing the inhibitory influence on LC neurons only in neuropathic pain states, leading to activation of the descending noradrenergic system.

Pregabalin, S-(+)-3-isobutylgaba, activates the descending noradrenergic system to alleviate neuropathic pain in the mouse partial sciatic nerve ligation model

We have previously demonstrated that gabapentin supraspinally activates the descending noradrenergic system to alleviate neuropathic pain. In this study, we investigated whether pregabalin, an antiepileptic and analgesic drug that is also designed as a structural analogue of gamma-aminobutyric acid (GABA), exhibits supraspinal analgesic effects similar to those of gabapentin involving the descending noradrenergic system. Both systemically (intraperitoneally; i.p.) and locally (intracerebroventricularly or intrathecally; i.c.v. or i.t.) injected pregabalin reduced thermal and mechanical hypersensitivity in a murine chronic pain model that was prepared by partial ligation of the sciatic nerve (the Seltzer model), suggesting that pregabalin acts at both supraspinal and spinal loci. The supraspinal analgesic action of pregabalin was observed only after peripheral nerve injury, and pregabalin (i.p. and i.c.v.) did not affect acute thermal and mechanical nociception. Depletion of spinal noradrenaline (NA) or pharmacological blockade of spinal alpha(2)-adrenoceptors with yohimbine (i.p. or i.t.), but not alpha(1)-adrenoceptors with prazosin (i.p.), reduced the analgesic effects of pregabalin (i.p. or i.c.v.) on thermal and mechanical hypersensitivity. Moreover, i.c.v.-administered pregabalin dose-dependently increased the spinal 4-hydroxy-3-methoxyphenylglycol (MHPG) content and the MHPG/NA ratio only in mice with neuropathic pain, whereas the concentrations of NA, serotonin, 5-hydroxyindoleacetic acid and dopamine were unchanged, demonstrating that supraspinal pregabalin accelerated the spinal turnover of NA. Together, these results indicate that pregabalin supraspinally activates the descending noradrenergic pain inhibitory system coupled with spinal alpha(2)-adrenoceptors to ameliorate neuropathic pain.

Glycine Transporter Inhibitors as a Potential Therapeutic Strategy for Chronic Pain with Memory Impairment

Background:Impaired excitatory and inhibitory balance in the spinal dorsal horn has a crucial role in the pathophysiology of chronic pain. The authors addressed the therapeutic impact of increasing spinal glycine applied exogenously or via blockade of glycine transporter 1 using its selective inhibitors sarcosine and N-[3-(4′-fluorophenyl)-3-(4′-phenylphenoxy)propyl]sarcosine on neuropathic and inflammatory pain in mice. Methods:Mice with thermal and mechanical hypersensitivity after partial ligation of the sciatic nerve (Seltzer model) or mice with mechanical hypersensitivity after streptozotocin injection received intrathecal injection of glycine, sarcosine, and N-[3-(4′-fluorophenyl)-3-(4′-phenylphenoxy)propyl]sarcosine. These drugs were also intrathecally injected in mice to assess their effects on formalin-evoked nociceptive behaviors. The supraspinal effect of blockade of glycine transporter 1 was studied on tetanus-induced long-term potentiation of the Schaffer-collateral synapses in hippocampal slices prepared from Seltzer model mice. Results:Glycine, sarcosine, and N-[3-(4′-fluorophenyl)-3-(4′-phenylphenoxy)propyl]sarcosine ameliorated thermal and mechanical hypersensitivity in Seltzer model mice, and reduced mechanical hypersensitivity in streptozotocin-injected diabetic mice. Moreover, they selectively inhibited the second phase of formalin-evoked licking/biting behavior. In hippocampal slices prepared from Seltzer model mice, long-term potentiation was maintained at a significantly lower level than that in sham-treated mice. Such impairment of long-term potentiation was never observed when it was induced in the presence of N-[3-(4′-fluorophenyl)-3-(4′-phenylphenoxy)propyl]sarcosine. Conclusions:An increase in endogenous glycine via glycine transporter 1 blockade not only results in a net inhibitory influence on pain transmission at the spinal level but also supraspinally relieves decreased synaptic efficacy presumably related to cognitive disturbance often described in patients with chronic pain.

Spinal α2-adrenergic and muscarinic receptors and the NO release cascade mediate supraspinally produced effectiveness of gabapentin at decreasing mechanical hypersensitivity in mice after partial nerve injury

1 After partial nerve injury, the central analgesic effect of systemically administered gabapentin is mediated by both supraspinal and spinal actions. We further evaluate the mechanisms related to the supraspinally mediated analgesic actions of gabapentin involving the descending noradrenergic system. 2 Intracerebroventricularly (i.c.v.) administered gabapentin (100 μg) decreased thermal and mechanical hypersensitivity in a murine chronic pain model that was prepared by partial ligation of the sciatic nerve. These effects were abolished by intrathecal (i.t.) injection of either yohimbine (3 μg) or idazoxan (3 μg), α2‐adrenergic receptor antagonists. 3 Pretreatment with atropine (0.3 mg kg−1, i.p. or 0.1 μg, i.t.), a muscarinic receptor antagonist, completely suppressed the effect of i.c.v.‐injected gabapentin on mechanical hypersensitivity, whereas its effect on thermal hypersensitivity remained unchanged. Similar effects were obtained with pirenzepine (0.1 μg, i.t.), a selective M1‐muscarinic receptor antagonist, but not with methoctramine (0.1 and 0.3 μg, i.t.), a selective M2‐muscarinic receptor antagonist. 4 The cholinesterase inhibitor neostigmine (0.3 ng, i.t.) potentiated only the analgesic effect of i.c.v. gabapentin on mechanical hypersensitivity, confirming spinal acetylcholine release downstream of the supraspinal action of gabapentin. 5 Moreover, the effect of i.c.v. gabapentin on mechanical but not thermal hypersensitivity was reduced by i.t. injection of L‐NAME (3 μg) or L‐NMMA (10 μg), both of which are nitric oxide (NO) synthase inhibitors. 6 Systemically administered naloxone (10 mg kg−1, i.p.), an opioid receptor antagonist, failed to suppress the analgesic actions of i.c.v. gabapentin, indicating that opioid receptors are not involved in activation of the descending noradrenergic system by gabapentin. 7 Thus, the supraspinally mediated effect of gabapentin on mechanical hypersensitivity involves activation of spinal α2‐adrenergic receptors followed by muscarinic receptors (most likely M1) and the NO cascade. In contrast, the effect of supraspinal gabapentin on thermal hypersensitivity is independent of the spinal cholinergic–NO system.

Spinal hyperpolarization-activated cyclic nucleotide-gated cation channels at primary afferent terminals contribute to chronic pain.

&NA; Hyperpolarization‐activated cyclic nucleotide‐gated cation channels (HCN channels) have large influences upon neuronal excitability. However, the participation of spinal HCN channels in chronic pain states, where pathological conditions are related to altered neuronal excitability, has not been clarified. Intraperitoneally (i.p.) or intrathecally (i.t.) administered ZD7288, a selective blocker of Ih channels, reduced thermal and mechanical hypersensitivity in mice under neuropathic conditions induced by the partial ligation of the sciatic nerve, while no analgesic effect was observed in naïve animals. Moreover, in the mouse formalin test, ZD7288 (i.p. and i.t.) reduced the licking/biting behavior observed during the second phase without affecting the first phase. To further explore the pain‐modulatory action of spinal HCN channels, whole‐cell patch clamp recordings were made from the visually identified substantia gelatinosa neurons in adult mouse spinal cord slices with an attached dorsal root, and A‐fiber‐ and/or C‐fiber‐mediated monosynaptic excitatory postsynaptic currents (EPSCs) were evoked by electrical stimulation of the L4 or L5 dorsal root using a suction electrode. Bath‐applied ZD7288 reduced A‐fiber‐ and C‐fiber‐mediated monosynaptic EPSCs more preferentially in slices prepared from mice after peripheral nerve injury. In addition, ZD7288 reduced the frequency of miniature EPSCs without affecting their amplitude in cells receiving monosynaptic afferent inputs, indicating that it inhibits EPSCs via presynaptic mechanisms. The present behavioral and electrophysiological data suggest that spinal HCN channels, most likely at the primary afferent terminals, contribute to the maintenance of chronic pain.

Gabapentin produces PKA-dependent pre-synaptic inhibition of GABAergic synaptic transmission in LC neurons following partial nerve injury in mice

We have previously demonstrated that gabapentin supraspinally activates the descending noradrenergic system to ameliorate pain hypersensitivity in mice with partial nerve ligation. To clarify the supraspinal mechanism of action of gabapentin, whole‐cell patch‐clamp recordings were performed on locus coeruleus (LC) neurons in brainstem slices prepared from mice after peripheral nerve injury or mice subjected to a sham‐operation, and the effects of gabapentin in the modulation of synaptic transmission were studied. Bath application of gabapentin (10, 30 and 100 μM) in a concentration‐dependent manner reduced the GABAA receptor‐mediated inhibitory post‐synaptic currents (IPSCs) in slices prepared from partially nerve‐ligated mice, whereas glutamate‐mediated excitatory post‐synaptic currents were hardly affected. By contrast, gabapentin did not reduce IPSCs in slices taken from mice given a sham operation. Although gabapentin altered neither the amplitude nor the frequency of miniature IPSCs, it reduced IPSCs together with an increase in the paired‐pulse ratio, suggesting that gabapentin acts on the pre‐synaptic GABAergic nerve terminals in the LC. As the protein kinase A (PKA) inhibitor H‐89 but not the protein kinase C inhibitor chelerythrine abolished the inhibitory action of gabapentin on IPSCs, PKA‐mediated phosphorylation seems to be important for supraspinal gabapentin responsiveness in neuropathic conditions. Together, gabapentin generates PKA‐dependent pre‐synaptic inhibition of GABAergic synaptic transmission, and thereby removes the inhibitory influence on LC neurons only under neuropathic pain states. These findings provide crucial evidence of how supraspinally acting gabapentin recruits the descending noradrenergic system.

Role of voltage-dependent calcium channel subtypes in spinal long-term potentiation of C-fiber-evoked field potentials

&NA; Activity‐dependent increases in the responsiveness of spinal neurons to their normal afferent input, termed central sensitization, have been suggested to play a key role in abnormal pain sensation. We investigated the role of distinct voltage‐dependent calcium channel (VDCC) subtypes in the long‐term potentiation (LTP) of C‐fiber‐evoked field potentials (FPs) recorded in the spinal dorsal horn of rats, that is, a synaptic model to describe central sensitization. When spinally applied, we observed that omega‐conotoxin GVIA (ω‐CgTx), an N‐type VDCC antagonist, produced a dose‐dependent and prolonged inhibition of basal C‐fiber‐evoked FPs in naïve animals. ω‐CgTx did not perturb the induction of LTP by high‐frequency stimulation (HFS) of the sciatic nerve; however, potentiation was maintained at a lower level. Following the establishment of spinal LTP in naïve animals, the inhibitory effect of ω‐CgTx on C‐fiber‐evoked FPs was significantly increased. Furthermore, in animals with chronic pain produced via peripheral nerve injury, where spinal LTP was barely induced by HFS, basal C‐fiber‐evoked FPs were strongly inhibited by ω‐CgTx. As a result, ω‐CgTx exerted a similar inhibitory profile on C‐fiber‐evoked FPs following the establishment of spinal LTP and chronic pain. In contrast, spinally administered omega‐agatoxin IVA (ω‐Aga‐IVA), a P/Q‐type VDCC antagonist, showed little effect on C‐fiber‐evoked FPs either before or after the establishment of LTP, but strongly suppressed LTP induction. These results demonstrate the requirement of N‐ and P/Q‐type VDCCs in the maintenance and induction of LTP in the spinal dorsal horn, respectively, and their distinct contribution to nociceptive synaptic transmission and its plasticity. In vivo electrophysiological studies demonstrate the distinct and predominant functions of voltage‐dependent calcium channel subtypes for spinal long‐term potentiation and chronic pain.

The synthetic TRH analogue taltirelin exerts modality-specific antinociceptive effects via distinct descending monoaminergic systems

Exogenously administered thyrotropin‐releasing hormone (TRH) is known to exert potent but short‐acting centrally‐mediated antinociceptive effects. We sought to investigate the mechanisms underlying these effects using the synthetic TRH analogue taltirelin, focusing on the descending monoaminergic systems in mice.

Pharmacological characterization of lysophosphatidic acid-induced pain with clinically relevant neuropathic pain drugs.

Lysophosphatidic acid (LPA), an initiator of neuropathic pain, causes allodynia. However, few studies have evaluated the pharmacological profile of LPA‐induced pain. In this study, a LPA‐induced pain model was developed and pharmacologically characterized with clinically relevant drugs used for neuropathic pain, including antiepileptics, non‐steroidal anti‐inflammatory agents, analgesics, local anaesthetics/antiarrhythmics and antidepressants. Gabapentin (1–30 mg/kg, p.o.) significantly reversed LPA‐induced allodynia, but neither indomethacin (30 mg/kg, p.o.) nor morphine (0.3–3 mg/kg, s.c.) did, which indicates that LPA‐induced pain consists mostly of neuropathic rather than inflammatory pain. Both pregabalin (0.3–10 mg/kg, p.o.) and ω‐CgTX MVIIA (0.01–0.03 μg/mouse, i.t.) completely reversed LPA‐induced allodynia in a dose‐dependent manner. Lidocaine (1–30 mg/kg, s.c.), mexiletine (1–30 mg/kg, p.o.) and carbamazepine (10–100 mg/kg, p.o.) significantly ameliorated LPA‐induced allodynia dose dependently. Milnacipran (30 mg/kg, i.p.) produced no significant analgesic effect in LPA‐induced allodynia. In LPA‐injected mice, expression of the α2δ1 subunit of the voltage‐gated calcium channel (VGCC) was increased in the dorsal root ganglion (DRG) and spinal dorsal horn. Furthermore, the VGCC current was potentiated in both the DRG from LPA‐injected mice and LPA (1 μM)‐treated DRG from saline‐injected mice, and the potentiated VGCC current was amended by treatment with gabapentin (100 μM). The LPA‐induced pain model described here mimics aspects of the neuropathic pain state, including the sensitization of VGCC, and may be useful for the early assessment of drug candidates to treat neuropathic pain.