Koichi Tan-No

The capsaicin test in mice for evaluating tachykinin antagonists in the spinal cord

A capsaicin test involving peripheral nociception, which produces behaviour similar to that elicited by formalin, is described in mice. Capsaicin was injected subcutaneously (s.c.) into the dorsal surface of a hindpaw and the time the animals spent licking the paw was recorded. Doses of capsaicin of 6.25-1600 ng induced nociception, during a period of 5 min, starting immediately after injection and disappearing completely at 10 min. Intrathecally (i.t.) administered [D-Arg1,D-Trp7,9,Leu11]substance P (spantide), a tachykinin antagonist and [D-Phe7,D-His9]substance P (6-11), a selective antagonist of substance P (SP), inhibited the capsaicin-induced behaviour, in a dose-dependent manner. This licking behaviour was also inhibited by intrathecal administration of SP antiserum but not by somatostatin (SOM) antiserum. Intrathecal pretreatment with capsaicin resulted in a marked reduction of the licking response, following subcutaneous injection of capsaicin into the paw. Capsaicin-induced licking was not affected by intrathecal administration of cyclo[7-aminoheptanoyl-Phe-D-Trp-Lys-(OBz)-Thr], a SOM antagonist and by intrathecal pretreatment with cysteamine, a SOM depletor. This nociceptive test may allow discrimination between SP- and SOM-mediated responses in the spinal cord of the mouse.

Characteristics of changes in cholinergic function and impairment of learning and memory-related behavior induced by olfactory bulbectomy.

Memory function after olfactory bulbectomy (OBX) was examined in two tasks, namely, step-through passive avoidance task and elevated plus-maze task. OBX mice showed a significant impairment of learning and memory-related behavior on the 7th and 14th day, as measured by passive avoidance task but not elevated plus maze task. The impairment of learning and memory-related behavior on the 14th day was improved by administration of the cholinesterase inhibitor physostigmine (0.1 mg/kg, i.p.), the non-selective muscarinic agonist oxotremorine (0.1 mg/kg, i.p.) or the selective muscarinic M(1) agonist McN-A-343 (10 microg/mouse, i.c.v.). In contrast, administration of the nicotinic agonist lobeline (5-9.8 mg/kg, i.p.) or the selective muscarinic M(2) antagonist methoctramine (2.25-18 microg/mouse, i.c.v.) has no effect on the impairment of learning and memory-related behavior induced by OBX. In addition, we have demonstrated that the intensity of choline acetyltransferase (ChAT) fluorescence is significantly decreased in the cortex, hippocampus and amygdala on the 14th day after OBX. These results suggest that the impairment of learning and memory-related behavior induced by OBX may be caused by degeneration of cholinergic neurons and muscarinic M(1) receptors play an important role in the improvement process.

Antinociception induced by CP 96,345, a non-peptide NK-1 receptor antagonist, in the mouse formalin and capsaicin tests

The non-peptide NK-1 receptor antagonist, CP 96,345, has been evaluated for antinociceptive activity in two chemical pain models in the mouse. CP 96,345, injected intrathecally (i.t.) 5 min prior to 2.0% formalin, produced significant antinociception in both the early and late phases of the formalin-induced paw licking procedure. Antinociception could also be observed during the late phase by treatment with CP 96,345 after formalin. In the capsaicin (CAP) test, i.t. injection of CP 96,345 produced a dose-dependent reduction of the paw-licking response at doses much less than antinociceptive doses in the formalin test. Naloxone did not affect antinociception in either test. CP 96,345 evoked a reversible deficit in motor performance as assayed by the rotarod test. The results indicate that i.t. CP 96,345 is antinociceptive in the capsaicin test at doses showing no overt behavioural effects but there is an overlap in doses producing antinociceptive and motor effects in the formalin test.

Differential antinociceptive effects induced by intrathecally administered endomorphin-1 and endomorphin-2 in the mouse

Two highly selective mu-opioid receptor agonists, endomorphin-1 and endomorphin-2, have been identified and postulated to be endogenous ligands for mu-opioid receptors. Intrathecal (i.t.) administration of endomorphin-1 and endomorphin-2 at doses from 0.039 to 5 nmol dose-dependently produced antinociception with the paw-withdrawal test. The paw-withdrawal inhibition rapidly reached its peak at 1 min, rapidly declined and returned to the pre-injection levels in 20 min. The inhibition of the paw-withdrawal responses to endomorphin-1 and endomorphin-2 at a dose of 5 nmol observed at 1 and 5 min after injection was blocked by pretreatment with a non-selective opioid receptor antagonist naloxone (1 mg/kg, s.c.). The antinociceptive effect of endomorphin-2 was more sensitive to the mu (1)-opioid receptor antagonist, naloxonazine than that of endomorphin-1. The endomorphin-2-induced paw-withdrawal inhibition at both 1 and 5 min after injection was blocked by pretreatment with kappa-opioid receptor antagonist nor-binaltorphimine (10 mg/kg, s.c.) or the delta(2)-opioid receptor antagonist naltriben (0.6 mg/kg, s.c.) but not the delta(1)-opioid receptor antagonist 7-benzylidine naltrexone (BNTX) (0.6 mg/kg s.c.). In contrast, the paw-withdrawal inhibition induced by endomorphin-1 observed at both 1 and 5 min after injection was not blocked by naloxonazine (35 mg/kg, s.c.), nor-binaltorphimine (10 mg/kg, s.c.), naltriben (0.6 mg/kg, s.c.) or BNTX (0.6 mg/kg s.c.). The endomorphin-2-induced paw-withdrawal inhibition was blocked by the pretreatment with an antiserum against dynorphin A-(1-17) or [Met(5)]enkephalin, but not by antiserum against dynorphin B-(1-13). Pretreatment with these antisera did not affect the endomorphin-1-induced paw-withdrawal inhibition. Our results indicate that endomorphin-2 given i.t. produces its antinociceptive effects via the stimulation of mu (1)-opioid receptors (naloxonazine-sensitive site) in the spinal cord. The antinociception induced by endomophin-2 contains additional components, which are mediated by the release of dynorphin A-(1-17) and [Met(5)]enkephalin which subsequently act on kappa-opioid receptors and delta(2)-opioid receptors to produce antinociception.

INVOLVEMENT OF NITRIC OXIDE IN SPINALLY MEDIATED CAPSAICIN- AND GLUTAMATE-INDUCED BEHAVIOURAL RESPONSES IN THE MOUSE *

The intrathecal (i.t.) injection of capsaicin (0.1 nmol/mouse) through a lumbar puncture elicited scratching, biting and licking responses. Pretreatment with the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) (320 nmol), by i.t. injection, resulted in a significant inhibition of the behavioural response produced by i.t. capsaicin (0.1 nmol/mouse). Similar behavioural responses were induced by i.t. injections of NMDA (0.4 nmol), kainate (0.05 nmol) or AMPA (0.05 nmol), which were all inhibited by co-administration of L-NAME (20-80 nmol). L-Arginine (600 mg/kg, i.p.) but not D-arginine (600 mg/kg, i.p.) reversed the inhibitory effect of L-NAME on capsaicin-, NMDA-, kainate- and AMPA-induced behavioural response. Scratching, biting and licking responses induced by tachykinin receptor agonists, substance P, [Sar9,Met(O2)11]substance P, neurokinin A and neurokinin B were not affected by co-administration of L-NAME (40 and 80 nmol). These results suggest that spinal nitric oxide may play a significant role in mechanisms of the behavioural response to capsaicin, probably through the release of glutamate, but not tachykinins.

Involvement of Spinal NMDA Receptors in Capsaicin-Induced Nociception

Intraplantar injection of capsaicin into the mouse hindpaw produced an acute nociceptive response. The involvement of N-methyl-D-aspartate (NMDA) receptors was examined by intrathecal administration of various excitatory amino acid (EAA) receptor antagonists. The selective and competitive NMDA receptor antagonists, D(-)-2-amino-5-phosphono-valeric acid (APV) and (+/-)-3-(2-carboxypiperazin-4-yl) propyl-1-phosphoric acid (CPP), were most potent in inhibiting the nociceptive response induced by capsaicin (ED50, 0.23 nmol and 0.12 nmol). The noncompetitive NMDA receptor antagonist dizocilpine (MK-801) and the non-NMDA antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) had similar effects on the capsaicin-induced nociception (ED50, 2.90 and 7.98 nmol), while ketamine and 7-chlorokynurenic acid were without effect. Ifenprodil, an antagonist at the receptor-coupled polyamine site, showed a significant reduction of the nociceptive response (ED50, 13.8 nmol). The inhibitory effects of APV, CPP, MK-801, and ifenprodil were reversed by co-administration of NMDA. Coadministration of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) or kainate resulted in a marked reduction of CNQX-induced antinociception. The present results suggest that the NMDA receptor plays a key role in spinal nociceptive processing as measured by the capsaicin test in mice. This nociceptive test may be useful for evaluating competitive NMDA antagonists.

Induction of nociceptive responses by intrathecal injection of interleukin-1 in mice.

Intrathecal (i.t.) injection (between lumbar vertebrae 5 and 6) into mice of a markedly low dose of IL-1alpha (3x10(-4) fmol or 5.4 fg in 5 microl per mouse) induced behaviors involving scratching, biting, and licking of non-stimulated hindpaws. The IL-1-induced behaviors appeared within 10 min of the injection of IL-1alpha, peaked at 20-40 min, and had disappeared 60 min after the injection. The IL-1-induced behaviors were similar to the nociceptive responses induced in mice by i.t. injection of substance P (SP) or subcutaneous (s.c.) injection of formalin into the footpad. The IL-1-induced behaviors were suppressed by intraperitoneal morphine, indicating that they are nociceptive responses. The nociceptive responses induced by 3x10(-4) (5.4 fg) of IL-1alpha were almost completely suppressed by co-injection of 0.3 fmol (7.2 pg) of an IL-1 receptor antagonist (IL-1ra). An antiserum against substance P, but not an antiserum against somatostatin, suppressed the IL-1-induced nociceptive responses. The nociceptive responses induced by s.c. injection of 2% formalin into the footpad were also inhibited by i.t. injection of 30 pmol (720 ng) of IL-1ra. These results suggest that IL-1 may play a role in hyperalgesia in mice by acting as a factor augmenting pain transmission in the spinal cord at least in part by either directly or indirectly releasing substance P.

The effects of substance P analogues on the scratching, biting and licking response induced by intrathecal injection of N‐methyl‐d‐aspartate in mice

1 Intrathecal (i.t.) administration of N‐methyl‐d‐aspartate (NMDA) elicited a dose‐dependent behavioural response consisting of licking, biting and scratching in mice. 2 Repeated i.t. injections of 0.4 nmol NMDA, at 5 min intervals, resulted in the rapid development of desensitization to this NMDA‐induced behavioural phenomenon. 3 The NMDA‐induced response was dose‐dependently inhibited by the simultaneous injection of a selective NMDA‐receptor antagonist, d‐2‐amino‐5‐phosphonovaleric acid. 4 The substance P (SP) analogues [d‐Pro2, d‐Trp7,9] SP and [d‐Arg1, d‐Trp7,9, Leu11] SP (spantide) inhibited NMDA‐induced behavioural responses in a dose‐dependent manner. However, [d‐Phe7, d‐His9] SP (6–11), a SP analogue selective for neurokinin1 (NK1) receptors, failed to inhibit NMDA‐induced responses even at a dose of 4.0 nmol. 5 These results indicate that NMDA‐induced behavioural responses are mainly mediated through NMDA receptors without affecting NK1 receptors in the spinal cord.

Antinociceptive effect of different types of calcium channel inhibitors and the distribution of various calcium channel α1 subunits in the dorsal horn of spinal cord in mice

To understand better which voltage-dependent calcium channels (VGCCs) are involved in nociceptive neurotransmission, we investigated the pharmacological properties and distribution of VGCCs in the mouse spinal cord. A behavioral assay revealed that intrathecal injections of omega-agatoxin TK, omega-agatoxin IVA, omega-conotoxin GVIA, and SNX-482, which block P/Q-, P/Q-, N-, and R-type calcium channels, respectively, produced analgesic effects, while an L-type channel blocker had no such effect. An electrophysiological study demonstrated the presence of various types of VGCCs within dorsal root ganglion (DRG) neurons. Immunohistochemistry revealed distinct localization of P/Q-, N-, L-, and R-type calcium channel subunits to the dorsal horn of the spinal cord. The results of this study revealed the localization and functions of several calcium channels that are involved in nociceptive neurotransmission within the dorsal horn of the mouse spinal cord.

A comparison between microwave irradiation and decapitation: basal levels of dynorphin and enkephalin and the effect of chronic morphine treatment on dynorphin peptides

Opioid peptides were analysed in tissue extracts of various brain structures and the pituitary gland from rats sacrificed by microwave irradiation, and compared with peptide levels in tissue extracts from decapitated rats. Dynorphin A, dynorphin B and Leu-enkephalinArg6, derived from prodynorphin, and Met-enkephalinArg6Phe7 from proenkephalin, were measured. Basal immunoreactive levels of dynorphin A and B were consistently higher in extracts from microwave-irradiated rats, whereas in these extracts immunoreactive levels of Leu-enkephalinArg6, an endogenous metabolite of dynorphin peptides, were either lower than, the same as or higher than in decapitated rats. Immunoreactive levels of Met-enkephalinArg6Phe7 were higher in microwave-irradiated rats. Effects of morphine treatment on prodynorphin peptide levels were evaluated and compared with previous findings in decapitated rats. Dynorphin immunoreactive levels were higher in the nucleus accumbens and striatum of morphine-tolerant rats than in corresponding areas in saline-treated rats. These results indicate tissue-specific metabolism of prodynorphin peptides and show that metabolism of opioid peptides occurs during the dissection procedure after decapitation of the rat even though precautions are taken to minimize degradation.