Toshikazu Nishimori

Effect of intrathecal administration of hemokinin-1 on the withdrawal response to noxious thermal stimulation of the rat hind paw

Hemokinin-1 (HK-1) is a new peptide described as a member of the tachykinin family. HK-1 has biological effects similar to substance P (SP), a representative of the tachykinin family, following central administration. However, the biological function of HK-1 at the spinal level has not been well characterized. Thus, we investigated the effect of intrathecal administration of HK-1 by comparing it with that of SP. Intrathecal administration of HK-1 as well as SP at 10(-3) M caused pain-related behavior such as scratching. The scratching by HK-1 administration was inhibited by pretreatment with an antagonist of substance P receptor. In addition, SP (10(-8)-10(-6) M) decreased the latency of the withdrawal response of the hind paw to noxious thermal stimulation 20-30 min after intrathecal administration, whereas administration of HK-1 had little effect on this response. These results suggest that there may exist a proper receptor related to HK-1.

Unilateral lesions of mesostriatal dopaminergic pathway alters the withdrawal response of the rat hindpaw to mechanical stimulation

To investigate the role mesostriatal dopamine system plays in pain processing, we examined the withdrawal response of rat hindpaws to mechanical stimulus at 1, 4, and 12 weeks after unilateral 6-hydroxydopamine (6-OHDA) lesions of the mesostriatal pathway. In all of the 6-OHDA rats examined, almost no tyrosine hydroxylase (TH) immunoreactivity was detected in the substantia nigra, ventral tegmental area, and striatum ipsilateral to 6-OHDA lesions. Alteration in the withdrawal response in this model animal was evaluated by comparing the latency of withdrawal reflex following the mechanical stimulus to the hindpaw. The latency of withdrawal response in the 6-OHDA rats was significantly reduced in the side ipsilateral to 6-OHDA lesions at all times observed, whereas that was not changed through the period observed in the contralateral side, indicating that dopamine depletion in the mesostriatal system has the influence on withdrawal response to the mechanical stimulus. These results show that the unilateral dopamine depletion causes hypersensitivity to the mechanical stimulus in the ipsilateral side, suggesting that, at least in part, dopamine in the mesostriatal system may be involved in sensory processing including pain sensation induced by mechanical stimulation.

Regional differences in desensitization of c-Fos expression following repeated self-stimulation of the medial forebrain bundle in the rat

The acute self-stimulation of the medial forebrain bundle was reported to induce the expression of c-Fos, the protein product of c-fos, an immediate early gene, in the central nervous system. In the present study, we examined regional changes in c-Fos expression in several reward-related areas of rat brain in response to short- and long-term exposure to self-stimulation of the medial forebrain bundle. Short-term one-hour stimulation of the medial forebrain bundle for one day after training, which evoked steady self-stimulation behavior, significantly increased the number of c-Fos-positive neurons bilaterally in all of 15 brain structures assayed, as compared to the non-stimulation control. Among them, structures showing a larger number of the stained neurons on the stimulated side were the anterior olfactory nucleus, amygdala, medial caudate-putamen complex, lateral septum, bed nucleus of the stria terminals, ventral pallidum, substantia innominata, lateral preoptic area, medial preoptic area, lateral hypothalamus rostral to the stimulating electrodes, and substantia nigra. Long-term stimulation of the medial forebrain bundle once daily for five successive days, which maintained consistently stable self-stimulation behavior, also increased the number of c-Fos-positive neurons in the aforementioned structures, as compared to the control. However, the long-term rewarding stimulation diminished the increased number of labeled neurons, as compared to the short-term rewarding stimulation. Seven areas, medial caudate-putamen complex, ventral pallidum, substantia innominata, lateral preoptic area, medial preoptic area, rostral lateral hypothalamus and substantia nigra, showed asymmetrical, ipsilateral predominance after the short- and long-term stimulation. However, the stained neuron count in those areas after the long-term stimulation was reduced to less than 50% of that found after the short-term stimulation with the exception of lateral preoptic area and rostral lateral hypothalamus. The results suggest that the development of desensitization of c-Fos response may differ among the reward-relevant brain regions as a consequence of repeated self-stimulation. They also indicate that a larger portion of neurons in the lateral preoptic area and rostral lateral hypothalamus may be implicated in both short- and long-term self-stimulations of the medial forebrain bundle.

Differential effects of NMDA and AMPA/KA receptor antagonists on c-Fos or Zif/268 expression in the rat spinal dorsal horn induced by noxious thermal or mechanical stimulation, or formalin injection.

The involvement of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate (KA) receptors in the induction of c-Fos and Zif/268 expression in spinal dorsal horn neurons following noxious thermal or mechanical stimulation, or formalin injection into the rat hind paw was examined by intrathecal administration of a competitive NMDA receptor antagonist, 2-amino-5-phosphonopentanoic acid (APV) or an AMPA/KA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), or both, 30 min prior to noxious stimulation. APV caused a significant reduction in the level of c-Fos expression in the superficial layer induced by each of these three noxious stimuli. The effects of APV on Zif/268 expression or of CNQX on c-Fos or Zif/268 expression in the superficial layer induced by these three noxious stimuli were dependent on the type of stimulus applied to the rat hind paw. The noxious thermal stimulus-evoked c-Fos expression level was reduced by APV and/or CNQX, while Zif/268 expression was hardly changed. Both c-Fos and Zif/268 expressions following formalin injection were reduced by APV alone and APV+CNQX, but not by CNQX alone. Zif/268 expression following noxious mechanical stimulation was significantly reduced only by APV+CNQX although APV or CNQX alone did not affect the expression, while c-Fos expression was reduced by APV and APV+CNQX but not by CNQX alone. These findings suggest that NMDA and AMPA/KA receptors are differentially involved in c-Fos and Zif/268 expression in the spinal dorsal horn following noxious thermal, formalin and mechanical stimulation.

Intrathecal administration of the common carboxyl-terminal decapeptide in endokinin A and endokinin B evokes scratching behavior and thermal hyperalgesia in the rat.

Endokinins are novel mammalian tachykinin peptides designated from a human preprotachykinin gene and consist of endokinin A (EKA), endokinin B (EKB), endokinin C (EKC) and endokinin D (EKD). A representative of the tachykinin peptide is substance P (SP), which functions as a pain modulator or transmitter and contributes to pain processing; however, little is known about the function of endokinins in pain processing. Therefore, we evaluated the effects of EKA/B (using the common C-terminal decapeptide in EKA and EKB) and EKC/D (using the common C-terminal duodecapeptide in EKC and EKD) on pain processing in rats. Intrathecal administration of 10(-3) M (10 nmol) EKA/B evoked pain-related behavior such as scratching while 10(-3) M EKC/D administration did not. This induction of scratching behavior following EKA/B administration was suppressed by pretreatment with an NK1 receptor antagonist. In addition to the induction of scratching behavior, intrathecal administration of 10(-7) - 10(-4) M (1 pmol-1 nmol) EKA/B decreased the latency of the paw withdrawal response to noxious thermal stimulation, whereas there was little effect of EKC/D administration on the latency of the withdrawal response. This effect of EKA/B was also suppressed by pretreatment with NK1 receptor antagonists. These results indicate that intrathecal administration of EKA/B but not EKC/D evokes scratching behavior and thermal hyperalgesia through the NK1 receptor.

Intracranial self-stimulation induces Fos expression in GABAergic neurons in the rat mesopontine tegmentum

The cholinergic neurons which originate in the mesopontine tegmentum and innervate the midbrain ventral tegmental area have been proposed to play a key role in intracranial self-stimulation reward. This mesopontine area also contains GABA neurons. Detailed information is still lacking, however, about the relationship of cholinergic and GABAergic neurons in this region to self-stimulation reward. Therefore, using double immunostaining for Fos as a marker of neuronal activity and choline acetyltransferase as a marker of cholinergic neurons, or for Fos and GABA, we investigated whether self-stimulation of the medial forebrain bundle induces Fos expression within cholinergic and GABAergic neurons in two regions of the mesopontine tegmentum, i.e., pedunculopontine tegmental nucleus and laterodorsal tegmental nucleus. Self-stimulation of the medial forebrain bundle for 1 h induced a large increase in the number of cells expressing Fos in both the pedunculopontine tegmental nucleus and laterodorsal tegmental nucleus, when compared to control brains. However, the self-stimulation-induced expression of Fos was restricted mostly to GABA-, but not choline acetyltransferase-, immunostained cells. We also examined, using microdialysis, whether self-stimulation increases acetylcholine efflux in the ventral tegmental area, a terminal region of the mesopontine tegmentum cholinergic pathway. One hour of self-stimulation significantly increased acetylcholine efflux from this terminal area. These results indicate that intracranial self-stimulation of the medial forebrain bundle may increase acetylcholine release without affecting expression of Fos in cholinergic neurons, while the same stimulation may induce Fos expression in GABAergic neurons of the mesopontine tegmentum. GABAergic as well as cholinergic neurons in this area appear to be activated by self-stimulation reward in the medial forebrain bundle.

Differential effects of substance P or hemokinin-1 on transient receptor potential channels, TRPV1, TRPA1 and TRPM8, in the rat

Two tachykinin peptides, substance P (SP) and hemokinin-1 (HK-1), and three transient receptor potential (TRP) channels, TRPV1, TRPA1 and TRPM8, are similarly localized in the spinal dorsal horn and dorsal root ganglion, suggesting that TRP channels may be related or modulated by these tachykinin peptides. Thus, to clarify whether the responses of TRP channels are modulated by SP or HK-1, the effects of pretreatment with SP or HK-1 on the induction of scratching behavior by TRP channel agonists were examined. Pretreatment with SP or HK-1 enhanced the induction of scratching behavior by resiniferatoxin, a TRPV1 agonist, whereas scratching behavior induced by menthol, a TRPM8 agonist, was suppressed by pretreatment with these peptides. On the other hand, pretreatment with SP, but not HK-1, suppressed the induction of scratching behavior by cinnamaldehyde, a TRPA1 agonist. Taken together, the present results indicate that SP or HK-1 differentially modulated the response of TRPV1, TRPA1 or TRPM8 channel.

Differential expression of Fos protein after transection of the rat infraorbital nerve in the trigeminal nucleus caudalis.

To determine the effects of nerve injury on Fos expression, temporal and spatial distributions of Fos-positive neurons in the trigeminal nucleus caudalis were examined after tissue injury for isolation of the infraorbital nerve as controls and transection of this nerve as well as noxious chemical stimulation by formalin injection in adult rats. Fos immunoreactivity was markedly elevated in laminae I and II of the only ipsilateral nucleus caudalis 2 h after these surgical procedures and noxious chemical stimulation. The distributions of Fos-positive neurons were restricted rostro-caudally following formalin injection and tissue injury compared to transection of the infraorbital nerve. One day after tissue injury and nerve transection, however, Fos-positive neurons were distributed bilaterally in laminae III and IV extending rostro-caudally and medio-laterally in this nucleus, and this persisted over the 2-week study period. The number of Fos-positive neurons in the side ipsilateral to nerve transection was markedly less than that in the contralateral side whereas positive neurons in the tissue injured rats were distributed symmetrically along the rostro-caudal axis. There was no difference in the contralateral sides between nerve transection and tissue injury groups. The rostro-caudal level showing reduction in Fos expression corresponded roughly to the sites of central termination of the injured nerve in this nucleus, suggesting a role for the primary afferents in the reduction of Fos expression in laminae III and IV neurons of the ipsilateral nucleus caudalis.

Involvement of NMDA receptors in Zif/268 expression in the trigeminal nucleus caudalis following formalin injection into the rat whisker pad

We investigated the involvement of N-methyl-D-aspartate (NMDA) glutamate receptor in the expression of the proteins Zif/268 and c-Fos elicited by painful stimuli. To this purpose, the effect of the administration of MK-801, an NMDA receptor antagonist, on Zif/268 and c-Fos expression following a noxious stimulus, represented by formalin injection into the whisker pad of rats, was examined in neurons of the trigeminal nucleus caudalis. Furthermore, the co-localization of formalin injection-evoked Zif/268 and c-Fos expression and subunit 1 of the NMDA receptor (NR1) was studied in this nucleus. Zif/268 or c-Fos immunoreactivity elicited by formalin injection was significantly reduced by pretreatment with MK-801 in the superficial layer of the trigeminal nucleus caudalis; more than 40% of the neurons expressing Zif/268 and c-Fos in this layer were also immunolabeled by NR1. On the other hand, there was little effect of MK-801 administration on Zif/268 and c-Fos immunoreactivity in the nucleus proprius and deep lamina V of the trigeminal nucleus caudalis, while most neurons expressing Zif/268 or c-Fos in these two regions were labeled by NR1. These results point out differences between the superficial and deeper layers of the trigeminal nucleus caudalis in the involvement of NMDA receptor in the mechanisms underlying the expression of protein products of immediate early genes induced by painful stimuli.

Simultaneous activation of N-methyl-d-aspartate and neurokinin-1 receptors modulates c-Fos and Zif/268 expression in the rat trigeminal nucleus caudalis

We examined the acute expression of c-Fos or Zif/268 by simultaneous activation of N-methyl-D-aspartate receptor and neurokinin-1 receptor of the trigeminal nucleus caudalis in anesthetized rats. A selective N-methyl-D-aspartate receptor agonist, N-methyl-D-aspartate, and/or a selective neurokinin-1 receptor agonist, substance P, was applied topically to the dorsal surface of the spinal trigeminal tract. Immunohistochemically stained nuclei for c-Fos and Zif/268 at laminae I and II of the trigeminal nucleus caudalis were counted. Ipsilateral c-Fos and Zif/268 were increased significantly dose-dependently by N-methyl-D-aspartate (at 136 and 340 microM, and at 68, 136 and 340 microM, respectively). On the contralateral side, only Zif/268 increased significantly (at 68, 136 or 340 microM). These increases were abolished by D-2-amino-5-phosphonovaleric acid (at 25 mM), a selective N-methyl-D-aspartate receptor antagonist. Substance P (at 3.7 or 7. 4 microM) significantly increased dose-dependently ipsilateral c-Fos and Zif/268. On the contralateral side, only c-Fos was significantly increased (at 3.7 and 7.4 microM). These increases were abolished by D-2-amino-5-phosphonovaleric acid (at 25 mM) and L-703,606 (at 10 microM), a selective neurokinin-1 receptor antagonist. The combined application of N-methyl-D-aspartate 340 microM + substance P (at 0.74 or 3.7 microM) significantly increased ipsilateral c-Fos compared to either agent alone. Combined application of N-methyl-D-aspartate 340 microM + substance P at 0.74, 3.7 or 7.4 microM significantly increased ipsilateral Zif/268 expression compared to either drug alone. Other combinations did not increase c-Fos and Zif/268. Our results indicate that activation of N-methyl-D-aspartate or neurokinin-1 receptor of the trigeminal nucleus caudalis contributes to the acute induction of both c-Fos and Zif/268 on the ipsilateral superficial layer of this nucleus and simultaneous activation of both receptors by their agonists with specific concentrations produces a marked expression of these proteins. Simultaneous activation of N-methyl-D-aspartate and neurokinin-1 receptors under some specific conditions may augment synaptic transmission, contributing to long-term neuronal change.