Kosuke Ebihara

Analgesic effect of milnacipran is associated with c-Fos expression in the anterior cingulate cortex in the rat neuropathic pain model

The objective of the present study was to examine whether milnacipran, a serotonin-noradrenaline reuptake inhibitor, has an analgesic effect in rats with neuropathic pain. In addition, the c-Fos expression was investigated in the supraspinal sites of the brain and in the spinal dorsal horn in association with the nociceptive processing in rats with neuropathic pain produced by chronic constriction injury (CCI) in the sciatic nerve. In the CCI-induced neuropathic rats, behavioral testing for determining the change in the withdrawal threshold to mechanical stimulation and immunohistochemical detection of c-Fos were both performed. The anti-allodynic effect derived from milnacipran gradually increased over the observation period, indicating that the delayed-onset analgesia might be elicited by the continuous administration of milnacipran. The increased level of c-Fos expression in the anterior cingulate cortex (ACC) induced by noxious mechanical stimulation was significantly inhibited by the continuous administration of milnacipran, indicating that milnacipran might cause a functional modification in the nociceptive processing in the ACC.

Immunohistochemical characterisation of Fos-positive cells in brainstem monoaminergic nuclei following intracranial self-stimulation of the medial forebrain bundle in the rat

Fos immunostaining was used as a marker of neuronal activity following intracranial self‐stimulation (ICSS) of the medial forebrain bundle (MFB) in the rat, and was combined with immunostaining for tyrosine hydroxylase (TH), serotonin (5‐HT), γ‐aminobutyric acid (GABA), or NR1 (one of the glutamate N‐methyl‐d‐aspartate receptor subunits) for purposes of neurochemical identification. ICSS induced a significant but different degree of increase in the number of Fos‐immunopositive (Fos+) cells in the six brainstem monoaminergic nuclei examined, which included the ventral tegmental area (VTA), substantia nigra pars compacta (SNc), dorsal raphe nucleus (DR), median raphe nucleus (MR), locus coeruleus (LC), and A7 noradrenaline cells. Densely labelled Fos+ cells were observed in the LC following ICSS, and many of these Fos+ cells were colocalized with TH. Similarly, many of Fos+ cells in the A7 and DR/MR were colocalized with TH and 5‐HT, respectively. By contrast, a smaller number of Fos+ cells was detected in the VTA and SNc following the ICSS, and in these regions the majority of Fos+ cells were not colocalized with TH. Although results among regions quantitatively differed, the ICSS induced a significant increase in the number of double‐labelled cells (GABA+/Fos+ or NR1+/Fos+) in all of the VTA, DR, and LC, in which the ICSS produced an ipsilaterally weighted increase in Fos‐like immunoreactivity. These results suggest that ICSS of the MFB induces differential Fos expression within monoaminergic and GABAergic neurons in brainstem monoaminergic nuclei under modulation by glutamatergic afferents.

Psychological prenatal stress reduced the number of BrdU immunopositive cells in the dorsal hippocampus without affecting the open field behavior of male and female rats at one month of age.

We examined whether prenatal psychological stress with little physical stress causes changes in the behavior and neurogenesis of the offspring of Sprague-Dawley rats at one month. Dams in the last trimester of gestation were psychologically stressed by placing them in a social communication box and shocking a rat on the other side of a transparent wall. They suffered little physical stress. Male and female offspring from the dams showed little change in an open field test at postnatal day (PND) 30. To evaluate neurogenesis in the brain, BrdU was intraperitoneally injected at PND 35 into offspring not used in the open field test. Immunohistochemical examinations of BrdU in their dorsal hippocampus at PNDs 42 and 112 revealed that the number of BrdU immunopositive cells in the offspring of prenatally stressed rats was significantly smaller than in the offspring of unstressed ones. These results together with our previous finding that prenatal psychological stress can alter specific behaviors suggest that prenatal psychological stress can suppress neurogenesis in the dorsal hippocampus of rats of both sexes at PND 35 even though impairment in the behavioral task has not yet appeared.

Intra-ventral tegmental area or intracerebroventricular orexin-A increases the intra-cranial self-stimulation threshold via activation of the corticotropin-releasing factor system in rats.

Although orexin‐A peptide was recently found to inhibit the brain reward system, the exact neural substrates for this phenomenon remain unclear. The aim of the present study was to investigate the role of orexin neurons in intra‐cranial self‐stimulation behavior and to clarify the pathways through which orexin‐A inhibits the brain reward system. Immunohistochemical examination using Fos, a neuronal activation marker, revealed that the percentage of activated orexin cells was very low in the lateral hypothalamus even in the hemisphere ipsilateral to self‐stimulation, suggesting that orexin neurons play only a small part, if any, in performing intra‐cranial self‐stimulation behavior. Intra‐ventral tegmental area administration of orexin‐A (1.0 nmol) significantly increased the intra‐cranial self‐stimulation threshold. Furthermore, the threshold‐increasing effects of intra‐ventral tegmental area or intracerebroventricular orexin‐A were inhibited by administration of the nonspecific corticotropin‐releasing factor receptor antagonist, d‐Phe‐CRF12–41 (20 μg). Following intra‐ventral tegmental area infusion of orexin‐A, the percentage of cells double‐labeled with corticotropin‐releasing factor and Fos antibodies increased in the central nucleus of the amygdala but not in the bed nucleus of the stria terminalis, and brain microdialysis analyses indicated that dopamine efflux in both the central nucleus of the amygdala and bed nucleus of the stria terminalis were enhanced. Taken together, the present findings suggest that intra‐ventral tegmental area or intracerebroventricular administration of orexin‐A exerts its threshold‐increasing effect via subsequent activation of the corticotropin‐releasing factor system.

Differential expression of FosB, c-Fos, and Zif268 in forebrain regions after acute or chronic l-DOPA treatment in a rat model of Parkinson's disease

A study was carried out to examine the effects of acute and chronic L-DOPA treatment on the distribution of the immediate-early gene (IEG) proteins (FosB, c-Fos, and Zif268) in forebrain regions in a unilateral 6-hydroxydopamine (6-OHDA) rat model of Parkinsons disease. During a course of chronic L-DOPA treatment (15 mg/day, 15 days), rats with a 6-OHDA lesion developed abnormal involuntary movements. Compared with the rats in the acute L-DOPA treatment group, those in the chronic treatment group had significantly more FosB-immunopositive cells in the anterior cingulate (Cg) and the dorsolateral caudate-putamen ipsilateral to the lesion and significantly fewer c-Fos-immunopositive cells in the Cg, the nucleus accumbens shell, and the basolateral nucleus of amygdala ipsilateral to the lesion. No significant difference was observed in the number of Zif268-immunopositive cells between the acute and chronic L-DOPA groups. In summary, differential expression of three IEG proteins was observed in the forebrain regions during a course of chronic L-DOPA treatment of 6-OHDA-treated hemiparkinsonian rats.

Hemokinin-1 mediates pruriceptive processing in the rat spinal cord

Hemokinin-1 (HK-1) is a new mammalian tachykinin peptide consisting of the amino acid sequence similar to substance P (SP). Although the function of SP, a representative tachykinin peptide, has been well established in the pain system, that of HK-1 has not yet been elucidated. [Leu(11)]-SP had an antagonistic effect on SP-induced scratching behavior, suggesting that [Leu(11)]-HK-1 may also attenuate the induction of scratching behavior by HK-1. Thus, the effects of a pretreatment with [Leu(11)]-HK-1 were evaluated to clarify the function of HK-1. The intrathecal administration of [Leu(11)]-HK-1 attenuated the induction of scratching by HK-1, but not SP, while [Leu(11)]-SP reduced the induction of scratching by SP, but not HK-1. These results indicated that [Leu(11)]-HK-1 may be a more specific antagonist of HK-1-preferred receptors and [Leu(11)]-SP has an antagonistic effect on the SP-preferred receptor, the neurokinin 1 receptor. In the formalin test for examining noxious response, the intrathecal administration of [Leu(11)]-SP, but not [Leu(11)]-HK-1, reduced the number of flinchings and c-Fos-positive cells in the spinal dorsal horn following formalin injection into the plantar region of the hind paw. These results indicated that SP, but not HK-1, is involved in nociceptive processing at the spinal level. To evaluate the involvement of HK-1 and SP in pruritic processing, the effect of [Leu(11)]-HK-1 and [Leu(11)]-SP on the induction of scratching behavior and c-Fos expression by serotonin (5-HT) and histamine was evaluated. The increased induction of scratching behavior and c-Fos expression by 5-HT and histamine was markedly attenuated by pretreatment with both [Leu(11)]-HK-1 and [Leu(11)]-SP, suggesting that HK-1 and SP may be involved in pruritic processing. These results indicate that HK-1 is involved in pruritic processing and [Leu(11)]-HK-1 is a valuable tool for clarifying the mechanisms underlying pruritic processing.

Effects of cabergoline and rotigotine on tacrine-induced tremulous jaw movements in rats.

OBJECTIVES We examined the effects of two dopamine agonists, cabergoline and rotigotine, on tacrine-induced tremor and c-Fos expression in rats. METHODS Rats received intraperitoneal injection of cabergoline (0.5, 1.0, or 5.0mg/kg), rotigotine (1.0, 2.5, or 10.0mg/kg), or vehicle 30min before intraperitoneal injection of tacrine (5.0mg/kg). The number of tremulous jaw movements (TJMs) after tacrine administration was counted for 5min. Animals were sacrificed 2h later under deep anesthesia, and the brain sections were immunostained in order to evaluate the c-Fos expression. RESULTS Induction of TJMs by tacrine was dose-dependently reduced by pretreatment with cabergoline and rotigotine. The number of c-Fos-positive cells was significantly enhanced in the medial striatum, nucleus accumbens core, and nucleus accumbens shell after tacrine administration, and the enhanced expression of c-Fos in these three regions was significantly attenuated by cabergoline, while rotigotine suppressed c-Fos expression in two regions except the nucleus accumbens core. CONCLUSIONS These results suggest that tacrine-induced TJMs would be relieved by either cabergoline or rotigotine and that anticholinesterase-induced TJMs and the ameliorating effects of dopamine agonists would relate to neuronal activation in the striatum and nucleus accumbens.

Differential expression of Fos and Zif268 in the nigrostriatal system after methamphetamine administration in a rat model of Parkinson's disease

The goal of this study was to examine the topological specificity of methamphetamine‐induced activation of the immediate‐early gene proteins, Fos and Zif268, in the nigrostriatal system in a unilateral 6‐hydroxydopamine (6‐OHDA) rat model of Parkinsons disease with or without intrastriatal grafts of fetal ventral mesencephalon. Methamphetamine (3 mg/kg, i.p.) induced Fos‐like immunoreactivity (FLI) dominantly in the striatum and the globus pallidus (GP) on the intact side as well as in the substantia nigra pars reticulata (SNr) on the lesioned side in the 6‐OHDA rats. Lower levels of methamphetamine‐induced FLI in the striatum and GP on the lesioned side were restored by intrastriatal grafts which could completely suppress the methamphetamine‐induced rotation. In the striatum, a similar tendency could be observed between Fos and Zif268 immunoreactivity following methamphetamine. However, sparse immunoreactivity of Zif268 could be detected in the GP and SNr on both sides in the 6‐OHDA rats. Intrastriatal grafts had little influence on Zif268 expression in these two regions. The differential expression of Fos and Zif268 was observed among the three regions of the nigrostriatal system following methamphetamine in the 6‐OHDA rats. This may suggest that Fos and Zif268 therefore possess gene‐specific and region‐specific functions in the basal ganglia nuclei. Synapse 62:920–926, 2008.

Yokukansan, a Traditional Japanese Medicine, Enhances the L-DOPA-Induced Rotational Response in 6-Hydroxydopamine-Lesioned Rats: Possible Inhibition of COMT.

The aim of the present study was to investigate the effects of the traditional Japanese medicine yokukansan (YKS) on the function of dopamine (DA) in the rat nigrostriatal system. Unilateral 6-hydroxydopamine lesions were produced in the rat nigrostriatal system. Despite a marked loss in the striatal immunoreactivity of tyrosine hydroxylase on the lesion side, striatal serotonin (5-HT) immunoreactivity was not affected. Treatment using L-3,4-dihydroxyphenylalanine (L-DOPA) in conjunction with benserazide for 15 d induced abnormal involuntary movements (AIMs) such as locomotive (rotational response), axial, forelimb, and orolingual movements in the lesioned rats. The L-DOPA-induced locomotive and axial, but not forelimb and orolingual, AIMs were significantly increased and prolonged by the pre-administration of YKS. We next investigated the effects of YKS on the production of DA from L-DOPA in 5-HT synthetic RIN 14B cells. RIN 14B cells produced DA and its metabolite, 3-methoxytyramine (3-MT), following L-DOPA treatment. YKS significantly augmented DA production and inhibited its metabolism to 3-MT in a manner similar to the catechol-O-methyltransferase (COMT) inhibitor entacapone. YKS and some alkaloids (corynoxeine: CX, geissoschizine methyl ether: GM) in Uncaria hook, a constituent herb of YKS, also inhibited COMT activity, indicating that the augmenting effect of YKS on L-DOPA-induced DA production in 5-HT synthetic cells was due to the inhibition of COMT by CX and GM. Our results suggest that YKS facilitates the DA supplemental effect of L-DOPA, and that COMT inhibition by CX and GM contributes, at least in part, to the effects of YKS.

Intrastriatal grafts of fetal ventral mesencephalon improve allodynia-like withdrawal response to mechanical stimulation in a rat model of Parkinson's disease.

We previously reported that a unilateral 6-hydroxydopamine (6-OHDA) rat model of Parkinsons disease showed allodynia-like withdrawal response to mechanical stimulation of the ipsilateral side of the rat hindpaw. The goal of this study was to investigate the effect of intrastriatal grafts of fetal ventral mesencephalon (VM) on the withdrawal response in 6-OHDA rats. The withdrawal threshold in response to the mechanical stimulation of the rat hindpaw was measured using von Frey filaments. In the ipsilateral side of the 6-OHDA lesions, the withdrawal threshold in response to mechanical stimulation significantly increased in 6-OHDA rats with VM grafts compared with those with sham grafts, but did not change in the contralateral side at 5 weeks after transplantation. The present results suggest that the intrastriatal grafts of fetal VM may relieve pain sensation induced by mechanical stimulation in 6-OHDA rats.