Fumimasa Amaya

Nitrergic neurons in the medial amygdala project to the hypothalamic paraventricular nucleus of the rat

We investigated nitric oxide (NO)-producing neurons in the amygdala which project to the hypothalamic paraventricular nucleus (PVN) of the rat using retrograde tracing and NADPH-diaphorase histochemistry. Numerous NADPH-diaphorase positive neurons with moderate staining were observed mainly in the medial amygdaloid nucleus. We confirmed that these NADPH-diaphorase positive neurons are identical to NO synthase (NOS)-immunoreactive neurons by double staining with NADPH-diaphorase histochemistry and NOS immunocytochemistry. Most neurons containing cholera toxin B subunit (CTb) - which was retrogradely transported from the PVN - were observed in the medial amygdaloid nucleus. In other amygdaloid nuclei, they were observed much less in the central nucleus, basomedial and anterior cortical nucleus. Double labeled neurons by NADPH-diaphorase and CTb were also identified mostly in the medial nucleus. Approximately 40% of the neurons projecting to the PVN were nitrergic neurons and 16% of NADPH-diaphorase positive neurons in the medial nucleus were revealed to project to the PVN. These results suggest that NO-producing neurons in the medial amygdala directly innervate PVN neurons and regulate neuroendocrine systems such as vasopressin and corticotropin releasing factor release.

The influence of salt loading on vasopressin gene expression in magno- and parvocellular hypothalamic neurons: An immunocytochemical andin situ hybridization analysis

Arginine vasopressin peptide and messenger RNA expression were examined at the cellular level in the magnocellular and parvocellular neurons in the rat paraventricular nucleus after dehydration and rehydration, employing immunocytochemistry and in situ hybridization histochemistry on the same tissue sections. Most magnocellular vasopressinergic neurons of control animals expressed both vasopressin-like immunoreactivity and messenger RNA. However, neurons negative for vasopressin-like immunoreactivity but expressing messenger RNA were also detected, and their number increased during dehydration. In contrast, almost all of the parvocellular vasopressinergic neurons of dehydrated animals expressed vasopressin messenger RNA alone, with continued increase in their number after rehydration, despite return of the number of magnocellular vasopressinergic neurons to the control level. Vasopressin messenger RNA and corticotropin releasing factor-like immunoreactivity were co-localized in the same parvocellular neurons, and vasopressin-immunoreactive nerve terminals were detected in the external zone of the median eminence. These findings suggest that magno- and parvocellular vasopressinergic neurons are differentially activated during dehydration/rehydration. Osmotic stimuli activate all magnocellular vasopressinergic neurons, but the effect is not simultaneous in all of these neurons. Parvocellular vasopressinergic neurons are also activated by the stress of dehydration which effect appears to last longer than in the magnocellular system.

Serotonin Modulates Expression of VIP and GRP mRNA via the 5-HT1B Receptor in the Suprachiasmatic Nucleus of the Rat

The expression of vasoactive intestinal peptide (VIP) and gastrin-releasing peptide (GRP) in the suprachiasmatic nucleus (SCN) changes depending on light. VIP mRNA increases and GRP mRNA decreases in the light phase, while they do not show change without light. In the present study we investigated the involvement of serotonin (5-HT) in the expression of VIP and GRP messenger RNA in the SCN of the rat. The decrease in VIP mRNA and the increase in GRP mRNA in the light phase were amplified by 5-HT depletion using 5,6-dihydroxytryptamine injected into the lateral ventricle. These enhancements due to 5-HT depletion were reversed to control levels by applying 5-HT(1B) agonists TFMPP and CGS12066A, but not a 5-HT(1A)/5-HT(7) agonist, 8-OH-DPAT. The 5-HT(1B) receptor is known to exist on the terminals of the retinohypothalamic tract (RHT). Therefore, next we investigated the morphological relationship of RHT and 5-HT terminals by double-labeling immunocytochemistry and demonstrated that 5-HT-immunoreactive fibers and cholera toxin B subunit-labeled RHT terminals were intermingled in the ventrolateral SCN, and 5-HT axon processes had close contact with RHT terminals. Collectively, these pharmacological and morphological results suggest that 5-HT afferents from raphe nuclei modulate VIP and GRP expression in neurons of the ventrolateral SCN by activating the 5-HT(1B) receptor in the RHT.

A sensitive assay for the biosynthesis and secretion of MANF using NanoLuc activity

Mesencephalic astrocyte-derived neurotrophic factor (MANF) has been reported to prevent neuronal cell death caused by certain stimuli. Accordingly, the molecular features of MANF have been intensively investigated since the reporting of its cytoprotective actions. In addition to the characterization of the transcriptional regulation of MANF under pathophysiological conditions, it is important to understand its intracellular transport and secretion after translation. In this study, we developed a convenient and quantitative assay to evaluate the post-translational regulation of MANF using NanoLuc, a highly active and small luciferase. We inserted NanoLuc after the putative signal peptide sequence (SP) of MANF to construct NanoLuc-tagged MANF (SP-NL-MANF). Similar to wild-type (wt) MANF, SP-NL-MANF was secreted from transiently transfected HEK293 cells in a time-dependent manner. The overexpression of mutant Sar1 or wild-type GRP78, which has been reported to decrease wt MANF secretion, also attenuated the secretion of SP-NL-MANF. Using INS-1 cells stably expressing SP-NL-MANF, we found that the biosynthesis and secretion of SP-NL-MANF can be evaluated quantitatively using only a small number of cells. We further investigated the effects of several stimuli responsible for the expression of ER stress-induced genes on the secretion of SP-NL-MANF from INS-1 cells. Treatment with thapsigargin and high potassium significantly increased NanoLuc activity in the culture medium, but serum withdrawal dramatically down-regulated luciferase activity both inside and outside of the cells. Collectively, these results demonstrate that our method for measuring NanoLuc-tagged MANF as a secretory factor is highly sensitive and convenient not only for characterizing post-translational regulation but also for screening useful compounds that may be used to treat ER stress-related diseases such as neurodegenerative disease, ischemia and diabetes.

A Comparative Analysis of the Molecular Features of MANF and CDNF

Cerebral dopamine neurotrophic factor (CDNF) is a paralogous protein of mesencephalic astrocyte-derived neurotrophic factor (MANF). Both proteins have been reported to show a common cytoprotective effect on dopaminergic neurons as a secretory protein containing the KDEL-like motif of the ER retrieval signal at the C-terminus, RTDL in MANF and [Q/K]TEL in CDNF among many species, although functions of paralogous proteins tend to differ from each other. In this study, we focused on post-translational regulations of their retention in the endoplasmic reticulum (ER) and secretion and performed comparative experiments on characterization of mouse MANF and mouse CDNF according to our previous report about biosynthesis and secretion of mouse MANF using a NanoLuc system. In this study, co-expression of glucose-regulated protein 78 kDa (GRP78), KDEL receptor 1 or mutant Sar1 into HEK293 cells similarly decreased MANF and CDNF secretion with some degree of variation. Next, we investigated whether CDNF affects the secretion of mouse cysteine-rich with EGF-like domains 2 (CRELD2) because mouse wild-type (wt) MANF but not its KDEL-like motif deleted mutant (ΔCMANF) was found to promote the CRELD2 release from the transfected cells. Co-expressing CRELD2 with wt or ΔC CDNF, we found that CDNF and ΔCMANF hardly elevated the CRELD2 secretion. We then investigated effects of the four or six C-terminal amino acids of MANF and CDNF on the CRELD2 secretion. As a result, co-transfection of mouse CDNF having the mouse MANF-type C-terminal amino acids (CDNFRTDL and CDNFSARTDL) increased the CRELD2 secretion to a small extent, but mouse CDNF having human CDNF-type ones (CDNFKTEL and CDNFHPKTEL) well increased the CRELD2 secretion. On the other hand, the replacement of C-terminal motifs of mouse MANF with those of mouse CDNF (MANFQTEL and MANFYPQTEL) enhanced the CRELD2 secretion, and the mouse MANF having human CDNF-type ones (MANFKTEL and MANFHPKTEL) dramatically potentiated the CRELD2 secretion. These results indicate that the secretion of mouse MANF and mouse CDNF is fundamentally regulated in the same manner and that the variation of four C-terminal amino acids in the MANF and CDNF among species might influence their intracellular functions. This finding could be a hint to identify physiological functions of MANF and CDNF.

Activation of p38 mitogen-activated protein kinase in the dorsal root ganglion contributes to pain hypersensitivity after plantar incision.

BACKGROUNDnThe phosphorylation of p38 mitogen-activated protein kinase (MAPK) in the dorsal root ganglion (DRG) promotes primary afferent sensitization. The role of p38MAPK signaling in the DRG in the pathogenesis of plantar incision hyperalgesia has not been investigated.nnnRESULTSnLevels of phosphorylated p38MAPK (p-p38MAPK) obviously increased in the DRG after plantar incision. Unmyelinated and myelinated DRG neurons that express p-p38MAPK contained small to medium cell bodies, suggesting that p-p38MAPK expression is induced in neurons with C- and Aδ-fibers. The p-p38MAPK inhibitors FR167653 or SB203580 inhibited incision-induced mechanical hypersensitivity and spontaneous pain behavior. The systemic administration of tumor necrosis factor-α (TNF-α) inhibitor prevented subsequent incision-induced activation of p38MAPK in the DRG and alleviated mechanical hypersensitivity after the incision.nnnCONCLUSIONSnp38MAPK signaling in the DRG plays a crucial role in the development of primary afferent sensitization and pain behavior caused by plantar incision.

Comparative Effects of Periarticular Multimodal Drug Injection and Single-Shot Femoral Nerve Block on Pain Following Total Knee Arthroplasty and Factors Influencing Their Effectiveness

Purpose This study compared the analgesic effects of local infiltration analgesia (LIA) and femoral nerve block (FNB) after total knee arthroplasty (TKA) and assessed factors associated with analgesia obtained by these two methods. Materials and Methods Study subjects included 66 patients (72 knees) who underwent TKA for osteoarthritis of the knee. Pain visual analogue scale (VAS), the amount of analgesics used, number of days to achieve 90° of flexion of the knee joint, date of initiating parallel-bar walking, range of motion of the knee joint at discharge, and adverse events were investigated. Results The VAS scores did not differ significantly between two groups, whereas the amount of analgesics used was significantly lower in the LIA group. Preoperative flexion contracture was significantly more severe in the LIA group with high VAS compared with low VAS. No serious adverse event occurred in the LIA or FNB group. Conclusions The lower analgesic usage in the LIA group than the FNB group indicates that the analgesic effect of LIA was greater than that of singleshot FNB after TKA. There were no serious complications in either group. The postoperative analgesic effect of LIA was smaller in patients with severe than less severe preoperative flexion contracture.

Acquired Exchange Protein Directly Activated by Cyclic Adenosine Monophosphate Activity Induced by p38 Mitogen-activated Protein Kinase in Primary Afferent Neurons Contributes to Sustaining Postincisional Nociception

Background: The molecular mechanisms responsible for sustained pain after tissue injury are largely unknown. The aim of this study was to clarify the role of exchange protein directly activated by cyclic adenosine monophosphate (EPAC) in sustained postincisional nociception, using tissue injury-induced nociceptor priming, and involvement of p38 mitogen-activated protein kinase (p38MAPK) in EPAC-mediated nociceptor priming. Methods: Plantar incisions were made in the hind paws of Sprague–Dawley rats (n = 144). Nociceptor priming was confirmed by behavior testing followed by prostaglandin E2 injection 14 to 21 days after the incision. ESI-09, a selective EPAC inhibitor, was administered to assess its effects on nociceptor priming. Expression of two isoforms of EPAC (EPAC1/EPAC2) in dorsal root ganglions from naive rats and those 14 days after the incision was detected by immunohistochemistry and Western blotting. Separately, FR167653, a selective p38MAPK inhibitor, was administered to assess its effect on EPAC1/EPAC2 expression and the development of nociceptor priming. Results: Prostaglandin E2 injection 14 to 21 days after the plantar incision induced persistent mechanical hyperalgesia for 7 days. EPAC1/EPAC2 expression in dorsal root ganglion neurons was trivial in naive rats (7.7u2009±u20094.8% for EPAC1; 6.3u2009±u20094.1% for EPAC2) but markedly increased 14 days after the incision (21.0u2009±u20099.4% and 20.1u2009±u20093.8%, respectively). ESI-09 treatment inhibited prostaglandin E2-induced persistent mechanical hypersensitivity but had no effect on incision-induced acute nociceptive hypersensitivity. Treatment with FR167653 before the incision inhibited the development of nociceptor priming and incision-induced EPAC1/EPAC2 expression (8.5u2009±u20095.4% and 7.6u2009±u20093.3%, respectively). Conclusions: Transient inflammatory stimulation causes long-lasting nociceptive hypersensitivity via nociceptor priming during the subacute period after incision. Acquired EPAC activity by p38MAPK in the dorsal root ganglion neurons is a key for this event.

mTOR signaling controls VGLUT2 expression to maintain pain hypersensitivity after tissue injury.

Mammalian target of rapamycin (mTOR) is a serine-threonine protein kinase that controls protein synthesis in the nervous system. Here, we characterized the role of protein synthesis regulation due to mTOR signaling in rat dorsal root ganglion (DRG) following plantar incision. The number of phosphorylated mTOR (p-mTOR)-positive neurons was increased 2-4days after the incision. Rapamycin inhibited p-mTOR expression in the DRG and thermal hypersensitivity 3days but not 1day after the incision. Vesicular glutamate transporter 2 (VGLUT2) expression was increased after the plantar incision, which was inhibited by rapamycin. These results demonstrated that tissue injury induces phosphorylation of mTOR and increased protein level of VGLUT2 in the DRG neurons. mTOR phosphorylation involves in maintenance of injury-induced thermal hypersensitivity.

Can Acute Pain Treatment Reduce Postsurgical Comorbidity after Breast Cancer Surgery? A Literature Review

Regional analgesia, opioids, and several oral analgesics are commonly used for the treatment of acute pain after breast cancer surgery. While all of these treatments can suppress the acute postsurgical pain, there is growing evidence that suggests that the postsurgical comorbidity will differ in accordance with the type of analgesic used during the surgery. Our current study reviewed the effect of analgesics used for acute pain treatments on the major comorbidities that occur after breast cancer surgery. A considerable number of clinical studies have been performed to investigate the relationship between the acute analgesic regimen and common comorbidities, including inadequate quality of recovery after the surgery, persistent postsurgical pain, and cancer recurrence. Previous studies have shown that the choice of the analgesic modality does affect the postsurgical comorbidity. In general, the use of regional analgesics has a beneficial effect on the occurrence of comorbidity. In order to determine the best analgesic choice after breast cancer surgery, prospective studies that are based on a clear definition of the comorbidity state will need to be undertaken in the future.