Role of neuronal nitric oxide synthase in slowly progressive dopaminergic neurodegeneration in the Zitter rat.

Neuronal nitric oxide synthase (nNOS) is involved in nigrostriatal dopaminergic (DA) neurodegeneration. However, little is known about the distribution patterns and functions of nNOS in slowly progressive DA neurodegeneration. Here we describe the spatiotemporal change in nNOS expression over the course of neurodegeneration and the effect of short- or long-term treatment with the nNOS inhibitor, 7-nitroindazole (7-NI), in zitter (zi/zi) rats. In the substantia nigra pars compacta (SNc), nNOS expression was significantly increased with progression of neurodegeneration. nNOS-immunoreactive (ir) cells were in the vicinity of tyrosine hydroxylase-ir (TH-ir) DA neurons, and some of these cells were also positive for calbindin. nNOS in the caudate-putamen (CPu) showed little difference during progression of neurodegeneration. However, immunoelectron microscopic analysis revealed that abundant TH-ir fibers in the CPu were degenerated due to compression by vacuoles that contained swollen neuronal and glial elements. Additionally, lipid peroxidation as a marker of membrane oxidation was significantly increased in zi/zi rats. Short-term 7-NI treatment attenuated the increase in lipid peroxidation and inhibited the vacuolation in the CPu. Moreover, long-term 7-NI treatment significantly protected TH-ir neurons in the SNc, and TH-ir fibers and DA contents in the CPu. These results show that nNOS exacerbates slowly progressive DA neurodegeneration, and the neuroprotective effects of 7-NI may result from suppression of membrane oxidation that causes abnormal membrane structures in zi/zi rats.

Involvement of serotonin 2C receptor RNA editing in accumbal neuropeptide Y expression and behavioural despair.

Serotonin 2C receptors (5-HT2 C Rs) are widely expressed in the central nervous system, and are associated with various neurological disorders. 5-HT2 C R mRNA undergoes adenosine-to-inosine RNA editing at five sites within its coding sequence, resulting in expression of 24 different isoforms. Several edited isoforms show reduced activity, suggesting that RNA editing modulates serotonergic systems in the brain with causative relevance to neuropsychiatric disorders. Transgenic mice solely expressing the non-edited 5-HT2 C R INI-isoform (INI) or the fully edited VGV-isoform exhibit various phenotypes including metabolic abnormalities, aggressive behaviour, anxiety-like behaviour, and depression-like behaviour. Here, we examined the behavioural phenotype and molecular changes of INI mice on a C57BL/6J background. INI mice showed an enhanced behavioural despair in the forced swimming test, elevated sensitivity to the tricyclic antidepressant desipramine, and significantly decreased serotonin in the nucleus accumbens (NAc), amygdala, and striatum. They also showed reduced expression of neuropeptide Y (NPY) mRNA in the NAc. In addition, by stereotactic injection of adeno-associated virus encoding NPY into the NAc, we demonstrated that accumbal NPY overexpression relieved behavioural despair. Our results suggest that accumbal NPY expression may be regulated by 5-HT2 C R RNA editing, and its impairment may be linked to mood disorders.

The Sarin-like Organophosphorus Agent bis (isopropyl methyl)phosphonate Induces Apoptotic Cell Death and COX-2 Expression in SK-N-SH Cells.

Organophosphorus compounds, such as sarin, are highly toxic nerve agents that inhibit acetylcholinesterase (AChE), but not cholinesterase, via multiple mechanisms. Recent studies have shown that organophosphorus compounds increase cyclooxygenase-2 (COX-2) expression and induce neurotoxicity. In this study, we examined the toxicity of the sarin-like organophosphorus agent bis(isopropyl methyl)phosphonate (BIMP) and the effects of BIMP on COX-2 expression in SK-N-SH human neuroblastoma cells. Exposure to BIMP changed cell morphology and induced caspase-dependent apoptotic cell death accompanied by cleavage of caspase 3, caspase 9, and poly (ADP-ribose) polymerase (PARP). It also increased COX-2 expression, while pretreatment with a COX inhibitor, ibuprofen, decreased BIMP-dependent cell death and COX-2 expression in SK-N-SH cells. Thus, our findings suggest that BIMP induces apoptotic cell death and upregulates COX-2 expression.

Pharmacological assessment of methamphetamine-induced behavioral hyperactivity mediated by dopaminergic transmission in planarian Dugesia japonica.

The freshwater planarian Dugesia japonica has a simple central nervous system (CNS) and can regenerate complete organs, even a functional brain. Recent studies demonstrated that there is a great variety of neuronal-related genes, specifically expressed in several domains of the planarian brain. We identified a planarian dat gene, named it D. japonica dopamine transporter (Djdat), and analyzed its expression and function. Both in situ hybridization and immunofluorescence revealed that localization of Djdat mRNA and protein was the same as that of D. japonica tyrosine hydroxylase (DjTH). Although, dopamine (DA) content in Djdat(RNAi) planarians was not altered, Djdat(RNAi) planarians showed increased spontaneous locomotion. The hyperactivity in the Djdat(RNAi) planarians was significantly suppressed by SCH23390 or sulpiride pretreatment, which are D1 or D2 receptor antagonists, respectively. These results suggest that planarians have a Djdat ortholog and the ability to regulate dopaminergic neurotransmission and association with spontaneous locomotion.

Enhancement of alcohol drinking in mice depends on alterations in RNA editing of serotonin 2C receptors.

Serotonin 2C receptors (5-HT(2C)R) are G-protein-coupled receptors with various actions, including involvement in drug addiction. 5-HT2CR undergoes mRNA editing, converting genomically encoded adenosine residues to inosines via adenosine deaminases acting on RNA (ADARs). Here we show that enhanced alcohol drinking behaviour in mice is associated with the degree of 5-HT(2C)R mRNA editing in the nucleus accumbens and dorsal raphe nuceus, brain regions important for reward and addiction. Following chronic alcohol vapour exposure, voluntary alcohol intake increased in C57BL/6J mice, but remained unchanged in C3H/HeJ and DBA/2J mice. 5-HT(2C)R mRNA editing frequency in both regions increased significantly in C57BL/6J mice, as did expressions of 5-HT(2C)R, ADAR1 and ADAR2, but not in other strains. Moreover, mice that exclusively express the unedited isoform (INI) of 5-HT(2C)R mRNA on a C57BL/6J background did not exhibit increased alcohol intake compared with wild-type mice. Our results indicate that alterations in 5-HT(2C)R mRNA editing underlie alcohol preference in mice.

Experimental studies of remarkable monoamine releases and neural resistance to the transient ischemia and reperfusion.

INTRODUCTION: The literature described that neural damage caused by ischemia definitely occurs in brain areas. However, few studies have shown real-time changes of extracellular monoamine levels at the time of transient ischemia. METHODS: We examined changes in the responses of dopamine (DA) and serotonin (5-HT) release in the nucleus accumbens (ACC) of rats treated with four-vessel occlusion (4VO) in experiment 1. In the second experiment, we investigated the selective neural vulnerabilities among the ACC, lateral hypothalamus (LH), and frontal cortex (FC) of rats treated with 4VO and four days of reperfusion. RESULTS: The extracellular levels of DA and 5-HT were remarkably increased 200- and 20-fold upon the 10-min clipping of both common carotid arteries in transient cerebral ischemia, respectively. Each increased monoamine release returned to the baseline levels immediately. The release of DA in the ACC and FC was significantly decreased in the rats treated with the coagulation of bilateral vertebral arteries (2VO), compared with that of sham-operated rats. K(+)-induced DA release in the ACC and FC of 4VO-treated rats was increased without alteration of DA content. DISCUSSION: Surviving dopaminergic neurons in the ACC and FC showed neural hyperfunction associated with the monoamine release, serotonergic neurons in particular these areas exhibiting functional resistance to the transient ischemic change. CONCLUSION: It is suggested that the remarkable extracellular release of DA and 5-HT was not the cause of the ischemic delayed neural degeneration in each brain area, and that the functions of neurotransmitter release involved remarkable resistance to the transient ischemia.

Therapeutic effects of human mesenchymal and hematopoietic stem cells on rotenone-treated parkinsonian mice.

To appreciate the potential applications of stem cell technology in neurodegenerative diseases, including Parkinson's disease (PD), it is important to understand the characteristics of the various types of stem cells. In this study, we designed a set of experiments to compare the ability of three types of human stem cells--mesenchymal stem cells (MSCs), bone marrow CD34(+) cells (BM), and cord blood CD34(+) cells (CB)--using rotenone-treated NOD/SCID mice. Rotenone was orally administered once daily at a dose of 30 mg/kg for 56 days to induce a parkinsonian phenotype. Intravenous delivery of CB into rotenone-treated mice was slightly more beneficial than that of MSCs or BM according to both histological and behavioral analyses. Human nucleus (hNu)(+) cells, which are a specific marker of human cells, were observed in the striatum of rotenone-treated mice transplanted with stem cells. These hNu(+) cells expressed tyrosine hydroxylase (TH). Additionally, α-synuclein(+)/TH(+) cells in the substantia nigra pars compacta decreased significantly following stem cell transplantation. Immunohistochemical analysis also revealed that chronic exposure to rotenone decreased glial cell line-derived neurotrophic factor immunoreactivity and that the reduction was improved by each stem cell transplantation. Gene expression analyses revealed that MSCs, BM, and CB expressed several neurotrophic factors. These results suggest that the beneficial effects of intravenous delivery of stem cells into rotenone-treated mice may result not only from a neurotrophic effect but also from endogenous brain repair mechanisms and the potential of intravenous delivery of stem cells derived from an autologous source for clinical applications in PD.

Mumefural prevents insulin resistance and amyloid-beta accumulation in the brain by improving lowered interstitial fluid pH in type 2 diabetes mellitus.

The present study tried to clarify if mumefural would prevent hyperglycemia, one of the typical symptoms of type 2 diabetes mellitus (T2DM), since mumefural is an extract from Japanese apricots preventing hyperglycemia. To clarify if mumefural would prevent T2DM pathogenesis, we used Otsuka Long-Evans Tokushima fatty (OLETF) rats, T2DM model. Mumefural diminished hyperglycemia, HOMA-IR and plasma triglyceride concentration in OLETF rats under fasting conditions. In addition, mumefural elevated protein expression of sodium-coupled monocarboxylate transporter 1 (SMCT1) in the distal colon participating in absorption of weak organic acids, which behave as bases but not acids after absorption into the body. Mumefural also increased the interstitial fluid pH around the brain hippocampus lowered in OLETF rats compared with non-T2DM LETO rats used as control for OLETF rats. Amyloid-beta accumulation in the brain decreased in accordance with the pH elevation. On the one hand, mumefural didn't affect plasma concentrations of glucagon, GLP-1, GIP or PYY under fasting conditions. Taken together, these observations indicate that: 1) mumefural would be a useful functional food improving hyperglycemia, insulin resistance and the lowered interstitial fluid pH in T2DM; 2) the interstitial fluid pH would be one of key factors influencing the accumulation of amyloid-beta.

Serotonin2C receptors in the nucleus accumbens are involved in enhanced alcohol-drinking behavior.

Dopamine and serotonin (5-HT) in the nucleus accumbens (ACC) and ventral tegmental area of the mesoaccumbens reward pathways have been implicated in the mechanisms underlying development of alcohol dependence. We used a C57BL/6J mouse model with increased voluntary alcohol-drinking behavior by exposing the mice to alcohol vapor for 20 consecutive days. In the alcohol-exposed mice, the expression of 5-HT(2C) receptor mRNA increased in the ACC, caudate nucleus and putamen, dorsal raphe nucleus (DRN), hippocampus and lateral hypothalamus, while the protein level of 5-HT(2C) receptor significantly increased in the ACC. The expression of 5-HT(7) receptor mRNA increased in the ACC and DRN. Contents of 5-HT decreased in the ACC shell (ACC(S) ) and DRN of the alcohol-exposed mice. The basal extracellular releases of dopamine (DA) and 5-HT in the ACC(S) increased more in the alcohol-exposed mice than in alcohol-naïve mice. The magnitude of the alcohol-induced ACC(S) DA and 5-HT release in the alcohol-exposed mice was increased compared with the control mice. Intraperitoneal (i.p.) administration or local injection into ACC(S) of the 5-HT(2C) receptor antagonist, SB-242084, suppressed voluntary alcohol-drinking behavior in the alcohol-exposed mice. But the i.p. administration of the 5-HT(7) receptor antagonist, SB-258719, did not have significant effects on alcohol-drinking behavior in the alcohol-exposed mice. The effects of the 5-HT(2C) receptor antagonist were not observed in the air-exposed control mice. These results suggest that adaptations of the 5-HT system, especially the upregulation of 5-HT(2C) receptors in the ACC(S) , are involved in the development of enhanced voluntary alcohol-drinking behavior.

Effect of selective serotonin reuptake inhibitors via 5-HT1A receptors on L-DOPA-induced rotational behavior in a hemiparkinsonian rat model.

L-Dihydroxyphenylalanine (L-DOPA) is considered the gold standard for the treatment of Parkinson's disease (PD). However, long-term administration of L-DOPA can induce abnormal side effects. On the other hand, selective serotonin reuptake inhibitors (SSRIs) including fluoxetine have gained tremendous popularity in the treatment of depression in PD. SSRIs are thought to influence motor function in PD via pharmacological modification of interactions between serotonergic and dopaminergic networks, which are complex and not yet fully understand. In this study, intranigral injection of 6-hydroxydopamine (6-OHDA) in rats caused a significant loss of tyrosine hydroxylase immunoreactivity in the striatum and substantia nigra. However, tryptophan hydroxylase immunoreactivity of the striatum and raphe nucleus was unaffected by 6-OHDA. Immunohistochemical analysis reveal that the serotonergic system was unaffected by the injection of 6-OHDA. We demonstrated also that pre-treatment with fluoxetine significantly suppressed L-DOPA-induced rotational behavior. Additionally, fluoxetine suppressed L-DOPA-induced ERK1/2 and histone H3 phosphorylation. These effects of fluoxetine were abolished by pre-treatment with WAY 100135, a 5-HT(1A) antagonist. These results suggest that fluoxetine may influence motor function in PD via pharmacological modification of interactions between serotonergic and dopaminergic neuronal networks.

Detoxification of 6-hydroxydopamine-induced dopaminergic neurodegeneration by 5,5-dimethyl-1-pyrroline N-oxide, a radical trapper, in hemiparkinsonian rats.

Several useful spin trap agents have been identified for the identification and quantification of biological oxygen radicals. Among them, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) has been used most frequently as a spin trap agent. The function of DMPO in vivo, however, is still unclear. Thus, the purpose of this study was to evaluate the effect of DMPO in an in vivo model of Parkinson's disease (PD). Rats were microinjected with 6-hydroxydopamine (6-OHDA, 32 nmol) in the presence or absence of DMPO (0.4, 4 nmol). We investigated behavioral and histochemical parameters in this rat model of PD. In addition, to examine the effect of DMPO against oxidative stress, we performed an electron spin resonance (ESR) analysis. Intranigral injection of 6-OHDA alone caused behavioral dysfunction and a massive loss of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra pars compacta (SNpc). Co-microinjection of 4 nmol DMPO, but not 0.4 nmol, significantly prevented 6-OHDA-induced behavioral impairments and dopaminergic neurodegeneration. In ESR analysis, DMPO directly trapped hydroxyl radical (·OH) generated from 6-OHDA and Fe2+ in a concentration-dependent manner. These results suggest that DMPO attenuates 6-OHDA-induced dopaminergic neurodegeneration in a rat model of PD via scavenging ·OH, and is a useful tool for biological research of oxidative stresses.

A method for determining valproic acid in human whole blood and urine via gas chromatography-mass spectrometry and small-scale inter-laboratory trial.

A simple and cost-effective method for analyzing valproic acid (VPA) in biological samples was developed. VPA was extracted in methyl tertiary-butyl ether (MTBE) and derivatized using trimethylsilyldiazomethane. The MTBE extract was analyzed by gas chromatography-mass spectrometry (GC-MS). The extraction recovery in human whole blood and urine was over 90 %, with good linearity in the range of 1.0 to 250 µg/mL of VPA. The RSD for 2.0, 20, and 200 µg/mL VPA in whole blood ranged from 0.9 to 4.7 % for intra-day and 1.5 to 5.9 % for inter-day. The RSD for 2.0, 20, and 200 µg/mL VPA in urine ranged from 1.9 to 2.6 % for intra-day and 1.2 to 2.9 % for inter-day. As a preliminary cross-validation study, a cross-check was conducted using blinded concentration samples. The results demonstrated that the assay data of the two laboratories were comparable.

Four cases of cytokine storm after COVID-19 vaccination: Case report.

The global coronavirus disease 2019 (COVID-19) pandemic has led to the rapid development of vaccines against this disease. Despite the success of the international vaccination program, adverse events following vaccination, and the mechanisms behind them, remain poorly understood. Here we present four cases of death following receipt of a second dose of COVID-19 vaccine, with no obvious cause identified at autopsy. Using RNA sequencing, we identified genes that were differentially expressed between our post-vaccination cases and a control group that died of blood loss and strangulation. Three hundred and ninety genes were found to be upregulated and 115 genes were downregulated in post-vaccination cases compared with controls. Importantly, genes involved in neutrophil degranulation and cytokine signaling were upregulated. Our results suggest that immune dysregulation occurred following vaccination. Careful observation and care may be necessary if an abnormally high fever exceeding 40°C occurs after vaccination, even with antipyretic drugs.

Regeneration of dopaminergic neurons after 6-hydroxydopamine-induced lesion in planarian brain.

Planarians have robust regenerative ability dependent on X-ray-sensitive pluripotent stem cells, called neoblasts. Here, we report that planarians can regenerate dopaminergic neurons after selective degeneration of these neurons caused by treatment with a dopaminergic neurotoxin (6-hydroxydopamine; 6-OHDA). This suggests that planarians have a system to sense the degeneration of dopaminergic neurons and to recruit stem cells to produce dopaminergic neurons to recover brain morphology and function. We confirmed that X-ray-irradiated planarians do not regenerate brain dopaminergic neurons after 6-OHDA-induced lesioning, suggesting that newly generated dopaminergic neurons are indeed derived from pluripotent stem cells. However, we found that the majority of regenerated dopaminergic neurons were 5-bromo-2'-deoxyuridine-negative cells. Therefore, we carefully analyzed when proliferating stem cells became committed to become dopaminergic neurons during regeneration by a combination of 5-bromo-2'-deoxyuridine pulse-chase experiments, immunostaining/in situ hybridization, and 5-fluorouracil treatment. The results strongly suggested that G(2) -phase stem cells become committed to dopaminergic neurons in the mesenchymal space around the brain, after migration from the trunk region following S-phase. These new findings obtained from planarian regeneration provide hints about how to conduct cell-transplantation therapy for future regenerative medicine.

Excessive expression of hippocampal ezrin is induced by intrastriatal injection of 6-hydroxydopamine.

Accumulating evidence in humans demonstrates that visuo-spatial deficits are the most consistently reported cognitive abnormalities in Parkinson's disease (PD). Ezrin, radixin, and moesin are collectively known as ERM proteins. Although ERM proteins have important implications in cell-shape determination and relevant signaling pathway, they have not been studied in the hippocampus in association with visuo-spatial memory impairments. The purpose of the present study is to examine whether the expression level of ERM proteins in the hippocampus is changed by an intrastriatal injection of 6-hydroxydopamine (6-OHDA) in mice. The intrastriatal injection of 6-OHDA induced partial dopaminergic deficits and spatial memory impairments. We also found that ezrin was increased in the hippocampus by the microinjection of 6-OHDA. On the other hand, protein levels of radixin and moesin were not influenced by 6-OHDA lesions. These results suggest that excessive ezrin may be related to visuo-spatial memory impairments.

BIMP affects tubulin structure and causes abnormalities in cell division.

Although organophosphorus agents are used worldwide as pesticides, there have been many reports of pesticide poisoning. Nerve agents are organophosphorus agents that interfere with neurotransmission and have been used as chemical weapons in wars. These agents mainly irreversibly inhibit the action of acetylcholinesterase, an enzyme that breaks down acetylcholine, a neurotransmitter, and are believed to cause acute symptoms of poisoning. However, in recent years, the presence of subacute, delayed toxicity independent of acetylcholinesterase inhibition has been reported for some organophosphorus agents. We analyzed the subacute and delayed toxicity of bis(isopropylmethyl)phosphonate (BIMP), which has the same phosphonate group as sarin. BIMP rounded out the morphology of the cells and reduced the proportion of cells in the G1 phase of the cell cycle over time. No DNA damage was observed, suggesting that BIMP may affect cell division.

Neural dysfunctions following experimental permanent occlusions of bilateral common carotid arteries cause an increase of rat voluntary alcohol drinking behavior.

We have previously reported that ischemic animal models treated with a respiratory inhibitor, rotenon, show an increased voluntary alcohol intake. Although it is clear that ischemic brain, as a result of reduced-blood flow, shows pathological events and/or neuro-degenerations apparently, little is known of causal relationship between the mechanism of neural dysfunction and voluntary alcohol consumption. Authors have investigated effects of permanent two-vessel occlusion (p2VO) on rat voluntary alcohol drinking behavior. In first experiment the p2VO-treated rats showed an increase of voluntary alcohol drinking behavior, as compared with sham controls. Using brain microdialysis technique, increases of only nucleus accumbens (ACC) dopamine (DA) releases were suppressed in the p2VO-treated rats significantly, following the high K+ (40 mM) perfusion through the microdialysis probe membrane. Alcohol (200 mM) perfusion-induced DA and serotonin (5-HT) releases in the ACC of the p2VO-treated rats were suppressed significantly in the second experiment, as compared with the sham-treated rats. In third experiment p2VO-treated rats showed significant decreases of the contents of DA, not 5-HT, in the ACC, caudate-putamen (C/P), ventral tegmental area-substantia nigra (VT/SN) and lateral hypothalamus (LH). Dopaminergic neurons in the ACC showed more functional vulnerability against the p2VO treatments, as compared with the serotonergic neurons. An increase of alcohol intake in the p2VO-treated rats means the compensation for the neural degeneration of the dopaminergic system in the ACC consisted brain rewarding system. It was likely suggested that neural disturbance of higher functions involved with incomplete global brain ischemia leads the risk of an abnormal alcohol drinking in human.

Regulation of relaxin 3 gene expression via cAMP-PKA in a neuroblastoma cell line.

Relaxin 3 is expressed in neurons of the brain stem that inneravate wide areas of the forebrain. Relaxin 3 mRNA levels in these neurons are increased in response to restraint stress, and by central administration of corticotropin-releasing factor (CRF). In the present study, we observed that relaxin 3 was expressed in a mouse neuroblastoma cell line, Neuro2a, and investigated the intracellular signaling that activated relaxin 3 gene transcription in vitro. By means of a clone stably transfected with a relaxin 3 promoter-EGFP gene, we observed that dibutyryl cyclic AMP and forskolin increased the relaxin 3 promoter activity. These increases were inhibited by pretreatment with PKA inhibitors, H89 and KT5720. Moreover, the promoter activity was enhanced by CRF treatment after expression of CRF-R1 receptor on the cells. Taken together, these results indicate that relaxin 3 transcription is activated via the cAMP-PKA pathway in the downstream of CRF-R1.

Behavioral, hormonal, and neurochemical outcomes of neonatal repeated shaking brain injury in male adult rats.

It is well known that an abusive environment in childhood is related to individual anxiety behavior in adulthood. Though an imbalance of adrenocorticosteroid receptors and a dysfunction of monoaminergic neuron systems have been proposed, the underlying mechanisms are not fully understood. To address these problems, we recently developed a new model of shaking brain injury (SBI) in neonatal rats. These model rats showed transient microhemorrhages in the gray matter of the cerebral cortex and hippocampus. Using this model, we assessed the effects of neonatal repeated mild SBI on subsequent behavior and the stress response, and we further examined the possible contribution of adrenocorticosteroid receptors in the hippocampus and central monoaminergic neuron systems mediating such abnormalities. Behavioral screening examination with a novel open-field test showed that the rats with postnatal day (P) 3-7 shaking had significantly reduced locomotor activity and exploration behaviors than those with late (P8-14) shaking periods, indicating a critical period for neonatal SBI. In the elevated plus maze (EPM) and the light/dark transition (L/D) tests, the model rats spent less time in the open arm of the EPM and the light box of the L/D test, indicating anxiety-like behavior as adults. In adults, the novel EPM-induced adrenocorticotrophic hormone (ACTH) and corticosterone (CORT) responses were significantly increased by neonatal SBI. Further experiments showed that the expression of mineralocorticoid receptor (MR), but not glucocorticoid receptor (GR), was significantly downregulated in the hippocampus of this model rat. These results suggest that neonatal SBI-induced downregulation of MRs in the hippocampus attenuates negative feedback of the hypothalamic-pituitary-adrenal (HPA) axis, which results in abnormal secretion of ACTH and CORT. Furthermore, the neurochemical analysis showed that shaken rats had higher dopamine (DA), serotonin (5-HT), 5-hydroxyindolacetic acid (5-HIAA), and noradrenaline (NA) levels in the dorsal part of the medial prefrontal cortex (dmPFC). In the amygdala, higher 5-HIAA and lower NA levels were observed. Both areas are known to be anxiety and stress-related. Taken together, the effects of neonatal SBI on the monoaminergic systems may also be involved in the changes of behavioral and hormonal responses in this model.

Characterization of tyramine beta-hydroxylase in planarian Dugesia japonica: cloning and expression.

The planarian Dugesia japonica has a relatively well-organized central nervous system (CNS) consisting of a brain and ventral nerve cords (VNCs), and can completely regenerate it CNS utilizing pluripotent stem cells present in the mesenchymal space. This remarkable capacity has begun to be exploited for research on neural regeneration. Recently, several kinds of molecular markers for labeling of neural subtypes have been reported in planarians. These molecular markers are useful for visualizing the distinct neural populations in planarians. In this study, we isolated a cDNA encoding tyramine beta-hydroxylase (TBH), an octopamine (OA) biosynthetic enzyme, by degenerate PCR in the planarian D. japonica, and named it DjTBH (D. japonica tyramine beta-hydroxylase). In order to examine whether DjTBH contributes to OA biosynthesis, we measured the OA content in DjTBH-knockdown planarians created by RNA interference. In addition, to examine the specificity of DjTBH for OA biosynthesis, we measured not only OA content but also noradrenaline (NA) content, because NA is synthesized by a pathway similar to that for OA. According to high-performance liquid chromatography analysis, the amount of OA, but not NA, was significantly decreased in DjTBH-knockdown planarians. In addition, we produced anti-DjTBH antibody to visualize the octopaminergic neural network. As shown by immunofluorescence analysis using anti-DjTBH antibody, DjTBH-immunopositive neurons were mainly distributed in the head region, and elongated their dendrites and/or axons along the VNCs. In order to visualize octopaminergic and dopaminergic nervous systems (phenolamine/catecholamine nervous system) in the planarian CNS, double-immunofluorescence analysis was carried out using both anti-DjTBH antibody and anti-DjTH (a planarian tyrosine hydroxylase) antibody. DjTBH-immunopositive neurons and DjTH-immunopositive neurons mainly formed distinct neural networks in the head region. Here, we demonstrated that DjTBH clearly contributes to OA biosynthesis, and DjTBH antibody is a useful tool for detecting octopaminergic neurons in planarians.