동물의 종 유지에 있어서 교미행동은 매우 중요한 위치를 차지함. 교미과정에서 수컷의 경우 모든 암컷에 대해 성행동을 보이는반면, 암컷은 가장 적절한 교미의 시기를 정하며, 적절한 수컷을 선택하고 교미행동을 보이기 때문에 암컷의 교미행동을 유도하는 과정은 생물학에서 매우 중요한 의미를 지님. 본 연구에서는 초파리 (Drosophila melanogaster) 전자현미경자료와 총신경망분석 (Connectomics)을 이용하여 초파리 뇌에서 암컷의 교미행동을 조절하는 것으로 알려진 pC1신경의 하부신경 pC1b,c가 성적 성숙과정에서 교미를 하고자 하는 욕구 (sexual drive)를 증가시키는 기능을 하는 것을 처음으로 밝힘. 본 연구에서는 처음으로 pC1b,c 신경 내의 cAMP 수준이 교미의 욕구변화를 보여주는 중요한 물질이라는 것을 밝혔고 cAMP의 수준이 오르기 위해서는 신경펩티 드인 Dh44와 그 수용체 GPCR인 Dh44R1과 Dh44R2가 필요하다는 것을 확인함. 또한 cAMP의 변화는 신경내의 CREBB를 통하여 하위 유전자인 pyx (pyrexia)의 발현을 유도한다는 것을 밝힘. 본 연구로 종 유지 메커니즘을 좀 더 이해할 수 있음.

From invertebrate to vertebrate, females uptake sperm for a specific duration post-copulation known as the ejaculate holding period (EHP) before expelling un-stored sperm and the mating plug through sperm ejection. Our study uncovered that encountering males or mated females after mating substantially shortens EHP, a phenomenon we term ‘male-induced EHP shortening (MIES)’. MIES requires Or47b+ olfactory and ppk23+ gustatory neurons, activated by 2-methyltetracosane and 7-tricosene, respectively. These odorants raise cAMP levels in pC1b and c neurons, which are responsible for processing male courtship and regulating female receptivity. Elevated cAMP levels in pC1b and c reduce EHP and reinstate their responsiveness to male courtship cues, promoting re-mating with faster sperm ejection. This study establishes MIES as a genetically tractable model of sexual plasticity with a conserved neural mechanism.

Harlequin ladybird (Harmonia axyridis (Pallas, 1773)) is an invasive species originating from Asia, posing a potential threat to the ecosystem and the wine industry in New Zealand due to wine taint, although it can also be a useful biocontrol agent. In this study, the response profiles of antennal olfactory receptor neurons (ORNs) to 32 plant volatiles were examined in male and female H. axyridis, using the single sensillum recording technique. Various types of ORNs were identified from four types of olfactory sensilla in both male and female H. axyridis, with no sexual dimorphism. The most abundant type of sensilla contained two ORNs exhibiting highly specialized responses to methyl benzoate and β-caryophyllene, respectively. Another type of sensilla also contained two specialized ORNs, one responsive to geranyl acetate and the other to some aromatic compounds such as 2-phenylethanol, benzyl acetate, methyl benzoate, and methyl phenylacetate. In contrast, two other types of sensilla contained broadly tuned ORNs, one containing ORN(s) responsive to six-carbon alcohols such as (Z)-3-hexen-1-ol, 1-hexanol, and isomers of 2-hexen-1-ol as well as some other non-alcohol green leaf volatiles, and the other containing ORN(s) exhibiting responses to β-myrcene, geraniol, linalool, nerol, benzyl acetate, and methyl phenylacetate. This study suggests that H. axyridis possesses a set of ORNs specialized for specific plant volatiles, providing insights into the olfactory communication system of this species and potential volatiles to be used for trapping this insect.

Ischemic stroke leads to severe brain damage and high mortality. Chlorogenic acid is a phenolic compound known to have neuroprotective properties. Bcl-2 family protein plays an important role in the regulation of apoptosis. We investigated whether chlorogenic acid exerts neuroprotective effects against ischemic injury by modulating Bcl-2 and Bax proteins. Middle cerebral artery occlusion (MCAO) was performed to induce cerebral ischemia and rats were injected intraperitoneally with phosphate buffered saline or chlorogenic acid (30 mg/kg) for 2 h after MCAO. Cortical tissues were collected 24 h after MCAO injury and reverse transcription-quantitative real time polymerase chain reaction and Western blot analyses were performed to investigate the expression of Bcl-2 and Bax. The regulation of Bcl-2 and Bax proteins by chlorogenic acid during glutamateinduced cell damage were examined. Cells were collected at 24 h after administration of glutamate (5 mM) and chlorogenic acid (10, 30, 50 μM). These results showed a decrease in Bcl-2 expression and an increase in Bax expression in MCAO animals, but chlorogenic acid treatment alleviated these changes by MCAO damage. Glutamate significantly reduced cell viability, and chlorogenic acid treatment alleviated this reduction in a dose-dependent manner. Glutamate induced a decrease in Bcl-2 expression and an increase in Bax expression, but chlorogenic acid treatment alleviated these changes. We found that chlorogenic acid alleviates changes in the expression of Bcl-2 and Bax proteins induced by brain injury. Therefore, our findings provide an evidence that chlorogenic acid has neuroprotective effects against MCAO damage by modulating Bcl-2 and Bax proteins.

Ischemic stroke causes brain damage and neuronal cell death by depriving oxygen and nutrients and releasing excessive levels of glutamate and intracellular calcium. Epigallocatechin gallate (EGCG) is a polyphenolic compound present in green tea. It has antioxidant, anti-inflammatory, and neuroprotective effects. Hippocalcin is a calcium binding protein that regulates calcium concentration, neuronal differentiation, neuronal excitability, and neuronal cell death. In this study, we investigated whether EGCG regulates the expression of hippocalcin in neurons and astrocytes after focal cerebral ischemia. Cerebral ischemia was induced by meddle cerebral artery occlusion (MCAO). EGCG (50 mg/kg) or PBS was injected into the abdominal cavity just before MCAO surgery. Neurobehavioral tests were performed to evaluate the effect of EGCG on neurological behavioral deficits 24 h after MCAO surgery. Immunofluorescence staining was performed to evaluate the positive response to hippocalcin in the cerebral cortex after MCAO surgery. We also detected the positive reactions of neuronal nuclear protein (NeuN) and glial fibrillary acidic protein (GFAP) as markers of neuron and astrocyte, respectively. MCAO caused severe neurological impairment and EGCG treatment attenuated these impairments. MCAO damage reduced the number of NeuN-positive cells and increased the number of GFAP-positive cells. This result indicates a decrease in neurons and an increase in astrocytes. However, EGCG alleviated these changes caused by MCAO damage. MCAO reduced the number of hippocalcin-positive cells in neurons and astrocytes, and EGCG treatment attenuated these reductions. Hippocalcin exerts neuroprotective effect through regulating intracellular calcium concentration. In conclusion, EGCG regulates the expression of hippocalcin in neurons and astrocytes and has neuroprotective effects in focal cerebral ischemia.

Activation of transient receptor potential vanilloid 1 (TRPV1), a calcium permeable channel expressed in primary sensory neurons, induces the release of glutamate from their central and peripheral afferents during normal acute and pathological pain. However, little information is available regarding the glutamate release mechanism associated with TRPV1 activation in primary sensory neurons. To address this issue, we investigated the expression of vesicular glutamate transporter (VGLUT) in TRPV1-immunopositive (+) neurons in the rat trigeminal ganglion (TG) under normal and complete Freund’s adjuvant (CFA)-induced inflammatory pain conditions using behavioral testing as well as double immunofluorescence staining with antisera against TRPV1 and VGLUT1 or VGLUT2. TRPV1 was primarily expressed in small and medium-sized TG neurons. TRPV1+ neurons constituted approximately 27% of all TG neurons. Among all TRPV1+ neurons, the proportion of TRPV1+ neurons coexpressing VGLUT1 (VGLUT1+/ TRPV1+ neurons) and VGLUT2 (VGLUT2+/TRPV1+ neurons) was 0.4% ± 0.2% and 22.4% ± 2.8%, respectively. The proportion of TRPV1+ and VGLUT2+ neurons was higher in the CFA group than in the control group (TRPV1+ neurons: 31.5% ± 2.5% vs. 26.5% ± 1.2%, VGLUT2+ neurons: 31.8% ± 1.1% vs. 24.6% ± 1.5%, p < 0.05), whereas the proportion of VGLUT1+, VGLUT1+/TRPV1+, and VGLUT2+/TRPV1+ neurons did not differ significantly between the CFA and control groups. These findings together suggest that VGLUT2, a major isoform of VGLUTs, is involved in TRPV1 activation-associated glutamate release during normal acute and inflammatory pain.

Dopamine (DA) receptor (D1 and D2-like receptors) agonists are known to affect expression levels of DA receptors. Rotigotine, a DA D2-like receptor agonist, has been developed for treating Parkinson’s disease (PD). However, its role in PD by acting through DA D2-like receptors has not been fully understood yet. The purpose of this study was to investigate neuroprotective effects of rotigotine through DA D2 and D3 receptors in 6-hydroxydopamine (6-OHDA) induced mouse model of PD. Expression level of tyrosine hydroxylase (TH) was examined using immunohistochemistry and Western blot analysis. Results revealed that unilateral injection of 6-OHDA into the midbrain caused significant loss of TH positive cells in the substantia nigra, whereas rotigotine inhibited such loss of TH cells in 6-OHDA-induced mouse model of PD. In vitro experiments demonstrated that rotigotine increased expression levels of TH against 6-OHDA-induced toxicity. The expression level of TH after treatment with L’741,626, a D2 receptor antagonist was decreased more than that after treatment with GR 103691, a D3 receptor antagonist. These results suggest that rotigotine can protect DA neurons against 6-OHDA induced toxicity and that the protective effect of rotigotine for DAergic neurons through a DA D2 receptor is stronger than that through a DA D3 receptor.

Sparassis latifolia is a fungus abundant in β-glucan and amino acids and is highly valued as a medicinal mushroom. Among amino acids, γ-aminobutyric acid (GABA) is a free amino acid and has biological effects, such as increase/decrease of hypertension, improvement of cerebral blood flow, and prevention of dementia. In this study, biological elicitors were used to increase bioactive substances as a biofortification method. Sodium alginate extracted from seaweed (Sargassum horneri, Sargassum fulvellum, Sargassum fusiforme) were used as the elicitor. The levels of β-glucan and GABA in the mycelium and fruiting body grown by adding the elicitor to the medium were investigated. Addition of sodium alginate positively affected GABA production and negatively affected the β-glucan production in these fungi. Sodium alginates extracted from S. fulvellum induced the highest increase in GABA in the mycelium and fruiting bodies. Moreover, we investigated the effects of the extracts from mycelium and fruiting bodies on dendrite development in primary cortical neurons. We found that the extract from the fruiting bodies of sodium alginate treated fungi with increased levels of GABA inhibited the dendrite outgrowth of excitatory neurons, but not inhibitory neurons.

Reactive oxygen species (ROS) and nitrogen species (RNS) are involved in cellular signaling processes as a cause of oxidative stress. According to recent studies, ROS and RNS are important signaling molecules involved in pain transmission through spinal mechanisms. In this study, a patch clamp recording was used in spinal slices of rats to investigate the action mechanisms of O2 ⦁- and NO on the excitability of substantia gelatinosa (SG) neuron. The application of xanthine and xanthine oxidase (X/XO) compound, a ROS donor, induced inward currents and increased the frequency of spontaneous excitatory postsynaptic currents (sEPSC) in slice preparation. The application of S-nitroso-N-acetyl-DLpenicillamine (SNAP), a RNS donor, also induced inward currents and increased the frequency of sEPSC. In a single cell preparation, X/XO and SNAP had no effect on the inward currents, revealing the involvement of presynaptic action. X/XO and SNAP induced a membrane depolarization in current clamp conditions which was significantly decreased by the addition of thapsigargin to an external calcium free solution for blocking synaptic transmission. Furthermore, X/XO and SNAP increased the frequency of action potentials evoked by depolarizing current pulses, suggesting the involvement of postsynaptic action. According to these results, it was estblished that elevated ROS and RNS in the spinal cord can sensitize the dorsal horn neurons via pre- and postsynaptic mechanisms. Therefore, ROS and RNS play similar roles in the regulation of the membrane excitability of SG neurons.

Dopaminergic neurons are one of the major neuronal components in the brain. Mesencephalon dopamine (DA) neurogenesis takes place in the ventricular zone of the floor plate, when DA progenitors divide to generate postmitotic cells. These cells migrate through the intermediate zone while they differentiate and become DA neurons on reaching the mantle zone. However, neurogenesis and neuronal migration on dopaminergic neurons remain largely unexplored in the mesencephalon development. This study presents neurogenesis and neuronal migration patterns of dopaminergic neurons during mesencephalic development of the mouse. Neurons from embryonic day (E) 10–14 were labelled by a single injection of 5-bromodeoxyuridine and immunohistochemistry was performed. The neurogenesis occurred mainly at the E10 and E11, which was uniformly distributed in the mesencephalic region, but neurons after E13 were observed only in the dorsal mesencephalon. At the postnatal day 0 (P0), E10 generated neurons were spread out uniformly in the whole mesencephalon whereas E11-originated neurons were clearly depleted in the red nucleus region. DA neurons mainly originated in the ventromedial mesencephalon at the early embryonic stage especially E10 to E11. DA neurons after E12 were only observed in the ventral mesencephalon. At E17, E10 labelled neurons were only observed in the substantia nigra (SN) region. Our study demonstrated that major neurogenesis occurred at E10 and E11. However, neuronal migration continued until neonatal period during mesencephalic development.

Recent studies indicate that mitochondria are an important source of reactive oxygen species (ROS) in the spinal dorsal horn. In our previous study, application of malate, a mitochondrial electron transport complex I substrate, induced a membrane depolarization, which was inhibited by pretreatment with ROS scavengers. In the present study, we used patch clamp recording in the substantia geletinosa (SG) neurons of spinal slices, to investigate the cellular mechanism of mitochondrial ROS on neuronal excitability. DNQX (an AMPA receptor antagonist) and AP5 (an NMDA receptor antagonist) decreased the malate-induced depolarization. In an external calcium free solution and addition of tetrodotoxin (TTX) for blockade of synaptic transmission, the malateinduced depolarization remained unchanged. In the presence of DNQX, AP5 and AP3 (a groupⅠ metabotropic glutamate receptor (mGluR) antagonist), glutamate depolarized the membrane potential, which was suppressed by PBN. However, oligomycin (a mitochondrial ATP synthase inhibitor) or PPADS (a P2 receptor inhibitor) did not affect the substrates-induced depolarization. These results suggest that mitochondrial substrate-induced ROS in SG neuron directly acts on the postsynaptic neuron, therefore increasing the ion influx via glutamate receptors.

Alzheimer’s disease (AD), a progressive neurodegenerative disorder that deprives the patient of memory, is associated mainly with extracellular senile plaque induced by the accumulation of amyloid β protein (Aβ). Silybum marianum (Asteraceae; SM) is a medicinal plant that has long been used in traditional medicine as a hepatoprotective remedy owing to its antioxidant and anti-inflammatory activities. The present study examined the methanol extract of the aerial parts of SM for neuroprotection against Aβ (25-35)-induced neuronal death in cultured rat cortical neurons to investigate a possible therapeutic role of SM in AD. The primary cortical neuron cultures were prepared using embryonic day 15 to 16 SD rat fetuses. Cultured cortical neurons exposed to 10 μM Aβ (25-35) for 36 h underwent neuronal cell death. At 10 and 50 μg/mL, SM prevented Aβ (25-35)-induced neuronal cell death and apoptosis in cultured cortical neurons. Furthermore, SM inhibited the Aβ (25-35)-induced decrease in anti-apoptotic protein, Bcl-2, and the increase in the proapoptotic proteins, Bax and active caspase-3. Cultured cortical neurons exposed to 1 mM N-methyl-D-aspartate (NMDA) for 14 h induced neuronal cell death. SM (10 and 50 μg/mL) prevented NMDA-induced neuronal cell death. These results suggest that SM inhibited Aβ (25-35)-induced neuronal apoptotic death via inhibition of NMDA receptor activation and that SM has a possible therapeutic role in preventing the progression of neurodegeneration in AD.

Introducing lineage-specific transcription factors (TFs) into somatic cells enables the induction of distinct cellular identities without the need to first pass through a pluripotent stem cell (PSC) state. We and others have demonstrated the direct conversion of somatic cells into adult stem cells or progenitor cells, such as angioblast-like progenitor cells, hematopoietic stem cells, and neural stem cells. The process underlying direct conversion is known to be relatively simpler and faster than that of induced pluripotent stem cell (iPSC) generation. Furthermore, directly converted cells have been shown to exhibit therapeutic potential following transplantation into respective disease models without obvious evidence for tumor formation. Thus, TF-mediated direct conversion technology has been considered as an alternative to iPSC technology for patient-specific cell- and tissue-replacement therapies. Here we show our recent findings describing the robust direct conversion of differentiated somatic cells into distinct cellular identities. Furthermore, we also show the recent 3D organoid technology for generating brain tissues from human pluripotent stem cells.

Electrophysiological responses of Queensland fruit fly, Bacterocera tryoni, were investigated to identify the olfactory receptor neurons (ORN) in the antennae and corresponding active volatile compounds, using the single sensillum recording technique and 52 test compounds including methyl methyl eugenol, cuelure and raspberry ketone. We found that different classes of olfactory sensilla and ORNs are present in the antennae of B. tryoni. Most of the ORNs appeared to have narrow response spectra, exhibiting specialized responses to one or a few volatile compounds among the 52 compounds tested. In this study, ORNs specialized for ammonia, nonanal, isopentanol, ethyl acetate, indole, phenol, 2,5-dimethylpyrazine and 2-coumaranone, respectively, have been identified. A class of ORNs exhibited highly specialized responses to methyl eugenol. A preliminary field trapping test conducted in New Caledonia to evaluate the behavioral activities of the olfactory active compounds indicated a blend of three synthetic compounds is attractive to female B. tryoni.

Reactive oxygen species (ROS) and nitrogen species (RNS) are both important signaling molecules involved in pain transmission in the dorsal horn of the spinal cord. Xanthine oxidase (XO) is a well-known enzyme for the generation of superoxide anions (O2 ⦁-), while S-nitroso-N-acetyl-DLpenicillamine (SNAP) is a representative nitric oxide (NO) donor. In this study, we used patch clamp recording in spinal slices of rats to investigate the effects of O2 ⦁- and NO on the excitability of substantia gelatinosa (SG) neurons. We also used confocal scanning laser microscopy to measure XO- and SNAP-induced ROS and RNS production in live slices. We observed that the ROS level increased during the perfusion of xanthine and xanthine oxidase (X/XO) compound and SNAP after the loading of 2′,7′-dichlorofluorescin diacetate (H2DCF-DA), which is an indicator of intracellular ROS and RNS. Application of ROS donors such as X/XO, β -nicotinamide adenine dinucleotide phosphate (NADPH), and 3-morpholinosydnomimine (SIN-1) induced a membrane depolarization and inward currents. SNAP, an RNS donor, also induced membrane depolarization and inward currents. X/XO-induced inward currents were significantly decreased by pretreatment with phenyl N-tert-butylnitrone (PBN; nonspecific ROS and RNS scavenger) and manganese(III) tetrakis(4-benzoic acid) porphyrin (MnTBAP; superoxide dismutase mimetics). Nitro-L-arginine methyl ester (NAME; NO scavenger) also slightly decreased X/XO-induced inward currents, suggesting that X/XO-induced responses can be involved in the generation of peroxynitrite (ONOO-). Our data suggest that elevated ROS, especially O2 ⦁-, NO and ONOO-, in the spinal cord can increase the excitability of the SG neurons related to pain transmission.

Antennal olfactory receptor neurons (ORNs) for pheromone and plant volatile compounds were identified and characterized in Sitona lepidus and S. discoideus, using the single sensillum recording technique with five pheromone-related compounds and 40 host and non-host plant volatile compounds. Different types of olfactory sensilla containing specialized ORNs were identified in these weevils. Different types of sensilla housed ORNs specialized for pheromone-related compounds, 4-methyl-3,5-heptanedione or one or more of four stereoisomers of 5-hydroxy-4-methyl-3-heptanone. In addition to the pheromone-related ORNs, several types of olfactory sensilla contained ORNs responsive to plant volatile compounds. Most of the ORNs showed high specificity to specific volatile compounds although some of the active compounds showed overlapping response spectra in the ORNs across different types of sensilla. Our study indicates that both S. lepidus and S. discoideus have species-specific set of highly sensitive and selective ORNs for pheromone and plant volatile compounds. The behavioral implication of these findings is discussed.

Vitis amurensis, Aralia cordata, and Glycyrrhizae radix have been widely used as oriental medicinal plants in Korea, China and Japan and found to possess anti-oxidative and anti-inflammatory activities. A previous study demonstrated a protection of an ethanol extract (SSB) of a mixture of three medicinal plants of Vitis amurensis, Aralia cordata, and Glycyrrhizae radix against β amyloid protein-induced memory impairment. The current study was conducted to investigate the neuroprotective effect of SSB against ischemiainduced brain injury. Transient focal cerebral ischemia was induced by 2 hr middle cerebral artery occlusion followed by 24 hr reperfusion (MCAO/reperfusion) in rats. Oral administration of SSB (5, 10 and 25 mg/kg) 30 min before and 1 h after MCAO, and 1 h after reperfusion reduced MCAO/ reperfusion-induced brain infarct and edema formation. SSB also inhibited development of behavioral disabilities in MCAO/reperfusion-treated rats. Exposure of cultured cortical neurons to 500 μM glutamate for 12 hr resulted in neuronal cell death. SSB (1-10 μg/mL) inhibited glutamateinduced neuronal death, elevation of intracellular calcium concentration ([Ca2+]i), and generation of reactive oxygen species (ROS). These results suggest that the neuroprotective effect of SSB against ischemia-induced brain damage might be associated with its anti-excitotoxic activity and that SSB may have a therapeutic role for prevention of neurodegeneration in stroke.

Nitric Oxide (NO) is an important signaling molecule in the nociceptive process. Our previous study suggested that high concentrations of sodium nitroprusside (SNP), a NO donor, induce a membrane hyperpolarization and outward current through large conductances calcium-activated potassium (BKca) channels in substantia gelatinosa (SG) neurons. In this study, patch clamp recording in spinal slices was used to investigate the sources of Ca²+ that induces Ca²+-activated potassium currents. Application of SNP induced a membrane hyperpolarization, which was significantly inhibited by hemoglobin and 2-(4-carboxyphenyl) -4,4,5,5- tetramethylimidazoline-1-oxyl-3-oxide potassium salt (c-PTIO), NO scavengers. SNP-induced hyperpolarization was decreased in the presence of charybdotoxin, a selective BKCa channel blocker. In addition, SNP-induced response was significantly blocked by pretreatment of thapsigargin which can remove Ca²+ in endoplasmic reticulum, and decreased by pretreatment of dentrolene, a ryanodine receptors (RyR) blocker. These data suggested that NO induces a membrane hyperpolarization through BKca channels, which are activated by intracellular Ca²+ increase via activation of RyR of Ca²+ stores.

Neuronal activities of taste-responsive cells in the nucleus of the solitary tract (NST) are affected by various physiological factors, such as blood glucose level or sodium imbalance. These phenomena suggest that NST taste neurons are under the influence of neural substrates that regulate nutritional homeostasis. In this study, we reviewed a series of in vivo electrophysiological investigations that demonstrate that forebrain nuclei, such as the lateral hypothalamus or central nucleus of the amygdala, send descending projections and modulate neuronal activity of gustatory neurons in the NST. These centrifugal modulations may mediate plasticity of taste response in the NST under different physiological conditions.