사춘기전 마우스 미측소핵의 아교질 신경세포에서 시트랄의 억제성 신경전달 조절에 대한 연구

Thao, Nguyen Thi Phuong 전북대학교 일반대학원 2020 국내석사

Citral (3,7-dimethyl-2,6-octadienal) is a monoterpene aldehyde consisting of isomers geranial and neral chemically. The substantia gelatinosa (SG) of the trigeminal subnucleus caudalis (Vc) is admitted as a key site of receiving and regulating orofacial nociceptive inputs. Although citral is known to be involved in antinociception, the action mechanism of citral on the SG neurons of the Vc has not been fully understood yet. In this study, I examined the direct membrane effects of citral and how citral mediates responses on the SG neurons of the Vc in juvenile mice using a whole-cell patch-clamp technique. Under high chloride pipette solution, citral showed repeatable inward currents that persisted in the presence of tetrodotoxin, a voltage-gated Na+ channel blocker, and 6-cyano-7-nitro-quinoxaline-2,3-dione, a non-N-methyl D-aspartate (NMDA) glutamate receptor antagonist, 2-amino-5-phosphonopentanoic acid, an NMDA receptor antagonist. However, the citral-induced inward currents were partially blocked by picrotoxin, a gamma-aminobutyric acid (GABA) type A receptor antagonist, or by strychnine, a glycine receptor antagonist. Further, the citral-induced responses were almost blocked by picrotoxin with strychnine. I also found that citral exhibited additive effect with GABA-induced inward currents and glycine-induced inward currents were potentiated by citral. In addition, citral suppressed the firing activities by positive current injection on the SG neurons of the Vc. Taken together, these results indicate that citral has glycine- and/or GABA-mimetic actions and suggest that citral might be a potential target for orofacial pain modulation by the activation of inhibitory neurotransmission in the SG area of the Vc. keywords : Citral, GABAA receptor, Glycine receptor, Patch-clamp, Substantia gelatinosa neuron

사람 치주인대 세포에서 ATP-의존성 칼륨 통로의 기능적 발현

트란티휀프엉 전북대학교 일반대학원 2016 국내석사

Adenosine-5’-triphosphate (ATP) has been mentioned in dental research on multiple levels, such as inflammation, mechanical strain and pain, making the system particularly relevant for the specific challenges in the oral cavity. There are studies showing human periodontal ligament (PDL) cells respond to mechanical stress by increasing ATP release, which participates in bone resorption or bone homeostasis. So, in this study, we used the RT-PCR and patch-clamp technique to investigate the presence of KATP channel subunits and influence of ATP on the KATP channel opening on PDL cells. We observed transcripts for Kir6.1, Kir6.2 and Sur2B mRNAs isolated from the human PDL cells. In inside-out patch mode, the single channel conductance was 163 pS at symmetrical K+ concentration of 140 mM and inward rectification was seen at the ATP-free bath solution. The reversal potential of the currents was found to be 0 mV at symmetrical concentration (140 mM) of K+ in bath solution. The single channel currents were almost blocked by adding 5 mM ATP in the bath solution. However, the currents were not blocked by 100 μM glibenclamide, a subunit specific KATP channel blocker. These results indicate that human PDL cells express KATP channels which are not sensitive to glibenclamide

마우스 삼차신경 미측소핵 아교질 신경세포에 대한 모노터펜의 억제성 작용

장선희 전북대학교 일반대학원 2022 국내박사

Monoterpenes, as a major constituent of essential oils extracted from plants, are known to have various physiological activities, including analgesic effects. Although many studies have demonstrated that various monoterpenes have anti-nociceptive effects and can modulate neuropathic and inflammatory pain, little is known the mechanism of action of monoterpene in the trigeminal subnucleus caudalis (Vc), which is a principal site that relays orofacial nociceptive information to higher brain centers. The Vc is also called medullary dorsal horn because its structure and function are similar to those of spinal dorsal horn, and the substantia gelatinosa (SG) area of Vc is known to be a key part for processing and transmitting orofacial pain information. Therefore, in this study, to investigate direct effect and action mechanism of monoterpene on SG neurons of the Vc, whole-cell patch clamp technique was applied and statistical analysis was performed on electrophysiological data. In the voltage-clamp mode, geraniol induced repeatable and concentration- dependent inward currents under the condition of high chloride pipette solution. Geraniol-induced responses were sustained in the presence of tetrodotoxin, a voltage-gated Na+ channel blocker, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and DL-2-Amino-5-phosphonovaleric acid (DL-AP5), ionotropic glutamate receptor antagonists. However, geraniol-induced responses were relatively attenuated by picrotoxin, a GABA_A receptor antagonist and/or strychnine, a glycine receptor antagonist. Furthermore, GABA- and glycine-induced responses were enhanced in the presence of low concentration of geraniol. α-Terpineol also induced reproducible and dose-dependent inward currents under the condition of high chloride pipette solution. α-Terpineol-induced responses were maintained in the presence of tetrodotoxin, CNQX and DL-AP5. However, α-terpineol-induced responses were partially suppressed by picrotoxin and/or strychnine. Moreover, GABA- and glycine-induced responses were considerably increased in the presence of low concentrations of α-terpineol. Equally, isopulegol induced reversible and repetitive inward currents under the condition of high chloride pipette solution. Isopulegol-induced responses were increased in a concentration-dependent manner and there were no significant changes in the presence of tetrodotoxin, CNQX and DL-AP5 or strychnine. However, isopulegol-induced responses were significantly inhibited by picrotoxin. In addition, GABA- and glycine-induced responses were enhanced in the presence of low concentrations of isopulegol. Taken together, three monoterpenes used in this study exerted inhibitory actions on SG neurons of Vc through activation of GABA_A and/or glycine receptor and acted as positive modulators of GABA or glycine receptors. These results suggest that monoterpenes have inhibitory neurotransmitter mimetic actions through the activation of GABA_A and/or glycine receptors in the SG area of the Vc, and provide new evidence that monoterpenes may be involved in orofacial pain modulation at the central level. 모노터펜은 식물에서 추출되는 에센셜 오일의 주성분으로서 진통 효과뿐만 아니라 항산화, 항염, 항균, 항암, 항경련, 항불안 등의 다양한 약리학적 활성을 가지고 있는 것으로 보고되었다. 비록 많은 연구들을 통해 여러 종류의 모노터펜에서 항통각수용성 효과가 있고 염증성 및 신경병증성 통증을 조절할 수 있음이 입증되었으나 구강안면 통증정보 전달의 핵심 중추로 알려진 삼차신경 미측소핵에서 모노터펜의 작용 기전은 거의 밝혀진 바가 없다. 삼차신경 미측소핵은 척수후각의 구조와 기능이 유사하여 연수후각이라고도 불리며, 연수후각 내 아교질 영역은 악안면 통증정보 처리 및 전달에 중요한 역할을 하는 부위로 알려져 있다. 따라서 본 연구에서는 마우스 미측소핵 아교질 신경세포에서 모노터펜의 직접적인 영향 및 작용 기전을 규명하기 위해 전세포 패치클램프 기법을 적용하였다. 게라니올은 연수후각 아교질 신경세포에서 반복적이고 농도의존적인 내향성 전류를 유발하였으며, Na+ 채널 차단제인 TTX 및 이온성 glutamate 수용체 길항제인 CNQX와 AP5 존재 시 게라니올에 의해 유발된 반응이 유지되었으나, GABA_A 수용체 길항제인 picrotoxin 또는/그리고 glycine 수용체 길항제인 strychnine 존재 시 게라니올 유도성 반응의 크기가 유의하게 감소하였다. 뿐만 아니라 저용량의 게라니올을 전처리하였을 때 GABA 및 glycine 유도성 전류의 크기가 증가하였다. 다음으로, 알파-테르피네올 역시 연수후각 아교질 신경세포에서 반복 가능하며 농도의존적인 내향성 전류를 유발하였으며, TTX 및 CNQX와 AP5 적용 시에도 알파-테르피네올 유도성 반응은 유지되었다. 그러나 picrotoxin 또는/그리고 strychnine 존재 하에서는 알파-테르피네올에 의해 유도된 반응이 부분적으로 억제되었으며, 저용량의 알파-테르피네올 전처리 시 GABA 및 glycine 유도성 반응이 유의하게 증가하였다. 마찬가지로, 이소풀레골도 연수후각 아교질 신경세포에서 가역적이고 반복적인 내향성 전류를 유발하였다. 이소풀레골 농도에 따라 반응의 크기가 증가하는 양상을 보였으며, TTX 및 CNQX와 AP5, strychnine 존재 하에서도 이소풀레골 유도성 반응은 유의한 차이를 보이지 않았다. 하지만 picrotoxin 존재 하에서는 이소풀레골에 의해 유발된 반응이 일부 감소하였고, 저용량의 이소풀레골 전처리 시 GABA 및 glycine 유도성 반응이 유의하게 증가하는 것을 확인할 수 있었다. 종합해보면 본 연구에 사용된 세 가지의 모노터펜은 연수후각의 아교질 신경세포에서 GABA_A 수용체 그리고/또는 glycine 수용체를 활성화시켜 억제성 효과를 보였으며, GABA 유도 반응 및 glycine 유도 반응을 향상시켰다. 이러한 결과는 모노터펜이 연수후각의 아교질 영역에서 GABA_A 그리고/또는 glycine 수용체 활성화를 통해 억제성 신경전달물질 모방 작용을 가지며, 중추 수준에서 모노터펜이 구강안면 통증 조절에 관여할 수 있다는 새로운 증거를 제공한다.

사춘기전 마우스 생식선자극호르몬 방출호르몬 신경세포에서 멜라토닌에 의한 kainate 수용체 매개 흥분성 신경전달 억제

Santosh, Rijal 전북대학교 일반대학원 2018 국내석사

Melatonin (N-acetyl-5-methoxy tryptamine), a pineal secretion regulates various physiological functions such as regulation of cardiac rhythms, disruption of neuroendocrine physiology, pubertal development and reproduction in mammals. The gonadotropin-releasing hormone (GnRH) neurons are a central regulator of hypothalamic pituitary gonadal (HPG) axis which forms the final common pathway for regulation of reproductive physiology. There are substantial studies regarding the action of melatonin at one or several levels of the HPG axis but its control over GnRH neurons at hypothalamic levels is still unclear. So, the action of melatonin on GABAergic and glutamatergic signaling on juvenile GnRH neurons were examined using patch clamp technique. GABA-and muscimol-mediated responses in presence of melatonin were not significantly different compared to control. Similarly, AMPA-and NMDA receptor-mediated responses were not affected by application of melatonin. However, kainate-induced responses were decreased by melatonin in both whole cell voltage clamp and gramicidin perforated current clamp mode. The mean relative amplitude of kainate-induced inward current in the presence of melatonin with respect to control under whole cell voltage clamp and gramicidin perforated current clamp mode were 0.57±0.11 (n=7) and 0.64±0.12 (n=5), respectively (P<0.05). These results indicate that melatonin has little effects on GABAergic transmissions on GnRH neuron but alters the kainate receptor-mediated glutamatergic transmission suggesting melatonin can modulate the HPG axis by kainate receptor–mediated glutamate action at hypothalamic level.

마우스 미측소핵의 아교질 신경세포에서 세로토닌, 아연, 비스페놀 A에 의한 글리신 반응 조절에 대한 연구

Hoang, Nguyen Thi Thanh 전북대학교 일반대학원 2020 국내박사

The lamina II, also called the substantia gelatinosa (SG), of the medullary dorsal horn (the trigeminal subnucleus caudalis, Vc), is thought to play an essential role in the control of orofacial nociceptive. Glycine, the main inhibitory neurotransmitter in the central nervous system (CNS), has been shown to contribute an essential role in the transference of nociceptive messages from the periphery to the higher brain regions. For this reason, if any compound alters the functional properties of this neurotransmitter in the SG neurons, it may modify significantly the pain-signaling messages proceeding from the orofacial region to the brain. This thesis consists of three parts which correspond with the study about the effect of serotonin, Zn2+, and bisphenol A to the glycine receptors on SG neurons of the Vc. Serotonin (also known as 5-hydroxytryptamine, 5-HT) is an important neurotransmitter in the CNS. 5-HT is concerned in nociceptive transmission through regulation of the descending pathways in the spinal level initiating in the rostral ventromedial medulla. In this study, the whole-cell patch-clamp technique was used to investigate the interaction of intracellular signal transduction between 5-HT and the glycine receptors on SG neurons of the Vc. In nine of 13 neurons tested (69.2%), pretreatment with 5-HT potentiated glycine-induced current (IGly). Among the classes of 5-HT receptor subtypes, my study demonstrated that the 5-HT2 receptor is responsible for the potentiation of 5-HT to the glycine responses. The next compound is Zn2+. Zn2+ is particularly abundant in the mammalian CNS. There are proofs demonstrated the role of Zn2+ in the modulation of voltage- and ligand-gated ion channels. However, little is known about the roles of Zn2+ in the modulation of the glycine responses in the SG neurons of the Vc. Also with the whole-cell patch-clamp technique, Zn2+ was proved to induce a potentiation of glycine receptor-mediated responses. This effect was concentration dependent, the mean inward currents induced by glycine increased correspond with the rise of Zn2+concentration. The last compound of my thesis is bisphenol A (BPA), one of the endocrine disrupter. BPA is reported to alter the morphological and functional characteristics of neuronal cells and to be an effector of a great number of ion channels in the CNS. However, the electrophysiological effects of BPA on the glycine receptors on SG neurons of the Vc have not been well studied yet. By the whole-cell patch-clamp technique, BPA was demonstrated to not show any effect on the glycine-induced inward current in the early neonatal mice (0-3 postnatal day mice). However, at the juvenile and adult groups, BPA enhanced the glycine–mediated responses. This modulation by BPA was proved to be involved in the heteromeric glycine receptors. In summary, the results have demonstrated that both 5-HT, Zn2+, and BPA can potentiate the glycine-induced responses in the SG neurons in mice. The interaction between these above compounds on the glycine receptor in the SG neurons provides additional knowledge about their role in the transmission of orofacial nociceptive information. 연수후각 아교질은 악안면 통증정보 전달 및 조절의 핵심 중추로 간주된다. 중추신경계에서 대표적인 억제성 신경전달물질로 알려진 glycine은 악안면 영역으로부터 상위 중추로 통증정보를 전달하는데 있어 중요한 신경전달물질로 작용한다. 따라서 연수후각 아교질 내에 존재하는 신경세포에서 glycine 반응성을 조절하는 물질은 악안면 통증정보의 전달 및 조절에 영향을 미칠 수 있다. 따라서 본 연구에서는 마우스 미측소핵 아교질 신경세포에서 세로토닌, Zn2+ 및 bispenol A가 glycine의 반응성에 미치는 영향 및 작용 기전을 뇌절편을 이용한 패치클램프 기법을 통해서 규명하고자 하였다. 세로토닌(5-hydroxytryptamine, 5-HT)은 중추신경계에서 중요한 신경전달물질중의 하나로서 rostral ventromedial medulla (RVM)에서 기시하여 척수 수준으로 하행성 회로를 통해서 통증정보를 조절한다고 알려져 있다. 기록된 대다수의 세포에서, 세로토닌 전처리 시 glycine 반응성이 증가되었으며, 이러한 세로토닌에 의한 glycine 반응성의 증가는 세로토닌 수용체 아형 중 5-HT2 수용체가 관여됨을 확인하였다. Zn2+는 특히 포유류 중추신경계에서 막전압 의존성 및 리간드 의존성 이온통로의 활성 조절에 관여한다는 다수의 보고가 있으나 연수후각 아교질 신경세포에서의 작용 양상은 잘 알려져 있지 않다. 본 연구를 통해서 Zn2+가 마우스 연수후각 아교질 신경세포에서 glycine 반응성을 증가시킨다는 사실을 패치클램프 기법을 통해서 확인하였다. Bisphenol A는 내분비 교란물질로 잘 알려진 물질로 신경세포의 형태학 및 기능적 특성을 변화시키고 중추신경계 내 다수 이온통로의 활성에 영향을 미친다고 보고되고 있으나 미측소핵 아교질 신경세포에서의 영향은 보고된 바가 거의 없다. 신생아 마우스(생후 0-3일) 미측소핵 아교질 신경세포에서 bisphenol A는 glycine 매개로 유발된 전류에 영향을 미치지 않았으나 사춘기 및 성체에서는 glycine 매개 반응성 증가를 유발함을 확인하였다. 본 연구를 통해서 얻은 결과로서 세로토닌, Zn2+ 및 bisphenol A가 중추신경계 내 대표적인 억제성 신경전달물질인 glycine의 반응성을 조절함으로서 악안면 통증조절의 잠재적인 표적물질이 될 수 있음을 시사해 주고 있다.

과산화수소에 의한 마우스 생식샘 자극호르몬 방출호르몬

리잘산토스 전북대학교 일반대학원 2022 국내박사

생식샘 자극호르몬 방출호르몬(Gonadotropin-releasing hormone, GnRH) 신경세포는 시상하부에 산발적으로 분포하며, 포유류에서 사춘기 유발 및 생식 과정에 필수적인 생식샘 자극호르몬 분비를 조절하는 핵심 중추이다. 또한 GnRH 신경세포는 생식선 자극호르몬 분비 및 GnRH 신경세포 자체의 활성조절에 관여하는 다양한 신경전달물질이나 신경펩티드를 분비하는 신경세포로부터 흥분성 및 억제성 신호를 받는다. 뇌를 비롯한 다양한 장기에서 활성산소종(Reactive Oxygen Species, ROS)은 스트레스 및 신호전달물질의 작용에 의해 세포내에 발생하는 산화대사의 부산물이다. ROS는 생식계에서도 중요한 생리적 역할을 담당하지만 과도한 ROS는 생식기계의 기능이상을 초래한다. 또한 ROS는 시상하부-뇌하수체-생식샘 축과 다른 내분비 관련 축 사이의 교차반응을 방해하여 생식샘 호르몬 분비를 감소시킬 수 있음이 보고되고 있다. 그러나, ROS가 GnRH 신경세포의 활성조절에 미치는 영향과 그 조절기전은 알려진 바가 적다. 따라서 본 연구에서는 뇌절편을 이용한 전기생리학적인 방법을 통해서 ROS 공급원 중의 하나인 과산화수소가 GnRH 신경세포의 활성에 미치는 직/간접적인 영향 및 그 기전을 확인하고자 하였다. 본 연구에서는 GnRH 신경세포가 녹색의 형광을 발하도록 제작된 형질전환 마우스를 이용하여 GnRH 신경세포를 확보하고 뇌절편 패치 클램프 기법을 이용하여 전기적 활성을 기록하고 분석하였다. GnRH 신경세포에 과산화수소를 적용하였을 때 흥분성, 억제성, 무반응 등 다양한 반응이 관찰되었다. 성숙한 마우스로부터 얻은 뇌절편에서, 고농도(mM 수준)의 과산화수소는 주로 GnRH 신경세포의 과분극(억제성)을 유발하였으나 저농도(pM~μM 수준)에서는 약간의 탈분극(흥분성)을 유발하였다. 미성숙 마우스에서 얻은 뇌절편에서는 과산화수소가 주로 GnRH 신경세포의 흥분성을 유발하여 과산화수소가 연령에 따라 다른 반응을 보일 수 있음을 확인하였다. 또한, 암컷 성체에서 과산화수소의 반응성은 생리주기에 따라 다른 양상을 보였다. 과산화수소에 의한 과분극 반응은 막전압 의존성 나트륨통로 억제제인 테트로도톡신(tetrodotoxin)의 존재하에서도 유지됨으로써 절전섬유의 활성여부에 관련성이 적었음을 확인하였고, 대표적으로 흥분성을 매개하는 glutamate 수용체, 대표적으로 억제성을 매개하는 GABAA 또는 glycine 수용체를 차단한 상태에서도 과산화수소에 의해 유발되는 과분극 반응이 유지됨을 확인함으로써 과산화수소가 GnRH 신경세포에 직접적으로 작용할 가능성을 시사하였다. 과산화수소에 의해 유발되는 세포막 과분극 반응은 세포내 ATP level에 민감한 칼륨 통로(KATP 통로) 차단제인 glibenclamide 전처리 시 억제되어 과산화수소에 의한 과분극에 KATP 통로가 밀접하게 관련되어 있음을 확인하였다. Glutathione peroxidase를 억제함으로써 내인성 과산화수소를 증가시켰을 경우 GnRH 신경세포에서 보이는 자발적 활성의 감소가 관찰되었다. 과산화수소 적용 시 자발적인 연접후 전류의 빈도수가 증가되었으며 이러한 빈도수 증가는 GABA 분비의 증가에 기인한 것임을 확인하였고, GnRH 신경세포에 미치는 GABA의 반응성도 과산화수소 농도 의존적으로 증가시킴을 확인하였다. 본 연구를 통해 과산화수소가 KATP 통로의 활성, GnRH 신경세포 연접부에서의 GABA의 분비 및 GnRH 신경세포에서의 GABA 반응성에 영향을 미칠 수 있음을 확인함으로써 ROS가 시상하부-뇌하수체-생식샘 축 조절에 관여하는 기전의 일부를 제시하였다. Keywords: GABA성 신경전달, 시상하부, ATP 민감성 칼륨 통로, 패치 클램프, 활성산소종 Gonadotropin-releasing hormone (GnRH) neurons are the major output cells of a complex neural network that maintains the pulsatile release of gonadotropins required for puberty and fertility in mammals. The population of these neurons ranges from 700 to 1200, with the majority clustered in an "inverted Y" pattern that runs from the rostral preoptic area to the caudal hypothalamus. GnRH neurons receive dense synaptic inputs around the soma and proximal dendrites, forming a complex neural network that maintains gonadotropin release rhythmicity. Synaptic inputs include both excitatory and inhibitory signals received from a variety of neurotransmitters and neuropeptides that affect the electrical activity of GnRH neurons and gonadotropins secretion. In the brain and other organs, reactive oxygen species (ROS) are by-products of normal cellular oxidative metabolism that operate as both stress and signaling molecules. ROS have both physiological and pathogenic significance in the reproductive system. Besides, ROS are signaling molecules that regulate reproductive physiology at a normal level, while excessive ROS have pathological consequences in the reproductive tract. ROS have been shown to interfere with the cross-talk between the hypothalamic-pituitary-gonadal (HPG) axis and other endocrine axes, disrupt reproductive function, and decrease gonadal hormones. Evidence shows that ROS play a signaling role in the hypothalamic arcuate-median eminence. In addition, it affects the physiology of hypothalamic neurons such as neuropeptide-Y/agouti-related protein neurons, pro-opiomelanocortin/cocaine-and-amphetamine responsive transcript neurons, and the paraventricular nucleus. However, its role in GnRH neuronal regulation remains unknown. Thus, the objective of this study was to determine the effect of hydrogen peroxide (H2O2), an ROS source, on GnRH neuronal excitabilities in transgenic GnRH-green fluorescent protein-tagged mice using the patch-clamp electrophysiology. Perfusion of H2O2 showed an impact on GnRH neuronal excitabilities. H2O2 at high concentrations (mM level) hyperpolarized most GnRH neurons tested, whereas low concentrations (pM to μM) caused slight depolarization. In immature GnRH neurons, H2O2 exposure induced excitation. The sensitivity of GnRH neurons to H2O2 was increased with postnatal development. The effect of H2O2 on adult female GnRH neurons was found to be estrous cycle-dependent. H2O2-induced hyperpolarization persisted in the presence of tetrodotoxin, a voltage-gated Na+ channel blocker, and an amino-acids receptor blocking cocktail, demonstrating that H2O2 can act directly on GnRH neurons. Furthermore, the ATP-sensitive K+ channel blocker glibenclamide prevented H2O2-mediated hyperpolarization completely. Increasing endogenous H2O2 by inhibiting glutathione peroxidase decreased the spontaneous activities of most GnRH neurons. This study suggests that ROS can act as signaling molecules for regulating GnRH neuron excitability and the adult GnRH neurons are sensitive to increased ROS concentration. Next, perfusion of H2O2 also had an impact on GABAergic signaling in GnRH neurons. The GABA receptor is well known for controlling the electrical excitability of GnRH neurons. GABAergic synaptic events and GABAA receptor-mediated responses were increased on GnRH neurons after H2O2 application. Furthermore, H2O2 action on GABAA receptors involves extracellular oxidative reaction and is independent of Fenton reaction. Beside, endogenous H2O2 has no effect on GABAA receptors on GnRH neurons. In addition, I observed that exogenous H2O2 suppressed the GnRH neuronal excitability via GABA release. This study provides initial findings for the H2O2 effect on the GABAergic neurotransmission on GnRH neurons, potentially influencing gonadotropins release from the hypothalamus. Taken together, this study identified inhibitory impact of ROS H2O2 on adult GnRH neuronal physiology, mediated via KATP channels and GABAergic signaling. These findings potentially explain how ROS disrupt the crosstalk of the HPG axis with another endocrine axis at hypothalamic levels and ROS-induced hormonal imbalance that leads to infertility. Keywords: GABAergic neurotransmission, Hypothalamus, KATP channels, Patch-clamp, Reactive oxygen species

마우스 연수후각 아교질 신경세포에서 글리신 수용체 및 시냅스외 GABAA 수용체를 통한 타우린의 작용

엔구엔 디탄홍 전북대학교 일반대학원 2015 국내석사

The substantia gelatinosa (SG) of the trigeminal subnucleus caudalis (Vc) is well known to be a critical site in the processing of orofacial nociceptive information because it receives the nociceptive inputs from primary afferents including myelinated Aδ- and unmyelinated C-fibers. Taurine, one of the most abundant free amino acids in human with various physiological functions, has proved to be involved in pain modulation. In this study, the whole-cell voltage and current clamp modes were used to investigate the direct membrane effects of taurine and the mechanism involved in taurine-mediated responses in the SG neurons of the Vc. Taurine induced non-desensitizing and repeatable membrane potential and current changes in SG neuron which persisted in the presence of mixture of amino acid receptors blocking cocktail (AARBC) and tetrodotoxin (a voltage dependent Na+ channel blocker) indicating that taurine directly acts on the postsynaptic SG neurons. Furthermore, the application of taurine at different concentrations ranging from 10 µM to 3 mM showed a concentration-dependent membrane potential changes and and inward currents with the EC50 of 84.3 µM and 723 µM, respectively. The responses induced by taurine (100 µM and/or 1 mM) were completely blocked by strychnine (2 µM) but not by gabazine (3 µM) indicating activation of the glycine receptor rather than synaptic GABAA receptor, and also partially blocked by picrotoxin (50 µM) indicating involvement of the homomeric glycine receptor, at least in part. In addition, responses induced by taurine (1 mM) were partially bloked by gabazine (50 µM) and picrotoxin (300 µM) suggesting involvement of extrasynaptic GABAA receptor at high concentration. Taken together, these results indicate that taurine can be a target molecule for orofacial pain modulation through the activation of glycine or/and extrasynaptic GABAA receptors in the SG neurons of the Vc.

사춘기전 마우스 생식선자극호르몬 유리호르몬 신경세포에서 Vitamin D3 에 의한 NMDA 및 kainate 매개 신경전달 억제 효과

바타라이프라빈 전북대학교 일반대학원 2016 국내석사

Vitamin D is a versatile fat soluble signaling molecule which plays a critical role in calcium homeostasis. There are a number of studies showing the genomic action of vitamin D in control of reproduction. However, the quick non-genomic action of vitamin D at hypothalamic level has not been well understood. So, in order to investigate the effect of vitamin D on juvenile GnRH neurons, excitatory neurotransmitters NMDA (30-μM) and Kainate (10-μM) were applied in the absence or the presence of vitamin D3 (VitaD3) 10-nM. The NMDA-mediated responses were decreased by VitaD3 in intact and in presence of TTX a sodium channel blocker with a mean relative inward current being 0.56±0.07 and 0.66±0.07 (p<0.05) respectively. In addition, VitaD3 induced decrease in the frequency of GABAergic spontaneous postsynaptic currents and spontaneous postsynaptic currents induced by NMDA application with a mean relative frequency of 0.59 ± 0.07 and 0.56±0.09 respectively. Further, VitaD3 also decreased the Kainate-induced inward currents in intact and in presence of TTX with a relative inward current 0.64±0.06 and 0.72± 0.05 respectively (p<0.05). These results demonstrate that VitaD3 has non-genomic action and partially inhibits the NMDA and kainite receptor-mediated actions on GnRH neurons suggesting that VitaD3 may regulate HPG axis at the time of pubertal development.

마우스 뇌간 부위에서 Kainate 수용체와 TRPV1 수용체의 악안면 통증정보 전달 조절에 미치는 영향에 대한 연구

박선아 전북대학교 대학원 2009 국내박사

Trigeminal primary afferent neurons relay the somatosensory information from the orofacial region to the second-order neurons located in the trigeminal sensory nuclei (Vsn). The Vsn is composed of the mesencephalic nucleus, principal sensory nucleus (Vp) and spinal trigeminal nucleus (Vsp). The Vsp is divided into three subnuclei; oralis (Vo), interpolaris (Vi) and caudalis (Vc). The substantia gelatinosa (SG) of trigeminal subnucleus caudalis (Vc; also called the medullary dorsal horn) receives nociceptive afferent inputs from the thin-myelinated Aδ fibers and unmyelinated C fibers and are implicated in the processing of nociceptive information. Particularly, frequently caused pain in orofacial region involves in various tissue types as tooth, periodontal ligament, and jaw joint. However, mechanism and function of nocice ptive processing in thetrigeminal subnucleus caudalis (Vc) is little known. Therefore, we investigated the receptors involved in nociceptive processing on SG neurons of mice Vc using electrophysiological and molecular studies; existence and functional response of GluR5 and GluR6 of kainate receptor subunits (KARs) between young and adult and existence of TRPV1 receptor in young. First, mRNA and protein expression of GluR5 and GluR6 subunits were decreased according to developmental maturity in lamina II of mice Vc. Also, KA and GluR5/6 agonists highly show the membrane depolarization on SG neurons of young than adult, suggesting that the change of GluR5 and GluR6 of KA receptor subunits can involve in different pain processing modulation of orofacial regions between young and adult. Second, the TRPV1 receptor expression has been reported in the primary afferent sensory fibers, additionally, we investigated the existence of TRPV1 receptor mRNA on postsynaptic SG neurons in young mice Vc. TRPV1 receptor agonist, capsaicin induced the membrane depolarization and acts directly on young SG neurons. These results suggest that endogenous vanilloid can directly mediate the excitatory response on postsynaptic SG neurons and regulate the nociceptive information of orofacial pain through TRPV1 receptors. In this study, we propose the most prominent potential for therapeutic targets and understanding orofacial pain modulation.

삼차신경 척수감각핵 미측소핵 아교질 신경세포에서 5-HT7 수용체의 기능적 발현에 관한 연구 : Functional expression of 5-HT7 receptor on the substantia gelatinosa neurons of the trigeminal subnucleus caudalis in mice

양은주 전북대학교 일반대학원 2013 국내석사

The spinal trigeminal nucleus is associated with transmission of orofacial sensory information, and is composed of three subnuclei (oralis, interpolaris, and caudalis). Nociceptive input from the orofacial region is initially processed in the trigeminal subnucleus caudalis (Vc). Especially, the substantia gelatinosa (SG, lamina II) of the Vc is a key element for modulating nociceptive transmission from the periphery to the central nervous system (CNS). Serotonin, 5-hydroxytryptamine (5-HT), influences diverse physiological functions such as sleep, cognition, sensory perception, motor activity, temperature regulation, appetite, hormone secretion, and nociception. At least, 14 subtypes of 5-HT receptors have been characterized and grouped into seven principal classes, from 5-HT1 to 5-HT7. 5-HT7 receptor, one of the most recently identified subtypes, is coupled to adenylate cyclase through the stimulatory Gs protein, and plays physiological roles such as circadian rhythm regulation, thermoregulation, and neuroendocrine regulation in the central nervous system. However, few studies have been conducted to demonstrate whether 5-HT7 receptors are involved in orofacial pain modulation. So, in this study, functional expression of 5-HT7 receptor on SG neurons of the Vc, which are involved in orofacial pain modulation was investigated using perforated patch clamp technique. The bath application of 30 M 8-OH-DPAT, 5-HT1A/7 agonist, induced membrane depolarization, no response, or hyperpolarization in SG neurons of the Vc. The majority of neurons tested were depolarizd in PND (postnatal days) 5-21 and PND 22-84, and 8-OH-DPAT induced different level of membrane potential changes in SG neurons of the Vc in both groups. Further, 8-OH-DPAT-induced depolarization was reversible and reproducible, indicating that the 5-HT receptors involved in 8-OH-DPAT-mediated depolarization was not desensitized in SG neuron by repeated stimulation. Moreover, the study about the working site of 8-OH-DPAT proved that 8-OH-DPAT acts on postsynaptic SG neurons, directly. In addition, 8-OH-DPAT-induced depolarization remained unaltered in the presence of WAY 100635, a selective 5-HT1A antagonist, but was largely blocked by SB 269970, a specific 5-HT7 antagonist. Interestingly, 8-OH-DPAT-induced non depolarization was suppressed by WAY 100635, and some of these neurons were instead depolarized to 8-OH-DPAT plus WAY 100635 pretreatment. These results suggest that the depolarization was mediated via 5-HT7 receptor, but not via 5-HT1A receptor. Finally, 5-HT7 receptor mRNA was detected in SG neurons of the Vc using single cell RT-PCR technique. Taken together, 5-HT7 receptors functionally exist in SG neurons, and SG neurons were depolarized by activation of 5-HT7 receptor in the Vc. In conclusion, this study suggests that the functional involvement of 5-HT7 receptors in descending serotonergic modulation over orofacial pain.