Role of nociceptin/orphanin FQ and nociceptin opioid peptide receptor in depression and antidepressant effects of nociceptin opioid peptide receptor antagonists

Nociceptin/orphanin FQ (N/OFQ) and its receptor, nociceptin opioid peptide (NOP) receptor, are localized in brain areas implicated in depression including the amygdala, bed nucleus of the stria terminalis, habenula, and monoaminergic nuclei in the brain stem. N/OFQ inhibits neuronal excitability of monoaminergic neurons and monoamine release from their terminals by activation of G protein-coupled inwardly rectifying K⁺ channels and inhibition of voltage sensitive calcium channels, respectively. Therefore, NOP receptor antagonists have been proposed as a potential antidepressant. Indeed, mounting evidence shows that NOP receptor antagonists have antidepressant-like effects in various preclinical animal models of depression, and recent clinical studies again confirmed the idea that blockade of NOP receptor signaling could provide a novel strategy for the treatment of depression. In this review, we describe the pharmacological effects of N/OFQ in relation to depression and explore the possible mechanism of NOP receptor antagonists as potential antidepressants.

The roles of habenula and related neural circuits in neuropsychiatric diseases / 浙江大学学报·医学版

The habenula is a small and bilateral nucleus above dorsal thalamus, which contains several different types of neurons. The habenula has extensive connections with the forebrain, septum and monoaminergic nuclei in the midbrain and brainstem. Habenula is known as an 'anti-reward' nucleus, which can be activated by aversive stimulus and negative reward prediction errors. Accumulating researchs have implicated that the habenula is involved in several behaviors crucial to survival. Meanwhile, the roles of the habenula in neuropsychiatric diseases have received increasing attention. This review summaries the studies regarding the roles of habenula and the related circuits in neuropathic pain, depression, drug addiction and schizophrenia, and discusses the possibility to use the habenula as a treatment target.

Three-Dimensional Analysis of Mouse Habenula Subnuclei Reveals Reduced Volume and Gene Expression in the Lipopolysaccharide-mediated Depression Model

The habenula (Hb) is small but important brain structure, anatomically and functionally links the forebrain with the midbrain to modulate various neuropsychiatric functions associated with drug addiction and emotion-associated dysfunctions. Several reports suggested that the dysfunction of Hb-related functions affected the Hb structurally and functionally. However, the technical limitation has awaited the solid conclusion of whether Hb change due to depression is likely to occur in certain subnuclei of the Hb. To probe this possibility, we developed 3-dimensional reconstruction methods for the high-resolution volumetric analysis of Hb and the mRNA levels at the given volume in normal or lipopolysaccharide (LPS)-mediated mouse model of depression. Notably, we discovered that the volume reduction was prominent in medial Hb but not in lateral Hb after LPS treatments. On the other hand, the RNA expression levels of known Hb regional markers such as Tac1 (dorsal part of medial Hb), ChAT (ventral part of medial Hb), and Tacr1 (medial and lateral Hb) were all decreased in all Hb subnuclei in LPS-injected mice. Accordingly, accurate volumetry with marker labeling was not feasible. Collectively, these established 3D analyses of mouse Hb successfully and precisely determine the volume-based changes of small brain structure, which should be applicable in a wider range of mouse models or pathological specimens.

Efectos de la estimulación eléctrica habenular en la modulación de respuestas emocionales en ratas Wistar / Efeitos da estimulação elétrica habenular na modulação de respostas emocionais em ratos Wistar / Effects of electrical stimulation of the habenula on the modulation of emotional responses in Wistar rats

Resumen A pesar del amplio uso de la estimulación cerebral profunda para controlar patologías neurológicas y neuropsiquiátricas, su mecanismo de acción aún no es claramente conocido, y existen pocos estudios sistemáticos que relacionen la variación de parámetros de estimulación eléctrica (frecuencia, intensidad, duración del pulso) y la ejecución comportamental. La habénula es una estructura reguladora de respuestas emocionales diana en tratamientos para dolor crónico y depresión, pero la relación entre su estimulación crónica y el desempeño animal en pruebas conductuales no se ha establecido con claridad. Con el objetivo de evaluar el efecto emocional de la estimulación habenular crónica, en este estudio se utilizaron ratas Wistar que recibieron estimulación habenular a intensidad baja (10-80 pA) o alta (120-260 pA) y frecuencia baja (80-150 Hz) o alta (240380 Hz): BIBF-AIBF-BIAF-AIAF, durante 15 minutos a lo largo de tres días consecutivos. Al cuarto día, se hizo la evaluación en un laberinto elevado en cruz y en campo abierto. Los resultados indican un efecto de tipo ansiolítico en el tratamiento BIAF, en comparación con BIBF y AIBF (aumento del número de entradas, porcentaje de tiempo en brazos abiertos y de la distancia recorrida en ellos), efecto que no se explica por cambios en la locomotricidad (distancia recorrida en los brazos cerrados y la exploración en el campo abierto). Se concluye que el parámetro frecuencia posee mayor impacto sobre el efecto comportamental que la intensidad -lo que puede explicar algunos hallazgos paradójicos previos-, que los parámetros utilizados no poseen efecto ansiogénico, y que los efectos potencialmente ansiogénicos de la estimulación a baja frecuencia y el papel de los sistemas dopaminérgicos y serotoninérgicos encontrados deben ser estudiados en futuras investigaciones.

Resumo Apesar do amplo uso da estimulação cerebral profunda para controlar patologias neurológicas e neuropsiquiátricas, seu mecanismo de ação ainda não é claramente conhecido e existem poucos estudos sistemáticos que relacionem a variação de parâmetros de estimulação elétrica (frequência, intensidade, duração do pulso) e a execução comportamental. A habênula é uma estrutura reguladora de respostas emocionais específicas em tratamentos para dor crònica e depressão, mas a relação entre sua estimulação crònica e o desempenho animal em testes comportamentais não foi claramente estabelecida. Com o objetivo de avaliar o efeito emocional da estimulação habenular crònica, neste estudo foram utilizados ratos Wistar que receberam estimulação habenular de intensidade baixa (10-80 pA) ou alta (120-260 pA) e frequência baixa (80-150 Hz) ou alta (240-380 Hz): BIBF-AIBF-BIAF-AIAF, durante 15 minutos ao longo de três dias consecutivos. No quarto dia, foi feita a avaliação em um labirinto em cruz elevado e em campo aberto. Os resultados indicam um efeito de tipo ansiolítico no tratamento BIAF, em comparação com BIBF e AIBF (aumento do número de entradas, porcentagem de tempo em braços abertos e da distância percorrida neles), efeito que não se explica por mudanças na locomotividade (distância percorrida nos braços fechados e a exploração no campo aberto). Conclui-se que o parâmetro "frequência" tem mais impacto sobre o efeito comportamental do que a "intensidade" - o que pode explicar algumas descobertas paradoxais prévias -, que os parâmetros utilizados não tenham efeito ansiogênico, e que os efeitos potencialmente ansiogênicos da estimulação de baixa frequência e o papel dos sistemas dopaminérgicos e serotoninérgicos encontrados devem ser estudados em pesquisas futuras.

Abstract Deep brain stimulation is a widely-used approach to the treatment of neurologic and neuropsychiatric diseases. However, its mechanisms remain unclear. There are few systematic studies relating variations on electrical stimulation parameters (frequency, intensity, pulse duration) and behavioral outcome. The habenula relates to emotional behavior and is a main target for chronic pain and depression stimulation treatment. The relation between habenular electrical stimulation and performance in behavioral tests has not been clearly defined. In order to assess the emotional effects of chronic habenular electrical stimulation, Wistar male rats were unilaterally implanted with electrodes aimed to the lateral habenula and assigned to low (10-80 pA) or high (120-260 pA) intensity and low (80-150 Hz) or high (240-380 Hz) frequency conditions: BIBF-AIBF-BIAF-AIAF. They received electrical stimulation 15 minutes/day for three consecutive days and on the fourth day were tested in the elevated plus maze and the open field. The results of these study show that BIAF stimulation has a possible anxiolytic-like effect when compared to BIBF and AIBF (increase in the percentage of open-arms time, entries into the open-arms and total-distance-run in the open-arms). This is not due to any changes in locomotion (total-distance-run and open field exploration). It is concluded that frequency is more important than intensity for behavioral modification. This could explain some previous inconsistent results. The data also suggest that these parameters of stimulation have no anxiogenic effects. The role for dopaminergic and serotonergic systems must be subsequently evaluated as well as potential anxiogenic-like effects of low frequency stimulation.

Functional Connectivity of Basolateral Amygdala Neurons Carrying Orexin Receptors and Melanin-concentrating Hormone Receptors in Regulating Sociability and Mood-related Behaviors

Chronic stress induces changes in neuronal functions in specific brain regions regulating sociability and mood-related behaviors. Recently we reported that stress-induced persistent upregulation of the neuropeptides orexin and melanin-concentrating hormone (MCH) in the basolateral amygdala (BLA) and the resulting activation of orexin receptors or MCH receptors within the BLA produced deficits in sociability and mood-related behaviors. In the present study, we investigated the neural targets that were innervated by BLA neurons containing orexin receptors or MCH receptors. The viral vector system AAV2-CaMKII-ChR2-eYFP was injected into the BLA to trace the axonal tracts of BLA neurons. This axon labeling analysis led us to identify the prelimbic and infralimbic cortices, nucleus accumbens (NAc), dorsal striatum, paraventricular nucleus (PVN), interstitial nucleus of the posterior limb of the anterior commissure, habenula, CA3 pyramidal neurons, central amygdala, and ventral hippocampus as the neuroanatomical sites receiving synaptic inputs of BLA neurons. Focusing on these regions, we then carried out stimulus-dependent c-Fos induction analysis after activating orexin receptors or MCH receptors of BLA neurons. Stereotaxic injection of an orexin receptor agonist or an MCH receptor agonist in the BLA induced c-Fos expression in the NAc, PVN, central amygdala, ventral hippocampus, lateral habenula and lateral hypothalamus, which are all potentially important for depression-related behaviors. Among these neural correlates, the NAc, PVN and central amygdala were strongly activated by stimulation of orexin receptors or MCH receptors in the BLA, whereas other BLA targets were differentially and weakly activated. These results identify a functional connectivity of BLA neurons regulated by orexin and MCH receptor systems in sociability and mood-related behaviors.

Effects and possible mechanism of cocaine on the neurons of lateral habenular nucleus / 中国应用生理学杂志

<p><b>AIM</b>To investigate the effects and the possible mechanism of cocaine on the neurons of lateral habenular nucleus (LHb).</p><p><b>METHODS</b>We observed the effects on c-Fos protein expression in lateral habenular nucleus and medial habenular nucleus after injecting cocaine into a belly cavity and spontaneous and evoked discharge of pain-correlative unit through iontophoresis of cocaine into LHb. The delayed rectifier K+ current was recorded in the acute isolated LHb neuron in whole-cell mode.</p><p><b>RESULTS</b>(1) The c-Fos protein expression was increased by cocaine treatment in LHb, but little effect in MHb. (2) Iontophoresis of cocaine into LHb increased the discharges of pain excitation unit and enhanced excitation response to noxious stimulation, but it decreased the discharges of pain inhibition unit and its responses to noxious stimulation in LHb. Cocaine inhibited the delayed rectifier K+ current.</p><p><b>CONCLUSION</b>Cocaine can excite the LHb and increase its sensitivity. The probable mechanism is that cocaine inhibits the delayed rectifier K+ channels.</p>

Effect and possible mechanism of melatonin on the firing rate of pain neurons in lateral habenular nucleus / 中国应用生理学杂志

<p><b>AIM</b>The effect and possible mechanism of Melatonin (MEL) on firing rate of pain neurons in lateral habenular nucleus of rats were investigated in the experiment.</p><p><b>METHODS</b>Single extracellular firing were recorded to study the firing rate changes of pain neurons and sensitivity changes to pain stimulation induced by MEL in LHb of rats. Reverse effect of naloxone on the analgesia induced by melatonin was also observed.</p><p><b>RESULTS</b>Melatonin showed the effects on the firing of pain neurons in the LHb and decreased the sensitivity of pain neurons to pain stimulation, which could be reversed by naloxone.</p><p><b>CONCLUSION</b>Melatonin can change the responses of pain neurons to pain stimulation via opioid receptor in the LHb, which might be one of analgesic mechanisms by MEL.</p>

The characteristics of opioid receptors distributed in the neurons of habenula / 中国应用生理学杂志

<p><b>AIM</b>To explore the types of receptors distributed in MHb and LHb.</p><p><b>METHODS</b>Recording the currents of potassium channels in Hb neurons isolated from the rats 10-15 days after birth. To distinguish the types of receptors distributed in MHb and LHb by using the agonists of mu receptor DAMGO, and sigma receptor DPDPE.</p><p><b>RESULTS</b>Two types of current of K+ channels were recorded, the transient rectifier and delayed rectifier potassium channels. DAMGO or DPDPE increased the intensity of current of K+ channels.</p><p><b>CONCLUSION</b>In MHb there was a higher density of sigma receptor, and in LHb a higher density of mu receptor distributed.</p>

The responses of pain-related neurons in habenula to nociceptive stimuli and morphine / 中国应用生理学杂志

<p><b>AIM</b>To observe the responses of pain-related neurons in habenula to the nociceptive stimuli and classic analgesic morphine for inquiring into its characteristics of pain.</p><p><b>METHODS</b>The experiment was proceeded with adult rats under light anesthetized. Through the cannula inserted by operation or the multielectrode injecting the morphine, naloxone, CCK-8 and etc into lateral cerebro-ventricule or habenula, the unit firings from the neurons of habenula were recorded.</p><p><b>RESULTS</b>The unit firings were recorded from pain-related neurons distributed in MHb or LHb. The pain-related neurons could be differentiated into pain excitatory or pain inhibitory neurons. After the morphine iontophoresed, the main response of the pain excitatory neurons was inhibited, the pain inhibitory neurons were excited. The naloxone iontophoresed could antagonize the analgesic effect of morphine on neurons of habenula. After the morphine injected (10 mg/kg, i. p) into morphine-tolerated rats, the analgesic efficacy of pain-related neurons in LHb was more stronger than in MHb. It showed that the neurons in LHb were suffered from morphine was higher than MHb. After injection of antagonist of CCK-8 into lateral cerebro-ventricle, morphine injected peritoneally could weaken the tolerance level of morphine. Conversely, after injection of morphine (10 mg/kg, i. p.) 10 min, second time injection of CCK-8 (15 ng/10 microl) into lateral cerebro-ventricle could antagonize the analgesic action of morphine on the neurons in LHb, but in MHb the antagonized action was not obviously.</p><p><b>CONCLUSION</b>The excitatory and inhibitory neurons in Hb were sensitive to the nociceptive stimuli and not easy to adapt to it. The sensitivity of the neurons in LHb to morphine was more higher than the neurons in MHb.</p>

Habenula participates the vasopressor response by stimulation of the insular cortex, central-, lateral amygdaloid nucleus respectively / 中国应用生理学杂志

<p><b>AIM</b>To investigate whether if the Habenula is the main relay involved in the vasopressor effect induced by the stimulus of insular cortex, central-, lateral amygdaloid nucleus respectively.</p><p><b>METHODS</b>Electrostimulation of the nuclei mention above respectively, and microinjection of lidocaine into Habenula unilaterally and bilaterally.</p><p><b>RESULTS</b>When INS or CeA was stimulated, inducing an obvious increase of blood pressure. To stimulate INS or CeA after microinjecting lidocaine into Hb 5 minutes, the amplitudes of the vasopressor responses were decreased significantly, and the decrease of the bilaterally was larger (decreased value: 41.7% in INS, 46.1% in CeA) than that of unilaterally (decreased value: 36.9% in INS, 39.6% in CeA).</p><p><b>CONCLUSION</b>Habenula is one of the main relays involved in the vasopressor effects induced by the stimulus of insular cortex, central-, lateral amygdaloid nucleus.</p>

Effect of losartan on arterial blood pressure and unit discharging of neurons in LHb and MHb of rat / 中国应用生理学杂志

<p><b>AIM AND METHODS</b>To investigate the effect of 2 mg/kg and 10 mg/kg losartan intraperitoneally (i.p) on arterial blood pressure (AP) and heart rate (HR) in rat and the involvement in the activity of habenulas neurons. Glass micropipette was used to record any changes of unit discharging of neurons in LHb and MHb before and after losartan was intraperitoneally injected.</p><p><b>RESULTS</b>AP and HR were not significantly changed by 2 mg/kg losartan (i.p). However, AP was apparently decreased by 10 mg/kg losartan (i.p), but HR was unchanged. After 10 mg/kg losartan (i.p), 66.66% (12/18) unit discharging of neurons in LHb were increased in frequency, and 61.90% (13/21) in MHb were decreased.</p><p><b>CONCLUSION</b>AP of rat was significantly decreased by 10 mg/kg losartan (i.p). Depressor effect of losartan (i.p) was involved in the excision of neurons in LHb and the inhibition in MHb.</p>

The role of dorsomedial hypothalamic nucleus in the cardiovascular responses induced by habenular nucleus stimulation and inhibitory effect of deep peroneal nerve / 中国应用生理学杂志

<p><b>AIM</b>To explore whether dorsomedial hypothalamic nucleus (DMH) is involved in the cardiovascular responses induced by habenular nucleus (Hb) stimulation and inhibitory effect of deep peroneal nerve (DPN) stimulation on above responses or not and to analysis transmitter mechanism.</p><p><b>METHODS</b>Experiments were performed on white male rabbits anesthetised with chloralose and urethan. Experimental data were collected by means of stimulating Hb and DPN, recording arterial blood pressure and ectal electrocardiogram (EECG) and microinjecting transmitter blocker.</p><p><b>RESULTS</b>Prominent pressor and ischemic EECG segment changes were elicited by Hb stimulation (P < 0.01) Microinjecting kynurenic acid into ipsilateral DMH had partial blocking effect on pressor and ischemic EECG-ST segment changes induced by Hb stimulation (P < 0.01). Stimulation of bilateral DPN prominently inhibited the pressor and ischemic EECG-ST changes induced by Hb stimulation (P < 0.01, P < 0.05). Microinjecting naloxone into ipsilateral DMH decreased the inhibitory effect of DPN stimulation on above cardiovascular responses elicited by Hb stimulation (P < 0.01, P < 0.05).</p><p><b>CONCLUSION</b>Glutamic acid receptor in DMH is involved in pressor and ischemic changes induced by Hb stimulation. DMH and opiate peptide receptors in DMH are involved in the inhibitory effect of DPN stimulation on pressor and ischemic changes induced by Hb stimulation.</p>

Nip2 Expression of Postnatal, Aged and Transient Focal Cerebral Ischemic Rat / 대한해부학회지

Nip2 mRNA expression of hippocampus was clarified in the postnatal, adult and aging rat. We observed the change of Nip2 mRNA expression in the ischemic rat hippocampus at 3, 6, 24 and 72 hours after reperfusion. And we investigated the relation between Nip2 and apoptosis after ischemic insults. The Rats were killed 2, 4, 7 days after birth and normal adult rats and aged rat were killed. Male F344 rats were subjected to transient middle cerebral artery occlusion. Reperfusion was achieved by withdrawing the filament after 90 min minutes, and rats were sacrificed 3, 6, 24, 72 hours after reperfusion. Hippocampal sections were stained for TUNEL using ApopTag kit following the protocol provided by the manufacturer, and stained with 2, 3, 5 -triphenyl tetrazolium chloride and cresyl violet We used quantitative reverse transcription and polymerase chain reaction to characterise changes in the mRNA expression of Nip2 in the rat models of transient focal ischemia and postnatal, adult and aged rat. Nip2 mRNA expression were increased in the rat of postnatal development and aging more than these of adult. After reperfusion, marked increase of Nip2 mRNA was observed after 3 and 6 hours. After that time mRNA expression of Nip2 was decreased gradually. The TUNEL staining detected DNA fragmentation in neurons of the entorhinal cortex, forcep major corpus callosum and secondary visual cortex at 24 hours. And TTC staining results showed the whitish infact changes of hippocampal CA1 region and lateral habenular nucleus. We hypothesize that the overexpression of Nip2 is concerned with sensitivity to the ischemic insult at postnatal period and aging period. And, early apoptotic events after cerebral ischemic insults relate to Nip2 mRNA overexpression.

Chromosomal localization and neural distribution of voltage dependent calcium channel alpha1A and alpha1E subunit genes / 대한해부학회지

Fluorescent in situ hybridization using human genomic DNA probes was performed to localize genes encoding the alpha1A and alpha1E of voltage dependent calcium channels (VDCCs) in the human chromosome and the mRNA expression of these two alpha1 subunits of VDCC was demonstrated in the 18 day old embryo (E18) and adult rat brain by in situ hybridization histochemistry. The genes for the VDCC alpha1A and alpha1E were specifically localized on human chromosome 19p13.1 and 1q25, respectively. In 18 days old rat embryos, the mRNAs of the VDCC alpha1A and alpha1E were predominently expressed in the nervous system including brain and spinal cord. In adult rat brain, the expression pattern of each subunit was extremely different. The expression of alpha1A mRNA was strong in the purkinje cells of cerebellum and CA3 area of hippocampus, relatively high level of expression was found in the dentate gyrus, CA1 area of hippocampus, superficial layer of cerebral cortex and olfactory mitral cells. Whereas alpha1E was highly expressed in the dentate gyrus, CA1-3 area of hippocampus, medial habenula nucleus of thalamus and olfactory mitral and internal granule cells and relatively high level of expression was found in the Purkinje cells of cerebellum, cerebral cortex and caudate-putamen. Until now, no neurological disorder has been mapped to 1q25, location of VDCC alpha1E gene. Recently, it has been reported that mutation of VDCC alpha1A gene causes episodic ataxia type 2 (EA-2) and spinocerebellar ataxia 6 (SCA6). These reports comfirm the our experimental results of chromosomal mapping and prominent cerebellar expression of VDCC a1A gene.

The developmental expression of voltage dependent calcium channel alpha1 subunits (alpha1D, alpha1B, alpha1A, alpha1E) mRNA in the rat brain / 대한해부학회지

Voltage dependent calcium channels (VDCCs) mediate Ca++ influx into cells and are responsible for regulation of a variety of physiological effects. The key functional property of VDCCs are attributed to the calcium-pore forming alpha1 subunit. In this study, distribution pattern of alpha1 subunit (alpha1D, alpha1B, alpha1A, alpha1E) mRNA of VDCCs in developing and adult rat brain was investigated by in situ hybridization histochemistry. In the adult rat brain, each alpha1 subunit mRNA displayed a specific and distinct distribution pattern. alpha1D was highly expressed in the olfactory bulb, dentate gyrus, pituitary gland, pineal gland, hypothalamus, superior colliculus and cerebellum. Relatively low level of alpha1B was expressed throughout the whole brain and strong expression of alpha1A was observed in CA3 area of Ammon's horn, medial geniculate body, inferior colliculus and cerebellum. High level of alpha1E was found in the olfactory bulb, hippocampus, dentate gyrus, medial habenular nucleus and cerebellum. Moreover, alpha1B, alpha1A and alpha1E were expressed only in the nervous system but alpha1D was expressed not only in the nervous system but also in other tissues including liver, heart, lung and skeletal muscle. Generally the expression of alpha1D, alpha1A, and alpha1E subunit was observed from E14 and thereafter the intensity of labeling was gradually increased to P14 and then decreased to the adult level. But the expression of alpha1B subunit was observed from E14 and gradually increased to E20 and P0 and then decresaed. From the differential expressions of VDCC alpha1 subunits in developing and adult rat brain, it is suggested that each type of VDCCs may play a distinct roles in neural and nonneural tissues, and the VDCCs may be related with development of nervous system.

Cloning and localization of two forms of voltage-dependent calcium channel beta3 subunit gene / 대한해부학회지

Voltage-dependent calcium channel (VDCC) is composed of at least four subunits: alpha1, alpha2, beta, delta. Four mammalian beta subunit isoforms (beta1, beta2, beta3 and beta4) have been identified from nervous system. beta subunit accelerates the kinetics of activation (channel openning) and inactivation (channel closure), and regulates the channel activity by phosphorylation through various signal transduction mechanisms. We have cloned three cDNAs (RB8, RB10, and RB11) encoding beta3 subunit of voltage-dependent calcium channel from rat cDNA library using the oligonucleotides of which sequences obtained from the highly conserved regions of rat b subunits. The RB8 and RB10 (rtB3a) encode a same protein of 484 amino acids with estimated Mr of 54,571 Da, which was identical to beta3 subunit gene previously reported. The RB11 (rtbBb) is diffferent from RB10 at N-terminal region but shares common amino acid sequences from the glycine, the 16th amino acid of RB10, to the end of the gene. Open reading frame of RB10 encodes a 483 amino-acid protein with a predicted Mr of 54,473 Da. The RB10 and RB11 are suspected to be alternatively spliced variants from a single b3 subunit gene. The existence of the variants was confirmed by RT-PCR using the oligonucleotide primers from the specific sequences of each variant. The expression patterns of VDCC beta3 (rtB3a) and its specific variant (rtB3b) were investigated in the rat brain by in situ hybridization histochemistry. The mRNAs for rtB3a and rtB3b were exclusively expressed in the nervous system. In the brain, strong expression of both mRNAs (rtB3a and rtB3b) was found in the medial habenular nucleus of thalamus, hippocampus, dentate gyrus, olfactory bulb and cerebellum. But significant discrepancy of expression was found in the lateral posterior thalamic nucleus and olfactory bulb. From these results, it is suspected that newly cloned VDCC variant (rtB3b) should be the alternatively spliced variant of VDCC beta3 gene.

The Changes of c-fos and c-jun after Capsaicine Treatment in the Rat Brain / 대한해부학회지

The expression of c-fos and c-jun in the brain of the rat after capsaicin treatment was investigated by in situ hybridization, dot blot hybridization and immunocytochemical methods. Adult male Sprague-Dawley rats[200g] were used for this study. The first set of rats received a single subcutaneous injection of capsaicin[50mg/Kg] dissolved in 10% Tween-80 and 10% ethanol in saline. The rats were decapitated 1, 3, 5, 10, 24, 72 hours and 1 week after capsaicin treatment. The control set of rats were treated with saline instead of capsaicin. In situ hybridization and dot blot hybridization were carried out. O1igonucleotide probe complimentary to c-fos mRNA sequences were used for this study and labeling of oligonucleotides was accomplished using the DNA tailing kit. The expression of c-fos mRNA on the nucleus of neurons in in situ hybridization was observed throughout the brain, and was especially abundant in the olfactory cortex, nucleus of diagonal band of Broca, habenular nuclei, periaqueductal gray, parabrachial nucleus, entopeduncular nucleus, ventral posterolateral nucleus of the thalamus and cerebellum. Compared to the control rats, c-fos mRNA were increased 24 hours after capsaicin injection and gradually decreased after 72 hours, returning to the normal control level 1 week after capsaicin injection. c-fos mRNA was detected only 1 week after capsaicin injection in the various areas of the brain. The fos protein-like immunoreactivity was initially somewhat decreased at 24 hours, but increased at 72 hours and reactions was maximally observed at 1 week after capsaicin treatment. But Jun protein immunoreactivity was not increased, on the contrary, it was even decreased both in numbers of reactive cells and immunoreactivity 1 week after capsaicin injection. From the above results, c-fos gene expression was pronounced in the nucleus concerned with pain, olfaction and taste such as VPL nucleus of the thalamus, olfactory cortex and parabrachial nucleus, in the limbic system concerned with stress and emotion such as nucleus of diagonal band of Broca, periaqueductal gray and habenular nucleus, in the structure concerned with somatic motor function such as entopeduncular nucleus and cerebellum. Also, the c-fos gene was activated by the capsaicin early in the course of effects, then the fos protein increased as a results of c-fos activation. On the other hand, c-jun did not respond to capsaicin treatment early in the course, but Jun protein decreased late in the course of capsaicin effects.

The Distribution of Brain-stem and Hypothalamus Fos like Immunoreactivity Associated with blockade of Superior Cervical Ganglion in Rats / 대한마취과학회지

BACKGROUND: Recently there have been many experiences regarding systemic effects of stellate ganglion block(SGB). During sympathetic hyperactivation, the SGB can be helpful to impaired microcirculation in brain-stem and hypothalamus. However, the exact mechanism and possible central action sites of SGB have not yet been investigated. In the present study, we traced central neural pathways following superior cervical ganglion block using the protein product(Fos) of c-fos protooncogene as a metabolic marker in a rat's brain. Method: The animals were divided into a superior cervical ganglion block group(n=5) using Marcaine 0.2 ml and a control block group(n=5) using saline 0.2ml. Medulla oblongata, pons, midbrain and hypothalamus were sectioned transversely with a sliding microtome. After imunohistochemical staining using rabbit polyclonal antibody we observed the distribution and grade of Fos expression under a light microscope. RESULTS: A blockade of superior cervical ganglion in rat led to the induction of c-fos in areas related to pain modulation sites and the autonomic nervous system; such as the parabrachial nucleus and central gray of the pons including dorsal raphe nucleus, as well as the substatia nigra of the midbrain, paraventricular hypothalamic nucleus, paraventricular thalamic nucleus posterior and habenular nucleus. CONCLUSIONS: Expressions of c-fos-like protein as a marker for neuronal activity following the blockade of the superior cervical ganglion in the rat have a part in the transcriptional control of the neurons which are related to the autonomic nervous system and endogenous pain modulation sites in the brain-stem.