Electroacupuncture improves insomnia by down-regulating peripheral benzodiazepine receptor expression in hippocampus, and up-regulating 5-HT, 5-HIAA, TNF-α and IL-1β contents in hypothalamus in insomnia rats / 针刺研究

OBJECTIVE: To observe the therapeutic effect of electroacupuncture (EA) at five Back-Shu points on sleep, hippocampal peripheral benzodiazepine receptor (PBR) expression and hypothalamic 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), tumor necrosis factor alpha (TNF-α) and interleukin (IL)-1β contents in insomnia rats, so as to explore its mechanisms underlying improvement of insomnia. METHODS: Forty male SD rats were randomly divided into control, model, EA and medication (Diazepam) groups (n=10 rats in each group). The insomnia model was established by intraperitoneal injection (i.p.) of para-Chlorophenylalanine (PCPA, 300 mg/kg) once daily for 2 days. EA (60 Hz, 1 mA) was applied to bilateral five Back-Shu points, i.e., Feishu (BL13), Xinshu (BL15), Ganshu (BL18), Pishu (BL20) and Shenshu (BL23) for 10 min, once daily for 6 days. Rats of the medication group were treated by gavage of Diazepam (0.92 mg/kg) once daily for 6 days. The sleep duration was recorded after i.p. of Pentobarbital Sodium (45 mg/kg). Histopathological changes of the hippocampus were displayed by H.E. staining. The contents of 5-HT, 5-HIAA, TNF-α and IL-1β in the hypothalamus were assessed by using ELISA. The expression levels of PBR mRNA and protein in the hippocampus were detected by quantitative real-time PCR, immunohistochemistry and Western blot, separately. RESULTS: Following modeling, the sleep duration was considerably shortened in rats of the model group relevant to the control group (P0.05), and significantly superior to that of the medication in increasing TNF-α and IL-1β levels (P<0.05), and considerably superior to that of medication in down-regulating PBR mRNA and protein expression (P<0.05). CONCLUSION: EA at five Back-Shu-points of the five Zang-organs can significantly improve the sleep in insomnia rats, which is closely associated with its effects in reducing the expression of PBR in hippocampus and up-regulating the levels of 5-HT, 5-HIAA, TNF-α and IL-1β in hypothalamus.

Impairment of locomotor activity induced by the novel N-acylhydrazone derivatives LASSBio-785 and LASSBio-786 in mice

The N-acylhydrazone (NAH) analogues N-methyl 2-thienylidene 3,4-benzoylhydrazine (LASSBio-785) and N-benzyl 2-thienylidene 3,4-benzoylhydrazine (LASSBio-786) were prepared from 2-thienylidene 3,4-methylenedioxybenzoylhydrazine (LASSBio-294). The ability of LASSBio-785 and LASSBio-786 to decrease central nervous system activity was investigated in male Swiss mice. LASSBio-785 or LASSBio-786 (30 mg/kg, ip) reduced locomotor activity from 209 ± 26 (control) to 140 ± 18 (P < 0.05) or 146 ± 15 crossings/min (P < 0.05), respectively. LASSBio-785 (15 or 30 mg/kg, iv) also reduced locomotor activity from 200 ± 15 to 116 ± 29 (P < 0.05) or 60 ± 16 crossings/min (P < 0.01), respectively. Likewise, LASSBio-786 (15 or 30 mg/kg, iv) reduced locomotor activity from 200 ± 15 to 127 ± 10 (P < 0.01) or 96 ± 14 crossings/min (P < 0.01), respectively. Pretreatment with flumazenil (20 mg/kg, ip) prevented the locomotor impairment induced by NAH analogues (15 mg/kg, iv), providing evidence that the benzodiazepine (BDZ) receptor is involved. This finding was supported by the structural similarity of NAH analogues to midazolam. However, LASSBio-785 showed weak binding to the BDZ receptor. LASSBio-785 or LASSBio-786 (30 mg/kg, ip, n = 10) increased pentobarbital-induced sleeping time from 42 ± 5 (DMSO) to 66 ± 6 (P < 0.05) or 75 ± 4 min (P < 0.05), respectively. The dose required to achieve 50% hypnosis (HD50) following iv injection of LASSBio-785 or LASSBio-786 was 15.8 or 9.5 mg/kg, respectively. These data suggest that both NAH analogues might be useful for the development of new neuroactive drugs for the treatment of insomnia or for use in conjunction with general anesthesia.

Abnormal Cerebral Benzodiazepine Receptors Binding Measured with (123)I-Iomazenil SPECT in Panic Disorder / 신경정신의학

OBJECTIVES: Researchers have long hypothesized that the benzodiazepine-GABA system plays a role in the pathophysiology of panic disorder (PD). However, previous neuroimaging studies have been inconclusive, possibly due to matching discrepancies between patients and controls, confounding medication factors, and/or image analyzing methods. This study aimed to compare benzodiazepine receptor binding between PD patients and healthy controls. METHODS: Via (123)I-iomazenil single-photon emission computed tomography (IMZ-SPECT), we obtained regional brain patterns of benzodiazepine receptor binding for 12 unmedicated PD patients and 8 healthy age-and sex-matched volunteer controls. To analyze the image data, we used statistical parametric mapping (SPM). RESULTS: Benzodiazepine receptor binding showed a decrease in both the parietal and occipital lobes in PD patients as compared to controls. The anterior cingulate cortex, left parahippocampal gyrus, and both temporal lobes showed increased binding in PD patients. CONCLUSION: Our findings provide evidence of an abnormal BZD-GABA system in PD patients, suggesting that basal and/or compensatory changes in inhibitory neurotransmissions contribute to the pathogenesis of human PD.

Apoptosis and Peripheral Benzodiazepin Receptor (PBR) Expression in Human Granulosa-Luteal Cells by GnRH-agonist / 대한불임학회지

OBJECTIVE: To investigate whether GnRH-agonist (GnRH-Ag) using in IVF-ET affects apoptosis of human granulosa-luteal cells and expression of peripheral benzodiazepine receptor (PBR) protein involved in the apoptosis of the cells. METHODS: Granulosa-luteal cells obtained during oocyte retrieval were cultured and treated with 10(-5) M GnRH-Ag. Apoptosis of the cells by the treatment was confirmed using DNA fragmentation analysis 24 h after culture. The presence of PBR protein within the cells was examined by immunofluorescence staining and the expression of the protein was analyzed by Western blotting. In addition, it was measured for progesterone and nitric oxide (NO) produced by granulosa-luteal cells after GnRH-Ag treatment. To evaluate the relationship between NO production and PBR expression, sodium nitroprusside (SNP) as a NO donor was added in media and investigated the expression of PBR protein by Western blotting. RESULTS: Apoptosis increased in the granulosa-luteal cells 24 h after GnRH-Ag treatment, whereas the expression of PBR protein significantly decreased. Furthermore, the production of progesterone and nitric oxide (NO) by the cells significantly fell from 12 h after the treatment. In the results of Western blotting after SNP treatment, the expression of PBR protein increased in the treatment with SNP alone to the granulosa-luteal cells, but was suppressed in the treatment with GnRH-Ag and SNP. Additionally, the staining result of PBR protein in the cells showed the even distribution of it through the cell. CONCLUSION: These results demonstrate that GnRH-Ag treatment induces apoptosis, decreasing expression of PBR protein and NO production in human granulosa-luteal cells. The present study suggests that one of the apoptosis mechanism of human granulosa-luteal cells by GnRH-Ag might be a signal transduction pathway via NO and PBR.

Glial Peripheral Benzodiazepine Receptor Mediates the Relief of Acute Stress-Induced Anxiety in Rats / 신경정신의학

OBJECTIVES: Peripheral benzodiazepine receptor has been suggested to be associated with the relief of anxiety response induced by stresses. This study was designed to observe the anxiolytic activity of peripheral benzodiazepine receptor. METHODS: Male Sprague-Dawley rats, weighing 200-250g were forced to suffer an immobilization stress for 2 hours. The level of anxiety by immobilization was performed by an elevated plus maze and was evaluated by the number of [3H]Ro5-4864 binding sites in the olfactory bulb. RESULTS: Saturation experiments followed by scatchard anlayses of the results showed that the density of peripheral benzodiazepine receptor increased and the affinity of the peripheral benzodiazepine receptor remained unchanged. It was found that there was no significant change in the cerebral cortex. Pretreatment with clonazepam, a central benzodiazepine receptor agonist, before an immobilization stress abolished the anxoius response on the performance of plus maze. In this group, upregulation of peripheral benzodiazepine receptor of olfactory bulb was not observed. Ro5-4864, a peripheral benzodiazepine receptor agonist, elicited an increase of anxiolytic response on the performance of plus maze. Progesterone, a precursor of neuroactive steroid, also increased anxiolytic response on the performance of plus maze. Pretreatment with PK11195, a peripheral benzodiazepine receptor antagonist, abolshed the anxiolytic effect of progesterone. CONCLUSIONS: From these results, it could be concluded that peripheral benzodiazepine receptor is closely associated with the relief of acute stress induced anxiety response via an increase of synthesis of neuroactive steroid.

Synthesis and Biodistribution of Flumazenil Derivative F-18(3-(2-Fluoro) flumazenil for Imaging Benzodiazepine Receptor / 대한핵의학회잡지

PURPOSE: Radiotracers that bind to the central benzodiazepine receptor are useful for the investigation of various neurological and psychiatric diseases. [C-11]Flumazenil, a benzodiazepine antagonist, is the most widely used radioligand for central benzodiazepine receptor imaging by PET. We synthesized 3-(2-[F- 18]fluoro)flumazenil, a new fluorine-18 (t1/2=110 min) labeled analogue of benzodiazepine receptor imaging agent, and evaluated in vivo for biodistribution in mice. MATERIALS AND METHODS: Flumazenil (Ro 15-1788) was synthesized by a modification of the reported method. Precursor of 3-(2-[F-18]fluoro)flumazenil, the tosylated flumazenil derivative was prepared by the tosylation of the ethyl ester by ditosylethane. [F-18] labeling of tosyl substitued flumazenil precursor was performed by adding F-18 ion at 85 degree C in the hot cell for 20 min. The reaction mixture was trapped by C18 cartridge, washed with 10% ethanol, and eluted by 40% ethanol. Bidistribution in mice was determined after intravenous injection. RESULTS: The total chemical yield of tosylated flumazenil derivative was ~40%. The efficiency of labeling 3-(2-[F-18]fluoro)flumazenil was 66% with a total synthesis time of 50 min. Brain uptakes of 3-(2-[F-18]fluoro)flumazenil at 10, 30, 60 min after injection, were 2.5+/-0.37, 2.2+/-0.26, 2.1+/-0.11 and blood activities were 3.7+/-0.43, 3.3+/-0.07, 3.3+/-0.09%ID/g, respectively. CONCLUSION: We synthesized a tosylated flumazenil derivative which was successfully labeled with no-carrier-added F-18 by nucleophilic substitution.

Brain Benzodiazepine-like Molecules and Stress-anxiety Response / 영남의대학술지

Benzodiazepines(BZDs) are among the widely prescribed drugs in the world. They are potent anxiolytic, antiepileptic, hypnotic, and muscle relaxing agents. There is an emerging model of the role of several neural systems in anxiety and their relation to the mechanism of action of BZDs. It has been postulated that BZD drugs exert their anxiolytic action by regulating GABAergic transmission in limbic areas such as the amygdala, in the posterior hypothalamus, and in the raphe nuclei. The involvement of the amygdala in the behaviors triggered by fear and stress has been suggested by many previous studies. In this review, reports about regulatory effects of endogenous BZD receptor ligands on the perception of anxiety and memory consolidation were summerized. These findings further support the contention that BZD receptor ligands modulate memory consolidation of averse learning tasks by influencing the level of stress and/or anxiety that accompanies a learning experience. The findings suggest that the decrease in the limbic levels of BZD-like molecules seen after the various behavioral procedures represent a general response to stress and/or anxiety, since it occurs in proportion to the level of stress and/or anxiety that accompany these tasks. In addition, these findings further support the hypothesis that the GABAA/BZD receptor complex in limbic structures plays a pivotal role in the stress and anxiety.

The Effect of Neuroactive Steroids on the GAVA-Benzodiazepine Receptor Complex / 대한정신약물학회지

Pregnolone[5beta-pregnan-3alpha-ol-one(5beta3alpha)] and allopregnanolone [(5alpha-pregnan-3alpha-ol-20-one(5alpha3alpha))] are neuroactive steroids that are reduced metabolites of progesterone. It was reported that Neuroactive steroids may have anxiolytic and anticonvulsant action similar to benzodiazepines and barbiturates. Therefore, the present study was designed to assess the interaction of steroids with GABAA-benzodiazepine receptor complex. The effect of steroids on the ligands binding to GABAA receptor complex was investigated using rat cortices. 5beta3alpha and 5alpha3alpha enhanced the binding of [3H] flunitrazepam to GABAA receptor, but testosterone, progesterone and dexamethasone did not. GABA also showed the enhancement of [3H] flunitrazepam binding, but did not show the additive effect. Unlike to GABA, 5beta3alpha and 5alpha3alpha did not affect on the [3H] muscimol binding to rat cortices. From these findings, it can be concluded that Neuroactive steroids are potent positive modulators of the GABA A receptor, and do not act at GABA binding site.

Benzodiazepine receptor ligand influences on learning: an endogenous modulatory mechanism mediated by benzodiazepines possibly of alimentary origin

In rats pre-but not post-training ip administration of either flumazenil, a central benzodiazepine (BSD) receptor antagonist, or of n-butyl-B-carboline-carboxylate (BCCB), an inverse agonist, enhanced retention of inhibitory avoidance learning. Flumazenil vlocked the enhancing effect of BCCB, and the inhibitory effect of the BZD agonists clonazepam and diazepam also given pre-training. Post-training administration of these drugs had no effects. The peripheral BZD receptor agonist/chloride channel blocker Ro5-4864 had no effect on the inhibitory avoidance task when given ip prior to training, buth it caused enhancement when given immediately post-training either ip or icv. This effect was blocked by PK11195, a competitive antagonist of Ro5-4864. These results suggest that ther is an endogenous mechanism mediated by BZD agonists, which is sensitive to inverse agonists and that normally down-regulates the formation of memories through a mechanism involving GABA-A receptors and the corresponding chloride channels. The most likely agonists for the endogenous mechanism suggested are the diazepam-like BZDs found in brain whose origin is possibly alimentary. Levels of these BZDs in the cortex were found to sharply decrease after inhibitory acoidance training or mere exposure to the training apparatus.

Involvement of peripheral benzodiazepine receptor in the regulation of rat cardiac mitochondria permeability transition / 中国药理学通报

Aim To investigate the role of peripheral benzodiazepine receptor in rat cardiac mitochondrial permeability transition.Methods The isolated rat cardiac mitochondria were incubated with different doses(50,100,200 ?mol?L-1) of PBR antagonist 1-(2-chlorophenyl-N-methyl-1-methylpropyl)-3-isoquinolinecarboxamide (PK 11195). In additional group(CsA group), 5 ?mol?L-1 cyclosporine A (CsA), an inhibitor of MPT was added 5 minutes before the addition of 100 ?mol?L-1 PK 11195. Negative control group(Con group) was given none treatment. Positive control group(Ca2+ group) was given 150 ?mol?L-1 CaCl2. The absorbanceat 520 nm(Abs 520 nm) was monitored with a split-beam spectrophotometer at 30℃ for 10 min. The mitochondrial ultrastructure was assessed by transmission electron microscopy. Mitochondrial cytochrome C release was demonstrated by Western Blotting.Results PK11195 triggered large-amplitude mitochondrial swelling in a dose dependent manner(vs Con group,P