Effects of mechanical stress and deficiency of dihydrotestosterone or 17ß-estradiol on Temporomandibular Joint Osteoarthritis in mice.

OBJECTIVE: To observe and analyze the interaction between excessive mechanical stress (MS) and decreased sex hormones on Temporomandibular Joint Osteoarthritis (TMJ-OA), and to discover TMJ-OA disease susceptibility genes by molecular biological analysis to elucidate part of the mechanism of TMJ-OA onset. DESIGN: For experimental groups, orchiectomy (ORX) or ovariectomy (OVX) was performed on sexually mature 8-week-old mice. A metal plate was attached to the posterior surface of the maxillary incisors to apply excessive MS on mandibular condyles. Male mice were divided into control, ORX, MS, and ORX + MS groups, while female mice were divided into control, OVX, MS, and OVX + MS groups. Mandibular condyles were evaluated by histology and molecular biology. RESULTS: Histomorphometric analysis of the TMJ in ORX + MS and OVX + MS groups revealed the thinnest chondrocyte layers, highest modified Mankin scores, and significant increases in the number of osteoclasts. Gene expression analysis indicated upregulation of Angptl7 and Car1 genes in the mandibular condyles of mice subjected to the combined effects of excessive MS and reduced sex hormones. In vitro analysis suggested that cartilage-like cells overexpressing Angptl7 enhanced calcification, and osteoblast-like cells overexpression Car1 suppressed cell proliferation and calcification. CONCLUSIONS: A severe TMJ-OA mouse model was successfully developed by applying excessive MS on the mandibular condyle of male and female mice with reduced sex hormones. Disease-susceptibility genes Angptl7 and Car1 were newly discovered in the experimental groups, suggesting their involvement in the onset mechanism of TMJ-OA.

Drebrin A regulates hippocampal LTP and hippocampus-dependent fear learning in adult mice.

Structural plasticity of dendritic spines, which underlies higher brain functions including learning and memory, is dynamically regulated by the actin cytoskeleton and its associated proteins. Drebrin A is an F-actin-binding protein preferentially expressed in the brain and localized in the dendritic spines of mature neurons. Isoform conversion from drebrin E to drebrin A and accumulation of the latter in dendritic spines occurs during synapse maturation. We have previously demonstrated that drebrin A plays a pivotal role in spine morphogenesis and plasticity. However, it is unclear whether drebrin A plays a specific role in processes required for structural plasticity, and whether drebrin E can substitute in this role. To answer these questions, we analyzed mutant mice (named DAKO mice), in which isoform conversion from drebrin E to drebrin A is disrupted. In DAKO mouse brain, drebrin E continues to be expressed throughout life instead of drebrin A. Electrophysiological studies using hippocampal slices revealed that long-term potentiation of CA1 synapses was impaired in adult DAKO mice, but not in adolescents. In parallel with this age-dependent impairment, DAKO mice exhibited impaired hippocampus-dependent fear learning in an age-dependent manner; the impairment was evident in adult mice, but not in adolescents. In addition, histological investigation revealed that the spine length of the apical dendrite of CA1 pyramidal cells was significantly longer in adult DAKO mice than in wild-type mice. Our data indicate that the roles of drebrin E and drebrin A in brain function are different from each other, that the isoform conversion of drebrin is critical, and that drebrin A is indispensable for normal synaptic plasticity and hippocampus-dependent fear memory in the adult brain.

Allopregnanolone increases mature excitatory synapses along dendrites via protein kinase A signaling.

Allopregnanolone (APα; 5α-pregnan-3α-ol-20-one) is synthesized in both the periphery and central nervous system and is known to be a potent positive allosteric modulator of the GABAA receptor. Because APα was suggested to improve the symptoms of depression and Alzheimer's disease (AD), which involve synaptic dysfunction and loss, we examined whether APα affects excitatory synapses. Drebrin, which is an actin-binding protein, forms a unique stable actin structure in dendritic spines, and drebrin levels correlate positively with cognitive levels in AD and mild cognitive impairment. We investigated whether APα increases excitatory synapse density along dendrites of mature hippocampal neurons using drebrin-imaging-based evaluation of mature synapses. We prepared primary cultures of hippocampal neurons and either transfected them with GFP or immunostained them against drebrin. Morphological analysis of GFP-transfected neurons revealed that a 24-h exposure to 0.3 or 1 µM APα significantly increased dendritic spine density without any morphological changes to spines. Drebrin cluster density was also increased by 0.3 and 1 µM APα. The protein kinase A (PKA) inhibitor H-89 inhibited the APα-induced increase in drebrin cluster density. These data demonstrate that APα increases mature excitatory synapses via activation of PKA. Therefore, the PKA-cAMP response element-binding protein (CREB) signaling pathway is likely to be involved in the APα-induced increase of mature excitatory synapses. Another possibility is that the PKA-dependent increase in AMPA receptors at dendritic spines mediates the APα function. In conclusion, our study indicates that APα may improve neuropsychiatric disorder outcomes via increasing the numbers of mature excitatory synapses.

Expression profiles of NOS isoforms in gingiva of nNOS knockout mice.

Nitric oxide is a gaseous molecule associated with many distinct physiological functions, and is derived from L-arginine catalyzed by nitric oxide synthase (NOS). Nitric oxide synthase has 3 isoforms: nNOS, iNOS and eNOS. Although these NOS isoforms are believed to play an important role in gingival tissue, little information is available on their morphological dynamics. The aim of this study was to investigate the profiles of NOS isoforms in deficiency of nNOS in gingiva of mice. Twelve male (6 normal (C57BL/6) and 6 nNOS knockout) mice were used. All mice were 5-week-old, weighing approximately 20-25 g each. After sacrifice, the jaws of the mice were removed by mechanical means and specimens analyzed by histology, in situ hybridization and immunohistochemistry. Immunohistochemical observation revealed positive staining for iNOS and eNOS, especially in lamina propria. Similar results in the mRNA expression levels were shown by in situ hybridization analysis. It may suggest that iNOS and eNOS compensated nNOS deficiency in the gingiva of nNOS knockout mice.

TMJ degeneration in SAMP8 mice is accompanied by deranged Ihh signaling.

The temporomandibular joint (TMJ) functions as a load-bearing diarthrodial joint during mastication, and its continuous use and stress can lead to degeneration over age. Using senescence-accelerated (SAMP8) mice that develop early osteoarthritis-like changes in synovial joints at high frequency, we analyzed possible molecular mechanisms of TMJ degeneration and tested whether and how malocclusion may accelerate it. Condylar articular cartilage in young SAMP8 mice displayed early-onset osteoarthritic changes that included reductions in superficial/chondroprogenitor cell number, proteoglycan/collagen content, and Indian hedgehog (Ihh)-expressing chondrocytes. Following malocclusion induced by tooth milling, the SAMP8 condyles became morphologically defective, displayed even lower proteoglycan levels, and underwent abnormal chondrocyte maturation compared with malocclusion-treated condyles in wild-type mice. Malocclusion also induced faster progression of pathologic changes with increasing age in SAMP8 condyles as indicated by decreased PCNA-positive proliferating chondroprogenitors and increased TUNEL-positive apoptotic cells. These changes were accompanied by steeper reductions in Ihh signaling and by expression of matrix metalloproteinase 13 at the chondro-osseous junction in SAMP8 articular cartilage. In sum, we show for the first time that precocious TMJ degeneration in SAMP8 mice is accompanied by--and possibly attributable to--altered Ihh signaling and that occlusal dysfunction accelerates progression toward degenerative TMJ disease in this model.

DNA methylation of the BMAL1 promoter.

We previously analyzed transcriptional regulation of the BMAL1 gene, a critical component of the mammalian clock system and found that the BMAL1 gene is expressed with circadian oscillation and that its regulatory region is located in hypomethylated CpG islands with an open chromatin structure. Here, we found that the BMAL1 gene is not expressed with circadian oscillation in CPT-K cells because the CpG islands located in the BMAL1 promoter are hypermethylated and that 5-aza-2'-deoxycytidine (aza-dC) recovered BMAL1 expression. In contrast, CpG islands in the PER2 promoter were hypomethylated, the PER2 gene was expressed and aza-dC enhanced PER2 gene expression in CPT-K cells. Reporter gene assays showed that intracellular transcriptional machinery for the BMAL1 gene is active, suggesting that BMAL1 inactivation is caused by DNA methylation and not by malfunctional promoter activity. Incubating CPT-K cells with aza-dC also increased CRY1 expression, whereas CLOCK expression was not altered and the CRY1 promoter was unmethylated. These results suggest that aza-dC induces BMAL1 expression via DNA demethylation in the BMAL1 promoter and enhances PER2 and CRY1 transcription. Finally, aza-dC recovered the circadian oscillation of BMAL1 transcription. These results suggest that DNA methylation of the BMAL1 gene is critical for interfering with circadian rhythms.

The influence of reduced glutathione in fertilization medium on the fertility of in vitro-matured C57BL/6 mouse oocytes.

It is well known that IVM oocytes show a decreased potential for fertility and development compared with in vivo-matured oocytes. In this study, we added reduced glutathione (GSH) to the fertilization medium during IVF to investigate its effect on the fertility and early embryo development of IVM oocytes. The fertilization rate for IVM oocytes and fresh sperm increased with the addition of GSH (0, 1.0, and 2.0 mM: 51%, 76%, and 70%). Moreover, the addition of GSH to the fertilization medium also improved the developmental potential compared with the control sample (0 mM). In addition, we performed IVF using IVM oocytes and frozen/thawed sperm that had been cryopreserved in a mouse bank. Results indicated a marked increase in the fertilization rate when 1.0 mM GSH was added to the fertilization medium compared with when no GSM was used (0.0 mM GSH: 2% (3/195); 1.0 mM GSH: 33% (156/468)). Furthermore, the fertilization rate improved dramatically via zona drilling using laser equipment (52%: 267/516), whereas normal offspring were obtainsed after transferring embryos created via IVF using IVM oocytes and frozen/thawed sperm. This is the first report in which offspring have been obtained via IVF using IVM oocytes and frozen/thawed sperm.

Gender difference in tooth autotransplantation with complete root formation: a retrospective survey.

Gender-related risk factors in the survival of transplanted teeth with complete root formation have not yet been identified. The purpose of this study was to investigate gender differences in tooth autotransplantation at dental clinics. We asked participating dentists to provide information on transplantations they had undertaken from 1 January 1990 to 1931 December 2010. The data were screened to exclude patients who underwent more than one transplantation, smokers or those whose smoking habits were unknown, patients under 30 or who were 70 years old and over, cases where the transplanted teeth had incomplete root formation or multiple roots and those with fewer than 20 present teeth post-operation. We analysed 73 teeth of 73 males (mean age, 47.2 years) and 106 teeth of 106 females (mean age, 45.3 years) in this study. The cumulative survival rate and mean survival time were calculated using the Kaplan-Meier method. The cumulative survival rate for males was 88.3% at the 5-year mark, 64.8% at 10 years and 48.6% at 15 years; for females, it was 97.2% at the 5-year mark, 85.9% at 10 years and 85.9% at 15 years. A log-rank test indicated the difference between males and females to be significant (P = 0.011). There was also a significant difference in the main causes for the loss of transplanted teeth: males lost more transplanted teeth due to attachment loss than females (P < 0.05). These results indicate that males require more attention during the autotransplantation process, particularly at the stage of pre-operation evaluation and that of follow-up maintenance.

AM-36 modulates the neutrophil inflammatory response and reduces breakdown of the blood brain barrier after endothelin-1 induced focal brain ischaemia.

BACKGROUND AND PURPOSE: Following transient focal stroke, rapid accumulation and activation of neutrophils in the ischaemic region is deleterious due to release of reactive oxygen species and myeloperoxidase (MPO). The purpose of this study was to examine whether AM-36, both a Na+ channel blocker and an antioxidant, afforded neuroprotection by modulating neutrophil accumulation into brain, following endothelin-1 (ET-1) induced middle cerebral artery occlusion (MCAo) in conscious rats. EXPERIMENTAL APPROACH: AM-36 was administered at 3 and 24 h after ET-1-induced MCAo. Functional recovery was determined using grid-walking and cylinder tests. Image analysis of brain sections was used to determine infarct volume. The effect of AM-36 on neutrophil infiltration and their interaction with macrophages was examined in rats at 48 h following MCAo by both an MPO assay and double-label immunofluorescence. Blood brain barrier (BBB) breakdown was measured by the area stained by intravenous Evans Blue. KEY RESULTS: AM-36 reduced functional deficits in both tests such that no difference existed from pre-ischaemic values at 48 h. Neutrophil infiltration, assessed by MPO activity, and infarct volume were significantly reduced following AM-36 administration by 54 and 60% respectively. Similarly, immunofluorescence revealed that AM-36 reduced neutrophil infiltration by approximately 50% in selected brain regions, when compared to controls, and also modulated macrophage phagocytosis of neutrophils. Breakdown of the BBB was significantly reduced by 60% following AM-36 treatment. CONCLUSIONS AND IMPLICATIONS: These findings suggest that AM-36 can directly modulate the neutrophil inflammatory response and reduce BBB breakdown following MCAo.

Plasma orexin-A is lower in patients with narcolepsy.

Recently identified hypothalamic peptides called orexins (or hypocretins) have been implicated in the sleep-wake cycle and in sleep disorder narcolepsy. Neuropathological studies have shown that in patients with narcolepsy, global reduction in the expression of orexins occurs due to the loss of orexin neurons in the hypothalamus. Cerebrospinal fluid analysis has confirmed a reduced or undetectable level of orexin-A in most narcolepsy patients. In this study, measurement of plasma orexin showed significantly lower concentrations in patients with narcolepsy than in age- and gender-matched normal controls. These data suggest that low levels of orexin-A in plasma could serve as a biological marker for narcolepsy.

[Tumor ablation with MRI navigation--a novel method of microwave coagulation therapy for hepatic tumor].

Fifty-eight patients with hepatic tumor which consisted of 22 hepatocellular carcinomas and 36 metastatic liver tumors were treated by microwave coagulation therapy with MRI navigation. The tumors were located in all segments of liver except S1. In 24 cases among them, the abdominal approach was difficult, because the tumors were located just below the diaphragm. These cases were selected for thoracoscope-assisted microwave ablation under MR-guidance across the diaphragm. All MR data were collected on a vertically oriented open MRI system (0.5 T SIGNA SP/i system: GE Medical Systems). The microwave electrode was introduced into the liver through a 14G needle via a percutaneous puncture with real-time MR image navigation. Microwave ablations at 60 W for 60 seconds were repeated several times depending on the tumor size. MR imaging may be employed as a reliable guide for percutaneous puncture. Moreover, sufficient safety margin could be obtained for hepatic tumor ablation. MR-guided microwave thermoablation therapy is a feasible method of treatment for hepatic tumors.

Cardiovascular changes induced by central hypertonic saline are accompanied by glutamate release in awake rats.

To elucidate neurochemical mechanisms responsible for cardiovascular responses induced by central salt loading, we directly perfused the paraventricular nucleus (PVN) of the hypothalamus region with hypertonic saline (0.3 or 0.45 M) by using an in vivo brain microdialysis technique. We then measured the extracellular concentrations of glutamate in the PVN region in conscious rats along with the blood pressure and heart rate. Blood pressure, heart rate, and glutamate levels were increased by perfusion of 0.45 M saline; however, they did not change by perfusion of 0.3 M saline. Next, we examined the possible involvement of glutamate in the cardiovascular responses induced by hypertonic saline. Dizocilpine, a noncompetitive antagonist of the N-methyl-D-aspartate (NMDA) receptor, attenuated the increases of blood pressure and heart rate, although 6-cyano-7-nitroquinoxaline-2,3-dione, an antagonist of the non-NMDA receptor, did not affect the blood pressure and heart rate. Our results show that local perfusion of the hypothalamic PVN region with hypertonic saline elicits a local release of glutamate, which may act via NMDA-type glutamate receptors to produce cardiovascular responses.

Micromere descendants at the blastula stage are involved in normal archenteron formation in sea urchin embryos.

Several lines of evidence suggest that micromere signaling plays a key role in endo-mesoderm differentiation along the animal-vegetal (A-V) axis in sea urchin embryos. A recent study has suggested that the activity of micromeres of inducing endoderm differentiation of mesomere descendants is, unexpectedly, maximal at the hatching blastula stage in the echinoids Scaphechinus mirabiris and Hemicentrotus pulcherrimus. In the present study, to confirm the inductive capacity of the micromere descendants in normal development, the timing of initiation of gastrulation and the elongation rate of the archenteron were examined in both micromereless embryos and in micromereless embryos cultured until the hatching blastula stage and then recombined with micromere descendants of the same age. The micromereless embryos consistently exhibited a delay in the initiation of gastrulation and a decrease in elongation rate of the archenteron, as compared with those in controls. In contrast, when the micromereless embryos cultured until the hatching blastula stage were recombined with micromere descendants of the same age, the recombinant embryos exhibited rescue of both the delay in initiation of gastrulation and a decrease in elongation rate of the archenteron. The delayed expression of alkaline phosphatase activity, an endoderm-specific marker, in the micromereless embryos was also rescued in the recombinant embryos. The recombined micromere descendants formed the larval spicules in the same schedule as that observed in the controls. These results indicate that at the hatching blastula stage, micromere descendants emanate a signal(s) required for normal gastrulation of the presumptive endo-mesodermal region.

[Transient nephrotic syndrome after allogeneic bone marrow transplantation for chronic myelogenous leukemia].

A 42-year-old man with chronic myelogenous leukemia underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) from an unrelated donor in January 1998. About 100 days later, he developed skin eruption and a diagnosis of chronic graft-versus-host disease (cGVHD) was made by skin biopsy. The eruption improved with steroid therapy, and the dose of steroid was gradually tapered. On day 151, the patient developed nephrotic syndrome with proteinuria up to 20 g/day. A renal biopsy carried out on day 160 showed minimal change in the glomeruli. The proteinuria disappeared 19 days after the onset of nephrotic syndrome without any additional therapy, and no recurrence was observed upon re-tapering of the steroid. In this case, cGVHD might have been related to development of the nephrotic syndrome. Nephrotic syndrome after allo-HSCT is a rare complication, and only ten cases have been reported. The histological findings were mainly membranous nephropathy, and immunosuppressive therapy was effective. As seen in this case, transient nephrotic syndrome with cGVHD may occur after allo-HSCT, and care is necessary to ensure that treatment of cGVHD is sufficient.

A homologue of N-ethylmaleimide-sensitive factor in the malaria parasite Plasmodium falciparum is exported and localized in vesicular structures in the cytoplasm of infected erythrocytes in the brefeldin A-sensitive pathway.

N-Ethylmaleimide-sensitive factor (NSF) and its homologues play a central role in vesicular trafficking in eukaryotic cells. We have identified a NSF homologue in Plasmodium falciparum (PfNSF). The reported PfNSF gene sequence (GenBank accession number CAB10575) indicated that PfNSF comprises 783 amino acids with a calculated molecular weight of 89,133. The overall identities of its gene and amino acid sequences with those of rat NSF are 50.9 and 48.8%, respectively. Reverse transcription-polymerase chain reaction analysis and Northern blotting with total P. falciparum RNA indicated expression of the PfNSF gene. Polyclonal antibodies against a conserved region of NSF specifically recognized an 89-kDa polypeptide in the parasite cells. After homogenization of the parasite cells, approximately 90% of an 89-kDa polypeptide is associated with particulate fraction, suggesting membrane-bound nature of PfNSF. PfNSF was present within both the parasite cells and the vesicular structure outside of the parasite cells. The export of PfNSF outside of the parasite cells appears to occur at the early trophozoite stage and to terminate at the merozoite stage. The export of PfNSF is inhibited by brefeldin A, with 9 microM causing 50% inhibition. Immunoelectromicroscopy indicated that intracellular PfNSF was associated with organelles such as food vacuoles and that extracellular PfNSF was associated with vesicular structures in the erythrocyte cytoplasm. These results indicate that PfNSF expressed in the malaria parasite is exported to the extracellular space and then localized in intraerythrocytic vesicles in a brefeldin A-sensitive manner. It is suggested that a vesicular transport mechanism is involved in protein export targeted to erythrocyte membranes during intraerythrocytic development of the malaria parasite.

Identification of cDNAs encoding two novel nuclear proteins, IMUP-1 and IMUP-2, upregulated in SV40-immortalized human fibroblasts.

Using a model system of young, senescent and SV40-immortalized WI-38 fibroblasts, we identified two mRNAs upregulated in immortalized cells (imup-1, immortalization-upregulated protein 1, and imup-2). Compared to normal tissues, both genes were more frequently expressed in cancer cells. The open reading frame of imup-1 spans 321bp, coding for a 10.9 kDa protein of 106 amino acids, while an insertion of 59bp in the otherwise identical mRNA of imup-2 leads to a frameshift, resulting in an 8.5 kDa protein of 85 amino acids. Database searches identified these genes on chromosome 19, which could account for the cloned imup-1 and imup-2 transcripts by alternative splicing. Southern blot analysis of digested genomic DNA confirmed that both transcripts are derived from a single locus. The expressed proteins IMUP-1 and IMUP-2 share 46 identical N-terminal amino acids, whereas the C-termini are unrelated. Green fluorescent protein-fusions of both IMUP-1 and IMUP-2 accumulated in the nucleus of HeLa cells. The C-terminus of IMUP-1 contains a bipartite nuclear localization signal, the deletion of which impaired nuclear translocation. In-vitro translated proteins bound to poly(rG), but did not interact with single-stranded DNA or double-stranded DNA. The nuclear localization of IMUP-1 and IMUP-2 as well as the upregulation of both underlying mRNAs in immortalized cells suggest a function in immortalization.

Fos expression in neurons immunoreactive for neuronal nitric oxide synthase in the rat paraventricular nucleus after intraperitoneal injection of interleukin-1 beta.

Double immunostaining for Fos and neuronal nitric oxide synthase (nNOS) was used to examine whether nNOS-immunoreactive neurons in the paraventricular hypothalamic nucleus (PVN) are activated to express Fos immunoreactivity by intraperitoneal injection of interleukin-1 beta (IL-1 beta) in the rat. Quantitative analysis revealed that some nNOS-positive PVN neurons are activated by IL-1 beta (4 microg/kg, i.p.) administration, but the majority of the IL-1 beta-activated PVN neurons do not express nNOS and are distributed mainly in the parvocellular part of the PVN.

Increase in norepinephrine but not nitric oxide metabolite levels in the hypothalamic paraventricular nucleus region in response to air jet and swing rotation in freely moving conscious rats.

Chronically instrumented, conscious rats were used to examine whether mild exteroceptive stress produces differential neurochemical changes in the hypothalamic paraventricular nucleus (PVN) region. We constructed systems for stress experiment of air jet and swing rotation that were conducted on freely moving conscious rats in a computer-controlled home cage. Concentration of extracellular norepinephrine (NE) and nitric oxide metabolites (NO(X)(-)), nitrite (NO(2)(-)) and nitrate (NO(3)(-)), in the PVN region was then measured by high-performance liquid chromatography with the respective detector; blood pressure (BP) and heart rate (HR) were also measured. Both stressors increased NE concentration in the PVN region as well as BP and HR. Neither stressor altered NO(X)(-) in the PVN region. Cardiovascular and NE changes showed reproducibility in intensity-dependent manner in response to repeated stressors. This finding demonstrated that exteroceptive stress produced different effects on the neurochemical mediators, NE and NO, in the PVN region.

Repeated administration of high dose levodopa enhances hydroxyl radical production in the rat striatum denervated with 6-hydroxydopamine.

We examined whether levodopa (L-DOPA) might increase production of hydroxyl radicals in intact and dopamine-denervated rat striatum. Salicylate trapping combined with in vivo microdialysis provided measurements of 2,3-dihydroxybenzoic acid (2,3-DHBA) as a marker of hydroxyl radical production. Acute administration of high-dose L-DOPA (200, 500 mg/kg, i.p.) did not alter 2,3-DHBA levels in intact striatum or in striatum denervated with 6-hydroxydopamine. On the other hand, L-DOPA administration (200 mg/kg, i.p.) transiently increased 2,3-DHBA in dopamine-denervated striatum of rats after repeated administration of L-DOPA (200 mg/kg, i.p., once daily for 16 days). The results indicated that repeated administration of high dose L-DOPA increased production of hydroxyl radicals in dopamine-denervated striatum.

GABA(A) and GABA(B) receptors modulating basal and footshock-induced nitric oxide releases in rat prefrontal cortex.

Using an in vivo brain microdialysis technique, we measured extracellular levels of nitric oxide (NO) metabolites (NO(x)(-)) in the medial prefrontal cortex (mPFC) upon perfusion of gamma-aminobutyric acid (GABA) receptor antagonists as well as agonists, and also examined the effects of GABA receptor agonists on mild intermittent footshock-induced NO releases in the mPFC in conscious rats. Perfusion of either bicuculline methiodide, a GABA(A) receptor antagonist, or saclofen, a GABA(B) receptor antagonist, through a microdialysis probe resulted in dose-dependent increases in NO(x)(-) levels. Higher-dose perfusion of either muscimol (50 microM), a GABA(A) receptor agonist, or baclofen (250 microM), a GABA(B) receptor agonist resulted in a significant decrease in NO(x)(-) levels. The elevated levels of NO(x)(-) after mild intermittent footshock were attenuated by perfusion of either muscimol (10 microM) or baclofen (50 microM), either of which alone did not affect basal NO(x)(-) levels. These findings are likely to provide helpful clues to our understanding of the inhibitory modulation of basal and footshock-induced NO metabolites releases by GABA(A) and GABA(B) receptors in the mPFC.