Inhibition of suplatast tosilate on EPSC in the single paratracheal ganglion neurons attached with presynaptic boutons.

Using single neurons of rat paratracheal ganglia (PTG) attached with presynaptic boutons, the effects of suplatast tosilate on excitatory postsynaptic currents (EPSCs) were investigated with nystatin-perforated patch-clamp recording technique. We found that suplatast concentration dependently inhibited the EPSC amplitude and its frequency in single PTG neurons attached with presynaptic boutons. EPSC frequency was higher sensitive to suplatast than EPSC amplitude. IC50 for EPSC frequency was 1.1 × 10-5 M, being similar to that for the effect on histamine release from mast cells and lower than that for the inhibitory effect on cytokine production. Suplatast also inhibited the EPSCs potentiated by bradykinin (BK), but it did not affect the potentiation itself by BK. Thus suplatast inhibited the EPSC of PTG neurons attached with presynaptic boutons at both the presynaptic and postsynaptic sites.NEW & NOTEWORTHY In this study, using single neurons of rat paratracheal ganglia (PTG) attached with presynaptic boutons, the effects of suplatast tosilate on excitatory postsynaptic currents (EPSCs) were investigated with patch-clamp recording technique. We found that suplatast concentration dependently inhibited the EPSC amplitude and its frequency in single PTG neurons attached with presynaptic boutons. Thus suplatast inhibited the function of PTG neurons at both of presynaptic and postsynaptic sites.

Dimethylglycine, a Methionine Metabolite, Participates in the Suppressive Effect of Methionine on 1-Fluoro-2,4-dinitrobenzene-Induced Dermatitis.

Allergic contact dermatitis (ACD) is a common skin disorder caused by contact with allergens. The optimal treatment for ACD is to avoid contact with allergens. However, in some cases, avoiding exposure is not possible when the allergens are unknown. Therefore, establishing treatment methods other than allergen avoidance is important. We previously reported that the continuous administration of methionine, an essential amino acid, in a mouse model of atopic dermatitis alleviated its symptoms. In the present study, we investigated the effect of methionine on a mouse model of ACD caused by 1-fluoro-2,4-dinitrobenzene (DNFB). Differences in the effect of methionine were observed in DNFB-induced ACD model mice based on the mouse strain used. This difference was attributed to the suppression of hepatic dimethylglycine (DMG) production, which is associated with the suppression of hepatic betaine-homocysteine methyltransferase (Bhmt) expression by ACD. Although we did not reveal the mechanism underlying DMG suppression, our study suggests the presence of interactions between the liver and skin in dermatitis, such as the regulation of hepatic metabolic enzyme expression in dermatitis and the alleviation of dermatitis symptoms by the hepatic metabolism status of DMG.

Estimation of dietary intake and sources of organohalogenated contaminants among infants: 24-h duplicate diet survey in Fukuoka, Japan.

The widespread occurrence of persistent organic pollutants (POPs) in the environment is a matter of concern. In this study, selected organohalogenated contaminants, including dichlorodiphenyltrichloroethane and its metabolites (DDTs) polychlorinated biphenyls (PCBs), chlordanes (CHLs), hexachlorobenzene (HCB), polybrominated diphenyl ethers (PBDEs), hexabromocyclododecanes (HBCDs), tetrabromobisphenol A (TBBPA), 2,4,6-tribromophenol (TBP), were measured in complete meal sets (24-h duplicate-diet) of Japanese infants to investigate the levels, profiles, and possible sources of contamination. In total, 46 whole-day meals of infants (7-24-months old) were collected during 2017 from Fukuoka, Japan. To the best of our knowledge, this is the first report based on the duplicate-diet method for infants. The median intakes among the POP groups were highest for ΣDDTs (18 ng/day, maximum 251 ng/day), followed by ΣPCBs (17 ng/day, maximum 198 ng/day), ΣCHLs (14 ng/day, maximum 105 ng/day), HCB (11 ng/day, maximum 64 ng/day), TBP (3.5 ng/day, maximum 109 ng/day), ΣHBCDs (1.9 ng/day, maximum 70 ng/day), TBBPA (0.72 ng/day, maximum 34 ng/day), and ΣPBDEs (0.11 ng/day, maximum 4.5 ng/day). Among the PCBs, PCB-138 and PCB-153 were the most abundant congeners (27% and 23%, respectively). p,p'-DDE, the major DDT metabolite, accounted for 96% of total DDTs. Among PBDEs, BDE47 was the only detected congener (present in 4% of the samples). The dietary intake of the targeted compounds was lower than the intake via breast milk, suggesting that the exposure from baby food was limited. In the principal component analysis, chlorinated and brominated compounds were separated on principal component 1, while TBP and α-HBCD were separated on principal component 2, likely suggesting a differing emission time trend or source. PCB-153, PCB-138, trans-chlordane, cis-chlordane, and trans-nonachlor were correlated with seafood consumption (Spearman's ρ = 0.45 to 0.57, p < 0.05), while TBP was correlated with seaweed consumption (Spearman's ρ = 0.46, p < 0.05). Also, four species of commercial edible seaweed in Japan were analyzed to confirm the findings of the duplicate-diet study. The relatively high concentration of TBP (5.5 ± 6.6 ng/g wet weight) was observed in the seaweed samples, indicating that seaweed is a potential exposure source of TBP.

Direct Potentiation of Capsaicin Current by Histamine and Its Effect by Suplatast on Rat Trigeminal Ganglia Neurons.

In this study, we investigated the effect of histamine on capsaicin-induced current and its influence by suplatast in rat trigeminal ganglia neurons using a patch-clamp technique. We found that histamine directly potentiated capsaicin-induced currents in rat sensory neurons, and suplatast had little effect on this potentiation. Since it has been known that suplatast suppresses histamine release from mast cells, it is possible that suplatast inhibits the activation of nociceptive fibers in the pathological condition via prevention of histamine-induced potentiation of the transient receptor potential vanilloid 1 receptor-mediated currents.

The effects of suplatast tosilate on acutely dissociated sensory and paratracheal ganglia neurons.

In this study, we investigated the effects of suplatast on acutely dissociated single neurons of sensory and paratracheal ganglia using a patch-clamp technique. Suplatast had little effect on various responses caused by capsaicin, acid, bradykinin, serotonin and adenosine 5'-triphosphate in rat sensory neurons. Suplatast, even at 10-3 M, also did not induce any current at various membrane potentials in rat and guinea pig paratracheal ganglia neurons. Further, acetylcholine- and bradykinin-induced depolarizations were not affected by suplatast. On the other hand, in rat paratracheal ganglia neurons, 10-5 M nicotine-induced current were inhibited by suplatast in a concentration-dependent manner with a 50% inhibitory concentration of 9.86x10-5 M. The effect was noncompetitive and voltage-dependent. Furthermore, the effect was use-independent and not affected by the pretreatment time of suplatast. The results suggested that suplatast may inhibit neurotransmission at the paratracheal ganglia via the inhibition of nicotinic current. Thus, suplatast may attenuate cough production through the improvement of pathological conditions of the lower airway via suppressed acetylcholine release from the postganglionic nerve terminal.

Novel antitussive effect of suplatast tosilate in guinea pigs.

We studied the antitussive effects of suplatast, a Th2 cytokine inhibitor, and compared them with the effects of codeine using an experimental cough model in guinea pigs. Suplatast and codeine dose-dependently inhibited cough caused by mechanical stimulation of the larynx, but they did not inhibit cough caused by mechanical stimulation of the bifurcation of the trachea. In guinea pigs with bronchitis, suplatast had an antitussive effect on cough caused by stimulation of the larynx, whereas codeine did not inhibit such cough. In SO2-exposed guinea pigs, suplatast tended to inhibit cough caused by mechanical stimulation of the tracheal bifurcation. Further, suplatast inhibited citric acid-induced cough augmented by pretreatment with an angiotensin-converting enzyme inhibitor, whereas codeine did not inhibit such cough. Suplatast also inhibited bradykinin-induced discharges of airway vagal afferent nerves and significantly inhibited 4-aminopyridine-induced discharges of airway vagal afferent nerves. These findings indicate that the antitussive effects of suplatast are mediated by a novel mechanism involving the peripheral nervous system.

Cholinergic EPSCs and their potentiation by bradykinin in single paratracheal ganglion neurons attached with presynaptic boutons.

We have found that bradykinin (BK) potentiates the nicotine-induced currents in airway paratracheal/parabronchial ganglia (PTG) neurons. In this study, we investigated if BK affects the cholinergic synaptic transmission in rat PTG neurons attached with synaptic buttons. Excitatory postsynaptic currents (EPSCs) were recorded in acutely dissociated PTG neurons attached with presynaptic boutons. EPSC frequency was increased in the high-K(+) external solution without affecting their amplitude. Activation and deactivation kinetics also did not change in the high-K(+) solution. Cd(2+) inhibited the EPSC frequency at 10(-7) M and also amplitude at higher concentrations without changing the kinetics. Mecamylamine inhibited both the amplitude and frequency of EPSCs and reduced the activation and deactivation kinetics. 10(-8) M BK potentiated the EPSC amplitude to 1.37 ± 0.19 times of preapplication control. In addition, its frequency was increased to 2.04 ± 0.41 times. BK did not affect the activation and deactivation kinetics. The effects of BK were mimicked by [Hyp(3)]-BK, a B2 kinin receptor agonist, whereas HOE 140, a B2 kinin receptor antagonist, abolished the effects of BK. In conclusion, BK potentiates the cholinergic synaptic transmission via B2 kinin receptors in the PTG. Since predominant control of airway function is thought to be exerted by cholinergic nerves arising from the PTG, the present findings might underlie at least partly the inflammatory pathological conditions of the lower airway.

Cloperastine rescues impairment of passive avoidance response in mice prenatally exposed to diethylstilbestrol.

We previously reported that prenatal exposure to diethylstilbestrol (DES) impaired passive avoidance responses in mice. Apart from the above, we also found that cloperastine, a centrally acting antitussive, ameliorated depression-like and anxiety-like behaviors in rodents at antitussive-effective doses. In this study, we investigated whether or not cloperastine rescues impairment of passive avoidance responses in mice prenatally exposed to DES. Male DES-exposed mice were subcutaneously administered cloperastine at 10 or 30 mg/kg twice a day from 32 to 41 days after birth and subjected to behavioral testing 42 to 46 days after birth. Cloperastine at 10 and 30 mg/kg ameliorated DES-induced impairment of passive avoidance responses. In addition, cloperastine affected the levels of 5-HT1A receptors, GIRK and BDNF in the hippocampus of DES-exposed mice. However, the number of BrdU-positive cells in the hippocampus of DES-exposed mice was not changed by chronic administration of cloperastine. These findings suggest that the action of endocrine disruptors in the brain may not always be irreversible, and that the symptoms caused by endocrine disruptors might be curable with drugs such as cloperastine.

Effects of enriched environment on micturition activity in freely moving C57BL/6J mice.

OBJECTIVES: An enriched environment (EE) has been known to promote structural changes in the brain and enhance learning and emotional performance. However, little is known about the effect of an EE on brain stem functions, such as the micturition function. In this study, we examined whether an EE affects micturition activity in mice. METHODS: Male C57BL/6J mice were used. We assessed the micturition activity of freely moving mice using a novel system developed in-house. RESULTS: During the dark period, but not light, the EE significantly increased voiding frequency, total voided volume, mean voided volume, voiding duration, mean flow rate, and maximum flow rate compared with the control environment. This EE effect on micturition function was associated with habituation to novel environments in the open-field test, but not with amelioration of motor coordination in the rotarod test. Interestingly, even after the mice were withdrawn from the EE, the improvements in micturition function persisted, while other behavioral changes were abolished. The relative value of voiding frequency and total voided volume during the light period, expressed as a percentage of 24 hours, increased with age when mice were reared in a standard environment. However, this age-related change was not observed in mice reared in an EE. CONCLUSIONS: These results suggest that an EE may promote micturition activity during the active phase of C57BL/6J mice, and its effects persist even after withdrawal from the EE. Furthermore, an EE may mitigate dysfunctions in micturition activity, such as polyuria, during the resting phase in aged mice.

[Novel antidepressant-like action of drugs possessing GIRK channel blocking action in rats].

We have previously found that antitussive drugs inhibit G protein-coupled inwardly rectifying potassium (GIRK) channel currents in brain neurons. Potassium efflux through GIRK channels causes membrane hyperpolarization, and thus plays an important role in the inhibitory regulation of neuronal excitability. Because GIRK channels are coupled to various G protein-coupled receptors including monoamine receptors, antitussives are possible to affect the levels of various neurotransmitters in the brain. Many currently available antidepressants have been developed based on the monoamine theory for the etiology of depression. We hypothesized that new drugs such as tipepidine may lead to changes in the balance of monoamine levels in the brain resulting in improvement in symptoms of depression. Therefore, we investigated whether or not the drugs have antidepressant activity in the animal models. Male Wistar rats (200-240 g) were used. Tipepidine, cloperastine and caramiphen significantly reduced the immobility in forced swimming test (FST) using normal rats. All drugs had little effect on loco-motor activity. The effects on the forced swimming were inhibited by treatment with AMPT, but not PCPA. Tipepidine also inhibited hyperactivity in olfactory bulbectomized rats. Interestingly, tipepidine also significantly reduced the immobility in FST using ACTH-treated rats which is a model of depression resistant to treatment with antidepressants. Given these results together with cumulated findings, it is suggested that tipepidine may have a novel antidepressant-like action, and that the effect may be caused at least partly through the action on the catecholaminergic system in the brain.

Ameliorating effects of cloperastine on dysfunction of the urinary bladder caused by cerebral infarction in conscious rats.

We investigated the effects of the centrally acting antitussives dextromethorphan and cloperastine on urinary bladder dysfunction 24 h after cerebral infarction in rats using the cystometry technique. First, cystometrography was performed in conscious male Sprague-Dawley rats. Cerebral infarction was then induced by occlusion of the left middle cerebral artery. Twenty-four hours after cerebral infarction, the effect of each drug on micturition disorder was estimated for 5 parameters: bladder capacity, maximum voiding pressure, micturition latency, flow rate, and urethral resistance. Cerebral infarction markedly reduced bladder capacity, micturition latency, and flow rate and increased urethral resistance. After cerebral infarction, intravenous dosing of saline had no effect on these parameters. Dextromethorphan (20 mg/kg) and cloperastine (2.5 and 5.0 mg/kg) at antitussive effective doses significantly increased bladder capacity and micturition latency. Unlike dextromethorphan, cloperastine ameliorated decreases in flow rate and increases in urethral resistance caused by cerebral infarction. These results suggest that cloperastine may have therapeutic value for the treatment of disorders of the micturition reflex associated with cerebral infarction, and that the drug may become a base compound from which to develop more active drugs for such disorders.

Effects of tipepidine on MK-801-induced cognitive impairment in mice.

We previously reported that centrally acting non-narcotic antitussives, including tipepidine, inhibit G-protein-coupled inwardly rectifying potassium (GIRK) channel-activated currents of neurons. In addition, when administered at a cough suppressant dose, the drugs ameliorated the symptoms of various models of intractable brain disease in rodents. In the current study, we investigated whether tipepidine causes recovery from schizophrenia-like cognitive dysfunction, which was induced by MK-801 (0.2 mg/kg, i.p.) in mice. We also examined the effect of tipepidine and clozapine co-administration on the dysfunction. Moreover, we studied whether clozapine inhibits GIRK channel activated currents in single brain neurons using the patch-clamp technique. Tipepidine elicited recovery from MK-801-induced cognitive impairment in the novel objective recognition test and Y-maze test. Further, co-administration of tipepidine and clozapine, at subthreshold doses of each drug, improved MK-801-induced cognitive impairment in the novel objective recognition test. Clozapine (3 × 10-5 M) had a minor effect on baclofen-induced currents in dopamine neurons of the ventral tegmental area.

Deletion of GIRK2 subunit containing GIRK channels of neurons expressing dopamine transporter decrease immobility time on forced swimming in mice.

We previously reported that non-narcotic antitussives possessing inhibitory actions on G protein-coupled inwardly rectifying potassium (GIRK) channels have antidepressant-like effects in the forced swimming test in normal and adrenocoticotropic hormone (ACTH) treated rats. Furthermore, the antidepressant-like effects of the antitussives such as tipepidine were blocked by dopamine D1 receptor antagonist, and inhibitory actions on GIRK channels of dopamine neurons may be involved in the antidepressant-like effects of tipepidine. In this study, we generated GIRK2DATKO mice with Girk2/Kcnj6 conditional deletion and assessed depression-related behavior of the mice. The Cre/loxP system was used to selectively delete GIRK2 subunit containing GIRK channels in the neurons expressing dopamine transporter. First, deletion of GIRK2 subunits in the ventral tegmental area (VTA) neurons expressing dopamine transporters was confirmed by hisitochemically and electrophysiologically. In the mice, a significant decrease in the immobility time of forced swimming test was observed. Locomotor activity of the mice was not changed compared to that of GIRK2floxed mice, when tested in the open field. These results suggest that the antidepressant-like effect of antitussives such as tipepidine may be caused partly through the inhibitory actions on GIRK channels in the dopamine neurons.

Potentiation of nicotinic currents by bradykinin in the paratracheal ganglia neurons of rats.

The effects of bradykinin on nicotine-induced responses were investigated in neurons dissociated from rat paratracheal ganglia using the nystatin-perforated patch-clamp recording technique. When bradykinin (10(-9) to 10(-8) M) was pretreated and then simultaneously applied with 10(-5) M nicotine, bradykinin potentiated the nicotine-induced currents. The potentiation was mimicked by [Hyp3]-bradykinin and inhibited by HOE-140, pertussis toxin, neomycin and U-73122, but not U-73433. These results suggest that bradykinin potentiates nicotinic currents via bradykinin B2 receptor, pertussis toxin-sensitive G-protein and phospholipase C. Since bradykinin inhibits the M-current via bradykinin B2 receptor and pertussis toxin-insensitive G-protein [Mochidome, T., Ishibashi, H., Takahama, K., 2001. Bradykinin activates airway parasympathetic ganglion neurons by inhibiting M-currents. Neuroscience 105, 785-791.], it seemed that bradykinin B2 receptor activated two distinct signal transduction pathways in the paratracheal ganglia neurons. This effect of bradykinin might cause enhanced synaptic transmission in paratracheal ganglia neurons and contribute to the aggravation of pathological conditions of the lower airway via enhanced acetylcholine release from the postganglionic nerve terminals.

Tipepidine, a non-narcotic antitussive, exerts an antidepressant-like effect in the forced swimming test in adrenocorticotropic hormone-treated rats.

We investigated whether tipepidine exerts an antidepressant-like effect in the forced swimming test in adrenocorticotropic hormone (ACTH)-treated rats, which is known as a treatment-resistant depression model, and we studied the pharmacological mechanisms of the effects of tipepidine. Male Wistar rats (5-7 weeks old) were used in this study. Tipepidine (20 and 40 mg/kg, i.p.) decreased the immobility time in the forced swimming test in ACTH-treated rats. The anti-immobility effect of tipepidine was blocked by a catecholamine-depleting agent, alpha-methyl-p-tyrosine (300 mg/kg, s.c.), but not by a serotonin-depleting agent, p-chlorophenylalanine. The anti-immobility effect of tipepidine was also blocked by a dopamine D1 receptor antagonist, SCH23390 (0.02 mg/kg, s.c.) and an adrenaline α2 receptor antagonist, yohimbine (2 mg/kg, i.p.). In microdialysis technique, tipepidine (40 mg/kg, i.p.) increased the extracellular dopamine level of the nucleus accumbens (NAc) in ACTH-treated rats. These results suggest that tipepidine exerts an antidepressant-like effect in the forced swimming test in ACTH-treated rats, and that the effect of tipepidine is mediated by the stimulation of dopamine D1 receptors and adrenaline α2 receptors. The results also suggest that an increase in the extracellular dopamine level in the NAc may be involved in the antidepressant-like effect of tipepidine in ACTH-treated rats.

Centrally acting non-narcotic antitussives prevent hyperactivity in mice: Involvement of GIRK channels.

We have previously reported that centrally acting non-narcotic antitussives inhibited G protein-coupled inwardly rectifying potassium (GIRK) channel-activated currents, and that the antitussives had multiple pharmacological actions on various models of intractable brain diseases in rodents. In this study, the question of whether these antitussives inhibit drug-induced hyperactivity in mice was investigated. Antitussives, such as cloperastine and tipepidine, at cough suppressant doses, inhibited an increase in ambulation of mice neonatally treated with 6-hydroxydopamine. In addition, all antitussives studied inhibited an increase in methamphetamine-induced hyperactivity in mice. Methylphenidate, which is used for treatment of ADHD, inhibited 6-hydroxydopamine-lesion-induced, but not methamphetamine-induced, hyperactivity in mice. By the rota-rod test, the drugs had little effect on motor coordination of the hyperactive mice. Significant correlation was found between the ameliorating effects of antitussives on methamphetamine-induced hyperactivity and their inhibitory actions on GIRK channel currents (coefficient factor, 0.998). Furthermore, tertiapin, a GIRK channel blocker, prevented an increase in methamphetamine-induced hyperactivity of mice. These results demonstrated that antitussive drugs (cloperastine, tipepidine and caramiphen) possessing inhibitory action on GIRK channels inhibit drug-induced hyperactivity in mice, suggesting that such antitussives may potentially be therapeutic for patients with ADHD.

Tipepidine increases dopamine level in the nucleus accumbens without methamphetamine-like behavioral sensitization.

We previously reported that the novel antidepressant-like effect of tipepidine may be produced at least partly through the activation of mesolimbic dopamine neurons via inhibition of G protein-coupled inwardly rectifying potassium channels. In this study, we investigated whether tipepidine increases dopamine levels in the nucleus accumbens (NAc) in rats using an in vivo microdialysis technique. We further assessed whether tipepidine at antidepressant-like effective doses induces behavioral- and cross-sensitization of locomotor activity in rats using the open field test. We found that acute administration of tipepidine increased dopamine levels in the NAc in freely moving rats without increasing locomotor activity. Tipepidine at antidepressant-like effective doses (20 and 40 mg/kg, i.p.) did not cause behavioral sensitization in rats. Furthermore, cross-sensitization between tipepidine and methamphetamine was not observed in rats. These results further support our working hypothesis that tipepidine may produce a novel antidepressant-like effect through activation of ventral tegmental area-NAc dopaminergic neurons whose mechanisms differ from those contributing to the reinforcing effects of addictive drugs.

delta-Opioid receptor antagonists inhibit GIRK channel currents in acutely dissociated brainstem neurons of rat.

In this study, we investigated the effects of delta-opioid receptor antagonists on the G protein-coupled inwardly rectifying potassium (GIRK) channel currents induced by serotonin (5-HT) and noradrenaline (NAd) in the dorsal raphe and the locus coeruleus neurons, respectively. Perforated patch and conventional whole-cell patch clamp recording techniques were used for the study. Neurons were acutely dissociated from neonatal rats. Both naltrindole (NTI) and naltriben (NTB), which are selective delta-antagonists possessing antitussive activity in in vivo animal studies, reversibly inhibited the 5-HT-induced GIRK channel currents (I(5-HT)) in dorsal raphe neurons. This inhibition was concentration-dependent and voltage-independent. The half-maximum inhibitory concentration (IC(50)) on I(5-HT) was 9.84x10(-5) M for NTI and 1.28x10(-5) M for NTB. The inhibition was not reversed by 10(-5) M DPDPE, a selective delta-opioid receptor agonist. NTI did not affect 50% effective concentration (EC(50)) on the concentration-response relationship for 5-HT but inhibited the maximum response. In neurons internally perfused with GTPgammaS, both NTI and NTB also inhibited the GIRK channel currents irreversibly activated by 5-HT. Furthermore, these antagonists concentration dependently inhibited 10(-6) M NAd-induced currents (I(NAd)) in locus coeruleus neurons. The IC(50) of NTI on I(NAd) was 8.44x10(-5) M, which was close to that on I(5-HT). The results suggest that NTI and NTB, which are delta-opioid receptor antagonists possessing antitussive activity, may inhibit GIRK channel currents through a non-opioid action, and give further support to our idea previously proposed that centrally acting non-narcotic antitussives have a common characteristic of the inhibitory action on GIRK channels.

Estimation of increased concentration of intracellular Cd(2+) by fluo-3 in rat thymocytes exposed to CdCl(2).

Cadmium, an environmental pollutant, has been reported to induce apoptosis in murine lymphocytes. To reveal the mechanism of cadmium-induced apoptosis, one of important questions is whether cadmium increases intracellular concentration of Ca(2+) ([Ca(2+)](i)), Cd(2+) ([Cd(2+)](i)) or both. It is difficult to detect the increase in [Ca(2+)](i) using Ca(2+)-chelator-based fluorescent Ca(2+) indicators in the presence of Cd(2+) because of their sensitivity to Cd(2+). Therefore, the study on membrane response such as Ca(2+)-dependent hyperpolarization gives a clue to reveal whether the [Ca(2+)](i) or [Cd(2+)](i) is increased. Cadmium at concentrations of 3 µM or more dose-dependently augmented fluo-3 fluorescence in rat thymocytes, presumably suggesting an increased [Ca(2+)](i). However, the membranes were not hyperpolarized although the cells possess Ca(2+)-dependent K(+) channels. One may argue that cadmium inhibits Ca(2+)-dependent K(+) channels so that cadmium fails to hyperpolarize the membranes. It is unlikely because the [Ca(2+)](i) increased by A23187, a calcium ionophore, elicited the hyperpolarization in the presence of Cd(2+). Furthermore, the profile of cytotoxicity induced by cadmium, examined by ethidium bromide and annexin V-FITC, was different from that induced by A23187. Taken together, it is concluded that the application of cadmium increases the [Cd(2+)](i) rather than the [Ca(2+)](i) in rat thymocytes, resulting in the induction of cytotoxicity.

Characterization of neural estrogen signaling and neurotrophic changes in the accelerated ovarian failure mouse model of menopause.

Accelerated ovarian failure (AOF) can be induced in young mice with low doses of 4-vinylcyclohexene diepoxide (VCD), modeling the hormone changes observed across menopause. We assessed markers of synaptic plasticity in the hippocampus, anxiety-like behavior, and spatial learning longitudinally at 4 time points across the AOF model: premenopause, early perimenopause, late perimenopause, and postmenopause (POST). As others have shown, VCD administration decreased ovarian follicle counts and increased acyclicity as the model progressed to POST but with no impact on organ or body weights. The morphology of Iba1 immunoreactive microglia did not differ between vehicle- and VCD-administered mice. Hippocampal postsynaptic density 95 levels were minimally altered across the AOF model but decreased at POST in CA3b 24 hours after exogenous estradiol benzoate (EB). In contrast, hippocampal phosphorylated AKT levels transiently decreased in premenopause but increased at POST after 24 hours of EB in select subregions. Electron microscopy revealed fewer estrogen receptor α containing dendritic spines and terminals in CA1 stratum radiatum at POST. mRNA levels of most brain-derived neurotrophic factor exons (except V and VI) were lower in POST compared with ovariectomized mice. Exon V was sensitive to 24 hours of EB administration in POST-VCD. Anxiety-like behavior was unaffected at any menopause phase. Spatial learning was unaffected in all groups, but POST-VCD mice performed below chance. Our results suggest that the AOF model is suitable for longitudinal studies of neurobiological changes across the menopause transition in mice. Our findings also point to complex interactions between estrogen receptors and pathways involved in synaptic plasticity.