Bumetanide blocks the acquisition of conditioned fear in adult rats.

BACKGROUND AND PURPOSE: Bumetanide has anxiolytic effects in rat models of conditioned fear. As a loop diuretic, bumetanide blocks cation-chloride co-transport and this property may allow bumetanide to act as an anxiolytic by modulating GABAergic synaptic transmission in the CNS. Its potential for the treatment of anxiety disorders deserves further investigation. In this study, we evaluated the possible involvement of the basolateral nucleus of the amygdala in the anxiolytic effect of bumetanide. EXPERIMENTAL APPROACH: Brain slices were prepared from Wistar rats. extracellular recording, stereotaxic surgery, fear-potentiated startle response, locomotor activity monitoring and Western blotting were applied in this study. KEY RESULTS: Systemic administration of bumetanide (15.2 mg·kg-1 , i.v.), 30 min prior to fear conditioning, significantly inhibited the acquisition of the fear-potentiated startle response. Phosphorylation of ERK in the basolateral nucleus of amygdala was reduced after bumetanide administration. In addition, suprafusion of bumetanide (5 or 10 µM) attenuated long-term potentiation in the amygdala in a dose-dependent manner. Intra-amygdala infusion of bumetanide, 15 min prior to fear conditioning, also blocked the acquisition of the fear-potentiated startle response. Finally, the possible off-target effect of bumetanide on conditioned fear was excluded by side-by-side control experiments. CONCLUSIONS AND IMPLICATIONS: These results suggest the basolateral nucleus of amygdala plays a critical role in the anxiolytic effects of bumetanide.

Unilateral stimulation of the lateral division of the dorsal telencephalon induces synaptic plasticity in the bilateral medial division of zebrafish.

This study was aimed to evaluate the synaptic plasticity in projections from the dorsal lateral region (Dl) to the bilateral dorsal medial region (Dm) of the zebrafish telencephalon. The results showed that unilateral electrical stimulation of the Dl evokes a negative field potential (FP) in both the contralateral and ipsilateral side of the Dm. We tested synaptic plasticity, including high-frequency stimulation-induced LTP (HFS-LTP) and low-frequency stimulation-induced LTD (LFS-LTD). We demonstrated that HFS-induced bilateral LTP is NMDAR-dependent by the application of an NMDAR antagonist, DL-AP5 (30 µM, suprafused for 10 min), which blocked the HFS-induced LTP in both the contralateral and ipsilateral Dm. In addition, LTP was restored after DL-AP5 was washed out by continuous aCSF suprafusion. These results suggested that the potentiation is NMDAR-dependent. Either LFS (1 Hz for 20 min) or applying the mGluR agonist, DHPG (40 µM, suprafused for 10 min) successfully induced bilateral LTD for at least 1 h. Furthermore, both the contralateral fEPSP and LTP vanished after ablation of the anterior commissure. In conclusion, the results of the present study suggested that the projection between the Dl and contralateral Dm in the telencephalon of zebrafish is via the anterior commissure and possesses synaptic plasticity.

Amygdaloid zif268 participated in the D-cycloserine facilitation effect on the extinction of conditioned fear.

RATIONALE: The involvement of glutamate in fear extinction is perhaps the most promising in terms of facilitating clinical interventions for posttraumatic stress disorder (PTSD). This study was aimed at elucidating the possible role of zif268 on the D-cycloserine (DCS) facilitation effect on extinction. OBJECTIVE: We investigated the association between zif268 and the extinction of conditioned fear by using antisense oligodeoxynucleotide (ODN) of zif268 and the fear-potentiated startle paradigm. METHODS: Male adult Wistar rats were injected DCS (15 mg/kg, IP) 15 min prior to the extinction training, administered with antisense or sense ODN (800 pmol) of zif268 and subjected for fear-potentiated startle paradigm (FPS) and Western blot. RESULTS: Either context exposure or cue exposure elevated the expression of zif268 in the basolateral nucleus of the amygdala (BLA) (p < 0.05 and p < 0.01, respectively) compared to the control group. Additionally, zif268 expression in BLA was further elevated by the glutamate NMDA receptor agonist DCS administration. Intra-amygdaloid injection of the antisense ODN of zif268 blocked the facilitation effect of DCS on the extinction of conditioned fear. Subsequent control experiments indicated that administration of vehicle or zif268 sense ODN did not alter the facilitation of DCS and that the blockage effect of zif268 antisense ODN was not due to lasting damage to the amygdala. CONCLUSIONS: Our results suggest that zif268 within the amygdala participates in the DCS facilitation effect on the extinction of conditioned fear.

Oxidative stress disruption of receptor-mediated calcium signaling mechanisms.

BACKGROUND: Oxidative stress increases the cytosolic content of calcium in the cytoplasm through a combination of effects on calcium pumps, exchangers, channels and binding proteins. In this study, oxidative stress was produced by exposure to tert-butyl hydroperoxide (tBHP); cell viability was assessed using a dye reduction assay; receptor binding was characterized using [3H]N-methylscopolamine ([3H]MS); and cytosolic and luminal endoplasmic reticulum (ER) calcium concentrations ([Ca2+]i and [Ca2+]L, respectively) were measured by fluorescent imaging. RESULTS: Activation of M3 muscarinic receptors induced a biphasic increase in [Ca2+]i: an initial, inositol trisphosphate (IP3)-mediated release of Ca2+ from endoplasmic reticulum (ER) stores followed by a sustained phase of Ca2+ entry (i.e., store-operated calcium entry; SOCE). Under non-cytotoxic conditions, tBHP increased resting [Ca2+]i; a 90 minute exposure to tBHP (0.5-10 mM ) increased [Ca2+]i from 26 to up to 127 nM and decreased [Ca2+]L by 55%. The initial response to 10 µM carbamylcholine was depressed by tBHP in the absence, but not the presence, of extracellular calcium. SOCE, however, was depressed in both the presence and absence of extracellular calcium. Acute exposure to tBHP did not block calcium influx through open SOCE channels. Activation of SOCE following thapsigargin-induced depletion of ER calcium was depressed by tBHP exposure. In calcium-free media, tBHP depressed both SOCE and the extent of thapsigargin-induced release of Ca2+ from the ER. M3 receptor binding parameters (ligand affinity, guanine nucleotide sensitivity, allosteric modulation) were not affected by exposure to tBHP. CONCLUSIONS: Oxidative stress induced by tBHP affected several aspects of M3 receptor signaling pathway in CHO cells, including resting [Ca2+]i, [Ca2+]L, IP3 receptor mediated release of calcium from the ER, and calcium entry through the SOCE. tBHP had little effect on M3 receptor binding or G protein coupling. Thus, oxidative stress affects multiple aspects of calcium homeostasis and calcium dependent signaling.

Zinc oxide nanoparticle disruption of store-operated calcium entry in a muscarinic receptor signaling pathway.

The influences of ZnO nanoparticles on cellular responses to activation of muscarinic receptors were studied in Chinese hamster ovary cells expressing the human M3 muscarinic acetylcholine receptor. ZnO particles (20 nm) induced cytotoxicity in a time and concentration-dependent manner: following a 24h exposure, toxicity was minimal at concentrations below 20 µg/ml but virtually complete at concentrations above 28 µg/ml. ZnO particles did not affect antagonist binding to M3 receptors or allosteric ligand effects, but increased agonist binding affinity while eliminating guanine nucleotide sensitivity. At a noncytotoxic concentration (10 µg/ml), ZnO increased resting [Ca(2+)](i) from 40 to 130 nM without compromising calcium homeostatic mechanisms. ZnO particles had minimal effects on IP3- or thapsigargin-mediated release of intracellular calcium from the endoplasmic reticulum, but strongly inhibited store-operated calcium entry (capacitive calcium entry). The latter effect was seen as (1) a decrease in the plateau phase of the response and (2) a decrease in Ca(2+) entry upon introduction of calcium to the extracellular medium following thapsigargin-induced depletion of calcium from the endoplasmic reticulum (EC50's ≈ 2 µg/ml). Thus, ZnO nanoparticles interfere with two specific aspects of the M3 signaling pathway, agonist binding and store-operated calcium entry.