Noradrenergic stimulation of α1 adrenoceptors in the medial prefrontal cortex mediates acute stress-induced facilitation of seizures in mice.

Stress is one of the critical facilitators for seizure induction in patients with epilepsy. However, the neural mechanisms underlying this facilitation remain poorly understood. Here, we investigated whether noradrenaline (NA) transmission enhanced by stress exposure facilitates the induction of medial prefrontal cortex (mPFC)-originated seizures. In mPFC slices, whole-cell current-clamp recordings revealed that bath application of picrotoxin induced sporadic epileptiform activities (EAs), which consisted of depolarization with bursts of action potentials in layer 5 pyramidal cells. Addition of NA dramatically shortened the latency and increased the number of EAs. Simultaneous whole-cell and field potential recordings revealed that the EAs are synchronous in the mPFC local circuit. Terazosin, but not atipamezole or timolol, inhibited EA facilitation, indicating the involvement of α1 adrenoceptors. Intra-mPFC picrotoxin infusion induced seizures in mice in vivo. Addition of NA substantially shortened the seizure latency, while co-infusion of terazosin into the mPFC inhibited the effect of NA. Finally, acute restraint stress shortened the latency of intra-mPFC picrotoxin infusion-induced seizures, whereas prior infusion of terazosin reversed this stress-induced shortening of seizure latency. Our findings suggest that stress facilitates the induction of mPFC-originated seizures via NA stimulation of α1 adrenoceptors.

[Evaluation of Simple and Rapid Shiga Toxin Gene Detection of Enterohemorrhagic Escherichia coliin Food Using 3MTM Molecular Detection Assay2-STEC Gene Screen Kit].

The sensitivity of the 3M TM Molecular Detection Assay 2-STEC Gene Screen (stx) assay (3M MDA2 STEC assay) was evaluated for verotoxin (VT) gene screening from food materials. The pure culture and foods such as sliced beef, tandoori paste, cucumber, etc. were used for this study. The sensitivity was obtained as 3 to 4 log CFU/mL in enrichment broth (BPW and mEC), which was cultured with food matrices. These results showed this detection kit was suitable the notification of standard methods from Ministry of health, which requires 4 log CFU/mL as detection limit in enrichment broth. This assay was useful as a rapid and simple screening method for VT gene from foods.

Synthesis of C-Plane Oriented Hexagonal Tungsten Oxide Membranes on Tubular Substrates and Their Acetic Acid/Water Separation Performances.

Hexagonal tungsten oxide (h-WO3) membrane is a novel candidate for dehydration of acetic acid (CH3COOH)/water mixtures owing to its molecular sieving property and acidic resistance. Meanwhile, c-plane orientation is an important factor for h-WO3 membranes because the pores of h-WO3 run along its c-axis. However, so far, high c-plane orientation has not been successful on tubular substrates. Here, the effect of synthesis conditions of h-WO3 membranes on tubular substrates against c-plane orientation and CH3COOH/water separation performance are investigated. The h-WO3 membranes were prepared by hydrothermal synthesis from a precursor sol containing various amounts of sodium tungstate (Na2WO4) in the presence of tubular substrates with seeds embedded on their outside surface. The seeding method and the amount of Na2WO4 in the precursor sol significantly affected both crystal orientation and densification of the membrane. A precursor sol with appropriate amounts of Na2WO4 was essential to simultaneously satisfy high c-plane orientation and densification of the membrane while excess Na2WO4 drastically decreased the degree of c-plane orientation. A highly c-plane oriented h-WO3 membrane was successfully obtained under the optimized condition, which exhibited a maximum separation factor of 40.0 and a water permeance of 1.53 × 10-7 mol·m-2·s-1·Pa-1 in a 90:10 wt % CH3COOH/water mixture. The water permeance approximately doubled compared to the previous report, possibly owing to the significantly higher degree of c-plane orientation. Furthermore, it was found that its separation ability can be maintained while stored in 90:10 wt % CH3COOH/water mixture with pH < 0 for more than 500 h.

Acute restraint stress augments the rewarding memory of cocaine through activation of α1 adrenoceptors in the medial prefrontal cortex of mice.

Stress augments the rewarding memory of cocaine, which plays a critical role in inducing cocaine craving. However, the neurobiological mechanisms underlying the enhancing effect of stress remain unclear. Here, we show that noradrenaline (NA) transmission in the medial prefrontal cortex (mPFC) mediates stress-induced enhancement of cocaine craving. When mice were exposed to acute restraint stress immediately before the posttest session of the cocaine-induced conditioned place preference (CPP) paradigm, the CPP score was significantly higher than that in non-stressed mice. Because extracellular NA levels have been reported to be increased in the mPFC during stress exposure, we assessed the effects of NA on mPFC layer 5 pyramidal cell activity. Whole-cell recordings revealed that NA application induces depolarization and a concomitant increase in spontaneous excitatory postsynaptic currents (sEPSCs). The NA effects were inhibited by terazosin, but not by yohimbine or timolol, and the sEPSC increase was not observed in the presence of tetrodotoxin, suggesting the involvement of postsynaptic α1, but not α2 or ß, adrenoceptors in the NA effects. Additionally, intra-mPFC injection of terazosin before stress exposure attenuated the stress-induced increase in cocaine CPP. Intra-mPFC injection of phenylephrine, an α1 adrenoceptor agonist, before the posttest session without stress exposure significantly enhanced cocaine CPP. Furthermore, chemogenetic suppression of mPFC pyramidal cells with inhibitory DREADD (designer receptors exclusively activated by designer drugs) also suppressed the stress-induced CPP enhancement. These findings suggest that the stress-induced increase in NA transmission activates mPFC pyramidal cells via α1 adrenoceptor stimulation, leading to enhancement of cocaine craving-related behavior.

Glutamatergic neurons in the medial prefrontal cortex mediate the formation and retrieval of cocaine-associated memories in mice.

In drug addiction, environmental stimuli previously associated with cocaine use readily elicit cocaine-associated memories, which persist long after abstinence and trigger cocaine craving and consumption. Although previous studies suggest that the medial prefrontal cortex (mPFC) is involved in the expression of cocaine-addictive behaviors, it remains unclear whether excitatory and inhibitory neurons in the mPFC are causally related to the formation and retrieval of cocaine-associated memories. To address this issue, we used the designer receptors exclusively activated by designer drugs (DREADD) technology combined with a cocaine-induced conditioned place preference (CPP) paradigm. We suppressed mPFC neuronal activity in a cell-type- and timing-dependent manner. C57BL/6J wild-type mice received bilateral intra-mPFC infusion of an adeno-associated virus (AAV) expressing inhibitory DREADD (hM4Di) under the control of CaMKII promotor to selectively suppress mPFC pyramidal neurons. GAD67-Cre mice received bilateral intra-mPFC infusion of a Cre-dependent AAV expressing hM4Di to specifically silence GABAergic neurons. Chemogenetic suppression of mPFC pyramidal neurons significantly attenuated both the acquisition and expression of cocaine CPP, while suppression of mPFC GABAergic neurons affected neither the acquisition nor expression of cocaine CPP. Moreover, chemogenetic inhibition of mPFC glutamatergic neurons did not affect the acquisition and expression of lithium chloride-induced conditioned place aversion. These results suggest that the activation of glutamatergic, but not GABAergic, neurons in the mPFC mediates both the formation and retrieval of cocaine-associated memories.

Activation of GABAergic Neurons in the Nucleus Accumbens Mediates the Expression of Cocaine-Associated Memory.

Cocaine-associated environmental cues elicit craving and relapse to cocaine use by recalling the rewarding memory of cocaine. However, the neuronal mechanisms underlying the expression of cocaine-associated memory are not fully understood. Here, we investigated the possible contribution of γ-aminobutyrate (GABA)ergic neurons in the nucleus accumbens (NAc), a key brain region associated with the rewarding and reinforcing effects of cocaine, to the expression of cocaine-associated memory using the conditioned place preference (CPP) paradigm combined with designer receptors exclusively activated by designer drugs (DREADD) technology. The inhibitory DREADD hM4Di was selectively expressed in NAc GABAergic neurons of vesicular GABA transporter-Cre (vGAT-Cre) mice by infusing adeno-associated virus (AAV) vectors. Ex vivo electrophysiological recordings revealed that bath application of clozapine-N-oxide (CNO) significantly hyperpolarized membrane potentials and reduced the number of spikes induced by depolarizing current injections in hM4Di-positive NAc neurons. Additionally, systemic CNO injections into cocaine-conditioned mice 30 min before posttest session significantly reduced CPP scores compared to saline-injected mice. These results indicate that chemogenetic inhibition of NAc GABAergic neurons attenuated the expression of cocaine CPP, suggesting that NAc GABAergic neuronal activation is required for the environmental context-induced expression of cocaine-associated memory.

The synthetic cannabinoid 5F-AMB changes the balance between excitation and inhibition of layer V pyramidal neurons in the mouse medial prefrontal cortex.

RATIONALE: 5F-AMB is one of the synthetic cannabinoids (SCs) designed to potentiate the ability to activate cannabinoid 1 (CB1) receptors and is abused worldwide. Although inhalation of 5F-AMB elicits serious adverse effects including impaired memory and consciousness, it is not known whether and how 5F-AMB affects the activity of pyramidal neurons in the medial prefrontal cortex (mPFC), a brain region associated with higher functions such as memory and cognition. OBJECTIVES: In the present study, we examined the effects of 5F-AMB on mPFC layer V (L5) pyramidal neurons using in vitro whole-cell patch-clamp recordings. RESULTS: Bath application of 5F-AMB attenuated the frequency but not the amplitude of spontaneous excitatory and inhibitory postsynaptic currents (sEPSCs and sIPSCs). The attenuating effects of 5F-AMB were abolished by the CB1 receptor antagonist AM251. 5F-AMB also attenuated the frequency of miniature EPSCs and IPSCs recorded in the presence of tetrodotoxin. Moreover, the extent of attenuating effects of 5F-AMB on stimulus-evoked EPSCs was significantly larger than that on evoked IPSCs. CONCLUSIONS: These findings suggest that 5F-AMB attenuates both excitatory and inhibitory transmission in mPFC L5 pyramidal neurons via the activation of CB1 receptors located in presynaptic terminals. Further, the net impact of 5F-AMB on L5 pyramidal neurons is inhibition due to the change in balance between excitation and inhibition. This inhibitory effect might at least partly contribute to the expression of the adverse effects induced by 5F-AMB inhalation.

Prevalence and Antimicrobial Resistance of Salmonella serotypes Isolated from Poultry Meat in Japan.

The prevalence and antimicrobial susceptibility of Salmonella in 512 poultry meat samples collected from retail stores and poultry-processing plants in Japan between 2015 and 2016 were investigated. The results showed that 55.9% of poultry meat samples were contaminated with Salmonella, with nine different serotypes represented. The most frequent serovar was Salmonella enterica serovar Infantis, followed by S. Schwarzengrund, together accounting for 78.2% of the isolates. High antimicrobial resistance rates were observed against tetracycline (80.9% S. Infantis and 83.9% S. Schwarzengrund), streptomycin (53.4% S. Infantis and 76.8% S. Schwarzengrund), and kanamycin (33.6% S. Infantis and 82.1% S. Schwarzengrund). All tested isolates were susceptible to colistin and ciprofloxacin. In addition, a high proportion (65.6% of S. Infantis, 85.7% of S. Schwarzengrund) of Salmonella isolates were resistant to two or more antimicrobials, and 22 and 17 different resistance patterns were observed in the two strains, respectively. The predominant antibiotic resistance patterns were streptomycin-tetracycline (32/131, 24.4% of S. Infantis) and streptomycin-kanamycin-tetracycline-sulfamethoxazole/trimethoprim (43/112, 38.4% of S. Schwarzengrund). These data indicate that multidrug-resistant S. Infantis and S. Schwarzengrund have spread among poultry meat in Japan.

Chronic cocaine exposure induces noradrenergic modulation of inhibitory synaptic transmission to cholinergic neurons of the laterodorsal tegmental nucleus.

The laterodorsal tegmental nucleus (LDT), which sends cholinergic efferent connections to dopaminergic (DA) neurons in the ventral tegmental area (VTA), plays a critical role in the development of addictive behavior and the reinstatement of cocaine-seeking behavior. Although repeated cocaine exposure elicits plastic changes in excitatory synaptic transmission and intrinsic membrane excitability in LDT cholinergic neurons, it remains unclear whether inhibitory synaptic transmission is modulated by cocaine exposure. The LDT receives fibers containing noradrenaline (NA), a neurotransmitter whose extracellular levels increase with cocaine exposure. Therefore, it is hypothesized that repeated cocaine exposure induces plastic changes in LDT cholinergic neurons via NA. Ex vivo electrophysiological recordings in LDT cholinergic neurons were obtained from rats repeatedly exposed to cocaine. Bath-application of NA induced similar levels of hyperpolarization in both saline- and cocaine-treated neurons. However, NA attenuated the amplitude of inhibitory postsynaptic currents (IPSCs) in cocaine- but not saline-treated neurons through α2 adrenoceptors. This NA-induced IPSC attenuation was observed in the presence of strychnine, but not gabazine, indicating that NA modulated GABAergic but not glycinergic neurotransmission. NA increased the paired-pulse ratios of evoked IPSCs and decreased the frequencies of miniature IPSCs (mIPSCs) without affecting their amplitudes, suggesting a presynaptic mechanism. These findings suggest that repeated cocaine exposure induces neuroplasticity in GABAergic synaptic transmission onto LDT cholinergic neurons by probably modulating presynaptic α2 adrenoceptors. This potentially increases the activity of LDT cholinergic neurons, which might contribute to the development of addictive behavior by enhancing VTA DA neuronal activity.

[Antimicrobial susceptibility tests and resistant strain of Helicobacter pylori].

The antimicrobial susceptibility test was necessary for the eradication therapy of Helicobacter pylori infections. This is because, clarithromycin resistant strains has became an increasing problem. In this study, we used the antimicrobial susceptibility test which was compare with the agar gradient method, Etest, and broth microdilution method (dry plate) with 4 antimicrobial agents. The results strongly suggested that broth microdilution method was the best method in order to test the antimicrobial susceptibility of H. pylori. On the other hand, 393 H. pylori stains isolated during 1994-1998 from clinical patients were tested for antimicrobial susceptibility test to amoxicillin, clarithromycin, metronidazole, and minomycin. There were no resistant strains to amoxicillin and minomycin. But clarithromycin and Metronidazole resistant strains were recognized in 85 (22.0%) and 36 (21.7%) strains.