High ethanol and acetaldehyde decrease extracellular glutamate in the frontal cortex of freely moving mice.

Our recent study demonstrated that local perfusion of ethanol (EtOH) and acetaldehyde (AcH) into the hippocampus via microdialysis decreased extracellular glutamate; however, it is not clear whether this effect occurs in the frontal cortex. To address this issue, we investigated the effects of local perfusion of EtOH and AcH on extracellular glutamate in the frontal cortex of Aldh2-knockout (Aldh2-KO) and C57BL/6 N [wild-type (WT)] mice. Dialysates were collected every 20 minutes, and extracellular glutamate was measured using HPLC coupled with electrochemical detector. We found local perfusion of 200 and 500 mM EtOH into the frontal cortex of WT and Aldh2-KO mice produced significant decreases in extracellular glutamate levels (P < 0.05). A dose of 500 mM EtOH induced a greater decrease in Aldh2-KO mice (P < 0.05) than in WT mice, indicating the action of AcH. Similarly, perfusion of 200 and 500 µM AcH decreased glutamate in the frontal cortex of Aldh2-KO mice (P < 0.05), but this decrease was not seen in WT mice at any AcH dose, due to the subsequent oxidation of AcH by mitochondrial aldehyde dehydrogenase 2. A low dose of EtOH (100 mM) or AcH (100 µM) had no effect on glutamate. These results showed that high doses of EtOH and AcH induces a significant decrease in extracellular glutamate in the frontal cortex of mice, replicating previous findings and providing further evidence that reduced glutamate is likely to be involved in the depressant effects of EtOH.

High Ethanol and Acetaldehyde Inhibit Glutamatergic Transmission in the Hippocampus of Aldh2-Knockout and C57BL/6N Mice: an In Vivo and Ex Vivo Analysis.

We aimed to investigate whether ethanol (EtOH) and acetaldehyde (AcH) can affect glutamate and its receptors GluN1 and GluA1 in the hippocampus of Aldh2-knockout (Aldh2-KO) and C57BL/6N (wild-type (WT)) mice. To do this, we first examined the effect of local administration of EtOH (100 mM, 200 mM, and 500 mM) and AcH (100 µM, 200 µM, and 500 µM) on extracellular glutamate levels in freely moving mice. Retrodialysis of 200 mM and 500 mM EtOH into the hippocampus of WT and Aldh2-KO mice produced significant decreases in extracellular glutamate levels (p < 0.05). A dose of 500 mM EtOH induced a greater decrease in Aldh2-KO mice (p < 0.05) than in WT mice, indicating the action of AcH. Similarly, perfusion of 200 µM and 500 µM AcH decreased glutamate in Aldh2-KO mice (p < 0.05), but this decrease was not seen in WT mice at any AcH dose. Second, we tested whether the EtOH- and AcH-induced decrease in glutamate was associated with decreases in GluN1 and GluA1 expression, as measured by real-time PCR and Western blot. We found a significant decrease in GluN1 (p < 0.05) and GluA1 (p < 0.05) subunits after a high dose of EtOH (4.0 g/kg) and AcH (200 mg/kg) in WT mice. However, a 2.0 g/kg dose of EtOH did not produce a consistent decrease in GluN1 or GluA1 between messenger RNA and protein. In Aldh2-KO mice, all three doses of EtOH (1.0 g/kg, 2.0 g/kg, and 4.0 g/kg) and AcH (50 mg/kg, 100 mg/kg, and 200 mg/kg) decreased GluN1 expression (p < 0.05), while moderate-to-high doses of EtOH (2.0 g/kg and 4.0 g/kg) and AcH (100 mg/kg and 200 mg/kg) decreased GluA1 expression (p < 0.05). Together, these in vivo and ex vivo data suggest that EtOH and AcH decrease extracellular glutamate in the hippocampus of mice with a concomitant decrease in GluN1 and GluA1 subunits, but these effects require relatively high concentrations and may, therefore, explain the consequences of EtOH intoxication.

COA-Cl induces dopamine release and tyrosine hydroxylase phosphorylation: In vivo reverse microdialysis and in vitro analysis.

We found that local perfusion of COA-Cl (0.1, 0.4, or 1.0 mM) into the dorsal striatum of living mice produced a significant and dose-dependent increase in extracellular DA levels, with the highest dose of 1.0 mM COA-Cl producing an approximately 5-fold increase in DA. Consistent with in vivo findings, 0.1 and 0.2 mM COA-Cl significantly and dose-dependently enhanced DA release 3.0 to 5.0-fold in PC12 cells, an in vitro model of DA-responsive neurons. Interestingly, the increase in striatal DA levels by COA-Cl in vivo was similar in magnitude to that observed in PC12 cells. Treatment with 0.1 mM COA-Cl significantly increased both Ser31 and Ser40 phosphorylation of tyrosine hydroxylase (TH) in PC12 cells, and Ser40 phosphorylation in iCell neurons, without altering total TH protein levels. Further, we examined whether COA-Cl could stimulate neurite outgrowth in PC12 cells and iCell neurons and found that COA-Cl significantly induced neurite outgrowth in both cell lines. Our results provide the first evidence that COA-Cl can stimulate dose-dependent DA release and activation of TH phosphorylation, suggesting that COA-Cl may be a promising therapeutic candidate for the treatment of neurological dysfunction associated with low DA.

Acetaldehyde administration induces salsolinol formation in vivo in the dorsal striatum of Aldh2-knockout and C57BL/6N mice.

Acetaldehyde (AcH) and salsolinol play important roles in the central effects of ethanol. This study aimed to investigate the effect of administration of AcH on dopamine (DA), DA-derived salsolinol and serotonin (5-HT) levels in the dorsal striatum of Aldh2-knockout (Aldh2-KO) and C57BL/6 N (WT) mice. Animals were treated with AcH (50, 100 and 200 mg/kg) intraperitoneally and dialysate levels of DA, 5-HT and salsolinol were determined using in vivo microdialysis coupled with HPLC-ECD. Salsolinol was first detected at 20 min after AcH administration, and reached its peak concentration (WT mice: 0.29 ± 0.22 pg/µl; Aldh2-KO mice: 0.63 ± 0.17 pg/µl) at 25 min in the 200 mg/kg AcH group, before decreasing rapidly and reaching zero at approximately 55-80 min. Treatment with 100 and 200 mg/kg AcH increased levels of salsolinol in both WT and Aldh2-KO mice, with 200 mg/kg AcH inducing a higher level of salsolinol in Aldh2-KO mice than in WT mice. Treatment with 50 mg/kg AcH produced a small increase in salsolinol levels in Aldh2-KO mice, whereas no elevation of salsolinol was detected in WT mice. The increase in salsolinol formation was found to occur a dose-dependent manner in both genotypes. Administration of AcH and the subsequent changes in salsolinol concentrations did not change DA or 5-HT levels in either genotype. Our study suggests that AcH dose-dependently increases the formation of salsolinol in the dorsal striatum of mice, which provides further support for the role of AcH in salsolinol formation in the animal brain.

Effect of the volatile anesthetic agent isoflurane on lateral diffusion of cell membrane proteins.

The volatile anesthetic isoflurane (ISO) has previously been shown to increase the fluidity of artificial lipid membranes, but very few studies have used biological cell membranes. Therefore, to investigate whether ISO affects the mobility of membrane proteins, fluorescence-labeled transferrin receptor (TfR) and glycosylphosphatidylinositol (GPI)-anchored protein were expressed in human embryonic kidney 293T cells and neural cells and lateral diffusion was examined using fluorescence recovery after photobleaching. Lateral diffusion of the TfR increased with ISO treatment. On the other hand, there was no effect on GPI-anchored protein. We also used GC/MS to confirm that there was no change in the concentration of ISO due to vaporization during measurement. These results suggest that ISO affects the mobility of transmembrane protein molecules in living cells.

The Role of Apolipoprotein E and Ethanol Exposure in Age-Related Changes in Choline Acetyltransferase and Brain-Derived Neurotrophic Factor Expression in the Mouse Hippocampus.

Disruption of apolipoprotein E (APOE) is responsible for age-dependent neurodegeneration and cognitive impairment. Elderly individuals are more sensitive than young individuals to the effects of ethanol (EtOH), particularly those affecting cognition. We investigated the role of APOE deficiency and EtOH exposure on age-dependent alterations in choline acetyltransferase (ChAT) and brain-derived neurotrophic factor (BDNF) mRNA and protein expression in the mouse hippocampus. Three-month-old (young) and 12-month-old (aged) ApoE-knockout (ApoE-KO) and wild-type (WT) mice were treated with saline or 2 g/kg EtOH, and the bilateral hippocampus was collected after 60 min for real-time PCR and western blotting analyses. ChAT (P < 0.01) and BDNF (P < 0.01) expression were significantly decreased in both young and aged saline- and EtOH-treated ApoE-KO mice versus young and aged saline- and EtOH-treated WT mice. Aged saline- and EtOH-treated ApoE-KO mice exhibited greater differences in ChAT and BDNF expression (P < 0.01) than young saline- and EtOH-treated ApoE-KO mice. Aged EtOH-treated WT mice also exhibited larger decreases in BDNF expression (P < 0.01)-but not in ChAT expression-than young EtOH-treated WT mice. EtOH decreased ChAT and BDNF expression in both young (P < 0.01) and aged (P < 0.01) ApoE-KO mice versus EtOH-free ApoE-KO mice of the same age. EtOH also decreased BDNF expression in aged (P < 0.01) WT mice versus EtOH-free aged WT mice. In summary, these results suggest that APOE deficiency and EtOH exposure cause age-dependent decreases in ChAT and BDNF in the hippocampus. Importantly, the decreases in ChAT and BDNF were greater in aged EtOH-treated mice, particularly those lacking APOE, raising the possibility that APOE-deficient individuals who consume alcohol may be at greater risk of memory deficit.

Perfusion with carbon monoxide does not affect extracellular glutamate in dialysates of the hippocampus of freely moving mice.

Carbon monoxide (CO) produces several neurological effects, including cognitive, mood, and behavioral disturbance. Glutamate is thought to play a particularly important role in learning and memory. Thus, the present study was aimed at investigating the local effect of CO on the glutamate level in the hippocampus of mice using in vivo reverse microdialysis. Mice were perfused with Ringer's solution (control) or CO (60-125 µM) in Ringer's solution into the hippocampus via microdialysis probe. Dialysate samples were collected every 20 min, and then analyzed with high-performance liquid chromatography coupled to an electrochemical detector. The result revealed that the perfusion with CO had no significant effect on glutamate levels (p = 0.316) as compared to the control group. This finding does not support a local CO rise as the cause of the increased glutamate level in the hippocampus of mice.

Comparison of histological findings and the results of energy-dispersive X-ray spectrometry analysis in experimental electrical injury.

The findings of histological examination and the results of energy-dispersive X-ray spectrometry (EDX) analysis were compared to identify skin metallization in experimental electrical injury. Rats were divided into three experimental groups (n = 5, each group): control, current exposure for five seconds, and current exposure for ten seconds. A relatively high peak of copper, which was used as an electrical conductor, was detected in formalin-fixed skin samples of the two current exposure groups by EDX. There was a significant increase of the specific X-ray intensity in the two current exposure groups compared to the control group. On histological examination, epidermal nuclear elongation was observed in all samples of the two current exposure groups. However, deposition of metal was observed in two samples of each current exposure group. Metallization is an important finding for the diagnosis of electrocution. The present results suggest that EDX analysis is useful for the proof of metallization in electrocution, even where it is not identified on morphological examination.

An autopsy case of death by combined use of benzodiazepines and diphenidine.

We present an autopsy case involving benzodiazepines and diphenidine. Quantitative toxicological analysis showed concentrations of 7-aminoflunitrazepam (a flunitrazepam metabolite), 7-aminonimetazepam (a nimetazepam metabolite), chlorpheniramine and diphenidine in femoral blood of 0.086 µg/ml, 0.027 µg/ml, 0.066 µg/ml, and 0.073 µg/ml, respectively. Death was attributed to combined toxicity due to the influence of multiple drug interactions.

Distinction between entrance and exit wounds by energy dispersive X-ray fluorescence spectrometry.

We investigated gunshot wounds in two autopsy cases using energy dispersive X-ray spectrometry (EDX). Lead and copper were detected in the entrance wound of one case and lead, antimony, and copper were detected in that of the other case. In the exit wounds of both cases, lead, antimony, and copper were below detection limits. These findings indicate that the detection of metallic elements, such as lead, antimony, and copper, which are found in bullets, may be useful for differentiating entrance from exit wounds using EDX.

Ethanol and Acetaldehyde After Intraperitoneal Administration to Aldh2-Knockout Mice-Reflection in Blood and Brain Levels.

This paper reports, for the first time, on the analysis of ethanol (EtOH) and acetaldehyde (AcH) concentrations in the blood and brains of Aldh2-knockout (Aldh2-KO) and C57B6/6J (WT) mice. Animals were administrated EtOH (1.0, 2.0 or 4.0 g/kg) or 4-methylpyrazole (4-MP, 82 mg/kg) plus AcH (50, 100 or 200 mg/kg) intraperitoneally. During the blood tests, samples from the orbital sinus of the eye were collected. During the brain tests, dialysates were collected every 5 min (equal to a 15 µl sample) from the striatum using in vivo brain microdialysis. Samples were collected at 5, 10, 15, 20, 25, 30 and 60 min intervals post-EtOH and -AcH injection, and then analyzed by head-space GC. In the EtOH groups, high AcH levels were found in the blood and brains of Aldh2-KO mice, while only small traces of AcH were seen in the blood and brains of WT mice. No significant differences in EtOH levels were observed between the WT and the Aldh2-KO mice for either the EtOH dose. EtOH concentrations in the brain were comparable to the EtOH concentrations in the blood, but the AcH concentrations in the brain were four to five times lower compared to the AcH concentrations in the blood. In the AcH groups, high AcH levels were found in both WT and Aldh2-KO mice. Levels reached a sharp peak at 5 min and then quickly declined for 60 min. Brain AcH concentrations were almost equal to the concentrations found in the blood, where the AcH concentrations were approximately two times higher in the Aldh2-KO mice than in the WT mice, both in the blood and the brain. Our results suggest that systemic EtOH and AcH administration can cause a greater increase in AcH accumulation in the blood and brains of Aldh2-KO mice, where EtOH concentrations in the Aldh2-KO mice were comparable to the EtOH concentrations in the WT mice. Furthermore, detection of EtOH and AcH in the blood and brain was found to be dose-dependent in both genotypes.

Ethanol and acetaldehyde differentially alter extracellular dopamine and serotonin in Aldh2-knockout mouse dorsal striatum: A reverse microdialysis study.

Dopamine (DA) and serotonin (5-HT) seem to be involved in several of the effects of ethanol (EtOH). Acetaldehyde (AcH), especially in the brain, induces effects that mimic those of EtOH. The purpose of this study was to investigate the effects of local perfusion of EtOH and AcH on extracellular DA and 5-HT in the dorsal striatum of Aldh2-knockout (Aldh2-KO) and wild-type (WT) mice. Aldh2-KO mice were used as a model of aldehyde dehydrogenase 2 deficiency in humans to examine the effects of AcH. Mice were perfused with Ringer's solution (control), EtOH (100, 200, or 500mM) and AcH (100, 200, or 500µM) into the dorsal striatum. Dialysate samples were collected every 5min, and then analyzed with HPLC coupled to an ECD. We found that local perfusion with 500mM EtOH increased extracellular levels of DA (p<0.05) in both Aldh2-KO and WT mice, while 5-HT levels remain unchanged. EtOH at a dose of 200mM also increased DA in WT mice, but this was limited to a 30-40-min time-point. In contrast, perfusion with 200 and 500µM AcH decreased both DA and 5-HT (p<0.05) in Aldh2-KO mice, but this decrease was not found in WT mice at any AcH dose, indicating an effect of AcH on DA and 5-HT levels. There were no genotype effects on the basal levels of DA and 5-HT. These results indicate that high EtOH can stimulate DA, whereas high AcH can depress both DA and 5-HT in the dorsal striatum of mice.

A color test for the convenient identification of an ingested surface activating agent.

Color tests are easy, simple and inexpensive methods for the qualitative identification of chemicals. A color test was applied to the stomach contents of a forensic autopsy case. The result of the test, using bromophenol blue reagent, indicated the ingestion of a commercial cleaning product containing a cationic surface activating agent. Our findings suggest that forensic investigators should consider the additives used in commercial chemical products, such as surface activating agents, when determining the cause of death.

Detection of chlorine and bromine in free liquid from the sphenoid sinus as an indicator of seawater drowning.

We have investigated the usefulness of elemental analysis by energy-dispersive X-ray spectroscopy (EDX) in the examination of free liquid from the sphenoid sinus of drowning victims. We detected both chlorine and bromine in liquid taken from the sphenoid sinus of seawater drowning victims. Because these elements were below the quantification limit in freshwater cases, we could easily distinguish seawater from freshwater drowning cases. Detection of these elements from the liquid in the sphenoid sinuses of drowning victims may be useful as a supportive measure for seawater drowning.

A fatal case of poisoning with ethanol and psychotropic drugs with putrefactive changes.

We present a fatal case involving poisoning with paroxetine, flunitrazepam, and ethanol, with putrefactive changes. Quantitative toxicological analysis showed that the concentrations of paroxetine and 7-aminoflunitrazepam, a metabolite of flunitrazepam, in the femoral blood were 0.28 µg/ml and 0.17 µg/ml, respectively. We also detected an ethanol level of 2.90 mg/ml and an n-propanol level of 0.10 mg/ml. We concluded that the cause of death was due to the interaction of paroxetine, flunitrazepam, and ethanol. The effects of putrefactive changes should be considered during forensic toxicological evaluation.

Aldehyde dehydrogenase 2 deficiency increases resting-state glutamate and expression of the GluN1 subunit of N-methyl-D-aspartate receptor in the frontal cortex of mice.

Our previous study showed that Aldh2-knockout (Aldh2-KO) mice, an animal model of inactive aldehyde dehydrogenase 2 (ALDH2), have better spatial memory when compared with wild-type (WT) mice. Given that the neurotransmitter glutamate has been associated with learning and memory, the goal of the present study was to investigate whether the strain-dependent difference in spatial memory was associated with changes in glutamate transmitter levels or receptor function in the frontal cortex of Aldh2-KO and WT mice. Thus, we first measured extracellular glutamate levels in free-moving mice using microdialysis. Second, we studied protein expression of the N-methyl-D-aspartate (NMDA) receptor (GluN1) subunit and the α-amino-3-hydroxy-5 methylisoxazole-4-propionic acid (AMPA) receptor (GluA1) subunit in lipid raft fractions using Western blot (WB). The samples were collected for WB, and lipid rafts were prepared from the insoluble fraction of homogenate tissue. Protein concentration was measured in the whole cell lysate (WCL) and in five separate lipid raft fractions. Cholesterol was also measured in all fractions 1-5. The microdialysis study revealed that basal glutamate concentration in the dialysates was approximately three-fold (0.27 ± 0.12 µM) higher in Aldh2-KO mice than in WT (0.10 ± 0.03 µM) mice. We also found an increase in the expression of GluN1 in Aldh2-KO mice compared with WT mice, both in the WCL and fraction 5, but GluA1 levels were unchanged as measured by WB. Our novel findings provide the first evidence for the role of ALDH2 in glutamate release and GluN1 protein expression in the frontal cortex. The observed strain differences in glutamate levels and GluN1 expression may suggest that enhanced glutamatergic function facilitates improved spatial memory in Aldh2-KO mice and such observation deserves further investigation.

Xylene; a useful marker for agricultural products ingestion.

Here we report about a case of malathion (an organophosphate insecticide) ingestion. Headspace analysis of stomach content provided useful information for screening of toxic chemicals. We should pay attention to solvents used in commercial industrial products.

Ethanol- and acetaldehyde-induced cholinergic imbalance in the hippocampus of Aldh2-knockout mice does not affect nerve growth factor or brain-derived neurotrophic factor.

Neurotrophins, including nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), play an important role in the maintenance of cholinergic-neuron function. The objective of this study was to investigate whether ethanol (EtOH)- and acetaldehyde (AcH)- induced cholinergic effects would cause neurotrophic alterations in the hippocampus of mice. We used Aldh2 knockout (Aldh2-KO) mice, a model of aldehyde dehydrogenase 2 (ALDH2)-deficiency in humans, to examine the effects of acute administration of EtOH and the role of AcH. Hippocampal slices were collected and the mRNA and protein levels of choline acetyltransferase (ChAT), acetylcholinesterase (AChE), NGF and BDNF were analyzed 30 min after the i.p. administration of EtOH (0.5, 1.0, or 2.0 g/kg). We show that treatment with 2.0 g/kg of EtOH decreased ChAT mRNA and protein levels in Aldh2-KO mice but not in wild-type (WT) mice, which suggests a role for AcH in the mechanism of action of EtOH. The administration of 2.0 g/kg of EtOH increased AChE mRNA in both strains of mice. EtOH failed to change the levels of NGF or BDNF at any dose. Aldh2-KO mice exhibited a distinctly lower expression of ChAT and a higher expression of NGF both at mRNA and protein levels in the hippocampus compared with WT mice. Our observations suggest that administration of EtOH and elevated AcH can alter cholinergic markers in the hippocampus of mice, and this effect did not change the levels of NGF or BDNF.

Findings for current marks: histopathological examination and energy-dispersive X-ray spectroscopy of three cases.

We describe herein three cases of electrocution. As most deaths caused by electricity are due to cardiac arrhythmia or paralysis of the respiratory muscles, autopsy findings in electrocution cases are generally non-specific, with the exception of the presence of current marks. We detected metallization by histological examination and energy-dispersive X-ray spectroscopy (EDX) analysis in tissues of typical or atypical current marks. In addition, myofiber break-up was observed in one case. One patient was hospitalized before death and revealed patchy contraction band necrosis, along with infiltration of leucocytes and vacuolation in the diaphragm. The presence of current marks is the hallmark for forensic diagnosis of electrocution. Although specific findings are lacking at autopsy in cases of electrocution, detailed histological examination and EDX analysis provide useful information for forensic diagnosis.

Application of energy dispersive X-ray fluorescence spectrometry (EDX) in a case of methomyl ingestion.

We applied energy dispersive X-ray fluorescence spectrometry (EDX) in a case of poisoning by methomyl, a carbamate pesticide. Quantitative GC/MS analysis showed that the concentration of methomyl-oxime in the femoral blood was 4.0 µg/ml. The elemental analysis by EDX identified the high peak of silicon and sulfur in the stomach contents. We concluded that the cause of his death was methomyl poisoning. This indicates that screening of stomach contents by EDX provides useful information for the forensic diagnosis.