Cannabinoid CB1 receptors in distinct circuits of the extended amygdala determine fear responsiveness to unpredictable threat.

The brain circuits underlying behavioral fear have been extensively studied over the last decades. Although the vast majority of experimental studies assess fear as a transient state of apprehension in response to a discrete threat, such phasic states of fear can shift to a sustained anxious apprehension, particularly in face of diffuse cues with unpredictable environmental contingencies. Unpredictability, in turn, is considered an important variable contributing to anxiety disorders. The networks of the extended amygdala have been suggested keys to the control of phasic and sustained states of fear, although the underlying synaptic pathways and mechanisms remain poorly understood. Here, we show that the endocannabinoid system acting in synaptic circuits of the extended amygdala can explain the fear response profile during exposure to unpredictable threat. Using fear training with predictable or unpredictable cues in mice, combined with local and cell-type-specific deficiency and rescue of cannabinoid type 1 (CB1) receptors, we found that presynaptic CB1 receptors on distinct amygdala projections to bed nucleus of the stria terminalis (BNST) are both necessary and sufficient for the shift from phasic to sustained fear in response to an unpredictable threat. These results thereby identify the causal role of a defined protein in a distinct brain pathway for the temporal development of a sustained state of anxious apprehension during unpredictability of environmental influences, reminiscent of anxiety symptoms in humans.

Fatal non-accidental alpha-lipoic acid intoxication in an adolescent girl.

The aim of our report is to increase awareness that the antioxidant alpha-lipoic acid, which is marketed primarily as weight loss and energy supplement, has potentially lethal effects. A 14-year-old girl ingested in suicidal intention a large amount of alpha-lipoic acid, which led to multiorgan failure and subsequent death within 24 h. Multiorgan failure consisted of decreased myocardial contractility, seizures, anuria, thrombocytopenia, and coagulopathy. Therapy consisted of ventilation, anticonvulsive treatment and circulatory support with high-dose catecholamines. According to alpha-lipoic acid serum levels following ingestion the girl must have ingested a minimum of 10 alpha-lipoic acid tablets of 600 mg each. This is the first report on a fatal case of alpha-lipoic acid ingestion, which is intended to inform physicians, pharmacists and patients about critical side effects of this allegedly innocuous drug.

Expression of freezing and fear-potentiated startle during sustained fear in mice.

Fear-potentiated acoustic startle paradigms have been used to investigate phasic and sustained components of conditioned fear in rats and humans. This study describes a novel training protocol to assess phasic and sustained fear in freely behaving C57BL/6J mice, using freezing and/or fear-potentiated startle as measures of fear, thereby, if needed, allowing in vivo application of various techniques, such as optogenetics, electrophysiology and pharmacological intervention, in freely behaving animals. An auditory Pavlovian fear conditioning paradigm, with pseudo-randomized conditioned-unconditioned stimulus presentations at various durations, is combined with repetitive brief auditory white noise burst presentations during fear memory retrieval 24 h after fear conditioning. Major findings are that (1) a motion sensitive platform built on mechano-electrical transducers enables measurement of startle responses in freely behaving mice, (2) absence or presence of startle stimuli during retrieval as well as unpredictability of a given threat determine phasic and sustained fear response profiles and (3) both freezing and startle responses indicate phasic and sustained components of behavioral fear, with sustained freezing reflecting unpredictability of conditioned stimulus (CS)/unconditioned stimulus (US) pairings. This paradigm and available genetically modified mouse lines will pave the way for investigation of the molecular and neural mechanisms relating to the transition from phasic to sustained fear.

Influence of chronic alcohol consumption on hepatic heme and porphyrin metabolism.

To study the effect of prolonged alcohol consumption on hepatic heme and porphyrin metabolism, female Wistar rats were fed for 60 days a nutritionally adequate liquid diet containing 36% of total calories as ethanol, whereas the control diet was isocaloric and contained no alcohol. Compared to pair-fed controls, the administration of the alcohol diet resulted in an increased hepatic activity of delta-aminolevulinic acid synthase by 223% (112.3 +/- 19.6 nmoles/hr/100 g b.wt. vs. 362.8 +/- 42.5; P less than 0.01), an enhanced urinary excretion of delta-aminolevulinic acid by 101% (64.8 +/- 11.8 nmoles/day vs. 130.8 +/- 22.4; P less than 0.05), and an augmented urinary output of total porphyrins by 142% (1.2 +/- 0.2 nmoles/day vs. 2.9 +/- 0.5; P less than 0.05). Concomitantly, the hepatic content of cytochrome P-450 was significantly enhanced and that of hepatic catalase activity marginally increased, whereas the hepatic iron content remained unaltered. In summary, the feeding of rats with a liquid alcohol diet for 60 days results in changes of hepatic heme and porphyrin metabolism which are associated and may be causally related with an induction of hepatic hemoproteins and subsequent derepression of hepatic delta-aminolevulinic acid synthase, whereas hepatic iron appears to play no pathogenic role.

Salsolinol and norsalsolinol in human urine samples.

The tetrahydroisoquinoline alkaloids salsolinol and norsalsolinol were found in human urine samples in concentrations ranging from 0.1 to 29.5 ng/ml. Great interindividual variation was found in urine levels of these alkaloids in a collection of chronic alcoholics and in a group of nonalcoholics. Thus, levels of the individual alkaloids are insufficient markers for distinguishing between alcoholics and nonalcoholics. However, by using the concentration ratio of norsalsolinol and salsolinol, the so-called dopamine-aldehyde adduct ratio (DAAR), significant differences between alcoholics (median 1.3) and nonalcoholics (median 0.6) were detected. This concentration ratio could serve as a marker for the processor state of the dopaminergic system.

Gas chromatographic/mass spectrometric determination of lysergic acid diethylamide (LSD) in serum samples.

A sensitive method for the detection and quantification of lysergic acid diethylamide (LSD) in serum samples is described. After liquid-liquid extraction the trimethylsilyl derivative of LSD is detected by gas chromatography-mass spectrometry. Experiments with spiked samples resulted in a recovery of 76%, the coefficient of variation was 9.3%. Excellent linearity was obtained over the range 0.1-10 ng ml-1. Additionally experiments demonstrating the light sensitivity of LSD are presented together with casuistics.

An analytical method for the rapid screening of organophosphate pesticides in human biological samples and foodstuffs.

Gas chromatography with nitrogen/phosphorus sensitive detection (GC/PND) and electron impact mass spectrometry (GC/MS) with selected ion monitoring provides a simple, rapid and sensitive method for the determination of organophosphate pesticides (OPs). A selective single-step extraction of 23 different OPs in urine, blood, serum and food samples (baby food, soft drinks and instant soups suspected of contamination from a blackmailing scare) is described. The OPs were extracted with 1ml toluene (with and without addition of mevinphos as internal standard), using a 0.7ml aliquot of urine, blood or serum sample. Food samples (0.2g) were homogenised with water (0.5ml) before extraction. An amount of 1microl of the toluene phase (extraction supernatant) was analysed directly by GC/PND and GC/MS.The method was validated using spiked human serum. The OPs were mixed with serum containing 10mg/ml disodium ethane diamine tetraacetic acid disodium salt (EDTA disodium salt) and stored up to 10 days at 4 and -20 degrees C, respectively. The recovery rates of OPs in freshly spiked human plasma ranged between 50% (dimethoate) and 133% (dialifos). OPs in plasma proved to be stable at -20 degrees C. Their levels decreased only slightly after storage at 4 degrees C.

Brain amino acid abnormalities in kidney disease and diabetes mellitus.

The present postmortem study examines whether specific amino acid abnormalities associated with renal diseases or diabetes mellitus in animal experiments and on clinical examination may also be found in human brain samples obtained at clinical autopsies. The material includes 12 deceased with renal insufficiency, 23 deceased with diabetes mellitus and 26 control cases with lethal cardiovascular diseases (without a history of hepatic, renal or metabolic disturbances). The autopsy and clinical records were retrospectively analyzed for age, sex, postmortem delay, cause of death, substantial preexisting diseases and histological findings. The analysis of free amino acid concentrations in human brain specimens was performed applying a Beckman amino acid analyzer. The results were evaluated using the U-test according to Mann, Willcox and Whitney. A P-value less than 0.05 was considered to be significantly different. Differences of amino acid concentrations attributable to sex, age and postmortem delay were not significant. The comparison of postmortem amino acid concentrations in the brains of patients with diabetes mellitus and controls did not reveal relevant changes. However, the patients with renal diseases, as compared to controls, showed a significant cerebral increase of urea, phenylethanolamine and gamma-aminobutyric acid. Thus, the postmortem amino acid analysis may contribute to the understanding of pathophysiological mechanisms of uremic encephalopathy and may supplement the conventional postmortem morphological diagnosis in kidney diseases by indication of functional impairment.

Acute poisoning with phosphamidon: determination of dimethyl phosphate (DMP) as a stable metabolite in a case of organophosphate insecticide intoxication.

Many organophosphate pesticides (OP) such as phosphamidon are unstable in aqueous solutions and especially in blood in the presence of esterases. In a case of intoxication, the phosphamidon concentration in serum decreased from 10 mg/L to 4.4 mg/L after storage at -20 degrees C for six months; nearly complete degradation was observed after three years. Dimethyl phosphate (DMP) is a metabolite of phosphamidon, mevinphos, dicrotophos, monocrotophos, dichlorvos, and trichlorfon. A gas chromatographic-mass spectrometric method with deuterated DMP-d6 as internal standard for the determination of DMP in biological material was validated. DMP was found in all of the patient's samples (3.9 and 4.9 mg/L in blood, 33.5 and 50.4 mg/L in urine, and 8.1 mg/L in gastric fluid), even after storage at -20 degrees C for up to 3 years. No hints for a degradation of DMP when spiked in fresh blood and stored at 4 degrees C for 1 week and stored in water over a time period of 10 months. Looking for the stable metabolites like DMP in cases of suspected OP intoxication is recommended.

Fatal accidental enflurane intoxication.

Among reported cases of abuse of volatile anesthetics there is only one of enflurane intoxication. We report another fatal enflurane intoxication. A 21-year-old man found dead seemed to have experimented with enflurane. Three and one-half days after death high amounts of enflurane were detected in blood, brain, and subcutaneous fat. Gas chromatographic quantification revealed the following high enflurane concentrations: blood: 130 mg/l-1, brain: 350 mg/l-1, and subcutaneous fat: 100 mg/l-1. Histologic signs of drug-induced damage were lacking. No suicide intentions became known. It was concluded that the young man died of an accidental intoxication while abusing enflurane.

[Ethanol-independent methanol elimination in chronic alcoholics]. / Ethanolunabhängige Methanolelimination bei chronischen Alkoholikern.

72% of a collective of chronic alcoholics (DSM-III-R, ICD 9), who were admitted under the influence of alcohol in order to undergo alcohol withdrawal, showed a serum methanol concentration (SMC) above 10 mg/l. This level is usually considered to be the one for the detection of regular alcohol consumption. The SMC values were considerably higher in cases where alcoholic beverages with a higher methanol content were consumed rather than the ones lower in methanol. In the majority of patients a decrease of the methanol concentration could only be detected once an individually varying limit concentration of ethanol (0-0.62 g/kg) was reached. There were, however, a few exceptions where the elimination of methanol independent from the ethanol concentration could be seen. Contrasting the general collective, these 'ethanol independent' methanol eliminators showed a much higher serum level of ethanol and methanol at the time of admission. As a sign of addiction, all patients showed increased beta 60 values for ethanol and preferred high proof beverages, which at the same time have high methanol contents.

[Anabolic steroids on the German black market]. / Anabole Steroide auf dem deutschen Schwarzmarkt.

Anabolic steroids found in the illegal market often do not contain ingredients declared on the label. 42 products found in the black market in Germany were analysed by gas chromatography/mass spectrometry. 15 of these products did not contain the expected ingredients. Authentic and counterfeit products are introduced.

[Occupation-related aspects of homicidal acts]. / Berufsbezogene Tönungen homizidaler Geschehensabläufe.

Several homicides characterized by peculiarities related to the occupation of the perpetrator are reported and compared with observations from literature. Common characteristics of the cases are determined by the following factors: availability of the equipment (i.e. drugs, poisons, tools) and application of special knowledge, skills or techniques provided by professional education (i.e. pharmacology, injection and dissection techniques in physicians and related professions, use of extraordinary, self-constructed weapons by technicians). Remarkable features of some cases are the long period of meticulous preparation and professional attempts to abolish or disguise suspicious findings. Thus, these homicides often present with uncommon modes of commitment and unusual forensic findings which are difficult to recognize and assess. Supplementing own experience by the cases reported in literature is an indispensable prerequisite to examine these cases properly and to provide professional and competent expert opinion for the police authorities.

Determination of hypoglycaemia induced by insulin or its synthetic analogues post mortem.

The determination of human insulin or its synthetic analogues in post-mortem specimens represents a challenge for forensic toxicologists due to its proven instability in post-mortem blood. We present two cases of an insulin-induced hypoglycaemia. In the first case, ante-mortem material was available for the detection of an injection with human insulin. Human insulin was detected by immunopurification with magnetic beads and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses at a concentration of 5180 µU/ml. The molar ratio human insulin:C-peptide was 111. The second case describes a suicide by self-injection of Insulin lispro and determination of the drug after pre-extraction with methanol and immunopurification by LC-MS/MS at the injection site, in vitreous humour and organs. Apart from the well-known matrices--femoral blood and urine--the specimen vitreous humour and the injection site promise the best possibilities for a proof of insulin at autopsy. In addition to insulin analyses, the parameters C-peptide, proinsulin, glucose, lactate, and sulfonylureas should be measured in case of suspected fatal hypoglycaemia.

[Practical experiences with the determination of cadaver age by evaluation of bacterial metabolic products]. / Praktische Erfahrungen mit der Leichenalterbestimmung durch Auswertung bakterieller Stoffwechselprodukte.

In an experiment a corpse had been kept at room temperature (16 degrees-23 degrees C) for 1163 h. At regular intervals brain samples were taken and the content of free amino acids and related compounds was determined by column chromatography. It could be demonstrated that in a period of 4-20 days postmortem the age of the corpse could be calculated from the concentrations of alpha-aminobutyrate (ABU), gamma-aminobutyrate (GABA), and glutamic acid (GLU) in brain employing the previously [5] presented formula: (formula; see text) T is the postmortem time lapse (days). ABU, GABA, and GLU are the concentrations (mumol/g) wet tissue of the corresponding amino acids. During this postmortem interval there is nearly a linear correlation; from this interval a correct assignment between concentrations and time cannot be given. The determination method is in the range of about 15 degrees-25 degrees C (guarantees bacterial growth and metabolism) independent of ambient temperature. The brain samples (cortex or parts of the putrified brain mush) can be taken without any special precaution during normal autopsy. The results of the experiment (in Fig. 1) were supported by some practical cases where the time lapse since death was well (in Fig. 1) or reasonably (in Fig. 1) known. These results suggest that this method allows in many cases the determination of the age of a corpse found in a warm environment (approximately 15 degrees-25 degrees C) approximately 4-20 days after death.

[The kinetics of the postmortal bacterial metabolism of the glutamic acid in brain (author's transl)]. / Zur Kinetik des postmortalen bakteriellen Glutaminsäurestoffwechsels im Gehirn.

This paper gives a brief report on investigations on bacterial enzyme activities in putrefied brain tissue. The effects of temperature changes (5--30 degrees C) and pH-changes (3--8) on the rate of the glutamic acid reactions, especially proteolysis and alpha- resp. gamma-decarboxylation, were examined to reveal to which extent a temperature and pH-independence is valid for the previously developed time of death formula: (formula: see text). The present investigation demonstrates the independence of the mentioned formula for the important pH-range of 6--7.2 and for a temperature range of 17.5--25 degrees C (room temperature). A second important result is that in these ranges a high proteolytic activity is observed so that the continuation of the reaction sequences will be maintained. These results reveal that the postmortal metabolism of the glutamic acid may be suitable to determine the time of death.

[Distribution of digoxin, digitoxin and their cardioactive metabolites in human heart and kidney tissue. A postmortem study]. / Verteilung von Digoxin, Digitoxin und ihren kardioaktiven Metaboliten im menschlichen Herz- und Nierengewebe. Eine post mortem-Untersuchung.

A method was developed for the specific determination of digoxin and digitoxin, as well as their semisynthetic derivatives and dependent cardioactive metabolites, in autopsy samples of heart and kidney. A collective of six patients on long-term treatment with therapeutic doses of beta-acetyldigoxin had a mean myocardial digoxin content of 46.1 +/- 25.0 ng/g (SD); kidney: 50.3 +/- 30.3 ng/g. Digoxigenin bisdigitoxoside represented the second most important metabolite in heart and kidney; digoxigenin monodigitoxoside and digoxigenin follow, respectively. In a collective of seven patients on maintenance treatment with digitoxin, the mean tissue levels were higher but the metabolic pattern was similar (myocardial digitoxin content: 78.9 +/- 38.4 ng/g, renal content: 104.1 +/- 44.1 ng/g). The amount of digoxin formed by hydroxylation under long-term treatment with digitoxin in heart and kidney were approximately 10 ng/g. A case of digoxin intoxication differed both in the tissue content and in the metabolic distribution.

Evaluation of a method for simultaneous quantification of codeine, dihydrocodeine, morphine, and 6-monoacetylmorphine in serum, blood, and postmortem blood.

A solid-phase extraction and gas chromatographic-mass spectrometric method for the simultaneous determination of codeine, dihydrocodeine, morphine, and 6-monoacetylmorphine in serum, blood or postmortem blood is described. The extraction technique allows the determination of free or total morphine (morphine plus morphine glucuronide). Experiments with spiked blood samples resulted in recoveries of 96.4% +/- 4.2% for codeine, 95.8% +/- 5.1% for dihydrocodeine, 90.3% +/- 7.8% for 6-monoacetylmorphine and 92.5% +/- 8.1% for morphine. Excellent linearity was obtained over the range 1-1500 ng/mL. The detection limit for all analytes is less than 1 ng/mL.

Detection of digoxin, digitoxin, their cardioactive metabolites and derivatives by high-performance liquid chromatography and high-performance liquid chromatography-radioimmunoassay.

High-performance liquid chromatographic (HPLC) systems are described for the separation of the cardioactive metabolites of the digoxin and digitoxin series that are formed by the splitting of digitoxose sugar residues from the aglycone steroid: isocratic separation of digoxin and its cardioactive metabolites; isocratic separation of digitoxin and its cardioactive metabolites; and gradient elution separation of the digoxin and digitoxin series including beta-acetyl- and beta-methyldigoxin. Separations were performed on a 10-microns bonded octadecyl phase column using various mixtures of acetonitrile--water and acetonitrile--methanol--water as the mobile phase. These methods provide high peak resolution and are well suited for collecting elution fractions, e.g. to link up with sensitive immunological measurements. An HPLC-radioimmunoassay method is described for the quantitation of digoxin, digitoxin and their metabolites in human tissues.