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.

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.

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.

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.

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.

Treatment of Tourette's syndrome with Delta 9-tetrahydrocannabinol (THC): a randomized crossover trial.

Anecdotal reports in Tourette's syndrome (TS) have suggested that marijuana (cannabis sativa) and delta-9-tetrahydrocannabinol (Delta(9)-THC), the major psychoactive ingredient of marijuana, reduce tics and associated behavioral disorders. We performed a randomized double-blind placebo-controlled crossover single-dose trial of Delta(9)-THC (5.0, 7.5 or 10.0 mg) in 12 adult TS patients. Tic severity was assessed using a self-rating scale (Tourette's syndrome Symptom List, TSSL) and examiner ratings (Shapiro Tourette's syndrome Severity Scale, Yale Global Tic Severity Scale, Tourette's syndrome Global Scale). Using the TSSL, patients also rated the severity of associated behavioral disorders. Clinical changes were correlated to maximum plasma levels of THC and its metabolites 11-hydroxy-Delta(9)-tetrahydrocannabinol (11-OH-THC) and 11-nor-Delta(9)-tetrahydrocannabinol-9-carboxylic acid (THC-COOH). Using the TSSL, there was a significant improvement of tics (p=0.015) and obsessive-compulsive behavior (OCB) (p = 0.041) after treatment with Delta(9)-THC compared to placebo. Examiner ratings demonstrated a significant difference for the subscore "complex motor tics" (p = 0.015) and a trend towards a significant improvement for the subscores "motor tics" (p = 0.065), "simple motor tics" (p = 0.093), and "vocal tics" (p = 0.093). No serious adverse reactions occurred. Five patients experienced mild, transient side effects. There was a significant correlation between tic improvement and maximum 11-OH-THC plasma concentration. Results obtained from this pilot study suggest that a single-dose treatment with Delta(9)-THC is effective and safe in treating tics and OCB in TS. It can be speculated that clinical effects may be caused by 11-OH-THC. A more long-term study is required to confirm these results.

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.

Severe and prolonged sedation in five neonates due to persistence of active diazepam metabolites.

UNLABELLED: Five neonates who suffered from an unexpected long period of respiratory failure, muscular hypotonia, and drowsiness were observed in a retrospective study. Prior to this general depression, unusually high doses of diazepam were administered to all patients via intravenous bolus injection. Serum levels of diazepam and its active metabolites were substantially elevated in the course of the disease. The persistence of the very long-acting N-desmethyldiazepam with considerable extension in neonates and even more exaggerated in premature infants is emphasized due to the reduced capacity of the hepatic biotransformation system. CONCLUSION: The intravenous application of diazepam imposes a risk of marked and prolonged general depression in neonates. Pronounced adverse effects are to be expected for prematures even after a single diazepam intravenous bolus if the dosage is not appropriate. Diazepam should not be used for short sedation and is not the drug of choice for anticonvulsant therapy in neonates.

[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 biological markers for alcohol abuse.

Alcoholism is one of the most frequent addictions and an important subject in forensic medicine and clinical toxicology. Several laboratory abnormalities are associated with excessive alcohol consumption. They are useful in the diagnosis of alcoholism especially during the follow-up of various treatment programs. The biological markers mostly used for diagnosis of alcoholism are presented. Especially, methods for the determination of the following diagnostic tools are reviewed: congener alcohols, gamma-glutamyltransferase, aspartate and alanine aminotransferase, beta-hexosaminidase, erythrocyte aldehyde dehydrogenase, alpha-amino-n-butyric acid to leucine ratio, macrocytosis, carbohydrate-deficient transferrin, (apo)lipoproteins, fatty acid ethyl esters, blood acetate, acetaldehyde adducts, 5-hydroxytryptophol, dolichol and condensation products. No laboratory test exists that is reliable enough for the exact diagnosis of alcoholism. The combination of physician interview, questionnaire and laboratory markers is necessary for the diagnosis of alcoholism.

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.

[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.

Formaldehyde-derived tetrahydroisoquinolines and tetrahydro-beta-carbolines in human urine.

Human urine samples were examined for the occurrence of formaldehyde-derived tetrahydroisoquinolines and tetrahydro-beta-carbolines generated by condensation of the methanol oxidation product with biogenic amines. Positive results were obtained for the tryptamine condensation product 1,2,3,4-tetrahydro-beta-carboline and the serotonine condensation product 6-hydroxy-1,2,3,4-tetrahydro-beta-carboline as well as for the condensation products with tyramine, dopamine, adrenaline and noradrenaline 1,2,3,4-tetrahydroisoquinoline, 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, N-methyl-4,6,7-trihydroxy-1,2,3,4-tetrahydroisoquinoline, 4,6,7-trihydroxy-1,2,3,4-tetrahydroisoquinoline, and the metabolite 6-methoxy-7-hydroxy-1,2,3,4-tetrahydroisoquinoline. Negative results were obtained for N-methyl-1,2,3,4-tetrahydroisoquinoline and 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline, N-methyl-1,2,3,4-tetrahydro-beta-carboline, 6-methyl-1,2,3,4-tetrahydro-beta-carboline, and 6-methoxy-1,2,3,4-tetrahydro-beta-carboline in samples of chronic alcoholics as well as in the urine of healthy volunteers. No correlation between alcohol ingestion or state of alcoholization could be demonstrated.

[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.

Gas chromatographic-mass spectrometric screening procedure for the identification of formaldehyde-derived tetrahydroisoquinolines in human urine.

A gas chromatographic-mass spectrometric method has been developed for the identification of 1,2,3,4-tetrahydroisoquinoline and six metabolites extracted from urine in the picogram range. The derivatization procedure for the substances, formed by reaction of formaldehyde with biogenic amines, employs propionic anhydride and can take place in aqueous medium. In this way artificial formation of these compounds via condensation of biogenic amines with aldehydes or alpha-keto acids during the work-up procedure is eliminated. The procedure results in hydrophobic compounds, which are quantitatively extractable by liquid-liquid extraction with organic solvents. Further clean-up was performed by solid-phase extraction on C18 sample preparation columns.

Gas chromatographic-mass spectrometric screening procedure for the identification of formaldehyde-derived tetrahydro-beta-carbolines in human urine.

A gas chromatographic-mass spectrometric method for the identification of 1,2,3,4-tetrahydro-beta-carboline and four metabolites extracted from urine is described. In a first step the substances, formed by reaction of formaldehyde with biogenic amines, were derivatized in aqueous solution with methyl chloroformate to eliminate an artificial formation of these compounds via condensation of endogenous indole ethylamines with aldehydes or alpha-keto acids during the work-up procedure. This initial derivatization formed stable hydrophobic compounds and improved the extractability for a liquid-liquid extraction. Further clean-up was performed by solid-phase extraction on C18 sample preparation columns. The method can identify these compounds in the picogram range.

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.

Suicidal yew leave ingestion--phloroglucindimethylether (3,5-dimethoxyphenol) as a marker for poisoning from Taxus baccata.

In a case of suicide in a depressive 19-year-old man with considerable ingestion of new leaves, resorption of yew ingredients could be demonstrated. The main substance could be identified as 3,5-dimethoxyphenol, the aglycone of taxicatine, which is a typical ingredient of yew leaves. 3,5-dimethoxyphenol was demonstrated in harvested yew leaves, stomach content and cardiac blood of the victim. Structure confirmation was achieved by means of HPLC, UV, GC-MS, IR and 1H-NMR spectroscopy. None of the Taxus alkoids could be identified. The components detected by TLC have not yet been identified. The results demonstrate that 3,5-dimethoxyphenol can be used as a marker in cases of intoxication by yew ingredients.