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Contrary to the large number of publications dealing with treatment of organophosphate poisoning in a variety of animal species, there is no logic reason in the preference of one species, for this purpose. Guinea pigs were reported to respond better to treatment by oximes, than mice and rats. However, in the analysis of data on the effect of obidoxim and atropine or benactyzine on sarin poisoning it is demonstrated, that guinea pigs do not respond differently from mice and rats. Subcutaneous LD50's of sarin in mice ranged from 0.06 to 0.207 mg/kg, and those of guinea pigs from 0.04 to 0.112 mg/kg. The difference in the LD50's may be related to the different susceptibility of various animal species. The importance of "in vivo" dosage, mode of application, kinetics of antagonists, in correlation to the ability to reactivate "in vitro" is discussed.

The pharmacokinetics and pharmacodynamics of the oxime obidoxime (Toxogonin, 50 mg/kg iv) were investigated in anesthetized normal rats and in sarin-poisoned (50 μg/kg iv) rats. The kinetics were described by a two-compartment open model. The elimination half-life ranged from 35 min in normal rats to 86 min in sarin-poisoned rats. Obidoxime excretion occurred predominantly by the renal route, amounting to 4.6% of the administered dose in normal rats and to 0.9% in sarin-poisoned rats within the first hour of administration. The significantly diminished glomerular filtration rate confirmed the retardation of obidoxime exoretion in sarin poisoning. The mean arterial blood pressure (MAP) response to obidoxime, measured in normal rats, was a transient hypotension, but to sarin an immediate hypertension. In sarin-poisoned rats the therapeutic sequence of administration of obidoxime and atropine (5 mg/kg iv) seemed to be important: the administration of atropine 10 min after and of obidoxime 20 min after sarin poisoning exerted a stabilizing effect on MAP. No serum albumin binding was found for obidoxime. Competition experiments at the isolated nicotinic receptor demonstrated the anticholinergic activity of obidoxime. The affinity of obidoxime was 1000 times smaller than that of acetylcholine. It is concluded that obidoxime, due to its prolonged residence time in the organism in sarin poisoning, exerts a “curare-like” inhibition and protection of the nicotinic acetylcholine receptor and, combined with atropine, a synergistic effect on blood pressure normalization.

Four synthetic THC derivatives (Δ911–8-hydroxy-THC (A), Δ7–8−9-hydroxyTHC (B), Δ8,9−3 (1′ dimethyl−3′ aza−4’keto)-THC (C), and Δ8,9-1′dimethylTHC (D) were investigated for their pharmacological activity with reference to barbiturate potentiation, temperature reduction, and analgesic properties and were compared with Δ9-THC.

1. The activities of some cholinergic compounds were investigated on the terminal ileum of the guinea-pig and their muscarinic potencies were found to be in the following order: acetylcholine (ACh) > diazoacetylcholine (DACh) > iodoacetylcholine (IACh) > azidoacetylcholine (AACh). 2. Protection experiments with atropine showed a decreased affinity of the four cholinergic compounds for the muscarinic receptor, demonstrating a direct interaction of the drugs and the receptor. 3. The maximal contractions (intrinsic activity) caused by DACh were greater than those caused by ACh, IACh and AACh. 4. Incubation of the ileum with ACh was followed by a reversible loss of its ability to contract (desensitization). The time course of recovery was similiar to that after incubation with AACh. 5. IACh caused a partial (50%), irreversible paralysis of the muscle. 6. The furaziridines seem to react partially irreversibly (30%), whereas, paranitrophenyldiazonium fluoborate (p-NDB) caused a complete, irreversible blockade of the muscarinic receptor.

The influences of various fixatives on the vesicle size of the electric organ of Torpedo marmorate were investigated. Thin section and freeze etched preparations were examined under the electron microscope. In thin section increased vesicle diameters were observed compared with the freeze etched preparations. The same experiments in different torpedo fish led to significantly different vesicle sizes observed. Variations of the molarity, the pH and osmolarities result in particularly high differences in vesicle diameters. Using Karnovsky's method (1965) and a fixative consisting of 2.5% glutaraldehyde in 0.2 M cacodylate buffer, pH 7.2, results in vesicle sizes comparable to those reported by other authors. Results obtained from freeze etched preparations are not comparable in general with results from thin section experiments with the same fixative.

1. d,l -Amphetamine sulfate, d -lysergic acid diethylamide tartrate (LSD) and delta-9-tetrahydrocannabinol (THC) were administered to ants in several doses either orally in food, or by injection into the abdominal cavity. 2. Amphetamine, injected in doses of 1 or 10 μg/ant caused an increase in intraspecific aggression. Sugar-water containing 1 or more mg/ml amphetamine was generally refused. 3. Application of 20 ng LSD or more by either route typically impaired locomotion. Dependent on the dose administered, these symptoms vanished, or led to death. 4. To dissolve THC, we mixed 10 vol% of Tween-80 with physiological NaCl-solution, or with sugar-water. Drug doses of 2 μg injected, or 3–4 μg orally did not cause conspicuous changes in behavior.

The uptake of 14C-choline into the axonal part of the motor endplate and muscle of mouse diaphragms was investigated by autoradiography. With i.v. doses of 0.1 microgram/g choline chloride, the uptake into the nerve endings is fast (less than 2 min) and into muscle slower (greater than 2 min). With higher doses (1.0 microgram/g) the uptake into muscle tissue is accelerated. The radioactivity in the endplates decreases with a halflife time of 20 min and remains constant in the muscle fibres over 60 min. Denervation by cutting the phrenic nerve reduces the uptake into endplates by 40% within 14 h, but probably induces uptake into regenerating Schwann cells during 30 days. Som compounds with choline-like structure (hemicholinium-3, decamethylen-dicholine, triethyl-choline) reduce the high-affinity uptake of choline into the nerve endings with sublethal doses, whereas tetraethylammonium and N-hydroxyethyl-4-(1-naphthylvinyl)-pyridinium, an inhibitor of cholinacetyltransferase, are less active. Half lethal doses of cocaine, imipramine and reserpine have no significant action on uptake of choline into the endplates. Chlorpromazine slightly diminishes the uptake into endplates. Chlorpromazine and imipramine reduce uptake into the muscle fibres.

1. 1. Absolute body weight of suckled mice, reared by dams fed with 3% hashish during the whole lactation period, was significantly reduced by 10–14% compared with controls on and after day 11. 2. 2. Sucklings' weight gains were significantly lowered from day 4–7, 7–11 and 11–15, whereas those from day 1–4 and 15–21 came up to the corresponding control values. 3. 3. Other parameters of development, such as projection of the ears (day 4), tail length (day 11), or eye opening (day 15) were slightly influenced only. 4. 4. These findings suggested a malnutrition of the pups rather than a developmental retardation. The drug effects might be caused directly by its passage into the milk, or indirectly by decreased lactation.

Choline acetyltransferase (EC 2.3.1.6) catalyzes the synthesis of the neurotransmitter acetylcholine from acetylcoenzyme A and choline. It has been purified from the electric organ of Torpedo marmorata by a new double-affinity chromatography. Our rapid and specific purification procedure includes affinity chromatography on CoA-Sepharose and then a second affinity chromatography on the enzyme's inhibitor [2-[3-(2-ammonioethoxy)-benzoyl]ethyl]trimethylammonium bromide coupled to Sepharose via a six-carbon spacer arm. The final enzyme preparation has been purified 7300-fold to a specific activity of 73 mumol acetylcholine formed min-1 mg protein-1. The isolated enzyme gave a single band on disc polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The relative molecular mass was determined to be 68,300 +/- 2100.

We investigated the effect of (+)-amphetamine on body temperature in the unrestrained and curarized rat. In the unrestrained animal kept at an ambient temperature of 30°C amphetamine had a dose-dependent hyperthermic action, whereas in animals kept at 7°C it decreased body temperature. An increase in body temperature was evident also in the curarized rat; after spinal cord transection at the level of C1; and in the pithed rat. Pretreatment with the alpha-adrenergic blocking agent azapetine attenuated amphetamine-hyperthermia in the curarized rat, but enhanced the hyperthermic response in the unrestrained rat. Pretreatment with the beta-adrenergic blocking agent propranolol abolished amphetamine-hyperthermia in the curarized rat, but did not affect the response in the unrestrained animal. In the hypophysectomized animal amphetamine had a hypothermic effect even at high ambient temperatures. It is concluded that the action of amphetamine on peripheral beta-receptors has a hyperthermic effect, but that additional mechanisms are involved in the thermal response of the freely moving animal.

The effects of three doses of caffeine and of chlordiazepoxide (CDX) on motor activity were tested in the chronic thalamic rat. In this preparation virtually all cortical, striatal and limbic structures were ablated. A small dose of caffeine had only a weak motor stimulant effect which was succeeded by sedation. Larger doses that are stimulatory in intact animals, depressed motor activity in the thalamic rat. Amphetamine, in contrast to caffeine, produced a substantial motor stimulation. CDX caused a dose-dependent reduction of motor activity, similar to its effect in the intact rat. It is concluded that (a) telencephalic structures are involved in mediating the stimulatory action of caffeine; (b) a sedative component of caffeine may be present, but masked, in the intact animal, and may be due to serotoninergic mechanisms; (c) the presence of limbic structures is not necessary for the sedative effect of CDX.

Institute of Pharmacology, Untversity of Ztirich, Gloriastrasse 32, CH-8006 Zurtch, Switzerland Introduction The aims of pharmacology include understanding the mechanisms underlying successful therapeutic interventions on the molecular level as well as laying down a rationale for use as a basis for new approaches. The recent and, one might safely say, major advances in our know- ledge of the central role of Ca 2÷ in the regu- lation of muscle activity have revealed a variety of interconnected pathways. These in turn help to explain the flexibility shown by different muscle tissues in their re- sponses to the natural neural and hormonal control as well as to pharmacological probes. In this short review we focus on selected topics arising from more recent progress in both the biochemical and phar- macological fields. We find, however, that the basic emphasis on these topics becomes two-fold as a result of the natural division between (1) skeletal muscles, where Ca2÷-control of contraction functions com- pletely within the cell and (2) smooth and cardiac muscles, where the extracellular Ca2*-supply is importanP. The first class presents us with the process of neuromuscu- lar transmission as the main area open to pharmacological intervention, whereas the latter offers many possibilities as a result of the polyfunctional control system existing within the cell. Levels of drug action It is now generally recognized that cytosolic-free Ca 2÷ is the messenger re- sponsible for initiating a wide range of cellu- lar activities, which cease to function as soon as its concentration falls below a cer- tain critical limit (around 10 -7 M). Besides contraction these activities include secre- tion, mitosis, various metabolic enzyme systems, membrane transports, microtubuli assembly-disassembly, phosphorylation reactions and many others. The extracellu- lar concentration of Ca 2+ is in the millimolar range, so that for a cell at rest an outside-to- inside ratio exceeding 104 must be estab- lished. The extremely low level of cytosolic free Ca ~ is maintained by Ca2+-pumps located in the cell outer membranes of heart and smooth muscles, in the endoplasmic reticulum system of smooth muscle cells 2'3 and in the sarcoplasmic reticulum (SR) sys- tem of sarcomeric muscles. Furthermore, Ca ~+ is extruded from the cell by a Na ~- Ca2+-exchange carrier under the control of the ion gradient established by the Na +- K+-pump in the outer membrane. All these pumps require energy to work against the ionic gradients. For contractile activity to begin, Ca ~ must flow from outside through the membrane via the voltage- dependent Ca2+-channels, reversal of the Na+-CaZ+-exchange carder and transfer by passive diffusion, or the release from the intracellular reticulum systems. Overflood- ing of the cytosolic space, however, leads to cell death. If the free Ca 2 t-concentration rises above 10 -4 M, the mitochondria begin to absorb it. In this function they play the role of a Ca2+-sink and are not usually con- sidered to be actively involved in regulation under normal conditions. Although the molecular mechanism by which actin and myosin bring about con- traction is thought to be the same in differ- ent types of muscles, and even in non- muscle cells, the way it is regulated by the Ca2+-messenger varies from tissue to tis- suek However, at the first step of the regu- lation process, i.e. the intracellular Ca 2+- target sites, we do find marked similarities. Evolution has produced a family of phylogenetically related proteins capable of binding Ca 2+ reversibly with high affinity (affinity constant around 106 M 1). They include calmodulin, troponin-C, myosin P-light-chain and parvalbumin, have molecular weights ranging from 12 000 to 19 000 and are found exclusively within the celP. Troponin-C is directly involved in triggering contraction in sarcomeric muscles whereas binding of Ca 2÷ to cal- modulin m smooth muscles stimulates cer- tain protein phosphorylation reactions and thus leads indirectly to contraction as dis- cussed earlier a. In addition, calmodulin is involved in the intracellular processes enu- merated above. It was found to be increased in all transformed tumorous cells. A clear picture of how parvalbumin functions has not yet emerged but it is found in appre- ciable amounts in fast contracting muscle fibres as well as nerve cells which use the" transmitter y-aminobutyric acid ~. It may act as a scavenger for available free Ca 2÷ and so enable cells, which are switched on by a pulse of Ca 2+ to a state of high activity, to be rapidly returned to rest. Thus parvalbu- min could function as a soluble cytosolic relaxing factor by depleting the triggering sites of their bound Ca 2+ faster than the ions are eventually removed from the cytosol by the membrane pumps. In short, one can say there are two main levels at which drugs may act. First, we have the level of the membranes, whether at the cell surface or ~ntemally on the intra- cellular reticuhim systems. Because mem- branes contain various specific constituents such as ionic pumps, channels and hormone receptors, they offer a diversity of targets for drug interference. Nevertheless, the effects must be ultimately on the concentra- tions of cytosolic ions, in particular the Ca2+-messenger. Second, drug action can be directed at the level of Ca2+-binding proteins themselves. Furthermore, in some tissues these two levels are interconnected, e.g. hormone receptor proteins in the mem- brane on the one hand and calmedulin the other play a decisive role in cyclic nucleotide metabolism. In this case, drug action on one level may have its effects extended to the other. Some specific drug effects on contraction in different muscle types will now be discussed. Skeletal muscles The voluntary striated skeletal muscles are under the control of neural activity. Their fibres are surrounded by excitable membranes which can respond specifically to that activity and at the same time shield the interior from extracellular electrolytic changes. All Ca 2~ needed for triggering internal activity is contained in the well developed SR system. Isolated skeletal muscles can perform hundreds of succes- sive contractions without any extracelhi- larly originated Ca 2+. The regulation of con- traction

The synthesis and characterization of some reversible acetylcholinesterase inhibitors are described in detail. They are structurally related to the natural substrate acetylcholine. All of them bear a trimethylammonium moiety as ‘cationic head’. Instead of an electrophilic ester group, the title compounds include a variety of functionalities, such as halide, ether, thioether, epoxide, amide, ketone and a double bond. The substances are thus suited as probes for the investigation of the esteratic subsite of the acetylcholinesterase active center. Most of the synthesized compounds inhibit the enzyme in a competitive manner with inhibition constants in the range of 10−5M to 10−3M. With respect to acetylcholine (KD ≤ 10−5M and Km = (1.6±0.5) 10−4M) their affinity to acetylcholinesterase is in the same order of magnitude.

Aggressive behavior of dominant and isolated mice was assessed in intermale encounters with nonaggressive intruder mice. After an attack period of 90 s, the aggressor was exposed to a footshock punishment and retested the next day. The shock treatment, independent of the intensity and duration, failed to inhibit spontaneous aggression in isolated DBA/2 mice, while it significantly suppressed spontaneous aggression in dominant C57BL/6 mice. The different effects of post-trial shock punishment were not due to a different shock sensitivity and did not depend on the type of opponent used. Strain differences have been ruled out by the use of dominant and isolated ICR mice. Again, shock punishment was ineffective in isolates, while it reduced aggression in dominant animals. The findings were discussed with reference to the impaired learning performance reported for isolated animals, and suggest a difference between the aggression of isolated and that of dominant mice.

The effects of hashish extract on adaptive behavior of male mice were studied in a paradigm which allows the investigation of learning mechanisms in a social context. Mice of the C3H strain, which were not submissive in a confrontation with a nonaggressive DBA mouse on day 1, were defeated on day 2 over 3 min by aggressive, isolated DBA mice, and showed conditioned submissive behavior upon mere contact with a nonaggressive DBA mouse on day 3. A hashish extract containing 38.6-39.4% delta 9-tetrahydrocannabinol (delta 9-THC), 11.6-12.0% cannabinol and 47.7-48.5% cannabidiol was administered orally in all experiments. Hashish extract given 90 min before defeat on day 2, in dosages corresponding to 1, 5, and 10 mg delta 9-THC/kg, impaired retention of defensive upright, defensive sideways and immobility on day 3 (experiment 1). Experiment 2 showed that the drug (5, and 10 mg delta 9-THC/kg) had no antinociceptive potency in mice and did not modify defeat-induced analgesia. Experiment 3, with drug (5 mg delta 9-THC/kg) or solvent administration on day 2 and day 3, showed that the retention deficit was neither due to state-dependent learning, nor to impaired retrieval. It is suggested that hashish extract administered before learning may interfere with memory processing.

Contrary to the overwhelming expectations in special papers and textbooks, the reactivation of the active centre of acetylcholinesterase, inhibited by organophosphorous compounds, with the help of so called “tailor-made-drugs”, e.g. the oximes, never could be realized in vivo. However, the concept of the tailor-made drugs gave us a thorough insight in the function of synapses. As the chemical background of the basic reactions of catalysis of the hydrolysis of acetylcholine, carbamates and organophosphates at the active centre is now investigated, the reason for the failure of the “tailor-made-drugs-concept” can be discussed. The rate limiting step in all naturally “reactivation-reactions” is the hydrolysis of the acetylated, carbamoylated and phosphorylated enzyme. Thus the acceleration of the hydrolysis of the phosphorylated enzyme proved to be the most promising way for reactivation in vitro. However, in vivo the oximes alone never proved to be successful. The only successful way in a very limi...

The influence of the excitotoxin kainic acid (KA) on cultivated explants of rat hippocampus was investigated. Addition of 3 microM KA to the culture medium over 24-48 h induced a destruction of the pyramidal cells in the CA3 region, whereas the CA1 pyramidal cells and the granule cells were left undamaged. Higher concentrations (10-100 microM) of KA destroyed also the latter cell groups. The selectivity of the KA lesion at 3 microM was further indicated by the fact that the acetylcholinesterase-positive neurons in the hippocampus were not destroyed through KA administration and that the stereoisomer dihydrokainic acid was ineffective in inducing lesions. Application of tetrodotoxin did not protect the CA3 pyramidal cells from KA lesion, whereas gamma-glutamylaminomethylsulphonic acid (GAMS) only offered a very small, statistically not significant, protection. Baclofen protected the cultures slightly from KA lesions but not when added together with GAMS. Possible mechanisms responsible for the KA lesions in these cultures are discussed.

The effects of 60-60 min light-dark (LD) cycles on sleep, feeding, drinking and motor activity were investigated in the rat. Waking, consummatory behaviour and motor activity were inhibited by light, while sleep was enhanced. The effects were dependent on the intensity of light. The percentage of slow wave sleep (SS) increased within the first 15 min of the light period and reached a plateau within 30 min. The reduction of SS during darkness showed a similar time-course. The percentage of paradoxical sleep (PS) started to increase only 15 min after the onset of light and continued to rise during the first 15 min of darkness. All parameters exhibited a free-running circadian rhythm. The LD-induced changes of SS were largest during the circadian phase in which the animal was active. The changes of PS which succeeded the onset or offset of light, were largest in the inactive circadian phase. The most immediate influence of changes in illumination is exerted on the slow wave component of sleep. Hypotheses to account for the temporary dissociation between the two sleep states are discussed.

Three animal models, based on genetic differences in endogenous opioid peptides and opioid receptors, are described. Obese mice and rats, whose pituitary opioid content is elevated, may be used to investigate eating disorders. Recombinant inbred strains of mice, which differ in brain opioid receptors and analgesic responsiveness, can be used for study of opioid- and nonopioid-mediated mechanisms of pain inhibition. Individual reactivity to opioids can be examined in C57BL/6 and DBA/2 inbred strains of mice. A model that combines a variety of opioid effects is offered and suggests the existence of a genetically determined dissociation of opioid effects on locomotor activity and pain inhibition. In addition, stimulatory locomotor responses in the C57BL/6 reaction type are linked to a high risk of drug addiction and facilitatory effects on adaptive processes, while high analgesic potency in the DBA/2 reaction type is accompanied by a low proneness to drug abuse and amnesic properties of opioids.

Increases in the viscosity of solutions of different proteins varying over a wide range of molecular shape and size have been studied in a cone and plate viscometer. The protein concentrations used were lower than those needed to produce viscosities of the solutions significantly greater than the solvent medium. The high values of shear stress finally reached are attributed to a shear-induced formation of an intermolecular structure resulting from protein-protein interactions at the air water interface. The rate of formation of this structure was found to increase with ionic strength, indicating that the rate of adsorption at the interface may influence the development of high shear stress values. However, this effect also shows that hydrophobic intermolecular interactions and the conformation of the protein molecules in solution are important factors in the development of the structure. The presence of small amounts of detergents was found to be able to prevent increases in shear stress. The fact that the double-chained lipid lecithin does not produce this effect, even in high concentrations, may be due to its ability to take part in protein-lipid-protein interactions.

To investigate the possible role of the amygdala in the phenomenon of eating induced by single waves of cortical spreading depression, 10 rats received bilateral and 5 rats unilateral electrolytic lesions in the amygdaloid complex. They were tested for spreading depression-induced eating by injection of 0.5–1.0 μl of 25% KCl into the neocortex. Bilateral lesions of the amygdala significantly attenuated, but did not completely abolish elicited eating. Unilateral lesions significantly decreased the incidence of elicited eating from the ipsilateral but not the contralateral hemisphere.

A single wave of unilateral spreading depression in the cortex, hippocampus or caudate nucleus of rats elicits eating after 2–6 min. The present experiments provide evidence that such spreading depression-induced eating can be classically conditioned to a complex conditioned stimulus. A wave of spreading depression was triggered by injection of 0.5–2.0 μl of 25 percent KC1 solution. In a first experiment successful conditioning was demonstrated in 20 rats, involving 7 cortical, 5 caudate and 8 hippocampal spreading depression sites. Four animals failed to show any conditioned eating. A control group of 8 animals, in which cortical spreading depression did not induce eating, showed no increase in eating in the presence of the CS after conditioning trials. A second experiment, which included pseudoconditioning and NaCl control groups, confirmed the results obtained in Experiment 1. Classical conditioning was successful in 11 animals, involving 6 cortical and 5 caudate spreading depression sites. Neither the pseudoconditioning (14 animals, involving 8 cortical and 6 caudate spreading depression sites) nor the NaCl control group (8 animals, all with cortical sites), showed eating in the presence of the CS. In both experiments the conditioned eating underwent gradual extinction.

1. 1. Ants were fed with sugar-water containing LSD-tartrate (100 μ/ml), or sugar-water with 10 vol% of Tween-80 containing Δ9-THC (1 mg/ml), using the two solvents as controls. 2. 2. Analyses of social contacts between one drug- or control-fed ant and 10 hungry nestmates showed decreased frequencies and durations of interactions (particularly food-sharing behavior) 9–24 hr following LSD-intake, whereas THC had little influence. 3. 3. Only slight differences from normal behavior were shown by LSD- and THC-fed worker ants in adopting queens from their own nest. 4. 4. With large groups of ants, multiple feedings with LSD always impaired the behavior of foraging workers, whereas domestic workers performed normally. When drugged food was offered, the activity between nest and feeding place was relatively lower in the morning. THC-feedings did not cause any distinct behavioral changes, but the activity between nest and feeding-place increased compared to that preceding and succeeding control periods.

The experience of winning or loosing fights plays an important role in subsequent aggressive or submissive behaviors. In this study agonistic behavior of male mice was chosen to investigate learning mechanisms in the context of a biologically meaningful situation. An ICR mouse introduced into a group of five C57BL/6 mice was attacked by mice of high social status (Fighter, F), but not by lower ranking animals (Non-Fighter, NF). On this basis the following model was developed to study learning of submissive behavior. Day 1 (baseline trial): An ICR mouse was introduced to a single NF-C57 mouse. Few submissive behaviors (crouch) were observed in naive ICR mice upon contact with NF-C57 mice. Day 2 (learning trial): The same ICR mouse was defeated by an F-C57 mouse until it showed defensive upright posture upon approach. This criterion was reached after a mean latency of 3.5 min and after being exposed to a mean number of 14 bites. Day 3 (retest trial): The same pairs as on day 1 confronted each other. Without being attacked, the ICR mouse showed a significant increase of submissive behavior (crouch, defensive sideways and upright) upon mere contact with the NF-C57 mouse when compared to day 1 and to control mice on day 3. Controls, confronted on all three days with NF-C57 mice, showed no increase in submissive behaviors. The results are discussed in terms of acquisition, memory, retrieval and extinction of learned submissive behavior. It is suggested that the mechanisms underlying learning of submissive behavior include generalization of conditioning and specific extinction processes. The further use of the learning scheme to assess drug effects is illustrated.

Within groups of three adult male mice the acute and subchronic effects of hashish extract (20 mg delta 9-THC/kg) on social dominance, food dominance, and sexual interactions with a female were investigated. An initial drug treatment of only the dominant male weakened his social position, but dominance was regained after treatment 2 or 3. In contrast, a persistent change in dominance was found when only the male which was dominant in the feeding test was treated with the extract. Simultaneous drug treatment of all three males did not affect the social dominance relationship but resulted in a reversible change in food dominance. The original feeding order was reestablished after drug treatment 3. Upon meeting an estrous female, no male of the group was distinctly dominant in mating. After treatment 1 was given to all members of the group, all types of behavior were impaired and total activity was significantly reduced. After treatment 2, animals showed tolerance to the sedative effects, and after treatment 3, sexual behavior was even more frequent in drugged animals than in controls. The results are discussed in relation to a possible dependence of behavioral drug effects and tolerance development on the experimental situation.

The relation of the structure of 31 quaternary ammonium compounds (28 inhibitors; 3 substrate analogues) with their effects on the activity of acetylcholinesterase (EC 3.1.1.7; AChE) was studied. The compounds were structurally related to the natural substrate acetylcholine (ACh). All bear a trimethylammonium moiety as cationic head. The inhibitors include a variety of functional groups instead of an electrophilic ester group, making these substances suitable to probe the esteratic subsite. The inhibition constants and Km values were determined in kinetic experiments under steady state conditions (pH-stat method). Most of the substances acted as reversible, competitive inhibitors with KI in the range of 10(-6)-10(-3) M. The substrate analogues had Km values between (1.2-2.2) X 10(-4) M. The data allow the following main conclusions: (1) The quaternary trimethylammonium group of ACh is of high importance for substrate binding to AChE. It mediates association at the anionic site. (2) A poorer contribution to binding (two orders of magnitude lower) is attributable to the apolar methylene chain in ACh. It can be related to a hydrophobic interaction of the hydrocarbon chain at a region neighbouring the anionic site. (3) The ester group (both C = O and O) does not contribute to substrate binding. It is only responsible for reactivity.

The tissue distribution of[14C]3-Methoxy-4-aminopyridine was studied after intravenous administration in mice using whole body and microautoradiography. Dense accumulation was found in cholinergically innervated, secretory organs. High radioactivity was detected in the adrenal medulla suggesting that the observed excitement and hyperglycemia are due to the stimulation of catecholamine secretion. [14C]3-Methoxy-4-aminoypridine quickly passes the blood brain barrier and predominantly accumulates in the hippo-campus, the thalamic nuclei and the cortex.

Distribution and uptake of 3 labeled colloidal carrier systems have been studied following i.v. injection in mice: [14C]diazepam liposomes, [14C]polyhexylcyanoacrylate nanoparticles and 125I-albumin nanoparticles. All systems accumulated in the organs of the reticuloendothelial system (RES). Targeting to a specific organ was not achieved. Concentration kinetics revealed that [14C]diazepam was eliminated together with the lipids without any redistribution of the drug in the body. After 10 days, only radioactivity of the nanoparticle systems remained on a relatively high level in the organs of the RES. Whole-body and light microscopic autoradiographs of [14C]polyhexylcyanoacrylate and 125I-albumin nanoparticles revealed an unusual spotted appearance of silver grains in liver, lungs and bone marrow. Ultrastructural investigations of liver and lung tissue demonstrated incorporation of polyhexylcyanoacrylate nanoparticles into hepatocytes and pulmonary cells 5 min after injection and possibly microembolism for albumin and cyanoacrylate nanoparticles.

Affinity chromatography of proteins requires a ligand covalently bound to a solid support separated by a spacer of sufficient length. In the specific case of acetyl-cholinesterase we have reduced the conventional spacer synthesis from five to three steps. For affinity chromatography of cholinergic proteins the ideal ligand would be acetylcholine which, however, could not be used because it is easily hydrolyzed. We synthesized hydrolysis-resistent ligands. Different specific ligands were synthesized for the affinity chromatography of serum esterase.

The incorporation of [3H]choline into acetylcholine and other choline-containing compounds was investigated in slice cultures of the septal area of newborn rats. At choline concentrations in the range of the high affinity transport mechanism (0.1-1 microM) most of the labeled choline was incorporated into phosphorylcholine, followed by lipids, acetylcholine and the free choline pool. Hemicholinium-3 (1-10 microM) lead to a marked decrease of acetylcholine synthesis, whereas choline accumulation or phosphorylcholine synthesis were not decreased. Both basal and K+-induced release of acetylcholine were Ca2+ dependent. The efflux of choline was not stimulated by high K+. When choline was absent from the incubation medium, the slices were able to liberate significant amounts of the [3H]choline previously incorporated into phospholipids, and were also able to synthesize some acetylcholine. In choline-free medium, acetylcholine synthesis was greatly enhanced by depolarization. During the period in culture, there was a decrease of the incorporation rate of [3H]choline into phosphorylcholine and an increase of the incorporation rate into acetylcholine. The tissue structure was well preserved after several weeks in culture. After staining for acetylcholinesterase, the cholinergic neurons in the cultures showed a similar morphology to that seen in situ. The main conclusions of the present study are: cholinergic neurons in slice cultures develop and behave in a manner which is very similar to their in situ counterparts; the main divergence from previous studies of choline metabolism in tissue culture is the substantial incorporation rate of choline into acetylcholine at choline concentrations in the range of the high affinity uptake mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Results of quantification of whole-body autoradiography have been checked by means of oxygen combustion followed by liquid scintillation counting. For this purpose, the radioactivity of tissue samples of animals first used for autoradiography was counted after combustion. A comparison of the results of both methods shows that semiquantitative determination of radioactivity by whole-body autoradiography is valid between 10 and 1000 nCi14C per g wet weight of tissue. This method should be applied only to organs in which the measurement of tissue densities is not influenced by radioactivity stemming from large amounts of blood.

Synthesis of specific labelled [methyl-14C]sarin. The synthesis of labelled [methyl-14C]sarin, [14C]methylphosphonofluoridic acid 1-methylethyl ester, was accomplished by another approach as for nonlabelled sarin in a tele-conducted reaction vessel. The purity was estimated by IR, GC, and GC-MS and the stability in different media was examined.

Macroscopic autoradiograms can generally be quantified by exposing previously calibrated standard sources together with the labeled sections and measuring the silver grain density by photometric methods. Variation in section thickness is a real problem with isotopes like 14 C, with β-particles of higher energy than those of 3 H. Differences in self-absorption between tissues may be neglected at low section thickness. A simple fitting procedure for non-linear relationship between radiation dose and optical density is described. The combined effect of the examined errors for the practical evaluation of autoradiograms is discussed.

Changes in the mono- and divalentcation-stimulated ATPase activities of myosin progressively labeled with N-ethyl-[2,3-14C2]-maleimide were used to classify the readily reacting thiol groups into 3 types. The results show that one thiol-1 and one thiol-2 group are associated with each of the 2 active sites of myosin. Concentrations of KCl higher than 0.4M and/or temperatures above 10 degrees C lead to exposure of a variable number of thiol groups of a third class not affecting the enzymic properties. Although modification of thiol groups itself results in changes in structure and function of the protein, the patterns of incorporation of N-ethyl-[14C2]-malemide under various conditions of temperature, ionic strength and ligands bound to the protein revealed 9 different conformations of intact myosin. These were distinguished on the basis of the relative reactivity of the 3 different classes of thiol groups. The sequence of blockage of thiol groups reveals that cooperativity between the 2 active sites is induced by binding of a magnesium nucleotide complex to the protein. In the conformation of the long-lived myosin-product intermediate occuring during hydrolysis of Mg-ATP at 25 degrees C, 4 thiol groups of the third class react as well as or even more readily than those of the first and second classes.

Muscarinic acetylcholine receptors in intact, cultured explants of rat hippocampus were investigated in binding experiments with tritiated quinuclidinyl benzilate ([ 3 H]QNB) as ligand. Dissociation constants (K d ) were determined to 320–575 pM and maximal binding capacity (B max ) to 67–87 fmol/explant. The K d S obtained in kinetic experiments were very similar. Hippocampal explants cultured alone contained more muscarinic receptors than hippocampal explants reinnervated by cholinergic fibers from co-cultured septal explants. Pretreatment of hippocampal explants with carbachol resulted in a down-regulation of receptor number which was counteracted by the simultaneous addition of atropine. Atropine added alone had no effect on receptor number in hippocampal explants cultured alone whereas it occasionally caused an up-regulation in co-cultured hippocampus. Displacement experiments with scopolamine and oxotremorine as competitors, showed that hippocampal explants cultured alone contain multiple types of muscarinic receptors. With atropine, pirenzepine and AF-DX 116, only one class of receptors could be detected.

The actions and interactions of sodium and calcium on S-A nodal activity were investigated using the isolated spontaneously beating guinea-pig atrium suspended in a Mg-free, bicarbonate-buffered physiological solution. Osmolarity was maintained by addition of sucrose to solutions of reduced sodium concentration. Increasing the calcium concentration at a fixed sodium concentration caused a rate increase (ascending limb of the calcium-concentration-response curve) in small concentrations and again a rate decrease with still higher calcium concentrations (descending limb of the calcium-concentration-response curve). Sodium depletion produced a parallel shift of the concentration-response curve of calcium on rate to smaller calcium concentrations, and a depression of the maximal rate of atrial beat. Rates of beat obtained at the same calcium concentration but at different sodium concentrations do not show a significant difference between each other (on ascending limb of the calcium-concentration-response curve only). Identical points on calcium-concentration-response curves obtained in different sodium concentrations are caracterized by a constant quotient [Ca++]/[Na+]2 of these ions in the bath solution. The rate of atrial beat obtained in solution with constant [Ca++]/[Na+]2 depends on the logarithm of the sodium concentration by linear regression, as long as rates on the ascending limb of the calcium response curve are considered. A competition of sodium and calcium for an anionic site is suggested. Calcium might exert its positive chronotropic effect by carrying a greater fraction of the depolarizing current during slow diastolic depolarization. S-A nodal rate is under these conditions mainly determined by two factors, 1. the electrochemical sodium potential, 2. the relative calcium concentration given by the quotient [Ca++]/[Na+]2.

The concentrations of noradrenaline, dopamine and serotonin were measured in the brain of male albino mice and rats after intraperitoneal injections of muscimol, ibotenic acid or LSD. All three drugs induced a generalized increase of serotonin. When muscimol was administered to rats after pretreatment with p-chlorophenylalanine, a serotonin synthesis inhibitor, the serotonin concentration was still increased in midbrain and hypothalamus. Muscimol also caused a reduced accumulation of 5-hydroxyindoleacetic acid in rats pretreated with probenecid. There were differences in the action of the three compounds on the catecholamine concentration. Muscimol and LSD caused a decrease of the catecholamines. Ibotenic acid increased the catecholamine concentration. Certain topographical differences were noted. The increase in the serotonin concentration in the hypothalamus and midbrain after muscimol may be due to a reduced turnover of serotonin. An increase in serotonin concentration and a decrease of 5-hydroxyindolacetic acid in the rat brain are effects observed also with other psychotomimetic drugs such as LSD or psilocybin.

Es wird gezeigt, dass in der Rinde von Strychnos Mitscherlichii die Calebassen-Curare-Alkaloide: C-Fluorocurinin, C-Fluorocurarin, C-Curarin, C-Calebassin, C-Alkaloid I und C-Alkaloide der B-, C-, D-Gruppe vorkommen, und zwar in ungefahr denselben Mengenverhaltnissen wie in vielen Calebassen. Die Eingeborenen mussen daher bei der Bereitung des Calebassen-Curare von dieser Rinde Gebrauch machen.

Aus der Rinde einer brasilianischen Pflanze, die nicht genau bestimmt werden konnte, liessen sich die als Calebassen-Alkaloide bekannten Verbindungen C-Xanthocurin und C-Alkaloid P isolieren. Daneben wurde in sehr kleiner Menge ein rotgelbes Alkaloid gefunden, welchem der Name Croceocurin zugeteilt werden soll. Als Hauptalkaloid enthalt die Rinde eine neue Verbindung vom Charakter eines quartaren Ammoniumsalzes, die Kryptocurin genannt werden soll. Dieses neue Alkaloid wird vorlaufig durch einige Eigenschaften charakterisiert und ist zurzeit in genauerer Bearbeitung.

Es wird eine gute praparative Methode beschrieben, urn aus dem WIELAND- GUMLICH-Aldehyd C-Toxiferin-I herzustellen. Diese beruht auf: 1 der Selbstkondensation des N(b)-Metho-WIELAND-GUMLICH-aldehyds zu C-Toxiferin-I, Diacetyl-C-toxiferin-I und N(b)-Dimethocaracurin-V; 2 der Umwandlung des im Reaktionsgemisch vorhandenen N(b) -Dimethocara-curins-V in C-Toxiferin-I bzw. Diacetyl-C-toxiferin-I durch p-Toluolsulfosaure in Eisessig; 3 Verscifung des vorhandenen Diacetyl-C-toxiferins-I. Im weiteren wurden die synthetisicrten Alkaloide C-Toxiferin-I, N(b)-Dimetho-caracurin-V, C-Dihydro-toxiferin und Dihydro-toxiferin-I* auf ihre Toxizitat an Mausen gepruft und mit den entsprechenden naturlichen Alkaloiden verglichen. Die Toxizitaten der synthetischen und der naturlichen Verbindungen zeigten gute Uber-einstimmung.