Peter G. Waser

A fluorometric micromethod for the simultaneous determination of serotonin, noradrenaline and dopamine in milligram amounts of brain tissue

A miniaturized method for the assay of serotonin, noradrenaline and dopamine in extracts from l.5 to 5 mg of brain tissue (rat and mouse) was developed. The method permitted quantification and spectral analysis of pmole amounts of the amines. The method is derived from the solvent extraction technique and uses the principles of the trihydroxyindole and o-phthaldialdehyde methods for the development of fluorophores. The increase in sensitivity was accomplished mainly by volume reduction accompanied by changes of reagent concentrations. The small size of the tissue pieces requires a standardized dissection technique for the control of topographical variations. The problem of an appropriate reference system for the calculation of concentrations was also studied in detail. Protein weight was judged superior to wet weight for this purpose. The method was tested in a series of amine determinations on various areas of rat and mouse brain. Some examples of amine determinations as well as spectral analyses in various areas of single mouse brains are discussed.

Defeat, learned submissiveness, and analgesia in mice: effect of genotype

Defeat-induced unconditioned and conditioned behaviors of C57BL/6 and DBA/2 mice were assessed in a social-learning paradigm. Upon bites, mice of the DBA strain reacted with significantly more escape reactions, while C57 mice showed more immobility, crouch, and defensive sideways and upright postures. Clear genotype-dependent patterns were also evident from the conditioned responses recorded 24 h after defeat. DBA mice displayed more escape and defensive sideways and upright postures upon contact with a nonaggressive partner mouse; in contrast, C57 mice reacted with more immobility and crouch. With an increasing number of bites the sum of learned responses increased in C57 mice while it decreased in mice of the DBA strain. This decrement was paralleled by an increase in the analgesic response measured on the hot plate in defeated DBA mice. The possible role of endogenous opioids in the genotype-dependent interaction of defeat-induced learned submissiveness and analgesia is discussed.

Control of sleep states in the rat by short light-dark cycles

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.

Opioids and behavior: genetic aspects

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.

Physiological and behavioral effects of parachlorophenylalanine in the rat.

The effect of parachlorophenylalanine (PCPA 300 mg/kg i.p.) on several physiological and behavioral parameters was investigated with telemetric methods in the unrestrained rat. Body temperature did not change with the exception of an immediate and short-lasting decrease after drug injection. Food and water intake were maximally depressed on the day following drug administration, and recovered gradually during the subsequent days, drinking more rapidly than feeding. Click-evoked potentials recorded from the auditory cortex and inferior colliculus maintained their typical waveforms during synchronized and desynchronized sleep indicating that PCPA does not produce a qualitative change of the sleep stages. A short-lasting increase of the potentials was observed after drug injection. PCPA exerted profound changes on motor activity. The activity during the light periods was significantly increased. However, motor behavior was altered more in its temporal pattern than in intensity, especially during the dark periods. The circadian rhythms of feeding, drinking and motor activity were attenuated. Since the time-course of these changes corresponds to that known for serotonin depletion in the brain, serotoninergic neuronal mechanisms may play a major role in the organization of behavioral rhythms.

THE CHOLINERGIC RECEPTOR

SINCE the time when a knowledge of anatomical structure first offered a contribution to the understanding of the functioning of the living body, advances in physiology, and later in biochemistry, helped to further our understanding of the basic principles of life. This approach in biological research has remained the same in modern times. As we try to solve more and more problems of ultrastructural dimensions we still follow the same road that leads from morphology to function. Perhaps one day we will come to the point where we can see molecules and ions in action in a living tissue or organ. Then the two routes will meet and we will have complete understanding of a biological phenomenon. The final goal of all our endeavours in research will therefore be to link morphology with function. Today good tools have been developed with which to attack the basic problems of this sort. Microscopy has been surpassed by electron microscopy ; simple electrodes for measuring action potentials by microelectrodes, which can be placed inside single cells. Chemical compounds can be labelled with radioactive atoms and followed in the body to their site of action. Enzymes can be measured in single cells or even in microsomes. These are only a few of the many ingenious methods used in modern laboratories for biological research. In the instance I propose to discuss we have applied some of these methods to the elucidation of the nature of the cholinergic receptor.

Effect of p-Chlorophenylalanine on Cerebral Serotonin Binding, Serotonin Concentration and Motor Activity in the Rat

SummaryThe effect of p-chlorophenylalanine (PCPA) on the specific binding of 3H-serotonin (5-HT) was investigated in crude membrane preparations of the rat forebrain. The level of cerebral 5-HT and 5-hydroxyindoleacetic acid were concomitantly determined. A single dose of PCPA (300 mg/kg i.p.) increased specific binding at a low concentration of 3H-5-HT within 24 h by more than 100%. The timecourse of the elevation of specific binding was mirrored by the reduction in the 5-HT level. However, 8–10 days after drug administration, specific binding was close to the control level, while 5-HT was still significantly reduced. A Scatchard analysis revealed that the apparent affinity was significantly increased (kD reduced) during the initial period after PCPA administration, whereas the number of binding sites (BMax) was little changed. In contrast, after repeated injections of PCPA (3 injections of 300 mg/kg at 6–8 day intervals, or 100 mg/kg/day for up to 12 days) leading to a prolonged 5-HT depletion, BMax was significantly increased, while kD tended to be restored towards the baseline level. Daily injections of PCPA (100 mg/kg) for 12 days resulted in a transitory increase in day-time motor activity with a maximum on drug day 3, and in a persistent reduction of night-time activity. It is concluded that the initial PCPA-induced decrease in kD was due to a reduced competition for binding sites by the endogenous 5-HT, whereas the increase of BMax after a prolonged 5-HT depletion may reflect a 5-HT receptor supersensitivity.

Facilitation of learning by reward of post-trial memory processes

Unter Verwendung einer passiven Vermeidungsreaktion mit Futterbelohnung nach verschiedenen Zeitintervallen zwischen 0 und 120 sec konnte gezeigt werden, dass belohnung 20, 30 und 50 sec nach dem Fusschock signifikant verbessertes Lernen bewirkt. Diese Daten unterstützen die Hypothese, dass Gedächtnisprozesse-konzeptionell als operante Reaktion aufgefasst-durch Belohnung modiiizierbar sind.

Facilitation of memory processing by posttrial morphine: possible involvement of reinforcement mechanisms?

Posttrial administration of 40 mg/kg and 100 mg/kg, but not of 1 mg/kg, of morphine hydrochloride facilitates learning of a one-trial passive avoidance task in drug-naive mice. The effect does not depend on the punishing properties of the morphine injection, since an injection of LiCl (a strong punisher) fails to enhance learning in a similar way. After the establishment of tolerance by several morphine administrations, the 100 mg/kg, but not the 40 mg/kg, dose level resulted in memory facilitation.The data are discussed in connection with the hypothesis that morphine acts directly on reinforcement mechanisms by activating the opiate receptor.

Development of cholinergic projections in organotypic cultures of rat septum, hippocampus and cerebellum

ChAT and AChE activity in the hippocampus originate primarily in axons from cholinergic neurons located in the medial septum. The development of cholinergic projections in organotypic explant cultures of rat septum, hippocampus, cerebellum and habenula was studied using AChE histochemistry and biochemical ChAT and AChE determinations. Hippocampal and cerebellar explants cultured without a septum contain negligible amounts of ChAT after 6 days of culture. When the hippocampus was cultured for several days in the presence of a septal explant, a massive increase in ChAT was observed in the hippocampal explant. When co-cultures were stained for AChE, AChE-positive projections were seen to grow out from the septum to invade the hippocampal explant. To a certain extent this ingrowth of septal cholinergic fibers into the hippocampus is target-specific, since cerebellar explants cultured with septum showed neither an ingrowth of AChE-containing septal fibers, nor an increase in ChAT activity. Also, habenular AChE-positive fibers fail to grow into a co-cultivated hippocampal explant. Further, in septal explants co-cultivated with hippocampal explants an increase in ChAT activity was seen as compared to septal explants cultivated alone. The possible factors responsible for the observed specificity and the increase in ChAT activity under co-culture conditions are discussed.