Brain 5beta-reductase: a correlate of behavioral sensitivity to androgen.

Testosterone is converted in the dove (Streptopelia risoria) brain to 5 beta-reduced metabolites that do not affect behavior. In long-term castrated birds, which are relatively insensitive to the behavioral effects of testosterone, the activity of preoptic 5 beta-reductase is increased. The increase, which is specific to the preoptic area, is reversed by estrogen. Inactivation of testosterone by 5 beta-reduction may be involved in the control of brain sensitivity to androgen.

Psychogenetically selected (Roman high- and low-avoidance) rats differ in 24-hour sleep organization.

A comparison of sleep organization in Roman high-(RHA/Verh) and low-(RLA/Verh) avoidance rats, which differ in the way they respond to environmental stimuli and in several neuroendocrine and neurochemical parameters, was carried out. EEG-sleep recordings were obtained from adult males over 12:12 light-dark periods to determine how these two psychogenetically selected rat lines might also differ in their sleep-wake cycle. There was no significant difference in total sleep time between the two lines. However, the (hypoemotional) RHA/Verh rats showed an overall increase (percentage of total sleep) in paradoxical sleep (PS) duration, with a concomitant decrease in slow-wave sleep (SWS). During the dark phase, RHA/Verh rats showed a shorter PS latency and a larger number of PS episodes. Hourly sleep scoring also revealed a more discontinuous pattern (total sleep and PS vs. SWS) during the dark phase in RHA/Verh rats. In relation to recognized neurochemical and neuroendocrine differences between them, these rat lines may prove useful in investigations of the neurobiological mechanisms underlying sleep regulation.

Formation of behaviorally effective 17 beta-estradiol in the dove brain: steroid control of preoptic aromatase.

The preoptic area (POA) of the male dove is a known target area for separable behavioral actions of testosterone and 17 beta-estradiol (E2) and contains an active aromatase system. We have examined the regulatory influence of gonadal hormones on aromatase using an in vitro microassay that measures conversion of [1 alpha, 2 alpha-3H]testosterone to E2 in anatomically defined brain samples of individual animals. Preoptic aromatase activity, which is higher in males than females, is decreased (77%) in both short (30 day) and long term (180 day) castrates, indicating that gonadal hormones maintain POA aromatase activity. Basal levels of POA activity are not influenced by the period of hormonal deficit. Low levels of aromatase activity detected in area basalis are also unaffected by castration. Intramuscular testosterone propionate rapidly increases aromatase activity (within 12 h) specifically in POA of castrated males. The inductive effect of testosterone propionate in castrated doves is not increased by limited behavioral interactions in a test situation with sexually active females. A nonaromatizable androgen, 5 alpha-dihydrotestosterone, has no effect on POA aromatase activity, whereas the activity of this enzyme is restored to levels of sexually active males by systemic E2. Diethylstilbestrol has a similar, though less potent effect, indicating an estrogenic action on the enzyme. We conclude that circulating androgen modulates preoptic aromatase activity. The product of the reaction, E2, is also likely to be involved as part of a positive feedback system.

Behavioural action of androgen in the dove: effects of long-term castration on response specificity and brain aromatization.

Differences in the effectiveness of oestradiol-17 beta and testosterone on male courtship and vocal behaviour were examined in long-term castrated doves. Nest-orientated behaviour was restored by intramuscular injection of oestradiol-17 beta. Testosterone was effective in restoring aggressive courtship and vocal behaviour, but not for the nest-orientated behaviour. The effects of these hormones were separable, therefore, under conditions of prolonged androgen deficit, suggesting differences in their specificity of action. In-vitro assay of brain enzyme activity indicated that aromatization of testosterone to oestradiol-17 beta occurred in the preoptic area of long-term castrated doves. Preoptic aromatase activity of long- and short-term castrated doves did not differ. The ineffectiveness of testosterone in restoring nest-orientated behaviour in long-term castrated doves did not appear, therefore, to be due to a difference between the groups in the basal rate of testosterone aromatization in the preoptic area.

Effects of photoperiod on formation of oestradiol-17 beta in the dove brain.

The effects of photoperiod and castration on brain aromatase activity have been examined using an in-vitro radioassay. Formation of oestradiol-17 beta was lower in the preoptic area of male Barbary doves on a short daylength (6 h light:18 h darkness) than in males on a long daylength (14 h light:10 h darkness). This was a specific effect of photoperiod which did not influence aromatase activity in the anterior or posterior hypothalamic areas, and was not accompanied by changes in hormone-sensitive vocal behaviour. Production of 5 beta-dihydrotestosterone, 5 beta-androstane-3 alpha, 17 beta-diol and 5 alpha-dihydrotestosterone by the preoptic area did not differ between birds on long or short days. Therefore, a short photoperiod does not appear to influence other pathways of androgen metabolism. In contrast to the effects of photoperiod, castration reduced oestradiol formation in both preoptic and hypothalamic areas. Intramuscular injection of testosterone propionate (TP) in intact males on short days did not restore the pattern of distribution of aromatase activity seen in males on long days. Preoptic aromatase activity was, however, restored by TP in castrated birds. We conclude that a short photoperiod influences both the activity of aromatase and the inductive effect of testosterone on enzyme activity in the preoptic area, which is known to be associated with the behavioural action of oestrogen in the dove. Photoperiod is likely to act both through changes in circulating androgen and by a direct action on preoptic cells.

Brain metabolism of progesterone, coping behaviour and emotional reactivity in male rats from two psychogenetically selected lines.

Brain metabolites of progesterone such as tetrahydroprogesterone (THP) act on GABAA receptors and have anxiolytic properties. The formation of THP and its 5 alpha-reduced precursor, dihydroprogesterone (DHP) was measured in vitro in various microdissected brain areas obtained from males of two psychogenetically selected rat lines, i.e. the Roman High-(RHA/Verh) and low-(RLA/Verh) Avoidance rats, which are known to differ in emotional reactivity and/or anxiety. The behavioural and neuroendocrine responses of these rats were also measured following exposure to a novel environment in two different test situations. The formation of DHP and THP was found to be significantly higher in the frontal cortex (FCX), and DHP in the bed nucleus of the stria terminalis (BST), of the hypoemotional RHA/Verh rats. In addition, enzymatic activity in the FCX was found to be inversely correlated with behavioural measures of anxiety. These results suggest that individual, possibly genetically-determined differences in brain production of endogenous anxiolytics derived from progesterone may account at least in part for the behavioural differences characterizing these two lines, and provide further evidence that neurosteroids acting on the GABAergic system may play an important role in modulating physiological and/or behavioural responses to environmental stressors.

Lower sex hormones in men during anticipatory stress.

Free cortisol, luteinizing hormone (LH), total testosterone and monoamines were measured in two successive nocturnal urine collections in 50 healthy men to assess the influence of anticipatory stress. The first collection (N-2) was two nights before and the second (N-1) was just on the night before a one-day experimental stressor consisting of participation in a one-day clinical research protocol. The mean cortisol level increased from 23.4 (N-2) to 66.6 micrograms (N-1), while mean LH level decreased from 2.68 (N-2) to 1.71 IU (N-1) and the mean testosterone level fell from 1.31 (N-2) to 0.70 microgram (N-1). There were no changes in monoamines. Inhibition of sex hormones is a relatively neglected area of stress research.

Hypothalamic uptake of tritiated testosterone in the sheep following intracerebroventricular infusion.

A method is described for the intracerebroventricular infusion of labelled testosterone in conscious sheep. Our objective was to establish whether the infused testosterone entered the nuclei of cells in the hypothalamic region. Since the results from castrated males showed testosterone to be taken up by cell nuclei, in the hypothalamic region, the method promises to be useful for the study of the hypothalamic metabolism of androgens in relation to the sexual behaviour and neuroendocrine function in large animals.

Is the androgen-dependent increase in preoptic estradiol-17 beta formation due to aromatase induction?

Aromatization of testosterone in the preoptic area of the male ring dove is required for the expression of specific estrogen-sensitive sexual behavior. Estradiol metabolism and/or binding of the aromatization product(s) to the preoptic estrogen receptors (ER) could interfere with the apparent increase in estradiol formation that occurs after androgen administration to castrated males. Therefore, we have re-examined the induction mechanism using a direct in vitro assay of aromatase activity. Since levels of aromatase activity and enzyme kinetics were similar irrespective of the assay used, we can conclude that increased estradiol formation after androgen stimulation is mainly due to enzyme induction and that estrogen binding and/or metabolism do not interfere significantly with the induction process.

Metabolic control of the behavioural action of androgens in the dove brain: testosterone inactivation by 5 beta-reduction.

In vitro studies of androgen metabolism in the dove brain show that testosterone is rapidly converted to 5 beta-reduced metabolites (5 beta-dihydrotestosterone and the two corresponding 3 alpha/3 beta diols). The preoptic region and the anterior ventromedial and posterior hypothalamus which are target areas for androgen action on behaviour and the neuroendocrine system show significantly less 5 beta-reduction than adjacent parolfactory, septal and striatal areas not directly involved in androgen action. In contrast to 5 alpha-reduction and aromatization, which lead to the formation of active metabolites in the dove brain, 5 beta-reduction is likely to be part of a testosterone (T) inactivation mechanism because: (a) intrahypothalamic implants of 5 beta-dihydrotestosterone were ineffective for male behaviour in the dove; (b) 5 beta-reduced metabolites of T were not detected in hypothalamic cell nuclei following intramuscular injection of tritiated testosterone, and were rapidly eliminated from brain cells; (c) the 4 beta-diols were also found to be a major product of T catabolism in the liver, a site of androgen inactivation. Long-term androgen deficit induced by castration increased 5 beta DHT formation in the preoptic area, but not in an adjacent parolfactory area. The increase was reversed by exogenous testosterone and oestradiol-17 beta indicating that the 5 beta-reduction pathway in an androgen-sensitive brain area is influenced by hormonal conditions. It is suggested that 5 beta-reduction is a testosterone inactivation pathway involved in the regulation of brain sensitivity to androgens.

Nuclear uptake of testosterone in the dove preoptic area.

After intramuscular injection of tritiated testosterone, the tracer and its active metabolites, 5 alpha-dihydrotestosterone and estradiol-17 beta were taken up in vivo by preoptic cell nuclei in the male dove brain. An inactive metabolite, 5 beta-androstane-3 alpha-17 beta-diol, occurred only in the non-nuclear compartment. Saturable nuclear uptake of testosterone was higher in the preoptic area than in adjacent nontarget areas. The results suggest that the metabolic pathways demonstrated previously in vitro are all active in vivo, and only testosterone and its active metabolites interact with the cell nucleus.

Preoptic formation of 17 beta-oestradiol is influenced by behavioural stimuli in the dove.

In vitro study of testosterone (T) metabolism shows that formation of 17 beta-oestradiol (E2) in the preoptic area is higher in male doves exposed to behavioural stimuli from interacting pairs than in visually isolated males or males that have interacted with females. Formation of 5 alpha-dihydrotestosterone, 5 beta-dihydrotestosterone and 5 beta-androstane-3 alpha, 17 beta-diol was similar in all groups, indicating that exposure to stimuli from interacting pairs influences aromatization of T and not other pathways of androgen metabolism. The results suggest that sexual stimuli, which do not involve tactile contact between male and female, affect local E2 formation in the brain.

Area-specific hormonal regulation of brain aromatase.

In vitro experiments were conducted to compare the steroid regulation of aromatase in preoptic and posterior hypothalamic areas. Kinetic analysis of aromatase activity in the preoptic area (POA) and posterior hypothalamus (AHP) of castrated and intact doves indicate that both areas have a similar, high substrate affinity (apparent Km less than 15 nM), but castration decreases the Vmax to a greater extent in POA than AHP. This differential effect was confirmed using a single substrate (10 nM) concentration representing the Km of the enzyme. Comparison of the effects of non-aromatisable androgens, methyltrienolone (R1881) and 5 alpha-dihydrotestosterone (DHT), on aromatase activity in 20-day castrates showed that neither R1881 nor DHT induced aromatase activity in POA. This was confirmed in 40-day castrates which also showed a reduced inductive effect of testosterone (T) on the brain enzyme activity. R1881 specifically increased aromatase activity in AHP, but DHT did not affect either area. The non-aromatisable androgens influenced androgen-dependent vocal behaviour to the same extent. Oestradiol (E2) increased aromatase activity in both POA and AHP. We conclude that non-aromatisable androgens affect hypothalamic, but not preoptic oestrogen formation. Aromatase activity in the male preoptic area associated with behaviour is specifically sensitive to the aromatisable androgen, T and E2. The results suggest that hormonal regulation of the aromatase differs locally within androgen target areas of the brain.

Extracellular serotonin variations during vigilance states in the preoptic area of rats: a microdialysis study.

Numerous studies have shown that serotonergic transmission decreases from waking (W) to slow wave sleep (SWS) to paradoxical sleep (PS), suggesting an active role of serotonin (5-HT) in W but not in sleep. Conversely, the inhibition of 5-HT activity produces insomnia. This insomnia can be reversed by injections of 5-hydroxytryptophan in the preoptic area (POA), suggesting that 5-HT is necessary in this cerebral structure for sleep. Using microdialysis, we studied, 5-HT variations in the POA of rats in relation to vigilance states. 5-HT levels were higher during W than during during SWS and PS. 5-HT increased just before the rats fell asleep and then decreased during sleep. A decreased 5-HT transmission was also observed from SWS to PS. These data document a positive correlation between 5-HT levels in POA and wakefulness. Moreover, these observations are in favour of a permissive role of 5-HT in the POA during PS. A comparison between the POA and the prefrontal cortex in the sleep-wake cycle is discussed.

Long-term behavioural and neuroendocrine changes in Roman high-(RHA/Verh) and low-(RLA-Verh) avoidance rats following neonatal handling.

Roman high-(RHA/Verh) and low-(RLA/Verh) avoidance rats, originally selected and bred for rapid vs poor acquisition of a two-way active avoidance response, differ in emotional reactivity and coping style. These differences are associated with particular neuroendocrine and neurochemical characteristics. New data are presented here to show that the behavioural changes specifically induced by neonatal handling, i.e. decreased emotional reactivity, are associated with marked changes in the neuroendocrine responses of (hyperemotional) RLA/Verh rats to a novel environment. Eight months after neonatal handling, self-grooming behaviour, a reliable marker of emotional reactivity in this line of rats, was significantly decreased in RLA/Verh rats. Defecation scores were also significantly reduced in both lines. Moreover, there was a significant reduction in prolactin and corticosterone release following exposure to a novel environment in neonatally-handled RLA/Verh rats as compared to control, non-handled rats. No effects on prolactin and corticosterone release were observed in RHA/Verh rats. There was also no apparent effect of neonatal handling on coping style i.e. RLA/Verh rats did not increase their spontaneous exploration of novel environments. Thus, the phenotypic expression of basic traits of (high) neuroendocrine/emotional reactivity was specifically modulated by neonatal handling in RLA/Verh rats, whereas both the (hypoemotional) RHA/Verh rats as well as coping style in both lines remained unaffected. Changes in emotional reactivity were still apparent at 12 months of age when rats from the same groups were tested for hyponeophagia. These results suggest that psychogenetically selected lines such as RHA/RLA rats are suitable animal models to investigate interactions between genes and the environment in determining individual sensitivity to stress and coping styles, as well as potential vulnerability (or resistance) to the development of maladaptive syndromes similar to anxiety and mood disorders in humans.

Differential changes of cAMP-dependent protein kinase activity and 3H-cAMP binding sites in rat hippocampus during maturation and aging.

The cyclic AMP-dependent protein kinase (PKA) has been involved in the brain aging process and recent papers have reported age-associated changes in enzyme activity in rat brain. The present study was undertaken to assess simultaneously PKA activity and regulatory (R) subunit levels during maturation and aging. Five cohorts of rats of different ages were used, namely pups of 1 week and 3 weeks old, mature rats (2 months), postmature rats (1 year) and old rats (2 years or more). PKA activity and 3H-cAMP binding sites were determined in cytosolic fractions of hippocampus. Results showed a low PKA activity in newborn rats which increased in mature and postmature rats and finally declined in old rats (ANOVA, P<0.001). The maximum binding sites (Bmax) of 3H-cAMP which measure the PKA R subunit levels were elevated in newborn rats and declined in mature and old rats (ANOVA; P<0.001). It is suggested the changes in PKA R subunit levels reflect an adaptative role in maturing process, a role which is lost in aging phase.

Stressor-specific increase of vasopressin mRNA in paraventricular hypophysiotrophic neurons.

Cellular levels of vasopressin (VP) and corticotropin-releasing factor (CRF) RNA transcripts were determined in hypophysiotrophic neurons after open-field and immobilization stress using quantitative in situ hybridization. We found that 8 min open-field stress is sufficient to produce a significant up-regulation of CRF mRNA, without any concomitant changes in the level of VP mRNA. In contrast, 8 min immobilization stress resulted in an increased labeling density of both CRF and VP mRNAs. These results suggest that the level of CRF and VP transcripts in parvicellular hypophysiotrophic neurons is differentially regulated in a stressor-specific manner.

Psychotropic medication, psychiatric disorders, and higher brain functions.

Conventional psychiatric diagnosis is founded on symptom description; this then governs the choice of psychotropic medication. This purely descriptive approach resembles a description of diphtheria from the premicrobiology era. Based on current advances in basic and clinical neuroscience, we propose inserting an intermediate level of analysis between psychiatric symptoms and pharmacologic modes of action. Paradigm 1 is to analyze psychiatric symptoms in terms of which higher brain function(s) is (are) abnormal, ie, symptoms should be analyzed as higher brain dysfunction: a case study in obsessive-compulsive disorder reveals pointers in four common symptoms to the higher functions of working memory, emotional overlay, absence of voluntary control, and the ability to evaluate personal mental phenomena. Paradigm 2 is to view psychotropic drugs as modifying normal higher brain functions, rather than merely treating symptoms, which they do only secondarily: thus depression may respond to agents that act on related aspects of mental life derived from higher brain functions, eg, the ability to enhance bonding. We advocate a strategy in which psychiatric illness is progressively reclassified through knowledge in clinical neuroscience and treatment targets are revised accordingly.

Stress revisited: a critical evaluation of the stress concept.

With the steadily increasing number of publications in the field of stress research it has become evident that the conventional usage of the stress concept bears considerable problems. The use of the term 'stress' to conditions ranging from even the mildest challenging stimulation to severely aversive conditions, is in our view inappropriate. Review of the literature reveals that the physiological 'stress' response to appetitive, rewarding stimuli that are often not considered to be stressors can be as large as the response to negative stimuli. Analysis of the physiological response during exercise supports the view that the magnitude of the neuroendocrine response reflects the metabolic and physiological demands required for behavioural activity. We propose that the term 'stress' should be restricted to conditions where an environmental demand exceeds the natural regulatory capacity of an organism, in particular situations that include unpredictability and uncontrollability. Physiologically, stress seems to be characterized by either the absence of an anticipatory response (unpredictable) or a reduced recovery (uncontrollable) of the neuroendocrine reaction. The consequences of this restricted definition for stress research and the interpretation of results in terms of the adaptive and/or maladaptive nature of the response are discussed.