Patrick J. Ronan

Does Serotonin Influence Aggression? Comparing Regional Activity before and during Social Interaction*

Serotonin is widely believed to exert inhibitory control over aggressive behavior and intent. In addition, a number of studies of fish, reptiles, and mammals, including the lizard Anolis carolinensis, have demonstrated that serotonergic activity is stimulated by aggressive social interaction in both dominant and subordinate males. As serotonergic activity does not appear to inhibit agonistic behavior during combative social interaction, we investigated the possibility that the negative correlation between serotonergic activity and aggression exists before aggressive behavior begins. To do this, putatively dominant and more aggressive males were determined by their speed overcoming stress (latency to feeding after capture) and their celerity to court females. Serotonergic activities before aggression are differentiated by social rank in a region‐specific manner. Among aggressive males baseline serotonergic activity is lower in the septum, nucleus accumbens, striatum, medial amygdala, anterior hypothalamus, raphe, and locus ceruleus but not in the hippocampus, lateral amygdala, preoptic area, substantia nigra, or ventral tegmental area. However, in regions such as the nucleus accumbens, where low serotonergic activity may help promote aggression, agonistic behavior also stimulates the greatest rise in serotonergic activity among the most aggressive males, most likely as a result of the stress associated with social interaction.

Central Monoamines in Free-Ranging Lizards: Differences Associated with Social Roles and Territoriality

During the breeding season different social classes of field-active lizards, Sceloporus jarrovi, exhibit regionally specific changes in central monoaminergic activation. Changes in serotonergic content and turnover between lizards from different social classes are seen in forebrain structures (telencephalon and diencephalon) and reflect events associated with reproductive behaviors, stress and aggression. Males without territories (satellite males) exhibit higher forebrain serotonin (5-HT) system activation compared to territorial males and adult females. This serotonergic activation includes increased 5-hydroxyindoleacetic acid (5-HIAA) and 5-HIAA/5-HT ratio, suggesting increased release and catabolism. Satellite males also exhibit higher 5-HIAA/5-HT ratios (serotonergic turnover) compared to territorial males following agonistic interactions. Territorial males, immediately following aggressive defense of territories against intruder males, exhibit increased 5-hydroxytryptophan (5-HTP) levels, higher 5-HIAA levels and 5-HIAA/5-HT ratio, higher epinephrine levels, greater MHPG/NE, more DOPAC and larger DOPAC/DA ratio compared to territorial males that did not have an aggressive encounter. These differences suggest activation of 5-HT, norepinephrine (NE), and dopamine (DA) systems by the synthesis and release of more 5-HT and the release of more NE and DA during aggressive defense of territory. The highest activity of serotonergic systems is exhibited by satellite males compared to territorial males, perhaps reflecting stress in subordinate animals from social and ecological sources.

Visible sympathetic activity as a social signal in Anolis carolinensis: changes in aggression and plasma catecholamines

Darkening of postorbital skin in Anolis carolinensis occurs during stressful situations and is stimulated by sympathetic activation of beta(2)-adrenergic receptors via adrenal catecholamines. This eyespot forms more rapidly in dominant males during social interaction. Eyespot darkening (green to black) appears to function as a social signal communicating sympathetic activation and limiting aggressive interaction. To assess the value of the eyespot as a social signal, males were painted postorbitally with green, black, or red paint. Each male was exposed to a mirror following acclimation to the cage. The total number of aggressive displays toward the mirror image was greatest when eyespots were masked by green paint. In contrast, black or red artificial eyespots, regardless of size, inhibited biting behavior toward the mirror image. The most aggressive males, those who saw a reflected opponent with no eyespot (hidden with green paint), had significantly higher levels of all plasma catecholamines. These results suggest that A. carolinensis use information from the eyespot to assess their opponents readiness to fight and thereby determine whether to be aggressive. Darkened eyespots are capable of inhibiting aggression, whereas aggressive displays from an opponent in the mirror without darkened eyespots do not. Darkened eyespots reflect rapid changes in plasma NE, DA, and Epi that may signal dominant social status.

Dynamics and mechanics of social rank reversal

Stable social relationships are rearranged over time as resources such as favored territorial positions change. We test the hypotheses that social rank relationships are relatively stable, and although social signals influence aggression and rank, they are not as important as memory of an opponent. In addition, we hypothesize that eyespots, aggression and corticosterone influence serotonin and N-methyl-D-aspartate (NMDA) systems in limbic structures involved in learning and memory. In stable adult dominant-subordinate relationships in the lizard Anolis carolinensis, social rank can be reversed by pharmacological elevation of limbic serotonergic activity. Any pair of specific experiences: behaving aggressively, viewing aggression or perceiving sign stimuli indicative of dominant rank also elevate serotonergic activity. Differences in the extent of serotonergic activation may be a discriminating and consolidating factor in attaining superior rank. For instance, socially aggressive encounters lead to increases in plasma corticosterone that stimulate both serotonergic activity and expression of the NMDA receptor subunit 2B (NR2B) within the CA3 region of the lizard hippocampus. Integration of these systems will regulate opponent recognition and memory, motivation to attack or retreat, and behavioral and physiological reactions to stressful social interactions. Contextually appropriate social responses provide a modifiable basis for coping with the flexibility of social relationships.

Stress induces rapid changes in serotonergic activity: restraint and exertion

Rapid activation of central serotonergic systems occurs in response to the social stress of aggression in dominant lizards. The most rapid expression of serotonergic activity occurs in nucleus accumbens, hippocampus and brainstem. To compare previously measured responses induced by social stressors with those provoked by physical stress, serotonergic activity was examined following restraint stress (handling) and forced physical exertion. After handling, some male Anolis carolinensis were placed on a race track and either run until there was no movement following 1 min of prodding, or half that time. Controls were killed without treatment. Lizards stressed by handling showed rapid (25 s) increases in serotonergic activity (5-HIAA/5-HT) in striatum, dorsal cortex, locus ceruleus, and nucleus accumbens. Other changes in serotonergic systems caused by stress occurred in raphe and hippocampus. Serotonergic changes induced by handling stress were reversed by exercise (to 50% maximal exertion time) in subiculum, striatum and nucleus accumbens. The serotonergic profile of lizards run until they would no longer respond to prodding (maximal exertion time) was significantly different from that for more acute exertion in hippocampus, subiculum, striatum, medial amygdala, locus ceruleus, area postrema, and raphe. Physical stress (handling) mimicked social stress by producing rapid serotonergic changes in hippocampus, subiculum, nucleus accumbens and locus ceruleus. In contrast, the medial amygdala, which has previously been demonstrated to respond serotonergically to social stress only after a temporal delay, did not show a rapid response to restraint stress.

Lipid-lowering effect of berberine in human subjects and rats.

Due to serious adverse effects and the limited effectiveness of currently available pharmacological therapies for obesity, many research efforts have focused on the development of drugs from natural products. Our previous studies demonstrated that berberine, an alkaloid originally isolated from traditional Chinese herbs, prevented fat accumulation in vitro and in vivo. In this pilot study, obese human subjects (Caucasian) were given 500 mg berberine orally three times a day for twelve weeks. The efficacy and safety of berberine treatment was determined by measurements of body weight, comprehensive metabolic panel, blood lipid and hormone levels, expression levels of inflammatory factors, complete blood count, and electrocardiograph. A Sprague-Dawley rat experiment was also performed to identify the anti-obesity effects of berberine treatment. The results demonstrate that berberine treatment produced a mild weight loss (average 5 lb/subject) in obese human subjects. But more interestingly, the treatment significantly reduced blood lipid levels (23% decrease of triglyceride and 12.2% decrease of cholesterol levels) in human subjects. The lipid-lowering effect of berberine treatment has also been replicated in the rat experiment (34.7% decrease of triglyceride and 9% decrease of cholesterol level). Cortisol, calcitriol, ACTH, TSH, FT4, and SHBG levels were not significantly changed following 12 weeks of berberine treatment. However, there was interestingly, an increase in calcitriol levels seen in all human subjects following berberine treatment (mean 59.5% increase, p=0.11). Blood inflammatory factors (CRP, IL-6, TNFα, COX-2) and erythrocyte sedimentation rate (ESR) were not significantly affected by treatment with berberine. Tests of hematological, cardiovascular, liver, and kidney function following berberine treatment showed no detrimental side effects to this natural compound. Collectively, this study demonstrates that berberine is a potent lipid-lowering compound with a moderate weight loss effect, and may have a possible potential role in osteoporosis treatment/prevention.

The role of monoaminergic nuclei during aggression and sympathetic social signaling

A social sign stimulus that is sympathetically induced affects aggressive approaches and influences serotonergic, dopaminergic and noradrenergic activity in the brainstem nuclei of Anolis carolinensis. Darkening of postorbital skin via sympathetic activation of adrenal catecholamines and β2-adrenergic receptors provides a visual signal that forms more rapidly in dominant than subordinate males during social interactions. This signal limits aggressive interactions. Males were painted postorbitally with green or black paint and then exposed to a mirror. Aggressive approaches to the mirror were inhibited in males viewing a reflection with darkened eyespots, and increased in males viewing a reflection without eyespots (hidden). Noradrenergic turnover in the raphe and locus ceruleus were greatest in test subjects that viewed a reflection with eyespots hidden by green paint. Perception of darkened eyespots stimulated greater serotonergic turnover in raphe, locus ceruleus and substantia nigra/ventral tegmental area (SN/VTA). Dopaminergic turnover was higher in the raphe and SN/VTA of Anolis that viewed a reflection with darkened eyespots. However, these animals had lower dopamine turnover in the locus ceruleus than isolated and hidden eyespot groups. Of the possible roles of perikarya on central function and behavior, our results suggest feedback, cross-nuclear regulation, and some independence of function between nuclei and the forebrain terminal fields. Decreased serotonergic activity corresponds with increased aggression only in the raphe, suggesting that the raphe nuclei might be important for this behavioral trait. Increased serotonergic, noradrenergic and dopaminergic activities in SN/VTA in Anolis that view a reflected opponent with dark eyespots suggests that the SN/VTA might be directly involved in recognition of this social sign stimulus and the resulting inhibition of aggression.

Monoaminergic Activity in Subregions of Raphé Nuclei Elicited by Prior Stress and the Neuropeptide Corticotropin-Releasing Factor

Corticotropin‐releasing factor (CRF) coordinates neuroendocrine responses to stressful stimuli; one mechanism through which CRF may modulate hypothalamic‐pituitary‐adrenal axis activity is via actions on neuromodulatory systems such as serotonergic systems. Recent electrophysiological studies and the distribution of CRF receptors within midbrain and pontine raphé nuclei suggest that stress and CRF may have actions on topographically organized subpopulations of serotonergic neurones. We compared the effects of vehicle or intracerebroventricular r/hCRF injections (0, 0.1, 1 or 10u2003µg) in rats previously maintained in home cages or restrained for 1u2003h, 24u2003h before injection, on monoamine and monoamine metabolite tissue concentrations in the dorsal (lateral wings, rostral midline, caudal midline), median (rostral, caudal) and interfascicular raphé subdivisions of the midbrain and pontine raphé nuclei, using brain microdissection and high‐performance liquid chromatography with electrochemical detection. At the lowest dose studied (0.1u2003µg), CRF infusions in previously stressed rats decreased 5‐hydroxytryptophan (5‐HTP) and 5‐hydroxyindoleacetic acid (5‐HIAA) concentrations only within the rostral median raphé nucleus. At higher doses, CRF infusions in previously stressed rats increased tissue concentrations of 5‐HTP, serotonin (5‐HT), or the serotonin metabolite, 5‐HIAA, within rostral (but not caudal) regions of the median and dorsal raphé nuclei. By contrast, restraint stress alone had no effect on tissue concentrations of 5‐HTP, 5‐HT or 5‐HIAA measured 24u2003h later in any subdivision, while CRF injections in rats not previously exposed to restraint stress, with few exceptions, also had no effect. These results suggest that the effects of CRF on serotonergic function are context‐dependent, dose‐dependent, and regionally specific within subdivisions of the brainstem raphé nuclei.

Depressive Behavior and Activation of the Orexin/Hypocretin System

The orexin/hypocretin peptide signaling system plays a neuromodulatory role in motivation and stress; two critical components of depression. Although work has been done to identify links between orexin and depression, few specific neuroanatomical associations have been made. These studies have not investigated the relationship between orexin and orexin receptor expression in specific brain regions associated with this disorder. To address this, we examined immobility during the forced swim test (FST) in mice, a commonly used measure of depressive behavior. We analyzed the variation in FST immobility with the distribution of orexin and its receptor mRNA. We found that animals that exhibited more robust depressive behavior had greater or lesser orexin system expression that depended on the limbic brain region analyzed. In the hippocampus there was a negative correlation between orexin expression and FST immobility. Animals that displayed relatively more depressive behavior had lower hippocampal expression of Orexin A (OrxA). In the amygdala, there was a curvilinear relationship between OrxA and FST performance. In addition there was a positive correlation with amygdalar Type I orexin receptor (Orx1) mRNA and depressive behavior. Despite the differences in limbic orexin expression, there was no correlation between immobility and hypothalamic orexin neuron activation as measured by c-Fos. Overall, more severe depressive behavior was associated with reduced hippocampal orexin expression, contrasted with increased orexin plus Orx1 receptor mRNA expression in the amygdala. This divergent pattern between the hippocampus and amygdala mirrors a neurobiological theme seen in depression resulting from reduced hippocampal, but increased amygdalar, size and function.

Effects of labor contractions on catecholamine release and breathing frequency in newborn rats.

Plasma catecholamines in newborn rats (0-2 hr old) were analyzed following vaginal birth, cesarean section with simulated labor contractions, or cesarean section without labor contractions. Upon delivery, pups were exposed to key elements of the rats natural birth process, that is, umbilical cord occlusion, tactile stimulation, and cooling. Only pups exposed to actual or simulated labor showed an immediate rise in norepinephrine and epinephrine. Initial postpartum respiratory frequencies were higher in vaginal than in cesarean delivered pups and, in all groups, inversely correlated with catecholamine titers, suggesting respiratory distress or transient tachypnea at lower catecholamine levels. These findings establish a rat model for analyzing effects of labor on neonatal adaptive response during the transition from prenatal to postnatal life.