Search

Salmonids establish social hierarchies as a result of aggressive social interactions. The establishment of dominant or subordinate status is strongly linked to neuroendocrine responses mediated through the stress axis. In this study, we tested the effects of introcerebroventricular (icv) corticotropin releasing factor (CRF) on the behavioral outcome, plasma cortisol and monoamine function in trout subjected to a socially aggressive encounter. Rainbow trout were treated with an icv injection of artificial cerebrospinal fluid (aCSF), 500 or 2000 ng ovine CRF, or not injected. Fish were allowed to interact with a similarly sized conspecific for 15 min. Following the behavioral interaction, plasma cortisol and central monoamine concentrations were analyzed. Trout treated with CRF were victorious in approximately 66% of the aggressive encounters against aCSF-treated opponents. Trout injected with CRF exhibited a reduction in the total number of attacks and decreased latency to attack. When trout were divided into winners and losers, only victorious CRF-treated fish exhibited a reduced latency to attack and fewer retreats. Social stress increased cortisol levels in both winners and losers of aggressive interaction. This effect was enhanced with the additional stress incurred from icv injection of aCSF. However, icv CRF in addition to social stress decreased plasma cortisol in both winners and losers. While aggression stimulated significant changes in serotonergic and dopaminergic activity, the magnitude and direction were dependent on limbic brain region, CRF dose, and outcome of social aggression. With broad effects on aggressive behavior, anxiety, stress responsiveness, and central monoaminergic activity, CRF plays an important role in modulating the behavioral components of social interaction.

Decreases in serotonergic activity in the central nucleus of the amygdala reduce responses to stressors, suggesting an important role for serotonin in this region of the amygdala in stress reactivity. However, it is not known whether exposure to stressors actually increases serotonin release in the central nucleus of the amygdala. The current experiment tested the hypothesis that restraint stress increases extracellular serotonin within the central nucleus of the amygdala and adjacent medial amygdala using in vivo microdialysis in awake male rats during the dark phase of the light-dark cycle. Serotonin release in the central nucleus increased immediately in response to restraint stress. In contrast, there was no change in serotonin release within the adjacent medial amygdala during or following restraint. Since corticotropin-releasing factor is an important mediator of both responses to stressors and serotonergic activity, subsequent experiments tested the hypothesis that central nucleus serotonergic response to restraint stress is mediated by central corticotropin-releasing factor receptors. Administration of the corticotropin-releasing factor type 1 and 2 receptor antagonist d-Phe-CRF (icv, 10 microg/5 microl) prior to restraint stress suppressed restraint-induced serotonin release in the central nucleus. The results suggest that restraint stress rapidly and selectively increases serotonin release in the central nucleus of the amygdala by the activation of central corticotropin-releasing factor receptors. Furthermore, the results imply that corticotropin-releasing factor mediated serotonergic activity in central nucleus of the amygdala may be an important component of a stress response.

During agonistic interactions between male Anolis carolinensis, perception of a visual sign stimulus (darkened eyespots) not only inhibits aggression and promotes initial attainment of dominant social status, but also evokes distinct neuroendocrine responses in each opponent. This study was designed to examine the effect of eyespot manipulation on behavior and social rank during a second interaction between opponents that had previously established a natural dyadic social hierarchy. Prior to a second interaction, eyespots of familiar size-matched combatants were manipulated to reverse information conveyed by this visual signal. Eyespots on the previously dominant male were masked with green paint to indicate low aggression and social status. Previously subordinate males had their eyespots permanently marked with black paint to convey high aggression and status. Opponents were then repaired for a second 10 min interaction following either one or three days of separation. Aggression was generally decreased and social status between pairs remained reasonably consistent. Unlike rapidly activated monoaminergic activity that occurs following the initial pairing, most brain areas sampled were not affected when animals were re-introduced, regardless of visual signal reversal or length of separation between interactions. However in males with “normal” eyespot color, dominant males had reduced serotonergic activity in CA3 and raphé, while subordinate males exhibited elevated CA3 dopaminergic activity. Reversing eyespot color also reversed serotonergic activity in raphé and dopaminergic activity in CA3 after three days of separation. The results suggest that males remember previous opponents, and respond appropriately to their previous social rank in spite of eyespot color.

At the onset of agonistic social challenge, individuals must assess the degree of threat the opponent represents in order to react appropriately. We aimed to characterize the neuroendocrine changes accompanying this period of initial social assessment using the lizard Anolis carolinensis. Conveyance of aggressive intent by male A. carolinensis is facilitated by rapid post-orbital skin darkening (eyespot), whereas eyespot presence inhibits opponent aggression. By manipulating this visual signal, we also investigated whether differing neuroendocrine changes were evoked by initial presentation of varying levels of social threat. Subjects were painted postorbitally either with black paint (high threat level), green paint (low threat level) or water (controls). Painted animals were presented with a mirror and sampled immediately upon exhibiting aggressive intent towards the reflected simulated opponent, but before producing behaviors such as motor pattern-based displays. Control animals (blank surface presented) were sampled at times derived from averaging response times of painted subjects. Brains and plasma were analyzed for monoamine activity and catecholamine levels using electrochemical HPLC. Social threat evoked increases in plasma catecholamine levels indistinguishable from those caused by brief environmental disturbance. However, brief social challenge caused distinct rapid increases in amygdala and nucleus accumbens (NAc) dopamine and serotonin levels. Amygdalar changes were associated with general social threat presence, but NAc monoamines were affected by both threat level and subject motivation to engage in confrontation. This suggests that specific rapid activity changes in key forebrain limbic nuclei differ according to the degree of social threat perceived at the start of the interaction.

Repeated social defeat of adolescent male rats results in adult mesocortical dopamine hypofunction, impaired working memory, and increased contextual anxiety-like behavior. Given the role of glutamate in dopamine regulation, cognition, and fear and anxiety, we investigated potential changes to N-methyl-D-aspartic acid (NMDA) receptors following adolescent social defeat. As both NMDA receptors and mesocortical dopamine are implicated in the expression and extinction of conditioned fear, a separate cohort of rats was challenged with a classical fear conditioning paradigm to investigate whether fear learning is altered by adolescent defeat. Quantitative autoradiography was used to measure 3H-MK-801 binding to NMDA receptors in regions of the medial prefrontal cortex, caudate putamen, nucleus accumbens, amygdala and hippocampus. Assessment of fear learning was achieved using an auditory fear conditioning paradigm, with freezing towards the auditory tone used as a measure of conditioned fear. Compared to controls, adolescent social defeat decreased adult NMDA receptor expression in the infralimbic region of the prefrontal cortex and central amygdala, while increasing expression in the CA3 region of the hippocampus. Previously defeated rats also displayed decreased conditioned freezing during the recall and first extinction periods, which may be related to the observed decreases and increases in NMDA receptors within the central amygdala and CA3, respectively. The alteration in NMDA receptors seen following adolescent social defeat suggests that dysfunction of glutamatergic systems, combined with mesocortical dopamine deficits, likely plays a role in the some of the long-term behavioral consequences of social stressors in adolescence seen in both preclinical and clinical studies.

The receptors that couple to the G protein Gz in vivo are still relatively unknown. In this study, we investigated the effects of various dopamine receptor agonists in a mouse deficient in the alpha subunit of Gz. The dopamine D1-like receptor agonist SKF38393 stimulated comparable locomotor activity in both wildtype mice and mice lacking Galphaz. In contrast, the dopamine D2-like receptor agonist quinpirole suppressed locomotor activity in both groups of mice, but this suppression was significantly smaller in Galphaz knockout mice. Consistent with these behavioural observations, quinpirole inhibition of dopamine release in the forebrain nucleus accumbens evoked by electrical stimulation of dopamine axons was significantly attenuated in mice lacking Galphaz. In addition, hypothermia and adrenocorticotropic hormone release resulting from activation of dopamine D2-like receptors were also significantly reduced in Galphaz knockout mice. However, adrenocorticotropic hormone secretion induced by corticotrophin releasing hormone and the serotonin 1A receptor agonist 8-hydroxy-dipropylamino-tetralin were similar between wildtype and Galphaz knockout mice. Western blot analysis showed that the expression levels of Galphai, Galphao, Galphas, Galphaq and Gbeta were the same in the brains of mice of both genotypes. Overall, our data suggest that Gz proteins are functionally coupled to dopamine D2-like receptors in vivo.

Socially aggressive interaction is stressful, and as such, glucocorticoids are typically secreted during aggressive interaction in a variety of vertebrates, which may both potentiate and inhibit aggression. The behavioral relationship between corticosterone and/or cortisol in non-mammalian (as well as mammalian) vertebrates is dependent on timing, magnitude, context, and coordination of physiological and behavioral responses. Chronically elevated plasma glucocorticoids reliably inhibit aggressive behavior, consistent with an evolutionarily adaptive behavioral strategy among subordinate and submissive individuals. Acute elevation of plasma glucocorticoids may either promote an actively aggressive response via action in specialized local regions of the brain such as the anterior hypothalamus, or is permissive to escalated aggression and/or activity. Although the permissive effect of glucocorticoids on aggression does not suggest an active role for the hormone, the corticosteroids may be necessary for full expression of aggressive behavior, as in the lizard Anolis carolinensis. These effects suggest that short-term stress may generally be best counteracted by an actively aggressive response, at least for socially dominant proactive individuals. An acute and active response may be evolutionarily maladaptive under chronic, uncontrollable and unpredictable circumstances. It appears that subordinate reactive individuals often produce compulsorily chronic responses that inhibit aggression and promote submissive behavior.

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.

Male green anoles convey territorial ownership to rivals through use of stereotyped aggressive displays, with intrasexual interactions often escalating to physical combat. In the laboratory, losers of such interactions significantly reduce their aggressive behaviour when interacting with the same opponent 7 days later. In the current study, we examined whether this behavioural modification is a function of opponent recognition. Pairs of size-matched male A. carolinensis were allowed to interact for 2 h. Following this, subjects were separated for varying intervals, and then participated in a second interaction with either the same or an unfamiliar opponent. Losers of the first interaction significantly reduced their total aggressive behaviour when re-paired with the same opponent after a 3-day interval but not after a 10-day interval between the first and second interaction. However, losers of the first interaction failed to show reduced aggression during their second interaction when paired 3 or 10 days later with an unfamiliar opponent. Therefore, A. carolinensis appear to learn information specific to their opponent during an initial agonistic interaction, with the salience of this information declining when the interval between interactions increases. Males also showed low levels of chemosensory taste behaviour during agonistic interactions, suggesting that opponent recognition does not rely on chemoreception. Similarities between our results and findings from other vertebrate species suggest that the motivation to behave aggressively during social interactions can be influenced by opponent recognition rather than by previous experience of social defeat alone.

Midbrain dopamine neurons are critical in mediating the rewarding effects of opiates in dependent rats, as well as modulating some manifestations of opiate withdrawal. Morphine is known to excite dopamine neurons and thereby facilitate forebrain dopamine transmission through inhibition of GABA neurons. Cholinergic neurons in the mesopontine laterodorsal and pedunculopontine tegmental nuclei provide the principal source of excitatory cholinergic input to ventral tegmental area and substantia nigra pars compacta dopamine-containing neurons, via actions on midbrain muscarinic and nicotinic acetylcholine receptors. The present study hypothesized that a reduction in tonic cholinergic input via blockade of midbrain muscarinic receptors would reduce the pharmacological effects of morphine on forebrain dopamine release. Using in vivo chronoamperometry, alterations in morphine-evoked dopamine efflux were monitored at stearate-graphite paste electrodes implanted unilaterally in the nucleus accumbens and striatum of urethane (1.5g/kg) anesthetized rats, following the pharmacological inhibition of ventral tegmental area/substantia nigra pars compacta muscarinic receptors. The facilitatory effects of morphine (2.0mg/kg, i.v.) on accumbens and striatal dopamine efflux were markedly reduced by prior infusion of the non-selective muscarinic receptor antagonist scopolamine (200μg/μl) into the ventral tegmental area or substantia nigra pars compacta, respectively. These findings demonstrate that decreased activation of midbrain muscarinic receptors attenuates the excitatory effects of morphine on mesoaccumbens and nigrostriatal dopaminergic transmission.

The dopamine (DA) precursor, l -DOPA (500 μg), was injected into living crickets, which were ingested (one each) by adult male Anolis carolinensis . This method of delivery elevated plasma l -DOPA and DA concentrations by ∼1000-fold. In contrast, plasma epinephrine (Epi) and norepinephrine (NE) were not influenced by l -DOPA treatment, although they were elevated following the consumption of the cricket. Lizards that ingested l -DOPA treated crickets had elevated l -DOPA in all brain regions measured, with DA and/or DOPAC also increased significantly in most brain regions studied. Despite increased DA levels in the striatum and nucleus accumbens as a response to l -DOPA, the treatment had no influence on general motor activity. Central serotonin, NE, and Epi systems were not affected in any brain region by oral l -DOPA treatment. In addition, aggression was inhibited by this dose of l -DOPA, even though there was no effect on serotonergic systems. This is surprising because controlling aggressive behavior is usually considered the province of serotonergic activity. Aggression was measured before and after treatment, and while saline-treated lizards retained the full vigor of aggressive activity, those fed a cricket injected with l -DOPA were only one-third as aggressive after treatment. As l -DOPA treatment did not affect general motor activity, the effect appears to be directly associated with aggression. This is supported by the observation that l -DOPA treatment delayed latency to eyespot darkening, which predicts the latency to aggression.

The pedunculopontine tegmental nucleus appears to influence striatal dopamine activity via cholinergic and glutamatergic afferents to dopaminergic cells of the substantia nigra pars compacta. We measured changes in striatal dopamine oxidation current (dopamine efflux) in response to electrical stimulation of the pedunculopontine tegmental nucleus using in vivo electrochemistry in urethane-anaesthetized rats. Pedunculopontine tegmental nucleus stimulation evoked a three-component change in striatal dopamine efflux, consisting of: (i) an initial rapid increase of 2 min duration; followed by (ii) a decrease below prestimulation levels of 9 min duration; then by (iii) a prolonged increase lasting 35 min. Intra-nigral infusions of the ionotropic glutamate receptor antagonist kynurenate (10 microg/ microL) or the nicotinic cholinergic receptor antagonist mecamylamine (5 microg/0.5 microL) selectively attenuated the rapid first component, while systemic injections of the muscarinic cholinergic antagonist scopolamine (5 mg/kg, i.p.) diminished the second and third components. In addition, intra-pedunculopontine tegmental nucleus infusions of the M2 muscarinic antagonist methoctramine (50 microg/ microL) selectively abolished the inhibitory second component, while intranigral infusions of scopolamine (200 microg/ microL) selectively abolished the prolonged third component. Intra-nigral infusions of the metabotropic glutamate receptor antagonist (+)-alpha-methyl-4-carboxyphenylglycine (2 microg/ microL) had no effect on pedunculopontine tegmental nucleus-elicited striatal dopamine efflux. These results suggest that the pedunculopontine tegmental nucleus utilizes nicotinic and ionotropic glutamate receptors in the substantia nigra to mediate rapid activation, M2-like muscarinic autoreceptors in the pedunculopontine tegmental nucleus to mediate decreased activation, and muscarinic receptors in the substantia nigra (probably of the M5 subtype) to mediate prolonged activation, of the nigrostriatal dopaminergic system.

Male jacky dragons, Amphibolurus muricatus, indicate territoriality to rivals during the mating season through the use of stereotyped motor displays. The relationship between corticosterone (B) and testosterone (T) and its effects on territorial display expression were investigated in captive lizards. Results demonstrated that territorial display production was most effectively predicted by elevated baseline T levels. This parallels the heightened T concentrations exhibited by males in the field during the spring mating period. The effect of social interaction on B and T levels was also examined by presenting territory-holding males with a size-matched male intruder. Resident males exhibited clear differences in the level of territorial response evoked by a male intruder, which were correlated with differences in physiological activity. Males that gave no territorial response had moderate B levels that did not change with social interaction, and significantly lower T levels than males that responded to the intruder with territorial displays. Among displaying males, those exhibiting low levels of territorial responses showed no change in B or T. In contrast, high-level territorial responders exhibited acute B increases when faced with an intruder, which might assist in supporting sustained metabolic activity and could possibly reflect differences in the perception of social stress. These same males also exhibited a reduction in T levels at such times, probably due to the acute rise in B. Combined, these results suggest that high circulating T is acting in a preparatory manner to increase the likelihood of producing a territorial response upon engaging in a social encounter. However, once this response has been initiated T may not need to remain elevated to allow continued expression of territorial behavior.

Increased depressive and anxiety-like behaviors are exhibited by rats and humans during withdrawal from psychostimulants. Anxiety-like behaviors observed during amphetamine withdrawal are mediated by increased expression and activity of corticotropin releasing factor type 2 (CRF 2 ) receptors in the dorsal raphe nucleus (dRN). Anxiety-like behavior of rats during withdrawal can be reversed by CRF 2 receptor antagonism in the dRN, but the efficacy of global central CRF 2 receptor antagonism is unknown. Rats were treated with amphetamine (2.5 mg/kg, ip.) or saline daily for 2 weeks, and were tested for anxiety-like behaviors during withdrawal. Rats undergoing withdrawal showed increased anxiety-like behavior, which was reduced by ventricular infusion of the CRF 2 antagonist antisauvagine-30 (ASV 2 μg/2 μl). Surprisingly, ventricular ASV increased anxiety-like behavior in rats pre-treated with saline, but had an anxiolytic effect in un-treated rats. Western blots were performed to determine whether differences in CRF receptor densities could explain ASV-induced behavioral results. Saline pre-treated rats showed reduced CRF 1 receptor expression in the lateral septum compared to amphetamine pre-treated and un-treated rats. Overall, these results suggest that central CRF 2 antagonism reduces anxiety states during amphetamine withdrawal, and that behavioral effects may be dependent upon the balance of CRF 1 and CRF 2 receptor activity in anxiety-related regions.

Withdrawal from amphetamine increases anxiety and reduces the ability to cope with stress, which are factors that are believed to contribute to drug relapse. Stress-induced serotonergic transmission in the central nucleus of the amygdala is associated with anxiety states and fear. Conversely, stress-induced increases in ventral hippocampal serotonin (5-HT) levels have been linked to coping mechanisms. The goal of this study was to investigate the neurobiological changes induced by amphetamine that contribute to stress sensitivity during withdrawal. We tested the hypothesis that limbic serotonergic responses to restraint stress would be altered in male Sprague-Dawley rats chronically pretreated with amphetamine (2.5 mg/kg, intraperitoneal) and then subjected to 2 weeks of withdrawal. Amphetamine withdrawal resulted in increased stress-induced behavioral arousal relative to control treatment, suggesting that drug withdrawal induced greater sensitivity to the stressor. When microdialysis was used to determine the effects of restraint on extracellular 5-HT, stress-induced increases in 5-HT levels were abolished in the ventral hippocampus and augmented in the central amygdala during amphetamine withdrawal. Reverse dialysis of the glucocorticoid receptor antagonist mifepristone into the ventral hippocampus blocked the stress-induced increase in 5-HT levels in saline-pretreated rats, suggesting that glucocorticoid receptors mediate stress-induced increases in 5-HT levels in the ventral hippocampus. However, mifepristone had no effect on stress-induced increases in 5-HT levels in the central amygdala, indicating that stress increases 5-HT levels in this region independently of glucocorticoid receptors. During amphetamine withdrawal, the absence of stress-induced increases in ventral hippocampal 5-HT levels combined with enhanced stress-induced serotonergic responses in the central amygdala may contribute to drug relapse by decreasing stress-coping ability and heightening stress responsiveness.

Cholinergic and glutamatergic neurons in the laterodorsal tegmentum (LDT) and neighbouring mesopontine nuclei are thought to influence mesolimbic dopaminergic neuronal activity involved in goal-directed behaviours. We measured the changes in dopamine oxidation current (corresponding with dopamine efflux) in the nucleus accumbens (NAc) in response to electrical stimulation of the LDT using in vivo chronoamperometry in urethane-anaesthetized rats. LDT stimulation (35 Hz pulse trains for 60 s, 1 s intertrain interval) evoked a three-component change in dopamine efflux in the NAc: (i) an initial stimulation time-locked increase in the dopamine signal above baseline, followed by (ii) an immediate decrease below baseline, and thereafter by (iii) a prolonged increase in the dopamine signal above baseline. Intra-VTA infusion of the nicotinic receptor antagonist mecamylamine (5 microg/0.5 microL) or the ionotropic glutamate receptor antagonist kynurenate (10 microg/microL) attenuated the first LDT-elicited component. The second suppressive component was abolished by intra-LDT infusions of either the nonselective or the M2-selective muscarinic receptor antagonists scopolamine (100 microg/microL) and methoctramine (50 microg/microL), respectively. In contrast, intra-VTA infusions of scopolamine (200 microg/microL) resulted in a selective attenuation of the third facilitatory component, whereas both second and third components were abolished by systemic injections of scopolamine (5 mg/kg). These results suggest that the initial increase, subsequent decrease, and final prolonged increase in extracellular dopamine levels in the NAc are selectively mediated by LDT-elicited activation of (i) nicotinic and glutamatergic receptors in the VTA, (ii) muscarinic M2 autoreceptors on LDT cell bodies, and (iii) muscarinic receptors in the VTA, respectively.

Background Adolescent social stress is associated with increased incidence of mental illnesses in adulthood that are characterized by deficits in cognitive focus and flexibility. Such enhanced vulnerability may be due to psychosocial stress-induced disruption of the developing mesocortical dopamine system, which plays a fundamental role in facilitating complex cognitive processes such as spatial working memory. Adolescent rats exposed to repeated social defeat as a model of social stress develop dopaminergic hypofunction in the medial prefrontal cortex as adults. To evaluate a direct link between adolescent social stress and later deficits in cognitive function, the present study tested the effects of adolescent social defeat on two separate tests of spatial working memory performance. Methods Adult rats exposed to adolescent social defeat and their controls were trained on either the delayed win-shift task or the delayed alternating T-Maze task and then challenged with various delay periods. To evaluate potential differences in motivation for the food reward used in memory tasks, consumption and conditioned place preference for sweetened condensed milk were tested in a separate cohort of previously defeated rats and controls. Results Compared to controls, adult rats defeated in adolescence showed a delay-dependent deficit in spatial working memory performance, committing more errors at a 90 s and 5 min delay period on the T-maze and win-shift tasks, respectively. Observed memory deficits were likely independent of differences in reward motivation, as conditioned place preference for the palatable food used on both tasks was similar between the adolescent social defeat group and control. Conclusions The results demonstrate that severe social stressors during adolescence can produce long term deficits in aspects of cognitive function. Given the dependence of spatial working memory on prefrontal dopamine, pharmacologically reversing dopaminergic deficiencies caused by adolescent social stress has the potential to treat such cognitive deficits.

Anxiety is a term often used to encompass feelings of apprehension, dread, unease or similarly unpleasant emotions. Trait anxiety defines the affect of an organism over time and across situations, whereas state anxiety is the response or adaptation to a given situation [1]. Anxiety can be differentiated from fear, both biologically and behaviorally [see 1 for an extensive review]. Converging theories and evidence from clinical psychology and comparative neuroscience suggest that fear can be considered a negatively-valenced emotion that is brief, focused on the present, occurs in situations of specific threat, and aids in avoidance or escape [1,2]. Anxiety, on the other hand, is a negatively-valenced emotion that is characterized by sustained hyperarousal in response to uncertainty, is thus futurefocused, and aids in defensive approach or risk assessment [1,2]. Both anxiety and fear are emotions experienced by all individuals and can serve to be adaptive in shaping decisions and behaviors related to survival of an organism [1,3]. However, when excessive, or pathological, or triggered inappropriately, fear and anxiety form the basis of a variety of anxiety disorders [3,4,5; Table 1]. As illustrated by Table 1, some anxiety disorders such as generalized anxiety disorder (GAD) or obsessive-compulsive disorder (OCD) are characterized by excessive anxiety as defined above [1]. However, other anxiety disorders are characterized, at least in part, by excessive and inappropriate fear, such as posttraumatic stress disorder (PTSD), specific phobias and social anxiety disorder [1,3; Table 1]. Thus, it is important to understand the neurobiology of both anxiety and fear to obtain a comprehensive picture of the physiological basis of anxiety disorders.

Maturation of mesocorticolimbic dopamine systems occurs during adolescence, and exposure to social stress during this period results in behavioral dysfunction including substance abuse disorders. Adult male rats exposed to repeated social defeat in adolescence exhibit reduced basal dopamine tissue content in the medial prefrontal cortex, altered dopamine tissue content in corticoaccumbal dopamine regions following acute amphetamine, and increased amphetamine conditioned place preference following repeated amphetamine treatment. Such changes may reflect altered amphetamine-induced extracellular dopamine release in the corticoaccumbal regions. Therefore, we used in vivo microdialysis to measure extracellular dopamine simultaneously within the medial prefrontal cortex and nucleus accumbens core of previously defeated rats and controls, in response to either acute or repeated (7 daily injections) of amphetamine (1.0 mg/kg). Locomotion responses to acute / repeated amphetamine were also assessed the day prior to taking dopamine measurements. Adolescent defeat potentiated adult locomotion responses to acute amphetamine, which was negatively correlated with attenuated amphetamine-induced dopamine release in the medial prefrontal cortex, but there was no difference in amphetamine-induced accumbal dopamine release. However, both locomotion and corticoaccumbal dopamine responses to repeated amphetamine were equivalent between previously defeated rats and controls. These data suggest adolescent defeat enhances behavioral responses to initial amphetamine exposure as a function of diminished prefrontal cortex dopamine activity, which may be sufficient to promote subsequently enhanced seeking of drug-associated cues. Interestingly, repeated amphetamine treatment appears to normalize amphetamine-elicited locomotion and cortical dopamine responses observed in adult rats exposed to adolescent social defeat, providing implications for treating stress-induced dopamine dysfunction.

Selective-breeding of house mice for increased voluntary wheel-running has resulted in multiple physiological and behavioral changes. Characterizing these differences may lead to experimental models that can elucidate factors involved in human diseases and disorders associated with physical inactivity, or potentially treated by physical activity, such as diabetes, obesity, and depression. Herein, we present ethological data for adult males from a line of mice that has been selectively bred for high levels of voluntary wheel-running and from a non-selected control line, housed with or without wheels. Additionally, we present concentrations of central monoamines in limbic, striatal, and midbrain regions. We monitored wheel-running for 8 weeks, and observed home-cage behavior during the last 5 weeks of the study. Mice from the selected line accumulated more revolutions per day than controls due to increased speed and duration of running. Selected mice exhibited more active behaviors than controls, regardless of wheel access, and exhibited less inactivity and grooming than controls. Selective-breeding also influenced the longitudinal patterns of behavior. We found statistically significant differences in monoamine concentrations and associated metabolites in brain regions that influence exercise and motivational state. These results suggest underlying neurochemical differences between selected and control lines that may influence the observed differences in behavior. Our results bolster the argument that selected mice can provide a useful model of human psychological and physiological diseases and disorders.

Previous correlational studies suggest that mild traumatic brain injury (TBI) is associated with an increase in the prevalence of anxiety disorders, including posttraumatic stress disorder (PTSD). By using a relevant animal model, this research demonstrates that mild TBI can result in increased fear learning and anxiety states that mimic the symptoms of generalized anxiety disorder and PTSD. Furthermore, this research suggests that heightened fear and anxiety states following mild TBI may result from alterations to cell death and neuronal number in limbic brain regions such as the hippocampus and amygdala. Recent data generated from this project also suggests that experiencing psychosocial stress concurrent with mild brain injury results in serotonin dysfunction in the limbic system (particularly the hippocampus) and exacerbates anxiety states. Therefore, changes to limbic system function may underlie anxiety states that manifest following mild TBI and stress. Overall, the current rodent model of mild TBI appears to be a useful model to explore the neurobiological link between mild TBI and anxiety states, to allow better understanding of the consequences of war injuries like mild TBI, and to explore how such injury could be treated to prevent long-term disability.

Previous studies have shown that the Wistar-Kyoto (WKY) rat strain may be a genetic model of depression when their behaviors are compared to Sprague-Dawley (SD) or Wistar (WIS) rats. Significant differences in dopamine (DA), serotonin (5-HT), and norepinephrine (NE) transporter site densities have been reported when comparing WKY to both SD and WIS rats. Susceptibility of WKY rats to anxiety and depressive behavior may be related to underlying differences in monoamine levels in various regions of the brain. Levels of monoamines (DA, 5-HT and NE) and their metabolites were measured in monoaminergic cell body, cortical and limbic brain regions using HPLC with electrochemical detection and compared between WKY, WIS and SD rats. In regions where strain differences in monoamine levels were observed (the basolateral amygdala, subregions of the hippocampus and the nucleus accumbens shell), WKY rats consistently had lower levels than SD rats. Similarly, WKY rats had lower monoamine levels compared to WIS, although these differences were observed in a more restricted number of brain regions. Interestingly, WIS rats showed reduced levels of the 5-HT metabolite 5-hydroxyindoleacetic acid (5-HIAA) in several regions including the prefrontal cortex, subregions of the hippocampus and subregions of the hypothalamus, suggesting decreased 5-HT turnover when compared to both WKY and SD rats. Overall, these results imply that decreased monoamine levels, combined with alterations in transporter sites, may be related to the predisposition of WKY rats towards depressive behavior.

Acute amphetamine administration activates monoaminergic pathways and increases systemic corticosterone, both of which influence anxiety states and adult dentate gyrus neurogenesis. Chronic amphetamine increases anxiety states in rats when measured at 24 hours and at 2 weeks of withdrawal. However, the effects of chronic amphetamine exposure and withdrawal on long term anxiety-like behavior and adult neurogenesis in the dentate gyrus are unknown. Adult male rats were administered amphetamine (2.5 mg/kg, ip.) daily for two weeks. Anxiety-like behaviors were increased markedly in amphetamine-treated rats following four weeks of withdrawal from amphetamine. Plasma corticosterone level was unaltered by amphetamine treatment or withdrawal. However, norepinephrine and serotonin concentrations were selectively reduced in the dentate gyrus 20 hours following amphetamine treatment. This effect did not persist through the four-week withdrawal period. In separate experiments, rats received bromodeoxyuridine to label cells in S-phase, prior to or immediately following amphetamine treatment. Newly generated cells were quantified to measure extent of progenitor cell proliferation and neurogenesis following treatment or withdrawal. Progenitor cell proliferation and neurogenesis were not significantly affected by amphetamine exposure when measured 20 hours following the last amphetamine treatment. However, neurogenesis in the dentate gyrus was reduced after four weeks of withdrawal when compared to saline-pretreated rats. Overall, our findings indicate that withdrawal from chronic amphetamine leads to persistent anxiety-like behavior which may be maintained by reduced neurogenesis in the dentate gyrus at this protracted withdrawal time point. However, neurogenesis is unaffected at earlier withdrawal time points where anxiety states emerge, suggesting different mechanisms may underlie the emergence of anxiety states during amphetamine withdrawal.

The mesocorticolimbic dopamine system, which governs components of reward and goal-directed behaviors, undergoes final maturation during adolescence. Adolescent social stress contributes to adult behavioral dysfunction, and is linked to adult psychiatric and addiction disorders. Here, behavioral, corticosterone, and limbic dopamine responses to amphetamine were examined in adult male rats previously exposed to repeated social defeat stress during mid-adolescence. Amphetamine (2.5 mg/kg, ip) was administered after a novel environment test, with behavior observed in the same context for 90 min thereafter. Adult rats that had been defeated in adolescence showed increased locomotion in the novel environment, but reduced amphetamine-induced locomotion relative to non-defeated age matched controls. Monoamine and corticosterone responses to amphetamine were examined following a second amphetamine injection 3 days later. In previously defeated rats, corticosterone and medial prefrontal cortex dopamine responses to amphetamine were blunted while dopamine responses in the nucleus accumbens core were elevated. Our results suggest that experience of social defeat stress during adolescent development can contribute to altered behavioral and endocrine responses to amphetamine in adulthood. Furthermore, these effects are paralleled by changes in amphetamine-induced dopamine responses in corticolimbic systems implicated in addiction disorders.

Amphetamine use is associated with dysphoric states, including heightened anxiety, that emerge within 24 h of withdrawal from the drug. Corticotropin-releasing factor increases serotonin release in the central nucleus of the amygdala, and this neurochemical circuitry may play a role in mediating fear and anxiety states. We have previously shown that chronic amphetamine treatment increases corticotropin-releasing factor receptor type-2 levels in the serotonergic dorsal raphe nucleus of the rat. Therefore, we hypothesized that chronic amphetamine treatment would enhance the amygdalar serotonergic response to corticotropin-releasing factor infused into the dorsal raphe nucleus. Male rats were injected once-daily with d-amphetamine (2.5 mg/kg i.p., or saline) for two weeks. Serotonin release within the central nucleus of the amygdala in response to intra-raphe infusion of corticotropin-releasing factor (100 ng) was measured 24 h after the last treatment in urethane-anesthetized (1.8 mg/kg, i.p.) rats using in vivo microdialysis. Rats pretreated with amphetamine showed significantly enhanced serotonin release in the central nucleus of the amygdala in response to corticotropin-releasing factor infusion when compared to saline pretreated rats. Furthermore, this enhanced response was blocked by the corticotropin-releasing factor type-2 receptor antagonist antisauvagine-30 (2 μg) infused into the dorsal raphe nucleus. These results suggest increased sensitivity to corticotropin-releasing factor as mediated by type-2 receptors following chronic amphetamine treatment, which may underlie dysphoric states observed during amphetamine withdrawal.

Withdrawal from psychostimulants increases anxiety states, and amphetamine-treated rats show increased CRF(2) receptors in the serotonergic cell body region, the dorsal raphe nucleus (dRN). In the current study, amphetamine (2.5 mg/kg, i.p., 14 days) pre-treated rats spent less time in open arms of the elevated plus maze compared saline pre-treated rats at both 24h or 2 weeks of withdrawal, and CRF(2) receptor antagonism (ASV-30; 2 microg/0.5 microl) within the dRN reversed the effects of amphetamine withdrawal on anxiety-like behavior. Overall, results suggest that CRF(2) receptor antagonism may be a novel pharmacological target for anxiety states during drug withdrawal.

The dorsomedial hypothalamus (DMH) has been implicated in the coordination of stress responses. Restraint stress or systemic corticosterone (CORT) treatment induces a rapid increase in tissue concentrations of serotonin (5-hydroxytryptamine; 5-HT) in the DMH. Although the mechanism for rapid changes in 5-HT concentrations in the DMH is not clear, earlier results suggest that stress-induced increases in CORT may inhibit 5-HT transport from the extracellular fluid by acting on corticosterone-sensitive organic cation transporters (OCTs). We tested the hypothesis that perfusion of the medial hypothalamus (MH), which includes the DMH, with the OCT blocker decynium 22 (D-22) would potentiate the effects of mild restraint on extracellular 5-HT. Male Sprague–Dawley rats, implanted with a microdialysis probe into the MH, were treated with reverse-dialysis of D-22 (20 µM; 40 min) or vehicle and subjected to either 40 min mild restraint or undisturbed control conditions. Perfusates collected from a separate group of rats were evaluated for the effect of restraint on extracellular CORT concentrations in the MH. Reverse-dialysis of D-22 induced an increase (200%) in extracellular 5-HT concentrations in the MH in undisturbed control rats. Restraint in the absence of D-22 did not significantly affect MH CORT or 5-HT concentrations. However, perfusion of the MH with D-22 during restraint led to an increased magnitude and duration of extracellular 5-HT concentrations, relative to D-22 by itself. These results are consistent with the hypothesis that OCTs in the DMH contribute to the clearance of 5-HT from the extracellular fluid under both baseline conditions and mild restraint.

Exposure to adverse experiences in early-life is implicated in the later vulnerability to development of psychiatric disorders, including anxiety and affective disorders in humans. Adverse early-life experiences likely impart their long-term consequences on mental health by disrupting the normal development of neural systems involved in stress responses, emotional behavior and emotional states. Neural systems utilizing the neurotransmitters serotonin, dopamine and the neuropeptide corticotropin-releasing factor (CRF) are implicated in mediating emotive behaviors, and dysfunction of these neurochemical systems is associated with mood/anxiety disorders. These neural systems continue maturing until early or mid-adolescence in humans, thus alterations to their development are likely to contribute to the long-term consequences of adverse early-life experiences. A large body of literature suggests that post-weaning isolation rearing of rodents models the behavioral consequences of adverse early-life experiences in humans. Overall, the majority findings suggest that post-weaning social isolation that encompasses pre-adolescence produces long-lasting alterations to anxiety behavior, while measures of monoaminergic activity in various limbic regions during social isolation suggest alterations to dopamine and serotonin systems. The goal of this review is to evaluate and integrate findings from post-weaning social isolation studies specifically related to altered fear and anxiety behaviors and associated changes in neuroendocrine function and the activity of monoaminergic systems.

The dorsomedial hypothalamus (DMH) plays an important role in coordinating physiological and behavioral responses to stress-related stimuli. In vertebrates, DMH serotonin (5-HT) concentrations increase rapidly in response to acute stressors or corticosterone (CORT). Recent studies suggest that CORT inhibits postsynaptic clearance of 5-HT from the extracellular fluid in the DMH by blocking organic cation transporter 3 (OCT3), a polyspecific CORT-sensitive transport protein. Because OCTs are low-affinity, high-capacity transporters, we hypothesized that CORT effects on extracellular 5-HT are most pronounced in the presence of elevated 5-HT release. We predicted that local application of CORT into the DMH would potentiate the effects of d-fenfluramine, a 5-HT-releasing agent, on extracellular 5-HT. These experiments were conducted using in vivo microdialysis in freely-moving male Sprague-Dawley rats implanted with a microdialysis probe into the medial hypothalamus (MH), which includes the DMH. In Experiment 1, rats simultaneously received intraperitoneal (i.p.) injections of 1 mg/kg D-fenfluramine or saline and either 200 ng/mL CORT or dilute ethanol (EtOH) vehicle delivered to the MH by reverse-dialysis for 40 min. In Experiment 2, 5 microM D-fenfluramine and either 200 ng/mL CORT or EtOH vehicle were concurrently delivered to the MH for 40 min using reverse-dialysis. CORT potentiated the increases in extracellular 5-HT concentrations induced by either i.p. or intra-MH administration of D-fenfluramine. Furthermore, CORT and D-fenfluramine interacted to alter home cage behaviors. Our results support the hypothesis that CORT inhibition of OCT3-mediated 5-HT clearance from the extracellular fluid contributes to stress-induced increases in extracellular 5-HT and 5-HT signaling.

Interactions between central corticotropin-releasing factor (CRF) and serotonergic systems are believed to be important for mediating fear and anxiety behaviors. Recently we demonstrated that infusions of CRF into the rat dorsal raphe nucleus result in a delayed increase in serotonin release within the medial prefrontal cortex that coincided with a reduction in fear behavior. The current studies were designed to study the CRF receptor mechanisms and pathways involved in this serotonergic response. Infusions of CRF (0.5 microg/0.5 microL) were made into the dorsal raphe nucleus of urethane-anesthetized rats following either inactivation of the median raphe nucleus by muscimol (25 ng/0.25 microL) or antagonism of CRF receptor type 1 or CRF receptor type 2 in the dorsal raphe nucleus with antalarmin (25-50 ng/0.5 microL) or antisauvagine-30 (2 microg/0.5 microL), respectively. Medial prefrontal cortex serotonin levels were measured using in-vivo microdialysis and high-performance liquid chromatography with electrochemical detection. Increased medial prefrontal cortex serotonin release elicited by CRF infusion into the dorsal raphe nucleus was abolished by inactivation of the median raphe nucleus. Furthermore, antagonism of CRF receptor type 2 but not CRF receptor type 1 in the dorsal raphe nucleus abolished CRF-induced increases in medial prefrontal cortex serotonin. Follow-up studies involved electrical stimulation of the central nucleus of the amygdala, a source of CRF afferents to the dorsal raphe nucleus. Activation of the central nucleus increased medial prefrontal cortex serotonin release. This response was blocked by CRF receptor type 2 antagonism in the dorsal raphe. Overall, these results highlight complex CRF modulation of medial prefrontal cortex serotonergic activity at the level of the raphe nuclei.

Social isolation of rodents during development is thought to be a relevant model of early-life chronic stress. We investigated the effects of early-life social isolation on later adult fear and anxiety behavior, and on corticosterone stress responses, in male rats. On postnatal day 21, male rats were either housed in isolation or in groups of 3 for a 3 week period, after which, all rats were group-reared for an additional 2 weeks. After the 5-week treatment, adult rats were examined for conditioned fear, open field anxiety-like behavior, social interaction behavior and corticosterone responses to restraint stress. Isolates exhibited increased anxiety-like behaviors in a brightly-lit open field during the first 10 min of the test period compared to group-reared rats. Isolation-reared rats also showed increased fear behavior and reduced social contact in a social interaction test, and a transient increase in fear behavior to a conditioned stimulus that predicted foot-shock. Isolation-reared rats showed similar restraint-induced increases in plasma corticosterone as group-reared controls, but plasma corticosterone levels 2 h after restraint were significantly lower than pre-stress levels in isolates. Overall, this study shows that isolation restricted to an early part of development increases anxiety-like and fear behaviors in adulthood, and also results in depressed levels of plasma corticosterone following restraint stress.

Psychostimulant use increases anxious behavior, likely through interactions between central corticotropin-releasing factor (CRF) and serotonergic systems. The current study examined whether chronic amphetamine treatment (2.5mg/kg, 14 days) or withdrawal altered CRF receptor densities in the serotonergic dorsal raphe nucleus (dRN). Amphetamine treatment increased CRF(2) receptor densities in most subregions of the dRN, and CRF(2) receptors were still elevated following 6 weeks of withdrawal. No changes in CRF(1) receptor densities were observed following amphetamine treatment or during withdrawal. Selective increases in dRN CRF(2) receptors may be related to increased anxiety-like behaviors following psychostimulant use.

Amphetamine withdrawal increases anxiety and stress sensitivity related to blunted ventral hippocampus (vHipp) and enhances the central nucleus of the amygdala (CeA) serotonin responses. Extracellular serotonin levels are regulated by the serotonin transporter (SERT) and organic cation transporter 3 (OCT3), and vHipp OCT3 expression is enhanced during 24 hours of amphetamine withdrawal, while SERT expression is unaltered. Here, we tested whether OCT3 and SERT expression in the CeA is also affected during acute withdrawal to explain opposing regional alterations in limbic serotonergic neurotransmission and if respective changes continued with two weeks of withdrawal. We also determined whether changes in transporter expression were confined to these regions. Male rats received amphetamine or saline for two weeks followed by 24 hours or two weeks of withdrawal, with transporter expression measured using Western immunoblot. OCT3 and SERT expression increased in the CeA at both withdrawal timepoints. In the vHipp, OCT3 expression increased only at 24 hours of withdrawal, with an equivalent pattern seen in the dorsomedial hypothalamus. No changes were evident in any other regions sampled. These regionally specific changes in limbic OCT3 and SERT expression may partially contribute to the serotonergic imbalance and negative affect during amphetamine withdrawal.

Corticotropin releasing factor (CRF) and serotonin (5-HT) are strongly linked to stress and anxiety in vertebrates. As a neuromodulator in the brain, CRF has anxiogenic properties often characterized by increased locomotion and stereotyped behavior in familiar environments. We hypothesized that expression of anxiogenic behavior in response to CRF will also be exhibited in a teleost fish. Rainbow trout were treated with intracerebroventricular (icv) injections of artificial cerebrospinal fluid (aCSF), 500 or 2000 ng ovine CRF, or not injected. Treatment with either dose of CRF elicited greater locomotion and pronounced head shaking behavior, but did not influence water column position. Locomotor and head-shaking behaviors may be analogous to the increased stereotypy evoked by icv CRF in rats, and may reflect the expression of stress/anxiety behavior. Injection with either aCSF or CRF produced significant increases in plasma cortisol. The absence of behavioral changes in aCSF injected fish suggests that the behavioral responses following CRF were not due to cortisol. Treatment with 2000 ng CRF significantly increased serotonin, 5-HIAA and dopamine concentrations in the subpallium and raphé, and increased 5-HIAA in the preoptic hypothalamus (POA). Concurrent effects of CRF on central monoamines, locomotion, and headshaking in trout suggest that anxiogenic properties of CRF are evolutionarily conserved. In addition, positive linear correlations between locomotion and serotonergic and dopaminergic function in the subpallium, POA, and raphé nuclei suggest a locomotory function for these monoamines.

Corticotropin-releasing factor (CRF) is a neurohormone that mediates stress, anxiety, and affects serotonergic activity. Studies have shown that CRF has dose-dependent opposing effects on serotonergic activity. This effect has been hypothesized to be differentially mediated by CRF(1) and CRF(2) receptors in the dorsal raphe nucleus. We directly tested this hypothesis by using in vivo microdialysis to determine the effects of CRF and CRF receptor antagonists in the dorsal raphe nucleus on serotonin (5-HT) release in the nucleus accumbens, a brain region implicated in the neuropathology of stress-related psychiatric disorders. Male urethane-anesthetized rats were implanted with a microdialysis probe into the nucleus accumbens, and CRF (0, 100 or 500 ng) was infused into the dorsal raphe. Infusion of CRF into the dorsal raphe nucleus had dose-dependent opposite effects, with 100 ng of CRF significantly decreasing 5-HT levels in the nucleus accumbens and 500 ng CRF significantly increasing accumbal 5-HT levels. In subsequent experiments, the raphe was pre-treated with the CRF(1) receptor antagonist antalarmin (0.25 microg) or the CRF(2) receptor antagonist antisauvagine-30 (ASV-30; 2 microg) prior to CRF infusion. Antagonism of CRF(1) receptors in the dorsal raphe nucleus abolished the decrease in accumbal 5-HT levels elicited by 100 ng CRF, and CRF(2) receptor antagonism in the raphe blocked the increase in accumbal 5-HT levels elicited by 500 ng CRF. These results suggest that the opposing effects of dorsal raphe CRF on 5-HT release in the nucleus accumbens are dependent on differential activation of CRF(1) and CRF(2) receptors in the dorsal raphe nucleus.

Male Anolis carolinensis that win aggressive interactions mobilize neuroendocrine responses to social stress more rapidly than defeated lizards. We initially examined temporal patterns of neuroendocrine response to restraint stress in lizards of unknown status, and then investigated whether winning males respond more rapidly to this non-social stressor. Size-matched male pairs interacted to establish social status, and then were returned to individual home cages for 3 days. Plasma and brains were collected from non-restrained dominants and subordinates, and from a non-interacting control group. Additional groups of dominants and subordinates underwent 90 s restraint stress, with plasma and brains collected either immediately or 300 s after restraint. In lizards of unknown social status restraint stimulated rapid monoaminergic responses in nucleus accumbens, hippocampus, amygdala, and locus ceruleus, with delayed responses seen in VTA and raphe. Non-restrained dominants and subordinates had lower levels of raphe serotonergic activity and lower hippocampal dopaminergic activity 3 days after interacting, compared to controls. Dominants had higher corticosterone levels, both immediately and 300 s after restraint, than either non-restrained dominants or restrained subordinates. Restraint induced higher raphe serotonergic activity in dominants. However, subordinates also showed rapid responses to restraint; exhibiting lower hippocampal dopamine (DA) levels than non-restrained subordinates. At 300 s after the stress, amygdalar serotonin levels increased in dominants, while subordinates showed higher amygdalar DA levels. These results suggest that stressful aggressive interactions will not only alter basal neurochemical activity, but also influence neuroendocrine responses to non-social stressors according to individual social status.

Social interaction may elicit aggression, establish social rank, and be influenced by changes in central dopaminergic activity. In the lizard Anolis carolinensis, a sign stimulus (darkening of postorbital skin or eyespots) inhibits aggressive response from opponents, in part because it forms more rapidly in dominant males. The authors report that artificially hiding or darkening eyespots influences central dopaminergic activity, social status, and aggression during dyadic social interaction. All males that viewed an opponent with eyespots painted black became subordinate and exhibited elevated dopamine in raphe, lateral amygdala, and medial amygdala but decreased dopamine in septum and locus ceruleus. In contrast, males that viewed opponents with hidden eyespots (painted green) became dominant and had increased dopamine in striatum, nucleus accumbens, hypothalamus, and combined substantia nigra/ventral tegmental area.

The neurotransmitters serotonin and corticotrophin-releasing factor are thought to play an important role in fear and anxiety behaviors. This study aimed to determine the relationship between corticotrophin-releasing factor-evoked changes in serotonin levels within discrete regions of the limbic system and the expression of fear behavior in rats. The effects of corticotrophin-releasing factor administration to the serotonin cell body regions of the dorsal raphe nucleus on fear behavior, behavioral activity, and extracellular serotonin levels were assessed in freely moving rats with microdialysis probes implanted into the central nucleus of the amygdala and the medial prefrontal cortex. Infusion of corticotrophin-releasing factor (0.5 microg) into the dorsal raphe rapidly induced freezing behavior, which was positively correlated with an immediate increase in serotonin release in the central nucleus of the amygdala. In contrast, cessation of freezing behavior correlated with a delayed and prolonged increase in serotonin release within the medial prefrontal cortex. Our findings suggest that corticotrophin-releasing factor-induced freezing behavior is associated with regionally and temporally distinct serotonergic responses in the limbic system that may reflect differing roles for these regions in the expression of fear/anxiety behavior.

Immobilization stress and physical activity separately influence monoaminergic function. In addition, it appears that stress and locomotion reciprocally modulate neuroendocrine responses, with forced exercise ameliorating stress-induced serotonergic activity in lizards. To investigate the interaction of forced physical activity and restraint stress on central dopamine (DA), norepinephrine (NE), and epinephrine (Epi), we measured these catecholamines and their metabolites in select brain regions of stressed and exercised male Anolis carolinensis lizards. Animals were handled briefly to elicit restraint stress, with some lizards additionally forced to run on a track until exhaustion, or half that time (50% of average time to exhaustion), compared to a control group that experienced no restraint or exercise. Norepinephrine concentrations in the hippocampus and locus ceruleus decreased with restraint stress, but returned to control levels following forced exhaustion. Levels of NE in the raphe nuclei and area postrema, and epinephrine in raphe became elevated following restraint stress, and returned to control levels following forced physical activity to 50% or 100% exhaustion. Striatal DA increased as animals were exercised to 50% of exhaustion, and returned to baseline with exhaustion. At exhaustion, striatal Epi levels were diminished, compared with controls. In the area postrema, exhaustion reversed a decline in epinephrine levels that followed forced physical activity. These results suggest that stress stimulates a rapid influence on central catecholamines. In addition, forced exercise, and even exhaustion, may alleviate the effects of restraint stress on central monoamines.

M5 muscarinic receptors are coexpressed with D2 dopamine receptors in the ventral tegmentum and striatum, and are important for reward in rodents. Previously, we reported that disruption of the M5 receptor gene in mice reduced dopamine release in the nucleus accumbens. In this study, we established a polymerase chain reaction (PCR) genotyping method for M5 mutant mice, and, using RT-PCR, found that M5 mRNA expression was highest in the ventral tegmentum, striatum, and thalamus in wild-type mice. In the M5 mutant mice, D2 mRNA expression was increased in several brain structures, including the striatum. Genome mapping studies showed the M5 gene is localized to chromosome 2E4 in mice, and to 15q13 in humans in the region that has been linked to schizophrenia. Amphetamine-induced locomotion, but not baseline locomotion or motor functions, decreased in M5 mutant mice, consistent with lower accumbal dopamine release. Previous reports found latent inhibition improvement in rats following nucleus accumbens lesions, or blockade of dopamine D2 receptors with neuroleptic drugs. Here, latent inhibition was significantly increased in M5 mutant mice as compared with controls, consistent with reduced dopamine function in the nucleus accumbens. In summary, our results showed that M5 gene disruption in mice decreased amphetamine-induced locomotion and increased latent inhibition, suggesting that increased M5 mesolimbic function may be relevant to schizophrenia.

Social stress is frequently used as a model for studying the neuroendocrine mechanisms underlying stress-induced behavioral inhibition, depression, and fear conditioning. It has previously been shown that social subordination may result in increased glucocorticoid release and changes in brain signaling systems. However, it is still an open question which neuroendocrine and behavioral differences are causes, and which are consequences of social status. Using juvenile rainbow trout of similar size and with no apparent differences in social history, we demonstrate that the ability to win fights for social dominance can be predicted from the duration of a behavioral response to stress, in this case appetite inhibition after transfer to a new environment. Moreover, stress responsiveness in terms of confinement-induced changes in plasma cortisol was negatively correlated to aggressive behavior. Fish that exhibited lower cortisol responses to a standardized confinement test were markedly more aggressive when being placed in a dominant social position later in the study. These findings support the view that distinct behavioral–physiological stress coping styles are present in teleost fish, and these coping characteristics influence both social rank and levels of aggression.

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 (NR(2B)) within the CA(3) 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.

The pedunculopontine tegmental nucleus appears to influence striatal dopamine activity via cholinergic and glutamatergic afferents to dopaminergic cells of the substantia nigra pars compacta. We measured changes in striatal dopamine oxidation current (dopamine efflux) in response to electrical stimulation of the pedunculopontine tegmental nucleus using in vivo electrochemistry in urethane-anaesthetized rats. Pedunculopontine tegmental nucleus stimulation evoked a three-component change in striatal dopamine efflux, consisting of: (i) an initial rapid increase of 2 min duration; followed by (ii) a decrease below prestimulation levels of 9 min duration; then by (iii) a prolonged increase lasting 35 min. Intra-nigral infusions of the ionotropic glutamate receptor antagonist kynurenate (10 microg/ microL) or the nicotinic cholinergic receptor antagonist mecamylamine (5 microg/0.5 microL) selectively attenuated the rapid first component, while systemic injections of the muscarinic cholinergic antagonist scopolamine (5 mg/kg, i.p.) diminished the second and third components. In addition, intra-pedunculopontine tegmental nucleus infusions of the M2 muscarinic antagonist methoctramine (50 microg/ microL) selectively abolished the inhibitory second component, while intranigral infusions of scopolamine (200 microg/ microL) selectively abolished the prolonged third component. Intra-nigral infusions of the metabotropic glutamate receptor antagonist (+)-alpha-methyl-4-carboxyphenylglycine (2 microg/ microL) had no effect on pedunculopontine tegmental nucleus-elicited striatal dopamine efflux. These results suggest that the pedunculopontine tegmental nucleus utilizes nicotinic and ionotropic glutamate receptors in the substantia nigra to mediate rapid activation, M2-like muscarinic autoreceptors in the pedunculopontine tegmental nucleus to mediate decreased activation, and muscarinic receptors in the substantia nigra (probably of the M5 subtype) to mediate prolonged activation, of the nigrostriatal dopaminergic system.

Cholinergic and glutamatergic projections from the laterodorsal tegmental nucleus (LDT) in the rat pons excite midbrain dopamine cells to directly modulate forebrain dopamine transmission. We show that LDT-lesioned rats express higher intensity stereotypy (including orofacial movements), and higher levels of accumbal dopamine release in response to d-amphetamine (1.5 mg/kg), as compared to sham-operated rats. In contrast, LDT-lesioned rats showed decreased stereotypy and attenuated accumbal dopamine efflux as compared to sham animals, in response to morphine (2.0 mg/kg). These results suggest that the LDT plays a critical role in mediating motoric and neurochemical effects of diverse drugs of abuse, and that the pharmacology of the drug may critically determine whether its efficacy will be enhanced or attenuated by alterations in LDT activity. We conclude that the LDT has functional connections with the nigrostriatal dopamine system to affect drug-evoked stereotypy, which has implications for motoric disorders that are characterized by nigrostriatal dysfunction.

Male jacky dragons, Amphibolurus muricatus, indicate territoriality to rivals during the mating season through the use of stereotyped motor displays. The relationship between corticosterone (B) and testosterone (T) and its effects on territorial display expression were investigated in captive lizards. Results demonstrated that territorial display production was most effectively predicted by elevated baseline T levels. This parallels the heightened T concentrations exhibited by males in the field during the spring mating period. The effect of social interaction on B and T levels was also examined by presenting territory-holding males with a size-matched male intruder. Resident males exhibited clear differences in the level of territorial response evoked by a male intruder, which were correlated with differences in physiological activity. Males that gave no territorial response had moderate B levels that did not change with social interaction, and significantly lower T levels than males that responded to the intruder with territorial displays. Among displaying males, those exhibiting low levels of territorial responses showed no change in B or T. In contrast, high-level territorial responders exhibited acute B increases when faced with an intruder, which might assist in supporting sustained metabolic activity and could possibly reflect differences in the perception of social stress. These same males also exhibited a reduction in T levels at such times, probably due to the acute rise in B. Combined, these results suggest that high circulating T is acting in a preparatory manner to increase the likelihood of producing a territorial response upon engaging in a social encounter. However, once this response has been initiated T may not need to remain elevated to allow continued expression of territorial behavior.

Cholinergic and glutamatergic cells in the pedunculopontine tegmental nucleus are a principal source of excitatory input to midbrain dopamine neurons projecting to the striatum. Disruption of these brainstem inputs has been shown to respectively enhance and reduce psychostimulant and opiate self-administration in rats. In the present study, d-amphetamine- and morphine-induced behaviors and dorsal striatal dopamine efflux, measured using in vivo chronoamperometry, were investigated 21 days after bilateral excitotoxic (ibotenate) lesions of the pedunculopontine in rats. Compared to sham-operated controls, pedunculopontine lesions enhanced stereotyped behaviors induced by a challenge injection of d-amphetamine (1.5 mg/kg, i.p.) to an extent that markedly interfered with the expression of locomotor behavior. A significant augmentation in striatal dopamine efflux was also observed in these lesioned animals under urethane anesthesia in response to a similar challenge injection of d-amphetamine (1.5 mg/kg, i.v.) 2 days following these behavioral observations. In direct contrast, pedunculopontine lesions in a separate group of rats significantly attenuated morphine-induced (2 mg/kg, i.p.) stereotyped activity, although no significant differences were observed in locomotion compared to sham-operated animals. Under urethane anesthesia, these lesions attenuated striatal dopamine efflux evoked by a similar challenge injection of morphine (2 mg/kg, i.v.). These findings indicate that the pedunculopontine differentially mediates the pharmacological actions of two diverse drugs of abuse on striatal dopamine neurotransmission and resultant behaviors. These results also imply that the pedunculopontine tegmental nucleus may serve as a major striatal-motor interface in the processing of salient environmental stimuli, and their incentive rewarding impact on dopamine-mediated behavioral responses.

Midbrain dopamine neurons are activated directly by cholinergic agonists or by stimulation of the cholinergic neurons in the laterodorsal tegmental nucleus (LDT) of the pons in rats. In urethane-anesthetized mice, electrical stimulation of the LDT resulted in a rapid, stimulus-time-locked increase in dopamine release in the nucleus accumbens (NAc), followed several minutes later by a prolonged increase in dopamine release. In mutant mice with truncated M5 receptors, the prolonged phase of dopamine release was absent, but the initial, rapid phase of dopamine release was fully observed. We conclude that M5 muscarinic receptors on midbrain dopamine neurons mediate a prolonged facilitation of dopamine release in the NAc. These results imply that M5 muscarinic receptors play an important role in motivational behaviors driven by dopamine activity in the accumbens.

Cholinergic and glutamatergic cells in the pedunculopontine tegmental nucleus (PPT) and adjacent laterodorsal tegmental nucleus (LDT) in the mammalian pons monosynaptically project onto midbrain dopaminergic neurons (Clements and Grant, 1990; Oakman et al., 1995). Electrical stimulation of the PPT results in increased dopamine efflux in the striatum, via excitatory actions on acetylcholine and glutamate receptors located in the midbrain substantia nigra (SN) (Forster and Blaha, in press). Similarly, electrical stimulation of the LDT increases dopamine efflux in the nucleus accumbens (NAc) by activating acetylcholine and glutamate receptors located in the midbrain ventral tegmental area (VTA) (Forster and Blaha, 2000; Forster et al., 2001; Yeomans et al., 2001). In addition, excitotoxic lesions of the PPT selectively attenuate striatal dopamine elevations evoked by cholinergic stimulation of the SN (Blaha and Winn, 1993). By comparison, lesions of the LDT, but not PPT, selectively attenuate the stimulatory effects of VTA cholinergic receptor activation on NAc dopamine efflux (Blaha et al., 1996). Together, these findings implicate the PPT in mediating striatal dopamine efflux via the nigrostriatal dopaminergic pathway, and the LDT in mediating NAc dopamine efflux via the mesolimbic dopaminergic pathway.

s / Drug and Alcohol Dependence 140 (2014) e2–e85 e11 Conclusions: The results of the present longitudinal study suggest that most smokers in this nationally representative sample of adolescentswhowere followed into early adulthood are characterized as being occasional users. Further, we found that depressive symptoms influence the groupof adolescentswho steadily increase the number of cigarettes smoked per day in early adulthood. The findings have implications for coordinated mental health and substance use treatment. Financial support: No financial support was received. http://dx.doi.org/10.1016/j.drugalcdep.2014.02.050 Adaptation of the hippocampus-accumbens pathway following repeated cocaine Jeffrey L. Barr1, G.L. Forster2, E.M. Unterwald1 1 Department of Pharmacology and Center for Substance Abuse Research, Temple University, Philadelphia, PA, United States 2 Division of Basic Biomedical Sciences, The University of South Dakota, Vermillion, SD, United