Neurochemical correlates of accumbal dopamine D2 and amygdaloid 5-HT 1B receptor densities on observational learning of aggression.

Social learning theory postulates that individuals learn to engage in aggressive behavior through observing an aggressive social model. Prior studies have shown that repeatedly observing aggression, also called "chronic passive exposure to aggression," changes accumbal dopamine D2 receptor (D2R) and amygdaloid 5-HT1B receptor (5-HT1BR) densities in observers. But, the association between these outcomes remains unknown. Thus, in our study, we used a rat paradigm to comprehensively examine the linkage between aggression, D2R density in the nucleus accumbens core (AcbC) and shell (AcbSh), and 5-HT1BR density in the medial (MeA), basomedial (BMA), and basolateral (BLA) amygdala following chronic passive exposure to aggression. Male Sprague-Dawley rats (N = 72) were passively exposed to either aggression or nonaggression acutely (1 day) or chronically (23 days). When observer rats were exposed to aggression chronically, they showed increased aggressive behavior and reduced D2R density in bilateral AcbSh. On the other hand, exposure to aggression, regardless of exposure length, increased the 5-HT1BR density in bilateral BLA. Finally, low D2R in the AcbSh significantly interacted with high 5-HT1BR density in the BLA to predict high levels of aggression in observer rats. Our results advance our understanding of the neurobiological mechanisms in the observational learning of aggression, highlighting that dopamine-serotonin interaction, or AcbSh-BLA interaction, may contribute to a risk factor for aggression in observers who chronically witness aggressive interactions.

Sex-specific impairment and recovery of spatial learning following the end of chronic unpredictable restraint stress: potential relevance of limbic GAD.

Chronic restraint stress alters hippocampal-dependent spatial learning and memory in a sex-dependent manner, impairing spatial performance in male rats and leaving intact or facilitating performance in female rats. Moreover, these stress-induced spatial memory deficits improve following post-stress recovery in males. The current study examined whether restraint administered in an unpredictable manner would eliminate these sex differences and impact a post-stress period on spatial ability and limbic glutamic acid decarboxylase (GAD65) expression. Male (n=30) and female (n=30) adult Sprague-Dawley rats were assigned to non-stressed control (Con), chronic stress (Str-Imm), or chronic stress given a post-stress recovery period (Str-Rec). Stressed rats were unpredictably restrained for 21 days using daily non-repeated combinations of physical context, duration, and time of day. Then, all rats were tested on the radial arm water maze (RAWM) for 2 days and given one retention trial on the third day, with brains removed 30min later to assess GAD65 mRNA. In Str-Imm males, deficits occurred on day 1 of RAWM acquisition, an impairment that was not evident in the Str-Rec group. In contrast, females did not show significant outcomes following chronic stress or post-stress recovery. In males, amygdalar GAD65 expression negatively correlated with RAWM performance on day 1. In females, hippocampal CA1 GAD65 positively correlated with RAWM performance on day 1. These results demonstrate that GABAergic function may contribute to the sex differences observed following chronic stress. Furthermore, unpredictable restraint and a recovery period failed to eliminate the sex differences on spatial learning and memory.

Aggression is suppressed by acute stress but induced by chronic stress: immobilization effects on aggression, hormones, and cortical 5-HT(1B)/ striatal dopamine D(2) receptor density.

Although it has been established by a number of investigators that a variety of stressors are associated with the induction of aggressive behavior, two specific issues remain unanswered. First, it is unclear whether the contexts surrounding stressors (e.g., stressor length and chance of winning over opponents) change outcomes regarding aggressive behavior. Second, if a relationship exists between stress and aggressive behavior, altered levels of stress-related hormone (e.g., corticosterone [CORT]), as well as aggression-related biomarkers (e.g., testosterone [T], density of prefronto-cortical 5-HT(1B) receptor and striatal dopamine D(2) receptor [D2r]) may contribute to changes in aggressive behavior. Thus, we examined how immobilization (with a 1-, 5-, or 10-day exposure) would impact (1) a longitudinal course of aggression toward different-sized opponents, (2) levels of CORT and T, and (3) densities of 5-HT(1B) receptor (5-HT1Br) in the prefrontal cortex (PFC) and D2r in the striatum. It was found that, regardless of small or large opponents, a single 2-h exposure to immobilization reduced aggressive behavior (stress-suppressed aggression) over time, whereas repeated (10-day) exposure to immobilization escalated aggressive behavior (stress-induced aggression). These stress effects persisted up to 1 week of recovery from immobilization stress. Moreover, immobilized rats demonstrated elevated levels of T, but not CORT, as compared with controls. Finally, acute immobilization altered D2r densities in the shell of the nucleus accumbens, and chronic immobilization changed 5-HT1Br in the PFC, including the downregulation of 5-HT1Br densities in the right prelimbic and orbitolateral cortices. The potential relationships among stress, aggression, and 5-HT1Br/D2r roles are discussed.

Effects of recovery from immobilization stress on striatal preprodynorphin- and kappa opioid receptor-mRNA levels of the male rat.

Previously, we have reported that brain regions that are thought to be involved in motivated behavior are altered in animals undergoing repeated exposures to immobilization stress. The goal of the present study was to determine the effects of recovery from this type of stress on these same mesolimbic brain regions. For this purpose, adult male Sprague-Dawley rats were initially exposed to immobilization stress either once (2 h) or repeatedly (2 h×10 days). Rats were then either allowed to recover from the stressor for a shorter (2 days) or longer period of time (9 days) in their home cages. At the end of this recovery period, rats were euthanized and trunk blood and brains were processed for serum corticosterone (CORT) and neurochemistry, respectively. Brain mRNA levels were determined via in situ hybridization for the opioid preprodynorphin (DYN) and its cognate receptor (kappa, KOR), in striatal and accumbal subregions. A pattern of selective transcriptional activation emerged in the four resultant treatment conditions where a short recovery from either a single or repeated exposure to immobilization produced increases in KOR-mRNA levels in striatal and nucleus accumbens (Acb) subregions. Relative to controls, these differences were diminished after a longer recovery period. Interestingly, DYN-mRNA levels were unchanged after the shorter recovery period and after single or repeated immobilizations but appeared to be induced after a longer recovery period after repeated immobilizations. A relative amount of weight loss occurred after immobilization following repeated but not single exposure to stress. In addition, only those rats recovering from repeated stress exposures had higher CORT levels compared with non-immobilized controls. These results suggest that recovery from immobilization stress may alter the motivational system after as little as a single immobilization and that a possible dysphoric effect on appetitive behavior may be reflected by an altered striatal dynorphin system.

Increased 5-HT1B receptor density in the basolateral amygdala of passive observer rats exposed to aggression.

Previous studies have shown that repeated observations of aggressive incidents (i.e., chronic passive exposure to aggression) increase aggressiveness of a passive observer and downregulate the densities of serotonin 5-HT(1B) receptors in some tegmental regions. However, other brain areas (e.g., medial- and basolateral amygdala, hypothalamus, and hippocampus) have been implicated in different types of aggressive behavior including fear-induced defensive rage, steroid-induced aggression, and other types of aggression (e.g., learned aggression). The present study analyzed 5-HT(1B) receptor densities in those brain regions of aggressive observers to compare them with neurochemical markers of the different types of aggression. It was hypothesized that passive exposure to aggression for 23 consecutive days would result in altered 5-HT(1B) receptor densities in the ventromedial hypothalamus, medial amygdaloid nucleus, CA1 of the hippocampus, globus pallidus, dentate gyrus, and/or basolateral amygdala. Here we report that observer rats exposed to aggression exhibited higher densities of 5-HT(1B) receptors in only the basolateral amygdala, compared with those exposed to the non-aggressive condition. These results suggest that chronic passive exposure to aggression may elicit a form of learned aggression rather than fear- or steroid-induced aggression among passive observers. In addition, our study implies that 5-HT(1B) receptors play brain-region specific roles in expressing aggression.

Chronic passive exposure to aggression decreases D2 and 5-HT 1B receptor densities.

It has been recently reported that passive exposure to aggression induces aggressive behavior in a rodent model. However, it remains unclear whether this response is correlated with neurochemical changes that correspond either to stress-induced aggression or non-stressed, learned aggression. Stress-induced aggression has been shown to result in increased brain dopamine D(2) receptor and serum corticosterone levels. In contrast, learned aggression is probably associated with reward deficiency syndrome, characterized by low dopamine D(2) receptor levels, without stress effects (i.e., high corticosterone levels). We hypothesized that chronic passive exposure to aggression would produce learned aggression, represented by low levels of dopamine D(2) receptor binding but normal levels of stress hormone. The present study additionally focused on serum testosterone and serotonin 5-HT(1B) receptor density that has been associated with aggression/reward circuits. Hormonal results indicated that there were no differences between the "observer" rats that had been passively exposed to aggression and non-aggression for 10 min/day for 23 consecutive days. However, receptor binding autoradiography identified lower densities of dopamine D(2) receptors in the cortical-accumbal regions (shell of the nucleus accumbens and cingulate and motor cortices) and lower 5-HT(1B) receptor densities in the tegmental regions (ventral tegmental area, substantia nigra pars compacta, and periaqueductal gray) among observers exposed to aggression, compared to controls. Changes in dopamine D(2) receptor densities due to chronic exposure to aggression do not resemble those patterns reported for stress-induced aggressive behavior. Our evidence suggests that the development of aggressive behavior among passive observers occurs through a learned, and not a stress-induced, mechanism.

Effects of acute diuresis stress on egr-1 (zif268) mRNA levels in brain regions associated with motivated behavior.

Stressors evoke a well-studied physiological stress-response, namely, an immediate systemic release of catecholamines from the adrenals followed shortly afterwards by the release of adrenal steroids. The intensity of that response can often be inferred by the amount of adrenal steroids released into the circulatory system. It is still unclear however how the intensity and duration of the stressor affect a number of brain regions, including those in the motivational system. The present study sought to determine whether a brief stressor, such as an isotonic saline injection, activated the brain's motivational system in mesolimbic regions compared with a more intense stressor exemplified by pharmacological challenges caused by the administration of a diuretic. Adult male Sprague-Dawley rats were either injected (s.c.) with isotonic saline or 5mg of the diuretic, furosemide. Controls did not receive any injections. Animals were sacrificed at 30, 60, 120, and 240 min after injection and trunk blood and brains were collected. Serum corticosterone and aldosterone levels were assessed through radioimmunoassay and mesolimbic brain activity was determined through in situ hybridization of mRNA expression of the immediate-early gene egr-1 in the caudate-putamen and nucleus accumbens. While both adrenal steroids demonstrated an initial peak in both stress groups, levels were higher and longer lasting in rats treated with furosemide. Interestingly, egr-1 mRNA levels were significantly higher only in the furosemide-treated group compared with controls. These findings suggest that a selective activation of motivational circuits occurs under thirst and salt-appetite-induced conditions such as those caused by diuresis.

Chronic passive exposure to aggression escalates aggressiveness of rat observers.

Previous studies have documented that exposure to aggression increases aggressiveness of human witnesses. However, the question of whether passive exposure to aggression can exclusively cause a risk of aggressive inclination for observers through a learning process, rather than mimicry effect, has not been readily addressed in the clinical literature. This study aimed to investigate this question by using a simple animal model to test the behavioral effect of chronic passive exposure to aggression. Our results indicate that observer rats that had been passively exposed to aggression for 10 min per day for 23 consecutive days exhibited more aggressive behavior than controls or those groups undergoing a single exposure to passive aggression. Furthermore, aggression levels in the group of 23-day chronic exposure to aggression lasted 16 days after the recovery from exposure to aggression. These data suggest that the development of aggression in this model occurred through a learning process because only chronic exposure to aggression resulted in this behavioral outcome in the long run.

Effects of immobilization stress on neurochemical markers in the motivational system of the male rat.

Mesolimbic regions involved in motivated behavior are altered in animals undergoing repeated exposure to social stress. Here we test the hypothesis that other forms of persistent stress would also influence these same endpoints. Adult male Sprague-Dawley rats were exposed to immobilization stress either once (2 h) or repeatedly (2 hx10 days) and brains were harvested immediately after the last immobilization. A trio of indirect markers associated with dopaminergic activity was measured including dopamine transporter (DAT) and dopamine D2 receptor subtype (D2r) ligand levels as well as mRNA levels of the endogenous opioid enkephalin (ENK-mRNA). A single 2-h session of immobilization stress produced an increase in striatal ENK-mRNA levels and DAT ligand binding compared with group-housed controls. In animals undergoing repeated immobilization stress and singly housed post-stress, we found a significant reversal in the direction of ENK-mRNA levels and DAT binding in the striatum, in addition to an increase in D2r-binding density in the shell of the nucleus accumbens compared with single-stress-exposed rats. In another experiment using the same stress paradigm but allowing pair-housing post-stress, we found no alteration of ENK-mRNA but significant increases in DAT and D2r binding in the dorsal striatum. A major difference between single and group housing is the habituation of the corticosterone (CORT) stress response over 10-day stress in group-housed rats. The present results parallel previous findings by our laboratory that repeated stress results in a relative reduction of ENK-mRNA levels and increased D2r-binding density in the striatum of rats. Furthermore, our data are consistent with the hypothesis that chronic stress induces an allostatic attenuation of the mesolimbic dopaminergic system in animals that do not habituate to the stressor, possibly due in part to persistent CORT elevations.

Salt appetite in sodium-depleted or sodium-replete conditions: possible role of opioid receptors.

BACKGROUND/AIMS: Acute sodium depletion by the combination of pharmacological natriuresis via furosemide administration and a sodium-deficient diet results in a strong induction of salt appetite in rats. Recent evidence suggests that acute furosemide decreases both dopamine uptake and striatal dopamine transporter density and increases enkephalin mRNA levels in the nucleus accumbens (Acb). Therefore, it has been hypothesized that the motivational/attentional circuit in the brain is activated in salt-appetitive rats. METHODS: To determine which loci along the dopaminergic circuit are responsible for this behavior, 10-15 min before furosemide-treated adult male Sprague-Dawley rats were allowed 2-hour access to 2% salt solution (2-bottle choice), we pharmacologically blocked dopamine receptor subtype 1 (D1r) and subtype 2 (D2r) with SCH23390 or raclopride, respectively, and stimulated D1r with SKF81297 or D2r with quinpirole in the shell of the Acb (AcbSh). Furthermore, delta opioid receptors were blocked with naltrindole in the AcbSh or ventral tegmental area (VTA). RESULTS: We found that microinjections (1 mug) of SCH23390, raclopride, SKF81297, quinpirole, or naltrindole into the AcbSh had no effect. However, infusion of naltrindole into the VTA attenuated salt intake, whereas [D-Ser(2),Leu(5),Thr(6)]-enkephalin had no effect. Additionally, in rats previously primed with furosemide to crave salt in a 'need-free' manner, salt intake was augmented in the VTA and reduced in the AcbSh after infusion of [D-Ser(2),Leu(5),Thr(6)]-enkephalin. CONCLUSION: These data provide evidence that mesolimbic opioid systems are involved in the facilitation of salt-appetitive behavior.

Topics in ultrasound education: Use of patient models to teach endovaginal ultrasound skills.

Learning how to perform endovaginal pelvic ultrasound is often challenging for novices in a busy clinical practice. In this article, we describe a program in which we hired female patient models to help residents acquire basic endovaginal scanning skills.

Involvement of mesolimbic structures in short-term sodium depletion: in situ hybridization and ligand-binding analyses.

Acute treatment with the diuretic furosemide (Lasix) produces a reduction in plasma Na(+) and volume as well as increased thirst and salt appetite. The resulting hypovolemia stimulates the well-known counter-regulatory physiological response from the renin-angiotensin-aldosterone system. However, the neurochemical players underpinning the behavioral responses of thirst and salt appetite are less clear. Previously, we have reported that salt-replete deoxycorticosterone (DOCA) treatment activates mesolimbic structures associated with reward and goal-seeking behavior. The present study was designed to test whether the same brain regions are affected in a salt-depleted state. In experiment 1, two groups of adult male Sprague-Dawley (SD) rats were injected with Lasix (10 mg/rat, s.c.) and 18 h later were allowed access either to 2% NaCl solution ('Lasix+salt') or only to tap water ('Lasixnosalt') for 2 h. For comparison purposes, a third group received an isotonic saline injection instead of Lasix and was allowed access to the 2% salt solution (Vehicle). All groups were permitted 24 h access to tap water. We found no differences in dynorphin-mRNA levels in any striatal and accumbal regions among any of the treatment groups. However, as found previously in DOCA-treated rats, there were increased enkephalin (ENK)-mRNA and decreased dopamine transporter (DAT) binding levels throughout the striatum in Lasix+salt and decreased ENK-mRNA in Lasixnosalt rats versus Vehicle. In experiment 2, the involvement of the ENKergic and/or dopaminergic system was tested in rats divided into the same three groups described in experiment 1. However, before access to salt or water, the Lasix+salt and the vehicle groups were administered either a delta-opioid, naltrindole or a dopamine D(2) antagonist, raclopride. Only the naltrindole-treated rats showed a blunted intake of salt solution. Thus, these findings along with our neurochemical results suggest that mesolimbic enkephalin might impact salt intake through dopaminergic systems.

Decreases in neurokinin-3 tachykinin receptor-immunoreactive and -mRNA levels are associated with salt appetite in the deoxycorticosterone-treated rat.

Tachykinins are a family of neuropeptides that inhibit salt appetite. Although decreased tachykinin-mRNA levels are observed in natriorexic sodium-deplete rats, no decrease is seen in natriorexic sodium-replete rats that are administered the aldosterone-mimetic deoxycorticosterone acetate (DOCA). Since reduced synthesis of tachykinins could not account for increased appetite, we hypothesized that increased salt appetite was due to a downregulation of tachykinin receptors. Thus, we injected rats with DOCA once daily for 11 days and analyzed tachykinin receptor subtype, neurokinin 3 (NK3r)-immunoreactivity by Western blot analysis since intracerebroventricular injection of senktide (NK3r agonist) attenuates salt intake in DOCA-treated animals. We examined NK3r-immunoreactivity in several brain regions thought to be associated with the control of water and electrolyte balance including the bed nucleus of the stria terminalis, central nucleus of the amygdala, diagonal band of Broca, hippocampus, nucleus tractus solitarius, parabrachial nucleus, paraventricular nucleus of the hypothalamus, and supraoptic nucleus. Consistent with our hypothesis, we found decreased NK3r-immunoreactivity in all brain regions analyzed except for increases in the amygdala and no changes in the paraventricular nucleus of the hypothalamus. To examine whether DOCA's effects on NK3r synthesis are direct, we used differentiated N1E-115 neuroblastoma cells that express NK3r and treated them with a range of concentrations of DOCA and found a dose-dependent decrease in NK3r-mRNA abundance via Northern blotting. The present results suggest that the tachykinin receptors are downregulated after subchronic DOCA treatment and this finding is consistent with the hypothesis that suppressed inhibition of salt appetite as mediated through the tachykininergic system.

Effect of dopamine uptake inhibition on brain catecholamine levels and locomotion in catechol-O-methyltransferase-disrupted mice.

Two different uptake processes terminate the synaptic action of released catecholamines in brain: the high-affinity uptake to presynaptic nerve terminals (uptake(1), followed by oxidation by monoamine oxidase, MAO) or glial cells uptake (uptake(2), followed by O-methylation by catechol-O-methyltransferase, COMT, and/or oxidation by MAO). For dopaminergic neurons, uptake by the high-affinity dopamine transporter (DAT) is the most effective mechanism, and the contribution of glial COMT remains secondary under normal conditions. In the present study we have characterized the role of COMT using COMT-deficient mice in conditions where DAT is inhibited by 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)-piperazine (GBR 12909) or cocaine. In mice lacking COMT, GBR 12909 results in total brain tissue dopamine levels generally higher than in wild-type mice but no such potentiation was ever seen in striatal extracellular fluid. Dopamine accumulation in nerve endings is more evident in striatum and hypothalamus than in cortex. Both GBR 12909 and cocaine induced hyperlocomotion in mice lacking COMT. Unexpectedly, hyperactivity induced by 20 mg/kg GBR 12909 was attenuated only in male COMT knockout mice, i.e., they had an inability to sustain the hyperactivity induced by DAT inhibition. Furthermore, attenuation of hyperlocomotion was observed also after cocaine treatment in both C57BL/6 (at 5 and 15 mg/kg) and 129/Sv (at 30 mg/kg) genetic background COMT-deficient male mice. Despite the possible interaction between DAT and extraneuronal uptake (and subsequently COMT), the role of COMT in dopamine elimination is still minimal in conditions when DAT is inhibited.

Preprotachykinin A gene expression after administration of 3,4-methylene dioxymethamphetamine (Ecstasy).

This study tested the effects of 8 days of subchronic administration of 3,4-methylene dioxymethamphetamine (MDMA) (5 mg/kg b.w.) on preprotachykinin A mRNA levels in discrete rat brain regions. In situ hybridization examined preprotachykinin A mRNA levels in the core and shell of the nucleus accumbens, the islands of Calleja, the olfactory tubercle, the dorsal and ventral caudate-putamen, the bed nucleus of the stria terminalis, the medial preoptic area, the medial habenular nucleus and in the postero-dorsal part of the medial amygdala. Higher levels of preprotachykinin A mRNA were found in the core and shell of the nucleus accumbens, in the islands of calleja, in the olfactory tubercle, in the bed nucleus of the stria terminalis, in the medial habenular nucleus and the postero-dorsal part of the medial amygdala, compared to control animals. Conversely, increased preprotachykinin A mRNA levels were observed in the dorsal and ventral caudate-putamen in MDMA treated when compared to control rats. In the social memory test, MDMA significantly impaired rats' short-term working memory. These results show that chronic exposure to MDMA strongly affects preprotachykinin A mRNA levels in discrete rat brain regions. These changes occur in experimental conditions in which working memory is markedly reduced, suggesting that changes in gene expression of tachykinin mechanisms may contribute to the effects of MDMA on memory function.

Repeated sucrose access influences dopamine D2 receptor density in the striatum.

A decrease in D2 dopamine receptor subtype (D2R) binding in the striatum has been reported in obese individuals and drug addicts. We examined D2R density in the striatum of food-restricted rats that had contingent access to food with different incentive values. Results showed that animals receiving limited access to 0.3 M sucrose paired 2 h with a chow meal for 7 days had a significantly lower D2R binding in nucleus accumbens shell and dorsolateral striatum compared with animals that had limited access to chow. There was no differential binding, however, in the accumbens core in any of the groups. These findings indicate that feeding conditions and sucrose intake influence D2R density specifically in subregions of the striatum.