The enduring impact of neurulation stage alcohol exposure: A combined behavioral and structural neuroimaging study in adult male and female C57BL/6J mice.

Prenatal alcohol exposure (PAE) can cause behavioral and brain alterations over the lifespan. In animal models, these effects can occur following PAE confined to critical developmental periods, equivalent to the third and fourth weeks of human gestation, before pregnancy is usually recognized. The current study focuses on PAE during early neurulation and examines the behavioral and brain structural consequences that appear in adulthood. On gestational day 8 C57BL/6J dams received two alcohol (2.8g/kg, i.p), or vehicle, administrations, four hours apart. Male and female offspring were reared to adulthood and examined for performance on the elevated plus maze, rotarod, open field, Morris water maze, acoustic startle, social preference (i.e. three-chambered social approach test), and the hot plate. A subset of these mice was later evaluated using magnetic resonance imaging to detect changes in regional brain volumes and shapes. In males, PAE increased exploratory behaviors on the elevated plus maze and in the open field; these changes were associated with increased fractional anisotropy in the anterior commissure. In females, PAE reduced social preference and the startle response, and decreased cerebral cortex and brain stem volumes. Vehicle-treated females had larger pituitaries than did vehicle-treated males, but PAE attenuated this sex difference. In males, pituitary size correlated with open field activity, while in females, pituitary size correlated with social activity. These findings indicate that early neurulation PAE causes sex specific behavioral and brain changes in adulthood. Changes in the pituitary suggest that this structure is especially vulnerable to neurulation stage PAE.

Acute alcohol exposure during neurulation: Behavioral and brain structural consequences in adolescent C57BL/6J mice.

Prenatal alcohol exposure (PAE) can induce physical malformations and behavioral abnormalities that depend in part on thedevelopmental timing of alcohol exposure. The current studies employed a mouse FASD model to characterize the long-term behavioral and brain structural consequences of a binge-like alcohol exposure during neurulation; a first-trimester stage when women are typically unaware that they are pregnant. Time-mated C57BL/6J female mice were administered two alcohol doses (2.8g/kg, four hours apart) or vehicle starting at gestational day 8.0. Male and female adolescent offspring (postnatal day 28-45) were then examined for motor activity (open field and elevated plus maze), coordination (rotarod), spatial learning and memory (Morris water maze), sensory motor gating (acoustic startle and prepulse inhibition), sociability (three-chambered social test), and nociceptive responses (hot plate). Regional brain volumes and shapes were determined using magnetic resonance imaging. In males, PAE increased activity on the elevated plus maze and reduced social novelty preference, while in females PAE increased exploratory behavior in the open field and transiently impaired rotarod performance. In both males and females, PAE modestly impaired Morris water maze performance and decreased the latency to respond on the hot plate. There were no brain volume differences; however, significant shape differences were found in the cerebellum, hypothalamus, striatum, and corpus callosum. These results demonstrate that alcohol exposure during neurulation can have functional consequences into adolescence, even in the absence of significant brain regional volumetric changes. However, PAE-induced regional shape changes provide evidence for persistent brain alterations and suggest alternative clinical diagnostic markers.

Aggression heightened by alcohol or social instigation in mice: reduction by the 5-HT(1B) receptor agonist CP-94,253.

RATIONALE: Models of heightened aggression may be particularly relevant in exploring pharmacological options for the clinical treatment of aggressive and impulsive disorders. OBJECTIVES: To investigate and compare the effects of a 5-HT(1B) selective agonist, CP-94,253, on aggression that was heightened as a result of 1) social instigation or 2) alcohol treatment. METHODS: Male CFW mice were administered 1.0 g/kg EtOH and were subsequently confronted by an intruder in their home cage. In a separate experimental procedure, resident male mice were instigated to aggressive behavior by brief exposure to a provocative stimulus male. To test the hypothesis that activation of the 5-HT(1B )receptor subtype would preferentially attenuate heightened aggression, in comparison to the moderate levels of species-typical aggressive behaviors, the selective agonist, CP-94,253 (1.0-30 mg/kg, IP), and antagonists to the 5-HT(1B) (GR 127935; 10 mg/kg, IP) and the 5-HT(1A) receptor (WAY 100,635; 0.1 mg/kg IP) were used. RESULTS: CP-94,253 suppressed non-heightened aggressive behavior (ED(50)=7.2 mg/kg ). GR 127935, but not WAY 100,635 shifted the ED(50) for CP-94,253 to 14.5 mg/kg. Importantly, the anti-aggressive effects of CP-94,253 were not accompanied by locomotor sedation. Alcohol-heightened and instigation-heightened aggression were suppressed at lower doses than those necessary to suppress non-heightened aggression (ED(50)=3. 8 and 2.7 mg/kg, respectively). CONCLUSIONS: The current results support the hypothesis that activation of 5-HT(1B) receptors modulates very high levels of aggressive behavior in a pharmacologically and behaviorally specific manner.

Alcohol, allopregnanolone and aggression in mice.

RATIONALE: Aggressive behavior of certain individual animals can be greatly increased when under the influence of low doses of alcohol. One of alcohol's neurochemical actions that may be relevant to alcohol-heightened aggression (AHA) is its positive modulation of the GABA(A) receptor complex. OBJECTIVE: The objective of this study was to investigate whether alcohol interacts with an endogenous modulator of the GABA(A) receptor complex, the neurosteroid allopregnanolone, in stimulating/heightening aggressive behavior. METHODS AND RESULTS: The first experiment was designed to test the hypothesis that neurosteroid modulators of the GABA(A) receptor complex will increase aggression and to compare these effects with alcohol. Male CFW mice were injected with allopregnanolone, alphaxalone (3-30 mg/kg, i.p.), or alcohol (1.0 g/kg, p.o.) 15 min prior to a 5-min confrontation with an intruder. Moderate doses of alcohol and the neurosteroids increased aggression by ca. 50% above baseline; impaired locomotion was seen only at the highest doses. A second experiment compared AHA and ANA (i.e. alcohol-non-heightened aggression) mice by giving allopregnanolone (1-10 mg/kg) with a simultaneous oral injection of alcohol (0.6 or 1.0 g/kg) or water. When administered with water and the 0.6 g/kg dose of alcohol, allopregnanolone increased the aggression of AHA and ANA mice. Administration of the 1.0 g/kg dose of alcohol in ANA mice prevented allopregnanolone-heightened aggression. In AHA mice, addition of allopregnanolone to 1.0 g/kg alcohol dose-dependently reduced alcohol-heightened aggression, suggesting potentiation of alcohol's suppressive effects on aggression. CONCLUSIONS: The neuroactive steroid allopregnanolone appears to play an important role in alcohol-heightened aggression. Moreover, the upward shift of the aggression-heightening effects of alcohol and the downward shift at the maximally effective alcohol dose by allopregnanolone point to a shared mechanism for both positive modulators of the GABA(A) receptor complex.

Distress vocalizations in maternally separated mouse pups: modulation via 5-HT(1A), 5-HT(1B) and GABA(A) receptors.

RATIONALE: Young rodents emit ultrasonic vocalizations (USVs) when separated from their dams and littermates. Pharmacological agents that act on GABA(A) and/or 5-HT receptors and that alleviate anxiety in humans reduce the emission of these calls. OBJECTIVES: 1) to investigate specific 5-HT1 receptor subtypes that modulate maternal separation-induced USVs in mice; 2) to assess the behavioral specificity of these effects; and 3) to compare 5-HT1 agonists with a positive neurosteroid modulator of the GABA(A) receptor complex. METHODS: Seven-day old CFW mouse pups were isolated from their littermates and placed onto a 20 degrees C surface for 4 min. USVs between 30 and 80 kHz, grid crossing, and rectal temperature were measured in separate groups of mouse pups following subcutaneous administration of 5-HT1A and 5-HT1B receptor agonists and antagonists, the neurosteroid allopregnanolone, or the benzodiazepine midazolam. RESULTS: The 5-HT1A agonists (+)8-OH-DPAT (0.01-0.1 mg/kg) and flesinoxan (0.3-1.0 mg/kg), the selective 5-HT1B agonist CP-94,253 (0.03-30.0 mg/kg), and the mixed 5-HT1B/2C receptor agonist TFMPP (0.1-10.0 mg/kg) dose-dependently reduced USVs. These effects were reversed by the 5-HT1A receptor antagonist WAY 100,635 (0.1 mg/kg) or the 5-HT1B/D receptor antagonist GR 127935 (0.1 mg/kg). The effects of TFMPP were biphasic; low doses (i.e. 0.01 and 0.03 mg/kg) increased the rate of vocalization. Midazolam and allopregnanolone also reduced USVs. The highest doses of flesinoxan, (+)8-OH-DPAT, and allopregnanolone suppressed locomotion, whereas CP-94,253, TFMPP, and midazolam stimulated motor activity. CONCLUSIONS: These experiments confirm that agonists at the 5-HT1 receptors and a positive allosteric modulator of the GABA(A) receptor complex decrease maternal separation-induced USVs in mice, with 5-HT1B manipulations dissociating the effects on vocalizations from sedative effects.

Zolmitriptan--a 5-HT1B/D agonist, alcohol, and aggression in mice.

RATIONALE: Zolmitriptan is an anti-migraine agent with action at 5-HT1B/D receptors. It penetrates into the central nervous system and, like other 5-HT1B/D agonists, its pharmacotherapeutic profile may include significant anti-aggressive effects. OBJECTIVES: To examine whether zolmitriptan has potential anti-aggressive effects by studying two kinds of aggressive behavior in mice--species-typical and aggression under the influence of alcohol. A second objective was to study whether pre- or post-synaptic receptors mediate these anti-aggressive effects. METHODS: Initially, the anti-aggressive effects of zolmitriptan were studied in male CFW mice during 5-min resident-intruder confrontations. To confirm the 5-HT1B receptor as a critical site of action for the anti-aggressive effects, the zolmitriptan dose-effect determinations were repeated after pretreatment with GR 127935 (10 mg/kg, i.p.). In further experiments, mice were treated concurrently with alcohol (1.0 g/kg, p.o.) and zolmitriptan (1-30 mg/kg, i.p.) in order to compare the effects of this agonist on species-typical and alcohol-heightened aggression. Finally, mice were infused with the neurotoxin 5,7-DHT (10 microg) into the raphé area to eliminate somatodendritic and presynaptic autoreceptors. The anti-aggressive effects of zolmitriptan (17 mg/kg, i.p.) or CP-94,253 (10 mg/kg, i.p.) were assessed 10 days after the lesion, and levels of 5-HT and 5-HIAA were measured in the hippocampus and prefrontal cortex. RESULTS: Zolmitriptan exerted behaviorally specific anti-aggressive effects. The reduction in aggression was antagonized by GR 127935, indicated by a rightward shift in the dose-effect curves of zolmitriptan, showing the specificity for the 5-HT1B receptors. Zolmitriptan also decreased alcohol-heightened aggression with equal efficacy. The anti-aggressive effects of CP-94,253 and zolmitriptan remained unaltered by 5,7-DHT lesions that depleted cortical and hippocampal 5-HT by 60-80%. CONCLUSIONS: Zolmitriptan proved to be an effective and behaviorally specific anti-aggressive agent in situations that engender moderate and alcohol-heightened levels of aggression. These effects are potentially due to activation of post-synaptic 5-HT1BD receptors.

Aggressive behavioral phenotypes in mice.

Aggressive behavior in male and female mice occurs in conflicts with intruding rivals, most often for the purpose of suppressing the reproductive success of the opponent. The behavioral repertoire of fighting is composed of intricately sequenced bursts of species-typical elements, with the resident displaying offensive and the intruder defensive acts and postures. The probability of occurrence as well as the frequency, duration, temporal and sequential patterns of aggressive behavior can be quantified with ethological methods. Classic selection and strain comparisons show the heritability of aggressive behavior, and point to the influence of several genes, including some of them on the Y chromosome. However, genetic effects on aggressive behavior critically depend upon the background strain, maternal environment and the intruder. These factors are equally important in determining changes in aggressive behavior in mice with a specific gene deletion. While changes in aggression characterize mutant mice involving a variety of genes, no pattern has emerged that links particular gene products (i.e. enzyme, peptide, receptor) to either an increase or a decrease in aggressive behavior, but rather emphasizes polygenic influences. A potentially common mechanism may be some components of the serotonin system, since alterations in 5-HT neurotransmission have been found in several of the KO mice that display unusual aggressive behavior.

Infralimbic and dorsal raphé microinjection of the 5-HT(1B) receptor agonist CP-93,129: attenuation of aggressive behavior in CFW male mice.

RATIONALE: Aggressive behavior and impaired impulse control have been associated with dysregulations in the serotonergic system and with impaired functioning of the prefrontal cortex. 5-HT(1B) receptors have been shown to specifically modulate several types of offensive aggression. OBJECTIVE: This study aims to characterize the relative importance of two populations of 5-HT(1B) receptors in the dorsal raphé nucleus (DRN) and infralimbic cortex (ILC) in the modulation of aggressive behavior. METHODS: Male CFW mice were conditioned on a fixed-ratio 5 schedule of reinforcement to self-administer a 6% (w/v) alcohol solution. Mice repeatedly engaged in 5-min aggressive confrontations until aggressive behavior stabilized. Next, a cannula was implanted into either the DRN or the ILC. After recovery, mice were tested for aggression after self-administration of either 1.0 g/kg alcohol or water prior to a microinjection of the 5-HT(1B) agonist, CP-93,129 (0-1.0 µg/infusion). RESULTS: In both the DRN and ILC, CP-93,129 reduced aggressive behaviors after both water and alcohol self-administration. Intra-raphé CP-93,129 dose-dependently reduced both aggressive and locomotor behaviors. However, the anti-aggressive effects of intra-cortical CP-93,129 were behaviorally specific. CONCLUSIONS: These findings highlight the importance of the serotonergic system in the modulation of aggression and suggest that the behaviorally specific effects of 5-HT(1B) receptor agonists are regionally selective. 5-HT(1B) receptors in a medial subregion of the prefrontal cortex, the ILC, appear to be critically involved in the attenuation of species-typical levels of aggression.

Potentiation of brain stimulation reward by morphine: effects of neurokinin-1 receptor antagonism.

RATIONALE: The abuse potential of opioids may be due to their reinforcing and rewarding effects, which may be attenuated by neurokinin-1 receptor (NK1R) antagonists. OBJECTIVE: This study was conducted to measure the effects of opioid and NK1R blockade on the potentiation of brain stimulation reward (BSR) by morphine using the intracranial self-stimulation method. METHODS: Adult male C57BL/6J mice (n = 15) were implanted with unipolar stimulating electrodes in the lateral hypothalamus and trained to respond for varying frequencies of rewarding electrical stimulation. The BSR threshold (θ(0)) and maximum response rate (MAX) were determined before and after intraperitoneal administration of saline, morphine (1.0-17.0 mg/kg), or the NK1R antagonists L-733,060 (1.0-17.0 mg/kg) and L-703,606 (1.0-17.0 mg/kg). In morphine antagonism experiments, naltrexone (0.1-1.0 mg/kg) or 10.0 mg/kg L-733,060 or L-703,606 was administered 15 min before morphine (1.0-10.0 mg/kg) or saline. RESULTS: Morphine dose-dependently decreased θ(0) (maximum effect = 62% of baseline) and altered MAX when compared to saline. L-703,606 and L-733,060 altered θ(0); 10.0 mg/kg L-733,060 and L-703,606, which did not affect θ(0) or MAX, attenuated the effects of 3.0 and 10.0 mg/kg morphine, and 1.0 and 0.3 mg/kg naltrexone blocked the effects of 10.0 mg/kg morphine. Naltrexone given before saline did not affect θ(0) or MAX. CONCLUSIONS: The decrease in θ(0) by morphine reflects its rewarding effects, which were attenuated by NK1R and opioid receptor blockade. These results demonstrate the importance of substance P signaling during limbic reward system activation by opioids.