Pharmacological activation of the Sonic hedgehog pathway with a Smoothened small molecule agonist ameliorates the severity of alcohol-induced morphological and behavioral birth defects in a zebrafish model of fetal alcohol spectrum disorder.

Ethanol exposure during the early stages of embryonic development can lead to a range of morphological and behavioral differences termed fetal alcohol spectrum disorders (FASDs). In a zebrafish model, we have shown that acute ethanol exposure at 8-10 hr postfertilization (hpf), a critical time of development, produces birth defects similar to those clinically characterized in FASD. Dysregulation of the Sonic hedgehog (Shh) pathway has been implicated as a molecular basis for many of the birth defects caused by prenatal alcohol exposure. We observed in zebrafish embryos that shh expression was significantly decreased by ethanol exposure at 8-10 hpf, while smo expression was much less affected. Treatment of zebrafish embryos with SAG or purmorphamine, small molecule Smoothened agonists that activate Shh signaling, ameliorated the severity of ethanol-induced developmental malformations including altered eye size and midline brain development. Furthermore, this rescue effect of Smo activation was dose dependent and occurred primarily when treatment was given after ethanol exposure. Markers of Shh signaling (gli1/2) and eye development (pax6a) were restored in embryos treated with SAG post-ethanol exposure. Since embryonic ethanol exposure has been shown to produce later-life neurobehavioral impairments, juvenile zebrafish were examined in the novel tank diving test. Our results further demonstrated that in zebrafish embryos exposed to ethanol, SAG treatment was able to mitigate long-term neurodevelopmental impairments related to anxiety and risk-taking behavior. Our results indicate that pharmacological activation of the Shh pathway at specific developmental timing markedly diminishes the severity of alcohol-induced birth defects.

Loss of tumor protein 53 protects against alcohol-induced facial malformations in mice and zebrafish.

BACKGROUND: Alcohol exposure during the gastrulation stage of development causes the craniofacial and brain malformations that define fetal alcohol syndrome. These malformations, such as a deficient philtrum, are exemplified by a loss of midline tissue and correspond, at least in part, to regionally selective cell death in the embryo. The tumor suppressor protein Tp53 is an important mechanism for cell death, but the role of Tp53 in the consequences of alcohol exposure during the gastrulation stage has yet to be examined. The current studies used mice and zebrafish to test whether genetic loss of Tp53 is a conserved mechanism to protect against the effects of early developmental stage alcohol exposure. METHODS: Female mice, heterozygous for a mutation in the Tp53 gene, were mated with Tp53 heterozygous males, and the resulting embryos were exposed during gastrulation on gestational day 7 (GD 7) to alcohol (two maternal injections of 2.9 g/kg, i.p., 4 h apart) or a vehicle control. Zebrafish mutants or heterozygotes for the tp53zdf1  M214K mutation and their wild-type controls were exposed to alcohol (1.5% or 2%) beginning 6 h postfertilization (hpf), the onset of gastrulation. RESULTS: Examination of GD 17 mice revealed that eye defects were the most common phenotype among alcohol-exposed fetuses, occurring in nearly 75% of the alcohol-exposed wild-type fetuses. Tp53 gene deletion reduced the incidence of eye defects in both the heterozygous and mutant fetuses (to about 35% and 20% of fetuses, respectively) and completely protected against alcohol-induced facial malformations. Zebrafish (4 days postfertilization) also demonstrated alcohol-induced reductions of eye size and trabeculae length that were less common and less severe in tp53 mutants, indicating a protective effect of tp53 deletion. CONCLUSIONS: These results identify an evolutionarily conserved role of Tp53 as a pathogenic mechanism for alcohol-induced teratogenesis.

Different contributions of dopamine D1 and D2 receptor activity to alcohol potentiation of brain stimulation reward in C57BL/6J and DBA/2J mice.

C57BL/6J (C57) and DBA/2J (DBA) mice respond differently to drugs that affect dopamine systems, including alcohol. The current study compared effects of D1 and D2 receptor agonists and antagonists, and the interaction between D1/D2 antagonists and alcohol, on intracranial self-stimulation in male C57 and DBA mice to determine the role of dopamine receptors in the effects of alcohol on brain stimulation reward (BSR). In the initial strain comparison, dose effects on BSR thresholds and maximum operant response rates were determined for the D1 receptor agonist SKF-82958 (±-6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine; 0.1-0.56 mg/kg) and antagonist SCH 23390 (+-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepinehydrochloride; 0.003-0.056 mg/kg), and the D2 receptor agonist quinpirole (0.1-3.0 mg/kg) and antagonist raclopride (0.01-0.56 mg/kg). For the alcohol interaction, SCH 23390 (0.003 mg/kg) or raclopride (0.03 mg/kg) was given before alcohol (0.6-2.4 g/kg p.o.). D1 antagonism dose-dependently elevated and SKF-82958 dose-dependently lowered BSR threshold in both strains; DBA mice were more sensitive to SKF-82958 effects. D2 antagonism dose-dependently elevated BSR threshold only in C57 mice. Low doses of quinpirole elevated BSR threshold equally in both strains, whereas higher doses of quinpirole lowered BSR threshold only in C57 mice. SCH 23390, but not raclopride, prevented lowering of BSR threshold by alcohol in DBA mice. Conversely, raclopride, but not SCH 23390, prevented alcohol potentiation of BSR in C57 mice. These results extend C57 and DBA strain differences to D1/D2 sensitivity of BSR, and suggest differential involvement of D1 and D2 receptors in the acute rewarding effects of alcohol in these two mouse strains.

Levetiracetam results in increased and decreased alcohol drinking with different access procedures in C57BL/6J mice.

The antiepileptic levetiracetam (LEV) has been investigated for the treatment of alcohol abuse. However, little is known about how LEV alters the behavioral effects of alcohol in laboratory animals. The acute effects of LEV on alcohol drinking by male C57BL/6J mice were investigated using two different drinking procedures, limited access [drinking-in-the-dark (DID)] and intermittent access (IA) drinking. In the first experiment (DID), mice had access to a single bottle containing alcohol or sucrose for 4 h every other day. In the second experiment (IA), mice had IA to two bottles, one containing alcohol or sucrose and one containing water, for 24 h on Monday, Wednesday, and Friday. In both experiments, mice were administered LEV (0.3-100 mg/kg intraperitoneally) or vehicle 30 min before access to the drinking solutions. In the DID mice, LEV increased alcohol intake from 4.3 to 5.4 g/kg, whereas in the IA mice LEV decreased alcohol intake from 4.8 to 3.0 g/kg in the first 4 h of access and decreased 24 h alcohol intake from 20 to ∼15 g/kg. These effects appear specific to alcohol, as LEV did not affect sucrose intake in either experiment. LEV appears to differentially affect drinking in animal models of moderate and heavier alcohol consumption.

The impact of prenatal alcohol, synthetic cannabinoid and co-exposure on behavioral adaptations in adolescent offspring and alcohol self-administration in adulthood.

Prenatal exposure to alcohol or cannabinoids can produce enduring neurobiological, cognitive, and behavioral changes in the offspring. Furthermore, prenatal co-exposure to alcohol and cannabinoids induces malformations in brain regions associated with reward and stress-related circuitry. This study examined the effects of co-exposure to alcohol and the synthetic cannabinoid (SCB) CP55,940 throughout gastrulation and neurulation in rats on basal corticosterone levels and a battery of behavioral tests during adolescence and alcohol self-administration in adulthood. Importantly, we find that prenatal alcohol exposure (PAE) caused lower baseline corticosterone levels in adolescent males and females. Co-exposure to alcohol + CP produced hyperactivity during open field test in males, but not females. During the two-bottle choice alcohol-drinking procedure, prenatal cannabinoid exposed male and female adolescent rats drank more alcohol than their vehicle-exposed controls. In adulthood, female rats treated with prenatal cannabinoid exposure (PCE), showed an overall total increase in alcohol intake during alcohol self-administration; but this was not found in males. When the reinforcer was changed to a 1% sucrose solution, male rats exposed to PCE, showed a reduced self-administration compared to vehicle-exposed males, potentially indicative of an anhedonic response. This lower self-administration persisted when 20% alcohol was reintroduced to the sucrose solution. Lastly, following an abstinence period, there were no changes due to prenatal drug exposure in either males or females. Overall, these data suggest lasting consequences of prenatal alcohol and cannabinoid exposure during adolescence and adulthood in male and female rats.

Gastrulation-stage alcohol exposure induces similar rates of craniofacial malformations in male and female C57BL/6J mice.

BACKGROUND: Prenatal alcohol exposure during gastrulation (embryonic day [E] 7 in mice, ~3rd week of human pregnancy) impairs eye, facial, and cortical development, recapitulating birth defects characteristic of Fetal Alcohol Syndrome (FAS). However, it is not known whether the prevalence or severity of craniofacial features associated with FAS is affected by biological sex. METHODS: The current study administered either alcohol (2.9 g/kg, two i.p. doses, 4 hr apart) or vehicle to pregnant C57BL/6J females on E7, prior to gonadal sex differentiation, and assessed fetal morphology at E17. RESULTS: Whereas sex did not affect fetal size in controls, alcohol-exposed females were smaller than both control females and alcohol-treated males. Alcohol exposure increased the incidence of eye defects to a similar degree in males and females. Together, these data suggest that females might be more sensitive to the general developmental effects of alcohol, but not effects specific to the craniofacies. Whole transcriptomic analysis of untreated E7 embryos found 214 differentially expressed genes in females vs. males, including those in pathways related to cilia and mitochondria, histone demethylase activity, and pluripotency. CONCLUSION: Gastrulation-stage alcohol induces craniofacial malformations in male and female mouse fetuses at similar rates and severity, though growth deficits are more prevalent females. These findings support the investigation of biological sex as a contributing factor in prenatal alcohol studies.

The impact of prenatal alcohol and synthetic cannabinoid exposure on behavioral adaptations in adolescent offspring and alcohol self-administration in adulthood.

Prenatal exposure to alcohol or cannabinoids can produce enduring neurobiological, cognitive, and behavioral changes in the offspring. Furthermore, prenatal co-exposure to alcohol and cannabinoids induces malformations in brain regions associated with reward and stress-related circuitry. This study examined the effects of co-exposure to alcohol and the synthetic cannabinoid (SCB) CP55,940 throughout gastrulation and neurulation in rats on basal corticosterone levels and a battery of behavioral tests during adolescence and alcohol self-administration in adulthood. Importantly, we find that prenatal alcohol exposure (PAE) caused lower baseline corticosterone levels in adolescent males and females. Co-exposure to alcohol + CP produced hyperactivity during open field test in males, but not females. During the two-bottle choice alcohol-drinking procedure, prenatal cannabinoid exposed male and female adolescent rats drank more alcohol than their vehicle-exposed controls. In adulthood, female rats treated with prenatal cannabinoid exposure (PCE), showed an overall total increase in alcohol intake during alcohol self-administration; but this was not found in males. When the reinforcer was changed to a 1% sucrose solution, male rats exposed to PCE, showed a reduced self-administration compared to vehicle-exposed males, potentially indicative of an anhedonic response. This lower self-administration persisted when 20% alcohol was reintroduced to the sucrose solution. Lastly, following an abstinence period, there were no changes due to prenatal drug exposure in either males or females. Overall, these data suggest lasting consequences of prenatal alcohol and cannabinoid exposure during adolescence and adulthood in male and female rats.

The pro-apoptotic Bax gene modifies susceptibility to craniofacial dysmorphology following gastrulation-stage alcohol exposure.

BACKGROUND: During early development, alcohol exposure causes apoptotic cell death in discrete regions of the embryo which are associated with distinctive patterns of later-life abnormalities. In gastrulation, which occurs during the third week of human pregnancy, alcohol targets the ectoderm, the precursor of the eyes, face, and brain. This midline tissue loss leads to the craniofacial dysmorphologies, such as microphthalmia and a smooth philtrum, which define fetal alcohol syndrome (FAS). An important regulator of alcohol-induced cell death is the pro-apoptotic protein Bax. The current study determines if mice lacking the Bax gene are less susceptible to the pathogenic effects of gastrulation-stage alcohol exposure. METHODS: Male and female Bax+/- mice mated to produce embryos with full (-/- ) or partial (+/- ) Bax deletions, or Bax+/+ wild-type controls. On Gestational Day 7 (GD 7), embryos received two alcohol (2.9 g/kg, 4 hr apart), or control exposures. A subset of embryos was collected 12 hr later and examined for the presence of apoptotic cell death, while others were examined on GD 17 for the presence of FAS-like facial features. RESULTS: Full Bax deletion reduced embryonic apoptotic cell death and the incidence of fetal eye and face malformations, indicating that Bax normally facilitates the development of alcohol-induced defects. An RNA-seq analysis of GD 7 Bax+/+ and Bax-/- embryos revealed 63 differentially expressed genes, some of which may interact with the Bax deletion to further protect against apoptosis. CONCLUSIONS: Overall, these experiments identify that Bax is a primary teratogenic mechanism of gastrulation-stage alcohol exposure.

Prenatal alcohol exposure disrupts Sonic hedgehog pathway and primary cilia genes in the mouse neural tube.

Neurulation-stage alcohol exposure (NAE; embryonic day [E] 8-10) is associated with midline craniofacial and CNS defects that likely arise from disruption of morphogen pathways, such as Sonic hedgehog (Shh). Notably, midline anomalies are also a hallmark of genetic ciliopathies such as Joubert syndrome. We tested whether NAE alters Shh pathway signaling and the number and function of primary cilia, organelles critical for Shh pathway transduction. Female C57BL/6 J mice were administered two doses of alcohol (2.9 g/kg/dose) or vehicle on E9. Embryos were collected 6, 12, or 24 h later, and changes to Shh, cell cycle genes, and primary cilia were measured in the rostroventral neural tube (RVNT). Within the first 24 h post-NAE, reductions in Shh pathway and cell cycle gene expression and the ratio of Gli3 forms in the full-length activator state were observed. RVNT volume and cell layer width were reduced at 12 h. In addition, altered expression of multiple cilia-related genes was observed at 6 h post-NAE. As a further test of cilia gene-ethanol interaction, mice heterozygous for Kif3a exhibited perturbed behavior during adolescence following NAE compared to vehicle-treated mice, and Kif3a heterozygosity exacerbated the hyperactive effects of NAE on exploratory activity. These data demonstrate that NAE downregulates the Shh pathway in a region of the neural tube that gives rise to alcohol-sensitive brain structures and identifies disruption of primary cilia function, or a "transient ciliopathy", as a possible cellular mechanism of prenatal alcohol pathogenesis.

Alcohol, cocaine, and brain stimulation-reward in C57Bl6/J and DBA2/J mice.

BACKGROUND: Pleasure and reward are critical features of alcohol drinking that are difficult to measure in animal studies. Intracranial self-stimulation (ICSS) is a behavioral method for studying the effects of drugs directly on the neural circuitry that underlies brain reward. These experiments had 2 objectives: first, to establish the effects of alcohol on ICSS responding in the C57Bl6/J (C57) and DBA2/J (DBA) mouse strains; and second, to compare these effects to those of the psychostimulant cocaine. METHODS: Male C57 and DBA mice were implanted with unipolar stimulating electrodes in the lateral hypothalamus and conditioned to spin a wheel for reinforcement by the delivery of rewarding electrical stimulation (i.e., brain stimulation-reward or BSR). Using the curve-shift method, the BSR threshold (theta(0)) was determined immediately before and after oral gavage with alcohol (0.3, 0.6, 1.0, 1.7 g/kg) or water. Blood alcohol concentration (BAC) was measured to determine the influence of alcohol metabolism on BSR threshold. Separately, mice were administered cocaine (1.0, 3.0, 10.0, 30.0 mg/kg) or saline intraperitoneally. RESULTS: In C57 mice, the 0.6 g/kg dose of alcohol lowered BSR thresholds by about 20%, during the rising (up to 40 mg/dl), but not falling, phase of BAC. When given to the DBA mice, alcohol lowered BSR thresholds over the entire dose range; the largest reduction was by about 50%. Cocaine lowered BSR thresholds in both strains. However, cocaine was more potent in DBA mice than in C57 mice as revealed by a leftward shift in the cocaine dose-response curve. For both alcohol and cocaine, effects on BSR threshold were dissociable from effects on operant response rates. CONCLUSIONS: In C57 and DBA mice, reductions in BSR threshold reflect the ability of alcohol to potentiate the neural mechanisms of brain reward. The DBA mice are more sensitive to the reward-potentiating effects of both alcohol and cocaine, suggesting that there are mouse strain differences in the neural mechanisms of brain reward that can be measured with the ICSS technique.

Cannabinoids Exacerbate Alcohol Teratogenesis by a CB1-Hedgehog Interaction.

We tested whether cannabinoids (CBs) potentiate alcohol-induced birth defects in mice and zebrafish, and explored the underlying pathogenic mechanisms on Sonic Hedgehog (Shh) signaling. The CBs, Δ9-THC, cannabidiol, HU-210, and CP 55,940 caused alcohol-like effects on craniofacial and brain development, phenocopying Shh mutations. Combined exposure to even low doses of alcohol with THC, HU-210, or CP 55,940 caused a greater incidence of birth defects, particularly of the eyes, than did either treatment alone. Consistent with the hypothesis that these defects are caused by deficient Shh, we found that CBs reduced Shh signaling by inhibiting Smoothened (Smo), while Shh mRNA or a CB1 receptor antagonist attenuated CB-induced birth defects. Proximity ligation experiments identified novel CB1-Smo heteromers, suggesting allosteric CB1-Smo interactions. In addition to raising concerns about the safety of cannabinoid and alcohol exposure during early embryonic development, this study establishes a novel link between two distinct signaling pathways and has widespread implications for development, as well as diseases such as addiction and cancer.

5-HT(1B) receptor inhibition of alcohol-heightened aggression in mice: comparison to drinking and running.

RATIONALE: Serotonin 5-HT(1B) receptors are promising targets for the management of several mood and impulse disorders. OBJECTIVE: These experiments examine a 5-HT(1B) agonist, CP-94,253, and attempt to distinguish between its effects on seeking to perform three rewarding behaviors: aggression, drinking, and wheel running. MATERIALS AND METHODS: Male CFW mice perform nose-poke responses that are maintained by a fixed interval schedules of 10-min (FI10) schedule to gain access to one of three rewarding activities. The first experiment studies mice reinforced by the opportunity to confront an intruder mouse after drinking water or alcohol; the second studies mice reinforced by the presentation of alcoholic or non-alcoholic solutions (i.e., 6% ethanol, 0.05% saccharin vs 0.05% saccharin); the third studies mice reinforced by access to a running wheel. RESULTS: CP-94,253 (1.0-10 mg/kg i.p.) dose-dependently reduces aggression, drinking, and wheel running. Of these behaviors, alcohol-heightened aggression is the most sensitive to the 5-HT(1B) receptor agonist (ED50 = 4.8 mg/kg). Responding for the opportunity to drink or engage in alcohol-heightened aggression is suppressed by the highest dose of CP-94,253, whereas CP-94,253 does not affect responding that is reinforced by wheel running or species-typical aggression. CONCLUSIONS: These results confirm the inhibitory effects of 5-HT(1B) receptor stimulation on aggressive performance and drinking. They also reveal an inhibition of voluntary wheel running, contrary to the stimulation of running in a novel, open arena. 5-HT(1B) receptor agonists may be particularly useful for the treatment of aggressive behavioral disorders, but their efficacy and potency appear to be sensitive to the intensity and context of the behavior.

Maternal influences on the sexual behavior and reproductive success of the female rat.

In many species, including humans, there is evidence for parental effects on within-sex variations in reproductive behavior. In the present studies we found that variations in postnatal maternal care were associated with individual differences in female sexual behavior in the rat. Females born to and reared by dams that showed enhanced pup licking/grooming (i.e., High LG mothers) over the first week postpartum showed significantly reduced sexual receptivity and alterations in the pacing of male mounting (i.e., longer inter-intromission intervals) observed in a paced mating test. There were minimal effects on the sexual behavior of the male offspring. The female offspring of High LG mothers showed a reduced lordosis rating, a decreased mount:intromission ratio, received fewer ejaculations and were less likely to achieve pregnancy following mating in the paced mating context. The data suggest maternal influences on the sexual development of the female rat that are functionally relevant for reproductive success. Together with previous studies these findings imply that maternal care can 'program' reproductive strategies in the female rat.

A novel method for determining sex in late term gestational mice based on the external genitalia.

In many experiments using fetal mice, it is necessary to determine the sex of the individual fetus. However, other than genotyping for sex-specific genes, there is no convenient, reliable method of sexing mice between gestational day (GD) 16.5 and GD 18.0. We designed a rapid, relatively simple visual method to determine the sex of mouse fetuses in the GD 16.5-GD 18.0 range that can be performed as part of a routine morphological assessment. By examining the genitalia for the presence or absence of key features, raters with minimal experience with the method were able to correctly identify the sex of embryos with 99% accuracy, while raters with no experience were 95% accurate. The critical genital features include: the presence or absence of urethral seam or proximal urethral meatus; the shape of the genitalia, and the presence or absence of an area related to the urethral plate. By comparing these morphological features of the external genitalia, we show a simple, accurate, and fast way to determine the sex of late stage mouse fetuses. Integrating this method into regular morphological assessments will facilitate the determination of sex differences in fetuses between GD 16.5 and GD 18.0.

Anti-aggressive effects of agonists at 5-HT1B receptors in the dorsal raphe nucleus of mice.

RATIONALE: In rodents, serotonin 1B (5-HT(1B)) agonists specifically reduce aggressive behaviors, including several forms of escalated aggression. One form of escalated aggression is seen in mice that seek the opportunity to attack another mouse by accelerating their responding during a fixed interval (FI) schedule. Responses preceding the opportunity to attack may reflect aggressive motivation. OBJECTIVE: This study investigated the effects of two 5-HT(1B) receptor agonists on the motivation to fight and the performance of heightened aggression. MATERIALS AND METHODS: Male mice were housed as "residents" and performed nose-poke responses on an FI 10-min schedule with the opportunity to briefly attack an "intruder" serving as the reinforcer. In the first experiment, the 5-HT(1B) receptor agonist, CP-94,253 (0-10 mg/kg, IP), was given 30 min before the FI 10 schedule. To confirm that CP-94,253 achieved its effects via 5-HT(1B) receptors, the 5HT(1B/1D) receptor antagonist, GR 127,935 (10 mg/kg, IP) was administrated before the agonist injection. In the second experiment, the 5-HT(1B) agonist CP-93,129 (0-1.0 microg) was microinjected into the dorsal raphe 10 min before the FI 10 schedule. RESULTS: The agonists had similar effects on all behaviors. CP-94,253 and CP-93,129 significantly reduced the escalated aggression towards the intruder at doses lower than those required to affect operant responding. The highest doses of CP-94,253 (10 mg/kg) and CP-93,129 (1.0 microg) decreased the rate and accelerating pattern of responding during the FI 10 schedule; lower doses were less effective. GR 127,935 antagonized CP-94,253's effects on all other behaviors, except response rate. CONCLUSIONS: These data extend the anti-aggressive effects of 5-HT(1B) agonists to a type of escalated aggression that is rewarding and further suggest that these effects are associated with actions at 5-HT(1B) receptors in the dorsal raphe.

Genetic vulnerabilities to prenatal alcohol exposure: Limb defects in sonic hedgehog and GLI2 heterozygous mice.

BACKGROUND: Genetic factors influence the physical and neurobehavioral manifestations of prenatal alcohol exposure (PAE). Animal models allow the investigation of specific genes that confer vulnerability to, or protection from, birth defects associated with fetal alcohol spectrum disorders (FASDs). The objective of the present experiments was to determine if genetic alterations in the Sonic Hedgehog (Shh) signaling pathways affect the vulnerability to PAE-induced skeletal defects involving the forelimbs and/or hindlimbs. METHOD: Wild-type C57BL/6J female mice were bred with males in which one copy of the Shh or Gli2 genes had been knocked out, to produce litters with both wild-type (+/+) and heterozygous (+/-) embryos. Alcohol doses (two injections of 2.9 g/kg, 4 hours apart) or vehicles were administered starting at gestational day (GD) 9.25, 9.5, or 9.75, a critical exposure time for inducing limb defects. Limb defects were examined at GD 17 using a dysmorphology scale based on abnormalities ranging from increased interdigital spacing to the deletion of multiple fingers and the ulna. RESULTS: Alcohol treatment caused a high incidence of forelimb defects, particularly on the right side, that was higher in Shh+/- and Gli2+/- fetuses compared to wild-type fetuses. Dysmorphology scores were also significantly higher in the Shh+/- and Gli2+/- mice. CONCLUSIONS: These results extend previous findings demonstrating enhanced sensitivity to PAE-induced craniofacial dysmorphology and support the hypothesis that genetic alterations in the Shh signaling pathway influences the vulnerability to alcohol-induced birth defects. Moreover, these results emphasize the importance of understanding the interactions between genes and prenatal exposure to alcohol or other teratogens. Birth Defects Research 109:860-865, 2017. © 2017 Wiley Periodicals, Inc.

Preaxial polydactyly following early gestational exposure to the smoothened agonist, SAG, in C57BL/6J mice.

BACKGROUND: While pharmacological activation of the Hedgehog (HH) signaling pathway may have therapeutic benefits for developmental and adult diseases, its teratogenic potential is of concern. The membrane molecule Smoothened (SMO) transduces HH signaling and can be acutely modulated by antagonists and agonists. The objective of the current experiments was to determine how maternal treatment with the Smo agonist, SAG, affects the developing limb. METHODS: Pregnant C57BL/6J mice received a single injection of SAG (15, 17, or 20 mg/kg, i.p.) or its vehicle on gestational day (GD) 9.25, the time of limb bud induction. Embryos were examined on GD 15 for gross dysmorphology and skeletal staining was performed to visualize the number and type of digits on the fore- and hindlimbs. Additionally, in situ hybridization was performed 4 hr after GD 9.25 SAG administration to determine SAG's effects on Gli1 and Gli2 mRNA expression. RESULTS: The most prevalent effect of SAG was the dose-dependent induction of pre-axial polydactyly; defects ranged from a broad thumb to the duplication of two finger-like digits on the preaxial side of the thumb. The highest SAG dose was effective in ca. 80% of the embryos and increased Gli1 and Gli2 mRNA expression in the limb bud, with Gli1 mRNA being the most upregulated. CONCLUSION: Preaxial polydactyly can be caused in the developing embryo by acute maternal administration of a Smo agonist that activates HH signaling. These results are consistent with the preaxial polydactyly induced in developmental disorders associated with mutations in HH signaling genes.Birth Defects Research 109:49-54, 2017. © 2016 Wiley Periodicals, Inc.

Dose-dependent teratogenicity of the synthetic cannabinoid CP-55,940 in mice.

Potent synthetic cannabinoids (SCBs) are illegally distributed drugs of abuse that are frequently consumed in spite of their adverse consequences. This study was designed to determine if the toxicity observed in adults also extends to the prenatal period by examining the developmental toxicity/teratogenicity of one of these SCBs, CP-55,940, in a mammalian model. First, immunohistochemistry was employed for cannabinoid receptor 1 (CB1) localization within gestational day (GD) 8 mouse embryos; this receptor was identified in the cranial neural plate, suggesting that the endogenous cannabinoid system may be involved in normal development. Based on this information and on previous avian teratogenicity studies, the current investigation focused on cannabinoid exposure during neurulation. The treatment paradigm involved acute i.p. administration of vehicle, 0.0625, 0.125, 0.25, 0.5, 1.0, or 2.0mg/kg CP-55,940 to time-mated C57Bl/6J mice on their 8th day of pregnancy (n>10 litters per treatment group). On GD 17, litters were harvested and examined for numbers of live, dead, or resorbed fetuses, as well as for fetal weight, length, and gross morphological abnormalities. No effect on litter size, fetal weight, or crown rump length was seen at any of the CP-55,940 dosages tested. Major malformations involving the craniofacies and/or eyes were noted in all drug-treated groups. Selected fetuses with craniofacial malformations were histologically sectioned and stained, allowing investigation of brain anomalies. Observed craniofacial, ocular, and brain abnormalities in drug-treated fetuses included lateral and median facial clefts, cleft palate, microphthalmia, iridial coloboma, anophthalmia, exencephaly, holoprosencephaly, and cortical dysplasia. With the most commonly observed defects involving the eyes, the incidence and severity of readily identifiable ocular malformations were utilized as a basis for dose-response analyses. Ocular malformation ratings revealed dose-dependent CP-55,940 teratogenicity within the full range of dosages tested. While examination of additional critical periods and in depth mechanistic studies is warranted, the results of this investigation clearly show the dose-dependent teratogenicity of this SCB.

The programming of individual differences in defensive responses and reproductive strategies in the rat through variations in maternal care.

There are profound maternal effects on individual differences in defensive responses and reproductive strategies in species ranging literally from plants to insects to birds. Maternal effects commonly reflect the quality of the environment and are most likely mediated by the quality of the maternal provision (egg, propagule, etc.), which in turn determines growth rates and adult phenotype. In this paper, we review data from the rat that suggest comparable forms of maternal effects on both defensive responses to threat and reproductive behavior and which are mediated by variations in maternal behavior. Ultimately, we will need to contend with the reality that neural development, function and health are defined by social and economic influences.

Escalated aggression as a reward: corticosterone and GABA(A) receptor positive modulators in mice.

RATIONALE: Individuals seek out the opportunity to fight, but the mechanisms behind this positively reinforcing effect of aggression have yet to be understood. OBJECTIVES: The aims of this study were to (1) describe behavioral and corticosterone elevations that occur in aggressive mice conditioned to respond for the opportunity to fight another mouse, (2) determine if corticosterone elevations are necessary for operant responding and escalated aggression, and (3) determine if corticosterone elevations alter the aggression-heightening effects of gamma-aminobutyric acid (GABA)(A) receptor positive modulators. METHODS AND RESULTS: Aggressive male CFW mice were conditioned to respond under the control of a fixed-interval 10-min (FI10) schedule that reinforced their operant behavior by the presentation of an intruder mouse into their home cage. After the FI10, aggressive behavior was ca. 75% higher than the species-typical levels of fighting and plasma corticosterone was more than twice as high after briefly fighting and/or responding on the FI10 schedule. Inhibition of corticosterone synthesis by metyrapone (30-100 mg/kg) reduced both conditioned responding as well as the aggressive behavior after the FI. Although the benzodiazepine midazolam (0.3-3 mg/kg) heightened species-typical aggressive behavior, it did not increase the high level of aggression engendered by the FI schedule. However, midazolam (0.3 mg/kg) and the neurosteroid allopregnanolone (17 mg/kg) both heightened aggression when given after corticosterone synthesis inhibition by metyrapone (56 mg/kg). CONCLUSIONS: These data suggest that corticosterone elevations are required for responding that is motivated by aggressive behavior and for escalated aggression that follows this responding. Corticosterone elevations also appear to inhibit the aggression heightening effect of GABA(A) receptor positive modulators.