C57BL/6J (B6) mice are susceptible to in utero growth retardation and a number of morphological malformations following prenatal alcohol exposure, while DBA/2J (D2) mice are relatively resistant. We have previously shown that genomic imprinting may play a role in differential sensitivity between B6 and D2 (Downing and Gilliam 1999). The best characterized mechanism mediating genomic imprinting is differential DNA methylation. In the present study we examined DNA methylation and gene expression, in both embryonic and placental tissue, at the mouse Igf2 locus following in utero ethanol exposure. We also examined the effects of a methyl-supplemented diet on methylation and ethanol teratogenesis. In embryos from susceptible B6 mice, we found small decreases in DNA methylation at four CpG sites in one of the differentially methylated regions of the Igf2 locus; only one of the four sites showed a statistically significant decrease. We observed no significant decreases in methylation in placentae. All Igf2 transcripts showed approximately 1.5 fold decreases following intrauterine alcohol exposure. Placing dams on a methyl-supplemented diet before pregnancy and throughout gestation brought methylation back up to control levels. Methyl-supplementation also resulted in lower prenatal mortality, greater prenatal growth, and decreased digit malformations; it dramatically reduced vertebral malformations. Thus, while prenatal alcohol had only small effects on DNA methylation at the Igf2 locus, placing dams on a methyl-supplemented diet partially ameliorated ethanol teratogenesis.

We examined gene expression in the lumbar spinal cord and the specific response of motoneurons, intermediate gray and proprioceptive sensory neurons after spinal cord injury and exercise of hindlimbs to identify potential molecular processes involved in activity dependent plasticity. Adult female rats received a low thoracic transection and passive cycling exercise for 1 or 4 weeks. Gene expression analysis focused on the neurotrophic factors; brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), and their receptors because of their potential roles in neural plasticity. We also examined expression of genes involved in the cellular response to injury; heat shock proteins (HSP) -27 and -70, glial fibrillary acidic protein (GFAP) and caspases -3, -7, and -9. In lumbar cord samples, injury increased the expression of mRNA for TrkB, all three caspases and the HSPs. Acute and prolonged exercise increased expression of mRNA for the neurotrophic factors BDNF and GDNF, but not their receptors. It also increased HSP expression and decreased caspase-7 expression, with changes in protein levels complimentary to these changes in mRNA expression. Motoneurons and intermediate grey displayed little change in mRNA expression following injury, but acute and prolonged exercise increased levels of mRNA for BDNF, GDNF and NT-4. In large DRG neurons, mRNA for neurotrophic factors and their receptors were largely unaffected by either injury or exercise. However, caspase mRNA expression was increased by injury and decreased by exercise. Our results demonstrate that exercise affects expression of genes involved in plasticity and apoptosis in a cell specific manner and that these change with increased post-injury intervals and/or prolonged periods of exercise.

The H-reflex habituates at relatively low frequency (10 Hz) stimulation in the intact spinal cord, but loss of descending inhibition resulting from spinal cord transection reduces this habituation. There is a return towards a normal pattern of low-frequency habituation in the reflex activity with cycling exercise of the affected hind limbs. This implies that repetitive passive stretching of the muscles in spinalized animals and the accompanying stimulation of large (Group I and II) proprioceptive fibers has modulatory effects on spinal cord reflexes after injury. To test this hypothesis, we induced pyridoxine neurotoxicity that preferentially affects large dorsal root ganglia neurons in intact and spinalized rats. Pyridoxine or saline injections were given twice daily (IP) for 6 weeks and half of the spinalized animals were subjected to cycling exercise during that period. After 6 weeks, the tibial nerve was stimulated electrically and recordings of M and H waves were made from interosseous muscles of the hind paw. Results show that pyridoxine treatment completely eliminated the H-reflex in spinal intact animals. In contrast, transection paired with pyridoxine treatment resulted in a reduction of the frequency-dependent habituation of the H-reflex that was not affected by exercise. These results indicate that normal Group I and II afferent input is critical to achieve exercise-based reversal of hyper-reflexia of the H-reflex after spinal cord injury.

DNA methylation at cytosines is a widely studied epigenetic modification. Methylation is commonly detected using bisulfite modification of DNA followed by PCR and additional techniques such as restriction digestion or sequencing. These additional techniques are either laborious, require specialized equipment, or are not quantitative. Here we describe a simple algorithm that yields quantitative results from analysis of conventional four-dye-trace sequencing. We call this method Mquant and we compare it with the established laboratory method of combined bisulfite restriction assay (COBRA). This analysis of sequencing electropherograms provides a simple, easily applied method to quantify DNA methylation at specific CpG sites.

Objective: Comparison of pedophilic individuals who do and do not refrain from sexually engaging with children may offer critically important information regarding the differential contributors to pedophilic attraction versus behavior. This study compared 5 traits that are potentially contributory to pedophilic attraction or behavior in both minor-attracted persons (MAPs) who refrain from sexually engaging with minors (nonacting MAPs) and those who have acted on pedophilic attractions and subsequently entered the criminal justice system (forensic MAPs). Methods: Subjects included 195 nonacting MAPs, 50 forensic MAPs, and 60 healthy controls. Data on nonacting MAPs were drawn from an online survey, and data on the other 2 groups were based on prior in-person evaluations. Measures included the Millon Clinical Multiaxial Inventory-II (MCMI-II), Barratt Impulsiveness Scale (BIS-11), the MAP Questionnaire, and the Sexual History Questionnaire (SHQ). Results: Both MAP groups scored higher than healthy controls on the domains of socially inhibited personality traits, propensity toward cognitive distortions, and subjects’ own childhood sexual abuse (CSA). Forensic MAPs scored higher than nonacting MAPs on the CSA domain, but the 2 MAP groups differed little on the other 2 domains. Forensic MAPs also scored higher than the other 2 groups on the antisocial domain, whereas nonacting MAPs did not differ from controls on this measure. Nonacting MAPs scored higher than controls on impulsivity. Conclusions: Antisocial personality traits may be a primary driver of pedophilic behavior that is unrelated to pedophilic attraction. Socially inhibited personality traits and propensity toward cognitive distortions are associated with pedophilic attraction, although the direction of causation is not clear. CSA seems to increase the risk of both attraction and behavior.