Maternal cocaine administration in mice alters DNA methylation and gene expression in hippocampal neurons of neonatal and prepubertal offspring

Previous studies documented significant behavioral changes in the offspring of cocaine-exposed mothers. We now explore the hypothesis that maternal cocaine exposure could alter the fetal epigenetic machinery sufficiently to cause lasting neurochemical and functional changes in the offspring. Pregnant CD1 mice were administered either saline or 20 mg/kg cocaine twice daily on gestational days 8-19. Male pups from each of ten litters of the cocaine and control groups were analyzed at 3 (P3) or 30 (P30) days postnatum. Global DNA methylation, methylated DNA immunoprecipitation followed by CGI(2) microarray profiling and bisulfite sequencing, as well as quantitative real-time RT-PCR gene expression analysis, were evaluated in hippocampal pyramidal neurons excised by laser capture microdissection. Following maternal cocaine exposure, global DNA methylation was significantly decreased at P3 and increased at P30. Among the 492 CGIs whose methylation was significantly altered by cocaine at P3, 34% were hypermethylated while 66% were hypomethylated. Several of these CGIs contained promoter regions for genes implicated in crucial cellular functions. Endogenous expression of selected genes linked to the abnormally methylated CGIs was correspondingly decreased or increased by as much as 4-19-fold. By P30, some of the cocaine-associated effects at P3 endured, reversed to opposite directions, or disappeared. Further, additional sets of abnormally methylated targets emerged at P30 that were not observed at P3. Taken together, these observations indicate that maternal cocaine exposure during the second and third trimesters of gestation could produce potentially profound structural and functional modifications in the epigenomic programs of neonatal and prepubertal mice

Up-regulation of neuronal calcium sensor-1 (NCS-1) in the prefrontal cortex of schizophrenic and bipolar patients

The delineation of dopamine dysfunction in the mentally ill has been a long-standing quest of biological psychiatry. The present study focuses on a recently recognized group of dopamine receptor- interacting proteins as possible novel sites of dysfunction in schizophrenic and bipolar patients. We demonstrate that the dorsolateral prefrontal cortex in schizophrenia and bipolar cases from the Stanley Foundation Neuropathology Consortium display significantly elevated levels of the D2 dopamine receptor desensitization regulatory protein, neuronal calcium sensor-1. These levels of neuronal calcium sensor-1 were not influenced by age, gender, hemisphere, cause of death, postmortem period, alcohol consumption, or antipsychotic and mood stabilizing medications. The present study supports the hypothesis that schizophrenia and bipolar disorder may be associated with abnormalities in dopamine receptor-interacting proteins

Neonatal local noxious insult affects gene expression in the spinal dorsal horn of adult rats

Neonatal noxious insult produces a long-term effect on pain processing in adults. Rats subjected to carrageenan (CAR) injection in one hindpaw within the sensitive period develop bilateral hypoalgesia as adults. In the same rats, inflammation of the hindpaw, which was the site of the neonatal injury, induces a localized enhanced hyperalgesia limited to this paw. To gain an insight into the long-term molecular changes involved in the above-described long-term nociceptive effects of neonatal noxious insult at the spinal level, we performed DNA microarray analysis (using microarrays containing oligo-probes for 205 genes encoding receptors and transporters for glutamate, GABA, and amine neurotransmitters, precursors and receptors for neuropeptides, and neurotrophins, cytokines and their receptors) to compare gene expression profiles in the lumbar spinal dorsal horn (LDH) of adult (P60) male rats that received neonatal CAR treatment within (at postnatal day 3; P3) and outside (at postnatal 12; P12) of the sensitive period. The data were obtained both without inflammation (at baseline) and during complete Freund's adjuvant induced inflammation of the neonatally injured paw. The observed changes were verified by real-time RT-PCR. This study revealed significant basal and inflammation-associated aberrations in the expression of multiple genes in the LDH of adult animals receiving CAR injection at P3 as compared to their expression levels in the LDH of animals receiving either no injections or CAR injection at P12. In particular, at baseline, twelve genes (representing GABA, serotonin, adenosine, neuropeptide Y, cholecystokinin, opioid, tachykinin and interleukin systems) were up-regulated in the bilateral LDH of the former animals. The baseline condition in these animals was also characterized by up-regulation of seven genes (encoding members of GABA, cholecystokinin, histamine, serotonin, and neurotensin systems) in the LDH ipsilateral to the neonatally-injured paw. The largest aberration in gene expression, however, was observed during inflammation of the neonatally injured hindpaws in the ipsilateral LDH, which included thirty-six genes (encoding numerous members of glutamate, serotonin, GABA, calcitonin gene-related peptide, neurotrophin, and interleukin systems). These findings suggest that changes in gene expression may be involved in the long-term nociceptive effects of neonatal noxious insult at the spinal level

Primers and probes used in bisulfite sequencing and for real-time PCR.

<p>For the nested PCR, the first row for each gene depicts the primers used in the first and second steps of the nested PCR with the size of the expected amplicons. Common abbreviated names of the target genes are shown in parentheses. F1 and F2, forward primers; R1, reverse primer; Pr, probe.</p

PCR primers and probes used for analysis of DNA methylases and as internal controls.

<p>PCR primers and probes used for analysis of DNA methylases and as internal controls.</p

Histology of hippocampal sections before and after laser microdissection.

<p>Photomicrograph of a typical HistoGene-stained coronal section through the hippocampus of a 3-day-old mouse (a), and the same section following excision of the hippocampal pyramidal layer by microdissection on a Leica ASLMD laser capture microdissection system (b). HIPpl, hippocampal pyramidal layer; DG, dentate gyrus; SUB, subiculum. Note the precision of the cut.</p

Global DNA methylation and DNMT expression levels in the hippocampal pyramidal layer of P3 offspring from saline-injected or cocaine-treated dams.

<p>(a) Global levels of cytosine methylation in DNA samples of control and cocaine groups each expressed as percentage relative to the methylation signal in Epigentek's methylated DNA standard. (b) Expression levels of DNA methyltransferases DNMT1, DNMT3a, and DNMT3b each calculated as a ratio relative to GAPDH as internal standard. Similar results were obtained with ß-actin used as internal standard. Each column represents mean±SEM of 10 samples. *p<0.05 compared to the saline-control group.</p

PCR probes used in verification of the linearity of MeDIP-based methylcytosine detection.

<p>PCR probes used in verification of the linearity of MeDIP-based methylcytosine detection.</p

Genomic characteristics of abnormally methylated CGIs in the hippocampal pyramidal layer of P3 cocaine-treatment pups.

<p>Information associating abnormally methylated CGIs with various genomic elements was obtained by BLAT analysis of the affected CGIs as determined from the MeDIP/CGI array profiling experiments. The data is limited to the genomic elements that are present in at least 5 CGIs. The number of CGIs associated with a particular genomic element is represented by horizontal bars across from the element's name. The length of the bar is proportional to the number of CGIs; this number is also given at the end of the row. The number of CGIs with sequences matching more than one position in the genome is provided in parenthesis. The tendency for CGIs to be associated with more than one genomic element is reflected in the partial overlap of the bars on most rows. Note that overall there are more hypomethylated CGIs than hypermethylated CGIs.</p