Rudolph Spangler

Detection of single nucleotide polymorphisms of the human mu opioid receptor gene by hybridization or single nucleotide extension on custom oligonucleotide gelpad microchips: Potential in studies of addiction

The human mu opioid receptor (MOR) plays a central role in mediating the effects of opioids, both endogenous and exogenous. Epidemiological studies have shown that addiction in general, and especially opiate addiction, has a heritable component. Clinical and laboratory studies suggest that the MOR gene may contribute to the heritable component of vulnerability to develop opiate addiction. Naturally occurring single nucleotide polymorphisms (SNPs) have been identified in the MOR gene by conventional methods. Two coding region SNPs, the A118G and C17T substitutions, occur at high allelic frequencies (10.5% and 6.6%, respectively, in our previous studies). These common SNPs cause amino acid changes in the receptor, and may have implications for differences in individual responses to opioids, as well as decreased or increased vulnerability to opiate addiction. The A118G substitution encodes a variant receptor with binding and signal transduction differences in response to beta-endorphin in cellular assays. Recent innovations in microchip technology offer new potential methods for SNP detection. We report here on the development of two separate approaches using custom oligonucleotide gelpad microarrays for detection of these two common SNPs of the MOR gene in human DNA samples. First, PCR-amplified genomic DNA samples were used to produce target sequences, which were labeled with fluorescent dye and hybridized to custom microchips. Oligonucleotides on these reusable microchips were designed to query nucleotide substitutions at positions 17 and 118 of the MOR gene. Thirty-six human DNA samples were assayed both on these custom microchips and by conventional automated gel sequencing, with highly concordant identification of both heterozygous and homozygous substitutions. A second approach was developed for the C17T SNP utilizing single nucleotide extension on custom microchips. These custom gelpad microchips have potential for the rapid and inexpensive detection of specific SNPs for genetic and genomic studies.

Acute intermittent morphine increases preprodynorphin and kappa opioid receptor mRNA levels in the rat brain.

We determined the effects of morphine on mRNA levels for the opioid ligands preprodynorphin (PPD) and preproenkephalin (PPE) and the kappa opioid receptor (KOR). Rats received six injections of morphine (6.25 mg/kg/injection) every 2 h, and were sacrificed 30 min later. mRNA levels were measured in brain tissue after removal of the cortex, cerebellum and brainstem. There were increases in PPD and KOR mRNA levels (P<0.05 and P<0.005, respectively), with no alteration of PPE. These alterations in the kappa/dynorphin system may counter morphine-induced effects on the brain.

Modulation of CRF-R1 mRNA in rat anterior pituitary by dexamethasone: correlation with POMC mRNA.

Glucocorticoids have been shown to decrease CRF receptor binding in the anterior pituitary. To determine whether glucocorticoids or CRF peptide modulate CRF-R1 gene expression, CRF-R1 mRNA levels in rat pituitary and brain were measured after administration of a synthetic glucocorticoid, dexamethasone (DEX) or rat/human CRF (r/hCRF), using a sensitive solution hybridization RNase protection assay. DEX (400 micrograms/day) or r/hCRF (100 micrograms/kg/day) was administered twice daily for 5 days to male rats. DEX treatment caused a significant decrease in CRF-R1 mRNA levels in the anterior pituitary. Also, a significant positive correlation was found between CRF-R1 and POMC mRNA levels in the anterior pituitary of the individual animals: levels of both mRNAs were reduced by DEX. However, r/hCRF treatment had no significant effect on CRF-R1 mRNA levels in the anterior pituitary. In extrahypothalamic brain regions, mRNA levels of CRF-R1 did not change following either DEX or r/hCRF administration. Our data suggest that in addition to POMC and CRF genes, CRF-R1 gene may also be subject to negative feedback control by glucocorticoids.

Chronic food restriction and streptozotocin-induced diabetes differentially alter prodynorphin mRNA levels in rat brain regions

It was previously reported that chronic food restriction and streptozotocin-induced diabetes lead to brain region-specific changes in levels of Prodyn-derived peptides. These changes parallel behavioral adaptations that are reversed by opioid antagonists. In the present study, effects of food restriction and diabetes on Prodyn gene expression were measured in rat brain regions using a quantitative solution hybridization mRNA assay. Picogram amounts of Prodyn mRNA were determined in extracts of five brain regions. The highest density of Prodyn mRNA was observed in extracts of nucleus accumbens (4.68 pg/microg total RNA), bed nucleus of the stria terminalis (4.18 pg/microg), and in caudate nucleus (3.51 pg/microg). Lower levels were observed in the lateral hypothalamus (1.87 pg/microg) and central nucleus of the amygdala (1.22 pg/microg). Food restriction and diabetes both markedly increased the levels of Prodyn mRNA in the central amygdala (163% and 93%, respectively). Levels in the lateral hypothalamus were also increased (35% and 29%, respectively), though only the food-restriction effect was statistically significant. Neither treatment altered prodynorphin mRNA levels in the caudate nucleus, nucleus accumbens or bed nucleus of the stria terminalis. These results suggest that dynorphin neurons in central amygdala and lateral hypothalamus may be involved in behavioral or physiological adaptations to sustained metabolic need.

Optimizing Taq Polymerase Concentration for Improved Signal-to-Noise in the Broad Range Detection of Low Abundance Bacteria

Background PCR in principle can detect a single target molecule in a reaction mixture. Contaminating bacterial DNA in reagents creates a practical limit on the use of PCR to detect dilute bacterial DNA in environmental or public health samples. The most pernicious source of contamination is microbial DNA in DNA polymerase preparations. Importantly, all commercial Taq polymerase preparations inevitably contain contaminating microbial DNA. Removal of DNA from an enzyme preparation is problematical. Methodology/Principal Findings This report demonstrates that the background of contaminating DNA detected by quantitative PCR with broad host range primers can be decreased greater than 10-fold through the simple expedient of Taq enzyme dilution, without altering detection of target microbes in samples. The general method is: For any thermostable polymerase used for high-sensitivity detection, do a dilution series of the polymerase crossed with a dilution series of DNA or bacteria that work well with the test primers. For further work use the concentration of polymerase that gave the least signal in its negative control (H2O) while also not changing the threshold cycle for dilutions of spiked DNA or bacteria compared to higher concentrations of Taq polymerase. Conclusions/Significance It is clear from the studies shown in this report that a straightforward procedure of optimizing the Taq polymerase concentration achieved “treatment-free” attenuation of interference by contaminating bacterial DNA in Taq polymerase preparations. This procedure should facilitate detection and quantification with broad host range primers of a small number of bona fide bacteria (as few as one) in a sample.

QUANTITATION OF DOPAMINE TRANSPORTER MRNA IN THE RAT BRAIN : MAPPING, EFFECTS OF BINGE COCAINE ADMINISTRATION AND WITHDRAWAL

Dopamine transporter (DAT) mRNA from selected brain regions of individual male Fischer rats was quantitated utilizing a sensitive solution hybridization assay in which the levels of RNase‐protected 32P‐labeled mRNA:cRNA hybrids were measured. DAT mRNA was detected in whole brain regions known to contain abundant DAT mRNA (mean picogram of DAT mRNA/microgram of total RNA ± SEM): substantia nigra, 7.17 ± 0.47; ventral tegmentum, 4.71 ± 0.38. In regions known to contain low levels of DAT mRNA, these levels were detected: central grey, 0.39 ± 0.06; hypothalamus, 0.14 ± 0.03. In addition, DAT mRNA was detected in areas where it had not previously been identified: amygdala, 0.19 ± 0.03; caudate‐putamen, 0.15 ± 0.03; nucleus accumbens, 0.13 ± 0.01; pons/medulla, 0.12 ± 0.02; globus pallidus, 0.09 ± 0.04; pituitary, 0.07 ± 0.01; frontal cortex, 0.05 ± 0.01. No DAT mRNA was detected in 150 μg of rat liver RNA. As cocaine binds to and inhibits the activity of the dopamine transporter, we sought to determine if there were differences in dopamine transporter mRNA levels between saline‐ and cocaine‐injected rats or rats withdrawn from a chronic “binge” pattern (15 mg/kg per dose i.p.; three doses at 1 h intervals each day) cocaine injection. Using trichloroacetic acid precipitation of mRNA:cRNA hybrids from RNA extracted from whole brain regions, we found no significant differences in the substantia nigra or the ventral tegmentum following subacute (3 days) binge, chronic (14 days) binge or 10 days withdrawal from a chronic binge pattern cocaine or saline administration. Synapse 26:55–61, 1997.

Steady-state methadone in rats does not change mRNA levels of corticotropin-releasing factor, its pituitary receptor or proopiomelanocortin.

Male Fischer rats received either methadone (a long-acting opioid agonist, 10 mg/kg/day) or saline (24 microliters/day) subcutaneously by osmotic minipumps for 7 days. Chronic steady-state methadone administration did not alter (a) corticotropin-releasing factor (CRF) mRNA in the hypothalamus, (b) proopiomelanocortin (POMC) and CRF type 1 receptor (CRF-R1) mRNAs in the anterior lobe and neurointermediate/posterior lobe of the pituitary, or (c) circulating levels of corticosterone. No change was found in levels of either POMC mRNA in the hypothalamus and amygdala, or CRF mRNA in the frontal cortex, olfactory bulb and amygdala. These results demonstrate that neither the activity of the hypothalamic-pituitary-adrenal axis, nor the beta-endorphin and CRF systems in the brain, are altered by steady-state occupancy of opioid receptors with the long-acting opioid agonist methadone.

Effects of Chronic ‘Binge’ Cocaine Administration on Plasma ACTH and Corticosterone Levels in Mice Deficient in DARPP-32

The product of the DARPP-32 gene mediates intracellular signals initiated by the binding of dopamine to its receptors. Cocaine administration leads to increased activation of dopamine receptors, and causes activation of the stress-responsive hypothalamic-pituitary-adrenal (HPA) axis. We determined the effects of chronic ‘binge’ pattern cocaine on HPA activity in mice containing a targeted disruption of the DARPP-32 gene. Mice received three daily injections of cocaine (15 mg/kg/injection) for 14 days, and were sacrificed 30 min after the last injection. We measured the levels of plasma adrenocorticotropin (ACTH) and corticosterone which reflect HPA activity. In wild-type controls, ‘binge’ cocaine administration significantly increased plasma ACTH and corticosterone levels. In contrast, DARPP-32-deficient mice failed to show a significant elevation of either plasma ACTH or corticosterone levels following ‘binge’ cocaine. The results indicate that DARPP-32 plays a role in mediating the stimulatory effects of cocaine on the HPA axis.

Tests of the single-hit DNA damage model.

The algebra of target theory for damage by radiation was laid out by Atwood and Norman in 1949. Their equations provide a widely embraced framework for distinguishing single-hit and multi-hit mechanisms of damage. The present work asks whether in vitro damage to DNA duplexes by different agents affects amplification by the polymerase chain reaction (PCR) in a single-hit manner. Real-time monitoring of fluorescent PCR product (qPCR) was used to measure the fraction of DNA (S) surviving doses (D) of three damaging agents: gamma irradiation, DNase I, and UV radiation. The log fraction surviving was compared to the best-fit straight line predicted for a random single-hit model (lnS=kD). Human DNA targets for analysis were segments of multiple (nested) DNA lengths from the nuclear and the mitochondrial genomes within 10% of 150, 250, 350, 450, 650, 1000 and 2000 bases. For gamma irradiation, the results were consistent with a single-hit model for all segment sizes. In the case of DNase I, the shortest segment (150 bp), for both genomic and mitochondrial DNA, experienced more damage at low concentrations of DNase than the random single-hit model predicted. Conversely, in the case of UV, all segments of the nuclear target gene were less damaged at low doses and more damaged at high doses than predicted by the one hit model. These deviations from the predictions of a random single-hit model were interpreted as evidence for concerted activity in the case of DNase and of a multi-hit, sequence-dependent mechanism in the case of UV, perhaps due to the accumulation of lesions that slowed but did not entirely block Taq polymerase elongation.

Attenuation of the hypothalamic-pituitary-adrenal response to chronic “binge” pattern cocaine administration corresponds to decreased CRH mRNA in rat hypothalamus

Abstract The effects of subacute or chronic “binge” pattern administration of cocaine on the levels of corticotropin-releasing hormone (CRH) mRNA in rat hypothalamus and corticosterone concentrations in blood were studied. On day 3 of cocaine administration (15 mg/kg, i.p. at 1 hour intervals × 3 /day), no significant change in hypothalamic CRH mRNA levels was observed in injection-adapted rats. In contrast, this subacute “binge” pattern cocaine administration caused a significant elevation in plasma corticosterone concentrations ( p ). Following 14 days of chronic cocaine administration using the “binge” pattern, the levels of CRH mRNA in the hypothalamus were significantly reduced by 32% ( p ), compared with control animals. This decrease was coupled with a reduction in levels of plasma corticosterone compared to corticosterone levels in the subacute cocaine administration group ( p ). These results suggest that chronic “binge” pattern cocaine administration alters the hypothalamic — pituitary — adrenal response through a hypothalamic CRH-mediated mechanism.