Barbara Hitzemann

Binding of 3H-naloxone in the mouse brain: Effect of ions and tolerance development☆

Abstract The binding of 3 H-naloxone (spec. act. 5.2 Ci/mmol) in a crude mitochondrial fraction of the whole mouse brain was examined. Binding was reversed by the narcotic agonists levorphanol, morphine and 1-methadone but not by dextrorphan. Levorphanol sensitive (specific) 3 H-naloxone binding was blocked by Na+, Li+, Ca++, Mg++ and Mn++ but not by K+. When the crude mitochondrial fraction was separated on a discontinuous sucrose gradient, the highest activity of specific binding was found in the nerve ending particle fraction. Animals made physically dependent by 3 day morphine pellet implantation did not show an increased binding affinity for 3 H-nalovxone. The implantation of a 10 mg naloxone pellet increased the apparent total number of binding sites on the second and third day of implantation.

On the Integration of Alcohol-Related Quantitative Trait Loci and Gene Expression Analyses

BACKGROUND Quantitative trait loci (QTLs) have been detected for a wide variety of ethanol-related phenotypes, including acute and chronic ethanol withdrawal, acute locomotor activation, and ethanol preference. This study was undertaken to determine whether the process of moving from QTL to quantitative trait gene (QTG) could be accelerated by the integration of functional genomics (gene expression) into the analysis strategy. METHODS Six ethanol-related QTLs, all detected in C57BL/6J and DBA/2J intercrosses were entered into the analysis. Each of the QTLs had been confirmed in independent genetic models at least once; the cumulative probabilities for QTL existence ranged from 10 to 10. Brain gene expression data for the C57BL/6 and DBA/2 strains (n = 6 per strain) and an F2 intercross sample (n = 56) derived from these strains were obtained by using the Affymetrix U74Av2 and 430A arrays; additional data with the U74Av2 array were available for the extended amygdala, dorsomedial striatum, and hippocampus. Low-level analysis was performed by using multiple methods to determine the likelihood that a transcript was truly differentially expressed. For the 430A array data, the F2 sample was used to determine which of the differentially expressed transcripts within the QTL intervals were cis-regulated and, thus, strong candidates for QTGs. RESULTS Within the 6 QTL intervals, 39 transcripts (430A array) were identified as being highly likely to be differentially expressed between the C57BL/6 and DBA/2 strains at a false discovery rate of 0.01 or better. Twenty-eight of these transcripts showed significant (logarithm of odds > or =3.6) to highly significant (logarithm of odds >7) cis-regulation. The process correctly detected Mpdz (chromosome 4) as a candidate QTG for acute withdrawal. CONCLUSIONS Although improvements are needed in the expression databases, the integration of QTL and gene expression analyses seems to have potential as a high-throughput strategy for moving from QTL to QTG.

Effects of withdrawal from chronic amphetamine intoxication on exploratory and stereotyped behaviors in the rat.

Rats were administered 3, 6, and 12 mg/kg of d-amphetamine s.c. twice daily on a weekly increasing staircase schedule. On days 1, 7, 14, and 28 after the last injection of amphetamine the animals were challenged with 1 and 3 mg/kg of d-amphetamine and their behavior was observed. The 7-, 14-, and 28-day withdrawn animals required less amphetamine than controls to induce stereotyped behaviors. However, it was found that withdrawn animals and control animals were equally sensitive to the effects of apomorphine. Reserpine pretreatment eliminated the differences between control and withdrawn animals. α-Methyl tyrosine pretreatment blocked the effects of 1 but not 3 mg/kg of d-amphetamine in the withdrawn animals. Possible chemical mechanisms underlying the change in amphetamine sensitivity in the withdrawn animals are discussed.

Influence of prenatal d-amphetamine administration on development and behavior of rats

Abstract Beginning on the fifth day of gestation, rats were administered 1 or 3 mg/kg of d -amphetamine sulfate s.c. twice daily until term. The administration of d -amphetamine caused a dose-related increase in pup mortality. However, the increase in pup death could not be correlated with any gross pathological signs. The surviving 3 mg/kg amphetamine pups were analyzed for changes in motor behavior and brain biogenic amine levels. It was found that the amphetamine offspring showed a marked reduction in the ability to habituate to new surroundings, and this effect persisted for at least three months after birth. On day 35, brain levels of norepinephrine in the “amphetamine” offspring were decreased 21 percent. On day 84, in the “amphetamine offspring,” norepinephrine levels were reduced 18 percent in both the diencephalon and brainstem; dopamine levels were reduced 21 percent in the brainstem compared to control offspring.

On the specificity of trypsin (EC 3.4.4.4) of nerve ending particles to inhibit norepinephrine transport.

The Na+ and energy dependent uptake of norepinephrine into cortical rat brain homogenates or purified nerve ending particles (NEP) is reduced by prior trypsin treatment. In contrast, the uptake of dopamine, serotonin, choline and γ‐aminobutyric acid is markedly less sensitive to the effect of trypsin. Kinetic analyses indicate that the trypsin‐induced decrease of norepinephrine uptake is non‐competitive. In the dose range studied, trypsin did not appreciably alter the protein content or morphology of NEP. However, in a dose related fashion, trypsin decreased the glycoprotein content of NEP measured as the loss of protein bound N‐acetylneuraminic acid.

Effect of pentobarbital on regional brain phospholipid synthesis

Abstract Rats were given 45 mg/kg i.p. sodium pentobarbital 15 min prior to the intraventricular injection of 200 μCi [ 32 P]phosphoric acid and 50 μCi [ 3 H]glycerol. The animals were sacrificed 1 hr later, subcellular fractions were prepared from four subcortical brain regions and phospholipids were extracted. Pentobarbital significantly increased the ratio of [ 3 H]- and [ 32 P]-triphosphatidylinositol (TPI) to diphosphatidylinositol (DPI) in the microsomal but not synaptosomal fractions. The possible relationship of this change to nicotinic receptor activity is discussed. Pentobarbital specifically decreased 32 Pi but not [ 3 H]glycerol incorporation into synaptosomal phosphatidylinositol (PI). Thus, pentobarbital induced the opposite of the “neurotransmitter effect” on PI turnover. Pentobarbital either decreased or had no effect on the incorporation of 32 Pi and [ 3 H]glycerol into phosphatidylserine (PS), phosphatidylcholine (PC) and phosphatidylethanolamine (PE).

Lithium antagonism of ethanol-induced intoxication: relationship to intracellular lithium levels

Seventeen detoxified chronic alcoholics participated in a double-blind trial comparing placebo and lithium (Li+) effects on acute ethanol (1 g/kg) intoxication. In a repeated measures, split-half crossover design, subjects were maintained for 7 days on Li+ or placebo before the ethanol challenge. Plasma Li+ levels on day 7 averaged 1.3 +/- 0.3 mM. Li+ was not more effective than placebo in attenuating ethanol effects across the subjective dimensions of intoxication, desire to drink, and control of drinking and across the cognitive dimensions measured by Trail Making A, Speed of Closure, and the Minnesota Clerical Test. Li+ was not significantly more effective than placebo in preventing the ethanol-induced rise in plasma prolactin. Subjects were divided according to high and low red blood cell (RBC) Li+ intracellular/extracellular Li+ ratios. In a comparison of the Li+ to placebo arms of the trial, the high ratio subjects (n = 9) showed a significant 44% decrease in ethanol-induced intoxication, while the low ratio subjects (n = 8) showed a 15% increase. Furthermore, the high ratio subjects performed better than the low ratio subjects, independent of the ethanol effect, on all tests of cognitive performance. These preliminary data suggest that the Li+ ratio may be a useful tool in defining unique subgroups of alcoholic patients.