Norepinephrine transporter: a candidate gene for initial ethanol sensitivity in inbred long-sleep and short-sleep mice

BACKGROUND: Altered noradrenergic neurotransmission is associated with depression and may contribute to drug abuse and alcoholism. Differential initial sensitivity to ethanol is an important predictor of risk for future alcoholism, making the inbred long-sleep (ILS) and inbred short-sleep (ISS) mice a useful model for identifying genes that may contribute to alcoholism. METHODS: In this study, molecular biological, neurochemical, and behavioral approaches were used to test the hypothesis that the norepinephrine transporter (NET) contributes to the differences in ethanol-induced loss of righting reflex (LORR) in ILS and ISS mice. RESULTS: We used these mice to investigate the NET as a candidate gene contributing to this phenotype. The ILS and ISS mice carry different DNA haplotypes for NET, showing eight silent differences between allelic coding regions. Only the ILS haplotype is found in other mouse strains thus far sequenced. Brain regional analyses revealed that ILS mice have 30 to 50% lower [3H]NE uptake, NET binding, and NET mRNA levels than ISS mice. Maximal [3H]NE uptake and NET number were reduced, with no change in affinity, in the ILS mice. These neurobiological changes were associated with significant influences on the behavioral phenotype of these mice, as demonstrated by (1) a differential response in the duration of ethanol-induced LORR in ILS and ISS mice pretreated with a NET inhibitor and (2) increased ethanol-induced LORR in LXS recombinant inbred (RI) strains, homozygous for ILS in the NET chromosomal region (44-47 cM), compared with ISS homozygous strains. CONCLUSIONS: This is the first report to suggest that the NET gene is one of many possible genetic factors influencing ethanol sensitivity in ILS, ISS, and LXS RI mouse strains.     

N-methyl-D-aspartate receptor responses are differentially modulated by noncompetitive receptor antagonists and ethanol in inbred long-sleep and short-sleep mice: behavior and electrophysiology

BACKGROUND: Short-sleep (SS) mice exhibit higher locomotor activity than do long-sleep (LS) mice when injected with low doses of ethanol or the noncompetitive N-methyl-D-aspartate receptor (NMDAR) antagonist MK-801 (dizocilpine). SS mice also have higher densities of brain NMDARs. However, two strains of LS X SS recombinant inbred (RI) mice also show differential activation to ethanol and MK-801, but have similar numbers of NMDARs. Here we used inbred LS (ILS) and SS (ISS) mice to investigate further the relationship between NMDARs and sensitivity to the stimulant effects of low doses of ethanol. METHODS: Open field activity and spontaneous alternations were measured after saline or drug injection. [3H]MK-801 binding parameters were determined in hippocampus, cortex, striatum, and nucleus accumbens. Extracellular field excitatory postsynaptic potentials (fEPSPs) were recorded in the CA1 region of hippocampal slices. RESULTS: Systemic injection of either ethanol or MK-801 increased locomotor activity to a greater extent in ISS mice than in ILS mice. The competitive NMDAR antagonist 2-carboxypiperazin-4-yl-propyl-1-lphosphonic acid (+/- CPP) depressed activity of ILS, but not ISS, mice. No strain differences were observed in spontaneous alternations or in the number or affinity of NMDARs in the brain regions examined. Likewise, the magnitudes of hippocampal NMDAR-mediated fEPSPs were similar in ILS and ISS mice and were inhibited to the same extent by a competitive NMDAR antagonist. However, both ethanol and the NMDAR NR2B receptor antagonist ifenprodil inhibited the late component of hippocampal NMDAR fEPSPs to a greater extent in ISS, than in ILS, mice. CONCLUSIONS: Differential ethanol- and MK-801-induced behavioral activation in ILS and ISS mice was not associated with differences in NMDAR number. Nonetheless, pharmacological differences in hippocampal NMDAR responsiveness suggest that ISS mice express NMDARs that have a greater sensitivity to noncompetitive, but not competitive, NMDAR antagonists. These differences, which may reflect differences in NMDAR subunit composition, could underlie the differential responsiveness to low doses of ethanol in ILS and ISS mice.     

Parental psychopathology and migraine headaches among adolescent girls

Migraine headaches and depression often co-occur within individuals, and both syndromes run in families. However, knowledge about how these disorders relate across generations, as well as how migraine relates to other forms of psychopathology, is sparse. This study examined risk for migraine among female adolescent offspring of parents with different types of psychopathology. The sample was drawn from the Minnesota Twin Family Study, a community-based study of adolescents and their families ( n  = 674, 17-year-old female adolescents and their biological parents). Diagnoses of maternal, paternal and offspring major depression, antisocial behaviour, alcohol dependence and drug dependence were based on structured interviews. Migraine headaches in each family member were assessed via interviews with the mother. Parental depression, antisocial behaviour and drug dependence were associated with offspring migraine. These associations mostly remained significant even when parental migraine and the corresponding type of psychopathology in offspring were adjusted for. In contrast, there were no significant associations between parental psychopathology and offspring stomach problems, indicating that these associations did not extend to all offspring somatic symptoms. These results emphasize the need to look at antisocial behaviour and substance-related problems when examining associations between migraine and psychopathology, and indicate that more research on inter-generational links between migraine and psychopathology is needed.     

Clinical study of multiple breath biomarkers of asthma and COPD (NO, CO(2), CO and N(2)O) by infrared laser spectroscopy

Breath analysis is a powerful non-invasive technique for the diagnosis and monitoring of respiratory diseases, including asthma and chronic obstructive pulmonary disease (COPD). Exhaled nitric oxide (NO) and carbon monoxide (CO) are markers of airway inflammation and can indicate the extent of respiratory diseases. We have developed a compact fast response quantum cascade laser system for analysis of multiple gases by tunable infrared absorption spectroscopy. The ARI breath analysis instrument has been deployed in a study of exhaled breath from patients with asthma or COPD. A total of 173 subjects participated, including both adult and pediatric patients. Patients in asthma or COPD exacerbations were evaluated twice-during the exacerbation and at a follow-up visit-to compare variations in breath biomarkers during these events. The change in exhaled NO levels between exacerbation and 'well' visits is consistent with spirometry data collected. Respiratory models are important for understanding the exchange dynamics of nitric oxide and other species in the lungs and airways. At each patient's visit, tests were conducted at four expiratory flow rates. We have applied a trumpet model with axial diffusion to the multi-flow exhaled nitric oxide data, obtaining NO alveolar concentrations and airway fluxes. We found higher airway fluxes for those with more severe asthma and during exacerbation events. The alveolar concentrations from the model were higher in adults with asthma and COPD, but this trend was less clear among the pediatric subjects.     

Modulation of hippocampal glutamatergic transmission by ATP is dependent on adenosine a(1) receptors

Excitatory glutamatergic synapses in the hippocampal CA1 region of rats are potently inhibited by purines, including adenosine, ATP, and ATP analogs. Adenosine A(1) receptors are known to mediate at least part of the response to adenine nucleotides, either because adenine nucleotides activate A(1) receptors directly, or activate them secondarily upon the nucleotides' conversion to adenosine. In the present studies, the inhibitory effects of adenosine, ATP, the purportedly stable ATP analog adenosine-5'-O-(3-thio)triphosphate (ATPgammaS), and cyclic AMP were examined in mice with a null mutation in the adenosine A(1) receptor gene. ATPgammaS displaced the binding of A(1)-selective ligands to intact brain sections and brain homogenates from adenosine A(1) receptor wild-type animals. In homogenates, but not in intact brain sections, this displacement was abolished by adenosine deaminase. In hippocampal slices from wild-type mice, purines abolished synaptic responses, but slices from mice lacking functional A(1) receptors showed no synaptic modulation by adenosine, ATP, cAMP, or ATPgammaS. In slices from heterozygous mice the dose-response curve for both adenosine and ATP was shifted to the right. In all cases, inhibition of synaptic responses by purines could be blocked by prior treatment with the competitive adenosine A(1) receptor antagonist 8-cyclopentyltheophylline. Taken together, these results show that even supposedly stable adenine nucleotides are rapidly converted to adenosine at sites close to the A(1) receptor, and that inhibition of synaptic transmission by purine nucleotides is mediated exclusively by A(1) receptors.     

Behavioural sensitivity to PIA in selectively bred mice is related to a number of A1 adenosine receptors but not to cyclic AMP accumulation in brain slices

In a dose of 0.1 mg/kg, PIA had marked behavioural effects in long-sleep mice (which show a high sensitivity to ethanol, while no significant effect was observed in short sleep mice (low sensitivity to ethanol). The number of [3H]PIA binding sites in cortex and subcortical brain regions was significantly higher in long-sleep than in short-sleep mice. The KD value was higher in cortex and cerebellum in the short-sleep mice, but there were no differences in the number of hippocampal beta-adrenoceptors or in the adenosine analogue-induced increase in cyclic AMP accumulation in slices of mouse hippocampus.     

Regulation of adrenergic receptors in intraocular hippocampal transplants: role of noradrenergic innervation

Hippocampal tissue transplanted into the anterior chamber of the eye offers a unique system in which development can be studied in the absence of the noradrenergic innervation. This system was used to determine the extent to which noradrenergic innervation regulates the development of adrenergic receptors. In addition to examining single denervated transplants, transplants grown with innervation from the superior cervical ganglia of the host rat or from locus coeruleus cotransplants were also examined to determine whether the source of norepinephrine and extent of innervation in oculo regulate the development and density of adrenergic receptors. In vitro autoradiographic analysis of ligand binding to both alpha 1- and beta-adrenergic receptors with 125I-BE 2254 and 125I-pindolol, respectively, was used to characterize adrenergic receptors in the intraocular transplants. Quantitative analysis of the receptors showed an up-regulation of both alpha 1- and beta-adrenergic receptors in tissue grown in the absence of norepinephrine, but in general there was not a high degree of correlation between norepinephrine content and receptor density. Although high-performance liquid chromatography (HPLC) analysis of catecholamines revealed higher than normal amounts of norepinephrine in hippocampal transplants innervated by the superior cervical ganglia or a locus coeruleus cotransplant, the density of alpha 1 and beta receptors was quite comparable with values found in the literature for normal adult hippocampus. These results suggest that the relationship between receptor number and density of innervation may differ significantly from what is observed in response to pharmacological manipulation of norepinephrine systems in the adult brain.     

Expression of phosphorylcholine-specific B cells during murine development

The TEPC 15 (T15) clonotype, a putatively germline antibody specificity, does not appear in the neonatal B-cell repertoire until approximately 1 wk of age. This report extends this observation by the demonstration that (a) the T15 clonotype follows similar kinetics of appearance in germfree as well as conventionally-reared mice; (b) maternal influences and genetic background play a minor role in the development of the T15 clonotype since CBFI neonates raised by C57BL/6 or BALB/c mothers acquire the T15 clonotype at the same time in ontogeny as BALB/c neonates; (c) the lack of phosphorylcholine (PC)-specific B cells shortly after birth is reflected in a dearth of PC-binding cells in the neonate as well; and (d) no PC-specifc B cells are found in 19-day fetal liver or in bone marrow until 7 days of life, coincident with their appearance in the spleen. These findings, along with a previous report that PC-specific splenic B cells are tolerizable as late as day 10 after birth, confirm the invariant, late occurrence of the T15 clonotype and support a highly- ordered, rigorously predetermined mechanism for the acquisition of the B- cell repertoire. The results are discussed in light of other studies on the ontogeny of B-cell specificity, and in terms of the implications on the mechanism by which antibody diversity is generated.