We describe the use of the matrix eigenvalue decomposition (EVD) and pseudoinverse projection and a tensor higher-order EVD (HOEVD) in reconstructing the pathways that compose a cellular system from genome-scale nondirectional networks of correlations among the genes of the system. The EVD formulates a genes × genes network as a linear superposition of genes × genes decorrelated and decoupled rank-1 subnetworks, which can be associated with functionally independent pathways. The integrative pseudoinverse projection of a network computed from a “data” signal onto a designated “basis” signal approximates the network as a linear superposition of only the subnetworks that are common to both signals and simulates observation of only the pathways that are manifest in both experiments. We define a comparative HOEVD that formulates a series of networks as linear superpositions of decorrelated rank-1 subnetworks and the rank-2 couplings among these subnetworks, which can be associated with independent pathways and the transitions among them common to all networks in the series or exclusive to a subset of the networks. Boolean functions of the discretized subnetworks and couplings highlight differential, i.e., pathway-dependent, relations among genes. We illustrate the EVD, pseudoinverse projection, and HOEVD of genome-scale networks with analyses of yeast DNA microarray data.

This article presents the proceedings of the symposium "Endogenous Opioids and Voluntary Ethanol Consumption: What Have We Learnt From Knock-out Mice?" presented at the meeting of the International Society for Biomedical Research on Alcoholism held in Heidelberg/Mannheim, Germany, in September/October 2004. The organizers and chairpersons were Michael S. Cowen and Carles Sanchis-Segura. The presentations were as follows: (1) Regulation of the Opioid System by Alcohol: Comparison of Alcohol-Preferring and -Nonpreferring Strains by Michael S. Cowen; (2) Endogenous Opioids and Alcohol: Lessons From Microdialysis and Knock-out Mice by M. Foster Olive; (3) From Neurochemistry to Neuroanatomy: The Hypothalamic Arcuate Nucleus as a Main Site for Ethanol-Opioids Interaction by Carles Sanchis-Segura; (4) Sensitivity to Ethanol Is Modulated by beta-Endorphin in Transgenic Mice by Judy E. Grisel, Amanda J. Roberts, and George F. Koob; and () The mu-Opioid Receptor Modulates Acute Ethanol Sensitivity and Ethanol Withdrawal Severity by Sandra Ghozland.

We recently reported a method where water-restricted mice were given scheduled access to ethanol followed by access to water. C57BL/6J mice would repeatedly self-administer ethanol in amounts that produced high and stable blood ethanol concentrations (BEC) [Finn DA, Belknap JK, Cronise K, Yoneyama N, Murillo A, Crabbe JC. A procedure to produce high alcohol intake in mice. Psychopharmacol 2005;178:471–480]. The studies reported here demonstrate that behavioral signs of motor impairment result from these high alcohol intakes, and that there was some evidence of tolerance development across repeated sessions. Female C57BL/6J mice were allowed 30 min access to ethanol (5% v/v) followed by 2.5 h access to water either: every 3rd day for 12 days; every 2nd day for 28 days; or every 2nd day for 9 days. On intervening days, mice had 3 h access to water. A control group had daily access to water only. Mice consumed 2–2.5 g/kg ethanol in 30 min, resulting in BECs of 1.4–1.5 mg/ml. Motor impairment was assessed using the accelerating or fixed speed rotarod, balance beam or screen test. In all studies, mice were tested for motor impairment immediately after 30 min access to ethanol or water. In Experiment 1, ethanol-exposed mice had shorter latencies to fall from the fixed speed rotarod and more foot slips on the balance beam than the control group, indicating motor impairment. After drinking ethanol, mice also fell from a screen more quickly than during sober pretraining. In Experiment 2, mice tested (without prior training) for motor impairment and tolerance on the fixed speed rotarod at 6.5 and 10 RPM showed repeated motor impairment in the ethanol group, but did not develop tolerance. In Experiment 3, mice were first given rotarod training at 10 RPM. Following each fluid access period, performance was impaired in mice self-administering ethanol at 10, but not 15 RPM, when compared to control mice. There was no evidence of tolerance across days. However, on the last day, all mice were tested at both RPM following an i.p. injection of 2 g/kg ethanol. Ethanol-experienced mice were less impaired at both RPM than the ethanol-naïve mice, indicating tolerance development according to this between-groups index. These results suggest that C57BL/6J mice will repeatedly consume alcohol in amounts that produce motor impairment under these scheduled fluid access conditions, and that a modest degree of tolerance can be detected using appropriate tests.

This report of the proceedings of a symposium presented at the 2004 Research Society on Alcoholism Meeting provides evidence linking stress during sobriety to craving that increases the risk for relapse. The initial presentation by Rajita Sinha summarized clinical evidence for the hypothesis that there is an increased sensitivity to stress-induced craving in alcoholics. During early abstinence, alcoholics who were confronted with stressful circumstances showed increased susceptibility for relapse. George Breese presented data demonstrating that stress could substitute for repeated withdrawals from chronic ethanol to induce anxiety-like behavior. This persistent adaptive change induced by multiple withdrawals allowed stress to induce an anxiety-like response that was absent in animals that were not previously exposed to chronic ethanol. Subsequently, Amanda Roberts reviewed evidence that increased drinking induced by stress was dependent on corticotropin-releasing factor (CRF). In addition, rats that were stressed during protracted abstinence exhibited anxiety-like behavior that was also dependent on CRF. Christopher Dayas indicated that stress increases the reinstatement of an alcohol-related cue. Moreover, this effect was enhanced by previous alcohol dependence. These interactive effects between stress and alcohol-related environmental stimuli depended on concurrent activation of endogenous opioid and CRF systems. A.D. Lê covered information that indicated that stress facilitated reinstatement to alcohol responding and summarized the influence of multiple deprivations on this interaction. David Overstreet provided evidence that restraint stress during repeated alcohol deprivations increases voluntary drinking in alcohol-preferring (P) rats that results in withdrawal-induced anxiety that is not observed in the absence of stress. Testing of drugs on the stress-induced voluntary drinking implicated serotonin and CRF involvement in the sensitized response. Collectively, the presentations provided convincing support for an involvement of stress in the cause of relapse and continuing alcohol abuse and suggested novel pharmacological approaches for treating relapse induced by stress.

The L1 cell adhesion molecule has been implicated in ethanol teratogenesis as well as NMDAR-dependent long-term potentiation (LTP) of synaptic transmission, a process thought to be critical for neural development. Ethanol inhibits LTP at least in part by interacting with NMDA receptors. Ethanol also inhibits L1-mediated cell adhesion in a manner that is prevented by an octapeptide, D-NAPVSIPQ (D-NAP), as well as long chain alcohols such as 1-octanol. Here we analyzed the effects of D-NAP and 1-octanol on ethanol modulation of LTP induced by theta burst stimulation in two subfields of the rat hippocampus, the dentate gyrus and area CA1. When theta burst stimulation was delivered in ethanol (50 mM), LTP was inhibited by about 50%. Surprisingly, when D-NAP (10(-7) M) and ethanol were co-applied or applied sequentially, LTP was completely absent. The effects of D-NAP were persistent, since delivery of a second theta burst stimulation following washout of D-NAP and ethanol elicited minimal plasticity. Application of D-NAP alone had no effect on LTP induction or expression. The synergistic effect of D-NAP on ethanol inhibition of LTP was concentration-dependent since D-NAP (10(-10) M) had an intermediate effect, while D-NAP (10(-13) M) had no effect on ethanol suppression of LTP. These observations were also replicated with a different ethanol antagonist, 1-octanol, in area CA1. To address the mechanisms underlying this long-lasting suppression of LTP, the sensitivity of pharmacologically isolated NMDAR extracellular field potentials to combinations of D-NAP and ethanol was determined. D-NAP (10(-7)M) alone had no effect on NMDA extracellular field potentials; however, the peptide significantly increased the inhibitory action of ethanol on NMDA extracellular field potential. The findings suggest that D-NAP and 1-octanol selectively interact with NMDA receptors in an ethanol-dependent manner, further implicating the L1 cell adhesion molecule in alcohol-related brain disorders.

{BACKGROUND: We previously mapped a quantitative trait locus (QTL) for ethanol preference drinking to mouse chromosome 2 (mapped with high confidence

Considerable evidence suggests that the synapse is the most sensitive CNS element for ethanol effects. Although most alcohol research has focussed on the postsynaptic sites of ethanol action, especially regarding interactions with the glutamatergic and GABAergic receptors, few such studies have directly addressed the possible presynaptic loci of ethanol action, and even fewer describe effects on synaptic terminals. Nonetheless, there is burgeoning evidence that presynaptic terminals play a major role in ethanol effects. The methods used to verify such ethanol actions range from electrophysiological analysis of paired-pulse facilitation (PPF) and spontaneous and miniature synaptic potentials to direct recording of ion channel activity and transmitter/messenger release from acutely isolated synaptic terminals, and microscopic observation of vesicular release, with a focus predominantly on GABAergic, glutamatergic, and peptidergic synapses. The combined data suggest that acute ethanol administration can both increase and decrease the release of these transmitters from synaptic terminals, and more recent results suggest that prolonged or chronic ethanol treatment (CET) can also alter the function of presynaptic terminals. These new findings suggest that future analyses of synaptic effects of ethanol should attempt to ascertain the role of presynaptic terminals and their involvement in alcohol's behavioral actions. Other future directions should include an assessment of ethanol's effects on presynaptic signal transduction linkages and on the molecular machinery of transmitter release and exocytosis in general. Such studies could lead to the formulation of new treatment strategies for alcohol intoxication, alcohol abuse, and alcoholism.

Exogenous administration of the gamma-aminobutyric acid (GABA)-ergic neurosteroid allopregnanolone (ALLO) can increase ethanol intake in rats and mice. To determine the contribution of endogenous neurosteroids (i.e., ALLO and related pregnane steroids) in the regulation of established ethanol consumption patterns in male C57BL/6J (B6) mice, the 5alpha-reductase (5alpha-R) enzyme inhibitor, finasteride (FIN), was chronically administered and then subsequently withdrawn. Mice were provided daily 2-h limited access to a 10% vol/vol ethanol solution (10E) and water in lickometer chambers during the dark phase. Following the establishment of stable 10E intake patterns, mice were injected intraperitoneally with either vehicle (20% wt/vol 2-hydroxypropyl-beta-cyclodextrin; n=8) or FIN (50 mg/kg; n=16) for 7 days. Effects of withdrawal from FIN treatment were subsequently assessed for an additional 7 days. Ethanol intakes were significantly decreased with acute FIN treatment (days 1-3) and during early withdrawal (days 1-3). Acute FIN treatment was also associated with an extended latency to first bout, reduced first bout size, and greatly attenuated sipper contact count during the initial 20-min interval of 10E access. These findings collectively indicated that acute FIN treatment markedly attenuated the initiation of 10E consumption during the limited access sessions. The influence of FIN on 10E intake patterns was largely dissipated with chronic treatment, suggesting that compensatory changes in neurosteroid modulation of inhibitory tone may have occurred. Thus, acute FIN treatment modulated ethanol intake patterns in a manner opposite to that previously demonstrated for a physiologically relevant, exogenous ALLO dose, consistent with the ability of a alpha-R inhibitor to block ALLO biosynthesis. Manipulation of endogenous neurosteroid activity via biosynthetic enzyme inhibition or antagonism of steroid binding to the GABA type A receptor may prove to be a beneficial pharmacotherapeutic strategy in the intervention of alcohol abuse and alcoholism.

RATIONALE: There is accumulating evidence that the neuropeptide urocortin 1 (Ucn1) is involved in alcohol consumption. Thus far, however, most studies have been performed in mice. OBJECTIVES: The purpose of the present study was to characterize Ucn1 expression in rats selectively bred for either high or low alcohol intake. METHODS: Brains from naive male rats of five pairs of independently selected lines (iP/iNP, AA/ANA, HARF/LARF, HAD1/LAD1, and HAD2/LAD2) were analyzed by immunohistochemistry. RESULTS: Significant differences were found between iP/iNP, HARF/LARF, and HAD2/LAD2 in number of Ucn1-containing cells in the Edinger-Westphal (EW) nucleus (the main source of Ucn1 in the brain), whereas no significant differences were found between HAD1/LAD1 and AA/ANA. Similarly, significant differences in the optical density of Ucn1 immunoreactivity in EW were found between iP/iNP, HARF/LARF, and HAD2/LAD2, whereas no differences on this measure were found between HAD1/LAD1 and AA/ANA. In the lateral septum (LS, the main projection area of Ucn1-containing neurons in the rat), significant differences were found only between AA/ANA and HAD2/LAD2; however, a meta-analysis indicated that across all five lines, preferring animals had a significantly greater number of Ucn1-positive fibers than nonpreferring animals. CONCLUSIONS: These results provide evidence that, in rats, Ucn1 may be involved in regulation of alcohol intake, and that this regulation may occur through the Ucn1 projections to LS.