Effects of adolescent substance use disorders on central cholinergic function.

Adolescence is a transitional period between childhood and adulthood, in which the individual undergoes significant cognitive, behavioral, physical, emotional, and social developmental changes. During this period, adolescents engage in experimentation and risky behaviors such as licit and illicit drug use. Adolescents' high vulnerability to abuse drugs and natural reinforcers leads to greater risk for developing substance use disorders (SUDs) during adulthood. Accumulating evidence indicates that the use and abuse of licit and illicit drugs during adolescence and emerging adulthood can disrupt the cholinergic system and its processes. This review will focus on the effects of peri-adolescent nicotine and/or alcohol use, or exposure, on the cholinergic system during adulthood from preclinical and clinical studies. This review further explores potential cholinergic agents and pharmacological manipulations to counteract peri-adolescent nicotine and/or alcohol abuse.

A Genetic Animal Model of Alcoholism for Screening Medications to Treat Addiction.

The purpose of this review is to present up-to-date pharmacological, genetic, and behavioral findings from the alcohol-preferring P rat and summarize similar past work. Behaviorally, the focus will be on how the P rat meets criteria put forth for a valid animal model of alcoholism with a highlight on its use as an animal model of polysubstance abuse, including alcohol, nicotine, and psychostimulants. Pharmacologically and genetically, the focus will be on the neurotransmitter and neuropeptide systems that have received the most attention: cholinergic, dopaminergic, GABAergic, glutamatergic, serotonergic, noradrenergic, corticotrophin releasing hormone, opioid, and neuropeptide Y. Herein, we sought to place the P rat's behavioral and neurochemical phenotypes, and to some extent its genotype, in the context of the clinical literature. After reviewing the findings thus far, this chapter discusses future directions for expanding the use of this genetic animal model of alcoholism to identify molecular targets for treating drug addiction in general.

D1 receptors in the nucleus accumbens-shell, but not the core, are involved in mediating ethanol-seeking behavior of alcohol-preferring (P) rats.

Clinical and preclinical research suggest that activation of the mesolimbic dopamine (DA) system is involved in mediating the rewarding actions of drugs of abuse, as well as promoting drug-seeking behavior. Inhibition of DA D1 receptors in the nucleus accumbens (Acb) can reduce ethanol (EtOH)-seeking behavior of non-selective rats triggered by environmental context. However, to date, there has been no research on the effects of D1 receptor agents on EtOH- seeking behavior of high alcohol-preferring (P) rats following prolonged abstinence. The objective of the present study was to examine the effects of microinjecting the D1 antagonist SCH 23390 or the D1 agonist A-77636 into the Acb shell or Acb core on spontaneous recovery of EtOH-seeking behavior. After 10 weeks of concurrent access to EtOH and water, P rats underwent seven extinction sessions (EtOH and water withheld), followed by 2 weeks in their home cages without access to EtOH or operant sessions. In the 2nd week of the home cage phase, rats were bilaterally implanted with guide cannula aimed at the Acb shell or Acb core; rats were allowed 7d ays to recover before EtOH-seeking was assessed by the Pavlovian Spontaneous Recovery (PSR) model. Administration of SCH23390 (1µg/side) into the Acb shell inhibited responding on the EtOH lever, whereas administration of A-77636 (0.125µg/side) increased responding on the EtOH lever. Microinfusion of D1 receptor agents into the Acb core did not alter responding on the EtOH lever. Responses on the water lever were not altered by any of the treatments. The results suggest that activation of D1 receptors within the Acb shell, but not Acb core, are involved in mediating PSR of EtOH-seeking behavior of P rats.

Convergent functional genomic studies of ω-3 fatty acids in stress reactivity, bipolar disorder and alcoholism.

Omega-3 fatty acids have been proposed as an adjuvant treatment option in psychiatric disorders. Given their other health benefits and their relative lack of toxicity, teratogenicity and side effects, they may be particularly useful in children and in females of child-bearing age, especially during pregnancy and postpartum. A comprehensive mechanistic understanding of their effects is needed. Here we report translational studies demonstrating the phenotypic normalization and gene expression effects of dietary omega-3 fatty acids, specifically docosahexaenoic acid (DHA), in a stress-reactive knockout mouse model of bipolar disorder and co-morbid alcoholism, using a bioinformatic convergent functional genomics approach integrating animal model and human data to prioritize disease-relevant genes. Additionally, to validate at a behavioral level the novel observed effects on decreasing alcohol consumption, we also tested the effects of DHA in an independent animal model, alcohol-preferring (P) rats, a well-established animal model of alcoholism. Our studies uncover sex differences, brain region-specific effects and blood biomarkers that may underpin the effects of DHA. Of note, DHA modulates some of the same genes targeted by current psychotropic medications, as well as increases myelin-related gene expression. Myelin-related gene expression decrease is a common, if nonspecific, denominator of neuropsychiatric disorders. In conclusion, our work supports the potential utility of omega-3 fatty acids, specifically DHA, for a spectrum of psychiatric disorders such as stress disorders, bipolar disorder, alcoholism and beyond.

Phenomic, convergent functional genomic, and biomarker studies in a stress-reactive genetic animal model of bipolar disorder and co-morbid alcoholism.

We had previously identified the clock gene D-box binding protein (Dbp) as a potential candidate gene for bipolar disorder and for alcoholism, using a Convergent Functional Genomics (CFG) approach. Here we report that mice with a homozygous deletion of DBP have lower locomotor activity, blunted responses to stimulants, and gain less weight over time. In response to a chronic stress paradigm, these mice exhibit a diametric switch in these phenotypes. DBP knockout mice are also activated by sleep deprivation, similar to bipolar patients, and that activation is prevented by treatment with the mood stabilizer drug valproate. Moreover, these mice show increased alcohol intake following exposure to stress. Microarray studies of brain and blood reveal a pattern of gene expression changes that may explain the observed phenotypes. CFG analysis of the gene expression changes identified a series of novel candidate genes and blood biomarkers for bipolar disorder, alcoholism, and stress reactivity.

The role of 5-HT3 receptors in drug abuse and as a target for pharmacotherapy.

Alcohol and drug abuse continue to be a major public health problem in the United States and other industrialized nations. Extensive preclinical research indicates the mesolimbic dopamine (DA) pathway and associated regions mediate the rewarding and reinforcing effects of drugs of abuse and natural rewards, such as food and sex. The serotonergic (5-HT) system, in concert with others neurotransmitter systems, plays a key role in modulating neuronal systems within the mesolimbic pathway. A substantial portion of this modulation is mediated by activity at the 5-HT3 receptor. The 5-HT3 receptor is unique among the 5-HT receptors in that it directly gates an ion channel inducing rapid depolarization that, in turn, causes the release of neurotransmitters and/or peptides. Preclinical findings indicate that antagonism of the 5-HT3 receptor in the ventral tegmental area, nucleus accumbens or amygdala reduces alcohol self-administration and/or alcohol-associated effects. Less is known about the effects of 5-HT3 receptor activity on the self-administration of other drugs of abuse or their associated effects. Clinical findings parallel the preclinical findings such that antagonism of the 5-HT3 receptor reduces alcohol consumption and some of its subjective effects. This review provides an overview of the structure, function, and pharmacology of 5-HT3 receptors, the role of these receptors in regulating DA neurotransmission in mesolimbic brain areas, and discusses data from animal and human studies implicating 5-HT3 receptors as targets for the development of new pharmacological agents to treat addictions.

Candidate genes, pathways and mechanisms for alcoholism: an expanded convergent functional genomics approach.

We describe a comprehensive translational approach for identifying candidate genes for alcoholism. The approach relies on the cross-matching of animal model brain gene expression data with human genetic linkage data, as well as human tissue data and biological roles data, an approach termed convergent functional genomics. An analysis of three animal model paradigms, based on inbred alcohol-preferring (iP) and alcohol-non-preferring (iNP) rats, and their response to treatments with alcohol, was used. A comprehensive analysis of microarray gene expression data from five key brain regions (frontal cortex, amygdala, caudate-putamen, nucleus accumbens and hippocampus) was carried out. The Bayesian-like integration of multiple independent lines of evidence, each by itself lacking sufficient discriminatory power, led to the identification of high probability candidate genes, pathways and mechanisms for alcoholism. These data reveal that alcohol has pleiotropic effects on multiple systems, which may explain the diverse neuropsychiatric and medical pathology in alcoholism. Some of the pathways identified suggest avenues for pharmacotherapy of alcoholism with existing agents, such as angiotensin-converting enzyme (ACE) inhibitors. Experiments we carried out in alcohol-preferring rats with an ACE inhibitor show a marked modulation of alcohol intake. Other pathways are new potential targets for drug development. The emergent overall picture is that physical and physiological robustness may permit alcohol-preferring individuals to withstand the aversive effects of alcohol. In conjunction with a higher reactivity to its rewarding effects, they may able to ingest enough of this nonspecific drug for a strong hedonic and addictive effect to occur.

Protein expression changes in the nucleus accumbens and amygdala of inbred alcohol-preferring rats given either continuous or scheduled access to ethanol.

Chronic ethanol (EtOH) drinking produces neuronal alterations within the limbic system. To investigate changes in protein expression levels associated with EtOH drinking, inbred alcohol-preferring (iP) rats were given one of three EtOH access conditions in their home-cages: continuous ethanol (CE: 24h/day, 7days/week access to EtOH), multiple scheduled access (MSA: four 1-h sessions during the dark cycle/day, 5 days/week) to EtOH, or remained EtOH-naïve. Both MSA and CE groups consumed between 6 and 6.5g of EtOH/kg/day after the 3rd week of access. On the first day of EtOH access for the seventh week, access was terminated at the end of the fourth MSA session for MSA rats and the corresponding time point (2300h) for CE rats. Ten h later, the rats were decapitated, brains extracted, the nucleus accumbens (NAcc) and amygdala (AMYG) microdissected, and protein isolated for 2-dimensional gel electrophoretic analyses. In the NAcc, MSA altered expression levels for 12 of the 14 identified proteins, compared with controls, with six of these proteins altered by CE access, as well. In the AMYG, CE access changed expression levels for 22 of the 27 identified proteins, compared with controls, with 8 of these proteins altered by MSA, as well. The proteins could be grouped into functional categories of chaperones, cytoskeleton, intracellular communication, membrane transport, metabolism, energy production, or neurotransmission. Overall, it appears that EtOH drinking and the conditions under which EtOH is consumed, differentially affect protein expression levels between the NAcc and AMYG. This may reflect differences in neuroanatomical and/or functional characteristics associated with EtOH self-administration and possibly withdrawal, between these two brain structures.

Low-dose stimulatory effects of ethanol during adolescence in rat lines selectively bred for high alcohol intake.

BACKGROUND: The low-dose stimulatory effect of ethanol (EtOH) in rats has been hypothesized to reflect its hedonic effects and to be associated with a genetic predisposition toward high alcohol preference. To test the hypothesis that phenotypes associated with high alcohol preference in adulthood are also present in adolescent rats at the time of onset of alcohol drinking, the current study examined the effects of EtOH on locomotor activity (LMA) during adolescence in lines of rats selectively bred for divergent alcohol intakes. METHODS: Subjects were adolescent (31-40 days of age) rats from the alcohol-preferring (P) and -nonpreferring (NP) lines and from the high-alcohol-drinking (HAD) and low-alcohol-drinking (LAD) replicate lines. On day 1, all subjects (n = 8-10/line/gender/dose) received intraperitoneal saline injections and were placed in the activity monitor for 30 min. On day 2, subjects received intraperitoneal saline or 0.25, 0.50, 0.75, 1.0, or 1.5 g EtOH/kg. RESULTS: The LMA of male and female P rats was increased with low doses (0.25-0.75 g/kg) and decreased at the highest dose (1.5 g/kg) of EtOH. Similar effects were observed with low doses of EtOH on the LMA of HAD-1 and HAD-2 rats. None of the EtOH doses stimulated LMA in the NP, LAD-1, or LAD-2 rats, although all of the low-alcohol-intake lines of rats showed decreased LMA at the highest dose of EtOH. Only the P rats among the high-alcohol-consuming lines of rats showed decreased LMA at the highest dose of EtOH. CONCLUSION: Selective breeding for high alcohol consumption seems to be associated with increased sensitivity to the low-dose stimulating effects of EtOH and reduced sensitivity to the high-dose motor-impairing effects of ethanol. The expression of these phenotypes emerges during adolescence by the age of onset of alcohol-drinking behavior.

Ethanol drinking and deprivation alter dopaminergic and serotonergic function in the nucleus accumbens of alcohol-preferring rats.

The alcohol deprivation effect is a temporary increase in the intake of, or preference for, ethanol after a period of deprivation that may result from persistent changes in key limbic regions thought to regulate alcohol drinking, such as the nucleus accumbens. The present study tested the hypothesis that chronic alcohol drinking under continuous 24-h free-choice conditions alters dopamine and serotonin neurotransmission in the nucleus accumbens and that these alterations persist in the absence of alcohol. Using the no-net-flux microdialysis method, the steady-state extracellular concentration (point of no-net-flux) for dopamine was approximately 25% higher in the adult female alcohol-preferring P rats given prior access to 10% ethanol, even after 2 weeks of ethanol abstinence, compared with the P rats gives access only to water. However, the extracellular concentration of serotonin was approximately 35% lower in animals given 8 weeks of continuous access to ethanol compared with water controls and animals deprived of ethanol for 2 weeks. The effect of local perfusion with 100 microM sulpiride (D(2) receptor antagonist) and 35 microM 1-(m-chlorophenyl)-biguanide (5-hydroxytryptamine(3) receptor agonist) on dopamine overflow were reduced approximately 33% in both groups of ethanol-exposed P rats compared with water controls. Free-choice alcohol drinking by P rats alters dopamine and serotonin neurotransmission in the nucleus accumbens, and many of these effects persist for at least 2 weeks in the absence of ethanol, suggesting that these underlying persistent changes may be in part responsible for increased ethanol drinking observed in the alcohol-deprivation effect.

Amphetamine-modified acoustic startle responding and prepulse inhibition in adult and adolescent alcohol-preferring and -nonpreferring rats.

Selective breeding has been used to develop the alcohol-preferring (P) and -nonpreferring (NP) rats, with the P rat having lower CNS levels of dopamine (DA) and reduced DA innervation in the nucleus accumbens compared with the NP rat. The acoustic startle response (ASR) and prepulse inhibition (PPI) of the ASR are experimental behaviors altered by DA agonists. We examined whether functional differences in amphetamine (AMPH)-modified ASR and PPI exist between P and NP rats. AMPH [0.0 (saline), 1.0, 2.0, or 4.0 mg/kg] was injected 15 min prior to placement into a startle apparatus. After a 5-min habituation period, rats were given approximately twelve 95-, 105-, or 115-dB white-noise burst (ASR) and PPI trials. As adults, P rats were sensitive to AMPH potentiation of the ASR to a greater extent than NP rats. During adolescence, P and NP rats had similar levels of AMPH-potentiated ASR. As adults, NP rats displayed potentiated, rather than disrupted, PPI at the 1.0-mg/kg dose, whereas P rats displayed the expected disrupted PPI at the 4.0-mg/kg dose. As adolescents, NP rats did not display significant differences in PPI after AMPH, whereas P rats displayed dose-dependent disruption of PPI, which was significant at the 4.0-mg/kg dose. The limited effect of AMPH on increasing the ASR and the presence of AMPH-potentiated PPI at the lowest dose in the adult NP rat suggests reduced functioning of the interactions between DA circuits and the neurocircuitry mediating the ASR and PPI, compared with P rats. However, the neurocircuitry mediating PPI does not appear to be fully developed in the adolescent NP rat. The present findings also indicate that lower levels of DA content and immunoreactive fibers in the P rat may not reflect reduced DA neuronal activity, because the P rat displayed AMPH-potentiated ASR, and, at the highest dose, AMPH disruption of PPI during both adulthood and adolescence.

Deoxycorticosterone acetate (DOCA)-induced sodium appetite in group-housed mice.

Research investigating sodium hunger in mice has failed to produce evidence that mineralocorticoids are involved in sodium appetite. In our own laboratory, doses of deoxycorticosterone acetate (DOCA) ranging from 1 mg/kg to 20 mg/kg have failed to induce a sodium appetite. In rats, glucocorticoids have been effective in potentiating mineralocorticoid-induced sodium appetite. Recent research has suggested that the mouse, like the rat, does possess mineralocorticoid sensitivity for sodium hunger but, unlike the rat, has a strong dependency on an accompanying glucocorticoid action. The present experiment was conducted to study the effects of DOCA on sodium appetite in mice under housing conditions that attempted to eliminate the reduction of corticosterone associated with social isolation. Therefore, male GHSC mice were group-housed and were tested within two counterbalanced treatment conditions. One condition consisted of an injection of 10 mg/kg DOCA on 2 consecutive days, and the other condition consisted of an injection of the vehicle on 2 consecutive days. Group-housed male GHSC mice showed a significantly larger amount of NaCl consumption after injections of DOCA than after injections of the vehicle.