α2A-Adrenergic Receptor Signaling Underlies Synergistic Enhancement of Ethanol-Induced Behavioral Impairment by Clonidine

Background: We tested the hypothesis that central α_2A‐adrenergic receptor (α_2AAR) signaling plays a key role in clonidine‐ethanol evoked synergistic behavioral impairment. Methods: Male Sprague‐Dawley rats, with intracisternal and jugular vein cannulae implanted 6 days earlier, were tested for drug‐induced behavioral impairment. The latter was assessed as the duration of loss of righting reflex (LORR) and rotorod performance every 15 minutes until the rat recovered to the baseline walk criterion (180 seconds). In a separate cohort, c‐Fos expression in locus coeruleus (LC) and cerebellum was determined as a marker of neuronal activity following drug treatment. Results: Rats that received clonidine (60 μg/kg, i.v.) followed by ethanol (1 g/kg, i.v.) exhibited synergistic impairment of rotorod performance and LORR. The mixed α_2AAR and I₁‐imidazoline receptor agonist clonidine (30, 60, and 90 μg/kg) synergistically and dose‐dependently enhanced behavioral impairment elicited by ethanol (1 g/kg). Possible involvement of I₁‐imidazoline receptors was ruled out because selective I₁‐agonist rilmenidine (300 μg/kg, i.v.) did not cause behavioral impairment alone or enhance ethanol‐evoked behavioral impairment. Pharmacological blockade of central α_2AAR (RX821002, 0.3 mg i.c.) abolished the synergy between clonidine and ethanol; the behavioral response caused by the drug combination was similar to that caused by ethanol alone. Conversely, involvement of central α_2BAR in the interaction was ruled out because blockade of central α_2BAR (ARC‐239) independently evoked a strong sedative effect. Clonidine (60 μg/kg) or ethanol (1 g/kg) alone increased, but their combination decreased, c‐Fos levels in LC, while inconsistent c‐Fos responses were observed in cerebellum. Conclusions: Central α_2AAR, but not I₁‐imidazoline or α_2BAR, signaling is implicated in the synergistic enhancement of ethanol‐evoked behavioral impairment by clonidine. Although the mechanism of c‐Fos response remains to be investigated, this neurochemical response highlights the LC as a neuroanatomical target for clonidine‐ethanol behavioral interaction.     

Ontogeny of the Enhanced Fetal‐Ethanol‐Induced Behavioral and Neurophysiologic Olfactory Response to Ethanol Odor

Background: Studies report a fundamental relationship between chemosensory function and the responsiveness to ethanol, its component orosensory qualities, and its odor as a consequence of fetal ethanol exposure. Regarding odor, fetal exposed rats display enhanced olfactory neural and behavioral responses to ethanol odor at postnatal (P) day 15. Although these consequences are absent in adults (P90), the behavioral effect has been shown to persist into adolescence (P37). Given the developmental timing of these observations, we explored the decay in the response to ethanol odor by examining ages between P37 and young adulthood. Moreover, we sought to determine whether the P15 neurophysiologic effect persists, at least, to P40. Methods: Behavioral and olfactory epithelial (OE) responses of fetal ethanol exposed and control rats were tested at P40, P50, P60, or P70. Whole‐body plethysmography was used to quantify each animal’s innate behavioral response to ethanol odor. We then mapped the odorant‐induced activity across the OE in response to different odorants, including ethanol, using optical recording methods. Results: Relative to controls, ethanol exposed animals showed an enhanced behavioral response to ethanol odor that, while significant at each age, decreased in magnitude. These results, in conjunction with previous findings, permitted the development of an ontologic odor response model of fetal exposure. The fitted model exemplifies that odor‐mediated effects exist at birth, peak in adolescence and then decline, becoming absent by P90. There was no evidence of an effect on the odor response of the OE at any age tested. Conclusions: Fetal exposure yields an enhanced behavioral response to ethanol odor that peaks in adolescence and wanes through young adulthood. This occurs absent an enhanced response of the OE. This latter finding suggests that by P40 the OE returns to an ethanol “neutral” status and that central mechanisms, such as ethanol‐induced alterations in olfactory bulb circuitry, underlie the enhanced behavioral response. Our study provides a more comprehensive understanding of the ontogeny of fetal‐ethanol‐induced olfactory functional plasticity and the behavioral response to ethanol odor.     

1H and 31P NMR Lipidome of Ethanol‐Induced Fatty Liver

Background: Hepatic steatosis (fatty liver), an early and reversible stage of alcoholic liver disease, is characterized by triglyceride deposition in hepatocytes, which can advance to steatohepatitis, fibrosis, cirrhosis, and ultimately to hepatocellular carcinoma. In the present work, we studied altered plasma and hepatic lipid metabolome (lipidome) to understand the mechanisms and lipid pattern of early‐stage alcohol‐induced‐fatty liver. Methods: Male Fischer 344 rats were fed 5% alcohol in a Lieber‐DeCarli diet. Control rats were pair‐fed an equivalent amount of maltose‐dextrin. After 1 month, animals were killed and plasma collected. Livers were excised for morphological, immunohistochemical, and biochemical studies. The lipids from plasma and livers were extracted with methyl‐tert‐butyl ether and analyzed by 750/800 MHz proton nuclear magnetic resonance (¹H NMR) and phosphorus (³¹P) NMR spectroscopy on a 600 MHz spectrometer. The NMR data were then subjected to multivariate statistical analysis. Results: Hematoxylin and Eosin and Oil Red O stained liver sections showed significant fatty infiltration. Immunohistochemical analysis of liver sections from ethanol‐fed rats showed no inflammation (absence of CD3 positive cells) or oxidative stress (absence of malondialdehyde reactivity or 4‐hydroxynonenal positive staining). Cluster analysis and principal component analysis of ¹H NMR data of lipid extracts of both plasma and livers showed a significant difference in the lipid metabolome of ethanol‐fed versus control rats. ³¹P NMR data of liver lipid extracts showed significant changes in phospholipids similar to ¹H NMR data. ¹H NMR data of plasma and liver reflected several changes, while comparison of ¹H NMR and ³¹P NMR data offered a correlation among the phospholipids. Conclusions: Our results show that alcohol consumption alters metabolism of cholesterol, triglycerides, and phospholipids that could contribute to the development of fatty liver. These studies also indicate that fatty liver precedes oxidative stress and inflammation. The similarities observed in plasma and liver lipid profiles offer a potential methodology for detecting early‐stage alcohol‐induced fatty liver disease by analyzing the plasma lipid profile.     

Ethanol‐Induced Conditioned Taste Aversion in Male Sprague‐Dawley Rats: Impact of Age and Stress

Background: Age‐specific characteristics may contribute to the elevation in ethanol intake commonly reported among adolescents compared to adults. This study was designed to examine age‐related differences in sensitivity to ethanol’s aversive properties using a conditioned taste aversion (CTA) procedure with sucrose serving as the conditioned stimulus (CS). Given that ontogenetic differences in responsiveness to stressors have been previously reported, the role of stressor exposure on the development of CTA was also assessed. Methods: Experiment 1 examined the influence of 5 days of prior restraint stress exposure on the expression of CTA in a 2‐bottle test following 1 pairing of a sucrose solution with ethanol. In Experiment 2, the effects of 7 days of social isolation on the development of CTA were observed using a 1‐bottle test following multiple sucrose–ethanol pairings. Results: This study revealed age‐related differences in the development of ethanol‐induced CTA. In Experiment 1, adolescents required a higher dose of ethanol than adults to demonstrate an aversion. In Experiment 2, adolescents required not only a higher ethanol dose but also more pairings of ethanol with the sucrose CS. No effects of prior stressor exposure were observed in either experiment. Conclusions: Together, these experiments demonstrate an adolescent‐specific insensitivity to the aversive properties of ethanol that elicit CTA, a pattern not influenced by repeated restraint stress or housing in social isolation. This age‐related insensitivity to the dysphoric effects of ethanol is consistent with other work from our laboratory, adding further to the evidence that adolescent rats are less susceptible to negative consequences of ethanol that may serve as cues to curb consumption.     

Effects of a Novel Cognition‐Enhancing Agent on Fetal Ethanol‐Induced Learning Deficits

Background: Drinking during pregnancy has been associated with learning disabilities in affected offspring. At present, there are no clinically effective pharmacotherapeutic interventions for these learning deficits. Here, we examined the effects of ABT‐239, a histamine H₃ receptor antagonist, on fetal ethanol‐induced fear conditioning and spatial memory deficits. Methods and Results: Long‐Evans rat dams stably consumed a mean of 2.82 g ethanol/kg during a 4‐hour period each day during pregnancy. This voluntary drinking pattern produced a mean peak serum ethanol level of 84 mg/dl. Maternal weight gain, litter size and birth weights were not different between the ethanol‐consuming and control groups. Female adult offspring from the control and fetal alcohol‐exposed (FAE) groups received saline or 1 mg ABT‐239/kg 30 minutes prior to fear conditioning training. Three days later, freezing time to the context was significantly reduced in saline‐treated FAE rats compared to control. Freezing time in ABT‐239‐treated FAE rats was not different than that in controls. In the spatial navigation study, adult male offspring received a single injection of saline or ABT‐239 30 minutes prior to 12 training trials on a fixed platform version of the Morris Water Task. All rats reached the same performance asymptote on Trials 9 to 12 on Day 1. However, 4 days later, first‐trial retention of platform location was significantly worse in the saline‐treated FAE rats compared control offspring. Retention by ABT‐239‐treated FAE rats was similar to that by controls. ABT‐239’s effect on spatial memory retention in FAE rats was dose dependent. Conclusions: These results suggest that ABT‐239 administered prior to training can improve retention of acquired information by FAE offspring on more challenging versions of hippocampal‐sensitive learning tasks. Further, the differential effects of ABT‐239 in FAE offspring compared to controls raises questions about the impact of fetal ethanol exposure on histaminergic neurotransmission in affected offspring.     

Interactions of Stress and CRF in Ethanol‐Withdrawal Induced Anxiety in Adolescent and Adult Rats

Background: Repeated stress or administration of corticotropin‐releasing factor (CRF) prior to ethanol exposure sensitizes anxiety‐like behavior in adult rats. Current experiments determined whether adolescent rats were more sensitive to these challenges in sensitizing ethanol withdrawal‐induced anxiety and altering CRF levels in brain during withdrawal. Methods: Male adult and adolescent Sprague–Dawley rats were restraint stressed (1 hour) twice 1 week apart prior to a single 5‐day cycle of ethanol diet (ED; stress/withdrawal paradigm). Other rats received control diet (CD) and three 1‐hour restraint stress sessions. Rats were then tested 5, 24, or 48 hours after the final withdrawal for anxiety‐like behavior in the social interaction (SI) test. In other experiments, adolescent rats were given two microinjections of CRF icv 1 week apart followed by 5 days of either CD or ED and tested in social interaction 5 hours into withdrawal. Finally, CRF immunoreactivity was measured in the central nucleus of the amygdala (CeA) and paraventricular nucleus (PVN) after rats experienced control diet, repeated ethanol withdrawals, or stress/withdrawal. Results: Rats of both ages had reduced SI following the stress/withdrawal paradigm, and this effect recovered within 24 hours. Higher CRF doses were required to reduce SI in adolescents than previously reported in adults. CRF immunohistochemical levels were higher in the PVN and CeA of CD‐exposed adolescents. In adolescent rats, repeated ethanol withdrawals decreased CRF in the CeA but was not associated with decreased CRF cell number. There was no change in CRF from adult treatments. Conclusions: In the production of anxiety‐like behavior, adolescent rats have equal sensitivity with stress and lower sensitivity with CRF compared to adults. Further, adolescents had higher basal levels of CRF within the PVN and CeA and reduced CRF levels following repeated ethanol withdrawals. This reduced CRF within the CeA could indicate increased release of CRF, and future work will determine how this change relates to behavior.