Neuropeptide Y Signaling in the Central Nucleus of Amygdala Regulates Alcohol‐Drinking and Anxiety‐Like Behaviors of Alcohol‐Preferring Rats

Background: The neuropeptide Y (NPY) system of the central nucleus of amygdala (CeA) has been shown to be involved in anxiety and alcoholism. In this study, we investigated the molecular mechanisms by which NPY in the CeA regulates anxiety and alcohol drinking behaviors using alcohol‐preferring (P) rats as an animal model. Methods: Alcohol‐preferring rats were bilaterally cannulated targeting the CeA and infused with artificial cerebrospinal fluid (aCSF) or NPY. Alcohol drinking and anxiety‐like behaviors were assessed by the 2‐bottle free‐choice paradigm and light/dark box (LDB) exploration test, respectively. The levels of NPY and related signaling proteins were determined by the gold immunolabeling procedure. The mRNA levels of NPY were measured by in situ RT‐PCR. Double‐immunofluorescence labeling was performed to observe the colocalization of NPY and Ca²⁺/calmodulin‐dependent protein kinase IV (CaMK IV). Results: We found that NPY infusion into the CeA produced anxiolytic effects, as measured by the LDB exploration test, and also decreased alcohol intake in P rats. NPY infusion into the CeA significantly increased levels of CaMK IV and phosphorylated cAMP responsive element‐binding (pCREB) protein and increased mRNA and protein levels of NPY, but produced no changes in protein levels of CREB or the catalytic α‐subunit of protein kinase A (PKA‐Cα) in the CeA. We also observed that alcohol intake produced anxiolytic effects in P rats in the LDB test and also increased NPY expression and protein levels of pCREB and PKA‐Cα without modulating protein levels of CREB or CaMK IV, in both the CeA and medial nucleus of amygdala. In addition, we found that CaMK IV‐positive cells were co‐localized with NPY in amygdaloid structures of P rats. Conclusions: These results suggest that NPY infusion may increase the expression of endogenous NPY in the CeA, which is most likely attributable to an increase in CaMK IV‐dependent CREB phosphorylation and this molecular mechanism may be involved in regulating anxiety and alcohol drinking behaviors of P rats.     

Prenatal Alcohol Exposure and Chronic Mild Stress Differentially Alter Depressive‐ and Anxiety‐Like Behaviors in Male and Female Offspring

Background: Fetal Alcohol Spectrum Disorder (FASD) is associated with numerous neurobehavioral alterations, as well as disabilities in a number of domains, including a high incidence of depression and anxiety disorders. Prenatal alcohol exposure (PAE) also alters hypothalamic‐pituitary‐adrenal (HPA) function, resulting in increased responsiveness to stressors and HPA dysregulation in adulthood. Interestingly, data suggest that pre‐existing HPA abnormalities may be a major contributory factor to some forms of depression, particularly when an individual is exposed to stressors later in life. We tested the hypothesis that exposure to stressors in adulthood may unmask an increased vulnerability to depressive‐ and anxiety‐like behaviors in PAE animals. Methods: Male and female offspring from prenatal alcohol (PAE), pair‐fed (PF), and ad libitum‐fed control (C) treatment groups were tested in adulthood. Animals were exposed to 10 consecutive days of chronic mild stress (CMS), and assessed in a battery of well‐validated tasks sensitive to differences in depressive‐ and/or anxiety‐like behaviors. Results: We report here that the combination of PAE and CMS in adulthood increases depressive‐ and anxiety‐like behaviors in a sexually dimorphic manner. PAE males showed impaired hedonic responsivity (sucrose contrast test), locomotor hyperactivity (open field), and alterations in affiliative and nonaffiliative social behaviors (social interaction test) compared to control males. By contrast, PAE and, to a lesser extent, PF, females showed greater levels of “behavioral despair” in the forced swim test, and PAE females showed altered behavior in the final 5 minutes of the social interaction test compared to control females. Conclusions: These data support the possibility that stress may be a mediating or contributing factor in the psychopathologies reported in FASD populations.     

β‐Endorphin Mediates Behavioral Despair and the Effect of Ethanol on the Tail Suspension Test in Mice

Background: The opioid peptide β‐endorphin (β‐E) is synthesized and released in response to stressful stimuli as well as acute alcohol administration. The release of β‐E following exposure to an inescapable aversive situation may mediate behaviors that contribute to allostasis of the stress response. The present study examines the effects of β‐E on immobility in assays involving inescapable stress, both under basal conditions and after acute administration of EtOH. Methods: Female and male transgenic mice with varying capacities to translate β‐E were subjected to either the forced swim (FST, Experiment 1) or the tail suspension test (TST, Experiment 2). In Experiment 3, mice were divided into three groups based on hormonal status (male, female‐estrous, and female‐nonestrous) and injected with either 1 g/kg EtOH or equivolume saline 14 minutes prior to behavioral assessment on the TST. Results: Experiments 1 and 2 demonstrated a direct relationship between β‐E levels and immobility. There were also sex differences in behavior in these tests, with males displaying more immobility than females. A main effect of genotype in Experiment 3 replicated findings in Experiments 1 and 2. There was also an effect of EtOH (increasing immobility) and a significant interaction reflecting a particularly robust effect of the drug in mice with low β‐E. In addition, there were interactions between β‐E, EtOH effects, and hormonal status. Conclusions: These findings support the contention that β‐E moderates behavioral responses to stressful stimuli and suggest a role for this peptide in coping behavior. Furthermore, the effects of EtOH on the response to stress may be mediated by β‐E. Sex differences in this influence may contribute to sex differences in disease susceptibility and expression.     

β-Endorphin Neuronal Cell Transplant Reduces Corticotropin Releasing Hormone Hyperresponse to Lipopolysaccharide and Eliminates Natural Killer Cell Functional Deficiencies in Fetal Alcohol Exposed Rats

Background: Natural killer (NK) cell dysfunction is associated with hyperresponse of corticotropin releasing hormone (CRH) to immune challenge and with a loss of β‐endorphin (BEP) neurons in fetal alcohol exposed animals. Recently, we established a method to differentiate neural stem cells into BEP neurons using cyclic adenosine monophosphate (cAMP)‐elevating agents in cultures. Hence, we determined whether in vitro differentiated BEP neurons could be used for reversing the compromised stress response and immune function in fetal alcohol exposed rats. Methods: To determine the effect of BEP neuron transplants on NK cell function, we implanted in vitro differentiated BEP neurons into the paraventricular nucleus of pubertal and adult male rats exposed to ethanol or control in utero. The functionality of transplanted BEP neurons was determined by measuring proopiomelanocortin (POMC) gene expression in these cells and their effects on CRH gene expression under basal and after lipopolysaccaride (LPS) challenge. In addition, the effectiveness of BEP neurons in activating NK cell functions is determined by measuring NK cell cytolytic activity and interferon‐γ (IFN‐γ) production in the spleen and in the peripheral blood mononuclear cell (PBMC) following cell transplantation. Results: We showed here that when these in vitro differentiated BEP neurons were transplanted into the hypothalamus, they maintain biological functions by producing POMC and reducing the CRH neuronal response to the LPS challenge. BEP neuronal transplants significantly increased NK cell cytolytic activity in the spleen and in the PBMC and increased plasma levels of IFN‐γ in control and fetal alcohol exposed rats. Conclusions: These data further establish the BEP neuronal regulatory role in the control of CRH and NK cell cytolytic function and identify a possible novel therapy to treat stress hyperresponse and immune deficiency in fetal alcohol exposed subjects.