Downregulation of mu opioid receptor by RNA interference in the ventral tegmental area reduces ethanol consumption in mice

Pharmacological and genetic studies have implicated the mu opioid receptor (MOR) in the regulation of ethanol intake in animal models and humans. Non-specific antagonists of opioid receptors have been shown to affect ethanol consumption when infused directly into the ventral tegmental area (VTA) of rats. However, administration of MOR-selective antagonists into the VTA has yielded mixed results. We used RNA interference (RNAi) to specifically decrease levels of MOR messenger RNA in the VTA of mice and examined the effect on ethanol consumption in a two-bottle choice paradigm. Mice were injected in the VTA with lentivirus expressing either a small hairpin RNA (shRNA) targeting MOR or a control shRNA. One week after virus injection, mice were examined for ethanol consumption in a two-bottle choice experiment with increasing concentrations of ethanol over the course of 1 month. Expression of an shRNA targeting MOR in the VTA led to a significant reduction in ethanol consumption. These results strengthen the hypothesis that MOR in the VTA is one of the key brain substrates mediating alcohol consumption. The RNAi combined with lentiviral delivery can be used successfully in brain to effect a sustained reduction in expression of specific genes for behavioral analysis.     

Mapping the midkine family of developmentally regulated signaling molecules

Midkine (Mdk) and heparin-binding neurotrophic factor (Hbnf)/pleiotrophin (Ptn) comprise the Midkine family of developmentally regulated signaling molecules. We have determined the chromosomal localization of these genes in the mouse by use of singlestrand conformation polymorphisms (SSCPs), which facilitated the typing of Mdk and Hbnf alleles in recombinant inbred (RI) strains and interspecific backcrosses. Mapping was performed relative to other cloned genes, as well as simple sequence length polymorphisms (SSLPs) in the interspecific backcrosses. Mdk maps to mouse Chromosome (Chr) 2, linked to the Hoxd gene cluster. Hbnf maps to proximal mouse Chr 6, linked to the Cftr and Cpa genes. Comparative mapping of human MDK and HBNF employing species-specific polymerase chain reaction (PCR) primers and human monochromosomal somatic cell hybrids assigns MDK to human Chr 11 and HBNF to human Chr 7q32-qter.     

Hypothalamic‐specific proopiomelanocortin deficiency reduces alcohol drinking in male and female mice

Opioid receptor antagonist naltrexone reduces alcohol consumption and relapse in both humans and rodents. This study investigated whether hypothalamic proopiomelanocortin (POMC) neurons (producing beta‐endorphin and melanocortins) play a role in alcohol drinking behaviors. Both male and female mice with targeted deletion of two neuronal Pomc enhancers nPE1 and nPE2 (nPE?/?), resulting in hypothalamic‐specific POMC deficiency, were studied in short‐access (4‐h/day) drinking‐in‐the‐dark (DID, alcohol in one bottle, intermittent access (IA, 24‐h cycles of alcohol access every other day, alcohol vs. water in a two‐bottle choice) and alcohol deprivation effect (ADE) models. Wild‐type nPE+/+ exposed to 1‐week DID rapidly established stable alcohol drinking behavior with more intake in females, whereas nPE?/? mice of both sexes had less intake and less preference. Although nPE?/? showed less saccharin intake and preference than nPE+/+, there was no genotype difference in sucrose intake or preference in the DID paradigm. After 3‐week IA, nPE+/+ gradually escalated to high alcohol intake and preference, with more intake in females, whereas nPE?/? showed less escalation. Pharmacological blockade of mu‐opioid receptors with naltrexone reduced intake in nPE+/+ in a dose‐dependent manner, but had blunted effects in nPE?/? of both sexes. When alcohol was presented again after 1‐week abstinence from IA, nPE+/+ of both sexes displayed significant increases in alcohol intake (ADE or relapse‐like drinking), with more pronounced ADE in females, whereas nPE?/? did not show ADE in either sex. Our results suggest that neuronal POMC is involved in modulation of alcohol ‘binge’ drinking, escalation and ‘relapse’, probably via hypothalamic‐mediated mechanisms, with sex differences.     

CRMP4 suppresses apical dendrite bifurcation of CA1 pyramidal neurons in the mouse hippocampus

Collapsin response mediator proteins (CRMPs) are a family of cytosolic phosphoproteins that consist of 5 members (CRMP 1–5). CRMP2 and CRMP4 regulate neurite outgrowth by binding to tubulin heterodimers, resulting in the assembly of microtubules. CRMP2 also mediates the growth cone collapse response to the repulsive guidance molecule semaphorin‐3A (Sema3A). However, the role of CRMP4 in Sema3A signaling and its function in the developing mouse brain remain unclear. We generated CRMP4?/? mice in order to study the in vivo function of CRMP4 and identified a phenotype of proximal bifurcation of apical dendrites in the CA1 pyramidal neurons of CRMP4?/? mice. We also observed increased dendritic branching in cultured CRMP4?/? hippocampal neurons as well as in cultured cortical neurons treated with CRMP4 shRNA. Sema3A induces extension and branching of the dendrites of hippocampal neurons; however, these inductions were compromised in the CRMP4?/? hippocampal neurons. These results suggest that CRMP4 suppresses apical dendrite bifurcation of CA1 pyramidal neurons in the mouse hippocampus and that this is partly dependent on Sema3A signaling. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2012     

Progress in a replicated selection for elevated blood ethanol concentrations in HDID mice

Drinking in the dark (DID) is a limited access ethanol‐drinking phenotype in mice. High Drinking in the Dark (HDID‐1) mice have been bred for 27 selected generations (S27) for elevated blood ethanol concentrations (BECs) after a 4‐h period of access to 20% ethanol. A second replicate line (HDID‐2) was started later from the same founder population and is currently in S20. An initial report of response to selection in HDID‐1 was published after S11. This article reports genetic and behavioral characteristics of both lines in comparison with the HS controls. Heritability is low in both replicates (h² = 0.09) but the lines have shown 4–5 fold increases in BEC since S0; 80% of HDID‐1 and 60% of HDID‐2 mice reach BECs greater than 1.0 mg/ml. Several hours after a DID test, HDID mice show mild signs of withdrawal. Although not considered during selection, intake of ethanol (g/kg) during the DID test increased by approximately 80% in HDID‐1 and 60% in HDID‐2. Common genetic influences were more important than environmental influences in determining the similarity between BEC and intake for HDID mice. Analysis of the partitioning of intake showed that 60% of intake is concentrated in the last 2 h of the 4 h session. However, this has not changed during selection. Hourly BECs during the DID test reach peak levels after 3 or 4 h of drinking. HDID mice do not differ from HS mice in their rate of elimination of an acute dose of alcohol .     

Cdh13 and AdipoQ gene knockout alter instrumental and Pavlovian drug conditioning

Genome‐wide association studies in humans have suggested that variants of the cadherin‐13 (CDH13) gene are associated with substance use disorder, subjective response to amphetamine, and attention deficit hyperactivity disorder. To examine the role of the Cdh13 and its peptide ligand adiponectin (AdipoQ) in addiction‐related behaviors, we assessed Cdh13 knockout (KO) rats and AdipoQ KO mice using intravenous cocaine self‐administration and conditioned place preference (CPP) paradigms. During intravenous cocaine self‐administration, male Cdh13 heterozygous (+/?) and KO (?/?) rats showed increased cue‐induced reinstatement compared with wild‐type (WT) rats when presented with a cocaine‐paired stimulus, whereas female Cdh13 rats showed no differences across genotype. Cdh13 ?/? rats showed higher responding for a saccharin reinforcer and learned the choice reaction time (RT) task more slowly than WTs. However, we found no differences between Cdh13 ?/? and +/+ rats in responding for sensory reinforcement, number of premature responses in the RT task, tendency to approach a Pavlovian food cue, CPP and locomotor activation to cocaine (10 or 20 mg/kg). In AdipoQ ?/? mice, there was a significant increase in CPP to methamphetamine (1 mg/kg) but not to a range of d‐amphetamine doses (0.5, 1, 2 and 4 mg/kg). Taken together, these data suggest that Cdh13 and AdipoQ regulate sensitivity to psychomotor stimulants and palatable rewards without producing major changes in other behaviors. In humans, these two genes may regulate sensitivity to natural and drug rewards, thus influencing susceptibility to the conditioned drug effects and relapse.     

Scn4b regulates the hypnotic effects of ethanol and other sedative drugs

The voltage‐gated sodium channel subunit β4 (SCN4B) regulates neuronal activity by modulating channel gating and has been implicated in ethanol consumption in rodent models and human alcoholics. However, the functional role for Scn4b in ethanol‐mediated behaviors is unknown. We determined if genetic global knockout (KO) or targeted knockdown of Scn4b in the central nucleus of the amygdala (CeA) altered ethanol drinking or related behaviors. We used four different ethanol consumption procedures (continuous and intermittent two‐bottle choice (2BC), drinking‐in‐the dark and chronic intermittent ethanol vapor) and found that male and female Scn4b KO mice did not differ from their wild‐type (WT) littermates in ethanol consumption in any of the tests. Knockdown of Scn4b mRNA in the CeA also did not alter 2BC ethanol drinking. However, Scn4b KO mice showed longer duration of the loss of righting reflex induced by ethanol, gaboxadol, pentobarbital and ketamine. KO mice showed slower recovery to basal levels of handling‐induced convulsions after ethanol injection, which is consistent with the increased sedative effects observed in these mice. However, Scn4b KO mice did not differ in the severity of acute ethanol withdrawal. Acoustic startle responses, ethanol‐induced hypothermia and clearance of blood ethanol also did not differ between the genotypes. There were also no functional differences in the membrane properties or excitability of CeA neurons from Scn4b KO and WT mice. Although we found no evidence that Scn4b regulates ethanol consumption in mice, it was involved in the acute hypnotic effects of ethanol and other sedatives.     

A method for mapping intralocus interactions influencing excessive alcohol drinking

Excessive alcohol (ethanol) consumption is the hallmark of alcohol use disorders. The F1 hybrid cross between the C57BL/6J (B6) and FVB/NJ (FVB) inbred mouse strains consumes more ethanol than either progenitor strain. The purpose of this study was to utilize ethanol-drinking data and genetic information to map genes that result in overdominant (or heterotic) ethanol drinking. About 600 B6 × FVB F2 mice, half of each sex, were tested for ethanol intake and preference in a 24-h, two-bottle water versus ethanol choice procedure, with ascending ethanol concentrations. They were then tested for ethanol intake in a Drinking in the Dark (DID) procedure, first when there was no water choice and then when ethanol was offered versus water. DNA samples were obtained and genome-wide QTL analyses were performed to search for single QTLs (both additive and dominance effects) and interactions between pairs of QTLs, or epistasis. On average, F2 mice consumed excessive amounts of ethanol in the 24-h choice procedure, consistent with high levels of consumption seen in the F1 cross. Consumption in the DID procedure was similar or higher than amounts reported previously for the B6 progenitor. QTLs resulting in heightened consumption in heterozygous compared to homozygous animals were found on Chrs 11, 15, and 16 for 24-h choice 30% ethanol consumption, and on Chr 11 for DID. No evidence was found for epistasis between any pair of significant or suggestive QTLs. This indicates that the hybrid overdominance is due to intralocus interactions at the level of individual QTL.     

NPY‐Y1 coexpressed with NPY‐Y5 receptors modulate anxiety but not mild social stress response in mice

The Y1 and Y5 receptors for neuropeptide Y have overlapping functions in regulating anxiety. We previously demonstrated that conditional removal of the Y1 receptor in the Y5 receptor expressing neurons in juvenile Npy1r^Y5R?/? mice leads to higher anxiety but no changes in hypothalamus‐pituitary‐adrenocortical axis activity, under basal conditions or after acute restraint stress. In the present study, we used the same conditional system to analyze the specific contribution of limbic neurons coexpressing Y1 and Y5 receptors on the emotional and neuroendocrine responses to social chronic stress, using different housing conditions (isolation vs. group‐housing) as a model. We demonstrated that control Npy1r^2lox male mice housed in groups show increased anxiety and hypothalamus‐pituitary‐adrenocortical axis activity compared with Npy1r^2lox mice isolated for six weeks immediately after weaning. Conversely, Npy1r^Y5R?/? conditional mutants display an anxious‐like behavior but no changes in hypothalamus‐pituitary‐adrenocortical axis activity as compared with their control littermates, independently of housing conditions. These results suggest that group housing constitutes a mild social stress for our B6129S mouse strain and they confirm that the conditional inactivation of Y1 receptors specifically in Y5 receptor containing neurons increases stress‐related anxiety without affecting endocrine stress responses.     

Absence of Tau triggers age‐dependent sciatic nerve morphofunctional deficits and motor impairment

Dementia is the cardinal feature of Alzheimer's disease (AD), yet the clinical symptoms of this disorder also include a marked loss of motor function. Tau abnormal hyperphosphorylation and malfunction are well‐established key events in AD neuropathology but the impact of the loss of normal Tau function in neuronal degeneration and subsequent behavioral deficits is still debated. While Tau reduction has been increasingly suggested as therapeutic strategy against neurodegeneration, particularly in AD, there is controversial evidence about whether loss of Tau progressively impacts on motor function arguing about damage of CNS motor components. Using a variety of motor‐related tests, we herein provide evidence of an age‐dependent motor impairment in Tau?/? animals that is accompanied by ultrastructural and functional impairments of the efferent fibers that convey motor‐related information. Specifically, we show that the sciatic nerve of old (17–22‐months) Tau?/? mice displays increased degenerating myelinated fibers and diminished conduction properties, as compared to age‐matched wild‐type (Tau+/+) littermates and younger (4–6 months) Tau?/? and Tau+/+ mice. In addition, the sciatic nerves of Tau?/? mice exhibit a progressive hypomyelination (assessed by g‐ratio) specifically affecting large‐diameter, motor‐related axons in old animals. These findings suggest that loss of Tau protein may progressively impact on peripheral motor system.     

Nicotinic acetylcholine receptors containing α6 subunits contribute to alcohol reward‐related behaviours

Evidence is emerging that neuronal nicotinic acetylcholine receptors (nAChRs) in the mesolimbic dopamine (DA) system are involved in mediating the reinforcing effects of alcohol. Midbrain DA neurons express high levels of α6 subunit‐containing nAChRs that modulate DA transmission, implicating their involvement in reward‐related behaviours. This study assessed the role of α6‐containing nAChRs in modulating alcohol reward using transgenic mice expressing mutant, hypersensitive α6 nAChR subunits (α6L9′S mice). α6L9′S mice and littermate controls were tested in three well‐established models of alcohol reward: 24‐h two‐bottle choice drinking, drinking in the dark (DID), and conditioned place preference (CPP). Confocal microscopy and patch‐clamp electrophysiology were used to show the localization and function of hypersensitive α6 subunit‐containing nAChRs. Results indicate that female α6L9′S mice showed significantly higher alcohol intake at low concentrations of alcohol (3% and 6%) in the two‐bottle choice procedure. Both male and female α6L9′S mice drank significantly more in the DID procedure and displayed an alcohol‐induced place preference using a low dose of alcohol (0.5 g/kg) that was ineffective in littermate controls. Confocal microscopy showed that α6 subunit‐containing nAChRs are selectively expressed on ventral tegmental area (VTA) DAergic, but not GABAergic neurons. Patch‐clamp electrophysiology showed that VTA DA neurons of α6L9′S mice are hypersensitive to ACh. Collectively, these results suggest that α6L9′S mice are more sensitive to the rewarding effects of alcohol, and suggest that VTA α6 subunit‐containing nAChRs modulate alcohol reward. Thus, α6 subunit‐containing nAChRs may be a promising therapeutic target for treatment of alcohol use disorders .     

Distinct ethanol drinking microstructures in two replicate lines of mice selected for drinking to intoxication

The High Drinking in the Dark (HDID) mice have been selectively bred for reaching high blood ethanol concentrations (BECs) following the limited access Drinking in the Dark (DID) test. We have shown previously that mice from the first HDID replicate line (HDID‐1) drink in larger, but not longer, ethanol drinking bouts than the low‐drinking HS/Npt control mice when consuming modest amounts in the DID test. Here, we assessed drinking microstructure in HDID‐1 mice during binge‐like levels of ethanol intake using a lickometer system. Mice from both HDID replicates (HDID‐1 and ‐2) and HS mice were also given three DID tests (single‐bottle ethanol, two‐bottle choice and single‐bottle saccharin) using a continuously recording BioDAQ system to determine whether there are selection‐dependent changes in drinking microstructure. Larger ethanol bout size in the HDID‐1 mice than the HS mice was found to be due to a larger lick volume in these mice. HDID‐1 and HDID‐2 mice were also seen to have different drinking microstructures that both resulted in high intake and high BECs. The HDID‐1 mice drank in larger ethanol bouts than HS, whereas HDID‐2 mice drank in more frequent bouts. This pattern was also seen in two‐bottle choice DID. The HDID‐2 mice had a high bout frequency for all fluid types tested, whereas the large bout size phenotype of the HDID‐1 mice was specific to alcohol. These findings suggest that selection for drinking to intoxication has resulted in two distinct drinking microstructures, both of which lead to high BECs and high ethanol intake.