Female C57BL/6J Mice Show Alcohol-Seeking Behaviour after Withdrawal from Prolonged Alcohol Consumption in the Social Environment.

AIMS: Recently we developed a model to study alcohol-seeking behaviour after withdrawal in a social context in female mice. The model raised several questions that we were eager to address to improve methodology. METHODS: In our model, female mice were group-housed in automated cages with three conditioned (CS+) corners and water in both sides of one separate non-conditioned corner. Water was available with opened doors at all the time of training. We established conditioning by pairing alcohol drinking with light cues. Here, we introduced prolonged access to increasing concentrations of alcohol instead of intermittent access. To study motivation to drink alcohol, we carried out the extinction tests on withdrawal days 1 (WD1) and 10 (WD10). During tests, the light cues were present in conditioned corners, but there was no liquid in the bottles. RESULTS: We found that the number of visits and nosepokes in the CS+ corner in the alcohol group was much higher than in the water group. Also, during training, the consumption of alcohol was increasing. In the extinction tests, we found that the number of nosepokes in the CS+ corner increased in the alcohol group on both WD1 and WD10. CONCLUSIONS: Our study supports that alcohol-seeking behaviour after withdrawal can be modelled and studied in group-housed animals and environments without social isolation.

The overexpression of GDNF in nucleus accumbens suppresses alcohol-seeking behavior in group-housed C57Bl/6J female mice.

BACKGROUND: Craving for alcohol, in other words powerful desire to drink after withdrawal, is an important contributor to the development and maintenance of alcoholism. Here, we studied the role of GDNF (glial cell line-derived neurotrophic factor) and BDNF (brain-derived neurotrophic factor) on alcohol-seeking behavior in group-housed female mice. METHODS: We modeled alcohol-seeking behavior in C57Bl/6J female mice. The behavioral experiments in group-housed female mice were performed in an automated IntelliCage system. We conducted RT-qPCR analysis of Gdnf, Bdnf, Manf and Cdnf expression in different areas of the female mouse brain after alcohol drinking conditioning. We injected an adeno-associated virus (AAV) vector expressing human GDNF or BDNF in mouse nucleus accumbens (NAc) after ten days of alcohol drinking conditioning and assessed alcohol-seeking behavior. Behavioral data were analyzed by two-way repeated-measures ANOVA, and statistically significant effects were followed by Bonferroni's post hoc test. The student's t-test was used to analyze qPCR data. RESULTS: The RT-qPCR data showed that Gdnf mRNA level in NAc was more than four times higher (p < 0.0001) in the mice from the sweetened alcohol group compared to the water group. Our data showed a more than a two-fold decrease in Manf mRNA (p = 0.04) and Cdnf mRNA (p = 0.02) levels in the hippocampus and Manf mRNA in the VTA (p = 0.04) after alcohol consumption. Two-fold endogenous overexpression of Gdnf mRNA and lack of CDNF did not affect alcohol-seeking behavior. The AVV-GDNF overexpression in nucleus accumbens suppressed alcohol-seeking behavior while overexpression of BDNF did not. CONCLUSIONS: The effect of increased endogenous Gdnf mRNA level in female mice upon alcohol drinking has remained unknown. Our data suggest that an increase in endogenous GDNF expression upon alcohol drinking occurs in response to the activation of another mesolimbic reward pathway participant.

Update of neurotrophic factors in neurobiology of addiction and future directions.

Drug addiction is a chronic brain disease and drugs of abuse cause long lasting neuroadaptations. Addiction is characterized by the loss of control over drug use despite harmful consequences, and high rates of relapse even after long periods of abstinence. Neurotrophic factors (NTFs) are well known for their actions on neuronal survival in the peripheral nervous system. Moreover, NTFs have been shown to be involved in synaptic plasticity in the brain. Brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) are two of the most studied NTFs and both of them have been reported to increase craving when administered into the mesocorticolimbic dopaminergic system after drug self-administration. Here we review recent data on BDNF and GDNF functions in addiction-related behavior and discuss them in relation to previous findings. Finally, we give an insight into how new technologies could aid in further elucidating the role of these factors in drug addiction.

A novel variant in SMG9 causes intellectual disability, confirming a role for nonsense-mediated decay components in neurocognitive development.

Biallelic loss-of-function variants in the SMG9 gene, encoding a regulatory subunit of the mRNA nonsense-mediated decay (NMD) machinery, are reported to cause heart and brain malformation syndrome. Here we report five patients from three unrelated families with intellectual disability (ID) and a novel pathogenic SMG9 c.551 T > C p.(Val184Ala) homozygous missense variant, identified using exome sequencing. Sanger sequencing confirmed recessive segregation in each family. SMG9 c.551T > C p.(Val184Ala) is most likely an autozygous variant identical by descent. Characteristic clinical findings in patients were mild to moderate ID, intention tremor, pyramidal signs, dyspraxia, and ocular manifestations. We used RNA sequencing of patients and age- and sex-matched healthy controls to assess the effect of the variant. RNA sequencing revealed that the SMG9 c.551T > C variant did not affect the splicing or expression level of SMG9 gene products, and allele-specific expression analysis did not provide evidence that the nonsense mRNA-induced NMD was affected. Differential gene expression analysis identified prevalent upregulation of genes in patients, including the genes SMOX, OSBP2, GPX3, and ZNF155. These findings suggest that normal SMG9 function may be involved in transcriptional regulation without affecting nonsense mRNA-induced NMD. In conclusion, we demonstrate that the SMG9 c.551T > C missense variant causes a neurodevelopmental disorder and impacts gene expression. NMD components have roles beyond aberrant mRNA degradation that are crucial for neurocognitive development.

Towards developing a model to study alcohol drinking and craving in female mice housed in automated cages.

It is about half a century ago when the so-called "Wise model" to study alcohol drinking behavior in rats was established. The model was based on voluntary intermittent access to increasing concentrations of alcohol. We aimed to establish a model of alcohol craving and used an extinction test on withdrawal days 1 and 10 to study motivation for alcohol. For this purpose, the alcohol drinking training was paired with light cues to establish conditioning. The extinction test was carried out without alcohol but in the presence of light cues and empty bottles. The outcome measures were number of visits, nosepokes, and licks in the conditioned corner where the number of nosepokes represents how much mice "want" alcohol and number of licks shows how much mice "like" alcohol. The number of nosepokes during withdrawal is a measure of craving. Late withdrawal craving was found when intermittent alcohol access was carried out in the automated cages. In this case, we observed a significant increase in the number of nosepokes on both withdrawal days 1 and 10 as compared to water control. The number of nosepokes in the withdrawal days did not correlate with alcohol dose, but number of nosepokes on withdrawal day 1 correlated with the number of nosepokes on the last training day. Although we did not observe incubation of alcohol craving after withdrawal, the craving was increased at the late time point. We conclude that we have established a new tool to study alcohol drinking behavior and craving in female mice.

Pre-α-pro-GDNF and Pre-ß-pro-GDNF Isoforms Are Neuroprotective in the 6-hydroxydopamine Rat Model of Parkinson's Disease.

Glial cell line-derived neurotrophic factor (GDNF) is one of the most studied neurotrophic factors. GDNF has two splice isoforms, full-length pre-α-pro-GDNF (α-GDNF) and pre-ß-pro-GDNF (ß-GDNF), which has a 26 amino acid deletion in the pro-region. Thus far, studies have focused solely on the α-GDNF isoform, and nothing is known about the in vivo effects of the shorter ß-GDNF variant. Here we compare for the first time the effects of overexpressed α-GDNF and ß-GDNF in non-lesioned rat striatum and the partial 6-hydroxydopamine lesion model of Parkinson's disease. GDNF isoforms were overexpressed with their native pre-pro-sequences in the striatum using an adeno-associated virus (AAV) vector, and the effects on motor performance and dopaminergic phenotype of the nigrostriatal pathway were assessed. In the non-lesioned striatum, both isoforms increased the density of dopamine transporter-positive fibers at 3 weeks after viral vector delivery. Although both isoforms increased the activity of the animals in cylinder assay, only α-GDNF enhanced the use of contralateral paw. Four weeks later, the striatal tyrosine hydroxylase (TH)-immunoreactivity was decreased in both α-GDNF and ß-GDNF treated animals. In the neuroprotection assay, both GDNF splice isoforms increased the number of TH-immunoreactive cells in the substantia nigra but did not promote behavioral recovery based on amphetamine-induced rotation or cylinder assays. Thus, the shorter GDNF isoform, ß-GDNF, and the full-length α-isoform have comparable neuroprotective efficacy on dopamine neurons of the nigrostriatal circuitry.