Alcohol craving after withdrawal in group-housed female mice : the role of neurotrophic factors

Excessive use of alcohol results in about 5.3% of all deaths worldwide (WHO, 2018). Alcohol consumption can have adverse social, legal, occupational, psychological, and medical consequences (WHO, 2018). Harmful alcohol consumption can result in diabetes, cancer, stroke, cardiovascular diseases, and mental and behavioral disorders (Shield et al., 2013). Craving for alcohol refers to an urge or conscious desire to drink alcohol-based solutions. It has been hypothesized to underlie alcohol-seeking and relapse in humans with alcohol use disorder. Only humans have been reported to experience cravings. Associative learning, a memorable event that occurs with drug consumption, is an important factor in craving. To model the craving for alcohol in animals with equivalence to humans is a challenging task. However, the knowledge that animal models provide is pivotal to understanding the mechanism of disease progression. Drug craving models are well established by using laboratory rats. Self-administration and following extinction and relapse paradigms in mice are still poorly developed. However, because of gene-manipulated strains, they still pose a possibility for studies that cannot be conducted in rats. The main aim of this thesis was to clarify whether GDNF and BDNF play a role in alcohol-seeking behavior in group-housed female mice. A novel methodology to study alcohol-seeking behavior in mice was established. The uniqueness of the model is that alcohol drinking is voluntary, and it occurs in a social environment with minimum handling by a human. This model was used to study the role of neurotrophic factors (GDNF, BDNF, MANF, and CDNF) in alcohol-seeking behavior. This thesis project resulted in a novel model to study alcohol-seeking behavior after alcohol withdrawal in group-housed female mice. This model provides the possibility to study the role of neurotrophic factors in alcohol-seeking behavior. The results showed that viral GDNF overexpression decreases alcohol-seeking behavior after withdrawal.Alkoholin haitallinen käyttö aiheuttaa maailmanlaajuisesti noin 5,3 % kaikista kuolemista (WHO, 2018). Alkoholin käytöllä voi olla haitallisia sosiaalisia, oikeudellisia, ammatillisia, psykologisia ja lääketieteellisiä seurauksia (WHO, 2018). Haitallinen alkoholinkäyttö voi johtaa moniin sairauksiin kuten diabetekseen, syöpiin, aivoinfarktin, sydän- ja verisuonisairauksiin sekä mielenterveys- ja käyttäytymishäiriöihin (Shield et al., 2013). Alkoholin himo viittaa haluun tai tietoiseen haluun juoda alkoholipohjaisia nesteitä. Alkoholin himo on oletettu olevan alkoholin etsimisen ja uusiutumisen taustalla ihmisillä, joilla on alkoholinkäyttöhäiriö. Vain ihmisten on raportoitu kokevan alkoholiin himoa. Assosiatiivinen oppiminen ja ikimuistoinen tapahtuma, joka tapahtuu huumeiden kulutuksen yhteydessä, on tärkeä tekijä himon kehittymisessä. Eläinten alkoholinhimon mallintaminen ihmisiä vastaavalla tavalla on haastava tehtävä. Eläinmallien tarjoama tieto on kuitenkin keskeistä taudin neurobiologisten mekanismien ymmärtämisessä. Huumeiden himomallit ovat vakiintuneet laboratoriorottien avulla. Itseannostelu sekä huumehakuisen käytöksen sammuttamiseen ja uusiutumiseen liittyvät paradigmat hiirillä eivät ole vielä kehittyneet, mutta lukuisten geenimanipuloitujen kantojen vuoksi ne tarjoavat mahdollisuuden tutkimuksiin, joita ei voida suorittaa rotilla. Tämän väitöskirjatutkimuksen päätavoitteena oli selvittää, onko GDNF:llä ja BDNF:llä roolia alkoholin himon kehittymisessä naarashiirillä jotka elivät ryhmässä. Tutkimuksessa kehitettiin uusi menetelmä tutkia alkoholiin liittyvää käyttäytymistä, jossa hiiret elivät sosiaalisessa ympäristössä. Kehitetyn mallin ainutlaatuisuus on siinä, että alkoholin juominen on vapaaehtoista, ja se tapahtuu sosiaalisessa ympäristössä, ja ihminen käsittelee eläimiä mahdollisimman vähän. Käytimme automatisoituja häkkejä, jotka mahdollistavat yksittäisten eläinten seurannan sosiaalisessa ympäristössä. Tätä mallia käytettiin tutkimaan neurotrofisten tekijöiden (GDNF, BDNF, MANF ja CDNF) roolia alkoholihakuisessa käyttäytymisessä. Tämä väitöskirjatutkimus johti uuden mallin kehittymiseen, jolla tutkittiin alkoholinhakukäyttäytymistä alkoholin vieroittamisen jälkeen naarashiirillä jotka elivät ryhmässä automatisoidussa häkissä. Tämä malli tarjoaa mahdollisuuden tutkia neurotrofisten tekijöiden roolia alkoholiahakuisessa käyttäytymisessä. Tulokset osoittivat, että GDNF:n yli-ilmentyminen vähentää alkoholinhakukäyttäytymistä vieroituksen jälkeen

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. Short Summary: We developed a model to study alcohol drinking behaviour in an enriched and social environment. Long-term conditioning coupling with alcohol reward results in cue-induced alcohol-seeking behaviour in group-housed female C57BL/6J mice. Moreover, a high number of nosepokes on the last day of alcohol drinking conditioning might potentiate alcohol-seeking after withdrawal response.Peer reviewe

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.Peer reviewe

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.Peer reviewe

Pre-α-pro-GDNF and Pre-β-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-alpha-pro-GDNF (u-GDNF) and pre-beta-pro-GDNF (beta-GDNF), which has a 26 amino acid deletion in the pro-region. Thus far, studies have focused solely on the u-GDNF isoform, and nothing is known about the in vivo effects of the shorter beta-GDNF variant. Here we compare for the first time the effects of overexpressed cx-GDNF and beta-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 u-GDNF enhanced the use of contralateral paw. Four weeks later, the striatal tyrosine hydroxylase (TH)-immunoreactivity was decreased in both u-GDNF and 1-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, beta-GDNF, and the full-length alpha-isoform have comparable neuroprotective efficacy on dopamine neurons of the nigrostriatal circuitry.Peer reviewe

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.Peer reviewe

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

Abstract 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 &gt; C p.(Val184Ala) homozygous missense variant, identified using exome sequencing. Sanger sequencing confirmed recessive segregation in each family. SMG9 c.551T &gt; 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 &gt; 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 &gt; 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