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

Nigral injection of a proteasomal inhibitor, lactacystin, induces widespread glial cell activation and shows various phenotypes of Parkinson's disease in young and adult mouse

Proteinaceous inclusions, called Lewy bodies, are used as a pathological hallmark for Parkinson's disease (PD). Lewy bodies contain insoluble alpha-synuclein (aSyn) and many other ubiquitinated proteins, suggesting a role for protein degradation system failure in the PD pathogenesis. Indeed, proteasomal dysfunction has been linked to PD but commonly used in vivo toxin models, such as 6-OHDA or MPTP, do not have a significant effect on the proteasomal system or protein aggregation. Therefore, we wanted to study the characteristics of a proteasomal inhibitor, lactacystin, as a PD model on young and adult mice. To study this, we performed stereotactic microinjection of lactacystin above the substantia nigra pars compacta in young (2 month old) and adult (12-14 month old) C57Bl/6 mice. Motor behavior was measured by locomotor activity and cylinder tests, and the markers of neuroinflammation, aSyn, and dopaminergic system were assessed by immunohistochemistry and HPLC. We found that lactacystin induced a Parkinson's disease-like motor phenotype 5-7 days after injection in young and adult mice, and this was associated with widespread neuroinflammation based on glial cell markers, aSyn accumulation in substantia nigra, striatal dopamine decrease, and loss of dopaminergic cell bodies in the substantia nigra and terminals in the striatum. When comparing young and adult mice, adult mice were more sensitive for dopaminergic degeneration after lactacystin injection that further supports the use of adult mice instead of young when modeling neurodegeneration. Our data showed that lactacystin is useful in modeling various aspects of Parkinson's disease, and taken together, our findings emphasize the role of a protein degradation deficit in Parkinson's disease pathology, and support the use of proteasomal inhibitors as Parkinson's disease models.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

Neuronal activation stimulates cytomegalovirus promoter-driven transgene expression

The cytomegalovirus (CMV) immediate early promoter has been extensively developed and exploited for transgene expression in vitro and in vivo, including human clinical trials. The CMV promoter has long been considered a stable, constitutive and ubiquitous promoter for transgene expression. Using two different CMV-based promoters, we found an increase in CMV-driven transgene expression in the rodent brain and in primary neuronal cultures in response to methamphetamine, glutamate, kainic acid, and activation of G-protein coupled receptor signaling using designer receptors exclusively activated by designer drugs (DREADDs). In contrast, promoters derived from human synapsin 1 (hSyn1) gene or elongation factor 1a (EF1a) did not exhibit altered transgene expression in response to the same neuronal stimulation. Overall, our results suggest that the long standing assertion that the CMV promoter confers constitutive expression in neurons should be reevaluated and future studies should evaluate the activity of the CMV promoter in a given application.Peer reviewe

Combination of CDNF and Deep Brain Stimulation Decreases Neurological Deficits in Late-stage Model Parkinson's Disease

Several neurotrophic factors ( NTF) are shown to be neuroprotective and neurorestorative in pre-clinical animal models for Parkinson's disease ( PD), particularly in models where striatal dopamine neuron innervation partially exists. The results of clinical trials on late-stage patients have been modest. Subthalamic deep brain stimulation ( STN DBS) is a proven treatment for a selected group of advanced PD patients. The cerebral dopamine neurotrophic factor ( CDNF) is a promising therapeutic protein, but its effects in animal models of late-stage PD have remained under-researched. The interactions of NTF and STN DBS treatments have not been studied before. We found that a nigral CDNF protein alone had only a marginal effect on the behavioral deficits in a late-stage hemiparkinsonian rat model ( 6-OHDA MFB). However, CDNF improved the effect of acute STN DBS on front limb use asymmetry at 2 and 3 weeks after CDNF injection. STN lesion-modeling chronic stimulation-had an additive effect in reducing front limb use in the cylinder test and apomorphine-induced rotation. The combination of CDNF and acute STN DBS had a favorable effect on striatal tyrosine hydroxylase. This study presents a novel additive beneficial effect of NTF and STN DBS, which might be explained by the interaction of DBS-induced endogenous NTFs and exogenously injected CDNF. SNpc can be reached via similar trajectories used in clinical STN DBS, and this interaction is an important area for future studies. (C) 2018 The Authors. Published by Elsevier Ltd on behalf of IBRO. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/).Peer reviewe

Systemic hypertonic saline enhances glymphatic spinal cord delivery of lumbar intrathecal morphine

The blood-brain barrier significantly limits effective drug delivery to central nervous system (CNS) targets. The recently characterized glymphatic system offers a perivascular highway for intrathecally (i.t.) administered drugs to reach deep brain structures. Although periarterial cerebrospinal fluid (CSF) influx and concomitant brain drug delivery can be enhanced by pharmacological or hyperosmotic interventions, their effects on drug delivery to the spinal cord, an important target for many drugs, have not been addressed. Hence, we studied in rats whether enhancement of periarterial flow by systemic hypertonic solution might be utilized to enhance spinal delivery and efficacy of i.t. morphine. We also studied whether the hyperosmolar intervention affects brain or cerebrospinal fluid drug concentrations after systemic administration. Periarterial CSF influx was enhanced by intraperitoneal injection of hypertonic saline (HTS, 5.8%, 20 ml/kg, 40 mOsm/kg). The antinociceptive effects of morphine were characterized, using tail flick, hot plate and paw pressure tests. Drug concentrations in serum, tissue and microdialysis samples were determined by liquid chromatography-tandem mass spectrometry. Compared with isotonic solution, HTS increased concentrations of spinal i.t. administered morphine by 240% at the administration level (T13-L1) at 60 min and increased the antinociceptive effect of morphine in tail flick, hot plate, and paw pressure tests. HTS also independently increased hot plate and paw pressure latencies but had no effect in the tail flick test. HTS transiently increased the penetration of intravenous morphine into the lateral ventricle, but not into the hippocampus. In conclusion, acute systemic hyperosmolality is a promising intervention for enhanced spinal delivery of i.t. administered morphine. The relevance of this intervention should be expanded to other i.t. drugs and brought to clinical trials.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

Cerebral dopamine neurotrophic factor-deficiency leads to degeneration of enteric neurons and altered brain dopamine neuronal function in mice

Cerebral dopamine neurotrophic factor (CDNF) is neuroprotective for nigrostriatal dopamine neurons and restores dopaminergic function in animal models of Parkinson's disease (PD). To understand the role of CDNF in mammals, we generated CDNF knockout mice (Cdnf(-/-)), which are viable, fertile, and have a normal life-span. Surprisingly, an age-dependent loss of enteric neurons occurs selectively in the submucosal but not in the myenteric plexus. This neuronal loss is a consequence not of increased apoptosis but of neurodegeneration and autophagy. Quantitatively, the neurodegeneration and autophagy found in the submucosal plexus in duodenum, ileum and colon of the Cdnf(-/-) mouse are much greater than in those of Cdnf(+/+) mice. The selective vulnerability of submucosal neurons to the absence of CDNF is reminiscent of the tendency of pathological abnormalities to occur in the submucosal plexus in biopsies of patients with PD. In contrast, the number of substantia nigra dopamine neurons and dopamine and its metabolite concentrations in the striatum are unaltered in Cdnf(-/-) mice; however, there is an age-dependent deficit in the function of the dopamine system in Cdnf(-/-) male mice analyzed. This is observed as D-amphetamine-induced hyperactivity, aberrant dopamine transporter function, and as increased D-amphetamine-induced dopamine release demonstrating that dopaminergic axon terminal function in the striatum of the Cdnf(-/-) mouse brain is altered. The deficiencies of Cdnf(-/-) mice, therefore, are reminiscent of those seen in early stages of Parkinson's disease.Peer reviewe