Ropinirole Cotreatment Prevents Perivascular Glial Recruitment in a Rat Model of L-DOPA-Induced Dyskinesia.

Dopamine replacement therapy for Parkinson's disease is achieved using L-DOPA or dopamine D2/3 agonists, such as ropinirole. Here, we compare the effects of L-DOPA and ropinirole, alone or in combination, on patterns of glial and microvascular reactivity in the striatum. Rats with unilateral 6-hydroxydopamine lesions were treated with therapeutic-like doses of L-DOPA (6 mg/kg), an equipotent L-DOPA-ropinirole combination (L-DOPA 3 mg/kg plus ropinirole 0.5 mg/kg), or ropinirole alone. Immunohistochemistry was used to examine the reactivity of microglia (ionized calcium-binding adapter molecule 1, IBA-1) and astroglia (glial fibrillary acidic protein, GFAP), as well as blood vessel density (rat endothelial cell antigen 1, RECA-1) and albumin extravasation. L-DOPA monotreatment and L-DOPA-ropinirole cotreatment induced moderate-severe dyskinesia, whereas ropinirole alone had negligible dyskinetic effects. Despite similar dyskinesia severity, striking differences in perivascular microglia and astroglial reactivity were found between animals treated with L-DOPA vs. L-DOPA-ropinirole. The former exhibited a marked upregulation of perivascular IBA-1 cells (in part CD68-positive) and IBA-1-RECA-1 contact points, along with an increased microvessel density and strong perivascular GFAP expression. None of these markers were significantly upregulated in animals treated with L-DOPA-ropinirole or ropinirole alone. In summary, although ropinirole cotreatment does not prevent L-DOPA-induced dyskinesia, it protects from maladaptive gliovascular changes otherwise associated with this disorder, with potential long-term benefits to striatal tissue homeostasis.

Comparison of dyskinesia profiles after L-DOPA dose challenges with or without dopamine agonist coadministration.

Many patients with Parkinson's disease (PD) experiencing l-DOPA-induced dyskinesia (LID) receive adjunct treatment with dopamine agonists, whose functional impact on LID is unknown. We set out to compare temporal and topographic profiles of abnormal involuntary movements (AIMs) after l-DOPA dose challenges including or not the dopamine agonist ropinirole. Twenty-five patients with PD and a history of dyskinesias were sequentially administered either l-DOPA alone (150% of usual morning dose) or an equipotent combination of l-DOPA and ropinirole in random order. Involuntary movements were assessed by two blinded raters prior and every 30 min after drug dosing using the Clinical Dyskinesia Rating Scale (CDRS). A sensor-recording smartphone was secured to the patients' abdomen during the test sessions. The two raters' CDRS scores were highly reliable and concordant with models of hyperkinesia presence and severity trained on accelerometer data. The dyskinesia time curves differed between treatments as the l-DOPA-ropinirole combination resulted in lower peak severity but longer duration of the AIMs compared with l-DOPA alone. At the peak of the AIMs curve (60-120 min), l-DOPA induced a significantly higher total hyperkinesia score, whereas in the end phase (240-270 min), both hyperkinesia and dystonia tended to be more severe after the l-DOPA-ropinirole combination (though reaching statistical significance only for the item, arm dystonia). Our results pave the way for the introduction of a combined l-DOPA-ropinirole challenge test in the early clinical evaluation of antidyskinetic treatments. Furthermore, we propose a machine-learning method to predict CDRS hyperkinesia severity using accelerometer data.

Dopamine Agonist Cotreatment Alters Neuroplasticity and Pharmacology of Levodopa-Induced Dyskinesia.

BACKGROUND: Current models of levodopa (L-dopa)-induced dyskinesia (LID) are obtained by treating dopamine-depleted animals with L-dopa. However, patients with LID receive combination therapies that often include dopamine agonists. OBJECTIVE: Using 6-hydroxydopamine-lesioned rats as a model, we aimed to establish whether an adjunct treatment with the D2/3 agonist ropinirole impacts on patterns of LID-related neuroplasticity and drug responses. METHODS: Different regimens of L-dopa monotreatment and L-dopa-ropinirole cotreatment were compared using measures of hypokinesia and dyskinesia. Striatal expression of ∆FosB and angiogenesis markers were studied immunohistochemically. Antidyskinetic effects of different drug categories were investigated in parallel groups of rats receiving either L-dopa monotreatment or L-dopa combined with ropinirole. RESULTS: We defined chronic regimens of L-dopa monotreatment and L-dopa-ropinirole cotreatment inducing overall similar abnormal involuntary movement scores. Compared with the monotreatment group, animals receiving the L-dopa-ropinirole combination exhibited an overall lower striatal expression of ∆FosB with a distinctive compartmental distribution. The expression of angiogenesis markers and blood-brain barrier hyperpermeability was markedly reduced after L-dopa-ropinirole cotreatment compared with L-dopa monotreatment. Moreover, significant group differences were detected upon examining the response to candidate antidyskinetic drugs. In particular, compounds modulating D1 receptor signaling had a stronger effect in the L-dopa-only group, whereas both amantadine and the selective NMDA antagonist MK801 produced a markedly larger antidyskinetic effect in L-dopa-ropinirole cotreated animals. CONCLUSIONS: Cotreatment with ropinirole altered LID-related neuroplasticity and pharmacological response profiles. The impact of adjuvant dopamine agonist treatment should be taken into consideration when investigating LID mechanisms and candidate interventions in both clinical and experimental settings. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Non-Apoptotic Caspase-3 Activation Mediates Early Synaptic Dysfunction of Indirect Pathway Neurons in the Parkinsonian Striatum.

Non-apoptotic caspase-3 activation is critically involved in dendritic spine loss and synaptic dysfunction in Alzheimer's disease. It is, however, not known whether caspase-3 plays similar roles in other pathologies. Using a mouse model of clinically manifest Parkinson's disease, we provide the first evidence that caspase-3 is transiently activated in the striatum shortly after the degeneration of nigrostriatal dopaminergic projections. This caspase-3 activation concurs with a rapid loss of dendritic spines and deficits in synaptic long-term depression (LTD) in striatal projection neurons forming the indirect pathway. Interestingly, systemic treatment with a caspase inhibitor prevents both the spine pruning and the deficit of indirect pathway LTD without interfering with the ongoing dopaminergic degeneration. Taken together, our data identify transient and non-apoptotic caspase activation as a critical event in the early plastic changes of indirect pathway neurons following dopamine denervation.

Influence of Age and Genetic Background on Ethanol Intake and Behavioral Response Following Ethanol Consumption and During Abstinence in a Model of Alcohol Abuse.

Genetic background and age at first exposure have been identified as critical variables that contribute to individual vulnerability to drug addiction. Evidence shows that genetic factors may account for 40-70% of the variance in liability to addiction. Alcohol consumption by young people, especially in the form of binge-drinking, is becoming an alarming phenomenon predictive of future problems with drinking. Thus, the literature indicates the need to better understand the influence of age and genetic background on the development of alcohol dependence. To this aim, the inbred rat strains Lewis (LEW, addiction prone) and Fischer 344 (F344, addiction resistant) were used as a model of genetic vulnerability to addiction and compared with the outbred strain Sprague-Dawley (SD) in a two-bottle choice paradigm as a model of alcohol abuse. During a 9-week period, adolescent and adult male rats of the three strains were intermittently exposed to ethanol (20%) and water during three 24-h sessions/week. Adult and adolescent SD and LEW rats escalated their alcohol intake over time reaching at stable levels, while F344 rats did not escalate their intake, regardless of age at drinking onset. Among adolescents, only F344 rats consumed a higher total amount of ethanol than adults, although only SD and LEW rats escalated their intake. Adult LEW rats, albeit having a lower ethanol consumption as compared to SD rats but greater than F344, showed a more compulsive intake, consuming higher amounts of ethanol during the first hour of exposure, reaching a higher degree of ethanol preference when start drinking as adolescents. Behavioral analysis during the first hour of ethanol consumption revealed significant strain differences, among which noticeable the lack of sedative effect in the LEW strain, at variance with F344 and SD strains, and highest indices of withdrawal (most notable jumping) in LEW rats during the first hour of abstinence days. The present results underscore the importance of individual genetic background and early onset of alcohol use in the progression toward abuse and development of alcohol addiction.

Sequential or Simultaneous Injection of Preformed Fibrils and AAV Overexpression of Alpha-Synuclein Are Equipotent in Producing Relevant Pathology and Behavioral Deficits.

BACKGROUND: Preclinical rodent models for Parkinson's disease (PD) based on viral human alpha-synuclein (h-αSyn) overexpression recapitulate some of the pathological hallmarks as it presents in humans, such as progressive cell loss and additional synucleinopathy in cortical and subcortical structures. Recent studies have combined viral vector-based overexpression of human wild-type αSyn with the sequential or simultaneous inoculation of preformed fibrils (PFFs) derived from human αSyn. OBJECTIVE: The goal of the study was to investigate whether sequential or combined delivery of the AAV vector and the PFFs are equipotent in inducing stable neurodegeneration and behavioral deficits. METHODS: Here we compare between four experimental paradigms (PFFs only, AAV-h-αSyn only, AAV-h-αSyn with simultaneous PFFs, and AAV-h-αSyn with sequential PFFs) and their respective GFP control groups. RESULTS: We observed reduction of TH expression and loss of neurons in the midbrain in all AAV (h-αSyn or GFP) injected groups, with or without additional PFFs inoculation. The overexpression of either h-αSyn or GFP alone induced motor deficits and dysfunctional dopamine release/reuptake in electrochemical recordings in the ipsilateral striatum. However, we observed a substantial formation of insoluble h-αSyn aggregates and inflammatory response only when h-αSyn and PFFs were combined. Moreover, the presence of h-αSyn induced higher axonal pathology compared to control groups. CONCLUSION: Simultaneous AAV and PFFs injections are equipotent in the presented experimental setup in inducing histopathological and behavioral changes. This model provides new and interesting possibilities for characterizing PD pathology in preclinical models and means to assess future therapeutic interventions.

Tracking Rats in Operant Conditioning Chambers Using a Versatile Homemade Video Camera and DeepLabCut.

Operant conditioning chambers are used to perform a wide range of behavioral tests in the field of neuroscience. The recorded data is typically based on the triggering of lever and nose-poke sensors present inside the chambers. While this provides a detailed view of when and how animals perform certain responses, it cannot be used to evaluate behaviors that do not trigger any sensors. As such, assessing how animals position themselves and move inside the chamber is rarely possible. To obtain this information, researchers generally have to record and analyze videos. Manufacturers of operant conditioning chambers can typically supply their customers with high-quality camera setups. However, these can be very costly and do not necessarily fit chambers from other manufacturers or other behavioral test setups. The current protocol describes how to build an inexpensive and versatile video camera using hobby electronics components. It further describes how to use the image analysis software package DeepLabCut to track the status of a strong light signal, as well as the position of a rat, in videos gathered from an operant conditioning chamber. The former is a great aid when selecting short segments of interest in videos that cover entire test sessions, and the latter enables analysis of parameters that cannot be obtained from the data logs produced by the operant chambers.

Role of genetic background in the effects of adolescent nicotine exposure on mesolimbic dopamine transmission.

Smoking during adolescence may increase the likelihood to develop nicotine dependence and to abuse other drugs such as cocaine. Despite great efforts to understand underlying neurobiological mechanisms of this progression, less attention has been paid to the role of genetic factors. Here, we investigated the influence of both genetic background and age at first nicotine exposure in the long-lasting effects on mesolimbic dopamine transmission including the increased cocaine-rewarding effect. Mid-adolescent and adult rats of inbred strains Lewis (addiction prone) and Fischer 344 (addiction resistant) were administered nicotine (0.4 mg/kg) or vehicle once daily for 5 days. Changes in dopamine transmission were investigated by in vivo microdialysis and electrophysiology after 30 days of withdrawal, whereas changes in cocaine-rewarding effect were assessed via conditioned place preference paradigm. Nicotine pre-exposure differentially changed mesolimbic dopamine transmission depending on strain and age of pre-exposure. A potentiation of dopamine response to nicotine was observed in nucleus accumbens (NAc) core of both strains and age groups, whereas dopamine response in NAc shell was enhanced exclusively in Lewis rats exposed to nicotine during adolescence. A similar response was observed following cocaine challenge at adulthood. Changes in VTA dopamine cell population and activity were observed only in adolescent nicotine-pretreated Lewis rats, which also showed an increased cocaine-rewarding effect at adulthood. These results highlight the influence of genetic background in the long-lasting effects of nicotine exposure and suggest that exposure during adolescence might increase nicotine and cocaine-rewarding properties in genetically vulnerable individuals, thereby facilitating progression toward dependence.

Seeding of protein aggregation causes cognitive impairment in rat model of cortical synucleinopathy.

BACKGROUND: Cortical α-synuclein pathology plays a role in the development of cognitive dysfunction in both Parkinson's disease and dementia with Lewy bodies, although the causative cellular lesions have remained unclear. We aimed to address causal links between α-synuclein-driven pathology in the cerebral cortex and the development of cognitive impairments using new experimental models. METHODS: Neuronal overexpression of human α-synuclein was induced in the rat medial prefrontal cortex using viral vectors. This was combined with inoculations of preformed fibrils of human α-synuclein in some animals. Rats were evaluated with tests probing prefrontal cognitive functions (delayed matching/nonmatching to position and 5-choice serial reaction time task). Patterns of neuropathology were characterized immunohistochemically. RESULTS: Neither α-synuclein overexpression nor the fibril seeds alone yielded any behavioral phenotype. In contrast, combining the 2 approaches produced significant impairments in working memory, attention, and inhibitory control. All animals injected with α-synuclein vectors exhibited high immunoreactivity for human α-synuclein in the medial prefrontal cortex and its primary projection targets. However, only when this overexpression was combined with fibril inoculations did animals exhibit large, proteinase K-resistant and Ser129 -phosphorylated α-synuclein intraneuronal inclusions in the medial prefrontal cortex and its closely interconnected brain regions. The inclusions were associated with distorted dendritic morphologies and partial neuronal loss in the targeted cortical areas. CONCLUSIONS: Cortical overexpression of human α-synuclein is not sufficient to produce cognitive dysfunction, whereas combining this overexpression with fibril seeds yields both cognitive and histopathological phenotypes that are relevant to human Lewy body disease. © 2019 International Parkinson and Movement Disorder Society.

Differential induction of dyskinesia and neuroinflammation by pulsatile versus continuous l-DOPA delivery in the 6-OHDA model of Parkinson's disease.

Neuroinflammation is associated with l-DOPA treatment in Parkinson's disease (PD), suggesting a role in l-DOPA-induced dyskinesia (LID), however it is unclear whether increased inflammation is specifically related to the dyskinetic outcome of l-DOPA treatment. Diversely from oral l-DOPA, continuous intrajejunal l-DOPA infusion is associated with very low dyskinetic outcome in PD patients. We reproduced these regimens of administration in 6-OHDA-lesioned hemiparkinsonian rats, where dyskinetic responses and striatal neuroinflammation induced by chronic pulsatile (DOPAp) or continuous (DOPAc) l-DOPA were compared. Moreover, we investigated the contribution of a peripheral inflammatory challenge with lipopolysaccharide (LPS), to DOPAp-induced dyskinetic and neuroinflammatory responses. Rats 6-OHDA-infused in the medial forebrain bundle received two weeks treatment with DOPAp, DOPAc via subcutaneous osmotic minipumps, or DOPAp followed by DOPAc. l-DOPA plasma levels were measured in all experimental groups. An independent group of rats received one peripheral dose of LPS 24h before DOPAp treatment. Abnormal involuntary movements (AIMs) were evaluated as a rat model of LID. Immunoreactivity (IR) for OX-42, microglial and neuronal TNF-α, iNOS and GFAP was quantified in denervated and contralateral striatum. In addition, serum TNF-α was measured. The 6-OHDA denervation induced a mild microgliosis in the striatum two weeks after neurotoxin infusion, and increased TNF-α IR in microglia. Rats receiving the DOPAp treatment developed AIMs and displayed increased striatal OX-42, microglial TNF-α, iNOS and GFAP. Moreover, TNF-α IR was also increased in a subpopulation of striatal neurons. Conversely, DOPAc did not induce AIMs or inflammatory responses in either drug-naïve animals or rats that were previously dyskinetic when exposed to DOPAp. Serum TNF-α was not altered by any l-DOPA treatment. LPS pre-treatment increased the degree of DOPAp-induced AIMs and striatal IR for OX-42, TNF-α, iNOS and GFAP. Altogether the present findings indicate that in the 6-OHDA model, chronic l-DOPA induces striatal inflammatory responses, which however depend upon the administration regimen and the dyskinetic outcome of drug treatment. The potentiation of dyskinetic responses by LPS suggests a reciprocal causal link between neuroinflammation and LID.

In vivo dopamine agonist properties of rotigotine: Role of D1 and D2 receptors.

Rotigotine acts in vitro as a full agonist of dopamine D1 receptors at concentrations almost superimposable to those at which it acts on D2 receptors. However in vivo evidence of the differences between the agonist activity of rotigotine at D1 receptors from that on the D2 receptors has not been provided yet. In order to test the ability of rotigotine to stimulate dopamine D1 and D2 receptors in vivo, we studied the effect of SCH39166 and eticlopride, selective dopamine D1 and D2/D3 receptor antagonists respectively, on rotigotine-induced contralateral turning behavior in 6-hydroxydopamine lesioned rats. Furthermore, the expression of the immediate-early gene c-fos in the caudate-putamen, was evaluated. As a comparison, we tested the D2/D3 agonist pramipexole. In primed rats, rotigotine (0.035, 0.1 and 0.35mg/kg) induced dose-dependent contralateral turning. Turning induced by 0.1mg/kg of rotigotine was reduced by pretreatment with the D1 antagonist SCH39166 and the D2 antagonist eticlopride. In drug-naive rats, rotigotine was less effective in eliciting turning but SCH39166 still reduced turning induced by rotigotine (0.35mg/kg). Pramipexole induced contralateral turning only in primed rats. SCH39166 potentiated and eticlopride abolished pramipexole-induced turning. Rotigotine induced Fos expression in the caudate-putamen and SCH39166 completely blocked it. Pramipexole failed to induce Fos. These results indicate that rotigotine acts in vivo as an agonist of D1 and D2 receptors while pramipexole is devoid of D1 activity in vivo. Given their differing DA receptor profiles, rotigotine and pramipexole might differ in their spectrum of application to the therapy of Parkinson's disease.

Withania somnifera Dunal (Indian ginseng) impairs acquisition and expression of ethanol-elicited conditioned place preference and conditioned place aversion.

Withania somnifera Dunal (Indian Ginseng) has recently been shown to impair ethanol self-administration. In order to gain further insights on the ability of the Withania somnifera standardised root extract (WSE) to affect the motivational properties of ethanol, this study investigated whether WSE may also affect ethanol (2 g/kg)-elicited conditioned place preference (CPP) and aversion (CPA). To this end male CD-1 mice were conditioned under two distinct schedules: in backward conditioning experiments ethanol was administered before mice were placed in the conditioning apparatus (CPP) while, in forward conditioning experiments, ethanol was administered immediately after removing mice from the apparatus (CPA). Following these schedules, mice developed significant CPP and CPA, respectively. Administration of WSE significantly impaired both the acquisition (50 and 100 mg/kg) and the expression (50 mg/kg) of CPP and CPA without affecting spatial memory (50 mg/kg), as determined by a two-trial memory recognition task. Overall, the study highlights the ability of WSE to interfere with both positive and negative motivational properties of ethanol and suggests that the effects of WSE may target both ethanol's motivational properties and underpinning associative learning mechanisms. In conclusion, these results cast new light on Withania somnifera as an agent potentially useful to counteract distinct aspects of ethanol effects.

Strain dependence of adolescent Cannabis influence on heroin reward and mesolimbic dopamine transmission in adult Lewis and Fischer 344 rats.

Adolescent Cannabis exposure has been hypothesized to act as a gateway to opiate abuse. In order to investigate the role of genetic background in cannabinoid-opiate interactions, we studied the effect of Δ(9) -tetrahydrocannabinol (THC) exposure of adolescent Lewis and Fischer 344 rats on the responsiveness of accumbens shell and core dopamine (DA), as monitored by microdialysis, to THC and heroin at adulthood. Heroin reward and reinstatement by heroin priming were studied by conditioned place preference (CPP) and cognitive and emotional functions by object recognition, Y maze and elevated plus maze paradigms. THC stimulated shell DA in Lewis but not in Fischer 344 rats. Adolescent THC exposure potentiated DA stimulant effects of heroin in the shell and core of Lewis and only in the core of Fischer 344 rats. Control Lewis rats developed stronger CPP to heroin and resistance to extinction compared with Fischer 344 strain. In Lewis rats, THC exposure did not affect heroin CPP but potentiated the effect of heroin priming. In Fischer 344 rats, THC exposure increased heroin CPP and made it resistant to extinction. Lewis rats showed seeking reactions during extinction and hedonic reactions in response to heroin priming. Moreover, adolescent THC exposure affected emotional function only in Lewis rats. These observations suggest that long-term effects of Cannabis exposure on heroin addictive liability and emotionality are dependent on individual genetic background.