Boris Ferger

Characterization of the striatal 6-OHDA model of Parkinson's disease in wild type and α-synuclein-deleted mice

Genetically modified mice models are increasingly used to study the pathophysiology of Parkinsons disease (PD), particularly in conditions where they are subjected to toxins specific for dopaminergic neurons. The most widely used toxin in these paradigms is 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), although it presents a number of drawbacks regarding (i) the kinetics of neurodegeneration, (ii) strain-specificity and (iii) partial lesion recovery. 6-hydroxydopamine (6-OHDA) may be an alternative tool since it leads to a partial damage of DA terminals and to a delayed and progressive loss of nigral DA neurons. It is frequently used in rats and well characterized in this species. In mice, however, this model has not been described in detail to date. The aim of the present study was to characterize the time course of intra-striatal 6-OHDA lesions in mice with regard to i) dopaminergic cell loss, ii) dopamine concentrations in the substantia nigra and the striatum, iii) hydroxylation products in substantia nigra and striatum and iv) behavioural impairment. Furthermore, we used alpha-synuclein-deleted mice, which have been studied extensively in MPTP paradigms, and examined their reactivity to intra-striatal 6-OHDA injections. Intra-striatally injected 6-OHDA leads to a long-lasting dopamine depletion of the nigro-striatal pathway, whereas behavioural parameters partially recovered over a two month period. Its toxicity seems to be influenced by alpha-synuclein, since alpha-synuclein-deleted mice are more resistant against 6-OHDA than their wild type littermates. In summary, we propose that the striatal 6-OHDA model may be a valuable addition and/or alternative in genetically modified mice models used in the study of PD pathophysiology.

Rapid analysis of GABA and glutamate in microdialysis samples using high performance liquid chromatography and tandem mass spectrometry

A liquid chromatography/tandem mass spectrometry (LC-MS/MS) method has been established for the rapid and reliable determination of gamma-aminobutyric acid (GABA) and glutamate in brain microdialysates. The microdialysis samples were analysed using a HILIC (hydrophilic interaction liquid chromatography) column, which is able to retain the polar amino acid neurotransmitters. The mobile phase consisted of a binary gradient elution profile comprising 0.1% formic acid in water and acetonitrile. GABA, glutamate as well as the respective internal standards [D(6)]-GABA and [D(5)]-glutamate were detected by a triple quadrupole mass spectrometer in the positive electrospray ionisation mode within a running time of 3 min. The linearity ranged from 1 nM to 10 microM for GABA and 10 nM to 10 microM for glutamate. The limit of quantitation was found to be 1 nM for GABA and 10nM for glutamate (injection volume 10 microl). The present LC-MS/MS method was compared to the classical method for analysis of GABA and glutamate using high performance liquid chromatography (HPLC) and fluorescence detection (FD). Eventually, the feasibility of the LC-MS/MS method was demonstrated using in vivo microdialysis in rats by monitoring changes of the extracellular concentrations of GABA and glutamate in the globus pallidus following stimulation with potassium.

Antidepressant effects of pramipexole, a dopamine D3/D2 receptor agonist, and 7-OH-DPAT, a dopamine D3 receptor agonist, in olfactory bulbectomized rats.

Treatment with pramipexole, a dopamine D(3)/D(2) receptor agonist, reduces depressive symptoms in patients suffering from Parkinsons disease. To test the putative antidepressant quality of pramipexole, its effects were assessed in one of the most attractive animal models of depression, the olfactory bulbectomized (OBX) rat. Two experiments studied the effects of pramipexole on bulbectomy-induced hyperactivity. In experiment I, pramipexole was tested at 0.3 and 1.0 mg/kg together with the reference dopamine D(3) receptor agonist 7-OH-DPAT (0.1 mg/kg) and the tri-cyclic antidepressant imipramine (10 mg/kg). In experiment II, pramipexole was tested at lower doses: 0.03 and 0.1 mg/kg, with the same reference compounds. All animals were tested in the open field on days one (acute), seven (sub-chronic) and fourteen (chronic) of administration, as well as one week after cessation of treatment. Pramipexole, in a U-shaped dose response, reduced bulbectomy-induced hyperactivity after (sub) chronic but not acute administration (like imipramine and 7-OH-DPAT). The highest dose of pramipexole (1.0 mg/kg) did not reduce OBX hyperactivity during treatment. However, one week after cessation of treatment, all pramipexole (including the 1.0 mg/kg dose), 7-OH-DPAT and imipramine groups showed a reduction in OBX-induced hyperactivity. Pramipexole and 7-OH-DPAT exert an antidepressant profile in the OBX-rat model in normalizing bulbectomy-induced hyperactivity during (sub) chronic treatment. Moreover, treatment with both these compounds induced long-lasting changes in the bulbectomized brain similar to established antidepressants, strongly predicting antidepressant activity in major depression.

ORIGINAL RESEARCH—BASIC SCIENCE: Acute and Repeated Flibanserin Administration in Female Rats Modulates Monoamines Differentially Across Brain Areas: A Microdialysis Study

INTRODUCTIONnHypoactive sexual desire disorder (HSDD) is defined as persistent lack of sexual fantasies or desire marked by distress. With a prevalence of 10% it is the most common form of female sexual dysfunction. Recently, the serotonin-1A (5-HT(1A)) receptor agonist and the serotonin-2A (5-HT(2A)) receptor antagonist flibanserin were shown to be safe and efficacious in premenopausal women suffering from HSDD in phase III clinical trials.nnnAIMnThe current study aims to assess the effect of flibanserin on neurotransmitters serotonin (5-HT), norepinephrine (NE), dopamine (DA), glutamate, and gamma-aminobutyric acid (GABA) in brain areas associated with sexual behavior.nnnMETHODSnFlibanserin was administered to female Wistar rats (280-350 g). Microdialysis probes were stereotactically inserted into the mPFC, NAC, or MPOA, under isoflurane anesthesia. The extracellular levels of neurotransmitters were assessed in freely moving animals, 24 hours after the surgery.nnnMAIN OUTCOME MEASURESnDialysate levels of DA, NE, and serotonin from medial prefrontal cortex (mPFC), nucleus accumbens (NAC), and hypothalamic medial preoptic area (MPOA) from female rats.nnnRESULTSnAcute flibanserin administration decreased 5-HT and increased NE levels in all tested areas. DA was increased in mPFC and MPOA, but not in the NAC. Basal levels of NE in mPFC and NAC and of DA in mPFC were increased upon repeated flibanserin administration, when compared to vehicle-treated animals. The basal levels of 5-HT were not altered by repeated flibanserin administration, but basal DA and NE levels were increased in the mPFC. Glutamate and GABA levels remained unchanged following either repeated or acute flibanserin treatment.nnnCONCLUSIONSnSystemic administration of flibanserin to female rats differentially affects the monoamine systems of the brain. This may be the mechanistic underpinning of flibanserins therapeutic efficacy in HSDD, as sexual behavior is controlled by an intricate interplay between stimulatory (catecholaminergic) and inhibitory (serotonergic) systems.

iTRAQ and multiple reaction monitoring as proteomic tools for biomarker search in cerebrospinal fluid of patients with Parkinson's disease dementia

About 30% of patients with Parkinsons disease (PD) develop Parkinsons disease dementia (PDD) in the course of the disease. Until now, diagnosis is based on clinical and neuropsychological examinations, since so far there is no laboratory marker. In this study we aimed to find a neurochemical marker which would allow a risk assessment for the development of a dementia in PD patients. For this purpose, we adopted a gel-free proteomic approach (iTRAQ-method) to identify biomarker-candidates in the cerebrospinal fluid (CSF) of patients with PD, PDD and non-demented controls (NDC). Validation of these candidates was then carried out by multiple-reaction-monitoring (MRM) optimised for CSF. Using the iTRAQ-approach, we were able to identify 16 differentially regulated proteins. Fourteen out of these 16 proteins could then be followed-up simultaneously in our optimised MRM-measurement protocol. However only Tyrosine-kinase-non-receptor-type 13 and Netrin-G1 differed significantly between PDD and NDC cohorts. In addition, a significant difference was found for Golgin-160 and Apolipoprotein B-100 between PD and NDC. Apart from possible pathophysiological considerations, we propose that Tyrosine-kinase non-receptor-type 13 and Netrin G1 are biomarker candidates for the development of a Parkinsons disease dementia. Furthermore we suggest that iTRAQ and MRM are valuable tools for the discovery of biomarker in cerebrospinal fluid. However further validation studies need to be done with larger patient cohorts and other proteins need to be checked as well.

The α2 adrenoceptor antagonist idazoxan alleviates l-DOPA-induced dyskinesia by reduction of striatal dopamine levels: an in vivo microdialysis study in 6-hydroxydopamine-lesioned rats

l‐DOPA‐induced dyskinesia is characterised by debilitating involuntary movement, which limits quality of life in patients suffering from Parkinson’s disease. Here, we investigate effects of the α2 adrenoceptor antagonist idazoxan on l‐DOPA‐induced dyskinesia as well as on alterations of extracellular l‐DOPA and dopamine (DA) levels in the striatum in dyskinetic rats. Male Wistar rats were unilaterally lesioned with 6‐hydroxydopamine and subsequently treated with l‐DOPA/benserazide to induce stable dyskinetic movements. Administration of idazoxan [(9u2003mg/kg, intraperitoneal (i.p.)] significantly alleviated l‐DOPA‐induced dyskinesia, whereas idazoxan (3u2003mg/kg, i.p.) did not affect dyskinetic behaviour. Bilateral in vivo microdialysis revealed that idazoxan 9u2003mg/kg reduces extracellular peak l‐DOPA levels in the lesioned and intact striatum as well as DA levels in the lesioned striatum. In parallel, the exposure to idazoxan in the striatum was monitored. Furthermore, no idazoxan and l‐DOPA drug–drug interaction was found in plasma, brain tissue and CSF. In conclusion, the decrease of l‐DOPA‐derived extracellular DA levels in the lesioned striatum significantly contributes to the anti‐dyskinetic effect of idazoxan.

An orally bioavailable positive allosteric modulator of the mGlu4 receptor with efficacy in an animal model of motor dysfunction

A high-throughput screening campaign identified 4-((E)-styryl)-pyrimidin-2-ylamine (11) as a positive allosteric modulator of the metabotropic glutamate (mGlu) receptor subtype 4. An evaluation of the structure-activity relationships (SAR) of 11 is described and the efficacy of this compound in a haloperidol-induced catalepsy rat model following oral administration is presented.

Simultaneous LC-MS/MS analysis of the biomarkers cAMP and cGMP in plasma, CSF and brain tissue.

The cyclic nucleotides cyclic adenosine-3,5-monophosphate (cAMP) and cyclic guanosine-3,5-monophosphate (cGMP) are important second messengers. They are useful biomarkers to indicate biological activity of drugs such as phosphodiesterase (PDE) inhibitors which block the degradation of these nucleotides. Here, we established a fast and sensitive method for the simultaneous analysis of cAMP and cGMP by LC-MS/MS with broad applicability. The limit of detection is 50 pM. Linearity is given in a range of 0.5-500 nM for both nucleotides, with a high intra- and inter-assay precision and accuracy and an analysis time of 3.5 min. We validated the suitability of the method by pharmacological modulation of cAMP or cGMP concentrations in mice with the PDE4 inhibitor rolipram and the PDE5 inhibitor zaprinast. Rolipram significantly increased cAMP concentrations in plasma, CSF and brain tissue. Zaprinast increased cGMP concentrations in plasma but not in brain tissue, which is in accordance with its blood brain barrier permeability. In conclusion, the LC-MS/MS method described here could be a valuable analytical tool for investigating pharmacodynamic effects of PDE inhibitors and to monitor disease-related changes of cAMP and cGMP in the periphery as well as in the central nervous system.

L-DOPA-induced dyskinesia in Parkinson's disease: a drug discovery perspective

L-DOPA-induced dyskinesia is a debilitating, treatment-limiting adverse effect that eventually occurs in the majority of Parkinsons disease patients. This review describes the strategy of biological testing, focusing on in vivo proof-of-concept animal models. We distinguish between symptomatic efficacy markers in predictive preclinical in vivo models and confounding factors that eventually produce false positive results. Clinical studies and predicted efficacy from preclinical studies are recapitulated and insights into the attempt to bridge preclinical and clinical studies are given. Furthermore, the mode of action that is to be involved in emerging therapy against L-DOPA-induced dyskinesia is reviewed.

The selective α1 adrenoceptor antagonist HEAT reduces L‐DOPA‐induced dyskinesia in a rat model of Parkinson's disease

In Parkinsons disease (PD), the long term use of L‐DOPA results in major adverse effects including dyskinesia or abnormal involuntary movements. The present study focuses on the effect of the selective α1 adrenoceptor antagonist HEAT (2‐[[β‐(‐4‐hydroxyphenyl)ethyl]aminomethyl]‐1‐tetralone) in the 6‐hydroxydopamine rat model of L‐DOPA‐induced dyskinesia. We demonstrate that the selective α1 adrenoceptor antagonist HEAT (1 and 2 mg kg−1), the α2 adrenoceptor antagonist idazoxan (9 mg kg−1), and the nonselective β1/β2 adrenoceptor antagonist propranolol (20 mg kg−1) alleviate dyskinetic movements induced by L‐DOPA. Furthermore, the adrenoceptor antagonists at the doses used did not influence exploratory behavior in the open field system indicating that the antidyskinetic effect is not due to a reduction in general motor activity. Intrastriatal administration of the selective α1 adrenoceptor agonist cirazoline via reverse in vivo microdialysis did not induce dyskinesia. Additionally, we measured plasma, brain, and CSF levels of HEAT. HEAT is a CNS active compound with a brain/plasma and CSF/plasma ratio of 4.29 and 0.15, respectively, which is appropriate for the investigation of α1‐mediated mechanisms in CNS disorders. In conclusion, these results demonstrated for the first time that a α1 adrenoceptor antagonist reduced L‐DOPA‐induced dyskinesia in a rat model. Further studies assessing the risk benefit in comparison to existing therapies are needed before considering α1 adrenoceptor antagonists as a target for the development of new antidyskinetic compounds. Synapse 64:117–126, 2010.