In synucleinopathies, including Parkinson's disease, partially ubiquitylated alpha-synuclein species phosphorylated on serine 129 (P(S129)-alpha-synuclein) accumulate abnormally. Parkin, an ubiquitin-protein ligase that is dysfunctional in autosomal recessive parkinsonism, protects against alpha-synuclein-mediated toxicity in various models.We analyzed the effects of Parkin deficiency in a mouse model of synucleinopathy to explore the possibility that Parkin and alpha-synuclein act in the same biochemical pathway. Whether or not Parkin was present, these mice developed an age-dependent neurodegenerative disorder preceded by a progressive decline in performance in tasks predictive of sensorimotor dysfunction. The symptoms were accompanied by the deposition of P(S129)-alpha-synuclein but not P(S87)-alpha-synuclein in neuronal cell bodies and neuritic processes throughout the brainstem and the spinal cord; activation of caspase 9 was observed in 5% of the P(S129)-alpha-synuclein-positive neurons. As in Lewy bodies, ubiquitin-immunoreactivity, albeit less abundant, was invariably co-localized with P(S129)-alpha-synuclein. During late disease stages, the disease-specific neuropathological features revealed by ubiquitin- and P(S129)-alpha-synuclein-specific antibodies were similar in mice with or without Parkin. However, the proportion of P(S129)-alpha-synuclein-immunoreactive neuronal cell bodies and neurites co-stained for ubiquitin was lower in the absence than in the presence of Parkin, suggesting less advanced synucleinopathy. Moreover, sensorimotor impairment and manifestation of the neurodegenerative phenotype due to overproduction of human alpha-synuclein were significantly delayed in Parkin-deficient mice.These findings raise the possibility that effective compensatory mechanisms modulate the phenotypic expression of disease in parkin-related parkinsonism.

Two principal component analyses of anxiety were undertaken investigating two strains of mice (ABP/Le and C57BL/6ByJ) in two different experiments, both classical tests for assessing anxiety in rodents. The elevated plus-maze and staircase were used for the first experiment, and a free exploratory paradigm and light-dark discrimination were used for the second. The components in the analyses produced definitions of four fundamental behavior patterns: novelty-induced anxiety, general activity, exploratory behavior, and decision making. We also noted that the anxious phenotype was determined by both strain and experimental procedure. The relationship between behavior patterns and the use of specific tests plus links with the genetic background are discussed.

Cerebral palsy remains a public health priority. Recognition of factors of susceptibility to perinatal brain lesions is key for the prevention of cerebral palsy. In most cases, the pathophysiology of these lesions is thought to involve prior exposure to predisposing factors that make the developing brain more vulnerable to perinatal events. The present study tested the hypothesis that exposure to chronic minimal stress throughout gestation would sensitize the offspring to neonatal excitotoxic brain lesions, which mimic lesions observed in cerebral palsy. Pregnant mice were exposed to chronic, ultramild stress, applied throughout gestation. Neonatal brain lesions were induced by intracerebral injection of glutamate analogs. Excitotoxic lesions were significantly worsened in pups exposed to gestational stress. Stress induced a significant rise of circulating corticosterone levels both in pregnant mothers and in newborn pups. The deleterious effects of stress on excitotoxicity were totally suppressed in mice with reduced levels of glucocorticoid receptors. Stress induced a significant increase of neopallial NMDA binding sites in the offspring. At adulthood, animals exposed to stress and neonatal excitotoxic challenge showed a significant impairment in the Morris water maze test when compared with animals exposed to the excitotoxic challenge but not the gestational stress. These findings suggest that stress during gestation, which may mimic low-level stress in human pregnancy, could be a novel risk factor for cerebral palsy.

Methyl-β-carboline-3-carboxylate (or β-CCM) is a benzodiazepine receptor ligand with inverse agonist properties. Two strains of mice were selected, one for sensitivity (BS) and one for resistance (BR) to a convulsive dose of β-CCM. These two strains were then shown to differ in several biochemical, pharmacological and behavioral characteristics; specifically BS mice were less anxious than BR mice. The present work provides evidence of differences in the learning abilities of the two strains. Three different learning tasks were used: spatial delayed discrimination on the 4-hole board, a learned choice between a lit and a dark compartment in a T-maze, and place-learning in an 8-arm radial maze. In all three tasks, BS mice had consistently better performance levels than BR mice.

In this study, we compared the effects of chronic ultramild stress (CUMS) exposure on decision-making behavior in a validated test, and on the stress responsive serotoninergic and dopaminergic systems in four age groups of B6D2F1 female mice (5-6, 11-12, 17-18 and 23-24 months old). The levels of serotonin (5-HT) and its metabolite 5-hydroxyindolacetic acid (5-HIAA) were measured in the brain stem, the cortex, the striatum and the hippocampus; the levels of dopamine (DA) and its metabolite dihydroxyphenylacetic acid (DOPAC) were measured in the brain stem and the striatum. The influence of a long-term treatment with the extract of Ginkgo biloba leaves EGb 761 (Tanakan) on age- and stress-related changes was also investigated in the two oldest age groups. In the absence of drug treatment, middle-age mice were the least efficient in making a decision, and senescent mice exhibited reduced levels of both 5-HT and DA and their metabolites in all the brain areas examined. CUMS facilitated evaluation and choice behavior in all age groups, but induced age-dependent reduction of hesitation, acceleration of information processing and reduction in serotoninergic neurotransmission. In senescent mice, EGb 761 reduced the impact of stress on evaluation and hesitation, and restored some stress-related neurobehavioral changes that were only seen in young mice, i.e. acceleration of information processing and reduction in brain 5-HIAA levels. Restoration of some plasticity of the serotoninergic systems might contribute to the stress alleviating influence of EGb 761 in old age.

The 32-kDa dopamine- and adenosine 3′,5′-monophosphate-regulated phosphoprotein (DARPP-32) is recognized to be critical to the pathogenesis of drug addiction. Opiates via the μ-receptor act on the dopaminergic system in the brain and modulates the expression of DARPP-32 phosphoprotein which is an important mediator of the activity of the extracellular signal-regulated kinase (ERK) signaling cascades, the activation of which represents an exciting nexus for drug-induced changes in neural long-term synaptic plasticity. Silencing of DARPP-32 using an siRNA against DARPP-32 may provide a novel gene therapy strategy to overcome drug addiction. In this study, we investigated the effect of the opiate (heroin) on D1 receptor (D1R) and DARPP-32 expression and additionally, evaluated the effects of DARPP-32-siRNA gene silencing on protein phosphatase-1 (PP-1), ERK, and cAMP response element-binding (CREB) gene expression in primary normal human astrocytes (NHA) cells in vitro. Our results indicate that heroin significantly upregulated both D1R and DARPP-32 gene expression, and that DARPP-32 silencing in the NHA cells resulted in the significant modulation of the activity of downstream effector molecules such as PP-1, ERK, and CREB which are known to play an important role in opiate abuse-induced changes in long-term neural plasticity. These findings have the potential to facilitate the development of DARPP32 siRNA-based therapeutics against drug addiction.

Mice (GR-i) bearing a transgene encoding a glucocorticoid receptor (GR) antisense RNA under the control of a neuron-specific neurofilament promoter were used to investigate the effects of a 4 week chronic mild stress (CMS) on the hypothalamo–pituitary–adrenocortical (HPA) axis and the serotoninergic system in a transgenic model of vulnerability to affective disorders. GR-i mice showed a decrease in both GR-specific binding (hippocampus and cerebral cortex) and GR mRNA levels [hippocampus, cerebral cortex, and dorsal raphe nucleus (DRN)] as well as a deficit in HPA axis feedback control (dexamethasone test) compared with paired wild-type (WT) mice. In the latter animals, CMS exposure caused a significant decrease in both GR mRNA levels and the density of cytosolic GR binding sites in the hippocampus, whereas, in the DRN, GR mRNA levels tended to increase. In contrast, in stressed GR-i mice, both GR mRNA levels and the density of GR binding sites were significantly increased in the hippocampus, cerebral cortex, and DRN. Electrophysiological recordings in brainstem slices and [γ- 35 S]GTP-S binding measurements to assess 5-HT 1A receptor functioning showed that CMS exposure produced a desensitization of DRN 5-HT 1A autoreceptors in WT, but not in GR-i, mice. In addition, CMS was found to facilitate choice behavior of WT, but not GR-i, mice in a decision-making task derived from an alternation paradigm. These results demonstrate that impaired GR functioning affects normal adaptive responses of the HPA axis and 5-HT system to CMS and alters stress-related consequences on decision-making behaviors.

Behavioral analyses of mice intoxicated by the parkinsonian toxin 1-methyl-4-phenyl-1,2,3,6,-tetrahydropyridine (MPTP) have generated conflicting results. We therefore analyzed the relationship between behavioral changes, loss of monoamine levels, and loss of dopaminergic cell bodies in groups of mice intoxicated with acute or subchronic MPTP protocols. Despite a higher degree of neuronal loss in the mice intoxicated using subchronic protocols, dopamine loss was severe and homogeneous in the striatum in all groups. Dopamine levels were less severely reduced in the frontal cortex in the three groups of MPTP-intoxicated mice. Norepinephrine and serotonin levels in the striatum were decreased only in the mice intoxicated with the acute protocol. The most surprising result was that the mice intoxicated with the subchronic protocols were more active than the saline-treated mice. As reported in rats with dopamine depletion in the prefrontal cortex, the hyperactivity observed in our mice could be due to the reduced dopamine levels detected in this structure.

Mutations of the parkin gene are the most frequent cause of early onset autosomal recessive parkinsonism (EO-AR). Here we show that inactivation of the parkin gene in mice results in motor and cognitive deficits, inhibition of amphetamine-induced dopamine release and inhibition of glutamate neurotransmission. The levels of dopamine are increased in the limbic brain areas of parkin mutant mice and there is a shift towards increased metabolism of dopamine by MAO. Although there was no evidence for a reduction of nigrostriatal dopamine neurons in the parkin mutant mice, the level of dopamine transporter protein was reduced in these animals, suggesting a decreased density of dopamine terminals, or adaptative changes in the nigrostriatal dopamine system. GSH levels were increased in the striatum and fetal mesencephalic neurons from parkin mutant mice, suggesting that a compensatory mechanism may protect dopamine neurons from neuronal death. These parkin mutant mice provide a valuable tool to better understand the preclinical deficits observed in patients with PD and to characterize the mechanisms leading to the degeneration of dopamine neurons that could provide new strategies for neuroprotection.

Anxiety disorders and balance disorders share common clinical features related to perception such as spatial disorientation or dizziness. The search for the mechanism underlying this core of symptoms led us to investigate impairments in multisensory integration. In mice, the 'rotating beam test' allows analysis of changes in balance control and posture in response to a multisensory challenge. We used the BALB/c and C57BL/6 inbred strains of mice, known for their contrasted anxiety-related behavior. The level of anxiety was also manipulated using anxiolytic and anxiogenic pharmacological compounds. Despite equal sensori-motor abilities, anxious mice were more prone to fall off the rotating beam and showed more imbalance than non-anxious mice. Striking inter-strain differences in posture were also observed. Diazepam and β-CCM reversed these strain-specific responses in opposite directions. We demonstrated that balance and postural strategies developed in response to a multisensory challenge vary as a function of the level of anxiety in mice.

We studied the capacity of adrenal medullary transplant to restore the deficits of GABAergic and dopaminergic neurons in mice injected with quinolinic acid (QA), using an open field test as well as pharmacological and immunohistochemical techniques. We analysed behavioural traits-total locomotor activity, peripheral and central activities, grooming, leaning and rearing in the QA-lesioned mice and mice that had undergone adrenal medulla (AM) transplantation. We found that the adrenal transplant recovered a loss of GABAergic neurons. It reduced QA-induced hyperactivity in locomotion and improved emotional indices. In addition, immunohistochemical studies of catecholaminergic markers-tyrosine hydroxylase (TH), dopamine (DA) and neuronal vesicular monoamine transporter type 2- and a single post-trial injection of tetrabenazine (TBZ; 5 mg/kg) indicated that catecholamines-synthesising chromaffin cells in the AM grafts were also involved in the beneficial effects. A likely interpretation of this behavioural pattern of results is that adrenal medullary transplants set into play an interaction between GABAergic and DAergic factors. Our results may contribute to the clarification of the beneficial effects of AM transplants in striatal function.

The loss of dopaminergic neurons in Parkinson's disease is associated with a glial reaction and the overproduction of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α). TNF-α acts via two different receptors, TNFR1 and TNFR2, and is believed to have both a neuroprotective and a deleterious role for neurons. In order to analyze the putative role of TNF-α in parkinsonism, we compared the effect of the parkinsonian drug 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice lacking TNFR1, TNFR2, or both receptors and in wild-type littermates. We show that MPTP does not affect spontaneous activity or anxiety in any of the groups and that it reduces motor activity on a rotarod in double knock out mice but not in mice lacking only one receptor. Postmortem analysis revealed no differences in the number of nigral dopaminergic neurons whatever the group. In contrast, striatal dopamine level was slightly decreased in double knock-out mice and more reduced by MPTP in this group than in the other groups of mice. In addition, dopamine turnover was significantly more increased in double knock out mice after MPTP injection. These data suggest that TNF-α does not participate in the death of dopaminergic neurons in parkinsonism but that it slightly alters dopamine metabolism or the survival of dopaminergic terminals by a mechanism involving both receptors.

Immunohistochemical and behavioral techniques were used to study the effects of adrenal medulla grafts, implanted in striatum after bilateral kainic acid (KA) lesions of this structure, on the open field behavior of mice. KA-induced behavioral changes in leaning, grooming and locomotor activity of the open field test were significantly improved after grafting of the adrenal medulla, and in some respects, fully restored. Immunohistochemical identification showed that grafts contained neuron-like cells with a tyrosine hydroxylase (TH), phenylethanolamine N-methyltransferase, gamma-aminobutyric acid (GABA), choline acetyltransferase (ChAT), and enkephalin-like immunostainings. A likely interpretation of this complex pattern of results is that adrenal medullary grafts may restore the deficits of GABAergic neurons which in turn reverse the abnormalities in emotionality and locomotion. Neurobiologically, these behavioral improvements probably involve GABAergic and catecholaminergic factors of adrenal medulla grafts, although other neuroactive substances, such as acetylcholine and enkephalins, cannot be excluded.

It is well recognized that Ginkgo biloba extract (EGb 761) exert beneficial effects against various age-related changes and is able to reduce the negative influence of stress. In view of the age-dependent increase in the activity of the B form of monoamine oxidase (MAO-B) and in view of the anti-stress action of EGb 761 hypothetically attributed to an inhibition of monoamine oxidase by this substance, we investigated the effects of long-term treatment with EGb 761 upon in vivo cerebral MAO-A and -B activities of stressed and unstressed 17- and 18-month-old mice. The stress was a 'chronic mild stress' regimen whose behavioral impact is known to be reduced by EGb 761. The results showed that: (1) EGb761 induced reductions in MAO activity in 18-month-old, but not in 17-month-old mice; the older animals having higher basal MAO activity; (2) in unstressed mice, EGb 761 appeared to reduce the age-induced increase in cerebral MAO activity; (3) MAO-A and -B activities of stressed and treated 18-month-old mice did not differ significantly from the levels observed in unstressed and untreated 17-month-old mice. These results may shed light on the anti-stress effects of Ginkgo biloba extract.

OBJECTIVE:To test the influence of a chronic ultra mild stress (CUMS) procedure, based solely on socio-environmental stressors, on cognitive-behavioural function in mice.DESIGN:Behavioural study.PARTICIPANTS:B6D2F1 mice.INTERVENTIONS:Mice were exposed to various stressors and then tested using a decision-making task.RESULTS:We observed that stress facilitated "choice" behaviour, with an absence of "no choice" behaviour. Stress also facilitated a more rapid capacity to process information, a decrease in the level of evaluation of the choice situation and less hesitation. These stress-related consequences on decision making may be attributed to a higher level of distractability in the stressed mice.CONCLUSIONS:The CUMS model may be useful for the study of stress-related disorders by proposing a new method for assessing gene-environment interactions in cognitive-affective behaviours.

BACKGROUND:Stress administered to pregnant rodents has been shown to lead to biological and behavioral alterations in both mother and pups. Most of these stress procedures use noxious stressors. Chronicity is obtained by simple repetition of one or two stressors and may be more representative of moderate daily stress experienced during normal life than stress during severe life events. The effects of this procedure were assessed by observing maternal pup-care behavior and testing maternal aggression.METHODS:The subjects included eight controls and eight stressed B6D2F1 females. Chronic ultramild stress was applied from mating to postpartum day (PD) 0. Pup-care behavior was observed on PD 1. Maternal aggression against a male intruder was tested on PD 8, which corresponds to the peak in the display of this behavior.RESULTS:Prenatal stress did not affect basic pup-care behavior, but dramatically impaired defense behavior designed to protect the pups from an external attacker.CONCLUSIONS:The results suggest that gestating females subjected to chronic ultramild stress suffer from a long-lasting decline in recognition of external distress cues either from a resident intruder and/or their own litter. It is assumed these effects are due to the chronicity of the stress rather than its severity.