Differential changes in hippocampal CaMKII and GluA1 activity after memory training involving different levels of adaptive forgetting.

Phosphorylation of CaMKII and AMPA receptor GluA1 subunit has been shown to play a major role in hippocampal-dependent long-term/reference memory (RM) and in the expression of long-term synaptic potentiation (LTP). In contrast, it has been proposed that dephosphorylation of these proteins could be involved in the opposite phenomenon of hippocampal long-term synaptic depression (LTD) and in adaptive forgetting. Adaptive forgetting allows interfering old memories to be forgotten to give new ones the opportunity to be stored in memory, and in particular in short-term/working memory (WM) that was shown to be very sensitive to proactive interference. To determine the role of CaMKII and GluA1 in adaptive forgetting, we adopted a comparative approach to assess the relative quantity and phosphorylation state of these proteins in the brain of rats trained in one of three radial maze paradigms: a RM task, a WM task involving a high level of adaptive forgetting, or a WM involving a low level of adaptive forgetting. Surprisingly, Western blot analyses revealed that training in a WM task involving a high level of adaptive forgetting specifically increased the expression of AMPA receptor GluA1 subunit and the activity of CaMKII in the dentate gyrus. These results highlight that WM with proactive interference involves mechanisms of synaptic plasticity selectively in the dentate gyrus.

REM Sleep-Dependent Bidirectional Regulation of Hippocampal-Based Emotional Memory and LTP.

Prolonged rapid-eye-movement (REM) sleep deprivation has long been used to study the role of REM sleep in learning and memory processes. However, this method potentially induces stress and fatigue that may directly affect cognitive functions. Here, by using a short-term and nonstressful REM sleep deprivation (RSD) method we assessed in rats the bidirectional influence of reduced and increased REM sleep amount on hippocampal-dependent emotional memory and plasticity. Our results indicate that 4 h RSD impaired consolidation of contextual fear conditioning (CFC) and induction of long-term potentiation (LTP), while decreasing density of Egr1/Zif268-expressing neurons in the CA1 region of the dorsal hippocampus. LTP and Egr1 expression were not affected in ventral CA1. Conversely, an increase in REM sleep restores and further facilitates CFC consolidation and LTP induction, and also increases Egr1 expression in dorsal CA1. Moreover, CFC consolidation, Egr1 neuron density, and LTP amplitude in dorsal CA1 show a positive correlation with REM sleep amount. Altogether, these results indicate that mild changes in REM sleep amount bidirectionally affect memory and synaptic plasticity mechanisms occurring in the CA1 area of the dorsal hippocampus.

COB231 targets amyloid plaques in post-mortem human brain tissue and in an Alzheimer mouse model.

Previous works have shown the interest of naturally fluorescent proflavine derivatives to label Abeta deposits in vitro. This study aimed to further characterize the properties of the proflavine 3-acetylamino-6-[3-(propargylamino)propanoyl]aminoacridine (COB231) derivative as a probe. This compound was therefore evaluated on human post-mortem and mice brain slices and in vivo in 18-month-old triple transgenic mice APPswe, PS1M146V and tauP301L (3xTgAD) mice presenting the main characteristics of Alzheimer's disease (AD). COB231 labelled amyloid plaques on brain slices of AD patients, and 3xTgAD mice at 10 and 0.1 µM respectively. However, no labelling of the neurofibrillary tangle-rich areas was observed either at high concentration or in the brain of fronto-temporal dementia patients. The specificity of this mapping was attested in mice using Thioflavin S and IMPY as positive controls of amyloid deposits. After intravenous injection of COB231 in old 3xTgAD mice, fluorescent amyloid plaques were detected in the cortex and hippocampus, demonstrating COB231 blood­brain barrier permeability. We also controlled the cellular localization of COB231 on primary neuronal cultures and showed that COB231 accumulates into the cytoplasm and not into the nucleus. Finally, using a viability assay, we only detected a slight cytotoxic effect of COB231 (< 10%) for the highest concentration (100 µM).

Mapping and kinetics of microglia/neuron cell-to-cell contacts in the 6-OHDA murine model of Parkinson's disease.

As neuroinflammatory processes are involved in the pathogenesis of Parkinson's disease (PD), we provide several key data describing the time-course of microglial accumulation in relation with behavioral alterations and neurodegeneration in a murine model of PD induced by intrastriatal injection of 6-hydroxydopamine (6-OHDA). Our study argues for a major role of microglia which accumulation is somehow early and transient in spite of the neuronal loss progression. Moreover, we observed less 6-OHDA-induced neurodegeneration associated with less inflammatory reaction in DAP-12 Knock-In mice. The direct cell-to-cell contacts that may support physical interactions between microglia and altered dopaminergic neurons are ill-defined, while it is currently hypothesized that microglia support an immune-mediated amplification of neurodegeneration by establishing a molecular cross talk with neurons. Indeed, we sought to map microglia/neuron appositions in substantia nigra (SN) of 6-OHDA injected C57Bl/6 mice and CX3CR1/(GFP/+) mice. Confocal immunofluorescence analyses followed by 3D reconstitutions reveal close appositions between the soma of TH+ neurons and microglial cell bodies and ramifications. Interestingly, some microglial ramifications penetrated TH(+) somas and about 40% of GFP(+) microglial cells in the injured SN harbored TH(+) intracytoplasmic inclusions. These results suggest a direct cross talk between neurons and microglia that may exert a microphagocytic activity toward TH+ neurons. Altogether, these results obtained in a murine PD model may participate in the understanding of microglial cells' function in neurodegenerative diseases.

ER stress inhibits neuronal death by promoting autophagy.

Endoplasmic reticulum (ER) stress has been implicated in neurodegenerative diseases but its relationship and role in disease progression remain unclear. Using genetic and pharmacological approaches, we showed that mild ER stress ("preconditioning") is neuroprotective in Drosophila and mouse models of Parkinson disease. In addition, we found that the combination of mild ER stress and apoptotic signals triggers an autophagic response both in vivo and in vitro. We showed that when autophagy is impaired, ER-mediated protection is lost. We further demonstrated that autophagy inhibits caspase activation and apoptosis. Based on our findings, we conclude that autophagy is required for the neuroprotection mediated by mild ER stress, and therefore ER preconditioning has potential therapeutic value for the treatment of neurodegenerative diseases.

The collapsin response mediator protein 5 onconeural protein is expressed in Schwann cells under axonal signals and regulates axon-Schwann cell interactions.

Collapsin response mediator protein 5 (CRMP5) is one of the rare peripheral nerve antigens that is a target of autoantibodies in a paraneoplastic peripheral neuropathy. The pattern of axonal and myelin alterations suggests that CRMP5 is involved in axon-Schwann cell interaction. We examined CRMP5 expression and function in primary cultures of Schwann cells and neurons and at various developmental and regenerating stages of rat sciatic nerve and in CRMP5-deficient mice in vivo. Collapsin response mediator protein 5 was strongly expressed during postnatal development and regeneration and decreased with myelination. It was mainly expressed by immature Schwann cells and persisted in Remak cells in the adult; however, a subpopulation of Schwann cells that were induced to myelinate also expressed CRMP5. We identified 2 axonal molecular cues regulating CRMP5 expression: human neuregulin type 1, which induces CRMP5 expression in immature and premyelinating Schwann cells, and cyclic adenosine monophosphate, which inhibits CRMP5 expression when Schwann cells begin myelination. Collapsin response mediator protein 5-deficient mice showed abnormal Schwann process extension resulting in abnormal cell-axon segregation, indicating that CRMP5 is involved in the morphologic adaptation of Schwann cells to surround axons. These results demonstrate the importance of CRMP5 in axon-Schwann cell cooperation during development and regeneration.

Quantification of iron-labeled cells with positive contrast in mouse brains.

PURPOSE: To quantify small amounts of iron-labeled cells in mouse brains with magnetic resonance imaging (MRI). PROCEDURES: Iron-labeled cells (from 500 to 7,500) were stereotaxically transplanted into the brain of living mice that were subsequently imaged with MRI at 4.7 T. We compared four quantitative methods: (1) T2 relaxometry, (2) T2* relaxometry, (3) the volume of the cloverleaf hypointense artifact generated on T2*-weighted images, and (4) the volume of the cloverleaf hyperintense artifact generated on positive contrast images. RESULTS: The methods based on relaxometry, whether T2 or T2*, did not correlate with the number of injected cells. By contrast, those based on measurement of cloverleaf artifact volume, whether using negative or positive enhancement, showed a significant linear relationship for the given range of cells (R [0.92-0.95], p < 0.05). CONCLUSIONS: T2* artifact volume imaging (negative or positive) appears promising for the quantification of magnetically labeled cells following focal injection in the brain.

Oligodendrocytes are damaged by neuromyelitis optica immunoglobulin G via astrocyte injury.

Devic's neuromyelitis optica is an inflammatory demyelinating disorder normally restricted to the optic nerves and spinal cord. Since the identification of a specific autoantibody directed against aquaporin 4, neuromyelitis optica-immunoglobulin G/aquaporin 4 antibody, neuromyelitis optica has been considered an entity distinct from multiple sclerosis. Recent findings indicate that the neuromyelitis optica-immunoglobulin G/aquaporin 4 antibody has a pathogenic role through complement-dependent astrocyte toxicity. However, the link with demyelination remains elusive. Autoantibodies can act as receptor agonists/antagonists or alter antigen density in their target cells. We hypothesized that the neuromyelitis optica-immunoglobulin G/aquaporin 4 antibody impairs astrocytic function and secondarily leads to demyelination. Rat astrocytes and oligodendrocytes from primary cultures and rat optic nerves were exposed long-term (24 h) to immunoglobulin G in the absence of complement. Immunoglobulin G was purified from the serum of patients with neuromyelitis optica who were either neuromyelitis optica-immunoglobulin G/aquaporin 4 antibody positive or negative, as well as from healthy controls. Flow cytometry analysis showed a reduction of membrane aquaporin 4 and glutamate transporter type 1 on astrocytes following contact with immunoglobulin G purified from neuromyelitis optica-immunoglobulin G/aquaporin 4 antibody positive serum only. The activity of glutamine synthetase, an astrocyte enzyme converting glutamate into glutamine, decreased in parallel, indicating astrocyte dysfunction. Treatment also reduced oligodendrocytic cell processes and approximately 30% oligodendrocytes died. This deleterious effect was confirmed ex vivo; exposed optic nerves showed reduction of myelin basic protein. Immunoglobulin G from neuromyelitis optica-immunoglobulin G/aquaporin 4 antibody seronegative patients and from healthy controls had no similar effect. Neuromyelitis optica-immunoglobulin G/aquaporin 4 antibody did not directly injure oligodendrocytes cultured without astrocytes. A toxic bystander effect of astrocytes damaged by neuromyelitis optica-immunoglobulin G/aquaporin 4 antibody on oligodendrocytes was identified. Progressive accumulation of glutamate in the culture medium of neuromyelitis optica-immunoglobulin G/aquaporin 4-antibody-treated glial cells supported the hypothesis of a glutamate-mediated excitotoxic death of oligodendrocytes in our models. Moreover, co-treatment of glial cultures with neuromyelitis optica-immunoglobulin G/aquaporin 4 antibody and d+2-amino-5-phosphonopentanoic acid, a competitive antagonist at the N-methyl-d-aspartate/glutamate receptor, partially protected oligodendrocytes. Co-immunolabelling of oligodendrocyte markers and neuromyelitis optica-immunoglobulin G/aquaporin 4 antibody showed that astrocytic positive processes were in close contact with oligodendrocytes and myelin in rat optic nerves and spinal cord, but far less so in other parts of the central nervous system. This suggests a bystander effect of neuromyelitis optica-immunoglobulin G-damaged astrocytes on oligodendrocytes in the nervous tissues affected by neuromyelitis optica. In conclusion, in these cell culture models we found a direct, complement-independent effect of neuromyelitis optica-immunoglobulin G/aquaporin 4 antibody on astrocytes, with secondary damage to oligodendrocytes possibly resulting from glutamate-mediated excitotoxicity. These mechanisms could add to the complement-induced damage, particularly the demyelination, seen in vivo.

Altered expression of CRMPs in the brain of bovine spongiform encephalopathy-infected mice during disease progression.

While recent studies suggest that synaptic alterations are first events in the mechanisms of prion-mediated neurodegeneration, little is known on the identity of the neuronal plasticity-related genes potentially concerned. Here the expression of 4 Collapsin Response Mediator Proteins (CRMPs), a family of signal transduction proteins involved in brain development and altered in Alzheimer's disease, was studied in the brain of C57Bl/6 mice infected with the BSE strain of prion agent, using RT-PCR and Western-blot methods. At the terminal stage of the disease, gene expression of each CRMP had decreased, while at the mid-stage of the disease only CRMP4 (mRNA and protein) expression had increased, concomitant to the start of PrP(Sc) accumulation in the brainstem. Altogether our findings picked out originally CRMPs, and especially CRMP4, as potential contributors to prion pathogenesis.

Paradoxical (REM) sleep deprivation causes a large and rapidly reversible decrease in long-term potentiation, synaptic transmission, glutamate receptor protein levels, and ERK/MAPK activation in the dorsal hippocampus.

STUDY OBJECTIVES: It has been shown that wake (W) and slow wave sleep (SWS) modulate synaptic transmission in neocortical projections. However the impact of paradoxical sleep (PS) quantities on synaptic transmission remains unknown. We examined whether PS modulated the excitatory transmission and expression of glutamate receptor subtypes and phosphorylated extracellular signal-regulated kinases (p-ERK1/2). DESIGN: PS deprivation (PSD) was carried out with the multiple platforms method on adult male Sprague-Dawley rats. LTP, late-LTP, and synaptic transmission were studied in the dorsal and ventral hippocampus of controls, 75-h PSD and 150-min PS rebound (PSR). GluR1 and NR1 protein and mRNA expression were evaluated by western blot and real-time PCR. p-ERK1/2 level was quantified by western blot and immunohistochemistry. MEASUREMENT AND RESULTS: PSD decreased synaptic transmission and LTP selectively in dorsal CA1 and PSR rescued these deficits. PSD-induced synaptic modifications in CA1 were associated with a decrease in GluR1, NR1, and p-ERK1/2 levels in dorsal CA1 without change in GluR1 and NR1 mRNA expression. Regression analysis shows that LTP is positively correlated with both PS quantities and SWS episodes duration, whereas synaptic transmission and late-LTP are positively correlated with PS quantities and negatively correlated with SWS quantities. CONCLUSIONS: These findings unveil previously unrecognized roles of PSD on synaptic transmission and LTP in the dorsal, but not in the ventral, hippocampus. The fact that the decrease in protein expression of GluR1 and NR1 was not associated with a change in mRNA expression of these receptors suggests that a sleep-induced modulation of translational mechanisms occurs in dorsal CA1.

Cerebrospinal fluid dendritic cells infiltrate the brain parenchyma and target the cervical lymph nodes under neuroinflammatory conditions.

BACKGROUND: In many neuroinflammatory diseases, dendritic cells (DCs) accumulate in several compartments of the central nervous system (CNS), including the cerebrospinal fluid (CSF). Myeloid DCs invading the inflamed CNS are thus thought to play a major role in the initiation and perpetuation of CNS-targeted autoimmune responses. We previously reported that, in normal rats, DCs injected intra-CSF migrated outside the CNS and reached the B-cell zone of cervical lymph nodes. However, there is yet no information on the migratory behavior of CSF-circulating DCs under neuroinflammatory conditions. METHODOLOGY/PRINCIPAL FINDINGS: To address this issue, we performed in vivo transfer experiments in rats suffering from experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. EAE or control rats were injected intra-CSF with bone marrow-derived myeloid DCs labeled with the fluorescent marker carboxyfluorescein diacetate succinimidyl ester (CFSE). In parallel experiments, fluorescent microspheres were injected intra-CSF to EAE rats in order to track endogenous antigen-presenting cells (APCs). Animals were then sacrificed on day 1 or 8 post-injection and their brain and peripheral lymph nodes were assessed for the presence of microspheres(+) APCs or CFSE(+) DCs by immunohistology and/or FACS analysis. Data showed that in EAE rats, DCs injected intra-CSF substantially infiltrated several compartments of the inflamed CNS, including the periventricular demyelinating lesions. We also found that in EAE rats, as compared to controls, a larger number of intra-CSF injected DCs reached the cervical lymph nodes. This migratory behavior was accompanied by an accentuation of EAE clinical signs and an increased systemic antibody response against myelin oligodendrocyte glycoprotein, a major immunogenic myelin antigen. CONCLUSIONS/SIGNIFICANCE: Altogether, these results indicate that CSF-circulating DCs are able to both survey the inflamed brain and to reach the cervical lymph nodes. In EAE and maybe multiple sclerosis, CSF-circulating DCs may thus support the immune responses that develop within and outside the inflamed CNS.

Abnormal expression of truncated CRMP-1 protein in the brain cortex of MPSIIIB mice.

Mucopolysaccharidosis IIIB is a lysosomal disease characterized by a severe neurological deterioration, the pathophysiological mechanisms of which are poorly understood. Recently FGF pathway was shown to be altered leading us to explore a downstream target involved in brain development: the collapsin response mediator protein-1 (CRMP-1). CRMP-1 transcript level was normal but a cleavage of CRMP-1 was observed with an abnormal expression of the truncated form until adult age. This truncated CRMP-1 protein could play a role in post-natal cortex maturation and be involved in neuronal alterations occurring in lysosomal diseases.

Glutamatergic alterations in the cortex of genetic absence epilepsy rats.

BACKGROUND: In absence epilepsy, the neuronal hyper-excitation and hyper-synchronization, which induce spike and wave discharges in a cortico-thalamic loop are suspected to be due to an imbalance between GABA and glutamate (GLU) neurotransmission. In order to elucidate the role played by GLU in disease outcome, we measured cortical and thalamic extracellular levels of GLU and GABA. We used an in vivo quantitative microdialysis approach (no-net-flux method) in an animal model of absence epilepsy (GAERS). In addition, by infusing labelled glutamate through the microdialysis probe, we studied in vivo glutamate uptake in the cortex and thalamus in GAERS and non-epileptic control (NEC) rats. Expression of the vesicular glutamate transporters VGLUT1 and VGLUT2 and a synaptic component, synaptophysin, was also measured. RESULTS: Although extracellular concentrations of GABA and GLU in the cortex and thalamus were not significantly different between GAERS and NEC rats, cortical GLU uptake was significantly decreased in unrestrained awake GAERS. Expression of VGLUT2 and synaptophysin was increased in the cortex of GAERS compared to NEC rats, but no changes were observed in the thalamus. CONCLUSION: The specific decrease in GLU uptake in the cortex of GAERS linked to synaptic changes suggests impairment of the glutamatergic terminal network. These data support the idea that a change in glutamatergic neurotransmission in the cortex could contribute to hyperexcitability in absence epilepsy.

Cortical glutamate metabolism is enhanced in a genetic model of absence epilepsy.

Disturbances in GABAergic and glutamatergic neurotransmission in the thalamocortical loop are involved in absence seizures. Here, we examined potential disturbances in metabolism and interactions between neurons and glia in 5-month-old genetic absence epilepsy rats from Strasbourg (GAERS) and nonepileptic rats (NER). Animals received [1-(13)C]glucose and [1,2-(13)C]acetate, the preferential substrates of neurons and astrocytes, respectively. Extracts from cerebral cortex, thalamus, and hippocampus were analyzed by (13)C nuclear magnetic resonance spectroscopy. Most changes were detected in the cortex. Pyruvate metabolism was enhanced as evidenced by increases of lactate, and labeled and unlabeled alanine. Neuronal mitochondrial metabolism was also enhanced as detected by elevated amounts of N-acetylaspartate and nicotinamide adenine dinucleotide as well as increased incorporation of label from [2-(13)C]acetyl CoA into glutamate, glutamine, and aspartate. Likewise, mitochondrial metabolism in astrocytes was increased. Changes in thalamus were restricted to increased concentration and labeling of glutamine. Changes in the hippocampus were similar to those in the cortex. This increase in glutamate-glutamine metabolism in cortical neurons and astrocytes accompanied by a decreased gamma aminobyturic acid level may lead to impaired thalamic filter function. Hence, reduced sensory input to cortex could allow the occurrence of spike-and-wave discharges in the thalamocortical loop. Increased glutamatergic output from the cortex to hippocampus may be the underlying cause of improved learning in GAERS.

Decreased expression of glutamate transporters in genetic absence epilepsy rats before seizure occurrence.

In absence epilepsy, epileptogenic processes are suspected of involving an imbalance between GABAergic inhibition and glutamatergic excitation. Here, we describe alteration of the expression of glutamate transporters in rats with genetic absence (the Genetic Absence Epilepsy Rats from Strasbourg: GAERS). In these rats, epileptic discharges, recorded in the thalamo-cortical network, appear around 40 days after birth. In adult rats no alteration of the protein expression of the glutamate transporters was observed. In 30-day-old GAERS protein levels (quantified by western blot) were lower in the cortex by 21% and 35% for the glial transporters GLT1 and GLAST, respectively, and by 32% for the neuronal transporter EAAC1 in the thalamus compared to control rats. In addition, the expression and activity of GLAST were decreased by 50% in newborn GAERS cortical astrocytes grown in primary culture. The lack of modification of the protein levels of glutamatergic transporters in adult epileptic GAERS, in spite of mRNA variations (quantified by RT-PCR), suggests that they are not involved in the pathogeny of spike-and-wave discharges. In contrast, the alteration of glutamate transporter expression, observed before the establishment of epileptic discharges, could reflect an abnormal maturation of the glutamatergic neurone-glia circuitry.