Lauren Deneyer

In-depth behavioral characterization of the corticosterone mouse model and the critical involvement of housing conditions

Depression and anxiety are disabling and highly prevalent psychiatric disorders. To better understand the neurobiological basis of mood and anxiety disorders, relevant animal models are needed. The corticosterone mouse model is frequently used to study depression. Chronic stress and accompanying glucocorticoid elevation causes pathological changes in the central nervous system, which are related to psychiatric symptoms. Exogenous administration of corticosterone is therefore often used to induce depressive-like behavior in mice and in some cases also features of anxiety-like behavior are shown. However, a thorough characterization of this model has never been conducted and housing conditions of the used subjects often differ between the implemented protocols. We chronically administered a subcutaneous corticosterone bolus injection to single- and group-housed mice, and we subsequently evaluated the face validity of this model by performing a battery of behavioral tests (forced swim test, mouse-tail suspension test, saccharin intake test, novelty-suppressed feeding test, elevated plus maze, light/dark paradigm and open field test). Our results show that corticosterone treatment has a substantial overall effect on depressive-like behavior. Increases in anxiety-like behavior on the other hand are mainly seen in single housed animals, independent of treatment. The current study therefore does not only show a detailed behavioral characterization of the corticosterone mouse model, but furthermore also elucidates the critical influence of housing conditions on the behavioral outcome in this model.

Absence of system xc⁻ on immune cells invading the central nervous system alleviates experimental autoimmune encephalitis

BackgroundMultiple sclerosis (MS) is an autoimmune demyelinating disease that affects the central nervous system (CNS), leading to neurodegeneration and chronic disability. Accumulating evidence points to a key role for neuroinflammation, oxidative stress, and excitotoxicity in this degenerative process. System xc− or the cystine/glutamate antiporter could tie these pathological mechanisms together: its activity is enhanced by reactive oxygen species and inflammatory stimuli, and its enhancement might lead to the release of toxic amounts of glutamate, thereby triggering excitotoxicity and neurodegeneration.MethodsSemi-quantitative Western blotting served to study protein expression of xCT, the specific subunit of system xc−, as well as of regulators of xCT transcription, in the normal appearing white matter (NAWM) of MS patients and in the CNS and spleen of mice exposed to experimental autoimmune encephalomyelitis (EAE), an accepted mouse model of MS. We next compared the clinical course of the EAE disease, the extent of demyelination, the infiltration of immune cells and microglial activation in xCT-knockout (xCT−/−) mice and irradiated mice reconstituted in xCT−/− bone marrow (BM), to their proper wild type (xCT+/+) controls.ResultsxCT protein expression levels were upregulated in the NAWM of MS patients and in the brain, spinal cord, and spleen of EAE mice. The pathways involved in this upregulation in NAWM of MS patients remain unresolved. Compared to xCT+/+ mice, xCT−/− mice were equally susceptible to EAE, whereas mice transplanted with xCT−/− BM, and as such only exhibiting loss of xCT in their immune cells, were less susceptible to EAE. In none of the above-described conditions, demyelination, microglial activation, or infiltration of immune cells were affected.ConclusionsOur findings demonstrate enhancement of xCT protein expression in MS pathology and suggest that system xc− on immune cells invading the CNS participates to EAE. Since a total loss of system xc− had no net beneficial effects, these results have important implications for targeting system xc− for treatment of MS.

Zonisamide attenuates lactacystin-induced parkinsonism in mice without affecting system xc−

&NA; Zonisamide (ZNS), an anticonvulsant drug exhibiting symptomatic effects in Parkinsons disease (PD), was recently reported to exert neuroprotection in rodent models. One of the proposed neuroprotective mechanisms involves increased protein expression of xCT, the specific subunit of the cystine/glutamate antiporter system xc−, inducing glutathione (GSH) synthesis. Here, we investigated the outcome of ZNS treatment in a mouse model of PD based on intranigral proteasome inhibition, and whether the observed effects would be mediated by system xc−. The proteasome inhibitor lactacystin (LAC) was administered intranigrally to male C57BL/6J mice receiving repeated intraperitoneal injections of either ZNS 30 mg kg− 1 or vehicle. Drug administration was initiated three days prior to stereotaxic LAC injection and was maintained until six days post‐surgery. One week after lesion, mice were behaviorally assessed and investigated in terms of nigrostriatal neurodegeneration and molecular changes at the level of the basal ganglia, including expression levels of xCT. ZNS reduced the loss of nigral dopaminergic neurons following LAC injection and the degree of sensorimotor impairment. ZNS failed, however, to modulate xCT expression in basal ganglia of lesioned mice. In a separate set of experiments, the impact of ZNS treatment on system xc− was investigated in control conditions in vivo as well as in vitro. Similarly, ZNS did not influence xCT or glutathione levels in naive male C57BL/6J mice, nor did it alter system xc− activity or glutathione content in vitro. Taken together, these results demonstrate that ZNS treatment provides neuroprotection and behavioral improvement in a PD mouse model based on proteasome inhibition via system xc− independent mechanisms. HighlightsZNS decreases LAC‐induced nigral DA‐ergic neurodegeneration.ZNS reduces the degree of LAC‐induced sensorimotor dysfunction.ZNS does not influence the expression or activity of system xc−.

Caloric Restriction Protects against Lactacystin-Induced Degeneration of Dopamine Neurons Independent of the Ghrelin Receptor

Parkinson’s disease (PD) is a neurodegenerative disorder, characterized by a loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNc). Caloric restriction (CR) has been shown to exert ghrelin-dependent neuroprotective effects in the 1-methyl-4-phenyl-1,2,3,6-tetrathydropyridine (MPTP)-based animal model for PD. We here investigated whether CR is neuroprotective in the lactacystin (LAC) mouse model for PD, in which proteasome disruption leads to the destruction of the DA neurons of the SNc, and whether this effect is mediated via the ghrelin receptor. Adult male ghrelin receptor wildtype (WT) and knockout (KO) mice were maintained on an ad libitum (AL) diet or on a 30% CR regimen. After 3 weeks, LAC was injected unilaterally into the SNc, and the degree of DA neuron degeneration was evaluated 1 week later. In AL mice, LAC injection significanty reduced the number of DA neurons and striatal DA concentrations. CR protected against DA neuron degeneration following LAC injection. However, no differences were observed between ghrelin receptor WT and KO mice. These results indicate that CR can protect the nigral DA neurons from toxicity related to proteasome disruption; however, the ghrelin receptor is not involved in this effect.

Plastic changes at corticostriatal synapses predict improved motor function in a partial lesion model of Parkinson’s disease

In Parkinsons disease, striatal dopamine depletion leads to plastic changes at excitatory corticostriatal and thalamostriatal synapses. The functional consequences of these responses on the expression of behavioral deficits are incompletely understood. In addition, most of the information on striatal synaptic plasticity has been obtained in models with severe striatal dopamine depletion, and less is known regarding changes during early stages of striatal denervation. Using a partial model of nigral cell loss based on intranigral injection of the proteasome inhibitor lactacystin, we demonstrate ultrastructural changes at corticostriatal synapses with a 15% increase in the length and 30% increase in the area of the postsynaptic densities at corticostriatal synapses 1 week following toxin administration. This increase was positively correlated with the performance of lactacystin-lesioned mice on the rotarod task, such that mice with a greater increase in the size of the postsynaptic density performed better on the rotarod task. We therefore propose that lengthening of the postsynaptic density at corticostriatal synapses acts as a compensatory mechanism to maintain motor function under conditions of partial dopamine depletion. The ultrastructure of thalamostriatal synapses remained unchanged following lactacystin administration. Our findings provide novel insights into the mechanisms of synaptic plasticity and behavioral compensation following partial loss of substantia nigra pars compacta neurons, such as those occurring during the early stages of Parkinsons disease.

Lack of effect of Theiler’s murine encephalomyelitis virus infection on system xc−

Changes in the expression of xCT, the specific subunit of system xc(-) or the cystine/glutamate antiporter, have been associated with several neurological disorders and system xc(-) was recently proposed as a potential target for the development of new treatment strategies for multiple sclerosis (MS). In this study we used Theilers murine encephalomyelitis virus (TMEV) infection, both in vitro and in vivo, as a model to further evaluate the involvement of system xc(-) in MS. Protein levels of xCT, as well as activity of system xc(-) were unaffected in RAW264.7 macrophages after infection with the demyelinating DA strain of TMEV. Also, protein expression of xCT remained stable in spinal cord and brain of FVB mice 1-2 and 6 weeks after intracranial injection of the DA strain of TMEV. These results demonstrate that TMEV infection of macrophages or FVB mice has no effect on system xc(-) and as such cannot be used as a model to study the involvement of system xc(-) in MS.

Slc7a11 (xCT) protein expression is not altered in the depressed brain and system xc- deficiency does not affect depression-associated behaviour in the corticosterone mouse model

Abstract Objectives: The cystine/glutamate antiporter (system xc-) is believed to contribute to nonvesicular glutamate release from glial cells in various brain areas. Although recent investigations implicate system xc- in mood disorders, unambiguous evidence has not yet been established. Therefore, we evaluated the possible role of system xc- in the depressive state. Methods: We conducted a protein expression analysis of the specific subunit of system xc- (xCT) in brain regions of the corticosterone mouse model, Flinders Sensitive Line rat model and post-mortem tissue of depressed patients. We next subjected system xc- deficient mice to the corticosterone model and analysed their behaviour in several tests. Lastly, we subjected additional cohorts of xCT-deficient and wild-type mice to N-acetylcysteine treatment to unveil whether the previously reported antidepressant-like effects are dependent upon system xc-. Results: We did not detect any changes in xCT expression levels in the animal models or patients compared to proper controls. Furthermore, loss of system xc- had no effect on depression- and anxiety-like behaviour. Finally, the antidepressant-like effects of N-acetylcysteine are not mediated via system xc-. Conclusions: xCT protein expression is not altered in the depressed brain and system xc- deficiency does not affect depression-associated behaviour in the corticosterone mouse model.

12.3 SYSTEM XC- AS A NOVEL MODULATOR OF CORTICOSTRIATAL NEUROTRANSMISSION

Abstract Background System xc- is a plasma membrane amino acid antiporter, of mainly glial origin, that couples the import of cystine with the export of glutamate. System xc- (specific subunit xCT) contributes substantially to ambient extracellular glutamate levels in various regions of the brain, including the striatum and hippocampus. Despite the fact that system xc- is highly expressed in the brain and is a proposed therapeutic target for various neurological disorders, its function under physiological conditions in the central nervous system remains poorly understood. By acting as a source of glial extrasynaptic glutamate, system xc- might modulate synaptic transmission as a mechanism of neuro-glial communication. Previous electrophysiological findings indicate that system xc- delivered glutamate can inhibit excitatory synaptic neurotransmission in the corticoaccumbens pathway and at hippocampal CA3-CA1 synapses. To gain further insight into the proposed function of system xc- as modulator of synaptic transmission, we here focus on corticostriatal synapses. Methods Single section electron microscopy was carried out on VGLUT1-pre-embed and glutamate immunogold post-embed labeled slices of the dorsolateral striatum of xCT+/+ and xCT-/- mice. Various parameters related to the pre- and post-synaptic compartments were integrated on the obtained electron micrographs, including glutamate immunogold density in the presynaptic terminal and spine, area of the terminal and spine, measures of the postsynaptic density (PSD) (length, area, thickness, and maximum thickness), percentage of PSDs showing perforations, and width of the synaptic cleft. Electrophysiological measures of corticostriatal transmission were obtained by recording the amplitude of field excitatory postsynaptic potentials (fEPSPs) after stimulation of corticostriatal fibers. Finally, grooming behavior was compared between xCT-/- and xCT+/+ littermates. Results Genetic deletion of xCT led to depletion of glutamate immunogold labeling from corticostriatal terminals and their corresponding dendritic spines. Absence of xCT did not, however, affect the morphology of corticostriatal synapses, as evaluated by the area of the terminals and spines, size of the PSD, and width of the synaptic cleft. Similarly, no changes could be observed in the density of VGLUT1-positive synapses, indicating normal cortical innervation and spine density. Electrophysiological recordings revealed decreased amplitude of fEPSPs in xCT-/- mice after stimulation of corticostriatal fibers. Preliminary investigations revealed that this reduced response can be rescued by restoring physiological levels of glutamate to xCT-/- slices. Changes in corticostriatal transmission were not reflected in aberrant grooming behavior in xCT-/- mice; we could not observe any difference in the total grooming duration, the number of grooming bouts, the average bout duration or the latency to onset to grooming between xCT-/- and xCT+/+ mice. Discussion Contrary to available evidence at hippocampal and corticoaccumbens pathways, our findings indicate a positive effect of system xc- on basal synaptic transmission at corticostriatal synapses. The decreased response we observed after stimulation of corticostriatal fibers in xCT-/- mice was accompanied by depletion of glutamate immunogold labeling from corticostriatal terminals, suggesting a possible defect in presynaptic glutamate handling. Given the strong decrease (70%) in extracellular glutamate levels previously reported in this strain of mice, we hypothesize that the decreased presynaptic glutamate labeling in xCT-/- mice is related to a loss of extracellular glutamate needed to supply terminals for proper excitatory transmission. This hypothesis is supported by our preliminary results showing increased responses in xCT-/- slices after restoring physiological levels of glutamate. Together, our findings shed new light on the role of system xc- in controlling synaptic transmission, and suggest that it may play an important role in supplying presynaptic terminals with glutamate as an alternative mechanism to the glutamate-glutamine cycle. As a novel modulator of corticostriatal transmission, system xc- may be of interest as a possible therapeutic target for disorders with a corticostriatal component, such as schizophrenia or obsessive-compulsive disorder.

Genetic deletion of xCT attenuates peripheral and central inflammation and mitigates LPS-induced sickness and depressive-like behavior in mice

The communication between the immune and central nervous system (CNS) is affected in many neurological disorders. Peripheral injections of the endotoxin lipopolysaccharide (LPS) are widely used to study this communication: an LPS challenge leads to a biphasic syndrome that starts with acute sickness and is followed by persistent brain inflammation and chronic behavioral alterations such as depressive‐like symptoms. In vitro, the response to LPS treatment has been shown to involve enhanced expression of system xc− . This cystine‐glutamate antiporter, with xCT as specific subunit, represents the main glial provider of extracellular glutamate in mouse hippocampus. Here we injected male xCT knockout and wildtype mice with a single intraperitoneal dose of 5 mg/kg LPS. LPS‐injection increased hippocampal xCT expression but did not alter the mainly astroglial localization of the xCT protein. Peripheral and central inflammation (as defined by cytokine levels and morphological activation of microglia) as well as LPS‐induced sickness and depressive‐like behavior were significantly attenuated in xCT‐deficient mice compared with wildtype mice. Our study is the first to demonstrate the involvement of system xc− in peripheral and central inflammation in vivo and the potential therapeutic relevance of its inhibition in brain disorders characterized by peripheral and central inflammation, such as depression.

Ketamine Does Not Exert Protective Properties on Dopaminergic Neurons in the Lactacystin Mouse Model of Parkinson’s Disease

Parkinson’s disease (PD) is an age-related neurodegenerative condition characterized by a progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). A loss of proteasome function participates to the pathogenesis of PD, leading to the development of rodent models in which a proteasome inhibitor is applied to the nigrostriatal pathway. We recently characterized the intranigral lactacystin (LAC) mouse model, leading to nigrostriatal degeneration, motor dysfunction and alpha-synuclein accumulation. In the present study, we compared the effect of two commonly used anesthetics for generating animal models of PD—i.e., ketamine (KET) and isoflurane (ISO)—on the vulnerability of mouse dopaminergic neurons to proteasome inhibition-induced degeneration. Both anesthetics have the potential to affect the susceptibility of the nigrostriatal pathway for toxin-induced degeneration, and are known to modulate dopamine (DA) homeostasis. Yet, their impact on nigrostriatal degeneration in the proteasome inhibition model has not been evaluated. Unilateral injection with LAC in the SNpc of mice induced motor impairment and significantly reduced the number of dopaminergic cells to ~55%, irrespective of the anesthetic used. However, LAC-induced striatal DA depletion was slightly affected by the choice of anesthetic, resulting in a significant increase in DA turnover in the ISO- but not in KET-treated mice. These results suggest that the extent of nigrostriatal dopaminergic neural loss caused by LAC is not influenced by the choice of anesthetic, and that compared to other PD models, KET is not neuroprotective in the LAC model.