Serotonin sensing by microglia conditions the proper development of neuronal circuits and of social and adaptive skills.

The proper maturation of emotional and sensory circuits requires fine-tuning of serotonin (5-HT) level during early postnatal development. Consistently, dysfunctions of the serotonergic system have been associated with neurodevelopmental psychiatric diseases, including autism spectrum disorders (ASD). However, the mechanisms underlying the developmental effects of 5-HT remain partially unknown, one obstacle being the action of 5-HT on different cell types. Here, we focused on microglia, which play a role in brain wiring refinement, and we investigated whether the control of these cells by 5-HT is relevant for neurodevelopment and spontaneous behaviors in mice. Since the main 5-HT sensor in microglia is the 5-HT2B receptor subtype, we prevented 5-HT signaling specifically in microglia by conditional invalidation of the Htr2b gene in these cells. We observed that abrogating the serotonergic control of microglia during early postnatal development affects the phagolysosomal compartment of these cells and their proximity to dendritic spines and perturbs neuronal circuits maturation. Furthermore, this early ablation of microglial 5-HT2B receptors leads to adult hyperactivity in a novel environment and behavioral defects in sociability and flexibility. Importantly, we show that these behavioral alterations result from a developmental effect, since they are not observed when microglial Htr2b invalidation is induced later, at P30 onward. Thus, a primary alteration of 5-HT sensing in microglia, during a critical time window between birth and P30, is sufficient to impair social and flexibility skills. This link between 5-HT and microglia may explain the association between serotonergic dysfunctions and behavioral traits like impaired sociability and inadaptability to novelty, which are prominent in psychiatric disorders such as ASD.

Aged xCT-Deficient Mice Are Less Susceptible for Lactacystin-, but Not 1-Methyl-4-Phenyl-1,2,3,6- Tetrahydropyridine-, Induced Degeneration of the Nigrostriatal Pathway.

The astrocytic cystine/glutamate antiporter system x c - (with xCT as the specific subunit) imports cystine in exchange for glutamate and has been shown to interact with multiple pathways in the brain that are dysregulated in age-related neurological disorders, including glutamate homeostasis, redox balance, and neuroinflammation. In the current study, we investigated the effect of genetic xCT deletion on lactacystin (LAC)- and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced degeneration of the nigrostriatal pathway, as models for Parkinson's disease (PD). Dopaminergic neurons of adult xCT knock-out mice (xCT-/-) demonstrated an equal susceptibility to intranigral injection of the proteasome inhibitor LAC, as their wild-type (xCT+/+) littermates. Contrary to adult mice, aged xCT-/- mice showed a significant decrease in LAC-induced degeneration of nigral dopaminergic neurons, depletion of striatal dopamine (DA) and neuroinflammatory reaction, compared to age-matched xCT+/+ littermates. Given this age-related protection, we further investigated the sensitivity of aged xCT-/- mice to chronic and progressive MPTP treatment. However, in accordance with our previous observations in adult mice (Bentea et al., 2015a), xCT deletion did not confer protection against MPTP-induced nigrostriatal degeneration in aged mice. We observed an increased loss of nigral dopaminergic neurons, but equal striatal DA denervation, in MPTP-treated aged xCT-/- mice when compared to age-matched xCT+/+ littermates. To conclude, we reveal age-related protection against proteasome inhibition-induced nigrostriatal degeneration in xCT-/- mice, while xCT deletion failed to protect nigral dopaminergic neurons of aged mice against MPTP-induced toxicity. Our findings thereby provide new insights into the role of system x c - in mechanisms of dopaminergic cell loss and its interaction with aging.

Systemic LPS-induced neuroinflammation increases the susceptibility for proteasome inhibition-induced degeneration of the nigrostriatal pathway.

INTRODUCTION: Besides proteasome dysfunction, neuroinflammation is a common feature in the pathogenesis of Parkinson's disease (PD). Accordingly, peripheral inflammation has been shown to increase the susceptibility of the brain for nigrostriatal degeneration by inducing activation of glial cells and release of pro-inflammatory cytokines in the brain. Given that current animal models of PD fail to recapitulate the pathophysiology occurring in idiopathic PD, the aim of this study was to combine two pathogenic mechanisms (i.e. neuroinflammation and proteasome inhibition) to create a dual-hit mouse model of PD. METHODS: We repeatedly injected mice with a low dose of LPS (250 µg/kg/day i. p. for four days) to induce neuroinflammation, followed by a unilateral intranigral injection of lactacystin (LAC; 3 µg). Seven days later, mice were evaluated behaviorally to assess locomotion, anxiety- and depressive-like behavior. Nigrostriatal degeneration was analyzed by measuring striatal dopamine loss as well as loss of nigral dopaminergic neurons. Neuroinflammation was confirmed by quantifying microglial cells in the substantia nigra (SN) and cytokine expression in the striatum. RESULTS: Repeated systemic LPS injections increase the number of microglial cells in the SN and induce a mixed profile of pro- and anti-inflammatory cytokines in the striatum without affecting the integrity of the nigrostriatal pathway. Systemic LPS-induced neuroinflammation, however, increases the susceptibility of the nigrostriatal pathway for LAC-induced degeneration. CONCLUSION: Recapitulating two relevant etiopathogenic mechanisms of PD - neuroinflammation and proteasome inhibition-, we propose this dual-hit model as a relevant mouse model for PD that could be used to investigate potential therapeutic targets.

Anticonvulsant and antiepileptogenic effects of system xc- inactivation in chronic epilepsy models.

OBJECTIVE: The cystine/glutamate antiporter system xc- could represent a new target for antiepileptogenic treatments due to its crucial roles in glutamate homeostasis and neuroinflammation. To demonstrate this, we compared epilepsy development and seizure susceptibility in xCT knockout mice (xCT-/- ) and in littermate controls (xCT+/+ ) in different chronic models of epilepsy. METHODS: Mice were surgically implanted with electrodes in the basolateral amygdala and chronically stimulated to develop self-sustained status epilepticus (SSSE); continuous video-electroencephalography monitoring was performed for 28 days after SE and hippocampal histopathology was assessed. Corneal kindling was induced by twice daily electrical stimulation at 6 Hz and maintenance of the fully kindled state was evaluated. Next, messenger RNA (mRNA) and protein levels of xCT and of the proteins involved in the phosphoinositide 3-kinase (PI3K)/Akt/glycogen synthase kinase 3ß (GSK-3ß)/eukaryotic initiation factor 2α (eIF2α)/activating transcription factor 4 (ATF4) signaling pathway were measured at different time points during epileptogenesis in NMRI mice treated with pilocarpine. Finally, the anticonvulsant effect of sulfasalazine (SAS), a nonselective system xc- inhibitor, was assessed against 6 Hz-evoked seizures in pilocarpine-treated mice. RESULTS: In the SSSE model, xCT-/- mice displayed a significant delayed epileptogenesis, a reduced number of spontaneous recurrent seizures, and less pronounced astrocytic and microglial activation. Moreover, xCT-/- mice showed reduced seizure severity during 6 Hz kindling development and a lower incidence of generalized seizures during the maintenance of the fully kindled state. In pilocarpine-treated mice, protein levels of the PI3K/Akt/GSK-3ß/eIF2α/ATF4 pathway were increased during the chronic phase of the model, consistent with previous findings in the hippocampus of patients with epilepsy. Finally, repeated administration of SAS protected pilocarpine-treated mice against acute 6 Hz seizure induction, in contrast to sham controls, in which system xc- is not activated. SIGNIFICANCE: Inhibition of system xc- could be an attractive target for the development of new therapies with a potential for disease modification in epilepsy.

Genetic and pharmacological manipulation of glial glutamate transporters does not alter infection-induced seizure activity.

The contribution of glial transporters to glutamate movement across the membrane has been identified as a potential target for anti-seizure therapies. Two such glutamate transporters, GLT-1 and system xc-, are expressed on glial cells, and modulation of their expression and function have been identified as a means by which seizures, neuronal injury, and gliosis can be reduced in models of brain injury. While GLT-1 is responsible for the majority of glutamate uptake in the brain, system xc- releases glutamate in the extracellular cleft in exchange for cystine and represents as such the major source of hippocampal extracellular glutamate. Using the Theiler's Murine Encephalomyelitis Virus (TMEV) model of viral-induced epilepsy, we have taken two well-studied approaches, one pharmacological, one genetic, to investigate the potential role(s) of GLT-1 and system xc- in TMEV-induced pathology. Our findings suggest that the methods we utilized to modulate these glial transporters, while effective in other models, are not sufficient to reduce the number or severity of behavioral seizures in TMEV-infected mice. However, genetic knockout of xCT, the specific subunit of system xc-, may have cellular effects, as we observed a slight decrease in neuronal injury caused by TMEV and an increase in astrogliosis in the CA1 region of the hippocampus. Furthermore, xCT knockout caused an increase in GLT-1 expression selectively in the cortex. These findings have significant implications for both the characterization of the TMEV model as well as for future efforts to discover novel and effective anti-seizure drugs.

The Barnes Maze Task Reveals Specific Impairment of Spatial Learning Strategy in the Intrahippocampal Kainic Acid Model for Temporal Lobe Epilepsy.

Temporal lobe epilepsy (TLE) is an acquired form of focal epilepsy, in which patients not only suffer from unprovoked, devastating seizures, but also from severe comorbidities, such as cognitive dysfunction. Correspondingly, several animal models of TLE exhibit memory dysfunction, especially spatial memory. The Morris water maze test is the most commonly used test for assessing spatial learning and memory in rodents. However, high stress and poor swimming abilities are common confounders and may contribute to misinterpretation. Particularly epileptic mice show altered behaviour during the test as they fail to understand the paradigm context. In the Barnes maze test, a dry-land maze test for spatial learning and memory that uses milder aversive stimuli, these drawbacks have not yet been reported. In the present study, we use this task to evaluate spatial learning and memory in the intrahippocampal kainic acid mouse model of TLE. We demonstrate that the epileptic mice understand the Barnes maze paradigm context, as they learn the location of the escape-chamber by using a serial search strategy but fail to develop the more efficient spatial search strategy. Our data indicate that the Barnes maze may be a better alternative to the Morris water maze for assessing search strategies and impairment of learning and memory in epileptic mice.

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.

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.

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 x c - . 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 x c - 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.

Inhibition of astroglial connexin43 hemichannels with TAT-Gap19 exerts anticonvulsant effects in rodents.

Accumulating evidence shows a key function for astrocytic connexin43 (Cx43) signaling in epilepsy. However, the lack of experimental distinction between Cx43 gap junction channels (GJCs) and hemichannels (HCs) has impeded the identification of the exact contribution of either channel configurations to epilepsy. We therefore investigated whether TAT-Gap19, a Cx mimetic peptide that inhibits Cx43 HCs but not the corresponding Cx43 GJCs, influences experimentally induced seizures in rodents. Dye uptake experiments in acute hippocampal slices of mice demonstrated that astroglial Cx43 HCs open in response to the chemoconvulsant pilocarpine and this was inhibited by TAT-Gap19. In vivo, pilocarpine-induced seizures as well as the accompanying increase in D-serine microdialysate levels were suppressed by Cx43 HC inhibition. Moreover, the anticonvulsant action of TAT-Gap19 was reversed by exogenous D-serine administration, suggesting that Cx43 HC inhibition protects against seizures by lowering extracellular D-serine levels. The anticonvulsive properties of Cx43 HC inhibition were further confirmed in electrical seizure mouse models, i.e. an acute 6 Hertz (Hz) model of refractory seizures and a chronic 6 Hz corneal kindling model. Collectively, these results indicate that Cx43 HCs play a role in seizures and underscore their potential as a novel and druggable target in epilepsy treatment.

6 Hz corneal kindling in mice triggers neurobehavioral comorbidities accompanied by relevant changes in c-Fos immunoreactivity throughout the brain.

OBJECTIVE: Besides seizures, patients with epilepsy are affected by a variety of cognitive and psychiatric comorbidities that further impair their quality of life. The present study provides an in-depth characterization of the behavioral alterations induced by 6 Hz corneal kindling. Furthermore, we correlate these behavioral changes to alterations in c-Fos protein expression throughout the brain following kindling. METHODS: Adolescent male Naval Medical Research Institute (NMRI) mice were kindled via repetitive subconvulsive 6 Hz corneal stimulations until they reached the fully kindled state (defined as 10 consecutive generalized seizures). Afterwards we performed an elaborate battery of behavioral tests and we evaluated c-Fos expression throughout the brain using immunohistochemistry. RESULTS: Fully kindled mice display an abnormal behavioral phenotype, characterized by basal and amphetamine-induced hyperlocomotion, anhedonia, social withdrawal, and deficits in short- and long-term memory. Moreover, 6 Hz corneal kindling enhances c-Fos immunoreactivity in the visual, parahippocampal, and motor cortices and the limbic system, whereas c-Fos+ cells are decreased in the orbital cortex of fully kindled mice. SIGNIFICANCE: The behavioral outcomes of 6 Hz corneal kindling cluster into 3 main categories: positive symptoms, negative symptoms, and cognitive impairment. These symptoms are accompanied by c-Fos activation in relevant brain regions once the fully kindled state is established. Based on the face validity of this model, we speculate that 6 Hz corneal kindling can be used to model not only pharmacoresistant limbic seizures, but also several neurobehavioral comorbidities that affect patients with epilepsy.

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.

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

BACKGROUND: Multiple 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. METHODS: Semi-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. RESULTS: xCT 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. CONCLUSIONS: Our 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.

Zonisamide (ZNS), an anticonvulsant drug exhibiting symptomatic effects in Parkinson's 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 30mgkg-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.

Disruption of the HPA-axis through corticosterone-release pellets induces robust depressive-like behavior and reduced BDNF levels in mice.

The corticosterone mouse model is widely used in preclinical research towards a better understanding of mechanisms of major depression. One particular administration procedure is the subcutaneous implantation of corticosterone slow-release pellets. In this report we want to provide basic evidence, regarding behavioral changes, neurotransmitter and -modulator levels and some other relevant biomolecules after hypothalamic-pituitary-adrenal-axis distortion. We show that three weeks of corticosterone pellet exposure robustly induces depressive-like but not anxiety-like behavior in mice, accompanied by a significant decrease in hippocampal brain-derived neurotrophic factor levels, at five weeks after the start of treatment. Furthermore there is an overall decrease in plasma corticosterone levels after three weeks of treatment that lasts up until the five weeks' time point. On the other hand, no differences are observed in total monoamine, glutamate or d-serine levels, nor in glucocorticoid receptor expression, in various depression-related brain areas. Altogether this characterization delivers vital information, supplementary to existing literature, regarding the phenotyping of pellet-induced hypothalamic-pituitary-adrenal-axis disruption in mice following three weeks of continuous corticosterone exposure.

Inhibition of Connexin43 Hemichannels Impairs Spatial Short-Term Memory without Affecting Spatial Working Memory.

Astrocytes are active players in higher brain function as they can release gliotransmitters, which are essential for synaptic plasticity. Various mechanisms have been proposed for gliotransmission, including vesicular mechanisms as well as non-vesicular ones, for example by passive diffusion via connexin hemichannels (HCs). We here investigated whether interfering with connexin43 (Cx43) HCs influenced hippocampal spatial memory. We made use of the peptide Gap19 that blocks HCs but not gap junction channels and is specific for Cx43. To this end, we microinfused transactivator of transcription linked Gap19 (TAT-Gap19) into the brain ventricle of male NMRI mice and assessed spatial memory in a Y maze. We found that the in vivo blockade of Cx43 HCs did not affect the locomotor activity or spatial working memory in a spontaneous alternation Y maze task. Cx43 blockade did however significantly impair the spatial short-term memory in a delayed spontaneous alternation Y maze task. These results indicate that Cx43 HCs play a role in spatial short-term memory.

Comparative analysis of antibodies to xCT (Slc7a11): Forewarned is forearmed.

The cystine/glutamate antiporter or system Xc- exchanges cystine for glutamate, thereby supporting intracellular glutathione synthesis and nonvesicular glutamate release. The role of system Xc- in neurological disorders can be dual and remains a matter of debate. One important reason for the contradictory findings that have been reported to date is the use of nonspecific anti-xCT (the specific subunit of system Xc-) antibodies. Often studies rely on the predicted molecular weight of 55.5 kDa to identify xCT on Western blots. However, using brain extracts from xCT knockout (xCT(-/-)) mice as negative controls, we show that xCT migrates as a 35-kDa protein. Misinterpretation of immunoblots leads to incorrect assessment of antibody specificity and thereby to erroneous data interpretation. Here we have verified the specificity of most commonly used commercial and some in-house-developed anti-xCT antibodies by comparing their immunoreactivity in brain tissue of xCT(+/+) and xCT(-/-) mice by Western blotting and immunohistochemistry. The Western blot screening results demonstrate that antibody specificity not only differs between batches produced by immunizing different rabbits with the same antigen but also between bleedings of the same rabbit. Moreover, distinct immunohistochemical protocols have been tested for all the anti-xCT antibodies that were specific on Western blots in order to obtain a specific immunolabeling. Only one of our in-house-developed antibodies could reveal specific xCT labeling and exclusively on acetone-postfixed cryosections. Using this approach, we observed xCT protein expression throughout the mouse forebrain, including cortex, striatum, hippocampus, midbrain, thalamus, and amygdala, with greatest expression in regions facing the cerebrospinal fluid and meninges.

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.

Alterations in the motor cortical and striatal glutamatergic system and D-serine levels in the bilateral 6-hydroxydopamine rat model for Parkinson's disease.

Parkinson's disease (PD) is hallmarked by progressive degeneration of the substantia nigra pars compacta (SNc) neurons and is associated with aberrant glutamatergic activity. However, studies on the glutamatergic system in the motor cortex and striatum, two motor loop-related areas, are lacking in the clinically relevant bilateral SNc 6-hydroxydopamine (6-OHDA) rat model, and therefore led to the rationale behind the present investigations. Using Western blotting, the expression levels of the glial glutamate transporters, GLT-1 and GLAST, as well as xCT, the specific subunit of system xc(-), and the vesicular glutamate transporters, VGLUT1 and 2 were investigated at two different time points (1 week and 2 weeks) post-lesion. In addition, the total content of glutamate was measured. Moreover, the total D-serine levels were, to the best of our knowledge, studied for the first time in these two PD-related areas in the bilateral 6-OHDA rat model. In the motor cortex, no significant changes were observed in the different glutamate transporter expression levels in the bilaterally-lesioned rats. In the striatum, GLAST expression was significantly decreased at both time points whereas VGLUT1 and 2 expressions were significantly decreased 2 weeks after bilateral 6-OHDA lesion. Interestingly, bilateral 6-OHDA SNc lesion resulted in an enhancement of the total d-serine content in both motor cortex and striatum at 1 week post-lesion suggesting its possible involvement in the pathophysiology of PD. In conclusion, this study demonstrates disturbed glutamate and D-serine regulation in the bilateral SNc-lesioned brain which could contribute to the behavioral impairments in PD.

Nigral proteasome inhibition in mice leads to motor and non-motor deficits and increased expression of Ser129 phosphorylated α-synuclein.

Parkinson's disease is a neurodegenerative disorder characterized by motor and non-motor disturbances. Various pathogenic pathways drive disease progression including oxidative stress, mitochondrial dysfunction, α-synuclein aggregation and impairment of protein degradation systems. Dysfunction of the ubiquitin-proteasome system in the substantia nigra of Parkinson's disease patients is believed to be one of the causes of protein aggregation and cell death associated with this disorder. Lactacystin, a potent inhibitor of the proteasome, was previously delivered to the nigrostriatal pathway of rodents to model nigrostriatal degeneration. Although lactacystin-treated animals develop parkinsonian motor impairment, it is currently unknown whether they also develop non-motor symptoms characteristic of this disorder. In order to further describe the proteasome inhibition model of Parkinson's disease, we characterized the unilateral lactacystin model, performed by stereotaxic injection of the toxin in the substantia nigra of mice. We studied the degree of neurodegeneration and the behavioral phenotype 1 and 3 weeks after lactacystin lesion both in terms of motor impairment, as well as non-motor symptoms. We report that unilateral administration of 3 µg lactacystin to the substantia nigra of mice leads to partial (~40%) dopaminergic cell loss and concurrent striatal dopamine depletion, accompanied by increased expression of Ser129-phosphorylated α-synuclein. Behavioral characterization of the model revealed parkinsonian motor impairment, as well as signs of non-motor disturbances resembling early stage Parkinson's disease including sensitive and somatosensory deficits, anxiety-like behavior, and perseverative behavior. The consistent finding of good face validity, together with relevant construct validity, warrant a further evaluation of proteasome inhibition models of Parkinson's disease in pre-clinical research and validation of therapeutic targets.

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 Theiler's 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.