Glial lipid droplets and ROS induced by mitochondrial defects promote neurodegeneration.

Reactive oxygen species (ROS) and mitochondrial defects in neurons are implicated in neurodegenerative disease. Here, we find that a key consequence of ROS and neuronal mitochondrial dysfunction is the accumulation of lipid droplets (LD) in glia. In Drosophila, ROS triggers c-Jun-N-terminal Kinase (JNK) and Sterol Regulatory Element Binding Protein (SREBP) activity in neurons leading to LD accumulation in glia prior to or at the onset of neurodegeneration. The accumulated lipids are peroxidated in the presence of ROS. Reducing LD accumulation in glia and lipid peroxidation via targeted lipase overexpression and/or lowering ROS significantly delays the onset of neurodegeneration. Furthermore, a similar pathway leads to glial LD accumulation in Ndufs4 mutant mice with neuronal mitochondrial defects, suggesting that LD accumulation following mitochondrial dysfunction is an evolutionarily conserved phenomenon, and represents an early, transient indicator and promoter of neurodegenerative disease.

eIF2α controls memory consolidation via excitatory and somatostatin neurons.

An important tenet of learning and memory is the notion of a molecular switch that promotes the formation of long-term memory1-4. The regulation of proteostasis is a critical and rate-limiting step in the consolidation of new memories5-10. One of the most effective and prevalent ways to enhance memory is by regulating the synthesis of proteins controlled by the translation initiation factor eIF211. Phosphorylation of the α-subunit of eIF2 (p-eIF2α), the central component of the integrated stress response (ISR), impairs long-term memory formation in rodents and birds11-13. By contrast, inhibiting the ISR by mutating the eIF2α phosphorylation site, genetically11 and pharmacologically inhibiting the ISR kinases14-17, or mimicking reduced p-eIF2α with the ISR inhibitor ISRIB11, enhances long-term memory in health and disease18. Here we used molecular genetics to dissect the neuronal circuits by which the ISR gates cognitive processing. We found that learning reduces eIF2α phosphorylation in hippocampal excitatory neurons and a subset of hippocampal inhibitory neurons (those that express somatostatin, but not parvalbumin). Moreover, ablation of p-eIF2α in either excitatory or somatostatin-expressing (but not parvalbumin-expressing) inhibitory neurons increased general mRNA translation, bolstered synaptic plasticity and enhanced long-term memory. Thus, eIF2α-dependent mRNA translation controls memory consolidation via autonomous mechanisms in excitatory and somatostatin-expressing inhibitory neurons.

Vestibular CCK signaling drives motion sickness-like behavior in mice.

Travel can induce motion sickness (MS) in susceptible individuals. MS is an evolutionary conserved mechanism caused by mismatches between motion-related sensory information and past visual and motion memory, triggering a malaise accompanied by hypolocomotion, hypothermia, hypophagia, and nausea. Vestibular nuclei (VN) are critical for the processing of movement input from the inner ear. Motion-induced activation of VN neurons recapitulates MS-related signs. However, the genetic identity of VN neurons mediating MS-related autonomic and aversive responses remains unknown. Here, we identify a central role of cholecystokinin (CCK)-expressing VN neurons in motion-induced malaise. Moreover, we show that CCK VN inputs onto the parabrachial nucleus activate Calca-expressing neurons and are sufficient to establish avoidance to novel food, which is prevented by CCK-A receptor antagonism. These observations provide greater insight into the neurobiological regulation of MS by identifying the neural substrates of MS and providing potential targets for treatment.

Microglial response promotes neurodegeneration in the Ndufs4 KO mouse model of Leigh syndrome.

Leigh syndrome is a mitochondrial disease characterized by neurodegeneration, neuroinflammation, and early death. Mice lacking NDUFS4, a mitochondrial complex I subunit (Ndufs4 KO mice), have been established as a good animal model for studying human pathology associated with Leigh syndrome. As the disease progresses, there is an increase in neurodegeneration and neuroinflammation, thereby leading to deteriorating neurological symptoms, including motor deficits, breathing alterations, and eventually, death of the animal. However, despite the magnitude of neuroinflammation associated with brain lesions, the role of neuroinflammatory pathways and their main cellular components have not been addressed directly as relevant players in the disease pathology. Here, we investigate the role of microglial cells, the main immune cells of the CNS, in Leigh-like syndrome pathology, by pharmacologically depleting them using the colony-stimulating factor 1 receptor antagonist PLX3397. Microglial depletion extended lifespan and delayed motor symptoms in Ndufs4 KO mice, likely by preventing neuronal loss. Next, we investigated the role of the major cytokine interleukin-6 (IL-6) in the disease progression. IL-6 deficiency partially rescued breathing abnormalities and modulated gliosis but did not extend the lifespan or rescue motor decline in Ndufs4 KO mice. The present results show that microglial accumulation is pathogenic, in a process independent of IL-6, and hints toward a contributing role of neuroinflammation in the disease of Ndufs4 KO mice and potentially in patients with Leigh syndrome.

Striatal GPR88 Modulates Foraging Efficiency.

The striatum is anatomically and behaviorally implicated in behaviors that promote efficient foraging. To investigate this function, we studied instrumental choice behavior in mice lacking GPR88, a striatum-enriched orphan G-protein-coupled receptor that modulates striatal medium spiny neuron excitability. Our results reveal that hungry mice lacking GPR88 (KO mice) were slow to acquire food-reinforced lever press but could lever press similar to controls on a progressive ratio schedule. Both WT and KO mice discriminated between reward and no-reward levers; however, KO mice failed to discriminate based on relative quantity-reward (1 vs 3 food pellets) or effort (3 vs 9 lever presses). We also demonstrate preference for the high-reward (3 pellet) lever was selectively reestablished when GPR88 expression was restored to the striatum. We propose that GPR88 expression within the striatum is integral to efficient action-selection during foraging.SIGNIFICANCE STATEMENT Evolutionary pressure driving energy homeostasis favored detection and comparison of caloric value. In wild and laboratory settings, neural systems involved in energy homeostasis bias foraging to maximize energy efficiency. This is observed when foraging behaviors are guided by superior nutritional density or minimized caloric expenditure. The striatum is anatomically and functionally well placed to perform the sensory and motor integration necessary for efficient action selection during foraging. However, few studies have examined this behavioral phenomenon or elucidated underlying molecular mechanisms. Both humans and mice with nonfunctional GPR88 have been shown to present striatal dysfunctions and impaired learning. We demonstrate that GPR88 expression is necessary to efficiently integrate effort and energy density information guiding instrumental choice.

Fertility-regulating Kiss1 neurons arise from hypothalamic POMC-expressing progenitors.

Hypothalamic neuronal populations are central regulators of energy homeostasis and reproductive function. However, the ontogeny of these critical hypothalamic neuronal populations is largely unknown. We developed a novel approach to examine the developmental pathways that link specific subtypes of neurons by combining embryonic and adult ribosome-tagging strategies in mice. This new method shows that Pomc-expressing precursors not only differentiate into discrete neuronal populations that mediate energy balance (POMC and AgRP neurons), but also into neurons critical for puberty onset and the regulation of reproductive function (Kiss1 neurons). These results demonstrate a developmental link between nutrient-sensing and reproductive neuropeptide synthesizing neuronal populations and suggest a potential pathway that could link maternal nutrition to reproductive development in the offspring.

Balanced NMDA receptor activity in dopamine D1 receptor (D1R)- and D2R-expressing medium spiny neurons is required for amphetamine sensitization.

Signaling through N-methyl-D-aspartate-type glutamate receptors (NMDARs) is essential for the development of behavioral sensitization to psychostimulants such as amphetamine (AMPH). However, the cell type and brain region in which NMDAR signaling is required for AMPH sensitization remain unresolved. Here we use selective inactivation of Grin1, the gene encoding the essential NR1 subunit of NMDARs, in dopamine neurons or their medium spiny neuron (MSN) targets, to address this issue. We show that NMDAR signaling in dopamine neurons is not required for behavioral sensitization to AMPH. Conversely, removing NMDARs from MSNs that express the dopamine D1 receptor (D1R) significantly attenuated AMPH sensitization, and conditional, virus-mediated restoration of NR1 in D1R neurons in the nucleus accumbens (NAc) of these animals rescued sensitization. Interestingly, sensitization could also be restored by virus-mediated inactivation of NR1 in all remaining neurons in the NAc of animals lacking NMDARs on D1R neurons, or by removing NMDARs from all MSNs. Taken together, these data indicate that unbalanced loss of NMDAR signaling in D1R MSNs alone prevents AMPH sensitization, whereas a balanced loss of NMDARs from both D1R and dopamine D2 receptor-expressing (D2R) MSNs is permissive for sensitization.

Neuron-specific RNA-sequencing reveals different responses in peripheral neurons after nerve injury.

Peripheral neurons are heterogeneous and functionally diverse, but all share the capability to switch to a pro-regenerative state after nerve injury. Despite the assumption that the injury response is similar among neuronal subtypes, functional recovery may differ. Understanding the distinct intrinsic regenerative properties between neurons may help to improve the quality of regeneration, prioritizing the growth of axon subpopulations to their targets. Here, we present a comparative analysis of regeneration across four key peripheral neuron populations: motoneurons, proprioceptors, cutaneous mechanoreceptors, and nociceptors. Using Cre/Ai9 mice that allow fluorescent labeling of neuronal subtypes, we found that nociceptors showed the greater regeneration after a sciatic crush, followed by motoneurons, mechanoreceptors, and, finally, proprioceptors. By breeding these Cre mice with Ribotag mice, we isolated specific translatomes and defined the regenerative response of these neuronal subtypes after axotomy. Only 20% of the regulated genes were common, revealing a diverse response to injury among neurons, which was also supported by the differential influence of neurotrophins among neuron subtypes. Among differentially regulated genes, we proposed MED12 as a specific regulator of the regeneration of proprioceptors. Altogether, we demonstrate that the intrinsic regenerative capacity differs between peripheral neuron subtypes, opening the door to selectively modulate these responses.

Interleukin-6-elicited chronic neuroinflammation may decrease survival but is not sufficient to drive disease progression in a mouse model of Leigh syndrome.

BACKGROUND: Mitochondrial diseases (MDs) are genetic disorders characterized by dysfunctions in mitochondria. Clinical data suggest that additional factors, beyond genetics, contribute to the onset and progression of this group of diseases, but these influencing factors remain largely unknown. Mounting evidence indicates that immune dysregulation or distress could play a role. Clinical observations have described the co-incidence of infection and the onset of the disease as well as the worsening of symptoms following infection. These findings highlight the complex interactions between MDs and immunity and underscore the need to better understand their underlying relationships. RESULTS: We used Ndufs4 KO mice, a well-established mouse model of Leigh syndrome (one of the most relevant MDs), to test whether chronic induction of a neuroinflammatory state in the central nervous system before the development of neurological symptoms would affect both the onset and progression of the disease in Ndufs4 KO mice. To this aim, we took advantage of the GFAP-IL6 mouse, which overexpresses interleukin-6 (IL-6) in astrocytes and produces chronic glial reactivity, by generating a mouse line with IL-6 overexpression and NDUFS4 deficiency. IL-6 overexpression aggravated the mortality of female Ndufs4 KO mice but did not alter the main motor and respiratory phenotypes measured in any sex. Interestingly, an abnormal region-dependent microglial response to IL-6 overexpression was observed in Ndufs4 KO mice compared to controls. CONCLUSION: Overall, our data indicate that chronic neuroinflammation may worsen the disease in Ndufs4 KO female mice, but not in males, and uncovers an abnormal microglial response due to OXPHOS dysfunction, which may have implications for our understanding of the effect of OXPHOS dysfunction in microglia.

Induction of atypical EAE mediated by transgenic production of IL-6 in astrocytes in the absence of systemic IL-6.

Interleukin (IL)-6 is crucial for the induction of many murine models of autoimmunity including experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. While IL-6-deficient mice (IL-6 KO) are resistant to EAE, we showed previously that in transgenic mice with astrocyte-targeted production of IL-6-restricted to the cerebellum (GFAP-IL6), EAE induced with MOG(35-55) was redirected away from the spinal cord to the cerebellum. To further establish the importance of IL-6 produced in the central nervous system, we have generated mice producing IL-6 essentially only in the brain by crossing the GFAP-IL6 mice with IL-6 KO mice. Interestingly, GFAP-IL6-IL-6 KO mice showed a milder but almost identical phenotype as the GFAP-IL6 mice, which correlated with a lower load of inflammatory cells and decreased microglial reactivity. These results indicate that not only is cerebellar IL-6 production and eventual leakage into the peripheral compartment the dominating factor controlling this type of EAE but that it can also facilitate induction of autoimmunity in the absence of normal systemic IL-6 production.

Complex I deficiency due to loss of Ndufs4 in the brain results in progressive encephalopathy resembling Leigh syndrome.

To explore the lethal, ataxic phenotype of complex I deficiency in Ndufs4 knockout (KO) mice, we inactivated Ndufs4 selectively in neurons and glia (NesKO mice). NesKO mice manifested the same symptoms as KO mice including retarded growth, loss of motor ability, breathing abnormalities, and death by approximately 7 wk. Progressive neuronal deterioration and gliosis in specific brain areas corresponded to behavioral changes as the disease advanced, with early involvement of the olfactory bulb, cerebellum, and vestibular nuclei. Neurons, particularly in these brain regions, had aberrant mitochondrial morphology. Activation of caspase 8, but not caspase 9, in affected brain regions implicate the initiation of the extrinsic apoptotic pathway. Limited caspase 3 activation and the predominance of ultrastructural features of necrotic cell death suggest a switch from apoptosis to necrosis in affected neurons. These data suggest that dysfunctional complex I in specific brain regions results in progressive glial activation that promotes neuronal death that ultimately results in mortality.

CPT1A in AgRP neurons is required for sex-dependent regulation of feeding and thirst.

BACKGROUND: Fatty acid metabolism in the hypothalamus has an important role in food intake, but its specific role in AgRP neurons is poorly understood. Here, we examined whether carnitinea palmitoyltransferase 1A (CPT1A), a key enzyme in mitochondrial fatty acid oxidation, affects energy balance. METHODS: To obtain Cpt1aKO mice and their control littermates, Cpt1a(flox/flox) mice were crossed with tamoxifen-inducible AgRPCreERT2 mice. Food intake and body weight were analyzed weekly in both males and females. At 12 weeks of age, metabolic flexibility was determined by ghrelin-induced food intake and fasting-refeeding satiety tests. Energy expenditure was analyzed by calorimetric system and thermogenic activity of brown adipose tissue. To study fluid balance the analysis of urine and water intake volumes; osmolality of urine and plasma; as well as serum levels of angiotensin and components of RAAS (renin-angiotensin-aldosterone system) were measured. At the central level, changes in AgRP neurons were determined by: (1) analyzing specific AgRP gene expression in RiboTag-Cpt1aKO mice obtained by crossing Cpt1aKO mice with RiboTag mice; (2) measuring presynaptic terminal formation in the AgRP neurons with the injection of the AAV1-EF1a-DIO-synaptophysin-GFP in the arcuate nucleus of the hypothalamus; (3) analyzing AgRP neuronal viability and spine formations by the injection AAV9-EF1a-DIO-mCherry in the arcuate nucleus of the hypothalamus; (4) analyzing in situ the specific AgRP mitochondria in the ZsGreen-Cpt1aKO obtained by breeding ZsGreen mice with Cpt1aKO mice. Two-way ANOVA analyses were performed to determine the contributions of the effect of lack of CPT1A in AgRP neurons in the sex. RESULTS: Changes in food intake were just seen in male Cpt1aKO mice while only female Cpt1aKO mice increased energy expenditure. The lack of Cpt1a in the AgRP neurons enhanced brown adipose tissue activity, mainly in females, and induced a substantial reduction in fat deposits and body weight. Strikingly, both male and female Cpt1aKO mice showed polydipsia and polyuria, with more reduced serum vasopressin levels in females and without osmolality alterations, indicating a direct involvement of Cpt1a in AgRP neurons in fluid balance. AgRP neurons from Cpt1aKO mice showed a sex-dependent gene expression pattern, reduced mitochondria and decreased presynaptic innervation to the paraventricular nucleus, without neuronal viability alterations. CONCLUSIONS: Our results highlight that fatty acid metabolism and CPT1A in AgRP neurons show marked sex differences and play a relevant role in the neuronal processes necessary for the maintenance of whole-body fluid and energy balance.

Excitatory neuron-specific suppression of the integrated stress response contributes to autism-related phenotypes in fragile X syndrome.

Dysregulation of protein synthesis is one of the key mechanisms underlying autism spectrum disorder (ASD). However, the role of a major pathway controlling protein synthesis, the integrated stress response (ISR), in ASD remains poorly understood. Here, we demonstrate that the main arm of the ISR, eIF2α phosphorylation (p-eIF2α), is suppressed in excitatory, but not inhibitory, neurons in a mouse model of fragile X syndrome (FXS; Fmr1-/y). We further show that the decrease in p-eIF2α is mediated via activation of mTORC1. Genetic reduction of p-eIF2α only in excitatory neurons is sufficient to increase general protein synthesis and cause autism-like behavior. In Fmr1-/y mice, restoration of p-eIF2α solely in excitatory neurons reverses elevated protein synthesis and rescues autism-related phenotypes. Thus, we reveal a previously unknown causal relationship between excitatory neuron-specific translational control via the ISR pathway, general protein synthesis, and core phenotypes reminiscent of autism in a mouse model of FXS.

Cell-type-specific isolation of ribosome-associated mRNA from complex tissues.

Gene profiling techniques allow the assay of transcripts from organs, tissues, and cells with an unprecedented level of coverage. However, most of these approaches are still limited by the fact that organs and tissues are composed of multiple cell types that are each unique in their patterns of gene expression. To identify the transcriptome from a single cell type in a complex tissue, investigators have relied upon physical methods to separate cell types or in situ hybridization and immunohistochemistry. Here, we describe a strategy to rapidly and efficiently isolate ribosome-associated mRNA transcripts from any cell type in vivo. We have created a mouse line, called RiboTag, which carries an Rpl22 allele with a floxed wild-type C-terminal exon followed by an identical C-terminal exon that has three copies of the hemagglutinin (HA) epitope inserted before the stop codon. When the RiboTag mouse is crossed to a cell-type-specific Cre recombinase-expressing mouse, Cre recombinase activates the expression of epitope-tagged ribosomal protein RPL22(HA), which is incorporated into actively translating polyribosomes. Immunoprecipitation of polysomes with a monoclonal antibody against HA yields ribosome-associated mRNA transcripts from specific cell types. We demonstrate the application of this technique in brain using neuron-specific Cre recombinase-expressing mice and in testis using a Sertoli cell Cre recombinase-expressing mouse.

Molecular and anatomical characterization of parabrachial neurons and their axonal projections.

The parabrachial nucleus (PBN) is a major hub that receives sensory information from both internal and external environments. Specific populations of PBN neurons are involved in behaviors including food and water intake, nociceptive responses, breathing regulation, as well as learning and responding appropriately to threatening stimuli. However, it is unclear how many PBN neuron populations exist and how different behaviors may be encoded by unique signaling molecules or receptors. Here we provide a repository of data on the molecular identity, spatial location, and projection patterns of dozens of PBN neuron subclusters. Using single-cell RNA sequencing, we identified 21 subclusters of neurons in the PBN and neighboring regions. Multiplexed in situ hybridization showed many of these subclusters are enriched within specific PBN subregions with scattered cells in several other regions. We also provide detailed visualization of the axonal projections from 21 Cre-driver lines of mice. These results are all publicly available for download and provide a foundation for further interrogation of PBN functions and connections.

Site-specific production of IL-6 in the central nervous system retargets and enhances the inflammatory response in experimental autoimmune encephalomyelitis.

IL-6 is crucial for the induction of many murine models of autoimmunity including experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. To establish the role of site-specific production of IL-6 in autoimmunity, we examined myelin oligodendrocyte glycoprotein immunization-induced EAE in transgenic mice (GFAP-IL6) with IL-6 production restricted to the cerebellum. Myelin oligodendrocyte glycoprotein-immunized (Mi-) GFAP-IL6 mice developed severe ataxia but no physical signs of spinal cord involvement, which was in sharp contrast to Mi-wild type (WT) animals that developed classical EAE with ascending paralysis. Immune pathology and demyelination were nearly absent from the spinal cord, but significantly increased in the cerebellum of Mi-GFAP-IL6 mice. Tissue damage in the cerebellum in the Mi-GFAP-IL6 mice was accompanied by increased total numbers of infiltrating leukocytes and increased proportions of both neutrophils and B-cells. With the exception of IL-17 mRNA, which was elevated in both control immunized and Mi-GFAP-IL6 cerebellum, the level of other cytokine and chemokine mRNAs were comparable with Mi-WT cerebellum whereas significantly higher levels of IFN-gamma and TNF-alpha mRNA were found in Mi-WT spinal cord. Thus, site-specific production of IL-6 in the cerebellum redirects trafficking away from the normally preferred antigenic site the spinal cord and acts as a leukocyte "sink" that markedly enhances the inflammatory cell accumulation and disease. The mechanisms underlying this process likely include the induction of specific chemokines, activation of microglia, and activation and loss of integrity of the blood-brain barrier present in the cerebellum of the GFAP-IL6 mice before the induction of EAE.

PF9601N [N-(2-propynyl)-2-(5-benzyloxy-indolyl) methylamine] confers MAO-B independent neuroprotection in ER stress-induced cell death.

Endoplasmic reticulum (ER) stress has recently been proposed as one of the factors contributing to apoptotic cell death in Parkinson's disease (PD). Although MAO-B inhibitors have been suggested to exert neuroprotective effects in several experimental models of PD, their effectiveness against ER stress has not been fully determined. Therefore, we have studied the potential usefulness of PF9601N, a non-amphetamine-like MAO-B inhibitor, in preventing cell death in a cell culture model of ER stress. Exposure of human dopaminergic cell line SH-SY5Y to the ER stressor brefeldin A led to Golgi disassembly, activation of the unfolded protein response (UPR), and subsequent expression of the proapoptotic mediator GADD153/CHOP. In this context, PF9601N pretreatment prevented brefeldin A-induced UPR responses, thus blocking the expression of GADD153/CHOP and resulting apoptotic features. In summary, our data suggests that PF9601N is able to block the responses elicited by ER stress, thus preventing apoptotic cell death in brefeldin A-treated cells.

Mitochondrial Proteome of Affected Glutamatergic Neurons in a Mouse Model of Leigh Syndrome.

Defects in mitochondrial function lead to severe neuromuscular orphan pathologies known as mitochondrial disease. Among them, Leigh Syndrome is the most common pediatric presentation, characterized by symmetrical brain lesions, hypotonia, motor and respiratory deficits, and premature death. Mitochondrial diseases are characterized by a marked anatomical and cellular specificity. However, the molecular determinants for this susceptibility are currently unknown, hindering the efforts to find an effective treatment. Due to the complex crosstalk between mitochondria and their supporting cell, strategies to assess the underlying alterations in affected cell types in the context of mitochondrial dysfunction are critical. Here, we developed a novel virus-based tool, the AAV-mitoTag viral vector, to isolate mitochondria from genetically defined cell types. Expression of the AAV-mitoTag in the glutamatergic vestibular neurons of a mouse model of Leigh Syndrome lacking the complex I subunit Ndufs4 allowed us to assess the proteome and acetylome of a subset of susceptible neurons in a well characterized model recapitulating the human disease. Our results show a marked reduction of complex I N-module subunit abundance and an increase in the levels of the assembly factor NDUFA2. Transiently associated non-mitochondrial proteins such as PKCδ, and the complement subcomponent C1Q were also increased in Ndufs4-deficient mitochondria. Furthermore, lack of Ndufs4 induced ATP synthase complex and pyruvate dehydrogenase (PDH) subunit hyperacetylation, leading to decreased PDH activity. We provide novel insight on the pathways involved in mitochondrial disease, which could underlie potential therapeutic approaches for these pathologies.

Functional and molecular heterogeneity of D2R neurons along dorsal ventral axis in the striatum.

Action control is a key brain function determining the survival of animals in their environment. In mammals, neurons expressing dopamine D2 receptors (D2R) in the dorsal striatum (DS) and the nucleus accumbens (Acb) jointly but differentially contribute to the fine regulation of movement. However, their region-specific molecular features are presently unknown. By combining RNAseq of striatal D2R neurons and histological analyses, we identified hundreds of novel region-specific molecular markers, which may serve as tools to target selective subpopulations. As a proof of concept, we characterized the molecular identity of a subcircuit defined by WFS1 neurons and evaluated multiple behavioral tasks after its temporally-controlled deletion of D2R. Consequently, conditional D2R knockout mice displayed a significant reduction in digging behavior and an exacerbated hyperlocomotor response to amphetamine. Thus, targeted molecular analyses reveal an unforeseen heterogeneity in D2R-expressing striatal neuronal populations, underlying specific D2R's functional features in the control of specific motor behaviors.

Monoamine oxidase-B activity is not involved in the neuroinflammatory response elicited by a focal freeze brain injury.

Cryolesion of the frontoparietal cortex in mice is a well-described brain injury paradigm that results in increased astrogliosis surrounding the lesion site and is accompanied by a prominent increase in the MAO-B levels in astrocytes. Whether these increased MAO-B levels contribute to cellular damage or modulate reactive astrocytosis remains unclear. MAO-B activity may contribute to cellular damage, since its metabolism products are highly toxic to the cells. Additionally, it has been suggested that MAO-B inhibition may regulate astrocytic reaction. In this study, we have determined the relative contribution of MAO-B activity to the outcome following freeze injury. Freeze injury induced a prominent increase of several inflammatory markers, including ICAM, Mac-1, EB22, and GFAP. Inhibition of MAO-B activity using the selective inhibitor PF9601N did not reduce this cryolesion-induced inflammatory response. Additional data revealed that the expression of several cryolesion-induced cell death genes, such as Fas, Rip, p53, and ICE, was not reduced in PF9601N-treated mice, evidencing that MAO-B activity did not contribute to cryolesion-induced cell death. Definitive functional analysis of the mice using the ladder beam task revealed that MAO-B inhibition did not improve the cryolesion-induced motor impairment. These data strongly suggest that, although MAO-B is highly expressed in the area surrounding the lesion site, its activity does not contribute to the cellular damage or play any role in regulating astrocytic reactivity.