Surfaceome mapping of primary human heart cells with CellSurfer uncovers cardiomyocyte surface protein LSMEM2 and proteome dynamics in failing hearts.

Cardiac cell surface proteins are drug targets and useful biomarkers for discriminating among cellular phenotypes and disease states. Here we developed an analytical platform, CellSurfer, that enables quantitative cell surface proteome (surfaceome) profiling of cells present in limited quantities, and we apply it to isolated primary human heart cells. We report experimental evidence of surface localization and extracellular domains for 1,144 N-glycoproteins, including cell-type-restricted and region-restricted glycoproteins. We identified a surface protein specific for healthy cardiomyocytes, LSMEM2, and validated an anti-LSMEM2 monoclonal antibody for flow cytometry and imaging. Surfaceome comparisons among pluripotent stem cell derivatives and their primary counterparts highlighted important differences with direct implications for drug screening and disease modeling. Finally, 20% of cell surface proteins, including LSMEM2, were differentially abundant between failing and non-failing cardiomyocytes. These results represent a rich resource to advance development of cell type and organ-specific targets for drug delivery, disease modeling, immunophenotyping and in vivo imaging.

Dysregulated cholesterol metabolism, aberrant excitability and altered cell cycle of astrocytes in fragile X syndrome.

Fragile X syndrome (FXS), the most prevalent heritable form of intellectual disability, is caused by the transcriptional silencing of the FMR1 gene. While neuronal contribution to FXS has been extensively studied in both animal and human-based models of FXS, the roles of astrocytes, a type of glial cells in the brain, are largely unknown. Here, we generated a human-based FXS model via differentiation of astrocytes from human-induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) and characterized their development, function, and proteomic profiles. We identified shortened cell cycle, enhanced Ca2+ signaling, impaired sterol biosynthesis, and pervasive alterations in the proteome of FXS astrocytes. Our work identified astrocytic impairments that could contribute to the pathogenesis of FXS and highlight astrocytes as a novel therapeutic target for FXS treatment.

Bottom-up proteomic analysis of human adult cardiac tissue and isolated cardiomyocytes.

The heart is composed of multiple cell types, each with a specific function. Cell-type-specific approaches are necessary for defining the intricate molecular mechanisms underlying cardiac development, homeostasis, and pathology. While single-cell RNA-seq studies are beginning to define the chamber-specific cellular composition of the heart, our views of the proteome are more limited because most proteomics studies have utilized homogenized human cardiac tissue. To promote future cell-type specific analyses of the human heart, we describe the first method for cardiomyocyte isolation from cryopreserved human cardiac tissue followed by flow cytometry for purity assessment. We also describe a facile method for preparing isolated cardiomyocytes and whole cardiac tissue homogenate for bottom-up proteomic analyses. Prior experience in dissociating cardiac tissue or proteomics is not required to execute these methods. We compare different sample preparation workflows and analysis methods to demonstrate how these can impact the depth of proteome coverage achieved. We expect this how-to guide will serve as a starting point for investigators interested in general and cell-type-specific views of the cardiac proteome.

N-Acetylcysteine Reverses Antiretroviral-Mediated Microglial Activation by Attenuating Autophagy-Lysosomal Dysfunction.

Successful suppression of viral replication by combined antiretroviral therapy (cART) in HIV-1 infected individuals is paradoxically also accompanied by an increased prevalence of HIV-associated neurocognitive disorders (HAND) in these individuals. HAND is characterized by a state of chronic oxidative stress and inflammation. Microglia are extremely sensitive to a plethora of stimuli, including viral proteins and cART. The current study aimed to assess the effects of cART-mediated oxidative stress on the induction of inflammatory responses in microglia. In the present study, we chose a combination of three commonly used antiretroviral drugs-tenofovir disoproxil fumarate, emtricitabine, and dolutegravir. We demonstrated that exposure of microglia to the chosen cART cocktail induced generation of reactive oxygen species, subsequently leading to lysosomal dysfunction and dysregulated autophagy, ultimately resulting in the activation of microglia. Intriguingly, the potent antioxidant, N-acetylcysteine, reversed the damaging effects of cART. These in vitro findings were further corroborated in vivo wherein cART-treated HIV transgenic (Tg) rats demonstrated increased microglial activation, exaggerated lysosome impairment, and dysregulated autophagy in the prefrontal cortices compared with HIV Tg rats not exposed to cART. Similar to in vitro findings, the treatment of HIV Tg rats with N-acetylcysteine also mitigated the deleterious effects of cART. Taken together, our findings suggest that oxidative stress-mediated lysosomal dysfunction plays a critical role in the pathogenesis of HAND in drug-treated HIV-infected individuals and that antioxidant-mediated mitigation of oxidative stress could thus be considered as an adjunctive therapeutic strategy for ameliorating/dampening some of the neurological complications of HAND.

KVA-D-88, a Novel Preferable Phosphodiesterase 4B Inhibitor, Decreases Cocaine-Mediated Reward Properties in Vivo.

Cocaine addiction remains a major public concern throughout the world especially in developed countries. In the last three decades, significant achievements have led to a greater understanding of the signaling pathways involved in the development of cocaine addiction; however, there are no FDA-approved treatments available to reverse or block this brain disease due to either the unsatisfactory therapeutic efficacy or severe side effects. Previous studies have demonstrated that chronic exposure to cocaine elevates levels of cyclic AMP (cAMP) as a neuroadaptative response in reward-related brain regions. Phosphodiesterase 4 (PDE4) inhibitors, which elevate cAMP levels, have been shown to block cocaine-mediated behavioral changes related to psychoactive and reinforcing properties. Unfortunately, previously studied PDE4 inhibitors induce severe side-effects, which limit their clinical usage. In this study, we identified a novel PDE4B inhibitor, KVA-D-88, with an improved selectivity profile compared to previous compounds (e.g., rolipram). Pharmacokinetic studies have shown that this compound is brain penetrant and preferably acts on PDE4B compared to PDE4D in vitro, alluding to less unwanted side effects with KVA-D-88 in vivo. Interestingly, pretreatment with KVA-D-88 significantly inhibited cocaine-induced hyperlocomotor activity. In cocaine self-administering mice with differential schedules, KVA-D-88 strikingly decreased the number of active nose-pokes and cocaine infusions and reduced the break point. Taken together, our findings demonstrate that this novel PDE4 inhibitor, KVA-D-88, could inhibit cocaine-mediated rewarding effects implying its potential clinical usage for cocaine addiction.

Cocaine self-administration differentially activates microglia in the mouse brain.

The evidence showing the involvement of microglial activation in the development of drug addiction remain scarce as microglia have not been systematically investigated in self-administered mice, a gold standard rodent model for drug addiction. Here we established the stable cocaine self-administration mice to examine microglial activation levels in various brain regions related to reward circuitry. Immunostaining for Iba1 showed a significant upregulation of intensity in the striatum but not in the medial prefrontal cortex (mPFc), hippocampus or thalamus. Further validation experiments showed that cocaine self-administered mice had significantly increased mRNA expression of ccl2 and IL1ß in the striatum but not the mPFc compared to saline controls. Consistently, we found elevated protein levels of Iba1, CCL2, TLR4 and mature IL1ß in the striatum, not in the mPFc of cocaine-receiving mice. In addition, cocaine-stimulated microglia had modified morphology including a reduced number of intersections, a shortened length and number of processes in the NAc. In summary, our results demonstrated that cocaine mediated microglial activation in a region-specific manner in vivo. These findings indicate that microglia could be activated in the early stage of cocaine addiction directly supporting the rationale that dysregulation on neuroimmune signaling is inherently involved in the development of drug addiction.

Antiretroviral-Mediated Microglial Activation Involves Dysregulated Autophagy and Lysosomal Dysfunction.

In the era of combined antiretroviral therapy (cART), as infected individuals continue to have longer lifespans, there is also an increased prevalence of HIV-associated neurocognitive disorders (HAND). Inflammation is one of the underlying features of HAND, with the role of viral proteins and antiretroviral drugs implicated in this process. Microglia are extremely sensitive to a plethora of stimuli, including viral products and cART. The current study was undertaken to understand the molecular mechanism(s) underlying cART-mediated activation of microglia. Herein we chose a combination of three commonly used drugs, tenofovir disoproxil fumarate (TDF), emtricitabine (FTC), and dolutegravir (DTG). We demonstrated that exposure of microglia to this cART cocktail induced lysosomal membrane permeabilization (LMP), which subsequently resulted in impaired lysosomal functioning involving elevated pH and decreased cathepsin D (CTSD) activity. cART exposure of microglia resulted in increased formation of autophagosomes as demonstrated by a time-dependent increase of autophagy markers, with a concomitant defect in the fusion of the lysosomes with the autophagosome. Taken together, our findings suggest a novel mechanism by which cART impairs lysosomal functioning, resulting in dysregulated autophagy and increased neuroinflammation. Interventions aimed at lysosome protection could likely be envisioned as promising therapeutic targets for abrogating cART-mediated microglia activation, which in turn, could thus be considered as adjunctive therapeutics for the treatment of HAND pathogenesis.

Caspase 1 activity influences juvenile Batten disease (CLN3) pathogenesis.

Juvenile Neuronal Ceroid Lipofuscinosis (JNCL) is an autosomal recessive lysosomal storage disease caused by loss-of-function mutations in CLN3. Symptoms appear between 5 and 10 years of age, beginning with blindness and seizures, followed by progressive cognitive and motor decline, and premature death. Glial activation and impaired neuronal activity are early signs of pathology in the Cln3Δex7/8 mouse model of JNCL, whereas neuron death occurs much later in the disease process. We previously reported that Cln3Δex7/8 microglia are primed toward a pro-inflammatory phenotype typified by exaggerated caspase 1 inflammasome activation and here we extend those findings to demonstrate heightened caspase activity in the Cln3Δex7/8 mouse brain. Based on the ability of caspase 1 to cleave a large number of substrates that have been implicated in JNCL pathology, we examined the functional implications of caspase 1 inflammasome activity by crossing Cln3Δex7/8 and caspase 1-deficient mice to create Cln3Δex7/8 /Casp-1-/- animals. Caspase 1 deletion influenced motor behavior deficits and astrocyte activation in the context of CLN3 mutation, since both were significantly reversed in Cln3Δex7/8 /Casp-1-/- mice, with phenotypes approaching that of wild-type animals. We also report a progressive age-dependent reduction in whisker length in Cln3Δex7/8 mice that was partially caspase 1-dependent. However, not all CLN3 phenotypes were reversed following caspase 1 deletion, since no significant differences in lysosomal accumulation or microglial activation were observed between Cln3Δex7/8 and Cln3Δex7/8 /Casp-1-/- mice. Although the molecular targets of aberrant caspase 1 activity in the context of CLN3 mutation remain to be identified, our studies suggest that caspase 1 may represent a potential therapeutic target to mitigate some attributes of CLN3 disease. This article is part of the Special Issue "Lysosomal Storage Disorders".

Age-dependent alterations in neuronal activity in the hippocampus and visual cortex in a mouse model of Juvenile Neuronal Ceroid Lipofuscinosis (CLN3).

Juvenile Neuronal Ceroid Lipofuscinosis (JNCL) is a fatal lysosomal storage disease caused by autosomal recessive mutations in CLN3. JNCL is typified by progressive neurodegeneration that has been suggested to occur from excessive excitatory and impaired inhibitory synaptic input; however, no studies to date have directly evaluated neuronal function. To examine changes in neuronal activity with advancing disease, electrophysiological recordings were performed in the CA1 hippocampus (HPC) and visual cortex (VC) of acute brain slices from Cln3Δex7/8 mice at 1, 4, 8, and 12months of age. Basic electrophysiological parameters, such as field excitatory post-synaptic potential (fePSP) and population spike (PS) amplitudes, were not altered in Cln3Δex7/8 CA1 and VC neurons at any age. However, fiber volley (FV) amplitudes were significantly increased in Cln3Δex7/8 neurons in the HPC at 1month as well as layer II/III of the VC at 1 and 4months, suggesting increased axonal excitability. In older Cln3Δex7/8 mice (8 and 12months), FV amplitude in the CA1 HPC and VC reached levels that were equal to or significantly lower than WT animals. Significant alterations in the synaptic strength of Cln3Δex7/8 CA1 neurons were also linked to age-dependent changes in axonal responses. Additionally, paired-pulse and 12-pulse facilitation responses calculated from PS recordings were significantly decreased in the CA1 HPC and layer II/III of the VC of Cln3Δex7/8 mice at all ages, suggesting permanent alterations in neuronal short-term plasticity. Collectively, our study has identified novel age- and region-dependent alterations in axonal excitability as well as synaptic and non-synaptic neuronal activity in Cln3Δex7/8 mice during disease progression, which may inform neurodegenerative mechanisms in JNCL.

Efficacy of phosphodiesterase-4 inhibitors in juvenile Batten disease (CLN3).

OBJECTIVE: Juvenile neuronal ceroid lipofuscinosis (JNCL), or juvenile Batten disease, is a pediatric lysosomal storage disease caused by autosomal recessive mutations in CLN3, typified by blindness, seizures, progressive cognitive and motor decline, and premature death. Currently, there is no treatment for JNCL that slows disease progression, which highlights the need to explore novel strategies to extend the survival and quality of life of afflicted children. Cyclic adenosine monophosphate (cAMP) is a second messenger with pleiotropic effects, including regulating neuroinflammation and neuronal survival. Here we investigated whether 3 phosphodiesterase-4 (PDE4) inhibitors (rolipram, roflumilast, and PF-06266047) could mitigate behavioral deficits and cell-specific pathology in the Cln3Δex7/8 mouse model of JNCL. METHODS: In a randomized, blinded study, wild-type (WT) and Cln3Δex7/8 mice received PDE4 inhibitors daily beginning at 1 or 3 months of age and continuing for 6 to 9 months, with motor deficits assessed by accelerating rotarod testing. The effect of PDE4 inhibitors on cAMP levels, astrocyte and microglial activation (glial fibrillary acidic protein and CD68, respectively), lysosomal pathology (lysosomal-associated membrane protein 1), and astrocyte glutamate transporter expression (glutamate/aspartate transporter) were also examined in WT and Cln3Δex7/8 animals. RESULTS: cAMP levels were significantly reduced in the Cln3Δex7/8 brain, and were restored by PF-06266047. PDE4 inhibitors significantly improved motor function in Cln3Δex7/8 mice, attenuated glial activation and lysosomal pathology, and restored glutamate transporter expression to levels observed in WT animals, with no evidence of toxicity as revealed by blood chemistry analysis. INTERPRETATION: These studies reveal neuroprotective effects for PDE4 inhibitors in Cln3Δex7/8 mice and support their therapeutic potential in JNCL patients. Ann Neurol 2016;80:909-923.

Self-Complementary AAV9 Gene Delivery Partially Corrects Pathology Associated with Juvenile Neuronal Ceroid Lipofuscinosis (CLN3).

UNLABELLED: Juvenile neuronal ceroid lipofuscinosis (JNCL) is a fatal lysosomal storage disease caused by autosomal-recessive mutations in CLN3 for which no treatment exists. Symptoms appear between 5 and 10 years of age, beginning with blindness and seizures, followed by progressive cognitive and motor decline and premature death (late teens to 20s). We explored a gene delivery approach for JNCL by generating two self-complementary adeno-associated virus 9 (scAAV9) constructs to address CLN3 dosage effects using the methyl-CpG-binding protein 2 (MeCP2) and ß-actin promoters to drive low versus high transgene expression, respectively. This approach was based on the expectation that low CLN3 levels are required for cellular homeostasis due to minimal CLN3 expression postnatally, although this had not yet been demonstrated in vivo One-month-old Cln3(Δex7/8) mice received one systemic (intravenous) injection of scAAV9/MeCP2-hCLN3 or scAAV9/ß-actin-hCLN3, with green fluorescent protein (GFP)-expressing viruses as controls. A promoter-dosage effect was observed in all brain regions examined, in which hCLN3 levels were elevated 3- to 8-fold in Cln3(Δex7/8) mice receiving scAAV9/ß-actin-hCLN3 versus scAAV9/MeCP2-hCLN3. However, a disconnect occurred between CLN3 levels and disease improvement, because only the scAAV9 construct driving low CLN3 expression (scAAV9/MeCP2-hCLN3) corrected motor deficits and attenuated microglial and astrocyte activation and lysosomal pathology. This may have resulted from preferential promoter usage because transgene expression after intravenous scAAV9/MeCP2-GFP injection was primarily detected in NeuN(+) neurons, whereas scAAV9/ß-actin-GFP drove transgene expression in GFAP(+) astrocytes. This is the first demonstration of a systemic delivery route to restore CLN3 in vivo using scAAV9 and highlights the importance of promoter selection for disease modification in juvenile animals. SIGNIFICANCE STATEMENT: Juvenile neuronal ceroid lipofuscinosis (JNCL) is a fatal lysosomal storage disease caused by CLN3 mutations. We explored a gene delivery approach using two self-complementary adeno-associated virus 9 (scAAV9) constructs to address CLN3 dosage effects using the methyl-CpG-binding protein 2 (MeCP2) and ß-actin promoters. hCLN3 levels were elevated 3- to 8-fold in Cln3(Δex7/8) mice receiving scAAV9/ß-actin-hCLN3 versus scAAV9/MeCP2-hCLN3 after a single systemic injection. However, only scAAV9/MeCP2-hCLN3 corrected motor deficits and attenuated glial activation and lysosomal pathology. This may reflect preferential promoter usage because transgene expression with scAAV9/MeCP2-green fluorescent protein (GFP) was primarily in neurons, whereas scAAV9/ß-actin-GFP drove transgene expression in astrocytes. This is the first demonstration of systemic delivery for CLN3 using scAAV9 and highlights the importance of promoter selection for disease modification in juvenile animals.

Evidence for aberrant astrocyte hemichannel activity in Juvenile Neuronal Ceroid Lipofuscinosis (JNCL).

Juvenile Neuronal Ceroid Lipofuscinosis (JNCL) is a lysosomal storage disease caused by an autosomal recessive mutation in CLN3 that leads to vision loss, progressive cognitive and motor decline, and premature death. Morphological evidence of astrocyte activation occurs early in the disease process and coincides with regions where neuronal loss eventually ensues. However, the consequences of CLN3 mutation on astrocyte function remain relatively ill-defined. Astrocytes play a critical role in CNS homeostasis, in part, by their ability to regulate the extracellular milieu via the formation of extensive syncytial networks coupled by gap junction (GJ) channels. In contrast, unopposed hemichannels (HCs) have been implicated in CNS pathology by allowing the non-discriminant passage of molecules between the intracellular and extracellular milieus. Here we examined acute brain slices from CLN3 mutant mice (CLN3Δex7/8) to determine whether CLN3 loss alters the balance of GJ and HC activity. CLN3Δex7/8 mice displayed transient increases in astrocyte HC opening at postnatal day 30 in numerous brain regions, compared to wild type (WT) animals; however, HC activity steadily decreased at postnatal days 60 and 90 in CLN3Δex7/8 astrocytes to reach levels lower than WT cells. This suggested a progressive decline in astrocyte function, which was supported by significant reductions in glutamine synthetase, GLAST, and connexin expression in CLN3Δex7/8 mice compared to WT animals. Based on the early increase in astrocyte HC activity, CLN3Δex7/8 mice were treated with the novel carbenoxolone derivative INI-0602 to inhibit HCs. Administration of INI-0602 for a one month period significantly reduced lysosomal ceroid inclusions in the brains of CLN3Δex7/8 mice compared to WT animals, which coincided with significant increases in astrocyte GJ communication and normalization of astrocyte resting membrane potential to WT levels. Collectively, these findings suggest that alterations in astrocyte communication may impact the progression of JNCL and could offer a potential therapeutic target.

IL-1RI (interleukin-1 receptor type I) signalling is essential for host defence and hemichannel activity during acute central nervous system bacterial infection.

Staphylococcus aureus is a common aetiological agent of bacterial brain abscesses. We have previously established that a considerable IL-1 (interleukin-1) response is elicited immediately following S. aureus infection, where the cytokine can exert pleiotropic effects on glial activation and blood-brain barrier permeability. To assess the combined actions of IL-1α and IL-1ß during CNS (central nervous system) infection, host defence responses were evaluated in IL-1RI (IL-1 receptor type I) KO (knockout) animals. IL-1RI KO mice were exquisitely sensitive to intracerebral S. aureus infection, as demonstrated by enhanced mortality rates and bacterial burdens within the first 24 h following pathogen exposure compared with WT (wild-type) animals. Loss of IL-1RI signalling also dampened the expression of select cytokines and chemokines, concomitant with significant reductions in neutrophil and macrophage infiltrates into the brain. In addition, the opening of astrocyte hemichannels during acute infection was shown to be dependent on IL-1RI activity. Collectively, these results demonstrate that IL-1RI signalling plays a pivotal role in the genesis of immune responses during the acute stage of brain abscess development through S. aureus containment, inflammatory mediator production, peripheral immune cell recruitment, and regulation of astrocyte hemichannel activity. Taken in the context of previous studies with MyD88 (myeloid differentiation primary response gene 88) and TLR2 (Toll-like receptor 2) KO animals, the current report advances our understanding of MyD88-dependent cascades and implicates IL-1RI signalling as a major antimicrobial effector pathway during acute brain-abscess formation.

Neuroinflammation alters voltage-dependent conductance in striatal astrocytes.

Neuroinflammation has the capacity to alter normal central nervous system (CNS) homeostasis and function. The objective of the present study was to examine the effects of an inflammatory milieu on the electrophysiological properties of striatal astrocyte subpopulations with a mouse bacterial brain abscess model. Whole cell patch-clamp recordings were performed in striatal glial fibrillary acidic protein (GFAP)-green fluorescent protein (GFP)(+) astrocytes neighboring abscesses at postinfection days 3 or 7 in adult mice. Cell input conductance (G(i)) measurements spanning a membrane potential (V(m)) surrounding resting membrane potential (RMP) revealed two prevalent astrocyte subsets. A1 and A2 astrocytes were identified by negative and positive G(i) increments vs. V(m), respectively. A1 and A2 astrocytes displayed significantly different RMP, G(i), and cell membrane capacitance that were influenced by both time after bacterial exposure and astrocyte proximity to the inflammatory site. Specifically, the percentage of A1 astrocytes was decreased immediately surrounding the inflammatory lesion, whereas A2 cells were increased. These changes were particularly evident at postinfection day 7, revealing increased cell numbers with an outward current component. Furthermore, RMP was inversely modified in A1 and A2 astrocytes during neuroinflammation, and resting G(i) was increased from 21 to 30 nS in the latter. In contrast, gap junction communication was significantly decreased in all astrocyte populations associated with inflamed tissues. Collectively, these findings demonstrate the heterogeneity of striatal astrocyte populations, which experience distinct electrophysiological modifications in response to CNS inflammation.

Neuroinflammation leads to region-dependent alterations in astrocyte gap junction communication and hemichannel activity.

Inflammation attenuates gap junction (GJ) communication in cultured astrocytes. Here we used a well-characterized model of experimental brain abscess as a tool to query effects of the CNS inflammatory milieu on astrocyte GJ communication and electrophysiological properties. Whole-cell patch-clamp recordings were performed on green fluorescent protein (GFP)-positive astrocytes in acute brain slices from glial fibrillary acidic protein-GFP mice at 3 or 7 d after Staphylococcus aureus infection in the striatum. Astrocyte GJ communication was significantly attenuated in regions immediately surrounding the abscess margins and progressively increased to levels typical of uninfected brain with increasing distance from the abscess proper. Conversely, astrocytes bordering the abscess demonstrated hemichannel activity as evident by enhanced ethidium bromide (EtBr) uptake that could be blocked by several pharmacological inhibitors, including the connexin 43 (Cx43) mimetic peptide Gap26, carbenoxolone, the pannexin1 (Panx1) mimetic peptide (10)Panx1, and probenecid. However, hemichannel opening was transient with astrocytic EtBr uptake observed near the abscess at day 3 but not day 7 after infection. The region-dependent pattern of hemichannel activity at day 3 directly correlated with increases in Cx43, Cx30, Panx1, and glutamate transporter expression (glial L-glutamate transporter and L-glutamate/L-aspartate transporter) along the abscess margins. Changes in astrocyte resting membrane potential and input conductance correlated with the observed changes in GJ communication and hemichannel activity. Collectively, these findings indicate that astrocyte coupling and electrical properties are most dramatically affected near the primary inflammatory site and reveal an opposing relationship between the open states of GJ channels versus hemichannels during acute infection. This relationship may extend to other CNS diseases typified with an inflammatory component.

Neuroprotective effects of interleukin-10 and tumor necrosis factor-alpha against hypoxia-induced hyperexcitability in hippocampal slice neurons.

In our previous experiments we have demonstrated that repeated exposures of rat hippocampal slices to brief episodes of hypoxia induce a sustained decrease in the threshold of stimulus-evoked epileptiform discharges in CA1 pyramidal neurons. The aim of this study was to investigate the comparative effects of interleukin-10 (IL-10) and tumor necrosis factor-alpha (TNF-alpha) on the hyperexcitability of CA1 pyramidal neurons induced by brief episodes of hypoxia in the rat hippocampal slices. The method of field potentials measurement in CA1 region of hippocampal slices have been described in our previous work [O. Godukhin, A. Savin, S. Kalemenev, S. Levin, Neuronal hyperexcitability induced by repeated brief episodes of hypoxia in rat hippocampal slices: involvement of ionotropic glutamate receptors and L-type Ca2+ channels, Neuropharmacology 42 (2002) 459-466]. The principal results of our work are summarized as follow. Pro-inflammatory cytokine TNF-alpha (0.8, 4 and 20 ng/ml) and anti-inflammatory cytokine IL-10 (1 and 10 ng/ml) significantly reduced the hyperexcitability in CA1 pyramidal neurons induced by brief episodes of hypoxia in the rat hippocampal slices. The neuroprotective effects of IL-10 and TNF-alpha against the hypoxia-induced hyperexcitability were mediated by anti-hypoxic actions of these cytokines through, possibly, mechanism of preconditioning.