Morphological differentiation of peritumoral brain zone microglia.

The Peritumoral Brain Zone (PBZ) contributes to Glioblastoma (GBM) relapse months after the resection of the original tumor, which is influenced by a variety of pathological factors. Among those, microglia are recognized as one of the main regulators of GBM progression and probably relapse. Although microglial morphology has been analyzed inside GBM and its immediate surroundings, it has not been objectively characterized throughout the PBZ. Thus, we aimed to perform a thorough characterization of microglial morphology in the PBZ and its likely differentiation not just from the tumor-associated microglia but from control tissue microglia. For this purpose, Sprague Dawley rats were intrastriatally implanted with C6 cells to induce a GBM formation. Gadolinium-based magnetic resonance imaging (MRI) was performed to locate the tumor and to define the PBZ (2 mm beyond the tumor border), thus delimitating the different regions of interest (ROIs: core tumoral zone and immediate interface; contralateral striatum as control). Brain slices were obtained and immunolabeled with the microglia marker Iba-1. Sixteen morphological parameters were measured for each cell, significative differences were found in all parameters when comparing the four ROIs. To determine if PBZ microglia could be morphologically differentiated from microglia in other ROIs, hierarchical clustering analysis was performed, revealing that microglia can be separated into four morphologically differentiated clusters, each of them mostly integrated by cells sampled in each ROI. Furthermore, a classifier based on linear discriminant analysis, including only three morphological parameters, categorized microglial cells across the studied ROIs and showed a gradual transition between them. The robustness of this classification was assessed through principal component analysis with the remaining 13 morphological parameters, corroborating the obtained results. Thus, in this study we provided objective and quantitative evidence that PBZ microglia represent a differentiable microglial morphotype that could contribute to the recurrence of GBM in this area.

Deleterious and protective effects of epothilone-D alone and in the context of amyloid ß- and tau-induced alterations.

Amyloid-ß (Aß) and hyperphosphorylated tau (P-tau) are Alzheimer's disease (AD) biomarkers that interact in a complex manner to induce most of the cognitive and brain alterations observed in this disease. Since the neuronal cytoskeleton is a common downstream pathological target of tau and Aß, which mostly lead to augmented microtubule instability, the administration of microtubule stabilizing agents (MSAs) can protect against their pathological actions. However, the effectiveness of MSAs is still uncertain due to their state-dependent negative effects; thus, evaluating their specific actions in different pathological or physiological conditions is required. We evaluated whether epothilone-D (Epo-D), a clinically used MSA, rescues from the functional and behavioral alterations produced by intracerebroventricular injection of Aß, the presence of P-tau, or their combination in rTg4510 mice. We also explored the side effects of Epo-D. To do so, we evaluated hippocampal-dependent spatial memory with the Hebb-Williams maze, hippocampal CA1 integrity and the intrinsic and synaptic properties of CA1 pyramidal neurons with the patch-clamp technique. Aß and P-tau mildly impaired memory retrieval, but produced contrasting effects on intrinsic excitability. When Aß and P-tau were combined, the alterations in excitability and spatial reversal learning (i.e., cognitive flexibility) were exacerbated. Interestingly, Epo-D prevented most of the impairments induced Aß and P-tau alone and combined. However, Epo-D also exhibited some side effects depending on the prevailing pathological or physiological condition, which should be considered in future preclinical and translational studies. Although we did not perform extensive histopathological evaluations or measured microtubule stability, our findings show that MSAs can rescue the consequences of AD-like conditions but otherwise be harmful if administered at a prodromal stage of the disease.

Perinatal inflammation and gestational intermittent hypoxia disturbs respiratory rhythm generation and long-term facilitation in vitro: Partial protection by acute minocycline.

Perinatal inflammation triggers breathing disturbances early in life and affects the respiratory adaptations to challenging conditions, including the generation of amplitude long-term facilitation (LTF) by acute intermittent hypoxia (AIH). Some of these effects can be avoided by anti-inflammatory treatments like minocycline. Since little is known about the effects of perinatal inflammation on the inspiratory rhythm generator, located in the preBötzinger complex (preBötC), we tested the impact of acute lipopolysaccharide (LPS) systemic administration (sLPS), as well as gestational LPS (gLPS) and gestational chronic IH (gCIH), on respiratory rhythm generation and its long-term response to AIH in a brainstem slice preparation from neonatal mice. We also evaluated whether acute minocycline administration could influence these effects. We found that perinatal inflammation induced by sLPS or gLPS, as well as gCIH, modulate the frequency, signal-to-noise ratio and/or amplitude (and their regularity) of the respiratory rhythm recorded from the preBötC in the brainstem slice. Moreover, all these perinatal conditions inhibited frequency LTF and amplitude long-term depression (LTD); gCIH even induced frequency LTD of the respiratory rhythm after AIH. Some of these alterations were not observed in slices pre-treated in vitro with minocycline, when compared with slices obtained from naïve pups, suggesting that ongoing inflammatory conditions affect respiratory rhythm generation and its plasticity. Thus, it is likely that alterations in the inspiratory rhythm generator and its adaptive responses could contribute to the respiratory disturbances observed in neonates that suffered from perinatal inflammatory challenges.

Configuration and dynamics of dominant inspiratory multineuronal activity patterns during eupnea and gasping generation in vitro.

The pre-Bötzinger complex (preBötC), located within the ventral respiratory column, produces inspiratory bursts in varying degrees of synchronization/amplitude. This wide range of population burst patterns reflects the flexibility of the preBötC neurons, which is expressed in variations in the onset/offset times of their activations and their activity during the population bursts, with respiratory neurons exhibiting a large cycle-to-cycle timing jitter both at the population activity onset and at the population activity peak, suggesting that respiratory neurons are stochastically activated before and during the inspiratory bursts. However, it is still unknown whether this stochasticity is maintained while evaluating the coactivity of respiratory neuronal ensembles. Moreover, the preBötC topology also remains unknown. In this study, by simultaneously recording tens of preBötC neurons and using coactivation analysis during the inspiratory periods, we found that the preBötC has a scale-free configuration (mixture of not many highly connected nodes, hubs, with abundant poorly connected elements) exhibiting the rich-club phenomenon (hubs more likely interconnected with each other). PreBötC neurons also produce multineuronal activity patterns (MAPs) that are highly stable and change during the hypoxia-induced reconfiguration. Moreover, preBötC contains a coactivating core network shared by all its MAPs. Finally, we found a distinctive pattern of sequential coactivation of core network neurons at the beginning of the inspiratory periods, indicating that, when evaluated at the multicellular level, the coactivation of respiratory neurons seems not to be stochastic.NEW & NOTEWORTHY By means of multielectrode recordings of preBötC neurons, we evaluated their configuration in normoxia and hypoxia, finding that the preBötC exhibits a scale-free configuration with a rich-club phenomenon. preBötC neurons produce multineuronal activity patterns that are highly stable but change during hypoxia. The preBötC contains a coactivating core network that exhibit a distinctive pattern of coactivation at the beginning of inspirations. These results reveal some network basis of inspiratory rhythm generation and its reconfiguration during hypoxia.

Interleukin 1-beta but not the interleukin-1 receptor antagonist modulates inspiratory rhythm generation in vitro.

Interleukin 1-beta (IL-1ß) is a cytokine that modulates breathing when applied systemically or directly into the brain. IL-1ß is expressed, along with its receptors, in IL-1ß-sensitive respiratory-related circuits, which likely include the inspiratory rhythm generator (the preBötzinger complex, preBötC). Thus, considering that IL-1ß might directly modulate preBötC function, we tested whether IL-1ß and its endogenous antagonist IL1Ra modulate inspiratory rhythm generation in the brainstem slice preparation containing the preBötC. We found that IL-1ß reduces, in a concentration-dependent manner, the amplitude of the fictive inspiratory rhythm generated by the preBötC, which is prevented by IL1Ra. Only a negligible effect on the rhythm frequency was observed at one of the concentrations tested (10 ng/mL). In sum, these findings indicate that IL-1ß modulates respiratory rhythm generation. In contrast, IL1Ra did not produce a major effect but slightly increased burst amplitude regularity of the fictive respiratory rhythm. Our findings show that IL-1ß modulates breathing by directly modulating the inspiratory rhythm generation. This modulation could contribute to the respiratory response to inflammation in health and disease.

Microglial modulators reduce respiratory rhythm long-term facilitation in vitro.

Inflammation inhibits the expression of some, but not all forms of respiratory motor plasticity. For example, systemic application of lipopolysaccharide (LPS) inhibits the phrenic long-term facilitation induced by moderate-intermittent hypoxia in vivo. There are multiple pro-inflammatory processes triggered by the systemic application of LPS, including neuroinflammation in the CNS. Considering that microglia can be activated by the systemic application of LPS, it is likely that this cell type influences the response of the respiratory circuits to intermittent hypoxia (IH). Thus, we aimed to test whether modulators of microglial function would affect the response to IH of the preBötzinger complex (preBötC) isolated in a brainstem slice preparation. This experimental approach avoids the systemic influences of these microglial modulators and limits their effects on cells, mostly microglia, included in the slice. First, we found that IH (3 × 5-min episodes of bubbling with 95% N2 and 5% CO2, mixed with 5-min normoxic intervals by bubbling with 95% O2 and 5% CO2) induces a long-lasting increase in the respiratory rhythm frequency recorded directly from the preBötC, called in vitro long-term facilitation (LTF), which occurs simultaneously with a long-lasting decrease in burst amplitude. Moreover, we found that bath applications of "microglial activators" (LPS and fractalkine), "microglial inhibitors" (minocycline and fucoidan) and a microgliotoxin (liposomal clodronate) partially reduce in vitro LTF. These findings reveal a complex scenario in which both the activation and the inhibition of microglia halts IH-induced preBötC plasticity and suggest that experimental or pathological conditions that affect this cell type, almost in any way, could affect breathing and its plastic responses.

Hydrogen peroxide extracellular concentration in the ventrolateral medulla and its increase in response to hypoxia in vitro: Possible role of microglia.

Hydrogen peroxide (H2O2) is a messenger involved in both damaging neuroinflammatory responses and physiological cell communication. The ventrolateral medulla, which regulates several vital functions including breathing and blood pressure, is highly influenced by hydrogen peroxide, whose extracellular levels could be determined by hypoxia and microglial activity, both of which modulate ventrolateral medulla function. Therefore, in this study we aimed to test whether different patterns of hypoxia and/or putative microglial modulators change extracellular hydrogen peroxide in the ventrolateral medulla by using an enzymatic reactor online sensing procedure specifically designed for this purpose. With this new technique, we detected extracellular levels of hydrogen peroxide in the ventrolateral medulla in vitro, which spontaneously fluctuated. These fluctuations are reduced by minocycline, a putative microglial inhibitor, and by the microglial toxin liposomal clodronate. Suitably, lipopolysaccharide increases extracellular hydrogen peroxide, while minocycline and liposomal clodronate reduce this increase. Application of blue light to slices with microglia expressing channelrhodopsin-2 also increases extracellular hydrogen peroxide. Moreover, long-lasting and intermittent hypoxia (as well as subsequent reoxygenation) increase extracellular hydrogen peroxide to similar levels, which is partially prevented by minocycline. The effect of long-lasting hypoxia was reproduced in vivo. Overall, our data show that changes in oxygen concentration, and possibly microglial function, modulate extracellular H2O2 levels in the ventrolateral medulla, which could influence the function of this neural circuit under normal and pathological conditions related to inflammation and/or hypoxia.

Pharmacological Tools to Study the Role of Astrocytes in Neural Network Functions.

Despite that astrocytes and microglia do not communicate by electrical impulses, they can efficiently communicate among them, with each other and with neurons, to participate in complex neural functions requiring broad cell-communication and long-lasting regulation of brain function. Glial cells express many receptors in common with neurons; secrete gliotransmitters as well as neurotrophic and neuroinflammatory factors, which allow them to modulate synaptic transmission and neural excitability. All these properties allow glial cells to influence the activity of neuronal networks. Thus, the incorporation of glial cell function into the understanding of nervous system dynamics will provide a more accurate view of brain function. Our current knowledge of glial cell biology is providing us with experimental tools to explore their participation in neural network modulation. In this chapter, we review some of the classical, as well as some recent, pharmacological tools developed for the study of astrocyte's influence in neural function. We also provide some examples of the use of these pharmacological agents to understand the role of astrocytes in neural network function and dysfunction.

Microglia modulate respiratory rhythm generation and autoresuscitation.

Inflammation has been linked to the induction of apneas and Sudden Infant Death Syndrome, whereas proinflammatory mediators inhibit breathing when applied peripherally or directly into the CNS. Considering that peripheral inflammation can activate microglia in the CNS and that this cell type can directly release all proinflammatory mediators that modulate breathing, it is likely that microglia can modulate breathing generation. It might do so also in hypoxia, since microglia are sensitive to hypoxia, and peripheral proinflammatory conditions affect gasping generation and autoresuscitation. Here, we tested whether microglial activation or inhibition affected respiratory rhythm generation. By measuring breathing as well as the activity of the respiratory rhythm generator (the preBötzinger complex), we found that several microglial activators or inhibitors, applied intracisternally in vivo or in the recording bath in vitro, affect the generation of the respiratory rhythms both in normoxia and hypoxia. Furthermore, microglial activation with lipopolysaccharide affected the ability of the animals to autoresuscitate after hypoxic conditions, an effect that is blocked when lipopolysaccharide is co-applied with the microglial inhibitor minocycline. Moreover, we found that the modulation of respiratory rhythm generation induced in vitro by microglial inhibitors was reproduced by microglial depletion. In conclusion, our data show that microglia can modulate respiratory rhythm generation and autoresuscitation.

Change in network connectivity during fictive-gasping generation in hypoxia: prevention by a metabolic intermediate.

The neuronal circuit in charge of generating the respiratory rhythms, localized in the pre-Bötzinger complex (preBötC), is configured to produce fictive-eupnea during normoxia and reconfigures to produce fictive-gasping during hypoxic conditions in vitro. The mechanisms involved in such reconfiguration have been extensively investigated by cell-focused studies, but the actual changes at the network level remain elusive. Since a failure to generate gasping has been linked to Sudden Infant Death Syndrome (SIDS), the study of gasping generation and pharmacological approaches to promote it may have clinical relevance. Here, we study the changes in network dynamics and circuit reconfiguration that occur during the transition to fictive-gasping generation in the brainstem slice preparation by recording the preBötC with multi-electrode arrays and assessing correlated firing among respiratory neurons or clusters of respiratory neurons (multiunits). We studied whether the respiratory network reconfiguration in hypoxia involves changes in either the number of active respiratory elements, the number of functional connections among elements, or the strength of these connections. Moreover, we tested the influence of isocitrate, a Krebs cycle intermediate that has recently been shown to promote breathing, on the configuration of the preBötC circuit during normoxia and on its reconfiguration during hypoxia. We found that, in contrast to previous suggestions based on cell-focused studies, the number and the overall activity of respiratory neurons change only slightly during hypoxia. However, hypoxia induces a reduction in the strength of functional connectivity within the circuit without reducing the number of connections. Isocitrate prevented this reduction during hypoxia while increasing the strength of network connectivity. In conclusion, we provide an overview of the configuration of the respiratory network under control conditions and how it is reconfigured during fictive-gasping. Additionally, our data support the use of isocitrate to favor respiratory rhythm generation under normoxia and to prevent some of the changes in the respiratory network under hypoxic conditions.