Stereological estimations and neurochemical characterization of neurons expressing GABAA and GABAB receptors in the rat pedunculopontine and laterodorsal tegmental nuclei

To better understand GABAergic transmission at two targets of basal ganglia downstream projections, the pedunculopontine (PPN) and laterodorsal (LDT) tegmental nuclei, the anatomical localization of GABAA and GABAB receptors was investigated in both nuclei. Specifically, the total number of neurons expressing the GABAA receptor γ2 subunit (GABAAR γ2) and the GABAB receptor R2 subunit (GABAB R2) in PPN and LDT was estimated using stereological methods, and the neurochemical phenotype of cells expressing each subunit was also determined. The mean number of non-cholinergic cells expressing GABAAR γ2 was 9850 ± 1856 in the PPN and 8285 ± 962 in the LDT, whereas those expressing GABAB R2 were 7310 ± 1970 and 9170 ± 1900 in the PPN and LDT, respectively. In addition, all cholinergic neurons in both nuclei co-expressed GABAAR γ2 and 95–98% of them co-expressed GABAB R2. Triple labeling using in situ hybridization revealed that 77% of GAD67 mRNA-positive cells in the PPT and 49% in the LDT expressed GABAAR γ2, while 90% (PPN) and 65% (LDT) of Vglut2 mRNA-positive cells also expressed GABAAR γ2. In contrast, a similar proportion (~2/3) of glutamatergic and GABAergic cells co-expressed GABAB R2 in both nuclei. The heterogeneous distribution of GABAAR and GABABR among non-cholinergic cells in PPN and LDT may give rise to physiological differences within each neurochemical subpopulation. In addition, the dissimilar proportion of GABAAR γ2-expressing glutamatergic and GABAergic neurons in the PPN and LDT may contribute to some of the functional differences found between the two nuclei

Dissemination of <i>Mycobacterium tuberculosis</i> is associated to a <i>SIGLEC1</i> null variant that limits antigen exchange via trafficking extracellular vesicles

The identification of individuals with null alleles enables studying how the loss of gene function affects infection. We previously described a non‐functional variant in SIGLEC1, which encodes the myeloid‐cell receptor Siglec‐1/CD169 implicated in HIV‐1 cell‐to‐cell transmission. Here we report a significant association between the SIGLEC1 null variant and extrapulmonary dissemination of Mycobacterium tuberculosis (Mtb) in two clinical cohorts comprising 6,256 individuals. Local spread of bacteria within the lung is apparent in Mtb‐infected Siglec‐1 knockout mice which, despite having similar bacterial load, developed more extensive lesions compared to wild type mice. We find that Siglec‐1 is necessary to induce antigen presentation through extracellular vesicle uptake. We postulate that lack of Siglec‐1 delays the onset of protective immunity against Mtb by limiting antigen exchange via extracellular vesicles, allowing for an early local spread of mycobacteria that increases the risk for extrapulmonary dissemination

Stereological estimates of glutamatergic, GABAergic, and cholinergic neurons in the pedunculopontine and laterodorsa tegmental nuclei in the rat

The pedunculopontine tegmental nucleus (PPN) and laterodorsal tegmental nucleus (LDT) are functionally associated brainstem structures implicated in behavioral state control and sensorimotor integration. The PPN is also involved in gait and posture, while the LDT plays a role in reward. Both nuclei comprise characteristic cholinergic neurons intermingled with glutamatergic and GABAergic cells whose absolute numbers in the rat have been only partly established. Here we sought to determine the complete phenotypical profile of each nucleus to investigate potential differences between them. Counts were obtained using stereological methods after the simultaneous visualization of cholinergic and either glutamatergic or GABAergic cells. The two isoforms of glutamic acid decarboxylase (GAD), GAD65 and GAD67, were separately analyzed. Dual in situ hybridization revealed coexpression of GAD65 and GAD67 mRNAs in ∼90% of GAD-positive cells in both nuclei; thus, the estimated mean numbers of (1) cholinergic, (2) glutamatergic, and (3) GABAergic cells in PPN and LDT, respectively, were (1) 3,360 and 3,650; (2) 5,910 and 5,190; and (3) 4,439 and 7,599. These data reveal significant differences between PPN and LDT in their relative phenotypical composition, which may underlie some of the functional differences observed between them. The estimation of glutamatergic cells was significantly higher in the caudal PPN, supporting the reported functional rostrocaudal segregation in this nucleus. Finally, a small subset of cholinergic neurons (8% in PPN and 5% in LDT) also expressed the glutamatergic marker Vglut2, providing anatomical evidence for a potential corelease of transmitters at specific target areas

Two-color fluorescence labeling in acrolein-fixed brain tissue

SUMMARY Acrolein is a potent fixative that provides both excellent preservation of ultrastructural morphology and retention of antigenicity, thus it is frequently used for immunocytochemical detection of antigens at the electron microscopic level. However, acrolein is not commonly used for fluorescence microscopy because of concerns about possible autofluorescence and destruction of the luminosity of fluorescent dyes. Here we describe a simple protocol that allows fine visualization of two fluorescent markers in 40-mm sections from acroleinperfused rat brain. Autofluorescence was removed by pretreatment with 1% sodium borohydride for 30 min, and subsequent incubation in a 50% ethanol solution containing 0.3% hydrogen peroxide enhanced fluorescence labeling. Thus, fluorescence labeling can be used for high-quality detection of markers in tissue perfused with acrolein. Furthermore, adjacent acrolein-fixed sections from a single experiment can be processed to produce high-quality results for electron microscopy or fluorescence labeling

Two-color fluorescence labeling in acrolein-fixed brain tissue

SUMMARY Acrolein is a potent fixative that provides both excellent preservation of ultrastructural morphology and retention of antigenicity, thus it is frequently used for immunocytochemical detection of antigens at the electron microscopic level. However, acrolein is not commonly used for fluorescence microscopy because of concerns about possible autofluorescence and destruction of the luminosity of fluorescent dyes. Here we describe a simple protocol that allows fine visualization of two fluorescent markers in 40-mm sections from acroleinperfused rat brain. Autofluorescence was removed by pretreatment with 1% sodium borohydride for 30 min, and subsequent incubation in a 50% ethanol solution containing 0.3% hydrogen peroxide enhanced fluorescence labeling. Thus, fluorescence labeling can be used for high-quality detection of markers in tissue perfused with acrolein. Furthermore, adjacent acrolein-fixed sections from a single experiment can be processed to produce high-quality results for electron microscopy or fluorescence labeling

Stereological estimations and neurochemical characterization of neurons expressing GABAA and GABAB receptors in the rat pedunculopontine and laterodorsal tegmental nuclei

To better understand GABAergic transmission at two targets of basal ganglia downstream projections, the pedunculopontine (PPN) and laterodorsal (LDT) tegmental nuclei, the anatomical localization of GABAA and GABAB receptors was investigated in both nuclei. Specifically, the total number of neurons expressing the GABAA receptor γ2 subunit (GABAAR γ2) and the GABAB receptor R2 subunit (GABAB R2) in PPN and LDT was estimated using stereological methods, and the neurochemical phenotype of cells expressing each subunit was also determined. The mean number of non-cholinergic cells expressing GABAAR γ2 was 9850 ± 1856 in the PPN and 8285 ± 962 in the LDT, whereas those expressing GABAB R2 were 7310 ± 1970 and 9170 ± 1900 in the PPN and LDT, respectively. In addition, all cholinergic neurons in both nuclei co-expressed GABAAR γ2 and 95–98% of them co-expressed GABAB R2. Triple labeling using in situ hybridization revealed that 77% of GAD67 mRNA-positive cells in the PPT and 49% in the LDT expressed GABAAR γ2, while 90% (PPN) and 65% (LDT) of Vglut2 mRNA-positive cells also expressed GABAAR γ2. In contrast, a similar proportion (~2/3) of glutamatergic and GABAergic cells co-expressed GABAB R2 in both nuclei. The heterogeneous distribution of GABAAR and GABABR among non-cholinergic cells in PPN and LDT may give rise to physiological differences within each neurochemical subpopulation. In addition, the dissimilar proportion of GABAAR γ2-expressing glutamatergic and GABAergic neurons in the PPN and LDT may contribute to some of the functional differences found between the two nuclei

CB2 Receptors and Neuron–Glia Interactions Modulate Neurotoxicity Generated by MAGL Inhibition

Monoacylglycerol lipase inhibition (MAGL) has emerged as an interesting therapeutic target for neurodegenerative disease treatment due to its ability to modulate the endocannabinoid system and to prevent the production of proinflammatory mediators. To obtain a beneficial response, it is necessary to understand how this inhibition affects the neuron–glia crosstalk and neuron viability. In this study, the effect of MAGL inhibition by KML29 was evaluated in two types of rat cortical primary cultures; mixed cultures, including neuron and glial cells, and neuron-enriched cultures. The risk of neuronal death was estimated by longitudinal survival analysis. The spontaneous neuronal risk of death in culture was higher in the absence of glial cells, a process that was enhanced by KML29 addition. In contrast, neuronal survival was not compromised by MAGL inhibition in the presence of glial cells. Blockade of cannabinoid type 2 (CB2) receptors expressed mainly by microglial cells did not affect the spontaneous neuronal death risk but decreased neuronal survival when KML29 was added. Modulation of cannabinoid type 1 (CB1) receptors did not affect neuronal survival. Our results show that neuron–glia interactions are essential for neuronal survival. CB2 receptors play a key role in these protective interactions when neurons are exposed to toxic conditions

Midbrain microglia mediate a specific immunosuppressive response under inflammatory conditions

Background: Inflammation is a critical process for the progression of neuronal death in neurodegenerative disorders. Microglia play a central role in neuroinflammation and may affect neuron vulnerability. Next generation sequencing has shown the molecular heterogeneity of microglial cells; however, the variability in their response to pathological inputs remains unknown. Methods: To determine the effect of an inflammatory stimulus on microglial cells, lipopolysaccharide (LPS) was administered peripherally to mice and the inflammatory status of the cortex, hippocampus, midbrain, and striatum was assessed. Microglial activation and interaction with the immune system were analyzed in single cell suspensions obtained from the different brain regions by fluorescence-activated cell sorting, next generation RNA sequencing, real-time PCR, and immunohistochemical techniques. Antigen-presenting properties of microglia were evaluated by the ability of isolated cells to induce a clonal expansion of CD4+ T cells purified from OT-II transgenic mice. Results: Under steady-state conditions, the midbrain presented a high immune-alert state characterized by the presence of two unique microglial subpopulations, one expressing the major histocompatibility complex class II (MHC-II) and acting as antigen-presenting cells and another expressing the toll-like receptor 4 (TLR4), and by the presence of a higher proportion of infiltrating CD4+ T cells. This state was not detected in the cortex, hippocampus, or striatum. Systemic LPS administration induced a general increase in classic pro-inflammatory cytokines, in coinhibitory programmed death ligand 1 (PD-L1), and in cytotoxic T lymphocyte antigen 4 (CTLA-4) receptors, as well as a decrease in infiltrating effector T cells in all brain regions. Interestingly, a specific immune-suppressive response was observed in the midbrain which was characterized by the downregulation of MHC-II microglial expression, the upregulation of the anti-inflammatory cytokines IL10 and TGFβ, and the increase in infiltrating regulatory T cells. Conclusions: These data show that the midbrain presents a high immune-alert state under steady-state conditions that elicits a specific immune-suppressive response when exposed to an inflammatory stimulus. This specific inflammatory tone and response may have an impact in neuronal viabilit

Midbrain microglia mediate a specific immunosuppressive response under inflammatory conditions

Background: Inflammation is a critical process for the progression of neuronal death in neurodegenerative disorders. Microglia play a central role in neuroinflammation and may affect neuron vulnerability. Next generation sequencing has shown the molecular heterogeneity of microglial cells; however, the variability in their response to pathological inputs remains unknown. Methods: To determine the effect of an inflammatory stimulus on microglial cells, lipopolysaccharide (LPS) was administered peripherally to mice and the inflammatory status of the cortex, hippocampus, midbrain, and striatum was assessed. Microglial activation and interaction with the immune system were analyzed in single cell suspensions obtained from the different brain regions by fluorescence-activated cell sorting, next generation RNA sequencing, real-time PCR, and immunohistochemical techniques. Antigen-presenting properties of microglia were evaluated by the ability of isolated cells to induce a clonal expansion of CD4+ T cells purified from OT-II transgenic mice. Results: Under steady-state conditions, the midbrain presented a high immune-alert state characterized by the presence of two unique microglial subpopulations, one expressing the major histocompatibility complex class II (MHC-II) and acting as antigen-presenting cells and another expressing the toll-like receptor 4 (TLR4), and by the presence of a higher proportion of infiltrating CD4+ T cells. This state was not detected in the cortex, hippocampus, or striatum. Systemic LPS administration induced a general increase in classic pro-inflammatory cytokines, in coinhibitory programmed death ligand 1 (PD-L1), and in cytotoxic T lymphocyte antigen 4 (CTLA-4) receptors, as well as a decrease in infiltrating effector T cells in all brain regions. Interestingly, a specific immune-suppressive response was observed in the midbrain which was characterized by the downregulation of MHC-II microglial expression, the upregulation of the anti-inflammatory cytokines IL10 and TGFβ, and the increase in infiltrating regulatory T cells. Conclusions: These data show that the midbrain presents a high immune-alert state under steady-state conditions that elicits a specific immune-suppressive response when exposed to an inflammatory stimulus. This specific inflammatory tone and response may have an impact in neuronal viabilit

Microglia and astrocyte activation is region-dependent in the alfa-synuclein mouse model of Parkinson's disease

Inflammation is a common feature in neurodegenerative diseases that contributes to neuronal loss. Previously, we demonstrated that the basal inflammatory tone differed between brain regions and, consequently, the reaction generated to a pro-inflammatory stimulus was different. In this study, we assessed the innate immune reaction in the midbrain and in the striatum using an experimental model of Parkinson's disease. An adeno-associated virus serotype 9 expressing the α-synuclein and mCherry genes or the mCherry gene was administered into the substantia nigra. Myeloid cells (CD11b+ ) and astrocytes (ACSA2+ ) were purified from the midbrain and striatum for bulk RNA sequencing. In the parkinsonian midbrain, CD11b+ cells presented a unique anti-inflammatory transcriptomic profile that differed from degenerative microglia signatures described in experimental models for other neurodegenerative conditions. By contrast, striatal CD11b+ cells showed a pro-inflammatory state and were similar to disease-associated microglia. In the midbrain, a prominent increase of infiltrated monocytes/macrophages was observed and, together with microglia, participated actively in the phagocytosis of dopaminergic neuronal bodies. Although striatal microglia presented a phagocytic transcriptomic profile, morphology and cell density was preserved and no active phagocytosis was detected. Interestingly, astrocytes presented a pro-inflammatory fingerprint in the midbrain and a low number of differentially displayed transcripts in the striatum. During α-synuclein-dependent degeneration, microglia and astrocytes experience context-dependent activation states with a different contribution to the inflammatory reaction. Our results point towards the relevance of selecting appropriate cell targets to design neuroprotective strategies aimed to modulate the innate immune system during the active phase of dopaminergic degeneration