Complexity of Ca2+ signals in astrocytes : Contribution of astroglial GABAB receptors

ZUSAMMENFASSUNG Neben Neuronen sind Astrozyten an der Signalübertragung der Synapse beteiligt. Dafür sind sie mit einer Vielzahl von Rezeptoren ausgestattet. Einer dieser Rezeptoren ist der metabotrope GABAB-Rezeptor, der durch den Transmitter GABA (Ɣ-Aminobuttersäure) aktiviert wird. In Neuronen führt die Aktivierung des GABAB-Rezeptors, bestehend aus GABAB1- und GABAB2-Untereinheit, zu einer Aktivierung des inhibitorischen G-Proteins. Abhängig von der Position des Rezeptors kommt es dadurch zu einer Inhibition von Kaliumkanälen oder zu einer Verlängerung der Öffnungswahrscheinlichkeit von Kalziumkanälen. Die Expression und auch die Funktionalität von GABAB-Rezeptoren auf Astrozyten wurde bereits beschrieben, aber die nachgeschaltete Signalkaskade und die Funktion in der Interaktion mit Neuronen ist bisher noch nicht geklärt. Eine Veränderung des intrazellulären Kalziums wird vermutet. Daher haben wir eine Mauslinie generiert, in der die essentielle GABAB1-Untereinheit spezifisch in Astrozyten deletiert wird. Hierfür wurde die Tamoxifen induzierbare GLAST-CreERT2-Mauslinie mit der gefloxten GABAB1-Linie verpaart (GLAST-CreERT2xGABAB1fl/fl). Durch den Verlust der GABAB1-Untereinheit kann kein GABAB-Rezeptor mehr gebildet werden. Ein Teil der GLAST-CreERT2xGABAB1fl/fl-Linie exprimiert außerdem den genetisch codierten Kalziumindikator GCaMP3, um in vivo Kalziumsignale von Kontroll- und Knockout-Tieren zu analysieren. In einen ersten Schritt wurde die genomische DNA Rekombinationseffizienz des gefloxten gabbr1 Alleles mittels quantitativer real-time PCR in verschiedenen Gehirnarealen drei Wochen nach Tamoxifen-Administration untersucht. Die Analyse der genomischen DNA in den verschiedenen Gehirnarealen; Hirnstamm, Zerebellum, Kortex, Hippocampus und optischer Nerv, zeigte eine Reduktion der gefloxten gabbr1 Allele (30%, 25%, 25%, 29%, 38%). Diese Reduktion spiegelt auch das relative Verhältnis der Astrozyten in den verschiedenen Regionen wieder. Außerdem konnte eine Reduktion um 50% an gefloxten gabbr1 Allelen in MACS-isolierten kortikalen Astrozyten beobachtet werden. Auf Grund der hohen Expression der GABAB-Rezeptoren in Neuronen und anderen Zellentypen konnten keine GABAB1 mRNA Reduktion in totalen Zellhomogenaten mittels RT-PCR identifiziert werden. In einer NGS-Analyse der GABAB1 mRNA in MACS-isolierten Astrozyten aus Zerebellum und Kortex konnte eine Reduktion von 50-70% gezeigt werden. Mittels Fluoreszenz in situ Hybridisierung (FISH) konnte der Verlust der mRNA spezifisch in Astrozyten in Zerebellum, Hippocampus und Kortex nachgewiesen werden. Eine genaue Analyse der Expression des GABAB1 Protein auf der Membran von Astrozyten mittels Immunhistochemie zeigte den Verlust des Proteins in Kortex, Hippocampus und Zerebellum. Die Sprache der Astrozyten sind die Kalziumsignale. Um die Auswirkung des Verlustes der astrozytären GABAB-Rezeptoren auf die Kalziumsignale zu evaluieren, wurden anästhesierten und wachen Mäusen mittels 2-Photonen-Mikroskopie untersucht. In vivo führt die Deletion von astrozytären GABAB-Rezeptoren zu kleineren und kürzeren Kalziumsignalen im Gliapil von anästhesierten Mäusen. In wachen Tieren sind neben den Signalen im Gliapil, auch somatische Kalziumsignale kleiner und kürzer in der Abwesenheit von GABAB-Rezeptoren in Astrozyten. Zusammenfassend konnte die Deletion der essentiellen GABAB1-Untereinheit gezeigt werden. Außerdem konnte gezeigt werden, dass GABAB-Rezeptoren einen Beitrag zu der Komplexität der Kalziumsignale beitragen.ABSTRACT Astrocytes are decisively involved in synaptic transmission and for this purpose equipped with transmitter receptors capable of sensing neuronal activity. One of these receptors is the metabotropic GABAB receptor, a target of the main inhibitory neurotransmitter γ-aminobutyric acid (GABA). In neurons, activation of GABAB receptors (formed by dimerization of GABAB1 and GABAB2 subunits) leads to an activation of inhibitory G proteins, resulting in prolonged of opening time of Ca2+ channels or inhibition of potassium channels, depending on the localization of the receptors. The presence and the functionality of astrocytic GABAB receptors are already described but the downstream signaling and the influence in communication of the astrocytes remains unclear. Recent publication suggest a link to intracellular Ca2+ signals. Therefore, we generated a genetically modified mouse model where an astroglia-specific deletion of the essential GABAB receptor subunit GABAB1 could be induced by crossbreeding GLAST-CreERT2 with floxed GABAB1 mice (GLAST-CreERT2 x GABAB1 fl/fl). After deleting the GABAB1 subunit no functional receptor can be formed. A subset of GLAST-CreERT2 x GABAB1 fl/fl also expressed the genetically encoded Ca2+ indicator GCaMP3 allowing in vivo Ca2+ imaging in cKO and control. First, we quantified the genomic DNA recombination efficiency of the floxed gabbr1 alleles in different brain region by real-time PCR three weeks after the tamoxifen injection. Analysis of genomic DNA purified from brainstem, cerebellum, cortex, hippocampus and optic nerve revealed a significant reduction of floxed gabbr1 alleles (30%, 25%, 25%, 29%, and 38%, respectively). This reflects the relative proportion of astroglia in the respective regions. Furthermore, we could detect loss of 50% of floxed gabbr1 alleles in MACS isolated cortical astrocytes. Due to the high expression of neuronal GABAB receptors and the expression of GABAB receptors on other cell types a reduction of GABAB1 mRNA could not be detected in brain homogenates by real-time PCR. However, using NGS we could detect a significant loss of the GABAB1 mRNA on MACS isolated cerebellar and cortical astrocytes (50% and 70%, respectively). Futhermore by using fluorescence in situ hybridization (FISH) we could visualize the loss of mRNA specifically in astrocytes in the cerebellum, hippocampus and cortex. A detailed analysis of GABAB1 protein expression in the astrocyte membrane revealed a loss of the protein on the membrane in cortex, hippocampus and cerebellum with immunohistochemistry. Astrocytes communicate through Ca2+ signals. To reveal the impact of GABAB receptors on Ca2+ signals, signals in anesthetized and awake animals were analyzed with the two-photon laser scanning microscopy. In vivo the deletion of astrocytic GABAB receptors resulted in only smaller and shorter Ca2+ signals in the gliapil of awake and anesthetized animals. In awake animals next to the signals in the gliapil, also somatic Ca2+ signals were smaller and shorter in the absence of astrocytic GABAB receptors. In summary, the deletion of the essential GABAB1 subunit could be proven and furthermore GABAB receptors play an important in the complexity of Ca2+ signals

Novel algorithms for improved detection and analysis of fluorescent signal fluctuations

Fluorescent dyes and genetically encoded fuorescence indicators (GEFI) are common tools for visualizing concentration changes of specifc ions and messenger molecules during intra- as well as intercellular communication. Using advanced imaging technologies, fuorescence indicators are a prerequisite for the analysis of physiological molecular signaling. Automated detection and analysis of fuorescence signals require to overcome several challenges, including correct estimation of fuorescence fuctuations at basal concentrations of messenger molecules, detection, and extraction of events themselves as well as proper segmentation of neighboring events. Moreover, event detection algorithms need to be sensitive enough to accurately capture localized and low amplitude events exhibiting a limited spatial extent. Here, we present two algorithms (PBasE and CoRoDe) for accurate baseline estimation and automated detection and segmentation of fuorescence fuctuations

Versatile Surface Electrodes for Combined Electrophysiology and Two-Photon Imaging of the Mouse Central Nervous System

Understanding and modulating CNS function in physiological as well as pathophysiological contexts remains a significant ambition in research and clinical applications. The investigation of the multifaceted CNS cell types including their interactions and contributions to neural function requires a combination of the state-ofthe-art in vivo electrophysiology and imaging techniques. We developed a novel type of liquid crystal polymer (LCP) surface micro-electrode manufactured in three customized designs with up to 16 channels for recording and stimulation of brain activity. All designs include spare central spaces for simultaneous 2P-imaging. Nanoporous platinumplated contact sites ensure a low impedance and high current transfer. The epidural implantation of the LCP micro-electrodes could be combined with standard cranial window surgery. The epidurally positioned electrodes did not only display long-term biocompatibility, but we also observed an additional stabilization of the underlying CNS tissue. We demonstrate the electrode’s versatility in combination with in vivo 2P-imaging by monitoring anesthesia-awake cycles of transgenic mice with GCaMP3 expression in neurons or astrocytes. Cortical stimulation and simultaneous 2P Ca2+ imaging in neurons or astrocytes highlighted the astrocytes’ integrative character in neuronal activity processing. Furthermore, we confirmed that spontaneous astroglial Ca2+ signals are dampened under anesthesia, while evoked signals in neurons and astrocytes showed stronger dependency on stimulation intensity rather than on various levels of anesthesia. Finally, we show that the electrodes provide recordings of the electrocorticogram (ECoG) with a high signal-to noise ratio and spatial signal differences which help to decipher brain activity states during experimental procedures. Summarizing, the novel LCP surface micro-electrode is a versatile, convenient, and reliable tool to investigate brain function in vivo

Microglial morphology in the somatosensory cortex across lifespan. A quantitative study

Background: Microglia are long-lived cells that constantly monitor their microenvironment. To accomplish this task, they constantly change their morphology both in the short and long term under physiological conditions. This makes the process of quantifying physiological microglial morphology difficult. Results: By using a semi-manual and a semi-automatic method to assess fine changes in cortical microglia morphology, we were able to quantify microglia changes in number, surveillance and branch tree starting from the fifth postnatal day to 2 years of life. We were able to identify a fluctuating behavior of most analyzed parameters characterized by a rapid cellular maturation, followed by a long period of relative stable morphology during the adult life with a final convergence to an aged phenotype. Detailed cellular arborization analysis revealed age-induced differences in microglia morphology, with mean branch length and the number of terminal processes changing constantly over time. Conclusions: Our study provides insight into microglia morphology changes across lifespan under physiological conditions. We were able to highlight, that due to the dynamic nature of microglia several morphological parameters are needed to establish the physiological state of these cells

Intra-accumbens injections of the adenosine A(2A) agonist CGS 21680 affect effort-related choice behavior in rats

Rationale: Nucleus accumbens dopamine (DA) participates in the modulation of instrumental behavior, including aspects of behavioral activation and effort-related choice behavior. Rats with impaired accumbens DA transmission reallocate their behavior away from food-reinforced activities that have high response requirements, and instead select less-effortful types of food-seeking behavior. Although accumbens DA is considered a critical component of the brain circuitry regulating effort-related processes, emerging evidence also implicates adenosine A2A receptors. Objective: The present work was undertaken to test the hypothesis that accumbens A2A receptor stimulation would produce effects similar to those produced by DA depletion or antagonism. Methods: Three experiments assessed the effects of the adenosine A2A agonist CGS 21680 on performance of a concurrent choice task (lever pressing for preferred food vs. intake of less preferred chow) that is known to be sensitive to DA antagonists and accumbens DA depletions. Results: Systemic injections of CGS 21680 reduced lever pressing but did not increase feeding. In contrast, bilateral infusions of the adenosine A2A receptor agonist CGS 21680 (6.0- 24.0 ng) into the nucleus accumbens decreased lever pressing for the preferred food, but substantially increased consumption of the less preferred chow. Injections of CGS 21680 into a control site dorsal to the accumbens were ineffective. Conclusions: Taken together, these results are consistent with the hypothesis that local stimulation of adenosine A2A receptors in nucleus accumbens produces behavioral effects similar to those induced by accumbens DA depletions. Accumbens adenosine A2A receptors appear to be a component of the brain circuitry regulating effort-related choice behavio

Epigenetic control of region-specific transcriptional programs in mouse cerebellar and cortical astrocytes

Astrocytes from the cerebral cortex (CTX) and cerebellum (CB) share basic molecular programs, but also form distinct spatial and functional subtypes. The regulatory epigenetic layers controlling such regional diversity have not been comprehensively investigated so far. Here, we present an integrated epigenome analysis of methylomes, open chromatin, and transcriptomes of astroglia populations isolated from the cortex or cerebellum of young adult mice. Besides a basic overall similarity in their epigenomic programs, cortical astrocytes and cerebellar astrocytes exhibit substantial differences in their overall open chromatin structure and in gene-specific DNA methylation. Regional epigenetic differences are linked to differences in transcriptional programs encompassing genes of region-specific transcription factor networks centered around Lhx2/Foxg1 in CTX astrocytes and the Zic/Irx families in CB astrocytes. The distinct epigenetic signatures around these transcription factor networks point to a complex interconnected and combinatorial regulation of region-specific transcriptomes. These findings suggest that key transcription factors, previously linked to temporal, regional, and spatial control of neurogenesis, also form combinatorial networks important for astrocytes. Our study provides a valuable resource for the molecular basis of regional astrocyte identity and physiology

Nucleus Accumbens Adenosine A2A Receptors Regulate Exertion of Effort by Acting on the Ventral Striatopallidal Pathway

Goal-directed actions are sensitive to work-related response costs, and dopamine in nucleus accumbens is thought to modulate the exertion of effort in motivated behavior. Dopamine-rich striatal areas such as nucleus accumbens also contain high numbers of adenosine A2A receptors, and, for that reason, the behavioral and neurochemical effects of the adenosine A2A receptor agonist CGS 21680 [2-p-(2-carboxyethyl) phenethylamino-5′-N-ethylcarboxamidoadenosine] were investigated. Stimulation of accumbens adenosine A2A receptors disrupted performance of an instrumental task with high work demands (i.e., an interval lever-pressing schedule with a ratio requirement attached) but had little effect on a task with a lower work requirement. Immunohistochemical studies revealed that accumbens neurons that project to the ventral pallidum showed adenosine A2A receptors immunoreactivity. Moreover, activation of accumbens A2A receptors by local injections of CGS 21680 increased extracellular GABA levels in the ventral pallidum. Combined contralateral injections of CGS 21680 into the accumbens and the GABAA agonist muscimol into ventral pallidum (i.e., “disconnection” methods) also impaired response output, indicating that these structures are part of a common neural circuitry regulating the exertion of effort. Thus, accumbens adenosine A2A receptors appear to regulate behavioral activation and effort-related processes by modulating the activity of the ventral striatopallidal pathway. Research on the effort-related functions of these forebrain systems may lead to a greater understanding of pathological features of motivation, such as psychomotor slowing, anergia, and fatigue in depression

Body Composition and Survival in Dialysis Patients: Results from an International Cohort Study

BACKGROUND AND OBJECTIVES: High body mass index appears protective in hemodialysis patients, but uncertainty prevails regarding which components of body composition, fat or lean body mass, are primarily associated with survival. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Data between April 2006 and December 2012 were extracted from the Fresenius Medical Care Europe subset of the international MONitoring Dialysis Outcomes initiative. Fresenius Medical Care Europe archives a unique repository of predialysis body composition measurements determined by multifrequency bioimpedance (BCM Body Composition Monitor). The BCM Body Composition Monitor reports lean tissue indices (LTIs) and fat tissue indices (FTIs), which are the respective tissue masses normalized to height squared, relative to an age- and sex-matched healthy population. The relationship between LTI and FTI and all-cause mortality was studied by Kaplan–Meier analysis, multivariate Cox regression, and smoothing spline ANOVA logistic regression. RESULTS: In 37,345 hemodialysis patients, median (25th–75th percentile) LTI and FTI were 12.2 (10.3–14.5) and 9.8 (6.6–12.4) kg/m(2), respectively. Median (25th–75th percentile) follow-up time was 266 (132–379) days; 3458 (9.2%) patients died during follow-up. Mortality was lowest with both LTI and FTI in the 10th–90th percentile (reference group) and significantly higher at the lower LTI and FTI extreme (hazard ratio [HR], 3.37; 95% confidence interval [95% CI], 2.94 to 3.87; P<0.001). Survival was best with LTI between 15 and 20 kg/m(2) and FTI between 4 and 15 kg/m(2) (probability of death during follow-up: <5%). When taking the relation between both compartments into account, the interaction was significant (P=0.01). Higher FTI appeared protective in patients with low LTI (HR, 3.37; 95% CI, 2.94 to 3.87; P<0.001 at low LTI–low FTI, decreasing to HR, 1.79; 95% CI, 1.47 to 2.17; P<0.001 at low LTI–high FTI). CONCLUSIONS: This large international study indicates best survival in patients with both LTI and FTI in the 10th–90th percentiles of a healthy population. In analyses of body composition, both lean tissue and fat tissue compartments and also their relationship should be considered