Relationship between oxygen consumption and neuronal activity in a defined neural circuit

Background: Neuronal computations related to sensory and motor activity along with the maintenance of spike discharge, synaptic transmission, and associated housekeeping are energetically demanding. The most efficient metabolic process to provide large amounts of energy equivalents is oxidative phosphorylation and thus dependent on O2 consumption. Therefore, O2 levels in the brain are a critical parameter that influences neuronal function. Measurements of O2 consumption have been used to estimate the cost of neuronal activity; however, exploring these metabolic relationships in vivo and under defined experimental conditions has been limited by technical challenges. Results: We used isolated preparations of Xenopus laevis tadpoles to perform a quantitative analysis of O2 levels in the brain under in vivo-like conditions. We measured O2 concentrations in the hindbrain in relation to the spike discharge of the superior oblique eye muscle-innervating trochlear nerve as proxy for central nervous activity. In air-saturated bath Ringer solution, O2 levels in the fourth ventricle and adjacent, functionally intact hindbrain were close to zero. Inhibition of mitochondrial activity with potassium cyanide or fixation of the tissue with ethanol raised the ventricular O2 concentration to bath levels, indicating that the brain tissue consumed the available O2. Gradually increasing oxygenation of the Ringer solution caused a concurrent increase of ventricular O2 concentrations. Blocking spike discharge with the local anesthetics tricaine methanesulfonate diminished the O2 consumption by ~ 50%, illustrating the substantial O2 amount related to neuronal activity. In contrast, episodes of spontaneous trochlear nerve spike bursts were accompanied by transient increases of the O2 consumption with parameters that correlated with burst magnitude and duration. Conclusions: Controlled experimental manipulations of both the O2 level as well as the neuronal activity under in vivo-like conditions allowed to quantitatively relate spike discharge magnitudes in a particular neuronal circuitry with the O2 consumption in this area. Moreover, the possibility to distinctly manipulate various functional parameters will yield more insight in the coupling between metabolic and neuronal activity. Thus, apart from providing quantitative empiric evidence for the link between physiologically relevant spontaneous spike discharge in the brain and O2-dependent metabolism, isolated amphibian preparations are promising model systems to further dissociate the O2 dynamics in relation to neuronal computations

Identification and characterization of Ca2+-activated K+ channels in granulosa cells of the human ovary

<p>Abstract</p> <p>Background</p> <p>Granulosa cells (GCs) represent a major endocrine compartment of the ovary producing sex steroid hormones. Recently, we identified in human GCs a Ca²⁺-activated K⁺channel (K_Ca) of big conductance (BK_Ca), which is involved in steroidogenesis. This channel is activated by intraovarian signalling molecules (e.g. acetylcholine) via raised intracellular Ca²⁺levels. In this study, we aimed at characterizing 1. expression and functions of K_Cachannels (including BK_Cabeta-subunits), and 2. biophysical properties of BK_Cachannels.</p> <p>Methods</p> <p>GCs were obtained from in vitro-fertilization patients and cultured. Expression of mRNA was determined by standard RT-PCR and protein expression in human ovarian slices was detected by immunohistochemistry. Progesterone production was measured in cell culture supernatants using ELISAs. Single channels were recorded in the inside-out configuration of the patch-clamp technique.</p> <p>Results</p> <p>We identified two K_Catypes in human GCs, the intermediate- (IK) and the small-conductance K_Ca(SK). Their functionality was concluded from attenuation of human chorionic gonadotropin-stimulated progesterone production by K_Cablockers (TRAM-34, apamin). Functional IK channels were also demonstrated by electrophysiological recording of single K_Cachannels with distinctive features. Both, IK and BK_Cachannels were found to be simultaneously active in individual GCs. In agreement with functional data, we identified mRNAs encoding IK, SK1, SK2 and SK3 in human GCs and proteins of IK and SK2 in corresponding human ovarian cells. Molecular characterization of the BK_Cachannel revealed the presence of mRNAs encoding several BK_Cabeta-subunits (beta2, beta3, beta4) in human GCs. The multitude of beta-subunits detected might contribute to variations in Ca²⁺dependence of individual BK_Cachannels which we observed in electrophysiological recordings.</p> <p>Conclusion</p> <p>Functional and molecular studies indicate the presence of active IK and SK channels in human GCs. Considering the already described BK_Ca, they express all three K_Catypes known. We suggest that the plurality and co-expression of different K_Cachannels and BK_Cabeta-subunits might allow differentiated responses to Ca²⁺signals over a wide range caused by various intraovarian signalling molecules (e.g. acetylcholine, ATP, dopamine). The knowledge of ovarian K_Cachannel properties and functions should help to understand the link between endocrine and paracrine/autocrine control in the human ovary.</p

Dopamine receptor repertoire of human granulosa cells

<p>Abstract</p> <p>Background</p> <p>High levels of dopamine (DA) were described in human ovary and recently evidence for DA receptors in granulosa and luteal cells has been provided, as well. However, neither the full repertoire of ovarian receptors for DA, nor their specific role, is established. Human granulosa cells (GCs) derived from women undergoing in vitro fertilization (IVF) are an adequate model for endocrine cells of the follicle and the corpus luteum and were therefore employed in an attempt to decipher their DA receptor repertoire and functionality.</p> <p>Methods</p> <p>Cells were obtained from patients undergoing IVF and examined using cDNA-array, RT-PCR, Western blotting and immunocytochemistry. In addition, calcium measurements (with FLUO-4) were employed. Expression of two DA receptors was also examined by in-situ hybridization in rat ovary. Effects of DA on cell viability and cell volume were studied by using an ATP assay and an electronic cell counter system.</p> <p>Results</p> <p>We found members of the two DA receptor families (D₁- and D₂ -like) associated with different signaling pathways in human GCs, namely D₁ (as expected) and D₅ (both are Gs coupled and linked to cAMP increase) and D₂, D₄ (Gi/Gq coupled and linked to IP3/DAG). D₃ was not found. The presence of the trophic hormone hCG (10 IU/ml) in the culture medium for several days did not alter mRNA (semiquantitative RT-PCR) or protein levels (immunocytochemistry/Western blotting) of D_1,2,4,5 DA receptors. Expression of prototype receptors for the two families, D₁ and D₂, was furthermore shown in rat granulosa and luteal cells by in situ hybridization. Among the DA receptors found in human GCs, D₂ expression was marked both at mRNA and protein levels and it was therefore further studied. Results of additional RT-PCR and Western blots showed two splice variants (D₂L, D₂S). Irrespective of these variants, D₂ proved to be functional, as DA raised intracellular calcium levels. This calcium mobilizing effect of DA was observed in the absence of extracellular calcium and was abolished by a D₂ blocker (L-741,626). DA treatment (48 h) of human GCs resulted in slightly, but significantly enlarged, viable cells.</p> <p>Conclusion</p> <p>A previous study showed D₂ in human GCs, which are linked to cAMP, and the present study reveals the full spectrum of DA receptors present in these endocrine cells, which also includes D₂-like receptors, linked to calcium. Ovarian DA can act thus via D_1,2,4,5, which are co-expressed by endocrine cells of the follicle and the corpus luteum and are linked to different signaling pathways. This suggests a complex role of DA in the regulation of ovarian processes.</p

Transcardial injection and vascular distribution of microalgae in Xenopus laevis as means to supply the brain with photosynthetic oxygen

Oxygen in vertebrates is generally provided through respiratory organs and blood vessels. This protocol describes transcardial injection, vascular distribution, and accumulation of phototrophic microalgae in the brain of Xenopus laevis tadpoles. Following tissue isolation, oxygen dynamics and neuronal activity are recorded in semi-intact whole-head preparations. Illumination of such microalgae-filled preparations triggers the photosynthetic production of oxygen in the brain that, under hypoxic conditions, rescues neuronal activity. This technology is potentially able to ameliorate consequences of hypoxia under pathological conditions. For complete details on the use and execution of this protocol, please refer to Özugur et al. (2021)

Cooperative population coding facilitates efficient sound-source separability by adaptation to input statistics

Our sensory environment changes constantly. Accordingly, neural systems continually adapt to the concurrent stimulus statistics to remain sensitive over a wide range of conditions. Such dynamic range adaptation (DRA) is assumed to increase both the effectiveness of the neuronal code and perceptual sensitivity. However, direct demonstrations of DRA-based efficient neuronal processing that also produces perceptual benefits are lacking. Here, we investigated the impact of DRA on spatial coding in the rodent brain and the perception of human listeners. Complex spatial stimulation with dynamically changing source locations elicited prominent DRA already on the initial spatial processing stage, the Lateral Superior Olive (LSO) of gerbils. Surprisingly, on the level of individual neurons, DRA diminished spatial tuning because of large response variability across trials. However, when considering single-trial population averages of multiple neurons, DRA enhanced the coding efficiency specifically for the concurrently most probable source locations. Intrinsic LSO population imaging of energy consumption combined with pharmacology revealed that a slow-acting LSO gain-control mechanism distributes activity across a group of neurons during DRA, thereby enhancing population coding efficiency. Strikingly, such "efficient cooperative coding" also improved neuronal source separability specifically for the locations that were most likely to occur. These location-specific enhancements in neuronal coding were paralleled by human listeners exhibiting a selective improvement in spatial resolution. We conclude that, contrary to canonical models of sensory encoding, the primary motive of early spatial processing is efficiency optimization of neural populations for enhanced source separability in the concurrent environment

Vesicle transport and growth dynamics in Aspergillus niger: Microscale modeling of secretory vesicle flow and centerline extraction from confocal fluorescent data

In this paper, we present a mathematical model to describe filamentous fungal growth based on intracellular secretory vesicles (SVs), which transport cell wall components to the hyphal tip. Vesicular transport inside elongating hyphae is modeled as an advection–diffusion–reaction equation with a moving boundary, transformed into fixed coordinates, and discretized using a high‐order weighted essentially nonoscillatory discretization scheme. The model describes the production and the consumption of SVs with kinetic functions. Simulations are subsequently compared against distributions of SVs visualized by enhanced green fluorescent protein in young Aspergillus niger hyphae after germination. Intensity profile data are obtained using an algorithm scripted in ImageJ that extracts mean intensity distributions from 3D time‐lapse confocal measurement data. Simulated length growth is in good agreement with the experimental data. Our simulations further show that a decrease of effective vesicle transport velocity towards the tip can explain the observed tip accumulation of SVs.DFG, 273937032, SPP 1934: Dispersitäts-, Struktur- und Phasenänderungen von Proteinen und biologischen Agglomeraten in biotechnologischen ProzessenTU Berlin, Open-Access-Mittel – 202

Comparison of Two Different Designs of a Scraped Surface Crystallizer for Desalination Effect and Hydraulic and Thermodynamic Numbers

The design of a desalination plant is most important if the desired product purity has to be as high as possible. This is also true for freeze crystallization plants. A correct solid-to-liquid ratio has to be ensured when pressing is used as a post-treatment. Thus, the dependence of the overall plant design on the achieved ice quality but also on different hydraulic and thermodynamic numbers is important. In this research, a scraped screw crystallizer plant is presented and examined for two different screw designs. Experiments with a low initial concentration, as for the usage to desalinate groundwater to gain it as process water, were conducted. Furthermore, solutions with high initial concentrations simulating seawater to produce potable water were used as another set of test solutions. The findings showed that neither of the screw designs is more favorable than the other, but it is important to have a plant design fitting the existing parameters on site

FSH regulates acetycholine production by ovarian granulosa cells

BACKGROUND: It has been previously shown that cultured granulosa cells (GCs) derived from human ovarian preovulatory follicles contain choline acetyltransferase (ChAT), the enzyme responsible for acetylcholine (ACh) synthesis. They also produce ACh and express functional muscarinic ACh receptors. ACh can act on GCs to increase proliferation, disrupt gap junctional communication, alter intracellular calcium levels, as well as expression of transcription factors, suggesting an unrecognized role of ACh in GC function. To gain further insights into the possible role of ACh in the ovary, we examined ChAT expression in the gland before and after birth, as well as in adults, and studied the regulation of ACh production by FSH. METHODS: ChAT immunohistochemistry was performed using ovarian samples of different species and ages (embryonic, postnatal and adult rats and mice, including embryonic ovaries from mice null for ChAT, neonatal and adult rhesus monkeys and adult humans). ACh was measured by HPLC and/or a fluorescence based method in rat ovaries and in a FSH receptor-expressing cell line (rat GFSHR-17) cultured with or without FSH. RESULTS: In adult rat, as well as in all other species, ovarian ChAT immunoreactivity is associated with GCs of antral follicles, but not with other structures, indicating that GCs are the only ovarian source of ACh. Indeed ACh was clearly detected in adult rat ovaries by two methods. ChAT immunoreactivity is absent from embryonic and/or neonatal ovaries (mouse/rat and monkey) and ovarian development in embryonic mice null for ChAT appears normal, suggesting that ACh is not involved in ovarian or follicular formation. Since ChAT immunoreactivity is present in GCs of large follicles and since the degree of the ChAT immunoreactivity increases as antral follicles grow, we tested whether ACh production is stimulated by FSH. Rat GFSHR-17 cells that stably express the FSH receptor, respond to FSH with an increase in ACh production. CONCLUSION: ACh and ChAT are present in GCs of growing follicles and FSH, the major driving force of follicular growth, stimulates ACh production. Since ACh stimulates proliferation and differentiation processes in cultured GCs, we suggest that ACh may act in the growing ovarian follicle as a local mediator of some of the actions ascribed to FSH

Cerebral attenuation on single-phase CT angiography source images: Automated ischemia detection and morphologic outcome prediction after thrombectomy in patients with ischemic stroke

Objectives: Stroke triage using CT perfusion (CTP) or MRI gained importance after successful application in recent trials on late-window thrombectomy but is often unavailable and time-consuming. We tested the clinical value of software-based analysis of cerebral attenuation on Single-phase CT angiography source images (CTASI) as CTP surrogate in stroke patients. Methods: Software-based automated segmentation and Hounsfield unit (HU) measurements for all regions of the Alberta Stroke Program Early CT Score (ASPECTS) on CTASI were performed in patients with large vessel occlusion stroke who underwent thrombectomy. To normalize values, we calculated relative HU (rHU) as ratio of affected to unaffected hemisphere. Ischemic regions, regional ischemic core and final infarction were determined on simultaneously acquired CTP and follow-up imaging as ground truth. Receiver operating characteristics analysis was performed to calculate the area-under-the-curve (AUC). Resulting cut-off values were used for comparison with visual analysis and to calculate an 11-point automated CTASI ASPECTS. Results: Seventy-nine patients were included. rHU values enabled significant classification of ischemic involvement on CTP in all ten regions of the ASPECTS (each p<0.001, except M4-cortex p = 0.002). Classification of ischemic core and prediction of final infarction had best results in subcortical regions but produced lower AUC values with significant classification for all regions except M1, M3 and M5. Relative total hemispheric attenuation provided strong linear correlation with CTP total ischemic volume. Automated classification of regional ischemia on CTASI was significantly more accurate in most regions and provided better agreement with CTP cerebral blood flow ASPECTS than visual assessment. Conclusions: Automated attenuation measurements on CTASI provide excellent performance in detecting acute ischemia as identified on CTP with improved accuracy compared to visual analysis. However, value for the approximation of ischemic core and morphologic outcome in large vessel occlusion stroke after thrombectomy was regionally dependent and limited. This technique has the potential to facilitate stroke imaging as sensitive surrogate for CTP-based ischemia