A massively multi-scale approach to characterizing tissue architecture by synchrotron micro-CT applied to the human placenta.

Multi-scale structural assessment of biological soft tissue is challenging but essential to gain insight into structure-function relationships of tissue/organ. Using the human placenta as an example, this study brings together sophisticated sample preparation protocols, advanced imaging and robust, validated machine-learning segmentation techniques to provide the first massively multi-scale and multi-domain information that enables detailed morphological and functional analyses of both maternal and fetal placental domains. Finally, we quantify the scale-dependent error in morphological metrics of heterogeneous placental tissue, estimating the minimal tissue scale needed in extracting meaningful biological data. The developed protocol is beneficial for high-throughput investigation of structure-function relationships in both normal and diseased placentas, allowing us to optimize therapeutic approaches for pathological pregnancies. In addition, the methodology presented is applicable in the characterization of tissue architecture and physiological behaviours of other complex organs with similarity to the placenta, where an exchange barrier possesses circulating vascular and avascular fluid spaces.

Purification and Application of Genetically Encoded Potassium Ion Indicators for Quantification of Potassium Ion Concentrations within Biological Samples.

Vital cells maintain a steep potassium ion (K+ ) gradient across the plasma membrane. Intracellular potassium ion concentrations ([K+ ]) and especially the [K+ ] within the extracellular matrix are strictly regulated, the latter within a narrow range of ∼3.5 to 5.0 mM. Alterations of the extracellular K+ homeostasis are associated with severe pathological alterations and systemic diseases including hypo- or hypertension, heart rate alterations, heart failure, neuronal damage or abnormal skeleton muscle function. In higher eukaryotic organisms, the maintenance of the extracellular [K+ ] is mainly achieved by the kidney, responsible for K+ excretion and reabsorption. Thus, renal dysfunctions are typically associated with alterations in serum- or plasma [K+ ]. Generally, [K+ ] quantifications within bodily fluids are performed using ion selective electrodes. However, tracking such alterations in experimental models such as mice features several difficulties, mainly due to the small blood volume of these animals, hampering the repetitive collection of sample volumes required for measurements using ion selective electrodes. We have recently developed highly sensitive, genetically encoded potassium ion indicators, the GEPIIs, applicable for in vitro determinations of [K+ ]. In addition to the determination of [K+ ] within bodily fluids, GEPIIs proved suitable for the real-time visualization of cell viability over time and the exact determination of the number of dead cells. © 2019 The Authors.

Brain-computer interfaces (BCIs): detection instead of classification.

Many studies over the past two decades have shown that people can use brain signals to convey their intent to a computer through brain-computer interfaces (BCIs). These devices operate by recording signals from the brain and translating these signals into device commands. They can be used by people who are severely paralyzed to communicate without any use of muscle activity. One of the major impediments in translating this novel technology into clinical applications is the current requirement for preliminary analyses to identify the brain signal features best suited for communication. This paper introduces and validates signal detection, which does not require such analysis procedures, as a new concept in BCI signal processing. This detection concept is realized with Gaussian mixture models (GMMs) that are used to model resting brain activity so that any change in relevant brain signals can be detected. It is implemented in a package called SIGFRIED (SIGnal modeling For Real-time Identification and Event Detection). The results indicate that SIGFRIED produces results that are within the range of those achieved using a common analysis strategy that requires preliminary identification of signal features. They indicate that such laborious analysis procedures could be replaced by merely recording brain signals during rest. In summary, this paper demonstrates how SIGFRIED could be used to overcome one of the present impediments to translation of laboratory BCI demonstrations into clinically practical applications.

Imprinting and cortical plasticity: a comparative review.

Results of research on imprinting and developmental neurobiology of the visual cortex are compared to evaluate the evidence for or against a frequently hypothesized linkage of the two phenomena. The comparison reveals striking similarities. In both paradigms a sensitive period exists. Once this sensitive period is over, the storage of early influences from the environment remains stable throughout life. Storage of "natural" stimuli is facilitated by a certain preorganisation of the receiving brain areas. It is stated that the two phenomena are not directly linked, but are two expressions of a developmental process, which may be common for the organisation of the connectivity of single cells as well as for complex neuronal networks as they are likely to be involved in imprinting. This process is basically self-organizing, but can be influenced by superimposed controls. Differences of the stability of storage of external influences might be explained by the difference in the overall amount of morphological alterations, which is large in the young and small in the adult animal. This holds for both the modifiability in the visual cortex and imprinting.

Delayed development of song control nuclei in the zebra finch is related to behavioral development.

The postnatal development of two visual areas (nucleus rotundus and ectostriatum) and two song control areas (hyperstriatum ventrale pars caudale, HVc, and nucleus robustus archistriatalis, RA) of the zebra finch brain was followed from birth to adulthood. The following parameters were investigated: (1) neuron size, (2) volume of the brain nuclei, and (3) myelination of axons. The nucleus rotundus, the diencephalic station of the tectofugal pathway, exhibits the fastest development: rotundal neurons reach their maximum size at 20 days of age; the volume of this structure reaches adult size at the same time. The process of myelination begins between day 5 and day 10 and is completed at 40 days of age. A similar temporal sequence of development is seen in the ectostriatum, except myelination starts some days later. Thus the development of these visual areas is completed at 40 days. In contrast, the development of the song control nuclei is delayed. Neurons in RA and HVc grow steadily up to 40 days of age, attaining a size larger than that observed in adults. Whereas the volume of HVc increases until day 40 and remains stable thereafter, RA volume increases until day 70 and evidences a decrease thereafter. It is not until postnatal day 20 (RA) and day 40 (HVc) that the myelination process starts in the song control areas. Adult myelin density is achieved by 70 days in RA and by 100 days in HVc. It can be demonstrated that the development of the visual system parallels the development of visual performance of the birds. Delayed growth of song control nuclei coincides with development of song.

Development of neurons in the ectostriatum of normal and monocularly deprived zebra finches: a quantitative Golgi study.

The postnatal development of the main neuron type in the ectostriatum, the telencephalic station of the tectofugal pathway, was followed in normally reared and monocularly deprived zebra finches by using the Golgi method. Three parameters were investigated: dendritic field radius, branching index, and spine density. The results show that all three exhibit the same developmental trend--namely, an increase from day 5 until day 20, followed by a subsequent reduction until adulthood (greater than 100 days). Monocular deprivation from birth until day 20, 40, or at least 100 does not seem to interfere with the development of the dendritic field radius or branching index. Clear changes in spine density result from depriving the birds for at least 40 days. In these birds, neurons in the deprived hemisphere bear significantly fewer spines than those in the nondeprived hemisphere, which is mainly due to a lack of normally occurring spine reduction in the nondeprived hemisphere rather than to spine reduction in the deprived hemisphere.

Posthatching development of synapses in the neuropil of nucleus rotundus of the zebra finch: a quantitative electron microscopic study.

The development and maturation of synapses in the nucleus rotundus of the zebra finch were examined at 1, 5, 10, 20, and 100 days posthatching. Quantitative ultrastructural techniques were applied to investigate synaptic density, size of presynaptic terminals, and length of postsynaptic thickenings. During development there is a steady increases in the number of synapses and an enlargement of the presynaptic terminals. The length of the postsynaptic thickenings, however, decreases significantly during posthatching development. All three parameters reach adult values within 20 days of age. A close relationship was found between the enlargement of the presynaptic terminals and the increase in the number of contact zones up to the 20th day. The general feature of this developmental progress is in good agreement with biochemical and, to a certain extent, behavioral studies.

GABAergic inputs to the nucleus rotundus (pulvinar inferior) of the pigeon (columba livia).

The avian nucleus rotundus, a nucleus that appears to be homologous to the inferior/ caudal pulvinar of mammals, is the major target of an ascending retino-tecto-thalamic pathway. Further clarification of the inputs to the rotundus and their functional properties will contribute to our understanding of the fundamental role of the ascending tectal inputs to the telencephalon in all vertebrates, including mammals. We found that the rotundus contains a massive plexus of glutamic acid decarboxylase (GAD)-immunoreactive axons using antibodies against GAD. The cells within the rotundus, however, were not immunoreactive for GAD. The retrograde tracer cholera toxin B fragment was injected into the rotundus to establish the location of the afferent neurons and determine the source of the gamma-aminobutyric acid (GABA) inputs into the rotundus. In addition to the recognized bilateral inputs from layer 13 of the tectum, we found intense retrograde labeling of neurons within the ipsilateral nuclei subpretectalis (SP), subpretectalis-caudalis (SPcd), interstitio-pretecto-subpretectalis (IPS), posteroventralis thalami (PV), and reticularis superior thalami (RS). All the neurons of the SP, SPcd, IPS, and PV were intensely GAD-immunoreactive. The neurons of layer 13 of the tectum were not immunoreactive for GAD. Following the destruction of the ipsilateral SP/IPS complex, we found a major reduction in the intensity of the GAD axonal immunoreactivity within the ipsilateral rotundus, but this destruction did not diminish the intensity of the GAD-immunoreactivity within the contralateral rotundus. Our studies indicated that the source of the massive GAD-immunoreactive plexus within the rotundus was from the ipsilateral SP, SPcd, IPS, and PV nuclei. These nuclei, in turn, received ipsilateral tectal input via collaterals of the neurons of layer 13 in the course of their projections upon the rotundus. We suggest that the direct bilateral tecto-rotundal projections are excitatory, whereas the indirect ipsilateral projections from the SP/IPS and PV are mainly inhibitory, possibly acting via a GABA-A receptor.

Coffee and cardiovascular risk: epidemiological findings in Austria.

The results of studies on the connection between the consumption of coffee and cardiovascular risk factors have been conflicting, particularly concerning serum lipids. We examined several associations in a representative sample of 1203 men and women between 25 and 64 years of age in the state of Vorarlberg, Austria. Coffee consumption correlated significantly (p less than 0.001) with the number of cigarettes smoked per day (men: r = 0.30; women: r = 0.26) and the prevalence of smokers (men: r = 0.27; women: r = 0.25). We found no adverse effect of coffee on blood lipids or lipoproteins. The lack of evidence was independent of smoking habits in both sexes. We suggest that the lack of evidence is due to the usual method of preparing coffee in Vorarlberg, ie percolated or as 'Espresso'.

Rearing conditions affect neuron morphology in a telencephalic area of the zebra finch.

The morphology of ANC (Archi-neostriatum caudale) neurons in zebra finches is affected by arousal and rearing conditions. Branching index and spine density of ANC neurons are decreased in isolated birds and enhanced in cage reared animals, compared to aviary reared animals. Chasing the birds around the cage, or seven days of social contact with a female, raises these indices in birds isolated until adulthood relative to those of the aviary reared animals. We conclude that branching index and spine density of ANC neurons are determined during development by the amount of social contact, arousal, and activation of ANC. The changes observed after short term treatments in adult bird may depend on the same factors.

Spine density changes in forebrain areas of the zebra finch by TEA-induced potentiation.

When young zebra finch males who have been reared in isolation court a female for the first time they develop a stable preference for females of this species (sexual imprinting). During this first courtship, two areas of the forebrain show a reversible enhancement of spine density, while in two other areas, spine density decreases irreversibly. Here we show that the same alterations can be induced by application of tetraethylammonium (TEA) to slices of adult, previously isolated males. TEA application induces an enhancement of spine density in two forebrain areas and leads to a decrease of spine density in two others. Although the exact mechanisms are unknown, our results indicate that potentiation phenomena are involved in the spine density changes induced by first courtship.

Morphological alterations of the visual system in white zebra finches.

Visual system anomalies in albino mammals are generally caused by a lack of retinal pigment and misrouting of retinofugal optic fibres. This study shows that the visual system of white zebra finches differs morphologically from that of normally coloured (wild type) birds, although eye pigmentation and retinofugal projection are normal. In white zebra finches, the recrossing tectorotundal projection is enhanced and a new (reciprocal) connection is established between the n. rotundi of both hemispheres. The morphological alterations found in this study may contribute to the previously described enhanced processing of ipsilateral stimuli in white zebra finches.

Testosterone-dependent plasticity of avian forebrain neurons is not restricted to the song control system.

Testosterone is acting on brain areas involved in the control of sexual behaviour, for example the preoptic area and the song system. We now demonstrate that it also affects other avian brain areas, as exemplified here by measurement of spine densities. Depletion of testosterone by castration or application of cyproterone acetate leads to a decrease in spine density in secondary sensory areas like lateral neo- and hyperstriatum and hyperstriatum accessorium and dorsale, or in associative areas such as the caudal archi- and neostriatum. We conclude that testosterone is acting directly on the spines, and suggest that the mechanism of spine density control by hormones may have arisen because of energy demands.

Arousal enhances [14C]2-deoxyglucose uptake in four forebrain areas of the zebra finch.

The activity pattern of the forebrain of male zebra finches was investigated by the [14C]2-deoxyglucose (2-DG) method in 4 different behavioral situations. Sitting alone in the cage (control); courtship by experienced birds; first courtship of inexperienced birds (100 days of age), and chasing the birds around the cage. The primary sensory areas (ectostriatum, field L) were active above background in each experiment. Vocal-motor control areas were at background activity (RA, HVc, MAN), or below background (area X), all unaffected by the type of experiment. In contrast, 4 different areas were active in Expts. 3 and 4, but not in 1 and 2: part of the neostriatum intermedium; part of the lateral neostriatum, both with adjacent parts of the hyperstriatum ventrale, hyperstriatum accessorium and hyperstriatum dorsale, a portion of the caudal neo/archistriatum. It is concluded that the enhanced activity of these areas is not due to distinct external stimulation or enhanced motor activity, but correlates with high arousal levels of the animals.

Phase specific morphological changes induced by social experience in two forebrain areas of the zebra finch.

We examined the changes of spine density in Golgi preparations of two different areas of the forebrain of the zebra finch, the ANC (Archi-Neostriatum caudale) and MNH (medial Neo-Hyperstriatum) during development, after transferring male birds from isolation to a social condition (exposure to a female for 1 week), and after a second isolation period. MNH and ANC are two of four brain regions which are strongly activated if a male bird is exposed to a female after some time of isolation. The results of our study can be summarized as follows. 1: a peak-decline trend is observed in ANC, but not in MNH. 2: rearing conditions do not affect the development of both areas until day 70. 3: from 80 days of age, isolation leads to reduced spine density within ANC, but to enhanced spine density within MNH. 4: short social contact after isolation diminishes or eliminates the effects of isolation by an enhancement of spine density in ANC and a reduction of spine density within MNH. 5: the effects of short social rearing after isolation are reversible within ANC, but not within MNH. We presume that the alterations of spine density, which are induced by changes in social conditions, are restricted to ages older than 70 days by hormonal factors. We propose that the complexity of the ANC neuronal net follows the complexity of the social environment, and that the level of arousal is the most important factor influencing the complexity.(ABSTRACT TRUNCATED AT 250 WORDS)

Behavioural and neurophysiological aspects of sexual imprinting in zebra finches.

Sexual imprinting has been defined as the process by which young animals learn the characteristics of their future sexual partners. It is a two stage process including an acquisition period where features of the social environment are learnt, and a stabilization process by which, under the guidance of the previously acquired social information, a preference for a sexual partner is established and stabilized, so that it cannot be altered again subsequently. The stabilization process is short (1 h) and can be controlled experimentally. This allows for the design of experiments to examine the physiological events accompanying the imprinting process. During the stabilization process, four areas of the forebrain are more activated than in any other behavioural context. These are the hyperstriatum accessorium/dorsale (HAD), the archi-neostriatum caudale (ANC), the medial neo/hyperstriatum (MNH) and the lateral neo/hyperstriatum (LNH). Isolation during development reduces the spine density of neurons in HAD and ANC and enhances it in MNH and LNH. Subsequent exposure to a female (which stabilizes the previously acquired preference in behavioural experiments) for 1 week leads to an enhancement of spine densities in HAD and ANC, and to a reduction in MNH and LNH. The enhancement in HAD and ANC is reversible by a second isolation period after the exposure to a female, the reduction within MNH and LNH is not. This irreversibility indicates that the reduction process within MNH and LNH may be the anatomical manifestation of the imprinting process. The examination of spine densities in the four brain areas after two experiments which have been shown previously to affect the stabilization process in behavioural experiments, confirms this idea.

Activity-dependent plasticity in visual forebrain areas of the zebra finch.

It has previously been shown that the activity of some area of the forebrain of birds is dependent on the arousal level of the animal. Other areas do not show this dependency. This paper, on the basis of 2-DG experiments and spine density measurements on Golgi-impregnated tissue, shows that primary telencephalic target areas of the two visual pathways of zebra finch males are not dependent on arousal for activation. In contrast, secondary areas of both visual pathways show arousal-dependent activation. Only the secondary visual areas also show effects of rearing conditions on the spine density: isolation of the birds from day 40 reduces spine density in the hyperstriatum accessorium (HA) of the thalamofugal pathway, and enhances spine density in the lateral neo/hyperstriatum (LNH, tectofugal pathway) significantly from day 80, if compared to aviary-reared birds. A 1-week exposure to a female eliminates the isolation effects in both areas. A second isolation period again reduces the spine density in the HA, but does not enhance it again in the LNH. By comparison with previous studies, we conclude that the spine density in the HA reflects the complexity of the social environment. The irreversible reduction of spine density in the LNH as consequence of the 7-day exposure to a female is interpreted as physiological correlate of an imprinting process, which has previously been shown to occur at the same time. The effects in both area, the HA and LNH, are dependent on arousal, which may be mediated by brainstem efferents innervating the secondary, but not the primary, visual areas in birds and in mammals.

Afferent connections of the ectostriatum and visual wulst in the zebra finch (Taeniopygia guttata castanotis Gould)--an HRP study.

Afferent connections of the two main areas in the telencephalon, the visual wulst and the ectostriatum, were traced in the zebra finch by injection of horseradish peroxidase and staining with tetramethylbenzidine (TMB). Nuclei projecting to the hyperstriatum accessorium (HA) or the HIS region (lamina hyperstriatica intercalatus superior) were: (1) ipsilaterally the n. dorsalis anterior pars lateralis (DLL) with its two subdivisions DLLd and DLLv, the n. dorsolateralis anterior pars magnocellularis (DLAmc), and the area pretectalis (AP); (2) bilaterally the nucleus of the septomesencephalic tract (SPC) with the ipsilateral component coming from the medial, the contralateral component from the lateral part of the nucleus. As in the pigeon or the owl the ectostriatum of the zebra finch receives massive input, which is topographically ordered, from the n. rotundus. In addition to this pathway the ectostriatum receives additional visual input from the ipsilateral area pretectalis, the n. subrotundus and eventually a bilateral projection from the n. tegmenti pedunculopontinus pars compacta (TPC).

Flash evoked responses in a song control nucleus of the zebra finch (Taeniopygia guttata castanotis).

The song of the zebra finch is facilitated and altered by the presence of a female. Thus, visual information should affect the song system of the bird. Visually evoked potentials can be recorded from n. hyperstriatum ventrale pars caudale (HVc). The long latency of this potential and its variability indicate several processing steps between primary sensory areas of the telencephalon and HVC. Within HVc, under these experimental conditions no interaction between acoustic and visual input could be demonstrated. However, at the dorsal border and within the shelf below HVc, visual information seems to enhance acoustically evoked potentials.

The sensitive period for the morphological effects of monocular deprivation in two nuclei of the tectofugal pathway of zebra finches.

Previous experiments with 2-deoxyglucose (2-DG) suggested the existence of a critical period for the effects of monocular deprivation in the nucleus rotundus of zebra finches. The present study concerns the time course of this sensitive period for the morphological effects of monocular deprivation in two areas of the tectofugal visual pathway of zebra finches, the nucleus rotundus of the thalamus and the telencephalic ectostriatum. Cell size and volume changes were measured in birds subjected to 40 days of unilateral eye closure starting at ages spaced regularly throughout the first 70 days of life. The results show that monocular deprivation markedly affects cell size in both areas if the treatment starts at one or 10 days posthatch. The differences between deprived and non-deprived neurons decline monotonically with increasing visual experience prior to deprivation. However, deprivation onset at day 40 again causes as severe effects as early monocular closure. Deprivation as from day 50 or later no longer leads to abnormalities. The measurements of the volume of the nucleus rotundus parallel the cell size measurements, with the exception that the second increase in sensitivity occurs with deprivation onset at day 50 instead of day 40. These data indicate that the time course of the sensitive period for the effects of monocular deprivation may be double-peaked: the sensitivity for external stimuli declines from hatch until day 30, but has another peak at 40-50 days of life. The definite end of the sensitive period, as determined with this method, can therefore be assumed to be at around day 50-60.