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In acute inhalation toxicity studies, animals inhale substances at given concentrations. Without additional information, however, appropriate starting concentrations for in-vivo inhalation studies are difficult to estimate. The goal of this project was the prevalidation of precision-cut lung slices (PCLS) as an ex-vivo alternative to reduce the number of animals used in inhalation toxicity studies. According to internationally agreed principles for Prevalidation Studies, the project was conducted in three independent laboratories. The German BfR provided consultancy in validation principles and independent support with biostatistics. In all laboratories, rat PCLS were prepared and exposed to 5 concentrations of 20 industrial chemicals under submerged culture conditions for 1 h. After 23 h post-incubation, toxicity was assessed by measurement of released lactate dehydrogenase and mitochondrial activity. In addition, protein content and pro-inflammatory cytokine IL-1α were measured. For all endpoints IC 50 values were calculated if feasible. For each endpoint test acceptance criteria were established. This report provides the final results for all 20 chemicals. More than 900 concentration–response curves were analyzed. Log 10 [IC 50 (μM)], obtained for all assay endpoints, showed best intra- and inter-laboratory consistency for the data obtained by WST-1 and BCA assays. While WST-1 and LDH indicated toxic effects for the majority of substances, only some of the substances induced an increase in extracellular IL-1α. Two prediction models (two-group classification model, prediction of LC 50 by IC 50 ) were developed and showed promising results. [ABSTRACT FROM AUTHOR]

Decreased mortality and disability after traumatic brain injury is a significant medical challenge. Desflurane, a widely used volatile anesthetic has proven to be neuroprotective in a variety of in vitro and in vivo models of ischemic brain injury. The aim of this study was to investigate whether desflurane exhibits neuroprotective properties in an in vitro model of traumatic brain injury. Organotypic hippocampal slice cultures were prepared from brains of 5-7-day-old C57/BL6 mouse pups. After 14 days of culture, the slices were subjected to a focal mechanical trauma and thereafter incubated with three different concentrations of desflurane (2, 4 and 6%) for 2, 24 and 72 hours. Cell injury was assessed with propodium iodide uptake. Our results showed that after 2 hours of desflurane exposure, no significant change in trauma intensity was observed. However, 2% and 4% desflurane could reduce the trauma intensity significantly in the no trauma group than in the no desflurane and trauma group. Incubation with 4% desflurane for 24 hours doubled the trauma intensity in comparison to the trauma control group and the trauma intensity further increased after 72 hours of incubation. Furthermore, a dose-dependent increase of trauma intensity after 24 hours exposure was observed. Our results suggest that a general neuroprotective attribute of desflurane in an in vitro model of traumatic brain injury was not observed. [ABSTRACT FROM AUTHOR]

Summary Grid cells, defined by their striking periodic spatial responses in open 2D arenas, appear to respond differently on 1D tracks: the multiple response fields are not periodically arranged, peak amplitudes vary across fields, and the mean spacing between fields is larger than in 2D environments. We ask whether such 1D responses are consistent with the system’s 2D dynamics. Combining analytical and numerical methods, we show that the 1D responses of grid cells with stable 1D fields are consistent with a linear slice through a 2D triangular lattice. Further, the 1D responses of comodular cells are well described by parallel slices, and the offsets in the starting points of the 1D slices can predict the measured 2D relative spatial phase between the cells. From these results, we conclude that the 2D dynamics of these cells is preserved in 1D, suggesting a common computation during both types of navigation behavior. [ABSTRACT FROM AUTHOR]

Multi time-point pseudo-continuous arterial spin labelling (pCASL) with a Look-Locker EPI readout can sample the signal curve of blood kinetics at multiple time points after the labelling pulse. However, due to signal relaxation of labelled blood, the number of readout slices is limited. The aim of this study is to employ a multiband excitation technique to triple the number of readout slices in multi time-point pCASL. The multiband technique, along with 2-fold in-plane parallel imaging, was incorporated into the Look-Locker EPI for the multi time-point sampling of blood kinetic behaviour following the pCASL labelling scheme. The performance evaluation of the multiband and the single-band techniques were performed on four healthy subjects using a 32-channel head RF coil at 3T. Quantitative perfusion maps were analysed using a combination of labelling with and without flow suppression gradients. The perfusion maps provided by the multiband accelerated multi time-point pCASL were in good agreement with the conventional single-band technique. Multiband acceleration caused SNR loss but offered quantitative perfusion maps in 6.23 min with 18 slices compared with 6 slices within the same time period for the single-band method. As conclusion, the multiband technique can successfully triple the number of readout slices while achieving comparable perfusion data in the same measurement time as the conventional single-band readout. [ABSTRACT FROM AUTHOR]

The effects of pulsed electric fields (PEF) on the extractability of phenolics from fresh spearmint leaves were studied. The circular tissue slices treated with PEF were subjected to a solid–liquid extraction process and the extracted phenolics were evaluated by measuring total phenolic content (TPC), antioxidant capacity (AC) and antioxidant activity (AA). The results obtained were compared with those of the extractions repeated with freezing/thawing, heat, microwave and solvent treatment. As measured from a conductivity-based tissue disintegration index, the highest disintegration (0.86 ± 0.02) was obtained at a PEF intensity corresponding to 99 pulses of 3 kV/cm with a specific energy input of 4102 ± 239 J/kg. The extraction with the PEF pre-treatment produced comparable TPC, AC and AA values with the heat and microwave pre-treatments, and a markedly higher AA value than the freezing/thawing. Furthermore, the combined effects of the PEF and other pre-treatments resulted in significant synergism in TPC and AC while showing antagonism in AA properties. [ABSTRACT FROM AUTHOR]

In the only bipolar cycling in vitro model, rat hippocampal slices are treated with the sodium pump inhibitor ouabain, which induces epileptiform activity, followed by refractory activity loss that recovers and cycles back to epileptiform activity. Thus, clinical cycling seen in patients with bipolar disorder is modeled on a cellular level as alternating hyperactivity and hypoactivity interspersed with normal activity. In this study, we tested the ability of cariprazine a new antipsychotic candidate to block ouabain-induced changes in rat hippocampal slices. Cycling of population spikes and epileptiform bursts was evoked using an extracellular stimulation electrode located in the Schaeffer collaterals of 400-µm-thick rat hippocampal slices treated with ouabain (3.3 μM) alone or in combination with cariprazine (1, 5, 25, and 50 µM). Responses were recorded using an extracellular electrode placed in the cell body layer of the CA1 region. Cariprazine 25 and 50 µM delayed ouabain-induced epileptiform burst onset and subsequent activity loss. Lower cariprazine concentrations were ineffective. Cariprazine delays the onset of ouabain-induced epileptiform bursts and the loss of spiking activity similarly to that previously demonstrated with the mood stabilizer lithium. These results suggest that cariprazine may have therapeutic potential for treatment of bipolar disorder. [ABSTRACT FROM AUTHOR]

A correction to the article "Increased Spread and Replication Efficiency of in Listeria Monocytogenes Organotypic Brain-Slices Is Related to Multilocus Variable Number of Tandem Repeat Analysis (MLVA) Complex" by Claudia Guildimann et al. that was published in a 2014 issue is presented.

Extracellular purines have multiple functional roles in development, plastic remodelling, and regeneration of the CNS by stimulating certain P2X/Y receptor (R) subtypes. In the present study we elucidated the involvement of P2YRs in neuronal fibre outgrowth in the developing nervous system. We particularly focused on the P2Y 1 R subtype and the dopaminergic system, respectively. For this purpose, we used organotypic slice co-cultures consisting of the ventral tegmental area/substantia nigra (VTA/SN) and the prefrontal cortex (PFC). After detecting the presence of the P2Y 1 R in VTA/SN, PFC, and on outgrowing fibres in the border region (e.g. on glial processes) connecting both brain slices, we could show that pharmacological modulation of the receptor influenced neuronal fibre outgrowth. Biocytin-tracing and tyrosine hydroxylase-immunolabelling together with quantitative image analysis revealed a significant increase in fibre growth in the border region of the co-cultures after treatment with ADPβS (P2Y 1,12,13 R agonist). The observed stimulatory potential of ADPβS was inhibited by pre-treatment with the P2X/YR antagonist PPADS. In P2Y 1 R knockout (P2Y 1 R −/− ) mice, the ADPβS-induced stimulatory effect was absent, while growth was significantly enhanced in the co-cultures of the respective wild-type. This observation was confirmed in entorhino-hippocampal co-cultures, an example of a different projection system, expressing the P2Y 1 R. Using wortmannin and PD98059 we further showed that PI3K/Akt and MAPK/ERK cascades are involved in the mechanism underlying ADPβS-induced fibre growth. In conclusion, the data of this study clearly indicate that activation of the P2Y 1 R stimulates fibre growth and thereby emphasises the general role of this particular receptor subtype during development and regeneration. [ABSTRACT FROM AUTHOR]

In vitro studies on living rat hippocampus slices addressed the role of Ca in the mechanism of deprivation potentiation of population responses (pop spikes) of neurons in field CA1 induced by prolonged (60 min) interruption of low-frequency test stimulation of Schaffer collaterals. Two phases were seen in deprivation potentiation, with presumptively different origins: an initial short-lived 'peak' (about 12 min) and a longer-lasting 'plateau' (more than 1 h). The experiments reported here showed that the presence of a penetrating Ca chelator (BAPTA-AM), decreasing the Ca concentration in the solution, and depletion of the intracellular calcium depot (presence of thapsigargin/cyclopiazonic acid in the solution) led to reductions in the transient phase and blockade of the longer-lasting phase of deprivation potentiation. These studies thus demonstrate the key roles of both extracellular and stored intracellular calcium in the mechanism of development of deprivation potentiation. [ABSTRACT FROM AUTHOR]

Cardiac tissue slices are becoming increasingly popular as a model system for cardiac electrophysiology and pharmacology research and development. Here, we describe in detail the preparation, handling, and optical mapping of transmembrane potential and intracellular free calcium concentration transients (CaT) in ventricular tissue slices from guinea pigs and rabbits. Slices cut in the epicardium-tangential plane contained wellaligned in-slice myocardial cell strands ("fibers") in subepicardial and midmyocardial sections. Cut with a high-precision slow-advancing microtome at a thickness of 350 to 400 m, tissue slices preserved essential action potential (AP) properties of the precutting Langendorff- perfused heart. We identified the need for a postcutting recovery period of 36 min (guinea pig) and 63 min (rabbit) to reach 97.5% of final steady-state values for AP duration (APD) (identified by exponential fitting). There was no significant difference between the postcutting recovery dynamics in slices obtained using 2,3-butanedione 2-monoxime or blebistatin as electromechanical uncouplers during the cutting process. A rapid increase in APD, seen after cutting, was caused by exposure to ice-cold solution during the slicing procedure, not by tissue injury, differences in uncouplers, or pH-buffers (bicarbonate; HEPES). To characterize intrinsic patterns of CaT, AP, and conduction, a combination of multipoint and field stimulation should be used to avoid misinterpretation based on source-sink effects. In summary, we describe in detail the preparation, mapping, and data analysis approaches for reproducible cardiac tissue slice-based investigations into AP and CaT dynamics. [ABSTRACT FROM AUTHOR]

Introduction: Lung cancer remains the leading cause of tumor-related deaths, despite advances in the understanding of the disease pathogenesis and in its clinical treatment. It is crucial to develop novel technologies to discover disease biomarkers and predict individual therapy response. Materials and methods: We established 48 patients-derived tumor xenografts (PDTXs) implanted in the subrenal capsule of immunodeficient mice using thin, precision-cut tumor tissue slices, derived from five patients affected by non-small cell lung cancer. Twenty-six tissue slices were immediately processed and implanted at sample recovery [patients-derived tumor xenografts derived from fresh tissue (dPDTX)], whereas the remaining sections were cultured on specific organotypic supports at 37°C and 5% CO2 for 24 h before grafting [patients-derived tumor xenografts derived from cultured tissue (cPDTX)]. At sacrifice, xenografts tissue morphology, proliferation (Ki67), and histotype markers were analyzed. Oncogenic miRNAs profiles were assessed in PDTXs, human tumors, and serum from one patient. Results: Xenografts retained the original cancer features and there were no differences between dPDTXs and cPDTXs. Squamous cell carcinoma (SCC) xenografts showed a higher engraftment rate than adenocarcinoma (AC)-derived tumors. At basal time, Ki67 levels were higher in SCCs than in ACs, and the expression levels of genes associated to a stem cell-like phenotype were also more expressed in SCC samples. The analysis of oncogenic miRNAs showed that circulating miR-19b, -21, and -210 levels were correlated with higher Ki67 expression in xenografts. Conclusion: Our study implemented the PDTX model with thin, precision-cut tumor slices from small tumors, which could be useful for clinical applications and predictive purposes. The different engraftment success is likely determined by tumor histotype, high proliferation index, and the expression of genes essential for cancer stem cells maintenance. Our PDTXs model could be a valid tool to expand primary tumors for the discovery of new biomarkers and explore therapeutic options. [ABSTRACT FROM AUTHOR]

The Xenopus tadpole optic tectum is a multisensory processing center that receives direct visual input as well as nonvisual mechanosensory input. The tectal neurons that comprise the optic tectum are organized into layers. These neurons project their dendrites laterally into the neuropil where visual inputs target the distal region of the dendrite and nonvisual inputs target the proximal region of the same dendrite. The Xenopus tadpole tectum is a popular model to study the development of sensory circuits. However, whole cell patch-clamp electrophysiological studies of the tadpole tectum (using the whole brain or in vivo preparations) have focused solely on the deep-layer tectal neurons because only neurons of the deep layer are visible and accessible for whole cell electrophysiological recordings. As a result, whereas the development and plasticity of these deep-layer neurons has been well-studied, essentially nothing has been reported about the electrophysiology of neurons residing beyond this layer. Hence, there exists a large gap in our understanding about the functional development of the amphibian tectum as a whole. To remedy this, we developed a novel isolated brain preparation that allows visualizing and recording from all layers of the tectum. We refer to this preparation as the "horizontal brain slice preparation." Here, we describe the preparation method and illustrate how it can be used to characterize the electrophysiology of neurons across all of the layers of the tectum as well as the spatial pattern of synaptic input from the different sensory modalities. [ABSTRACT FROM AUTHOR]

Background Ex vivo acute living brain slices are a broadly employed and powerful experimental preparation. Most new technology regarding this tissue has involved the chamber used when performing electrophysiological experiments. Alternatively we instead focus on the creation of a simple, versatile aerator designed to allow maintenance and manipulation of acute brain slices and potentially other tissue in a multi-well cell culture plate. New method Here we present an easily manufactured aerator designed to fit into a 24-well cell culture plate. It features a nylon mesh and a single microhole to enable gas delivery without compromising tissue stability. The aerator is designed to be individually controlled, allowing both high throughput and single well experiments. Results The aerator was validated by testing material leach, dissolved oxygen delivery, brain slice viability and neuronal electrophysiology. Example experiments are also presented, including a test of whether β1-adrenergic receptor activation regulates gene expression in ex vivo dorsal striatum using qPCR. Comparison with existing methods Key differences include enhanced control over gas delivery to individual wells containing brain slices, decreased necessary volume, a sample restraint to reduce movement artifacts, the potential to be sterilized, the avoidance of materials that absorb water and small biological molecules, minimal production costs, and increased experimental throughput. Conclusion This new aerator is of high utility and will be useful for experiments involving brain slices and other potentially tissue samples in 24-well cell culture plates. [ABSTRACT FROM AUTHOR]

Background In recent years, 3D printing technology has become inexpensive and simple enough that any lab can own and use one of these printers. New method We explored the potential use of 3D printers for quickly and easily producing in vitro slice chambers for patch clamp electrophysiology. Slice chambers were produced using five available plastics: ABS, PLA, Nylon 618, Nylon 680, and T-glase. These “lab-made” chambers were also made using stereolithography through a professional printing service (Shapeways). This study measured intrinsic membrane properties of neurons in the brain stem pedunculopontine nucleus (PPN) and layer V pyramidal neurons in retrosplenial cortex. Results Nylon 680 and T-glase significantly hyperpolarized PPN neurons. ABS increased input resistance, decreased action potential amplitude, and increased firing frequency in pyramidal cortical neurons. To test long term exposure to each plastic, human neuroblastoma SHSY5Y cell cultures were exposed to each plastic for 1 week. ABS decreased cell counts while Nylon 618 and Shapeways plastics eliminated cells. Primary mouse pituitary cultures were also tested for 24-h exposure. ABS decreased cell counts while Nylon 618 and Shapeways plastics dramatically decreased cell counts. Comparison to existing methods Chambers can be quickly and inexpensively printed in the lab. ABS, PLA, Nylon 680, and T-glase plastics would suffice for many experiments instead of commercially produced slice chambers. Conclusions While these technologies are still in their infancy, they represent a powerful addition to the lab environment. With careful selection of print material, slice chambers can be quickly and inexpensively manufactured in the lab. [ABSTRACT FROM AUTHOR]

To follow up our previous investigation of Sudan Red staining in plastinated brain specimen (Vibulchan et al., 2012), the present study aimed to examine staining dyes that can withstand the plastination procedures and be reserved in the specimen thereafter. Three staining methods, Mulligan's method, Alston's method and Prussian blue reaction method were tested in dog brain transverse slices. After the staining procedures the brain slices were dehydrated then forced impregnated using a polymer as described previously. The plastinated brain slices were compared for staining retention and analyzed for shrinkage. Results showed that Alston's method of staining was preferable to dye preservation after plastination procedures. However, the shrinkage occurred with all three staining methods. [ABSTRACT FROM AUTHOR]

Background The medulla is capable of controlling and modulating ingestive behavior and gastrointestinal function. These two functions, which are critical to maintaining homeostasis, are governed by an interconnected group of nuclei dispersed throughout the medulla. As such, in vitro experiments to study the neurophysiologic details of these connections have been limited by spatial constraints of conventional slice preparations. New method This study demonstrates a novel method of sectioning the medulla so that sensory, integrative, and motor nuclei that innervate the gastrointestinal tract and the oral cavity remain intact. Results Immunohistochemical staining against choline-acetyl-transferase and dopamine-β-hydroxylase demonstrated that within a 450 μm block of tissue we are able to capture sensory, integrative and motor nuclei that are critical to oromotor and gastrointestinal function. Within slice tracing shows that axonal projections from the NST to the reticular formation and from the reticular formation to the hypoglossal motor nucleus (mXII) persist. Live-cell calcium imaging of the slice demonstrates that stimulation of either the rostral or caudal NST activates neurons throughout the NST, as well as the reticular formation and mXII. Comparison with existing methods This new method of sectioning captures a majority of the nuclei that are active when ingesting a meal. Tradition planes of section, i.e. coronal, horizontal or sagittal, contain only a limited portion of the substrate. Conclusions Our results demonstrate that both anatomical and physiologic connections of oral and visceral sensory nuclei that project to integrative and motor nuclei remain intact with this new plane of section. [ABSTRACT FROM AUTHOR]

Cortical spreading depression (CSD) occurs during various forms of brain injury such as stroke, subarachnoid hemorrhage, and brain trauma, but it is also thought to be the mechanism of the migraine aura. It is therefore expected to occur over a range of conditions including the awake behaving state. Yet it is unclear how such a massive depolarization could occur under relatively benign conditions. Using a microfluidic device with focal stimulation capability in a mouse brain slice model, we varied extracellular potassium concentration as well as the area exposed to increased extracellular potassium to determine the minimum conditions necessary to elicit CSD. Importantly, we focused on potassium levels that are physiologically plausible (≤145 mM; the intracellular potassium concentration). We found a strong correlation between the threshold concentration and the slice area exposed to increased extracellular potassium: minimum area of exposure was needed with the highest potassium concentration, while larger areas were needed at lower concentrations. We also found that moderate elevations of extracellular potassium were able to elicit CSD in relatively small estimated tissue volumes that might be activated under noninjury conditions. Our results thus show that CSD may be inducible under the conditions that expected in migraine aura as well as those related to brain trauma. [ABSTRACT FROM AUTHOR]

Glutamate is thought to be the most important excitatory neurotransmitter in the CNS, while glutamine predominantly serves as a precursor and metabolite in the glutamate-glutamine cycle. To verify the interaction between intrinsic extracellular glutamate, y-aminobutyric acid (GABA) levels and glial glutamine outflow in human tissue, fresh brain slices from human frontal cortex were incubated in superfusion chambers in vitro. Human neocortical tissue was obtained during surgical treatment of subcortical brain tumors. For superfusion experiments, the white matter was separated and discarded from the gray matter, which finally contained all six neocortical layers. Outflows of endogenous glutamate, GABA and glutamine were established after a 40-min washout period and amounts were simultaneously quantified after two-phase derivatization by high-performance liquid chromatography with electrochemical detection. Under basal conditions, amounts of glutamate could be found 20-fold in comparison to the inhibitory neurotransmitter GABA, whereas this excitatory predominance markedly declined after veratridine-induced activation. The basal glutamate:glutamine ratio of extracellular levels was approximately 1:2. Blockade or activation of the voltage-gated sodium channel by tetrodotoxin or veratridine significantly modulated glutamate levels, but the glutamate:glutamine ratio was nearly constant with 1:2. When the EAAT blocker TBOA was employed, glutamine remained nearly unchanged whereas glutamate significantly enhanced. These results led us to suggest that glutamine release through glial SN1 is related to EAAT activity that can be modulated by intrinsic extracellular glutamate in human cortical slices. [ABSTRACT FROM AUTHOR]

The study and use of decellularized extracellular matrix (dECM) in tissue engineering, regenerative medicine, and pathophysiology have become more prevalent in recent years. To obtain dECM, numerous decellularization procedures have been developed for the entire organ or tissue blocks, employing either perfusion of decellularizing agents through the tissue's vessels or submersion of large sections in decellularizing solutions. However, none of these protocols are suitable for thin tissue slices (less than 100 µm) or allow side-by-side analysis of native and dECM consecutive tissue slices. Here, we present a detailed protocol to decellularize tissue sections while maintaining the sample attached to a glass slide. This protocol consists of consecutive washes and incubations of simple decellularizing agents: ultrapure water, sodium deoxycholate (SD) 2%, and deoxyribonuclease I solution 0.3 mg/mL (DNase I). This novel method has been optimized for a faster decellularization time (2-3 h) and a better correlation between dECM properties and native tissue-specific biomarkers, and has been tested in different types of tissues and species, obtaining similar results. Furthermore, this method can be used for scarce and valuable samples such as clinical biopsies. This protocol was validated in: Front Bioeng Biotechnol (2022), DOI: 10.3389/fbioe.2022.832178., Competing Interests: Competing interests There are no conflicts of interest or competing interests., (Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC.)

Excessive accumulation of reactive oxygen species (ROS) underlies oxidative damage. We find that in hippocampal slices, decreased activity of glucose-based antioxidant system induces a massive, abrupt, and detrimental change in cellular functions. We call this phenomenon metabolic collapse (MC). This collapse manifested in long-lasting silencing of synaptic transmission, abnormal oxidation of NAD(P)H and FADH2 associated with immense oxygen consumption, and massive neuronal depolarization. MC occurred without any preceding deficiency in neuronal energy supply or disturbances of ionic homeostasis and spread throughout the hippocampus. It was associated with a preceding accumulation of ROS and was largely prevented by application of an efficient antioxidant Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl). The consequences of MC resemble cortical spreading depression (CSD), a wave of neuronal depolarization that occurs in migraine, brain trauma, and stroke, the cellular initiation mechanisms of which are poorly understood. We suggest that ROS accumulation might also be the primary trigger of CSD. Indeed, we found that Tempol strongly reduced occurrence of CSD in vivo, suggesting that ROS accumulation may be a key mechanism of CSD initiation. [ABSTRACT FROM AUTHOR]

Registration of histological slices to volumetric imaging of the prostate is an important task that can be used to optimize imaging for cancer detection. Such registration is challenging due to physical changes of the specimen during excision and fixation, and misalignment of the histological slices during preparation and digital scanning. In this work, we consider a multi-slice to volume registration method in which a stack of sparse, unaligned 2-D whole-mount histological slices is registered to a 3-D volumetric imaging of the prostate. We propose a particle filtering framework to contend with the high dimensionality of the search space and multimodal nature of the optimization. Such framework allows modeling of the uncertainty in the pose of the slices and in the imaged information, in order to derive optimal registration parameters in a Bayesian approach. Intensity-, region-, and point-based similarity metrics were incorporated into the registration algorithm to account for different imaging modalities. We demonstrate and evaluate our method on a diverse set of data that includes a synthetic volume, ex vivo and in vivo magnetic resonance imaging, and in vivo ultrasound. [ABSTRACT FROM AUTHOR]

An increasing number of U.S. soldiers are diagnosed with traumatic brain injury (TBI) subsequent to exposure to blast. In the field, blast injury biomechanics are highly complex and multi-phasic. The pathobiology caused by exposure to some of these phases in isolation, such as penetrating or inertially driven injuries, has been investigated extensively. However, it is unclear whether the primary component of blast, a shock wave, is capable of causing pathology on its own. Previous in vivo studies in the rodent and pig have demonstrated that it is difficult to deliver a primary blast (i.e., shock wave only) without rapid head accelerations and potentially confounding effects of inertially driven TBI. We have previously developed a well-characterized shock tube and custom in vitro receiver for exposing organotypic hippocampal slice cultures to pure primary blast. In this study, isolated primary blast induced minimal hippocampal cell death (on average, below 14% in any region of interest), even for the most severe blasts tested (424 kPa peak pressure, 2.3 ms overpressure duration, and 248 kPa*ms impulse). In contrast, measures of neuronal function were significantly altered at much lower exposures (336 kPa, 0.84 ms, and 86.5 kPa*ms), indicating that functional changes occur at exposures below the threshold for cell death. This is the first study to investigate a tolerance for primary blast-induced brain cell death in response to a range of blast parameters and demonstrate functional deficits at subthreshold exposures for cell death. [ABSTRACT FROM AUTHOR]

Neuronal firing sequences that occur during behavioral tasks are precisely reactivated in the neocortex and the hippocampus during rest and sleep. These precise firing sequences are likely to reflect latent memory traces, and their reactivation is believed to be essential for memory consolidation and working memory maintenance. However, how the organized repeating patterns emerge through the coordinated interplay of distinct types of neurons remains unclear. In this study, we monitored ongoing spatiotemporal firing patterns using a multi-neuron calcium imaging technique and examined how the activity of individual neurons is associated with repeated ensembles in hippocampal slice cultures. To determine the cell types of the imaged neurons, we applied an optical synapse mapping method that identifies network connectivity among dozens of neurons. We observed that inhibitory interneurons exhibited an increase in their firing rates prior to the onset of repeating sequences, while the overall activity level of excitatory neurons remained unchanged. A specific repeating sequence emerged preferentially after the firing of a specific interneuron that was located close to the neuron first activated in the sequence. The times of repeating sequences could be more precisely predicted based on the activity patterns of inhibitory cells than excitatory cells. In line with these observations, stimulation of a single interneuron could trigger the emergence of repeating sequences. These findings provide a conceptual framework that interneurons serve as a key regulator of initiating sequential spike activity. [ABSTRACT FROM AUTHOR]

Highlights: [•] 95% O2 increases production of superoxide and nitric oxide in medullary slices relative to 40% O2 (control). [•] 67–81% of solitary complex neurons maintained were stimulated by 95% O2 (hyperoxia). [•] We suggest using 40% O2 as the control level of O2 in brain slices to avoid confounding effects of cellular oxidation. [•] Neuronal CO2-chemosensitivity is retained in 40% O2 (control O2) in the caudal solitary complex. [•] A greater incidence of CO2-inibition occurs in slices exposed to O2 manipulations. [ABSTRACT FROM AUTHOR]

Highlights: [•] Both adult and aged α7-nAChR KO mice exhibit impaired basic synaptic transmission. [•] Aged α7-nAChR KO mice exhibit significant deficit of hippocampal LTP. [•] Adult α7-nAChR KO mice do not show deficit of hippocampal LTP. [•] Both adult and aged α7-nAChR KO mice show normal paired-pulse facilitation. [ABSTRACT FROM AUTHOR]

Abstract: Brain tissue slices can be maintained within metabolically stable conditions for long periods of time (hours). This experimental setting has been productive for investigating long-term neural function in vitro. Here, we utilize this experimental approach to describe the recovery of functional connectivity in slices from the mouse hippocampus. Hippocampal slices were cut up bisecting the CA1 region (parietal cut) and each severed half placed adjacent to the other. Stimulation and recording electrodes were placed on each side of the cut; with one electrode stimulating one hemi-slice (20V, 0.033Hz) and the other electrode recording the evoked response from the adjacent hemi-slice. As expected, no evoked response was observed shortly after the beginning of stimulation. However, 20–40min after the initiation of stimulation a large depolarization signal was detected. Right after that, fiber volley potentials were observed in the adjacent hemi-slice. After 1h excitatory postsynaptic potentials (EPSP) were detected. Based on this observation, we hypothesize that recovery of functional connectivity is enhanced by constant delivery of electrical pulses at low frequency to the damaged neural tissue. The described in vitro slice system may become a very suitable experimental method to investigate strategies to enhance the recovery of neural connectivity after brain injury. [Copyright &y& Elsevier]

We used phase resetting methods to predict firing patterns of rat subthalamic nucleus (STN) neurons when their rhythmic firing was densely perturbed by noise. We applied sequences of contiguous brief (0.5–2 ms) current pulses with amplitudes drawn from a Gaussian distribution (10–100 pA standard deviation) to autonomously firing STN neurons in slices. Current noise sequences increased the variability of spike times with little or no effect on the average firing rate. We measured the infinitesimal phase resetting curve (PRC) for each neuron using a noise-based method. A phase model consisting of only a firing rate and PRC was very accurate at predicting spike timing, accounting for more than 80% of spike time variance and reliably reproducing the spike-to-spike pattern of irregular firing. An approximation for the evolution of phase was used to predict the effect of firing rate and noise parameters on spike timing variability. It quantitatively predicted changes in variability of interspike intervals with variation in noise amplitude, pulse duration and firing rate over the normal range of STN spontaneous rates. When constant current was used to drive the cells to higher rates, the PRC was altered in size and shape and accurate predictions of the effects of noise relied on incorporating these changes into the prediction. Application of rate-neutral changes in conductance showed that changes in PRC shape arise from conductance changes known to accompany rate increases in STN neurons, rather than the rate increases themselves. Our results show that firing patterns of densely perturbed oscillators cannot readily be distinguished from those of neurons randomly excited to fire from the rest state. The spike timing of repetitively firing neurons may be quantitatively predicted from the input and their PRCs, even when they are so densely perturbed that they no longer fire rhythmically. [ABSTRACT FROM AUTHOR]

Highlights: [•] We investigated the effect of respiration on motion correction of EPI images. [•] Respiration introduces additional noise after motion correction for long TR. [•] We proposed a new segmented motion correction for sequential acquisition order. [•] The method works best for superior slices with performance comparable to RETROICOR. [ABSTRACT FROM AUTHOR]

Cellular mechanisms for antinociception produced by oxytocin in the spinal dorsal horn have not yet been investigated thoroughly. We examined how oxytocin affects synaptic transmission in substantia gelatinosa neurons, which play a pivotal role in regulating nociceptive transmission, by applying the whole-cell patch-clamp technique to the substantia gelatinosa neurons of adult rat spinal cord slices. Bathapplied oxytocin did not affect glutamatergic spontaneous, monosynaptically- evoked primary-afferent Aδ-fiber and C-fiber excitatory transmissions. On the other hand, oxytocin produced an inward current at -70 mV and enhanced GABAergic and glycinergic spontaneous inhibitory transmissions. These activities were repeated with a slow recovery from desensitization, concentration-dependent and mimicked by oxytocin-receptor agonist. The oxytocin current was inhibited by oxytocin-receptor antagonist, intracellular GDPβS, U-73122, 2-aminoethoxydiphenyl borate, but not dantrolene, chelerythrine, dibutyryl cyclic-AMP, CNQX, Ca2+-free and tetrodotoxin, while the spontaneous inhibitory transmission enhancements were depressed by tetrodotoxin. Current-voltage relation for the oxytocin current reversed at negative potentials more than the equilibrium potential for K+, or around 0 mV. The oxytocin current was depressed in high-K+, low-Na+ or Ba2+-containing solution. Vasopressin V1A- receptor antagonist inhibited the oxytocin current, but there was no correlation in amplitude between a vasopressin-receptor agonist [Arg8]vasopressin and oxytocin responses. It is concluded that oxytocin produces a membrane depolarization mediated by oxytocin but not vasopressin-V1A receptors, which increases neuronal activity, resulting in the enhancement of inhibitory transmission, a possible mechanism for antinociception. This depolarization is due to a change in membrane permeabilities to K+ and/or Na+, which is possibly mediated by phospholipase C and inositol 1,4,5-triphosphate-induced Ca2+-release. [ABSTRACT FROM AUTHOR]

Numerous biomaterials based on extracellular matrix-components have been developed. It was our aim to investigate whether a hyaluronic acid–based hydrogel improves neuronal survival and tissue preservation in organotypic spinal cord slice cultures. Organotypic spinal cord slice cultures were cultured for 4 days in vitro (div), either on hyaluronic acid–based hydrogel (hyaluronic acid–gel group), collagen gel (collagen group), directly on polyethylene terephthalate membrane inserts (control group), or in the presence of soluble hyaluronic acid (soluble hyaluronic acid group). Cultures were immunohistochemically stained against neuronal antigen NeuN and analyzed by confocal laser scanning microscopy. Histochemistry for choline acetyltransferance, glial fibrillary acidic protein, and Griffonia simplicifolia isolectin B4 followed by quantitative analysis was performed to assess motorneurons and different glial populations. Confocal microscopic analysis showed a 4-fold increase in the number of NeuN-positive neurons in the hyaluronic acid–gel group compared to both collagen (p < 0.001) and control groups (p < 0.001). Compared to controls, organotypic spinal cord slice cultures maintained on hyaluronic acid–based hydrogel showed 5.9-fold increased survival of choline acetyltransferance-positive motorneurons (p = 0.008), 2-fold more numerous resting microglial cells in the white matter (p = 0.031), and a 61.4% reduction in the number of activated microglial cells within the grey matter (p = 0.05). Hyaluronic acid–based hydrogel had a shear modulus (G′) of ≈1200 Pascals (Pa), which was considerably higher than the ≈25 Pa measured for collagen gel. Soluble hyaluronic acid failed to improve tissue preservation. In conclusion, hyaluronic acid–based hydrogel improves neuronal and – most notably – motorneuron survival in organotypic spinal cord slice cultures and microglial activation is limited. The positive effects of hyaluronic acid–based hydrogel may at least in part be due to its mechanical properties. [ABSTRACT FROM AUTHOR]

Although drug induced steatosis represents a mild type of hepatotoxicity it can progress into more severe non-alcoholic steatohepatitis. Current models used for safety assessment in drug development and chemical risk assessment do not accurately predict steatosis in humans. Therefore, new models need to be developed to screen compounds for steatogenic properties. We have studied the usefulness of mouse precision-cut liver slices (PCLS) as an alternative to animal testing to gain more insight into the mechanisms involved in the steatogenesis. To this end, PCLS were incubated 24 h with the model steatogenic compounds: amiodarone (AMI), valproic acid (VA), and tetracycline (TET). Transcriptome analysis using DNA microarrays was used to identify genes and processes affected by these compounds. AMI and VA upregulated lipid metabolism, whereas processes associated with extracellular matrix remodelling and inflammation were downregulated. TET downregulated mitochondrial functions, lipid metabolism, and fibrosis. Furthermore, on the basis of the transcriptomics data it was hypothesized that all three compounds affect peroxisome proliferator activated-receptor (PPAR) signaling. Application of PPAR reporter assays classified AMI and VA as PPARγ and triple PPARα/(β/δ)/γ agonist, respectively, whereas TET had no effect on any of the PPARs. Some of the differentially expressed genes were considered as potential candidate biomarkers to identify PPAR agonists (i.e. AMI and VA) or compounds impairing mitochondrial functions (i.e. TET). Finally, comparison of our findings with publicly available transcriptomics data showed that a number of processes altered in the mouse PCLS was also affected in mouse livers and human primary hepatocytes exposed to known PPAR agonists. Thus mouse PCLS are a valuable model to identify early mechanisms of action of compounds altering lipid metabolism. [ABSTRACT FROM AUTHOR]

Highlights: [•] Effect of 50Hz magnetic field exposure was tested on rat brain development. [•] Pups were treated in utero or as newborn for 7 days at 0.5 or 3mT. [•] Field potentials were recorded from brain slices at young adult age. [•] Basic excitability of hippocampal and cortical slices increased due to treatment. [•] Fetal exposure inhibited plasticity phenomena in neocortex. [ABSTRACT FROM AUTHOR]

1. Chiral polychlorinated biphenyls (PCBs) such as PCB 136 enantioselectively sensitize the ryanodine receptor (RyR). In light of recent evidence that PCBs cause developmental neurotoxicity via RyR-dependent mechanisms, this suggests that enantioselective PCB metabolism may influence the developmental neurotoxicity of chiral PCBs. However, enantioselective disposition of PCBs has not been fully characterized. 2. The effect of sex and cytochrome P450 (P450) enzyme induction on the enantioselective metabolism of PCB 136 was studied using liver tissue slices prepared from naïve control (CTL), phenobarbital (PB; CYP2B inducer) or dexamethasone (DEX; CYP3A inducer) pretreated adult Sprague-Dawley rats. PCB 136 metabolism was also examined in hippocampal slices derived from untreated rat pups. 3. In liver tissue slices, hydroxylated PCB (OH-PCB) profiles depended on sex and inducer pretreatment, and OH-PCB levels followed the rank orders male > female and PB > DEX > CTL. In contrast, the enantiomeric enrichment of PCB 136 and its metabolites was independent of sex and inducer pretreatment. Only small amounts of PCB 136 partitioned into hippocampal tissue slices and no OH-PCB metabolites were detected. 4. Our results suggest that enantioselective metabolism, sex and induction status of P450 enzymes in the liver may modulate the neurotoxic outcomes of developmental exposure to chiral PCBs. [ABSTRACT FROM AUTHOR]

Abstract: Humans recognize faces exceptionally well. However, the neural correlates of face recognition are still elusive. Accumulated evidence in recent years suggests that the anterior temporal lobe (ATL), in particular face-selective region in the ATL, is a probable locus of face recognition. Unfortunately, functional MRI (fMRI) studies encounter severe signal drop-out in the ventral ATL, where that ATL face area resides. Consequently, all previous studies localized this region in no more than half of the subjects and its volume was relatively small. Thus, a systematic exploration of the properties of the ATL face area is scarce. In the current high-resolution fMRI study we used coronal slice orientation, which permitted us to localize the ATL face area in all the subjects. Furthermore, the volume of the area was much larger than was reported in previous studies. Direct within subjects comparison with data collected with the commonly used axial slice orientation confirmed that the advantage of the coronal slice orientation in revealing a reliable and larger face-selective area in the ATL. Finally, by displaying the face-selective activations resultant from coronal and axial scanning together, we demonstrate an organization principle of a chain of face-selective regions along the posterior–anterior axis in the ventral temporal lobe that is highly reproducible across all subjects. By using the procedure proposed here, a significant progress can be made in studying the neural correlates of face recognition. [Copyright &y& Elsevier]

Highlights: [•] A microfluidic device for perfusion of drugs to brain slices is presented. [•] This device administers drugs to the slices with high spatio-temporal resolution. [•] The tool delivers solution to cells 20× faster than conventional perfusion systems. [•] The device is able to store and deliver up to 4 solutions in series. [•] The tool is free standing and can be used in combination with other probes. [ABSTRACT FROM AUTHOR]

Abstract: The antioxidant and antimicrobial efficacy of grape seed extract (GSE) was studied in restructured mutton slices (RMS) under aerobic and vacuum packaging conditions during refrigerated storage. The RMS treated with grape seed extract (GSE) had significantly (P<0.05) lower thiobarbituric acid reactive substance (TBARS) values and free fatty acids (FFA) % compared to control (C) and butylated hydroxy anisole (BHA) treated RMS during storage at 4±1°C. Addition of GSE significantly (P<0.05) reduced the total psychrophilic and coliform counts in RMS during refrigerated storage. The GSE treated mutton slices recorded significantly (P<0.05) superior scores of color, flavor, juiciness and overall palatability than C and BHA treated RMS. The TBARS values, FFA % and microbial counts increased significantly (P<0.05) during storage. It can be concluded that GSE has excellent antioxidant and antimicrobial properties compared to control and BHA treated RMS during refrigerated storage under aerobic and vacuum conditions. [Copyright &y& Elsevier]

Abstract: Hormones regulate the mechanism of plant growth and development, senescence, and plants’ adaptation to the environment; studies of the molecular mechanisms of plant hormone action are necessary for the understanding of these complex phenomena. However, there is no measurable signal for the hormone signal transduction process. We synthesized and applied a quantum dot-based fluorescent probe for the labeling of jasmonic acid (JA) binding sites in plants. This labeling probe was obtained by coupling mercaptoethylamine-modified CdTe quantum dots with JA using N-hydroxysuccinimide (NHS) as a coupling agent. The probe, CdTe–JA, was characterized by transmission electron microscopy, dynamic light scattering, and fluorescent spectrum and applied in labeling JA binding sites in tissue sections of mung bean seedlings and Arabidopsis thaliana root tips. Laser scanning confocal microscopy (LSCM) revealed that the probe selectively labeled JA receptor. The competition assays demonstrated that the CdTe–JA probe retained the original bioactivity of JA. An LSCM three-dimensional reconstruction experiment demonstrated excellent photostability of the probe. [Copyright &y& Elsevier]

Abstract: Intracellular NAD(P)H oxidoreductases are a class of diverse enzymes that are the key players in a number of vital processes. The method we present and validate here is based on the ability of many NAD(P)H oxidoreductases to reduce the superoxide probe lucigenin, which is structurally similar to flavins, to its highly fluorescent water-insoluble derivative dimethylbiacridene. Two modifications of the method are proposed: (i) an express method for tissue homogenate and permeabilized cells in suspensions and (ii) a standard procedure for cells in culture and acute thin tissue slices. The method allows one to assess, visualize, and localize, using fluorescent markers of cellular compartments, multiple NADH and NADPH oxidoreductase activities. The application of selective inhibitors (e.g., VAS2870, a NOX2 inhibitor; plumbagin, a NOX4 inhibitor) allows one to distinguish and compare specific NAD(P)H oxidoreductase activities in cells and tissues and to attribute them to known enzymes. The method is simple, rapid, and flexible. It can be easily adapted to a variety of tasks. It will be useful for investigations of the role of various NAD(P)H oxidoreductases in a number of physiological and pathophysiological processes. [Copyright &y& Elsevier]

Grossing, or 'gross examination', is the inspection and dissection of surgical specimens into slices, which are subsequently processed and sectioned for eventual microscopic analysis. The goal of grossing is to produce a uniform tissue slice to allow adequate tissue processing and sectioning. Accelerated microwave technology has made uniformity a necessity for quality tissue processing. However, the basic principle of slicing biopsies is that the thickness of the slice is sacrificed for the sake of providing the most representative sample, while maintaining the uniformity of serial slices. A Biopsy Uniform Section (BUS) device is proposed as a technical solution, allowing uniform slices and adjustable immobilization. The core of the design is based on three features: a specific horizontal sidebar for a target slice thickness (e.g. 2 mm, 3 mm, and 4 mm), a vertical sideboard to immobilize the specimen, and a V-notched slot for the cutting blade. The thickness of the gross slice is adjusted, so that the visualized area of diagnostic interest is fully presented, while the specimen remains immobilized in the slot and against the vertical sideboard. [ABSTRACT FROM AUTHOR]

Highlights: [•] NMDA-induced theta oscillations in hippocampal slices were observed. [•] Enrichment of NMDA solution with baclofen increases the probability of inducing theta. [•] NMDA and baclofen-induced theta resembles cholinergically induced oscillations. [Copyright &y& Elsevier]

Highlights: [•] Novel nanoscale 2D and 3D probes for action potential recording in cultured cells, tissue slices, whole organs, and 3D synthetic nanoelectronic/tissue constructs are reviewed. [•] The uniqueness enabled by the use of nanometer scale functional elements is highlighted. Exciting future applications of these new probes in biophysical, electrophysiological, and neural activity mapping studies are discussed. [ABSTRACT FROM AUTHOR]

In complex organisms chemical sensation at the olfactory epithelium in the nose is translated into electrical signals that propagate toward the brain for odor identification. In this study the olfactory epithelium was coupled to an 8×8 multi-electrode array (MEA) for spatial odor detection. Sagittal slices of rat olfactory epithelium with intact connections to the olfactory bulb were analyzed for their electrical responses to odorants and stimulants. In the absence of odorants or stimulants, spontaneous spiking activity could be observed at multiple sites (on average 15 out of 59) with a mean frequency of 22±5Hz. When perfusing the slices with 0.1mM isoamyl acetate or l-carvone, the mean spike frequency increased to 41±6Hz and 44±7Hz respectively. Spike frequency was a function of isoamyl acetate concentration and increased from 38±6Hz at 0.05mM to 49±7Hz at 0.5mM. In the presence of FSK+IBMX the mean spike frequency increased to 94±10Hz. In addition, while FSK+IBMX increased the spike frequency at all sites displaying spontaneous activity, the induced responses to isoamyl acetate and l-carvone displayed a different but partially overlapping spatial distribution pattern. In conclusion, parallel multi-site extracellular recordings by MEA in olfactory slices potentially provide a powerful tool for odorant identification and quantification. [ABSTRACT FROM AUTHOR]

There are currently no practical systems that allow extended regions (>5 mm²) of a tissue slice in vitro to be exposed, in isolation, to changes in ionic conditions or to pharmacological manipulation. Previous work has only achieved this at the expense of access to the tissue for recording electrodes. Here, we present a chamber that allows a tissue slice to be maintained in multiple solutions, at physiological temperatures, and preserves the ability to record from the slice. We demonstrate the effectiveness of the tissue bath with respect to minimizing the mixing of the solutions, maintaining the viability of the tissue, and preserving the ability to record from the slice simultaneously. [ABSTRACT FROM AUTHOR]

Rapamycin is a selective inhibitor of the mammalian target of rapamycin (mTOR), a regulator kinase that integrates growth factors signaling via the phosphoinositide-3-kinase pathway and that has emerged as a novel therapeutic modality in breast cancer (BC). We propose a pre-clinical 'ex-vivo' personalized organotypic culture of BC that preserves the microenvironment to evaluate rapamycin-mediated gene expression changes. Freshly excised ductal invasive BC slices, 400 μm thick ( n=30), were cultured in the presence or absence (control) of rapamycin (20 nM) for 24 h. Some slices were formalin-fixed for immunohistochemical determinations and some were processed for microarray analysis. Control slices in culture retained their tissue morphology and tissue viability (detected by BrdU uptake). The percentage of proliferating cells (assessed by Ki67) did not change up to 24 h of treatment. Immunohistochemical evaluation of p-AKT, p-mTOR, p-4EBP1 and p-S6K1 indicated that AKT/mTOR pathway activation was maintained during cultivation. For microarray analysis, slices were divided into two groups, according to the presence/absence of epidermal growth factor receptor-type 2 and analyzed separately. Limited overlap was seen among differentially expressed genes after treatment ( P<0.01) in both groups suggesting different responses to rapamycin between these BC subtypes. Ontology analysis indicated that genes involved in biosynthetic processes were commonly reduced by rapamycin. Our network analysis suggested that concerted expression of these genes might distinguish controls from treated slices. Thus, breast carcinoma slices constitute a suitable physiological tool to evaluate the short-term effects of rapamycin on the gene profile of individual BC samples. [ABSTRACT FROM AUTHOR]

Abstract: By using brain slice preparations and extracellular recordings, the effect of histamine on spontaneous firing activities of neurons in the inferior vestibular nucleus (IVN), a key structure responsible for integration of vestibular, multisensory, and cerebellar inputs, in rats was investigated. Perfusing slices with histamine (1–10μM) elicited an excitatory response on IVN neurons. The responses were not blocked by low Ca2+/high Mg2+ medium, indicating a direct postsynaptic effect of the amine. Furthermore, the histamine-induced excitation was partially blocked by selective histamine H1 receptor antagonist mepyramine (1μM) and H2 receptor antagonist ranitidine (1μM), respectively. Co-application of mepyramine and ranitidine nearly totally antagonized the histamine-induced excitation. Additionally, both selective H1 receptor agonist 2-pyridylethylamine (30–300μM) and H2 receptor agonist dimaprit (10–100μM) effectively mimicked the excitatory action of histamine on IVN neurons. Moreover, selective H4 antagonist JNJ7777120 (10μM) and agonist VUF8430 (30–300μM) had no effect on IVN neurons. These results demonstrate that histamine excites IVN neurons via postsynaptic H1 and H2 rather than H4 receptors, and suggest that the central histaminergic system actively modulate all four major vestibular nuclei including the IVN and may subsequently influence the vestibular nuclei-related reflexes and functions. [Copyright &y& Elsevier]