Parallel action preparation has previously been

shown in PMd (Cisek and Kalaska, 2005) and PRR (Scherberger and Andersen, 2007), but in those studies the actions were specified by distinct stimulus cues. Here, Klaes et al. show that a single stimulus can specify two actions, revealing the simultaneous application of two different transformation rules in parallel. Interestingly, the direct goal engaged neural activity earlier than the inferred, consistent with prior studies showing that responses oriented directly toward stimuli are processed more quickly than responses requiring remapping (Crammond and Kalaska, 1994). This suggests that the information for specifying the direct goal may be processed along a simple parietal-to-frontal route, while information for www.selleckchem.com/products/Bortezomib.html find protocol specifying the inferred goal may need to pass through prefrontal cortex and then be sent back to premotor and parietal regions. Indeed, an earlier study from the same lab showed that unlike direct goals, inferred goals

were represented in PMd before appearing in PRR (Westendorff et al., 2010). Of course, in many situations, we make decisions that are unrelated to any particular action. When choosing between university courses, one presumably is not planning routes for walking to class. Obviously the brain is capable of making abstract decisions that do not involve action, and many studies have examined the neural mechanisms which may be involved. For example, in a paradigm similar to that used in Klaes et al., 2011 and Bennur and Gold, 2011 compared how monkeys judged the direction of visual motion when they either did or did not know what saccadic response would be used to report their decision. It was found that even before a saccade plan could be made, some cells in parietal cortex were selective for the motion direction of the visual stimulus. In the reach-planning system, Nakayama et al. (2008) showed that premotor activity is selective even when monkeys are only given a “virtual” action plan, specifying whether the rightmost

or leftmost of two stimuli will be the target for movement but the locations of the stimuli themselves are still not known. In fact, the very same monkeys studied by Klaes et al. were very familiar with this kind of situation, having previously been trained on most tasks in which the rule was indicated before the spatial target (Westendorff et al., 2010). In those cases, one might imagine the competition took place between the rules, and then later, also between the actions (Figure 1C). Since animals are clearly capable of making decisions between abstract rules, then why should they, in situations such as the experiment of Klaes et al., bother to simultaneously apply two rules to prepare two actions, only one of which can physically be performed? One answer, as Klaes et al. suggest, may be that doing so allows animals to make more informed choices.

Flash applied 0.4 s before the electrical stimulation of VIIIth nerves significantly increased the amplitude of both synaptic components (p < 0.001; Figures 5C–5E). Thus, a preceding visual stimulus increases the S/N ratio of sound-evoked spiking

activity in the VIIIth nerve ( Figure 4) and the efficacy of synapses made by the VIIIth nerve on the M-cell ( Figure 5), leading to facilitated sound detection in the M-cell. Vorinostat supplier In teleosts, exogenous DA elevates the firing threshold of VIIIth nerves (Curti and Pereda, 2010), and the activation of D1Rs increases the efficacy of VIIIth nerve-Mauthner cell synapses (Pereda et al., 1992, 1994). We therefore examined the role of the DA receptor in the visual enhancement of audiomotor functions, including sound-evoked M-cell response and C-start behavior. Using focal application of pharmacological agents in the vicinity of M-cell lateral dendrites (Figure 6A), which are innervated by VIIIth nerves (Eaton et al., 1977; Korn and Faber, 2005), we found that the total integrated charge of a-CSCs in M-cells was reduced by the D1R antagonist SCH-23390 (p < 0.001), and increased by the DA receptor Palbociclib chemical structure agonist apomorphine (p < 0.05). Importantly, the preceding

flash-induced enhancement of a-CSCs in M-cells was largely impaired by SCH-23390 application (Figure 6B), and was totally occluded by apomorphine application (Figure 6C). These findings indicate that D1R activation is required for the visual modulation of auditory responses in Mauthner cells. We then examined whether D1Rs mediate flash-induced increases in both the S/N ratio of VIIIth nerves and the efficacy of VIIIth nerve-Mauthner cell synapses. SCH-23390 application prevented flash-induced

increase in the S/N ratio of Levetiracetam VIIIth nerves (Figure 6D), whereas apomorphine application mimicked the flash-induced effects as it significantly suppressed the spontaneous spiking activity of VIIIth nerves (p < 0.01; Figure S3A1) and increased the S/N ratio (p < 0.01; Figure S3A2). Furthermore, local application of the persistent sodium channel blocker riluzole largely suppressed the spontaneous spiking activity of VIIIth nerves (p < 0.05; Figure S3B1) and prevented SCH-23390-induced increase in the spontaneous spiking activity of VIIIth nerves (Figure S3B2). These results suggest that D1R activation mediates visual modulation of the S/N ratio of VIIIth nerves possibly through suppressing persistent sodium channels (see also Curti and Pereda, 2010).

In our opinion, the key to understanding tinnitus pathophysiology lies in understanding how the auditory and limbic systems interact. The present study reports, for the first time, functional differences in the NAc of patients with chronic tinnitus. Furthermore, this hyperactivity in NAc correlates with the magnitude of structural changes in the vmPFC in these same patients. We conclude, therefore, that a dysregulation

of limbic and auditory networks may be at the heart of chronic tinnitus. A complete understanding and ultimate cure of tinnitus may depend on a detailed understanding of the nature and basis of this dysregulation. Given PLX-4720 supplier the paucity of effective treatments for tinnitus, this field of research is in need of new and testable ideas, and the model we propose will certainly benefit and evolve from future research. For example, although we report moderate correlations between functional activity in primary auditory cortex and limbic regions in tinnitus patients, additional studies are needed to directly assess the nature of connectivity between these and other limbic and auditory regions.

We have proposed topographic inhibitory influence HA-1077 datasheet of the thalamic reticular nucleus (TRN) on auditory thalamic (i.e., MGN) transmission as a candidate noise-cancellation site in this network (Mühlau et al., 2006 and Rauschecker et al., 2010); however, further research is needed to test the site(s) of limbic-auditory interaction relevant for tinnitus, particularly next in animal models of tinnitus. Limbic corticostriatal structures (i.e., vmPFC and NAc) have also been linked to disordered appraisal of hedonic state in drug addiction (Ahmed and Koob, 1998) and emotional state in mood disorders (Mayberg, 1997). Both these conditions are associated with structural abnormalities in vmPFC

(Drevets et al., 1997, Koenigs and Grafman, 2009 and Tanabe et al., 2009) similar to the ones we report in individuals with chronic tinnitus. Adjacent mPFC and cingulate structures, along with other limbic regions, have also been implicated in chronic pain (DaSilva et al., 2008, Geha et al., 2008 and Kuchinad et al., 2007), which too may involve the inability to suppress unwanted sensory signals. Converging evidence regarding common mechanisms shared between these and similar disorders will further our understanding of the limbic system and its influence on perception. Tinnitus, as a relatively circumscribed condition, may facilitate better understanding of limbic dysregulation in many of these disorders. Twenty-two volunteers (11 tinnitus patients, 6 female; 11 controls, 7 female) were recruited from the Georgetown University Medical Center community and gave informed written consent to participate in this study. Tinnitus patients ranged widely in age (20–64 years; SD = 16.0 years) and were on average 44.

(2008). Further experiments using conditional MyoVa alleles to disrupt MyoVa at later stages of development are needed to address this discrepancy. In presynaptic terminals, synaptic vesicle fusion is triggered by influx of Ca2+, which directly binds C2 domains of synaptotagmin 1, thereby directly coupling elevated Ca2+ to SNARE-mediated exocytosis (Chapman, 2008). A recent study demonstrated that disrupting a different synaptotagmin family member, synaptotagmin 4 (Syt4), blocks retrograde selleckchem signal-mediated

plasticity at the Drosophila NMJ. Yoshihara et al. (2005) demonstrated that high-frequency stimulation of muscle cells triggers an increase in the probability of presynaptic vesicle release. Animals null for Syt4 lack this form of retrograde signaling, which can be rescued by expressing Syt4 in muscle, suggesting that Ca2+ influx is coupled to postsynaptic vesicular trafficking. Interestingly, BDNF release from cultured mouse hippocampal neurons is also regulated by Syt4 ( Dean et al., 2009). Syt4 localizes to BDNF-containing vesicles in dendrites. Expression of a pHluorin-tagged version of Syt4 allowed visualization of Syt4-containing vesicle fusion events, which increased upon depolarization. Moreover, neurons from Syt4 knockout mice displayed increased BDNF release compared to wild-type neurons suggesting that Syt4 may

actually play a negative role in postsynaptic exocytosis. Using an elegant coculture

method, this study also demonstrated that WT presynaptic terminals connected to Syt4 null neurons exhibit increased vesicle Bumetanide release probability, providing strong evidence that, as in Drosophila, Selleckchem PF 2341066 Syt4 regulates retrograde signaling to modify presynaptic release probability ( Dean et al., 2009 and Yoshihara et al., 2005). Intriguingly, the quantal response amplitude was higher in Syt4 null neurons, indicating higher postsynaptic glutamate receptor content and raising the possibility that Syt4/BDNF positive vesicles also harbor AMPA receptors. Interestingly, although Syt4 plays a negative role in BDNF secretion in mammalian neurons, it appears to play a positive role in retrograde signaling at the Drosophila NMJ. It is notable that even though mammalian and Drosophila Syt4 are ∼50% identical at the amino acid level, mammalian Syt4 does not show enhanced binding to phospholipids upon elevated Ca2+ while the Drosophila version does, providing a potential explanation for this difference ( Wang and Chapman, 2010). Alternatively, in the absence of Syt4, a different, more efficient Ca2+ sensor could take its place, resulting in enhanced BDNF release and giving the appearance of a negative regulatory role for mammalian Syt4. While Ca2+-influx through NMDA receptors is required to mobilize postsynaptic membrane fusion for LTP, it remains unknown whether Ca2+ acts directly at the level of postsynaptic membrane fusion.

All solutions mTOR inhibitor contained TTX 1 μM and Picrotoxin 100 μM. The structure of the process was extracted from the rest of the image (red channel, TxR fluorescence) by a segmentation paradigm combining tools

of mathematical morphology implemented in Matlab. The process was then subdivided in many contiguous subregions (SRs, 5–16 μm2), roughly corresponding to the spatial extent of the [Ca2+]i elevation evoked by a local 2MeSADP puff. The amplitude of [Ca2+]i transients was measured in the selected responding SR by dividing the Ca2+ signal by the TxR signal in order to correct possible transient z motion. Events’ duration was calculated as the time-interval between the point at which the transient reached 50% of its maximal amplitude and the point at which it declined back to 50% (full width at half maximum [FWHM]). Rise-time was calculated from 10% to 90% of the peak amplitude. In the few cases when the [Ca2+]i elevation invaded more than one SR, kinetics of the event were calculated on the first responding SR. Recordings showing any drift (x, y, or z) were discarded.

Traces were subjected to median filter before analysis. In a set of electrophysiological experiments in hippocampal slices, BAPTA was dialyzed into the astrocytes. To indirectly follow the process, we monitored diffusion of a fluorescent dye, Alexa 488 hydrazide or Alexa 594, from the whole-cell patched astrocyte to the astrocytic syncytium in the dentate ML. Within 15–30 min tens of gap EX-527 junction-coupled astrocytes were labeled by the dye (Shigetomi et al., 2008). After removal of the pipette, florescence in the syncytium remained stable for at least 1 hr and during the whole experiment. Images were normally observed on an Olympus BX51WI microscope (20× water-immersion objective).

The excitation light beam (488 nm/590 nm, monochromator, Visichrome, Visitron; controlled by Metafluor software, Universal Imaging) was introduced through the objective by a long-pass filter (Olympus U-N31001); fluorescence emission was collected (cooled CCD camera, CoolSNAP-HQ, Roper Scientific) with a 1 frame/s acquisition rate. Some of the experiments (Figures 1A–1D) were performed under a two-photon laser scanning microscope with a 40× water-immersion objective. For in situ visualization of astrocytes and granule Org 27569 cells loaded respectively with SR-101 and Alexa 488, or Alexa 594 and Alexa 488, excitation was provided at 800–830 nm. Efflux of endogenous glutamate from cell cultures was monitored in continuous by use of an enzymatic assay as previously described (Bezzi et al., 1998); see Supplemental Experimental Procedures. We thank R.H. Edwards and S. Voglmaier for providing VGLUT1pHluorin and VGLUT1mCherry constructs, N. Liaudet for developing the custom-made program for two-photon Ca2+ imaging analysis, H. Stubbe, C. Calì, J. Marchaland, P. Spagnuolo, and J. Gremion for help on experiments on cultured astrocytes, and C. Duerst and M.

1/V5-His-TOPO plasmid (control) or 1 μg of the pIPNV-PP plasmid. For comparison with a DNA vaccine of known effectiveness learn more [23] and [24], other trout received a similar injection with the empty pMCV1.4 plasmid or the pMCV1.4-G vaccine. After 2, 7 or 14 days, muscle (area surrounding the injection site), spleen and head kidney from 5 fish were sampled. Fragments of each tissue were pooled in TRIzol Reagent (Invitrogen), in two tubes serving as duplicates, for RNA isolation. RNA was extracted from TRIzol Reagent (Invitrogen) frozen samples following the manufacturer’s indications. Pooled

organs from trout in the different groups were homogenised in 1 ml of Trizol in an ice bath. We performed these studies in pooled samples which assures us that our results are consistent in an entire population, something really important when dealing with vaccines. Homogenates were then mixed with 200 μl of chloroform, centrifuged at

12,000 × g for 15 min and the upper phases placed in clean tubes. Five hundred microlitres of isopropanol were then added, and the samples were again centrifuged at 12,000 × g for 10 min. The RNA pellet was washed with 75% ethanol, dissolved in diethylpyrocarbonate (DEPC)-treated water and stored at −80 °C. Five micrograms of RNA were treated with DNAse I (Promega) to remove any genomic DNA traces that might interfere with the PCR reactions Selleckchem Androgen Receptor Antagonist and then used to obtain cDNA using the Superscript III reverse transcriptase (Invitrogen). Briefly, RNA was incubated with 1 μl of oligo (dT)12–18 (0.5 μg ml−1) and 1 μl 10 mM dinucleoside triphosphate (dNTP) mix for 5 min at 65 °C. After the incubation,

isothipendyl 4 μl of 5× first strand buffer, 1 μl 0.1 M dithiothreitol (DTT) and 1 μl of Superscript III reverse transcriptase were added, mixed and incubated for 1 h at 50 °C. The reaction was stopped by heating at 70 °C for 15 min, and the resulting cDNA was diluted and used as template. Real-time PCR was performed an Mx3005P™ QPCR instrument (Stratagene) and SYBR Green PCR Core Reagents (Applied Biosystems). Reaction mixtures (containing 10 μl of 2× SYBR Green supermix, 5 μl of primers (0.6 mM each) and 5 μl of cDNA template) were incubated for 10 min at 95 °C, followed by 40 amplification cycles (30 s at 95 °C and 1 min at 60 °C) and a dissociation cycle (30 s at 95 °C, 1 min 55 °C and 30 s at 95 °C). For each mRNA, gene expression was corrected by the endogenous control (elongation factor 1-α; EF1-α) expression in each sample and expressed as 2−ΔCt, where ΔCt is determined by subtracting the EF1-α Ct value from the target Ct. All amplifications were performed in duplicate. Trout specimens were vaccinated with 50 μl of PBS containing 1 μg of the pIPNV-PP vaccine, or its respective empty plasmid, and sampled after 30 days.

25 A variable rarely explored in literature, TTT was also determined because it quantitatively assesses the capacity of the athlete when jumping quickly as opposed to maximally.20 TTT has been found to be a practical descriptive performance measure, as it has previously been found to be significantly correlated to vertical jumping SAR405838 price height performance in highly trained athletes.20 Indeed, jumping

quickly as compared to maximally are mutually exclusive tasks that may be of priority, because in sports where athletes generate quick bursts of movement the culminating outcome to success typically goes to that athlete who responds to a given situation in as little time as possible. In this regard, TTT was found to be significantly decreased 1 min after DS and increased 1 min after SS. Hence, the sport specific DS session in the present investigation was the preferable mode of stretching to female volleyball athletes, because it improved upon how quickly they jumped, as opposed to as maximally and forcefully as possible, but only for a short time (i.e., 1 min post-stretch). Therefore, the current findings suggest that when training to jump quickly the

female athlete should incorporate DS instead of SS as part of their warm-up, but conduct performance within 15 min of their warm-up to elicit maximal gains. A timing signature buy Capmatinib was another priority in the present investigation which, as previously mentioned, needs further attention in female athletic populations. In the present investigation, it was found that SS for 7 min of all the major muscle groups of the lower extremity elicited acute either decrements in kinetic parameters 1 min after stretching, but returned to baseline by 15 min. These findings are comparable with previous findings in male subjects, where the deleterious effects of SS are evident up to 10 min11 and 12 and 15 min13 after stretching. However, females are well known to differentially alter how their MTU operates in response maximal force producing tasks as compared to male counterparts.14 This is further obscured as athletes

are known to exhibit a stiffer MTU complex compared to that of non-trained individuals.17 and 18 Although these differences have obvious performance implications to the female athlete, based on the abovementioned evidence as well as current research findings, it might otherwise be interpreted that female athletes exhibit similar MTU features as males, and that the stretch-induced force deficit appears to be evident within a similar time frame (10–15 min after stretching). Despite the apparent lack of current knowledge regarding stretching strategies in the female athlete, from a practical standpoint the present investigation also may offer novel mechanistic insights towards proper placement strategies in a sport-specific DS regimen among other sports.

They act as prime movers of the glenohumeral joint rotating it internally and check details externally (Basmajian and DeLuca 1985, Jenp et al 1996, Kelly et al 1996). They also stabilise the glenohumeral joint by providing a medial (Inman et al 1944, Sharkey et al 1994), inferior (Hurschler et al 2000, Inman et al 1944, Sharkey and Marder 1995), anterior, and posterior force (Kronberg et al

1990) on the humeral head keeping it central in the glenoid fossa during shoulder joint movement. Adduction exercises are commonly recommended in the diagnosis and treatment of rotator cuff dysfunction (Allingham 1995, Allingham 2000, Morrison et al 1997, Reinold et al 2004). This is based on clinical observation, which suggests that adduction activates and strengthens the rotator cuff (Allingham 1995, Allingham 2000, Morrison et al 1997), increasing the depressive role of the rotator cuff on the head of the humerus without activating the superior translation forces of deltoid (Morrison et al 1997, Reinold et al 2004).

Additionally, when adduction is combined with external rotation it is thought to increase the contraction of the posterior cuff MEK inhibitor (supraspinatus, infraspinatus, teres minor) in their rotational role, providing greater potential for strengthening this portion of the rotator cuff (Wilk et al 2002). Adduction with external rotation also reduces activity in middle deltoid

(Bitter et al 2007). Data from magnetic resonance imaging during active shoulder adduction indicate that muscle activity leads to a significant increase in the size of the subacromial space due to inferior translation of the humeral head (Graichen et al 2005, Hinterwimmer et al 2003). It is not known, however, whether this inferior humeral head translation is due to rotator cuff muscle activity because rotator cuff activity during adduction has not been directly measured using electromyography. Force studies indicate that latissimus dorsi, pectoralis major and teres major have much larger depressive moment arms during adduction than the rotator cuff muscles (Hughes Mephenoxalone and An 1996, Kuechle et al 1997). Furthermore, we are unaware of any clinical trials evaluating the effectiveness of isolated adduction exercises in the treatment of rotator cuff dysfunction. Therefore, the validity of the use of adduction exercises to diagnose and treat rotator cuff dysfunction remains unknown. Thus the aim of this study was to electromyographically compare activity in the rotator cuff and other shoulder muscles during adduction. The specific questions addressed in this study were: 1.

We also examined whether an aversive stimulus affected these same sets of synapses in a similar manner. Our results suggest that the long-lasting modulation of synapses on DA cells caused in vivo by rewarding and aversive stimuli is not uniform but rather differs dramatically depending on GW786034 the respective target structures to which DA neurons project. Most previous in vitro electrophysiological studies of

midbrain DA neurons appear to have targeted DA neurons in the anterior lateral VTA, predominantly the parabrachial pigmented nucleus (PBP) (Brischoux et al., 2009 and Ungless et al., 2010). In addition, putative DA cells were commonly identified by the presence of a large hyperpolarization-activated current (Ih) while cells that lacked this current were considered nondopaminergic (Ungless et al., 2001, Gutlerner et al., 2002, Saal et al., 2003, Borgland et al., 2004, Faleiro et al., 2004, Bellone and Lüscher, 2006, Margolis et al., 2006, Hommel et al., 2006, Argilli et al., 2008, Stuber et al., 2008 and Zweifel et al., 2008) even though Enzalutamide this criterion does not unequivocally identify DA neurons

(Johnson and North, 1992, Ford et al., 2006, Margolis et al., 2006, Margolis et al., 2008 and Zhang et al., 2010a). Therefore, one major goal of this study was to identify and record from DA cell subpopulations that have largely been neglected. By using in vivo Retrobead injections to identify the projection target of individual DA neurons, we first determined the percentage of retrogradely labeled neurons in the posterior VTA that are dopaminergic as defined by immunoreactivity for TH. Injections were made in the mPFC, NAc medial shell, and NAc lateral shell to label VTA DA neurons as well as the dorsolateral striatum for labeling of nigrostriatal because DA cells (Figure 1A).

In agreement with previous results (Lammel et al., 2008) we found that retrogradely labeled neurons that project to the mPFC and medial shell of the NAc are mainly located in the medial posterior VTA, medial paranigral nucleus and adjacent medial aspects of the PBP nucleus (Figure 1B). In contrast, neurons that project to the lateral shell of the NAc were located in the lateral VTA, mainly in the lateral PBP nucleus. Nigrostriatal neurons were almost exclusively found in the SNc. Approximately 80%–95% of the retrogradely labeled cells in the posterior VTA and SN also were immunopositive for TH indicating that they were dopaminergic (Figure 1C, n = 49–140 cells for each group). Recordings from retrogradely labeled neurons revealed significant differences in the magnitude of Ih depending on the neurons’ projection targets. Cells projecting to the mPFC or NAc medial shell exhibited an Ih that was dramatically smaller than those recorded from neurons projecting to the NAc lateral shell or dorsal striatum (Figures 1D and 1E, mesocortical neurons: 24.2 ± 9.4 pA, n = 8; mesolimbic medial shell neurons: 10.7 ± 0.

(Paisley, UK). Bovine plasma derived serum (BPDS) was from First Link (UK) Ltd. (Birmingham, UK). RO-20-1724 was purchased from Merck Chemicals Ltd. (Nottingham, UK). Ko143 and MK571 were purchased from Tocris Bioscience (Bristol, UK). [3H] propranolol, [3H] vinblastine, [3H] naloxone and Optiphase HiSafe 2 scintillation cocktail were purchased from PerkinElmer Life & Analytical Sciences (Buckinghamshire, UK). [14C] acetylsalicylic acid was from

Sigma–Aldrich (Dorset, UK). [14C] sucrose was purchased from Amersham (UK). [3H] dexamethasone (from PerkinElmer, UK) was kindly provided by Dr. Sarah Thomas (BBB Group, King’s College London). Tariquidar and PSC833 were kindly provided by Dr. Maria Feldman and GlaxoSmithKline (Hertfordshire, UK) respectively.

INK-128 All other materials were purchased from Sigma–Aldrich (Dorset, UK). Rat-tail collagen was prepared according to Strom and Michalopoulos (1982). The protocol used was as reported in Skinner et al. check details (2009) and Patabendige et al., 2013a and Patabendige et al., 2013b, with slight modifications. In brief, brains from six pigs were transported from the abattoir to the lab on ice in Iscove’s medium with added penicillin (100 U/ml) and streptomycin (100 μg/ml). The hemispheres were washed, the cerebellum removed, and meninges peeled off. The white matter was removed and the gray matter homogenized, then filtered successively through 150 and 60 μm nylon meshes. The meshes with retained microvessels were kept separate, and immersed in medium containing collagenase, DNAse and Ketanserin trypsin to digest the microvessels. The microvessels were washed off the meshes, resuspended and centrifuged. The final pellets were

resuspended in freezing medium, aliquoted and stored in liquid nitrogen. Six brains generated 12 cryovials each of ‘150s’ and ‘60s’ microvessel fragments, named according to the mesh filter used (150 and 60 μm pore sizes). Cells derived from both 150s and 60s were used for permeability assays described in the present study. The cryopreserved microvessel fragments were thawed and cultured according to Patabendige et al., 2013a and Patabendige et al., 2013b to obtain primary porcine brain endothelial cells. Puromycin was used to kill contaminating cells such as pericytes. The in vitro BBB model using the primary porcine brain endothelial cells (PBEC) was set up on rat-tail collagen/fibronectin (7.5 μg/ml)-coated Corning Transwell® filter inserts (12 mm membrane diameter, 1.12 cm2 growth surface area, 0.4 μm pore size), transparent polyester (catalog no. 3460) or translucent polycarbonate membrane (catalog no. 3401), in 12-well plate. The PBEC were seeded onto Transwell® inserts at a density of 1 × 105 cells per insert. Confluency was reached within 3–4 days.