Hubs, in an intuitive sense, are nodes with special importance in

Hubs, in an intuitive sense, are nodes with special importance in a network by virtue of Buparlisib supplier their many, often diverse, connections. The quantitative importance of hubs has been demonstrated in a series of graph theoretic studies (Albert et al., 1999, Albert et al., 2000, Barabasi and Albert, 1999, Jeong et al., 2000 and Jeong et al., 2001). Graphs are mathematical models of complex systems (e.g., air traffic) in which the items

in a system become a set of nodes (e.g., airports) and the relationships in the system become a set of edges (e.g., flights). Hubs are defined as nodes with many edges or with edges that place them in central positions for facilitating traffic over a network. The number of edges on a node is called

the node’s degree, and degree is the simplest and most commonly used means of identifying hubs in graphs. Over the past decade it has become clear that many real-world networks contain nodes that vary by many orders of magnitude in their degree such that a handful of nodes have very powerful roles in networks (e.g., Google learn more in the World Wide Web) (Albert et al., 1999, Barabasi and Albert, 1999 and Jeong et al., 2000). The loss of such well-connected hubs can be particularly devastating to network function (Albert et al., 2000, Jeong et al., 2000 and Jeong et al., 2001). Given the role of hubs and their importance to networks, the locations and functions of hubs in the brain are of clear interest to neuroscientists. Over the past 15 years, advances in MRI techniques have enabled comprehensive estimates of structural

and functional connectivity in the living human brain, leading to the first estimates of hub locations in human brain networks. In an influential study, Buckner and colleagues (Buckner et al., 2009) examined voxelwise resting-state functional connectivity MRI (RSFC) networks, identifying hubs (high-degree nodes) in portions of the default mode system, as well as some regions of the anterior cingulate, anterior insula, and frontal and parietal cortex. Other investigations targeting “globally connected” regions in RSFC data Methisazone have converged on similar sets of regions (Cole et al., 2010 and Tomasi and Volkow, 2011). These “hubs” have garnered much interest because they are principally located in the default mode system, a collection of brain regions that are implicated in various “high-level” cognitive processes and that often degenerate in Alzheimer disease, thereby seeming to fit ideas about information integration and vulnerability to attack. In this article we outline reasons to suspect that degree-based hubs reported in functional connectivity networks may not be hubs in the interesting and intuitive sense outlined at the beginning of this article, but rather that they might simply be members of the largest subnetwork(s) (systems) of the brain. We follow two separate lines of argumentation to this conclusion.

According to this framework, continuous neurogenesis results in a

According to this framework, continuous neurogenesis results in a combination of buy AP24534 signals from the DG to the CA3 that consists of two separate populations (Figure 3): (1) A population of broadly tuned GCs that weakly encode most of the features of the environment (Figure 3A). By itself, the latter population is most similar to the classic pattern-separating DG network; however, while its encoding may be nearly orthogonal, it may not relay enough information to allow

subsequent discrimination (Figure 2). Likewise, on its own, the first population may contain information about the remembered event, but this information is in a sense “noisy” in that it lacks specificity. Combined, however, the two populations are capable of maximizing

the information encoded while preserving the sparse coding of the overall active population (Figure 3C). We propose that neurogenesis is actually capable of affecting this process in several ways. Clearly, the presence of “hyperexcitable” immature neurons provides a population of broadly tuned neurons, such as shown in Figure 3A. Due to their physiology and low connectivity, these immature neurons will be responsive to a wide range of inputs and overlap considerably with one another. While individually they are PCI-32765 cell line SPTLC1 not as informative as mature cells (by virtue of their responding to many inputs), as a population they can still contain some specificity about their inputs. Importantly, because these neurons are responsive to a wide range of inputs, not as many young neurons

are required to ensure that at least a few are responsive to any potential input to the DG. For this reason, the population of immature neurons does not need to be very large relative to the more sparsely active, sharply tuned mature neurons. Less obvious, but equally as important, is the proposed role of neurogenesis in forming the sharply tuned GC population (Figure 3B). A sparse population is thought to be necessary for memory encoding in the hippocampus: attractor formation in the CA3 requires fairly separate inputs to adequately form memories that do not interfere with one another (Treves and Rolls, 1992). However, although the DG is large relative to other regions, there are not enough neurons available to ensure an ability to encode every possible input that may be experienced. For this reason, the experience-dependent specialization of maturing neurons to features of their environment is important to ensure that the mature GC population consists of neurons that are capable of responding to the key features of most environments.

To account for CAF-induced changes in

To account for CAF-induced changes in PI3K inhibitor temporal song structure,

the post-CAF spectrogram was warped to the baseline spectrogram, using the same DTW warping routine as described above. Warping estimates for each interval were calculated as the ratio of post-CAF to pre-CAF interval duration. The warping paths thus derived were applied to the average post-CAF neural trace, yielding the green traces in Figure 7A. The same DTW routine was also applied to the neural traces to compare the warping in the underlying neural signal to warping in the song (Figure 7C). To make the warping estimates for the neural data more reliable, we flagged salient points in the neural trace (i.e., well-defined peaks and troughs) and calculated the time shifts in these points over the course of the CAF drive. Since

these points did not always line up with the interval boundaries in the song, we took the weighted average of the time shifts in the points within 10 ms of the interval boundary, AT13387 each point being weighted inversely to its distance from the boundary. The estimate for the neural warping in a given interval was then derived from the difference in the estimated time shifts corresponding to the start and end points of the interval. To quantify the degree and temporal specificity of the changes in neural power induced by CAF, we calculated running Pearson’s correlations (50 ms boxcar window, 1 ms advance) between Adenylyl cyclase the neural power in baseline and post-CAF conditions. For each analyzed CAF drive, we compared the mean correlation of nontargeted song intervals (motif onset to 50–100 ms prior to CAF target) with those in the targeted interval (pCAF) or targeted interval plus 100 ms (tCAF). All statistics presented in the main text refer to mean ± SD, while error bars in the figures all represent SEM. All statistical tests assessing significance across manipulations in the same birds were done using paired-samples t tests or one-sample t tests against mean zero unless otherwise noted.

We thank Ed Soucy for assistance with the CAF software and Stephen Turney and the Harvard University Neurobiology Department and the Neurobiology Imaging Facility for imaging consultation and equipment use. We acknowledge Jesse Goldberg, Aaron Andalman, Rajesh Poddar, Naoshige Uchida, Markus Meister, Evan Feinberg, Maurice Smith, and Kenneth Blum for helpful discussions and feedback on the manuscript. This work was supported by a grant from NINDS (R01 NS066408), a McKnight Scholar Award and Klingenstein Fellowship to B.P.Ö., and a Swartz Foundation postdoctoral fellowship to C.P. “
“Imagination, defined as the ability to interpret reality in ways that diverge from past experience, is fundamental to normal, adaptive behavior. This can be seen at a very simple level in our capacity to predict novel outcomes in new situations, unbound from our past experience with any particular static element or feature.

The log-normal distribution of sensitivities also suggests that m

The log-normal distribution of sensitivities also suggests that more synapses will be matched to the luminance values most prevalent in the image falling on the retina. The tuning curve of a sensory neuron is a key determinant of the information that it can transmit about a stimulus. Several theoretical studies have suggested that sharper tuning curves within individual neurons can improve the overall efficiency of population

codes, in part because the finest discrimination occurs over the range of stimulus strengths that most rapidly alter the neurons response (Brunel and Nadal, 1998, Pouget et al., 1999, Seriès et al., 2004 and Butts and Venetoclax molecular weight Goldman, 2006). Tuning curves similar to Hill functions or Gaussians can only provide this advantage at the cost of signaling over a narrower range of stimulus strengths, but we found a subset of bipolar cell synapses in which the dynamic range of signaling was increased by an unexpected mechanism: switching the polarity of the exocytic response as a function of luminance. Examples of sypHy signals from such terminals are shown in Figure 6A (ON) and Figure 6B (OFF): the response to a dim light was of the opposite polarity to the larger response to a brighter light. We

examined the tuning curves of linear and nonlinear synapses more closely by normalizing the relation measured in individual terminals to I1/2 and then averaging within the linear and nonlinear classes (Euler and Masland, 2000). The response of nonlinear ON synapses did not saturate check details as light Resminostat intensity increased but passed through a minimum (transition from phase one to two) and then a maximum (transition from phase two to three) before reaching a steady state (Figure 6C). The response of nonlinear OFF synapses was roughly an inversion of this triphasic shape (Figure 6D). A good empirical description of triphasic tuning curves could be obtained by considering them as the sum of two components, which we termed “intrinsic” (black traces in Figures 6E and 6F), and “antagonistic” (blue traces). The expression fitted to these curves is equation(Equation 3) Vexo=A+Int(I′hI′h+1)+Antagσ2π∫0I′exp[−(ln(I′)2σ)2]dI′where

I′ is the intensity normalized to I1/2, A is an offset, Int is a scaling factor for the “intrinsic” component described by a Hill function, Antag is the scaling factor for the “antagonistic” component, described by the cumulative density function of a log-normal distribution, and 2σ is the width of that distribution in log units. The value of σ varied between 3.0 and 4.5 log units and was therefore similar to the distribution of sensitivities across the population of terminals shown in Figure 5C. The growth of the antagonistic component in parallel with the number of bipolar cells activated suggests that this signal may originate from neighboring bipolar cells that are progressively recruited as the light intensity increases.

A N and a NARSAD grant from the Brain and Behavior


A.N. and a NARSAD grant from the Brain and Behavior

Research Fund to B.E.H. C.-Y.Z. and K.W.R. were supported by the Intramural Research Program of NINDS. Y.H.S. was supported by the Basic Science Research Program (2011-0011694) and MRC (2012048183) through the National Research Foundation GSK-3 phosphorylation of Korea. “
“Phosphoinositides are important cellular signaling lipids, but they are present at very low concentrations in the nervous system (Di Paolo and De Camilli, 2006). While based on their phosphorylation status, seven different phosphoinositides are known at the presynaptic terminal, and phosphatidylinositol 4,5 bisphosphate (PI(4,5)P2) has been best studied and is involved in a growing number of processes, including the spatial and temporal recruitment of cytosolic proteins that mediate synaptic vesicle cycling and synaptic growth (Cremona et al., 1999; Khuong et al., 2010; Martin, 2012; Verstreken et al., 2009; Wenk et al., 2001). PI(4,5)P2-dependent

recruitment of proteins to specific membrane domains occurs via specific motifs but also by electrostatic interactions with unstructured protein regions that are rich in basic amino acids, inducing the formation of protein-lipid Duvelisib mw microdomains (Heo et al., 2006; van den Bogaart et al., 2011). In contrast to PI(4,5)P2, phosphatidylinositol 3,4,5 trisphosphate (PI(3,4,5)P3) is much less abundant (Clark et al., 2011) and the lipid has been implicated in the clustering of glutamate receptors and postsynaptic density protein-95 in the plasma membrane of postsynaptic terminals (Arendt et al., 2010); however, the mechanism was not elucidated. Contrary to this postsynaptic role, the function of PI(3,4,5)P3 at presynaptic terminals remains

enigmatic. Here, using transgenic imaging probes based on split Venus, we show that PI(3,4,5)P3 concentrates in discrete foci and that these foci largely colocalize with presynaptic release sites that are also rich in Syntaxin1A, a SNARE protein essential for synaptic vesicle fusion (Gerber et al., 2008; Schulze et al., 1995). Non-specific serine/threonine protein kinase Although phosphorylated phosphoinositides have been implicated in synaptic vesicle endocytosis by interacting with adaptors and other proteins (Cremona et al., 1999; Di Paolo et al., 2004), we find that, unlike reducing PI(4,5)P2 availability, reducing PI(3,4,5)P3 levels at presynaptic terminals does not result in significant defects in synaptic vesicle formation. Instead, based on in vitro and in vivo assays, we find that PI(3,4,5)P3 is critical to induce the clustering of Syntaxin1A and this feature is dependent on the positively charged residues in the Syntaxin1A juxtamembrane domain, suggesting that electrostatic interactions mediate this effect. Either reducing PI(3,4,5)P3 availability or expressing a Syntaxin1A with a mutated juxtamembrane domain results in reduced neurotransmitter release, similar to partial loss of Syntaxin1A function.

, 2012), and in responses of hypothalamic neurons to leptin to co

, 2012), and in responses of hypothalamic neurons to leptin to control energy homeostasis (Liao et al., 2012). What function would a dendritically

released neuropeptide play? The most probable role would be that the neuropeptide acts to signal other nearby neurons to either increase or decrease activity. In the olfactory CP 868596 bulb, most of the neurons, including mitral, periglomerular, and granule cells possess dendrites that release either GABA or glutamate at presynaptic specializations (Shepherd et al., 2004). Many of the presynaptic dendrites are organized in a reciprocal manner; for instance, mitral cell dendritic release of glutamate activates a presynaptic granule cell dendrite that releases GABA back onto the mitral cell, resulting in feedback inhibition. In contrast, most dendrites in the brain are not presynaptic to other cells, and dendritic release of peptides appears to be independent of synaptic specializations. Nonsynaptic release of oxytocin or vasopressin could serve to recruit or inhibit click here neighboring cells, or to synchronize activity. Oxytocin receptors are expressed by oxytocin neurons (Freund-Mercier et al., 1994), and vasopressin receptors by vasopressin cells (Hurbin et al., 2002). During

lactation, oxytocin is released in an orchestrated burst where many or most oxytocin neurons fire rapidly for a brief period of about a second (Armstrong and Hatton, 2006; Leng et al., 2008). Intermittent bursts of oxytocin release

may prevent oxytocin receptors in the mammary gland from desensitizing if oxytocin levels were to remain at statically raised L-NAME HCl levels. The burst of oxytocin potentially appears to be dependent on dendritic release of oxytocin that primes the cells for subsequent massive oxytocin release induced by an increase in spike frequency, as described above. Dendritically released peptides can act to initiate retrograde signals to modulate subsequent release of fast amino acid neurotransmitters from local axons. Oxytocin released by magnocellular cell bodies and dendrites reduces presynaptic glutamate and GABA release; although this was initially thought to be mediated by presynaptic peptide receptors, it appears more likely that oxytocin release activates receptors on oxytocin cells, resulting in release of an endocannabinoid that diffuses in a retrograde direction to activate CB1 receptors on presynaptic axons and thereby reducing fast transmitter release (Kombian et al., 1997, 2002; Hirasawa et al., 2001, 2004; Leng et al., 2008). Oxytocin release appears to be obligatory to achieve this presynaptic inhibition after depolarization of oxytocin neurons ( Hirasawa et al., 2004). Blockade of synaptic activity transiently isolates oxytocin cells from external influences, potentially amplifying local cellular interactions.

Layer I GABAergic inhibitory interneurons, which are believed to

Layer I GABAergic inhibitory interneurons, which are believed to mediate feedforward inhibition by receiving direct mitral/tufted cell input (Stokes and Isaacson, 2010) are more broadly tuned to odors than pyramidal cells (Miyamichi et al., 2011 and Poo and Isaacson, 2009). These interneurons are hypothesized to have either a lower threshold or receive greater convergence of mitral/tufted

cell inputs than pyramidal cells (Poo and Isaacson, 2009). Thus, while pyramidal cells express excitatory responses to relatively few odors in a test stimulus set, the same cells show broadly tuned inhibitory responses. Thus, as in other systems, inhibition can play an important role in shaping stimulus receptive fields. Interneurons in layers II and III are more typically targets of intracortical association fiber inputs or input from nonpiriform sources. These Talazoparib supplier GABAergic interneurons tend to terminate on pyramidal cell proximal dendrites, soma, or axon initial segments and can be highly effective at blocking pyramidal cell output either via shunting inhibition or action potential blockade (Luna and

Schoppa, 2008). GABAergic interneurons in check details each layer also show a dichotomy in their response to excitatory synaptic input. A subset of interneurons in each layer show strong initial response to excitatory input evoking spiking output, while another subset show weaker initial responses but facilitation over repeated stimulation (Suzuki and Bekkers, 2010a). Suzuki and Bekkers suggest these differences in synaptic physiology could allow a temporal segregation of activity, with different interneurons producing output at different phases of the respiratory cycle. The respiratory cycle is a strong source of oscillations throughout the olfactory pathway; however, several other spontaneous and induced oscillations are also prominent. For example, beta (15–35 Hz) and gamma (35–90 Hz) frequency oscillations can be robustly

evoked in the piriform cortex, generally in phase with the 2–4 Hz respiratory cycle. Current source density analyses suggest that these higher frequency oscillations derive from the cyclical afferent-association fiber activity loop, shaped by synaptic inhibition (Ketchum and Haberly, 1993). More recently, in vivo whole-cell recordings from Parvulin piriform cortex pyramidal cells supported this by showing that pyramidal cell spiking was phase locked to beta frequency oscillations and that this phase locking was partially governed by synaptic inhibition (Poo and Isaacson, 2009). As mentioned above, precise timing of pyramidal cell activity can reinforce temporal convergence of afferent synaptic excitation driven by the current odor input with association fiber synaptic excitation which reflects both ongoing sensory input and previous experience (due to experience-dependent synaptic potentiation during past odor stimuli).

, 2004) We performed extensive trial simulations (data not shown

, 2004). We performed extensive trial simulations (data not shown) allowing various rate constants on the loop to covary with d2– (see Figure 7A). A tolerable representation of the observed kinetic behavior of our mutant panel was only obtained if the rate of channel closure

(α) was varied with d2–. This regime allowed shifts in the recovery rate of more than 2 orders of magnitude ( Figure 7B). The deactivation rate was altered in the same range as our mutant series ( Obeticholic Acid Figure 7C). Slower rates of channel closure for slower recovering channels also predict longer individual channel openings, as we observed for the A2 TR mutant. Despite the range of efficacies (β/α) being greater than 1,000-fold, the model predicted only limited effects on the peak open probability and extent of steady-state desensitization (because these properties are principally determined by the ratios β / d1+ and d2∗+ / d2∗−, respectively). At slow recovery rates, the foot of the concentration response relation was distorted ( Figure 7E), but the shifts in glutamate potency were modest, as for the A2 TR mutant ( Figure 4A). Predicted recovery and deactivation rates were positively Ribociclib cell line correlated, and approximately fit by a power law relation, with exponent about 1.5 ( Figure 7G).

As in our mutant series, the predicted desensitization rate was barely altered across the entire range of recovery rates. We investigated if our original model ( Figure 2A) could describe the observed data, if both α and d2– were varied without a connection between the desensitized and open states, but rate of entry to desensitization

varied strongly with recovery rate in this case (data not shown), in direct opposition to our observations (Figures 4D and 6F). The deviation from the linear correlation observed in the mutant panel may be due to the oversimplification of our model, relative to the true activation mechanism, and is one indication that further work to refine these activation mechanisms is necessary. Nonetheless, this simple reaction scheme shows that covariation of open and desensitized state lifetimes, due to reversibility constraints or other mechanisms, can lead to the correlations and that we observed for our mutant series. Our chimeras and mutant screens demonstrate that domain 2 of the AMPA and kainate receptor ligand binding domains determine both the lifetime of the desensitized state and the deactivation decay. These surprising results augment the established idea that the chemistry and dynamics of the ligand binding domains are central in determining glutamate receptor kinetic behavior. Our results exclude agonist potency as the basis of the difference in recovery rate between wild-type receptors. Consistent with this observation, none of the mutations that shift recovery contribute directly to the glutamate-binding pocket.

Finally, we tested whether ASO treatment might rescue the suscept

Finally, we tested whether ASO treatment might rescue the susceptibility to the glutamate excitotoxicity observed in C9ORF72 iPSNs. iPSNs treated with ASOs targeting the repeat sequence or targeting C9ORF72 exon 2 were exposed to 30 μM glutamate for 4 hr and monitored for cell selleck chemicals death. ASOs targeting the repeat, without altering C9ORF72

RNA levels, significantly rescued the glutamate toxicity phenotype by up to 30%. ASOs targeting exon 2, which reduced C9ORF72 mRNA levels, still significantly protected the iPSN by 16% (Figures 7E and S9J). This suggests that the loss of C9ORF72 mRNA and subsequent loss of C9ORF72 protein do not play a role in the observed vulnerability to glutamate, but instead implies that RNA toxicity causes C9ORF72 cells to be highly sensitive to excitotoxicity. This is further supported by the fact that RAN products were still detected in the C9ORF72 iPSNs after ASO treatment, through either immunocytochemistry or protein blotting, despite a rescue of the described phenotypes, including glutamate toxicity (Figures 7F and S9K). Notably, whether the ASO altered

a population of newly synthesized RAN or other RAN products that are not detected with the present antibodies is not known. Taken together, the current studies provide evidence that RNA toxicity plays a key role in C9ORF72 ALS based on the molecular, biochemical, and functional studies described here. Specifically, we have (1) demonstrated that patient fibroblasts and iPSNs contain intranuclear Sorafenib cost GGGGCC RNA foci similar to those found in vivo (DeJesus-Hernandez et al., 2011), (2) identified numerous proteins

that interact with the C9ORF72 GGGGCCexp RNA, (3) confirmed interaction of ADARB2 with the RNA expansion in vitro and in vivo, else (4) described atypical gene expression in C9ORF72 ALS tissue and cell lines that match C9ORF72 CNS patient tissue, and (5) determined that C9ORF72 iPSC neurons are highly susceptible to glutamate toxicity. Most importantly, by using these various pathological and physiological readouts in human iPSC neurons, we were able to identify antisense oligonucleotides that can abrogate C9ORF72 RNA expansion-dependent pathology, RNA binding protein aggregation, aberrant gene expression, and neurotoxicity. Furthermore, ASO that selectively blocked the hexanucleotide expansion without lowering C9ORF72 RNA levels could minimize pathology and toxicity (Figure 8). Notably, iPSCs derived from ALS patients appear to accurately recapitulate the pathological and genomic abnormalities found in the C9ORF72 ALS brain. Modeling this expansion mutation in animals can be particularly challenging in part due to the fact that the vast majority of human disease is caused by very large numbers of G:C-rich repeats that prove difficult to artificially express.

Based on the 17 studies uniquely identified in this investigation

Based on the 17 studies uniquely identified in this investigation, 23 data points were derived for the analysis of

the relative bioavailability between CR and IR formulations, 8 of which were directly given in the reports whilst the rest were calculated from the information given in the reports. The detailed information in terms of AUC ratios, 90% confidence intervals and their references are shown in Table S2 of the Supplementary Material. The simulated parameters and their ranges are summarized in Table 2. Solubility varied from 10−5 to 104 mg/mL as derived from Eq. (2). The range of solubility values was truncated to a minimum of 0.001 mg/mL and a maximum of 100 mg/mL in order to improve the computational

performance of the simulations. Human Peff ranged from 0.04 to 10 × 10−4 cm/s. Calculated Papp,Caco-2 values (Eq. (3)) varied find more from 0.01 to 80 × 10−6 cm/s, covering the range from low to highly permeable compounds ( Lennernas, 2007). The Vmax,CYP3A4 and Km,CYP3A4 range varied from 1 to 10,000 pmol/min/mg microsomal protein and 1–10,000 μM, respectively. Jmax,P-gp and Km,P-gp ranges were 1–1500 pmol/min and 1–2,000 μM, respectively. The values that defined the limits for high and low solubility were 10 mg/mL (Dn = 1.2) and 1.0 mg/mL (Dn = 0.12), respectively. Likewise, the value for high permeability was 5 × 10−6 cm/s (fa ≈ 0.89) DAPT chemical structure whereas for low permeability, the value was 0.5 × 10−6 cm/s (fa ≈ 0.34). For both solubility and permeability, the selected cut-off values coincided with the 25th and 50th percentile of their selected range (values 2 and 3 in Fig. 1). In general, a reduction in release rate, i.e., changing from an IR formulation to a CR formulation, was associated with a decrease in AUC for a majority of the CYP3A4 substrates (Figs. 3A and S1A–S3A). However, in certain cases, the AUC either remained constant as compared to the IR formulation or increased when the CR formulations were Libraries employed; dependent on both BCS class and CLint,CYP3A4. When Vmax,CYP3A4 was kept fixed (scenarios Ia and IIa in Table 1), Cediranib (AZD2171) the increase in exposure was only observed

for BCS class 1 CYP3A4 substrates with CLint,CYP3A4 values equal to or greater than 250 μL/min/mg ( Figs. 3A and S1A). A similar situation was observed when Km,CYP3A4 was fixed to the ‘medium’ value (scenario Ib in Table 1) though the CLint,CYP3A4 necessary to observe a similar change in exposure was reduced to 50 μL/min/mg (Fig. S2). The use of a low Km,CYP3A4 in scenario IIb, i.e., high affinity for CYP3A4, resulted in a similar outcome. However, the AUC also remained constant for CR formulations of highly cleared (CLint,CYP3A4 ⩾ 2500 μL/min/mg) BCS classes 2 and 3 drugs ( Fig. S3A). For scenarios Ia-IIb the BCS classification had an effect on fa, where fa decreased when moving from BCS class 1 to class 4. CLint,CYP3A4 had no impact on fa.