Our study suggests that the Imc circuit can, by itself, mediate categorization in the midbrain network. We propose a simpler and faster circuit motif for implementing flexible categorization and, possibly, winner-take-all decisions: reciprocal inhibition of feedforward lateral inhibition within the Imc. Anatomical support for such a motif has been found in a study of the projection patterns of Imc neurons

(Figure 4B; Wang et al., 2004). Future experiments will be needed to determine the contribution of the Imc to categorization in controlling gaze and attention. The computations explored in this study that account for explicit and compound screening assay flexible categorization of relative stimulus strengths in the midbrain network may generalize to other examples of categorical decisions and, therefore, to other brain areas (Wang, 2008). Classification of direction of stimulus motion with respect to a flexible reference (Freedman and Assad, 2006), of speed of stimulus motion with respect to a flexible reference (Ferrera et al., 2009), of odor based on Trichostatin A relative odor strengths in

a mixture (Niessing and Friedrich, 2010), and of tactile stimulus frequency relative to a prior sample frequency (Machens et al., 2005) can each be thought of as decisions based on such categorization. Indeed, the model that was proposed to account for neural responses in the monkey prefrontal cortex during the

discrimination of tactile stimulus frequency relative to a prior sample frequency employed feedback inhibition (Machens et al., 2005). In this task, the decision of whether the test frequency was higher or lower than the sample frequency can be thought of as a form of flexible Non-specific serine/threonine protein kinase categorization, in which the comparison of stimulus representations occurs over time rather than space. Like other models of decision, the model that was proposed was purely computational and without neural correlates, and the specific computational contributions of the different circuit elements to the decision were not explored. Recently, parallels between such potentially abstract decision-making processes and competitive stimulus selection have been recognized (Cisek and Kalaska, 2010 and Freedman and Assad, 2011). We propose that reciprocal inhibition of feedforward lateral inhibition, which works in various brain areas, could serve as a highly efficient motif for flexible categorization for decisions, as well as for flexible normalization. The transition range of a CRP was defined as the range of competitor strengths over which responses dropped from 90% to 10% of the total range of responses. Switch-like CRPs were defined as those for which the CRP transition range was ≤4°/s (Mysore et al., 2011).

, 2010). Indeed FGF22 has been shown to interact in vitro with both FGFR1b and FGFR2b while FGF7 only interacts with FGFR2b, suggesting that these FGFs control the specificity of presynaptic nerve terminal-postsynaptic target recognition in part through differential binding to FGFR isoforms. Not surprisingly given their widespread involvement in neural development, FGFs have been associated with multiple neurological disorders. Postmortem studies have shown that several FGF ligands and receptors are downregulated in the

prefrontal cortex and hippocampus of patients with major depression, suggesting that dysregulation of FGF Sorafenib datasheet signaling is involved in the disease, e.g., by contributing to the atrophy of the hippocampus and prefrontal cortex reported in depressed patients (Evans et al., 2004 and Riva et al., 2005). A role of FGFs in the action of antidepressants has also been proposed, based on the findings that treating patients and rodents

with specific serotonin reuptake inhibitors increases Fgf expression levels in the prefrontal cortex and Anti-diabetic Compound Library mw other brain regions, and that acute or chronic administration of FGF2 reduces anxiety and depression-like behaviors in rats (Evans et al., 2004, Perez et al., 2009 and Turner et al., 2008). FGF2 could contribute to the action of antidepressants by reversing hippocampal and cortical atrophy as well as through its mitogenic effect on hippocampal progenitors, since some of the behavioral effects of antidepressants require the stimulation of neurogenesis in the adult hippocampus Adenylyl cyclase (Duman and

Monteggia, 2006, Perez et al., 2009 and Zhao et al., 2007). Dysregulation of FGF signaling during development has also been proposed to increase vulnerability to neuropsychiatric disorders such as autism spectrum disorder (ASD) (Rubenstein, 2010 and Vaccarino et al., 2009). According to this hypothesis, mutations in autism susceptibility candidate genes might interfere with FGF signaling and produce defects in brain growth and cortical circuit formation that predispose affected individuals to the disease. This hypothesis has received some support from animal studies, in particular in Fgf17 mutant mice where patterning defects of the frontal cortex during development result in specific deficits in social behaviors and working memory in adults (Scearce-Levie et al., 2008). Besides neuropsychiatric disorders and Kallman syndrome (see above), FGF signaling deficiencies have been implicated in neurodegenerative diseases, including a progressive spinocerebellar ataxia (Laezza et al., 2007 and van Swieten et al., 2003) and Parkinson’s disease (PD).

84, p = 0.0003; GM concentration, patients < controls: t(13) = 4.68, p = 0.0004; WM concentration, patients > controls: t(13) = 4.97, p = 0.0003). In a masked analysis restricted to voxels within auditory-sensory regions, including auditory cortex, MGN, and IC, no significant differences were found between tinnitus patients and controls (p > 0.01). In a masked VBM analysis restricted to NAc voxels that demonstrated a significant functional difference between participant groups, there was no significant corresponding anatomical difference (p > 0.01). Similarly, in a masked fMRI analysis restricted to vmPFC voxels that demonstrated significant anatomical

between-group differences, we saw no significant functional difference Selleckchem LY294002 between tinnitus patients and controls (p > 0.01). So, no Selleckchem Dolutegravir single brain region exhibited both structural and functional differences. There was, however, a correlation between NAc fMRI signal and vmPFC VBM values in tinnitus patients (r = 0.73, t(8) = 2.99, p = 0.02; outlier removed; see Experimental Procedures), such that patients with the highest degree of NAc hyperactivity also had correspondingly greater anatomical differences (i.e.,

decreases in GM concentration and amount, with increased WM amount compared to controls; Figure 4A). This relationship was not present in control participants (r = −0.03, t(9) = −0.10, p = 0.919). Moreover, there was moderate correspondence between limbic abnormalities and primary auditory cortex hyperactivity in tinnitus patients (NAc x mHG: r = 0.51, t(8) = 1.67, p = 0.13, Figure 4B; vmPFC x mHG: r = 0.61, t(8) = 2.17, p = 0.06, Figure 4C). Correlations between limbic and posterior auditory areas were first not significant (NAc x pSTC; r = 0.17, t(8) = 0.49, p = 0.64, Figure 4D; vmPFC × pSTC: r = 0.42, t(8) = 1.30, p = 0.23, Figure 4E), nor was activity in primary and posterior auditory cortex related (mHG × pSTC: r = −0.13, t(8) = 0.38, p = 0.72, Figure 4F). This suggests that the degree of functional and structural differences in the limbic system (i.e., NAc and vmPFC, respectively) and primary auditory cortex may be directly related

in tinnitus patients. In this paper, we report both functional and structural markers of chronic tinnitus in limbic and auditory regions of the human brain. The most robust of these tinnitus-related differences were located in limbic areas previously shown to evaluate the significance of stimuli (Kable and Glimcher, 2009), including the nucleus accumbens (NAc; part of the ventral striatum) as well as the ventromedial prefrontal cortex (vmPFC). In tinnitus patients, the NAc exhibited hyperactivity specifically for stimuli matched to each patient’s tinnitus frequency (i.e., TF-matched). Corresponding anatomical differences were identified in the vmPFC, which is strongly connected to the ventral striatum (Di Martino et al., 2008 and Ferry et al., 2000).

Single pulse step depolarization (to 0mV, 10 ms) was used to evoke presynaptic Ca2+ currents and EPSCs. Experiments were made at room temperature (25°C–27°C) or at physiological temperature (35°C–37°C). Presynaptic pipette solutions containing MNI-caged-glutamate (10 mM, (S)-a-amino2,3-dihydro-4-methoxy-7-nitro-d-oxo-1H-indole-1-pentanoic acid, Tocris Cookson) were loaded into calyces through

whole-cell pipettes. MNI-glutamate was dissolved in the presynaptic pipette solution on the day of the experiment. A UV light flash was applied from a mercury lamp light source (100 μW) by opening a shutter (Uniblitz, Vincent Associate) for 1 s under the control of a shutter driver (JML Optical Industries). Data were analyzed using IGOR Pro 6.2J (WaveMatrics) and MS Excel 2003 (Microsoft) softwares. All values are given as mean ± SEM, and p < 0.05 was taken as a IOX1 mw significant difference in Student’s paired or unpaired t test. In figures, error bars indicate ± SEM. We thank Naoto Saitoh for technical advice, Takeshi Sakaba and Shigeo Takamori for their comments, Kevin Hunt for his

English editing, and Masahiro Kaneko for his collaboration in the early stages of this study. This study was supported by the Core Research for Evolutional Science and Technology of Japan Science and Technology Agency (to T.T.) and Grant-in-Aid for Young Trichostatin A order Scientists from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (to T.H.). “
“Direction-selective retinal ganglion cells (DSGCs) respond strongly to an image moving in the preferred direction (PD) and weakly to an image moving in the opposite, or null, direction (ND). The primary circuit model for generating this direction selectivity in the retina claims that directional responses arise by asymmetric inhibition, i.e., that stimulation in the ND leads to stronger inhibition than

stimulation in the PD. This inhibition is thought to arise through starburst amacrine cells (SACs) already that release GABA onto and costratify with DSGC processes (Borst and Euler, 2011; Vaney et al., 2012; Wei and Feller, 2011). Consistent with this hypothesis, paired recordings from SACs and DSGCs reveal that depolarization of a SAC on the null side induces significantly larger GABAergic inhibitory currents in the DSGC than depolarization of a SAC on the preferred side (Fried et al., 2002; Vaney et al., 2012; Wei et al., 2011). Serial electron microscopy (EM) reconstructions of the SAC-DSGC circuit conclude that this asymmetry is due to a specific wiring of SAC processes that tend to form synapses onto a DSGC whose PD is oriented antiparallel to the SAC process (Briggman et al., 2011). Hence, the predominant model for retinal direction selectivity claims that the circuit is hard wired and that the wiring predicts the function.

, 2008), and hence both forms should share the majority of in vivo interacting proteins. Human and/or murine fl-Htt complexes were immunoaffinity purified from the cortex, striatum, and cerebellum of BACHD and WT mice at 2 or 12 months of age using HDB4E10 (Figure 1B). As a negative control, a mock IP for each sample condition (defined by specific brain region, age, and genotype) was performed in the absence of the Htt antibody. Therefore, a total of 30 independent IP experiments were performed, and approximately 765 trypsin-digested gel slices

were subjected to LC-MS/MS analysis (Figure 1C). Using the MASCOT (Matrix Science) sequence database-searching tool, we identified a total of 747 high-scoring, putative Htt-interacting proteins Compound Library from BACHD and WT NVP-AUY922 ic50 mouse brains (Table S1 available online). Consistent with the claim that we are surveying fl-Htt-interacting proteins, we identified Htt peptides spanning the entire sequence of Htt in both BACHD and WT samples (Figure 1D

and Table S2). As confirmation that HDB4E10 more efficiently immunoprecipitates human Htt than murine Htt, we observed more Htt peptides and more extensive sequence coverage of fl-Htt in BACHD mice compared to WT mice. We next performed several standard bioinformatic analyses to determine the validity of our approach in isolating Htt interactors in vivo and to define potential biological pathways that Htt may be engaged in at various ages within different brain regions. We compared our list of 747 candidates

with previously reported Htt interactors. Among a curated list of 876 proteins previously identified as putative ex vivo Htt interactors, most of which were obtained from Y2H experiments (Goehler et al., 2004 and Kaltenbach et al., Thymidine kinase 2007; http://HDbase.org), we found 139 proteins also present in our in vivo Htt interactome (Figure 1E and Table S3). This represents a highly significant enrichment (p = 7.00 × 10−50; hypergeometric test). The disease specificity of our interactome is supported by the comparison of our data set with that of another polyQ disease protein, Ataxin-1 (Lim et al., 2006). Despite the fact that one-third of our samples originated from the cerebellum, the same tissue source as the Ataxin-1 interactome study, only 38 proteins were present in both data sets with relatively modest enrichment (p = 0.0005; Figure 1E and Table S3). Thus, our in vivo fl-Htt interactome appears to be specific to Htt and provides a valuable list of Htt-interacting proteins, both in vivo and ex vivo, for further investigation to determine their roles in Htt biology and HD pathogenesis. Gene Ontology (GO) analysis (Huang et al., 2009a and Huang et al.

Thus, IR84a and IR8a are together both necessary and sufficient to reconstitute a cilia-localized and physiologically active olfactory receptor in Drosophila neurons. We extended our investigation of the sufficiency of IR84a and IR8a to form a functional receptor by determining their ability to confer phenylacetaldehyde responsiveness in an ex vivo non-neuronal system. We chose Xenopus laevis oocytes, which are commonly used for functional expression of iGluRs ( Walker et al., 2006). In these cells, single or combinations of IR complementary RNAs (cRNAs) can be injected, and odor-evoked current responses across the oocyte

membrane can be measured by two-electrode voltage clamp. When cRNAs for IR84a Neratinib or IR8a were injected alone into oocytes, we observed no responses to phenylacetaldehyde (Figures 4D and 4E). By contrast, when IR84a and IR8a cRNAs were coinjected, phenylacetaldehyde induced an inward current of several hundred nA in these cells (Figures 4D and 4E). We further tested the functional properties of a different odor-specific receptor, IR75a, which is expressed in the IR8a-dependent propionic acid-sensitive neuron in ac3 sensilla (Figures 2B and 2C). Oocytes

expressing IR75a and IR8a together, but not either Olaparib solubility dmso receptor alone, exhibited robust propionic acid-evoked current responses (Figures 4D and 4E). Odor-induced current responses were highly specific to each receptor pair and displayed concentration dependency (Figures 4D–4F). The concentration response curves for both phenylacetaldehyde and propionic acid did not saturate at the highest concentrations obtainable without changing the osmolarity of the solution, preventing our determination of 50% effective concentration (EC50) values. Baseline currents measured in the absence of either agonist were similar between IR84a+IR8a-expressing, IR75a+IR8a-expressing, and uninjected oocytes (data Tryptophan synthase not shown), suggesting that these receptors do not have detectable constitutive activity, at least in these cells. The codependency of IR84a and IR8a for cilia localization and

odor-evoked responses suggested that these proteins might form a complex. We tested this possibility through optical imaging of fluorescent protein-tagged receptors. We first generated an mCherry-tagged IR8a fusion protein and confirmed that this promotes cilia targeting of EGFP:IR84a in OR22a neurons (Figure 5A). In these cells, we observed precise colocalization and consistent relative intensities of EGFP and mCherry fluorescence throughout the cell bodies, inner dendrites, and cilia (Figure 5A). Odor-evoked responses conferred on these neurons by the fluorescent protein-tagged IRs were indistinguishable from those generated by untagged receptors (Figure 5B), indicating that the fluorescent tags do not interfere with their function.

Dr. O’Malley received honoraria as a member

of the American College of Neuropsychopharmacology workgroup, the Alcohol Clinical Trial Initiative, sponsored by Eli Lilly, Janssen, Schering Plough, Lundbeck, Glaxo-Smith Kline and Alkermes. She received travel reimbursement for talks at the Controlled Release Society, the Drug Information Association and the Association for Medical Education and Research in Substance Abuse, and an honorarium from the Medical Education Speaker Network. She has consulted to the University of Chicago, Brown University, and the Medical University of South Carolina on studies of naltrexone. She is a partner in Applied Behavioral Research, and a Scientific Panel Member, Butler Center for Research at Hazelden. All other authors declare that they have no conflicts Akt inhibitor of interest. We would like to thank Dr. Michele Levine for assistance in creating the smoking cessation protocol for this study, Elaine LaVelle for assistance with data management, and Denise Romano, Amy Blakeslee, Susan Neveu, Vanessa Leary, Jessica Hopkins, Laura

Holt, Lisa Fucito, and Aesoon Park for assistance in implementing this project. We also want to thank members of the Data Safety and Monitoring Board: Bruce Nintedanib order Rounsaville, M.D., Rajita Sinha, Ph.D., and David Fiellin, M.D. “
“It is well documented that long-term alcohol use disorders (AUDs: alcohol abuse or alcohol dependence) are associated with brain atrophy and cognitive impairments such as reduced working memory, verbal memory, visuospatial abilities, and impaired response inhibition (Moselhy et al., 2001; for a review see Sullivan et al., 2000). Similar cognitive impairments have been found in patients suffering

from pathological gambling (PG) or problem gambling (e.g., Goudriaan et al., 2006; for a review see van Holst et Bay 11-7085 al., 2010). Because of clinical, neuropsychological, and neurobiological similarities between PG and substance dependence (Holden, 2001, Petry, 2007 and Potenza, 2006), the DSM-IV classification of PG as an impulse-control disorder not otherwise categorized is challenged and PG is likely to be classified in the Addiction and Related Disorders section in DSM-V (http://www.dsm5.org). In contrast to AUDs, gambling behaviour does not entail brain exposure to toxic agents. However, in theory regional grey matter (GM) volume abnormalities in problem gamblers could result from neuroadaptations due to chronic, repetitive gambling behaviour, and/or the existence of a common underlying neurobiological vulnerability for addictive behaviours. Moreover, if GM volume reductions would also be present in pathological gamblers, comparable to GM reductions in subjects with AUDs (Fein et al., 2002b, Fein et al., 2006, Fein et al., 2009 and Jang et al., 2007) this might explain similarities in neurocognitive impairments found in both disorders.

, 2003; Ohl et al., 2001; Russ PLX4032 ic50 et al., 2007). To monitor network activity in the auditory cortex of the mouse with single cell resolution, we used two-photon calcium imaging, a technique which gives the possibility to simultaneously record the activity of a large number of neurons

in vivo (Garaschuk et al., 2006). We injected isoflurane anaesthetized mice (1%) with the calcium-sensitive dye Oregon Green Bapta 1 AM (OGB1) in the region functionally identified as the AC using intrinsic imaging recordings (Figures 1A and 1B; Kalatsky et al., 2005). Neurons labeled with OGB1 were imaged using two-photon microscopy in single focal planes at a depth of ∼150–300 μm below the pia in cortical layers II/III (Figure 1C). KU-55933 in vitro The typical field of view was a 200 μm square, in which calcium signals from 46–99 individual neurons

were recorded using line scans (Figures 1C and 1D). To estimate the neuronal firing rate based on OGB1 fluorescence measurements, we performed loose-patch recordings of individual OGB1 loaded neurons in vivo. The electrically recorded neuron was simultaneously imaged together with its neighbors using our typical line scan settings (Figures 1E and 1F). Consistently with a previous report (Yaksi and Friedrich, 2006), we observe that the temporal deconvolution of the raw calcium signals using an exponential kernel matched the time course of the neuron’s instantaneous firing rate (Figures 1G–1H, and see Figure S1 available online). An estimate of the absolute firing rate amplitude was obtained by linearly scaling the deconvolved signal to fit the actual firing rate. The average scaling factor corresponding Montelukast Sodium to the change in

fluorescence elicited by a single action potential across all recordings was 1.80% ± 0.44% (mean ± SD, n = 5). Typical spontaneous and sound evoked AC activity was dominated by short population events in which a large fraction of neurons fired synchronously (Figure 1I). This observation is in agreement with previous reports based on multisite or intracellular current recordings (DeWeese and Zador, 2006; Luczak et al., 2009; Sakata and Harris, 2009). Additionally, it is consistent with the high noise correlations between neurons observed in previous calcium imaging studies (Bandyopadhyay et al., 2010; Rothschild et al., 2010). To evaluate qualitatively how different sounds might generate different types of local population events, we plotted single trial response vectors (∼15–20 trials per sound) obtained by averaging the activity for each neuron in a 250 ms time window following sound onset. An example of such plots for four distinct short pure tones (50 ms) at different sound levels is shown in Figure 2A.

This value is in agreement with the amount of residual baclofen-induced current in the Kir3.1 knockout mouse and in Kir3.2 knockout mouse (26%), as well as in the Kir3.2/Kir3.3 double knockout mice (15%) (Koyrakh et al., 2005). The relative contribution Dabrafenib mw of Kir3 and TREK1 channels depends, in part, on membrane

voltage. Since Kir3 is an inward rectifier and TREK1 is an outward rectifier, a holding potential of −55mV favors the TREK1 channel component, while at more negative potentials, such as −70mV, the Kir3 component will be favored. Photoswitched tethered ligands have opened the door to the selective, rapid and reversible optical control of membrane signaling through proteins that are normally insensitive to light (Szobota and Isacoff, 2010 and Fehrentz et al., 2011). We have developed a scheme for targeting optical control via a PTL to native proteins without the requirement for genetic knockin. The approach is to express a PCS that contains an anchoring site for the photoswitch as well as a mutation that retains the subunit inside the cell. The engineered subunit does not traffic and so has no function unless native subunits SCH 900776 cell line are present,

coassemble with it, and carry it to the cell surface. The PCS/WT complex is rendered light-sensitive once an externally applied membrane-impermeant photoswitch is attached to the PCS, something possible only at the plasma membrane. To generate Cytidine deaminase a PCS requires the fulfillment of three conditions. First, it is necessary to identify an appropriate site for cysteine modification on the extracellular face of the protein complex where the PTL will be covalently anchored so that its photoisomerization between trans and

cis states results in liganding, and hence modulation of signaling, in one isomer state but not the other. Second, this cysteine-substituted subunit must be mutated in a manner that eliminates its cell-surface trafficking as a homomultimer, but which allows for its surface targeting when it is coassembled with the wild-type subunit. Finally, the function of the heteromeric complex between the cysteine-substituted, trafficking-deficient (PCS) subunit and the wild-type subunit must be efficiently gated by PTL(s) on the PCS subunit(s). We describe here how these conditions can be met. The first step in developing a PCS—that of anchoring a PTL for photocontrol—has been successfully accomplished in a variety of proteins with a variety of ligands. The two main approaches have been steric block of an active site (either a pore of an ion channel or a catalytic site of an enzyme) or allosteric regulation by the PTL attached to a receptor’s ligand binding domain (Gorostiza and Isacoff, 2008, Szobota and Isacoff, 2010 and Fehrentz et al., 2011).

First, a Teal insert was generated by PCR amplification of Teal (Allele Biotech, San Diego, CA, USA) with added 5′ NheI and 3′ EcoRI restriction sites and subcloned into the pLL3.7syn lentiviral expression plasmid. Next, Gephyrin with 5′ BsrGI and 3′ MfeI restriction sites was generated by PCR amplification from a GFP-Gephyin expression plasmid ( Fuhrmann et al., 2002) and subcloned into the Teal expression plasmid using the BsrGI and EcoRI sites to generate a Teal-Gephryin fusion protein. Finally, Teal-Gephyrin with 5′ BsiWI and 3′ NheI restriction sites was

PCR amplified from this plasmid and subcloned into the Cre-dependent eYFP expression plasmid described above, replacing eYFP in the dio expression cassette. All animal work was approved by the Massachusetts Institute of Technology Committee on Animal Care; it conforms to the National Institutes of Health 17-AAG guidelines for the use and care of vertebrate animals. L2/3 cortical pyramidal neurons were labeled by in utero electroporation

on E16 timed pregnant FG-4592 cost C57BL/6J mice (Charles River, Wilmington, MA, USA) as previously described (Tabata and Nakajima, 2001). pFUdioeYFPW, pFUdioTealGephyrinW, pFUCreW plasmids were dissolved in 10 mM Tris ± HCl (pH 8.0) at a 10:5:1 molar ratio for a final concentration of 1 μg/μl along with 0.1% of Fast Green (Sigma-Aldrich, St. Louis, MO, USA). The solution, containing 1-2 μl of plasmid, was delivered into the lateral ventricle with a 32 gauge Hamilton syringe (Hamilton Company, Reno, NV, USA). Five pulses of 35–40 V (duration 50 ms, frequency 1 Hz) were delivered, targeting the visual cortex, using 5 mm diameter tweezer-type platinum electrodes connected to a square wave electroporator (Harvard Apparatus, Holliston, MA, USA). Mice born after in utero electroporation were bilaterally implanted with cranial windows at postnatal days Mephenoxalone 42–57 as previously described (Lee et al., 2008). Sulfamethoxazole (1 mg/ml) and trimethoprim (0.2 mg/ml) were chronically administered in the drinking water through the final imaging session to maintain optical clarity of implanted windows. For functional identification of monocular and binocular visual cortex,

optical imaging of intrinsic signal and data analysis were performed as described previously (Kalatsky and Stryker, 2003). Mice were anesthetized and maintained on 0.5%–0.8% isofluorane supplemented by chloroprothixene (10 mg/kg, i.m.) and placed in a stereotaxic frame. Heart rate was continuously monitored. For visual stimuli, a horizontal bar (5° in height and 73° in width) drifting up with a period of 12 s was presented for 60 cycles on a high refresh rate monitor positioned 25 cm in front of the animal. Optical images of visual cortex were acquired continuously under 610 nm illumination with an intrinsic imaging system (LongDaq Imager 3001/C; Optical Imaging Inc., New York, NY, USA) and a 2.5×/0.075 NA (Zeiss, Jena, Germany) objective.