NPFF Decreases Activity of Human Arcuate NPY Neurons: A Study in Embryonic-Stem-Cell-Derived Model.

Restoring the control of food intake is the key to obesity management and prevention. The arcuate nucleus (ARC) of the hypothalamus is extensively being studied as a potential anti-obesity target. Animal studies showed that neuropeptide FF (NPFF) reduces food intake by its action in neuropeptide Y (NPY) neurons of the hypothalamic ARC, but the detailed mode of action observed in human neurons is missing, due to the lack of a human-neuron-based model for pharmacology testing. Here, we validated and utilized a human-neural-stem-cell-based (hNSC) model of ARC to test the effects of NPFF on cellular pathways and neuronal activity. We found that in the human neurons, decreased cAMP levels by NPFF resulted in a reduced rate of cytoplasmic calcium oscillations, indicating an inhibition of ARC NPY neurons. This suggests the therapeutic potential of NPFFR2 in obesity. In addition, we demonstrate the use of human-stem-cell-derived neurons in pharmacological applications and the potential of this model to address functional aspects of human hypothalamic neurons.

The central fibroblast growth factor receptor/beta klotho system: Comprehensive mapping in Mus musculus and comparisons to nonhuman primate and human samples using an automated in situ hybridization platform.

Central activation of fibroblast growth factor (FGF) receptors regulates peripheral glucose homeostasis and reduces food intake in preclinical models of obesity and diabetes. The current work was undertaken to advance our understanding of the receptor expression, as sites of ligand action by FGF19, FGF21, and FGF1 in the mammalian brain remains unresolved. Recent advances in automated RNAscope in situ hybridization and droplet digital PCR (ddPCR) technology allowed us to interrogate central FGFR/beta klotho (Klb) system at the cellular level in the mouse, with relevant comparisons to nonhuman primate and human brain. FGFR1-3 gene expression was broadly distributed throughout the CNS in Mus musculus, with FGFR1 exhibiting the greatest heterogeneity. FGFR4 expression localized only in the medial habenula and subcommissural organ of mice. Likewise, Klb mRNA was restricted to the suprachiasmatic nucleus (SCh) and select midbrain and hindbrain nuclei. ddPCR in the rodent hypothalamus confirmed that, although expression levels are indeed low for Klb, there is nonetheless a bonafide subpopulation of Klb+ cells in the hypothalamus. In NHP and human midbrain and hindbrain, Klb + cells are quite rare, as is expression of FGFR4. Collectively, these data provide the most robust central map of the FGFR/Klb system to date and highlight central regions that may be of critical importance to assess central ligand effects with pharmacological dosing, such as the putative interactions between the endocrine FGFs and FGFR1/Klb, or FGF19 with FGFR4.

Integrated Brain Atlas for Unbiased Mapping of Nervous System Effects Following Liraglutide Treatment.

Light Sheet Fluorescence Microscopy (LSFM) of whole organs, in particular the brain, offers a plethora of biological data imaged in 3D. This technique is however often hindered by cumbersome non-automated analysis methods. Here we describe an approach to fully automate the analysis by integrating with data from the Allen Institute of Brain Science (AIBS), to provide precise assessment of the distribution and action of peptide-based pharmaceuticals in the brain. To illustrate this approach, we examined the acute central nervous system effects of the glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide. Peripherally administered liraglutide accessed the hypothalamus and brainstem, and led to activation in several brain regions of which most were intersected by projections from neurons in the lateral parabrachial nucleus. Collectively, we provide a rapid and unbiased analytical framework for LSFM data which enables quantification and exploration based on data from AIBS to support basic and translational discovery.

Surgical specialty and preoperative medical consultation based on commercial health insurance claims.

BACKGROUND: Surgical patients are sometimes referred for preoperative evaluations by consultants in other medical specialties, although consultations are unnecessary for many patients, particularly for healthy patients undergoing low-risk surgeries. Surgical specialty has been shown to predict usage of preoperative consultations. However, evidence is generally limited regarding factors associated with preoperative consultations. This study evaluates surgical specialty and other predictors of preoperative consultations. METHODS: This retrospective cohort study analyzed surgery claims of 7400 privately insured patients in Washington, United States, from eight surgical specialties. We estimated log-Poisson generalized estimating equation models that regress whether a patient received a consultation on surgical specialty and covariates accounting for the data's hierarchical structure with patients nesting within surgeons, and surgeons nesting within provider organizations. Covariates include age, gender, Deyo comorbidity index, surgical risk, and geographic factors. RESULTS: Overall, 485 (6.6%) patients had a preoperative consultation. The incidence of preoperative consultation varied significantly by surgical specialty. Orthopedics, neurosurgery, and ophthalmology had 3.9 (95% CI 2.4, 6.5), 2.3 (95% CI 1.1, 4.5), and 2.3 (95% CI 1.1, 4.6) times greater adjusted likelihoods of preoperative consultation than general surgery, respectively. The adjusted likelihoods of consultation for gynecology, urology, otolaryngology, and vascular surgery were not statistically different from general surgery. The following covariates were associated with greater likelihood of preoperative consultation: greater age, higher surgical risk, having one or more comorbidities vs. none, and small rural towns vs. urban areas. More than 75% of all consultations were provided to patients with a Deyo comorbidity index of 0 or 1. Low surgical risk patients had 0.3 (95% CI 0.3, 0.5) times the likelihood of preoperative consultation of intermediate and high-risk patients overall. CONCLUSIONS: The likelihood of preoperative consultation varied fourfold (an absolute 9% points) across surgical specialties. Most consultations were provided to patients with low comorbidity and with low or intermediate surgical risk. To improve usage of preoperative consultations as an evidence-based practice, future research should determine how the health outcomes effects of preoperative consultations vary depending on comorbidity burden and surgical risk.

An ultrastructural analysis of the effects of ethanol self-administration on the hypothalamic paraventricular nucleus in rhesus macaques.

A bidirectional relationship between stress and ethanol exists whereby stressful events are comorbid with problematic ethanol use and prolonged ethanol exposure results in adaptations of the physiological stress response. Endocrine response to stress is initiated in the hypothalamic paraventricular nucleus (PVN) with the synthesis and release of corticotropin-releasing hormone (CRH) and arginine-vasopressin (AVP). Alterations in CRH and AVP following long-term ethanol exposure in rodents is well demonstrated, however little is known about the response to ethanol in primates or the mechanisms of adaptation. We hypothesized that long-term ethanol self-administration in nonhuman primates would lead to ultrastructural changes in the PVN underlying adaptation to chronic ethanol. Double-label immunogold electron microscopy (EM) was used to measure presynaptic gamma-aminobutyric acid (GABA) and glutamate density within synaptic terminals contacting CRH- and AVP-immunoreactive dendrites. Additionally, pituitary-adrenal hormones (ACTH, cortisol, DHEA-s and aldosterone) under two conditions (low and mild stress) were compared before and after self-administration. All hormones were elevated in response to the mild stressor independent of ethanol consumption. The presynaptic glutamate density in recurrent (i.e., intra-hypothalamic) CRH terminals was highly related to ethanol intake, and may be a permissive factor in increased drinking due to stress. Conversely, glutamate density within recurrent AVP terminals showed a trend-level increase following ethanol, but was not related to average daily consumption. Glutamate density in non-recurrent AVP terminals was related to aldosterone under the low stress condition while GABAergic density in this terminal population was related to water consumption. The results reveal distinct populations of presynaptic terminals whose glutamatergic or GABAergic density were uniquely related to water and ethanol consumption and circulating hormones.

3D structure tensor analysis of light microscopy data for validating diffusion MRI.

Diffusion magnetic resonance imaging (d-MRI) is a powerful non-invasive and non-destructive technique for characterizing brain tissue on the microscopic scale. However, the lack of validation of d-MRI by independent experimental means poses an obstacle to accurate interpretation of data acquired using this method. Recently, structure tensor analysis has been applied to light microscopy images, and this technique holds promise to be a powerful validation strategy for d-MRI. Advantages of this approach include its similarity to d-MRI in terms of averaging the effects of a large number of cellular structures, and its simplicity, which enables it to be implemented in a high-throughput manner. However, a drawback of previous implementations of this technique arises from it being restricted to 2D. As a result, structure tensor analyses have been limited to tissue sectioned in a direction orthogonal to the direction of interest. Here we describe the analytical framework for extending structure tensor analysis to 3D, and utilize the results to analyze serial image "stacks" acquired with confocal microscopy of rhesus macaque hippocampal tissue. Implementation of 3D structure tensor procedures requires removal of sources of anisotropy introduced in tissue preparation and confocal imaging. This is accomplished with image processing steps to mitigate the effects of anisotropic tissue shrinkage, and the effects of anisotropy in the point spread function (PSF). In order to address the latter confound, we describe procedures for measuring the dependence of PSF anisotropy on distance from the microscope objective within tissue. Prior to microscopy, ex vivo d-MRI measurements performed on the hippocampal tissue revealed three regions of tissue with mutually orthogonal directions of least restricted diffusion that correspond to CA1, alveus and inferior longitudinal fasciculus. We demonstrate the ability of 3D structure tensor analysis to identify structure tensor orientations that are parallel to d-MRI derived diffusion tensors in each of these three regions. It is concluded that the 3D generalization of structure tensor analysis will further improve the utility of this method for validation of d-MRI by making it a more flexible experimental technique that closer resembles the inherently 3D nature of d-MRI measurements.

Enhancement of the pulmonary arteries and thoracic aorta: comparison of a biphasic contrast injection and fixed delay protocol with a monophasic injection and a timing bolus protocol.

The definitive diagnosis of pulmonary embolism, a significant cause of morbidity and mortality, relies on imaging. In this study, we compare the conventional computed tomography pulmonary angiogram (CTPA) protocol to a double-rule out CT angiogram (DRO CTA) protocol in terms of vascular enhancement, radiation dose, and contrast volume delivered. The CTPA protocol involves injection of a timing bolus for localization of the pulmonary artery, whereas the DRO CTA protocol involves a biphasic contrast. We analyzed 248 consecutive CTPA studies and 242 consecutive DRO CTA studies. Vessel enhancement using region of interest (ROI) measurements, radiation dose delivered, and total contrast volume administered was recorded. The enhancement of all vessels measured was statistically significantly higher with the biphasic DRO CTA protocol than the CTPA protocol. The difference in mean vascular enhancement for the two protocols was greatest in the descending aorta (DA, P < 0.001) and least in the main pulmonary artery (MPA, P = 0.001). The percent of studies with vascular enhancement ≥250 Hounsfield units (HU) was significantly greater in all vascular beds except the MPA when the DRO CTA protocol was used. Studies performed with the DRO CTA protocol led to less radiation exposure and used less contrast than those performed with the CTPA protocol (P < 0.001 for both). According to the final radiology report, 35.08 % of studies in the CTPA group and 22.31 % of studies in the DRO CTA group were considered indeterminate (P = 0.001). In conclusion, the biphasic DRO CTA protocol leads to statistically significantly higher opacification of all pulmonary arterial and aortic vessels studied, with no greater delivery of radiation or contrast, than the monophasic CTPA protocol.

Cell-autonomous heterogeneity of nutrient uptake in white adipose tissue of rhesus macaques.

Phenotypic diversity may play an adaptive role by providing graded biological responses to fluctuations in environmental stimuli. We used single-cell imaging of the metabolizable fluorescent fatty acid analog 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)-C12 and fluorescent 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG) to explore cellular heterogeneity in nutrient uptake in white adipose tissue (WAT) explants of rhesus macaques. Surprisingly, WAT displayed a striking cell size-independent mosaic pattern, in that adjacent adipocytes varied with respect to insulin-stimulated BODIPY-C12 and 2-NBDG uptake. Relative free fatty acid (FFA) transport activity correlated with the cellular levels of FFA transporter protein-1 and the scavenger receptor CD36 in individual adipocytes. In vitro incubation of WAT explants for 24 hours caused partial desynchronization of cellular responses, suggesting that adipocytes may slowly alter their differential nutrient uptake activity. In vitro-differentiated human adipocytes also exhibited a mosaic pattern of BODIPY-C12 uptake. WAT from animals containing a homogeneous population of large adipocytes was nonmosaic, in that every adipocyte exhibited a similar level of BODIPY-C12 fluorescence, suggesting that the development of obesity is associated with the loss of heterogeneity in WAT. Hence, for the first time, we demonstrate an intrinsic heterogeneity in FFA and glucose transport activity in WAT.

Spatiotemporal dynamics of triglyceride storage in unilocular adipocytes.

The spatiotemporal dynamics of triglyceride (TG) storage in unilocular adipocytes are not well understood. Here we applied ex vivo technology to study trafficking and metabolism of fluorescent fatty acids in adipose tissue explants. Live imaging revealed multiple cytoplasmic nodules surrounding the large central lipid droplet (cLD) of unilocular adipocytes. Each cytoplasmic nodule harbors a series of closely associated cellular organelles, including micro-lipid droplets (mLDs), mitochondria, and the endoplasmic reticulum. Exogenously added free fatty acids are rapidly adsorbed by mLDs and concurrently get esterified to TG. This process is greatly accelerated by insulin. mLDs transfer their content to the cLD, serving as intermediates that mediate packaging of newly synthesized TG in the large interior of a unilocular adipocyte. This study reveals novel cell biological features that may contribute to the mechanism of adipocyte hypertrophy.

A genome-wide map of adeno-associated virus-mediated human gene targeting.

To determine which genomic features promote homologous recombination, we created a genome-wide map of gene targeting sites. We used an adeno-associated virus vector to target identical loci introduced as transcriptionally active retroviral vectors. A comparison of ~2,000 targeted and untargeted sites showed that targeting occurred throughout the human genome and was not influenced by the presence of nearby CpG islands, sequence repeats or DNase I-hypersensitive sites. Targeted sites were preferentially located within transcription units, especially when the target loci were transcribed in the opposite orientation to their surrounding chromosomal genes. We determined the impact of DNA replication by mapping replication forks, which revealed a preference for recombination at target loci transcribed toward an incoming fork. Our results constitute the first genome-wide screen of gene targeting in mammalian cells and demonstrate a strong recombinogenic effect of colliding polymerases.

Fluorescence-based laser capture microscopy technology facilitates identification of critical in vivo cytomegalovirus transcriptional programs.

Cytomegalovirus gene expression in highly permissive, cultured fibroblasts occurs in three kinetic classes known as immediate early, early, and late. Infection of these cells results in a predictable transcriptional program leading to high levels of virus production. Infection of other, so-called, nonpermissive cell types results in a transcriptional program that either fails to produce virus particles or production is substantially reduced compared to fibroblasts. We have found that CMV gene expression profiles in tissues from infected hosts differ greatly from those observed in infected tissue culture cells. The number of viral genes expressed in tissues is much more limited, and the number of highly active genes does not correlate with viral DNA load. Additionally, viral gene expression in vivo is tissue selective with no two tissues expressing the exact same viral gene profile. Thus, in vivo CMV gene expression appears to be governed by mechanisms that are still uncharacterized. Cytomegalovirus remains in a persistent phase for the lifetime of the host. During this phase only a limited number of host cells are infected, and it is very difficult to detect CMV gene expression in whole tissues without sub-fractionating infected vs. uninfected cells. Herein, we describe the development of a fluorescence-based laser capture microscopy technique coupled with small sample size microarray analysis to determine the viral gene expression in 50-100 infected cells isolated from frozen RCMV-infected tissue sections.

Measuring cone density in a Japanese macaque (Macaca fuscata) model of age-related macular degeneration with commercially available adaptive optics.

The aim of this study was to assess the feasibility of using a commercially available high-resolution adaptive optics (AO) camera to image the cone mosaic in Japanese macaques (Macaca fuscata) with dominantly inherited drusen. The macaques examined develop drusen closely resembling those seen in humans with age-related macular degeneration (AMD). For each animal, we acquired and processed images from the AO camera, montaged the results into a composite image, applied custom cone-counting software to detect individual cone photoreceptors, and created a cone density map of the macular region. We conclude that flood-illuminated AO provides a promising method of visualizing the cone mosaic in nonhuman primates. Future studies will quantify the longitudinal change in the cone mosaic and its relationship to the severity of drusen in these animals.

Effects of androgens on early post-ischemic neurogenesis in mice.

Although androgens are reported to affect stroke outcomes by altering ischemic tissue damage, their effect on post-injury repair is unknown. Since neurogenesis has recently been recognized as contributing to stroke outcomes, we investigated the role of androgens on stroke-induced neurogenesis. Adult male mice were subjected to transient middle cerebral artery occlusion (MCAO) and neurogenesis was examined 1 week later by quantifying BrdU/doublecortin-positive and BrdU/NeuN-positive neurons in brain germinal regions as well as the injured striatum. To elucidate the role of endogenous androgens, post-MCAO neurogenesis was examined in gonadally intact males, intact males implanted with the androgen receptor antagonist flutamide, and surgically castrated males. Surgical castration or pharmacologic androgen receptor blockade had no effects on post-ischemic neurogenesis, except that continuous androgen receptor blockade unexpectedly suppressed maturation of newborn neurons (BrdU/NeuN-positive cells) in the dentate gyrus. Post-MCAO neurogenesis was also examined in surgically castrated mice treated with continuous release implants containing testosterone or dihydrotestosterone (DHT). Testosterone and DHT robustly inhibited post-ischemic neurogenesis in the dentate gyrus, and the more potent androgen DHT virtually abolished the presence of immature newborn neurons (BrdU/doublecortin-positive cells) in the injured striatum. Our data suggest that endogenous androgens do not alter post-stroke neurogenesis quantitatively, but the presence of supra-physiological androgen stimulation profoundly suppresses early neurogenesis in germinal brain areas and reduces cellular repair in injured tissue after cerebral ischemia. These results advance the understanding of the role that androgens play in stroke outcomes.

Restoration of testis function in hypogonadotropic hypogonadal mice harboring a misfolded GnRHR mutant by pharmacoperone drug therapy.

Mutations in receptors, ion channels, and enzymes are frequently recognized by the cellular quality control system as misfolded and retained in the endoplasmic reticulum (ER) or otherwise misrouted. Retention results in loss of function at the normal site of biological activity and disease. Pharmacoperones are target-specific small molecules that diffuse into cells and serve as folding templates that enable mutant proteins to pass the criteria of the quality control system and route to their physiologic site of action. Pharmacoperones of the gonadotropin releasing hormone receptor (GnRHR) have efficacy in cell culture systems, and their cellular and biochemical mechanisms of action are known. Here, we show the efficacy of a pharmacoperone drug in a small animal model, a knock-in mouse, expressing a mutant GnRHR. This recessive mutation (GnRHR E(90)K) causes hypogonadotropic hypogonadism (failed puberty associated with low or apulsatile luteinizing hormone) in both humans and in the mouse model described. We find that pulsatile pharmacoperone therapy restores E(90)K from ER retention to the plasma membrane, concurrently with responsiveness to the endogenous natural ligand, gonadotropin releasing hormone, and an agonist that is specific for the mutant. Spermatogenesis, proteins associated with steroid transport and steroidogenesis, and androgen levels were restored in mutant male mice following pharmacoperone therapy. These results show the efficacy of pharmacoperone therapy in vivo by using physiological, molecular, genetic, endocrine and biochemical markers and optimization of pulsatile administration. We expect that this newly appreciated approach of protein rescue will benefit other disorders sharing pathologies based on misrouting of misfolded protein mutants.

Diffusion MRI of the developing cerebral cortical gray matter can be used to detect abnormalities in tissue microstructure associated with fetal ethanol exposure.

Fetal alcohol spectrum disorders (FASDs) comprise a wide range of neurological deficits that result from fetal exposure to ethanol (EtOH), and are the leading cause of environmentally related birth defects and mental retardation in the western world. One aspect of diagnostic and therapeutic intervention strategies that could substantially improve our ability to combat this significant problem would be to facilitate earlier detection of the disorders within individuals. Light microscopy-based investigations performed by several laboratories have previously shown that morphological development of neurons within the early-developing cerebral cortex is abnormal within the brains of animals exposed to EtOH during fetal development. We and others have recently demonstrated that diffusion MRI can be of utility for detecting abnormal cellular morphological development in the developing cerebral cortex. We therefore assessed whether diffusion tensor imaging (DTI) could be used to distinguish the developing cerebral cortices of ex vivo rat pup brains born from dams treated with EtOH (EtOH; 4.5 g/kg, 25%) or calorie-matched quantities of maltose/dextrin (M/D) throughout gestation. Water diffusion and tissue microstructure were investigated using DTI (fractional anisotropy, FA) and histology (anisotropy index, AI), respectively. Both FA and AI decreased with age, and were higher in the EtOH than the M/D group at postnatal ages (P)0, P3, and P6. Additionally, there was a significant correlation between FA and AI measurements. These findings provide evidence that disruptions in cerebral cortical development induced by EtOH exposure can be revealed by water diffusion anisotropy patterns, and that these disruptions are directly related to cerebral cortical differentiation.

Selective control of SNARE recycling by Golgi retention.

Two distinct sets of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) catalyze membrane fusion in the cis-Golgi and trans-Golgi. The mechanism that controls Golgi localization of SNAREs remains largely unknown. Here we tested three potential mechanisms, including vesicle recycling between the Golgi and the endoplasmic reticulum, partitioning in Golgi lipid microdomains, and selective intra-Golgi retention. Recycling rates showed a linear relationship with intra-Golgi mobility of SNAREs. The cis-Golgi SNAREs had higher mobility than intra-Golgi SNAREs, whereas vesicle SNAREs had higher mobility than target membrane SNAREs. The differences in SNARE mobility were not due to preferential partitioning into detergent-resistant membrane microdomains. We propose that intra-Golgi retention precludes entropy-driven redistribution of SNAREs to the endoplasmic reticulum and endocytic compartments.

Quality control protocol for confocal systems.

Quantitative analysis of confocal imaging experiments require more stringent quality control of instrument function than qualitative imaging. Unfortunately, there are no standard procedures for quality control that are uniformly implemented, and, in multi user facilities experimenters rarely have access to the QC information. This paper proposes an easy and very efficient protocol that could be performed at the beginning of each day, experiment or even slide. It takes only a few minutes to assess laser stability, stage stability, channel registration in 3 dimensions and flatness of field. The information may be used either to calibrate data or, in more severe cases to request servicing the instrument.

Rescue of misrouted GnRHR mutants reveals its constitutive activity.

G protein-coupled receptors (GPCR) play central roles in almost all physiological functions, and mutations in GPCR are responsible for over 30 hereditary diseases associated with loss or gain of receptor function. Gain of function mutants are frequently described as having constitutive activity (CA), that is, they activate effectors in the absence of agonist occupancy. Although many GPCR have mutants with CA, the GnRH receptor (GnRHR) was not, until 2010, associated with any CA mutants. The explanation for the failure to observe CA appears to be that the quality control system of the cell recognizes CA mutants of GnRHR as misfolded and retains them in the endoplasmic reticulum. In the present study, we identified several human (h)GnRHR mutants with substitutions in transmembrane helix 6 (F(272)K, F(272)Q, Y(284)F, C(279)A, and C(279)S) that demonstrate varying levels of CA after being rescued by pharmacoperones from different chemical classes and/or deletion of residue K(191), a modification that increases trafficking to the plasma membrane. The movement of the mutants from the endoplasmic reticulum (unrescued) to the plasma membrane (after rescue) is supported by confocal microscopy. Judging from the receptor-stimulated inositol phosphate production, mutants F(272)K and F(272)Q, after rescue, display the largest level of CA, an amount that is comparable with agonist-stimulated activation. Because mutations in other GPCR are, like the hGnRHR, scrutinized by the quality control system, this general approach may reveal CA in receptor mutants from other systems. A computer model of the hGnRHR and these mutants was used to evaluate the conformation associated with CA.

Determination of axonal and dendritic orientation distributions within the developing cerebral cortex by diffusion tensor imaging.

As neurons of the developing brain form functional circuits, they undergo morphological differentiation. In immature cerebral cortex, radially-oriented cellular processes of undifferentiated neurons impede water diffusion parallel, but not perpendicular, to the pial surface, as measured via diffusion-weighted magnetic resonance imaging, and give rise to water diffusion anisotropy. As the cerebral cortex matures, the loss of water diffusion anisotropy accompanies cellular morphological differentiation. A quantitative relationship is proposed here to relate water diffusion anisotropy measurements directly to characteristics of neuronal morphology. This expression incorporates the effects of local diffusion anisotropy within cellular processes, as well as the effects of anisotropy in the orientations of cellular processes. To obtain experimental support for the proposed relationship, tissue from 13 and 31 day-old ferrets was stained using the rapid Golgi technique, and the 3-D orientation distribution of neuronal processes was characterized using confocal microscopic examination of reflected visible light images. Coregistration of the MRI and Golgi data enables a quantitative evaluation of the proposed theory, and excellent agreement with the theoretical results, as well as agreement with previously published values for locally-induced water diffusion anisotropy and volume fraction of the neuropil, is observed.

Single-cell analysis of insulin-regulated fatty acid uptake in adipocytes.

Increased body fat correlates with the enlargement of average fat cell size and reduced adipose tissue insulin sensitivity. It is currently unclear whether adipocytes, as they accumulate more triglycerides and grow in size, gradually become less insulin sensitive or whether obesity-related factors independently cause both the enlargement of adipocyte size and reduced adipose tissue insulin sensitivity. In the first instance, large and small adipocytes in the same tissue would exhibit differences in insulin sensitivity, whereas, in the second instance, adipocyte size per se would not necessarily correlate with insulin response. To analyze the effect of adipocyte size on insulin sensitivity, we employed a new single-cell imaging assay that resolves fatty acid uptake and insulin response in single adipocytes in subcutaneous adipose tissue explants. Here, we report that subcutaneous adipocytes are heterogeneous in size and intrinsic insulin sensitivity. Whereas smaller adipocytes respond to insulin by increasing lipid uptake, adipocytes with cell diameters larger than 80-100 microm are insulin resistant. We propose that, when cell size approaches a critical boundary, adipocytes lose insulin-dependent fatty acid transport. This negative feedback mechanism may protect adipocytes from lipid overload and restrict further expansion of adipose tissue, which leads to obesity and metabolic complications.