A quantitative analysis of ipsilateral tectal unit responses to moving stimuli in frogs

Ipsilateral retino-tecto-tectal (IRTT) units were recorded extracellularly in the rostral optic tectum of the frog (Rana esculenta). The activity of 79 superficial units (II type) was quantified in response to black disks of various sizes, moved vertically at various angular velocities and against a white background. The contrast ¦C¦ was constant during the experiments. Neuronal activity was analysed by two methods, yielding identical results:

Invariants in the ipsilateral retinotectal visual projection of frogs

We determined whether the sensitivity of the ipsilateral type II units of Rana esculenta to prey (W/H-oriented bars) and non-prey (A/V-oriented bars)-like targets remains invariant under various experimental conditions. We show that the shape of the 'discrimination' curve is largely unaffected by the level of general illumination and by the background texture. An increase in the stimulus velocity and in the width of the bars moderately affects the salient points (negative peak and preference reversal) of the curve, but does not alter the overall configurational preference of these units. As for retinal ganglion cells: (i) this curve expresses better a 'contrast' between two vertically oriented edges of different dimensions than a 'contrast' between two edges of equal dimension but of different orientation; and (ii) the experimentally induced variations can be explained on the basis of the spatial and temporal properties of the neuronal elements forming the antagonistic center-surround arrangement of the receptive field.     

Responses of class R3 retinal ganglion cells of the frog to moving configurational bars: effect of the stimulus velocity

Discrimination of 'prey' (bars elongated in the direction of movement; W- or H-orientation) and 'non-prey' (bars perpendicular to the direction of movement; A- or V-orientation) stimuli in freely moving amphibians is velocity-invariant. Whether or not this phenomenon is present in cells belonging to a general decision making neuronal process remains questionable. Present investigations report the effect of the angular velocity of the stimulus on the discrimination function of class R3 (transient ON-OFF) retinal ganglion cells. The main conclusions of this work are the following: (1) irrespective of the angular velocity, class R3 neurons always prefer vertically (A-) to horizontally (W-) oriented stripes as long as the stimulus length remains inferior to the receptive field size; (2) this preference for small A-stimuli is best expressed when stimuli are moved at V = 7.6 degrees/s; (3) a preference reversal is induced by stripes longer than the receptive field via a dual process involving both spatial and temporal mechanisms; (4) this preference reversal is velocity-dependent: the longer the bar, the faster the velocity should be.     

Measuring nicotinic receptors with characteristics of alpha4beta2, alpha3beta2 and alpha3beta4 subtypes in rat tissues by autoradiography

Comparison of [125I]epibatidine and 5-[125I]iodo-3-(2-azetidinylmethoxy)pyridine ([125I]A-85380) autoradiography showed evidence for nicotinic receptor heterogeneity. To identify the receptor subtypes, we performed [125I]epibatidine autoradiography in the presence of cytisine or A-85380. By comparing these results with binding data from human embryonic kidney (HEK) 293 cells stably transfected with different combinations of rat nicotinic receptor subunits, we were able to quantify three distinct populations of [125I]epibatidine binding sites with characteristics of alpha4beta2, alpha3beta2 and alpha3beta4 receptors. Although the predominant subtype in rat brain was alpha4beta2, non-alpha4beta2 binding sites were prominent in many regions. In the habenulo-peduncular system, cerebellum, substantia gelatinosa, and many medullary nuclei, alpha3beta4-like binding accounted for more than 40% of [125I]epibatidine binding, and nearly all binding in superior cervical ganglion and pineal gland. Other regions enriched in alpha3beta4-like binding included locus ceruleus, dorsal tegmentum, subiculum and anteroventral thalamic nucleus. Regions enriched in alpha3beta2-like binding included the habenulo-peduncular system, many visual system structures, certain geniculate nuclei, and dopaminergic regions. The combination of autoradiography using a broad spectrum radioligand in the presence of selective competitors, and data from binding to defined receptor subtypes in expression systems, allowed us to quantify the relative populations of these three subtypes.     

Amino acids in segment IVS6 and beta-subunit interaction support distinct conformational changes during Ca(v)2.1 inactivation

Ca(v)2.1 mediates voltage-gated Ca2+ entry into neurons and the release of neurotransmitters at synapses of the central nervous system. An inactivation process that is modulated by the auxiliary beta-subunits regulates Ca2+ entry through Ca(v)2.1. However, the molecular mechanism of this alpha1-beta-subunit interaction remains unknown. Herein we report the identification of new determinants within segment IVS6 of the alpha(1)2.1-subunit that markedly influence channel inactivation. Systematic substitution of residues within IVS6 with amino acids of different size, charge, and polarity resulted in mutant channels with rates of fast inactivation (k(inact)) ranging from a 1.5-fold slowing in V1818I (k(inact) = 0.98 +/- 0.09 s(-1) compared with wild type alpha(1)2.1/alpha2-delta/beta1a k(inact) = 1.35 +/- 0.25 s(-1) to a 75-fold acceleration in mutant M1811Q (k(inact) = 102 +/- 3 s(-1). Coexpression of mutant alpha(1)2.1-subunits with beta(2a) resulted in two different phenotypes of current inactivation: 1) a pronounced reduction in the rate of channel inactivation or 2) an attenuation of a slow component in I(Ba) inactivation. Simulations revealed that these two distinct inactivation phenotypes arise from a beta2a-subunit-induced destabilization of the fast-inactivated state. The IVS6- and beta2a-subunit-mediated effects on Ca(v)2.1 inactivation are likely to occur via independent mechanisms.     

Isolation of a novel integrin receptor mediating Arg-Gly-Asp-directed cell adhesion to fibronectin and type I collagen from human neuroblastoma cells. Association of a novel beta 1-related subunit with alpha v

We report the isolation from two human neuroblastoma cell lines of an Arg-Gly-Asp-dependent integrin complex capable of binding to vitronectin, fibronectin, and type I collagen. The two neuroblastoma cell lines, SK-N-SH and IMR-32, exhibit specific attachment to fibronectin and type I collagen. SK-N-SH cells exhibit a much stronger attachment to vitronectin than the IMR-32 cells, which attach poorly to this substrate. Affinity chromatography of octylglucoside extracts of 125I surface-labeled cells on GRGDSPK-Sepharose columns resulted in the specific binding and elution with GRGDSP of three radiolabeled polypeptides with relative molecular masses of 135, 115, and 90 kD when analyzed by SDS-PAGE under nonreducing conditions. In the SK-N-SH cells the 135- and 90-kD polypeptides were more abundant whereas in the IMR-32 cells the 135- and 115-kD polypeptides were more highly expressed. Liposomes prepared from fractions containing all three polypeptides bound to vitronectin, fibronectin, and type I collagen, whereas liposomes prepared from the 135- and 115-kD polypeptides bound only to fibronectin and type I collagen. Polyclonal antibodies against the alpha/beta complexes of both the vitronectin receptor and the fibronectin receptor immunoprecipitated all three polypeptides. A monoclonal antibody against beta 1 immunoprecipitated only the 135- and the 115-kD polypeptides, whereas a monoclonal antibody against beta 3 subunit immunoprecipitated the 135- and 90-kD polypeptides. Although, the 115-kD polypeptide could be recognized by an anti-beta 1 antibody, a comparison of peptide maps generated by V8 protease digestion of the 115-kD polypeptide and beta 1 subunit immunoprecipitated from GRGDSPK-Sepharose flow-through material indicated that these two polypeptides are distinct. Depletion of the 90-kD polypeptide with an anti-beta 3 monoclonal antibody did not effect the ability of the 115- and 135-kD polypeptides to bind to GRGDSPK-Sepharose. These data indicate that the SK-N-SH and IMR-32 neuroblastoma cells express a novel "beta 1-like" integrin subunit that can associate with alpha v and can bind to RGD. We propose to name this beta 1-like subunit beta n. The data reported here thus demonstrate that in these two cell lines alpha v associates with two beta subunits, beta n and beta 3, forming two heterodimers. The alpha v beta n complex mediates binding to fibronectin and type I collagen, whereas the alpha v beta 3 complex mediates binding to vitronectin.     

Human soleus single muscle fiber function with exercise or nutrition countermeasures during 60 days of bed rest

The soleus muscle has been consistently shown to atrophy more than other leg muscles during unloading and is difficult to protect using various exercise countermeasure paradigms. However, the efficacy of aerobic exercise, a known stimulus for oxidative adaptations, has not been tested in combination with resistance exercise (RE), a known hypertrophic stimulus. We hypothesized that a concurrent exercise program (AE + RE) would preserve soleus fiber myosin heavy chain (MHC) I size and function during 60 days of bed rest. A secondary objective was to test the hypothesis that a leucine-enriched high protein diet would partially protect soleus single fiber characteristics. Soleus muscle biopsies were obtained before and after bed rest from a control (BR; n = 7), nutrition (BRN; n = 8), and exercise (BRE; n = 6) group. Single muscle fiber diameter (Dia), peak force (Po), contractile velocity, and power were studied. BR decreased (P < 0.05) MHC I Dia (-14%), Po (-38%), and power (-39%) with no change in contractile velocity. Changes in MHC I size (-13%) and contractile function (approximately 30%) from BRN were similar to BR. BRE decreased (P < 0.05) MHC I Dia (-13%) and Po (-23%), while contractile velocity increased (P < 0.05) 26% and maintained power. These soleus muscle data show 1) the AE + RE exercise program maintained MHC I power but not size and strength, and 2) the nutrition countermeasure did not benefit single fiber size and contractile function. The divergent response in size and functional MHC I soleus properties with the concurrent exercise program was a unique finding further highlighting the challenges of protecting the unloaded soleus.     

Isolation and characterization of cell subpopulations from the monkey corpus luteum of the menstrual cycle

This study was designed to test the hypothesis that the corpus luteum of primate species consists of cell subpopulations that differ in physical characteristics, function, and regulation by endocrine and paracrine factors. The corpus luteum (n = 25) was removed from rhesus monkeys at the mid-luteal phase of the menstrual cycle (Days 7-8 after the surge of luteinizing hormone, LH) and enzymatically dispersed. Freshly dispersed cells were analyzed and sorted on the basis of their forward and 90 degrees light scatter (FLS and 90LS, respectively) properties using an EPICS C flow cytometer. Freshly dispersed and sorted cells were fixed, stained histochemically for the presence of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), and measured to determine their diameters. Freshly dispersed (MIX) and sorted cells from corpora lutea during the early (Days 4-5 after the LH surge; n = 4) and mid-luteal phases of the cycle were incubated in vitro and steroid production was assessed. The size distribution of dispersed cells revealed four peaks that corresponded to small (10-15 microns in diameter) 3 beta-HSD-negative, and small, medium (16-20 microns), and large (greater than 20 microns) 3 beta-HSD-positive cells. Analysis of dispersed cells for FLS and 90LS demonstrated two continua (C alpha and C beta). C alpha contained single cells and cell clusters; 99.7 +/- 0.3% (n = 3) of the cells were less than or equal to 15 microns in diameter and 96.7 +/- 0.3% were 3 beta-HSD-negative. C alpha cells produced low levels of progesterone (0.2 +/- 0.1 ng/ml per 5 x 10(4) cells; n = 3) in vitro under basal conditions. C beta consisted of single cells from 10 microns to 40 microns in diameter and contained the lipid-filled and 3 beta-HSD-positive cells. Two regions (R1 and R3) of C beta were defined and their cells separated. In R1, 96 +/- 2% (n = 3) of the cells had diameters of less than or equal to 15 microns, whereas 82 +/- 4% (n = 3) of those in R3 were greater than or equal to 20 microns. Basal progesterone production by R3 cells from early luteal phase of the cycle was 12 times greater than that by R1 cells (n = 3 per group).(ABSTRACT TRUNCATED AT 400 WORDS)     

A sensory map based on velocity threshold of sensory neurones from a chordotonal organ in the tailfan of the crayfish

The central projections of sensory neurones innervating a strand chordotonal organ (CO) in the tailfan of the crayfish, Procambarus clarkii (Girard) have been investigated. The CO monitors movement of the exopodite of the tailfan relative to the endopodite. Intracellular recording and staining were used to characterise the response of the sensory neurones to applied stretches of the chordotonal organ and to reveal their morphology. Two gross morphological types of afferents were found: those that terminated in the terminal (6th) abdominal ganglion on the side ipsilateral to the sensory receptor, and those that had branches in the terminal ganglion and an intersegmental axon that ascended rostrally. Afferents responded to position, velocity and direction of imposed CO displacement. Afferents with particular physiological properties had similar morphologies in different crayfish. Irrespective of their directional responses, afferents had central projection areas dependent upon their velocity thresholds. Many afferents responded only during movement of the CO, and those with the lowest velocity thresholds (2°/s) had branches that projected most anteriorly, while those with progressively higher velocity thresholds (up to 200°/s) projected progressively more posteriorly. Afferents that responded to low velocity ramp movements and spiked tonically projected to more posterior areas of the ganglion than those that responded only to movements.Abbreviations A6SCI sixth abdominal sensory commissure I - CO chordotonal organ - DMT dorsal medial tract - G6 sixth abdominal ganglion - LDT lateral dorsal tract - MDT medial dorsal tract - MVT medial ventral tract - R1–4 nerve roots 1–4 - VLT ventral lateral tract - VMT ventral medial tract     

Are the dynamic response characteristics of brachial artery flow-mediated dilation sensitive to the magnitude of increase in shear stimulus?

The purpose of this study was to determine the dynamic characteristics of brachial artery dilation in response to step increases in shear stress [flow-mediated dilation (FMD)]. Brachial artery diameter (BAD) and mean blood velocity (MBV) (Doppler ultrasound) were obtained in 15 healthy subjects. Step increases in MBV at two shear stimulus magnitudes were investigated: large (L; maximal MBV attainable), and small (S; MBV at 50% of the large step). Increase in shear rate (estimate of shear stress: MBV/BAD) was 76.8 +/- 15.6 s(-1) for L and 41.4 +/- 8.7 s(-1) for S. The peak %FMD was 14.5 +/- 3.8% for L and 5.7 +/- 2.1% for S (P < 0.001). Both the L (all subjects) and the S step trials (12 of 15 subjects) elicited a biphasic diameter response with a fast initial phase (phase I) followed by a slower final phase. Relative contribution of phase I to total FMD when two phases occurred was not sensitive to shear rate magnitude (r(2) = 0.003, slope P = 0.775). Parameters quantifying the dynamics of the FMD response [time delay (TD), time constant (tau)] were also not sensitive to shear rate magnitude for both phases (phase I: TD r(2) = 0.03, slope P = 0.376, tau r(2) = 0.04, slope P = 0.261; final phase: TD r(2) = 0.07, slope P = 0.169, tau r(2) = 0.07, slope P = 0.996). These data support the existence of two distinct mechanisms, or sets of mechanisms, in the human conduit artery FMD response that are proportionally sensitive to shear stimulus magnitude and whose dynamic response is not sensitive to shear stimulus magnitude.     

Axonal transmission in the retina introduces a small dispersion of relative timing in the ganglion cell population response

Background

Visual stimuli elicit action potentials in tens of different retinal ganglion cells. Each ganglion cell type responds with a different latency to a given stimulus, thus transforming the high-dimensional input into a temporal neural code. The timing of the first spikes between different retinal projection neurons cells may further change along axonal transmission. The purpose of this study is to investigate if intraretinal conduction velocity leads to a synchronization or dispersion of the population signal leaving the eye.

Methodology/Principal Findings

We ‘imaged’ the initiation and transmission of light-evoked action potentials along individual axons in the rabbit retina at micron-scale resolution using a high-density multi-transistor array. We measured unimodal conduction velocity distributions (1.3±0.3 m/sec, mean ± SD) for axonal populations at all retinal eccentricities with the exception of the central part that contains myelinated axons. The velocity variance within each piece of retina is caused by ganglion cell types that show narrower and slightly different average velocity tuning. Ganglion cells of the same type respond with similar latency to spatially homogenous stimuli and conduct with similar velocity. For ganglion cells of different type intraretinal conduction velocity and response latency to flashed stimuli are negatively correlated, indicating that differences in first spike timing increase (up to 10 msec). Similarly, the analysis of pair-wise correlated activity in response to white-noise stimuli reveals that conduction velocity and response latency are negatively correlated.

Conclusion/Significance

Intraretinal conduction does not change the relative spike timing between ganglion cells of the same type but increases spike timing differences among ganglion cells of different type. The fastest retinal ganglion cells therefore act as indicators of new stimuli for postsynaptic neurons. The intraretinal dispersion of the population activity will not be compensated by variability in extraretinal conduction times, estimated from data in the literature.     

The influence of self-MHC and non-MHC antigens on the selection of an antigen-specific T cell receptor repertoire

We have examined the influence of self-Ag on TCR expression and specificity in the immune response to the Ag pigeon cytochrome c. Previous work has shown that most Ek-restricted cytochrome c-specific T cells from B10 background mice express TCR alpha beta-heterodimers encoded by V beta 3 and V alpha 11 genes, but that T cells expressing V beta 3 proteins are eliminated due to self-tolerance in Mls-2a mouse strains. Thus, EK-restricted cytochrome c-specific T cells from Mls-2a mice fail to express any V beta 3. In the current study the influence of self-MHC and non-MHC Ag on TCR usage in the immune response to cytochrome c was further examined. First, it was demonstrated that the absence of V beta 3 expression in Mls-2a mice does not alter Ir gene function. Specifically, Mls-2a/Eb haplotype V beta 3- [C3H.SW x B10.A(5R)]F1 mice were high responders to cytochrome c despite the fact that previous structure function analyses have shown a very close correlation between Eb-restricted cytochrome c recognition and V beta 3 expression. This demonstration of the plasticity of TCR expression suggests that relatively few Ir gene defects result from tolerance induced by self-Ag. We also examined differences in V alpha 11 expression among cytochrome c-specific T cells from various H-2k haplotype mouse strains. In particular, the low level of expression of V alpha 11 in cytochrome c-specific T cells from C57BR (H-2k) mice was shown not to be due to self-tolerance. Rather, evidence for limited strain polymorphism of V alpha 11 genes, plus the fact that cytochrome c-specific T cells from F1 hybrids between H-2k, Mls-2b identical C57BR and B10.BR mice express high levels of V alpha 11, suggested the possibility that the variable V alpha 11 usage in the cytochrome c-specific responses of these two strains reflected differences in positive selection during ontogeny by non-MHC non-Mls self-Ag.     

Stimulation of lacrimal secretion by sympathetic nerve impulses in the rabbit

Direct electrical stimulation of the preganglionic nerve trunk of the ipsilateral superior cervical sympathetic ganglion induced fluid secretion from the cannulated main excretory duct of the non-stimulated rabbit lacrimal gland. The optimum strength and frequency of stimulation were 4 volts and 25 Hz. At this stimulus parameter, the rate of secretion was 2.1 +/- 0.2 microliter/10 min. The sympathetic-induced lacrimal secretion was markedly depressed after intravenous administration of hexamethonium (1 mg/kg) and by post-ganglionic neurectomy. The results suggest that, in addition to parasympathetic nerve activity, sympathetic nerve impulses, which pass through the superior cervical sympathetic ganglion, also play a role in initiating fluid secretion in the rabbit lacrimal gland.     

Synaptic mechanisms of monaural and binaural processing in the locust

(1) Responses of auditory interneurones were recorded intracellularly within the metathoracic ganglion of the locust when stimulating each tympanic membrane with a piezoelectric transducer. Thus, in contrast to conventional sound stimulation, each of the two ears could be activated independently from the other at variable intensities, duration and stimulus onsets. By means of this ‘earphone-like’ stimulation technique the binaural integration properties of auditory interneurons could be analysed. (2) A minority of units (3 out of 43) was affected by input from one side only. Their synaptic input was purely excitatory and the intensity characteristics reflected those of auditory receptor fibres. (3) Most interneurones received input from both ears, each being excitatory or one excitatory or one excitatory and one inhibitory. In some units the unilateral synaptic response already included both an EPSP and an IPSP. As a result of varying temporal interactions between the EPSP and the IPSP within the unilaterally evoked complex response the intensity characteristics differed widely from unit to unit. (4) With binaural simultaneous stimulation the complexity of the postsynaptic responses of most interneurones increased as the synaptic input from both ears coincided at the level of the recorded interneurone. Although both ears were stimulated symmetrically (at the same time and intensity), units were recorded where the latencies of ipsilateral and contralateral synaptic input were different. Contralateral inhibition could either follow or precede ipsilateral excitation and in some cases both EPSP and IPSP had the same latency. On the basis of these findings the binaural synaptic mechanisms of directional coding are discussed and compared with corresponding results under free field stimulus conditions.     

GABAergic inhibition shapes temporal and spatial response properties of pyramidal cells in the electrosensory lateral line lobe of gymnotiform fish

1. The amplitude-coding pyramidal neurons of the first-order nucleus in weakly electric gymnotiform fish (Eigenmannia), the electrosensory lateral line lobe (ELL), exhibit 2 major physiological transformations of primary afferent input. Pyramidal cells rapidly adapt to a step change in amplitude, and they have a center/surround receptive-field organization. This study examined the physiological role of GABAergic inhibition on pyramidal cells. GABAergic synapses onto the somata of pyramidal cells primarily originate from granule-cell interneurons along with descending input. 2. Pyramidal cells fall into two physiologically distinct categories: E units, which are excited by a rise in stimulus amplitude, and I units, which are inhibited by a rise in stimulus amplitude. Microiontophoretic application of bicuculline methiodide onto both types of pyramidal cells increased the time constant of adaptation, defined as the time required for the neuron's response to decay to 37% of its maximum value, by 70-90%. The peak firing rate of E units to a step increase in stimulus amplitude increased by 49%, while the firing rate of I units did not change significantly. 3. Bicuculline application demonstrated that GABAergic inhibition may contribute to the strict segregation of E and I response properties. In the presence of bicuculline, many E units (normally excited only by stimulus amplitude increases) became excited by both increases and decreases; many I units (normally excited only by amplitude decreases) also became excited to increases. 4. The size of the excitatory receptive-field of E units was not affected by bicuculline, although response magnitude increased. The inhibitory surround increased in spatial extent by 175% with bicuculline administration. Neither the size of the I unit receptive-field center nor the response magnitude changed in the presence of bicuculline. The antagonistic surround of I units, however, increased by 49%. 5. The anatomy of the ELL is well understood (see Carr and Maler 1986). The physiological results obtained in this study, along with the results of Bastian (1986a, b), further our understanding of the functional role of the ELL circuitry. Our results suggest that spatial and temporal response properties of pyramidal cells are regulated by different but interacting inhibitory interneurons, some of which use GABA as a neurotransmitter. The activity of these interneurons is in turn controlled by descending feedback systems.     

Motility evaluation of bull spermatozoa by photon correlation spectroscopy.

Quasi-elastic light scattering techniques are employed to evaluate motility of bovine spermatozoa. The electric field correlation function CE(k, tau) has two components CE(k, tau) = alphafm+(1-alpha)fd, where fm is the correlation function due to motile cells, fd is due to dead cells and alpha is the fraction of motile cells. A function which is a linear combination of the experimentally measured fd and an empirically determined function fm is fit to the CE(k, tau) data. In this way, alpha and the approximate analytic form of fm are determined. The distribution of swimming speeds P(v) is derived from fm using an inverse Fourier sine transform procedure. Results for the time dependence of motility of bull sperm in Hank's balanced salt solution are presented.     

Effects of Dopamine D2-Like Receptor Antagonists on Light Responses of Ganglion Cells in Wild-Type and P23H Rat Retinas

In animal models of retinitis pigmentosa the dopaminergic system in the retina appears to be dysfunctional, which may contribute to the debilitated sight experienced by retinitis pigmentosa patients. Since dopamine D2-like receptors are known to modulate the activity of dopaminergic neurons, I examined the effects of dopamine D2-like receptor antagonists on the light responses of retinal ganglion cells (RGCs) in the P23H rat model of retinitis pigmentosa. Extracellular electrical recordings were made from RGCs in isolated transgenic P23H rat retinas and wild-type Sprague-Dawley rat retinas. Intensity-response curves to flashes of light were evaluated prior to and during bath application of a dopamine D2-like receptor antagonist. The dopamine D2/D3 receptor antagonists sulpiride and eticlopride and the D4 receptor antagonist L-745,870 increased light sensitivity of P23H rat RGCs but decreased light sensitivity in Sprague-Dawley rat RGCs. In addition, L-745,870, but not sulpiride or eticlopride, reduced the maximum peak responses of Sprague-Dawley rat RGCs. I describe for the first time ON-center RGCs in P23H rats that exhibit an abnormally long-latency (>200 ms) response to the onset of a small spot of light. Both sulpiride and eticlopride, but not L-745,870, reduced this ON response and brought out a short-latency OFF response, suggesting that these cells are in actuality OFF-center cells. Overall, the results show that the altered dopaminergic system in degenerate retinas contributes to the deteriorated light responses of RGCs.     

Angiogenesis in collagen I requires alpha2beta1 ligation of a GFP*GER sequence and possibly p38 MAPK activation and focal adhesion disassembly

Angiogenesis depends on proper collagen biosynthesis and cross-linking, and type I collagen is an ideal angiogenic scaffold, although its mechanism is unknown. We examined angiogenesis using an assay wherein confluent monolayers of human umbilical vein endothelial cells were overlain with collagen in a serum-free defined medium. Small spaces formed in the cell layer by 2 h, and cells formed net-like arrays by 6-8 h and capillary-like lumens by 24 h. Blocking of alpha2beta1, but not alpha1 or alpha(v)beta3 integrin function halted morphogenesis. We found that a triple-helical, homotrimeric peptide mimetic of a putative alpha2beta1 binding site: alpha1(I)496-507 GARGERGFP*GER (where single-letter amino acid nomenclature is used, P* = hydroxyproline) inhibited tube formation, whereas a peptide carrying another putative site: alpha1(I)127-138 GLP*GERGRP*GAP* or control peptides did not. A chemical inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), SB202190, blocked tube formation, and p38 MAPK activity was increased in collagen-treated cultures, whereas targeting MAPK kinase (MEK), focal adhesion kinase (FAK), or phosphatidylinositol 3-kinase (PI3K) had little effect. Collagen-treated cells had fewer focal adhesions and 3- to 5-fold less activated FAK. Thus capillary morphogenesis requires endothelial alpha2beta1 integrin engagement of a single type I collagen integrin-binding site, possibly signaling via p38 MAPK and focal adhesion disassembly/FAK inactivation.     

Increased flow precedes remote arteriolar dilations for some microapplied agonists

This study asks which occurs first in time for remote responses: a dilation or a remote change in flow. Arteriolar diameter (approximately 20 microm) and fluorescently labeled red blood cell (RBC) velocity were measured in the cremaster muscle of anesthetized (pentobarbital sodium, 70 mg/kg) hamsters (n = 51). Arterioles were locally stimulated for 60 s with micropipette-applied 10 microg/ml LM-609 (alpha(v)beta(3)-integrin agonist), 10(-3) M adenosine, or 10(-3) M 3-morpholinosydnonimine (SIN-1, nitric oxide donor) as remote response agonists or with 10(-3) M papaverine, which dilates only locally. Observations were made at a remote site 1,200 microm upstream. With LM-609 or adenosine, the RBC velocity increased first (within 5 s), and the remote dilation followed 5-7 s later. N-nitro-L-arginine (100 microM) blocked the LM-609 (100%) and adenosine (60%) remote dilations. SIN-1 induced a concurrent remote dilation and decrease in RBC velocity (approximately 10 s), suggesting the primary signal was to dilate. Papaverine had no remote effects. This study suggests that, although remote responses to some agonists are induced by primary signals to dilate, additionally, network changes in flow can stimulate extensive remote changes in diameter.