C-kinase manipulations disrupt activity-driven retinotopic sharpening in regenerating goldfish retinotectal projection

Regenerating optic axons initially branch over a wide area in tectum to form a crude retinotopic map. The map is sharpened, and retinotopically appropriate synapses are stabilized via NMDA receptors that detect, via summation of EPSPs, the coincident activity of neighboring ganglion cells that make synapses onto common tectal cells. Sharpening shares a number of properties with long-term potentiation (LTP) in hippocampus. This study tested whether protein kinase C (PKC) activation is necessary for sharpening as it is for LTP. Intracular (IO) or intracranial (IC) injections of kinase inhibitors or activators were made every other day from 19 to 37 days postcrush (sensitive period), and the projections formed were later recorded. Retinotopic sharpening was prevented by IC injection of the following agents: (1) general kinase inhibitors sphingosine and H7 (100-200 μM in fluid above brain), (2) active but not inactive phorbols (TPA, 1 μM), and (3) calphostin C (1 μM), a specific and irreversible PKC inhibitor. The mature projection on the opposite tectum, however, when examined was not unsharpened. Lack of sharpening was reflected in multiunit fields at each tectal point that averaged 27°–30° versus 11° in Ringers and inactive phorbol control regenerates. Intraocular injections of either TPA (1 μM), or calphostin C (1 μM) also prevented sharpening (26° and 32° multiunit fields), suggesting action on PKC axonally transported to the presynaptic terminals. Calphostin C had no noticeable effect on the firing patterns of retinal ganglion cells. The endogenous activator of PKC, arachidonic acid (AA), disrupted sharpening at 20 μM or higher (IC injection, 32° multiunit fields), while a control fatty acid, elaidic acid, had no effect. Although AA at 5 μM showed no effect, and diacylglycerol at 5 μM exhibited only small effects, together they produced a large synergistic effect (32° multiunit fields). Such synergy mirrors the synergy in the activation of several isoforms of PKC. Actual concentrations in the extradural fluid around the brain were assayed via injections of ³H-AA. Levels fell about sixfold after a day and by an additional fivefold the second day before the next injection. The results confirm that activity-driven retinotopic sharpening is very sensitive to manipulations of kinases, especially PKC. © 1994 John Wiley & Sons, Inc.     

A new approach for on-line enrichment in electrophoresis of dilute protein solutions

A method is described for on-line enrichment/zone sharpening of a sample of negatively charged proteins (an analogous method for cationic proteins can be designed). The sample is applied on the top of a 5-mm thick layer of a neutral polyacrylamide gel which rests on another 5-mm thick, large-pore polyacrylamide gel which contains positively charged groups. The latter gel layer is attached to the neutral gel column, used for the electrophoretic separation of the proteins. When a voltage is applied the proteins start migrating and become electrostatically adsorbed at the top of the charged, large-pore gel layer (pH 5.4). With the upper electrode vessel filled with a buffer of a pH higher (pH 7.7) than that employed in the enrichment step and with a voltage between the electrodes, these enriched proteins are released (because the enrichment gel is non-charged at pH 7.7) with zone sharpening and migrate into the 5-cm long column (i.d. 5 mm) of a neutral, large-pore polyacrylamide gel for electrophoretic analysis. Upon the electrophoretic migration from the enrichment gel into the separation gel a second zone sharpening may occur, if the increase in pH from 5.4 to 7.7 in the separation gel is not close to momentary. By employing colored test proteins the efficiency of the enrichment step is visually illustrated by a picture. The principle of the concentration method described has been employed also in chromatographic experiments and can with appropriate modifications also be used in other electrophoretic methods, such as capillary electrophoresis.     

Role for cell adhesion and glycosyl (HNK-1 and oligomannoside) recognition in the sharpening of the regenerating retinotectal projection in goldfish

Cell-adhesion molecules (CAMs) are thought to play crucial roles in development and plasticity in the nervous system. This study tested for a role for cell adhesion and in particular, the recognition of two glycosyl epitopes (HNK-1 and oligomannoside) in the activity-driven sharpening of the retinotopic map formed by the regenerating retinal fibers of goldfish. HNK-1 is a prominent glycosyl epitope on many CAMs and extracellular matrix (ECM) molecules, including NCAM, L1, ependymin, and integrins, which have all been implicated in synaptic plasticity. To test for a role of HNK-1 in the sharpening process, we used osmotic minipumps to infuse HNK-1 antibodies for 7–21 days into the tectal ventricle starting at 18 days after optic nerve crush. Retinotopic maps recorded at 76–86 days postcrush showed a lack of sharpening similar to that seen previously with two antibodies to ependymin, an HNK-1–positive ECM component present in cerebrospinal fluid. The multiunit receptive fields at each point averaged 26° versus 11–12° in regenerates infused with control antibodies or Ringer's alone. The HNK-1 epitope also binds to the G2 domain of laminin to mediate neuron-ECM adhesion. To test for a role for laminin, a polyclonal antibody was similarly infused and also prevented sharpening to approximately the same degree. The results support a role for the HNK-1 epitope and laminin in retinotectal sharpening. The oligomannoside epitope (recognized by monoclonal antibody L3) on the CAM L1 interacts with NCAM on the same cell to promote stronger L1 homophilic interactions between cells. Both an L1-like molecule and NCAM are prominently reexpressed in the regenerating retinotectal system of fish. Infusion of oligomannosidic glycopeptides resulted in decreased sharpening, with multiunit receptive fields that averaged 22.7°. Infusions of mannose-poor glycopeptides less prominently disrupted sharpening, with average multiunit receptive fields of 18°. Thus, oligomannosidic glycans in particular may play a role in retinotopic sharpening. Blocking glycan-mediated interactions between CAMs and ECM molecules could decrease the extent of exploratory growth of retinal axon collaterals, preventing them from finding their retinotopic sites, or could interfere with L1 or NCAM and laminin binding at the synaptic densities preventing stabilization of retinotopically appropriate synapses. Together, these results support a prominent role for cell adhesion and glycan epitopes in visual synaptic plasticity. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 659–671, 1998     

Crystal structure of a novel non-Pfam protein PF2046 solved using low resolution B-factor sharpening and multi-crystal averaging methods

Sometimes crystals cannot diffract X-rays beyond 3.0 ? resolution due to the intrinsic flexibility associated with the protein. Low resolution diffraction data not only pose a challenge to structure determination, but also hamper interpretation of mechanistic details. Crystals of a 25.6 kDa non-Pfam, hypothetical protein, PF2046, diffracted X-rays to 3.38 ? resolution. A combination of Se-Met derived heavy atom positions with multiple cycles of B-factor sharpening, multi-crystal averaging, restrained refinement followed by manual inspection of electron density and model building resulted in a final model with a R value of 23.5 (R_free=24.7). The asymmetric unit was large and consisted of six molecules arranged as a homodimer of trimers. Analysis of the structure revealed the presence of a RNA binding domain suggesting a role for PF2046 in the processing of nucleic acids.     

Temperature and the Two-Point Threshold

Studies dating back to 1834 have shown that the temperature of objects contacting the skin can substantially intensify their apparent pressure on the skin. Later research demonstrated qualitatively that object temperature can also sharpen the spatial acuity of the skin as revealed by gap perception (two-point and two-edge thresholds). Pressure intensification and sharpening probably relate intimately. The present experiments sought to provide several more accurate and parametric extensions of thermal sharpening: (1) sharpening can improve tactile spatial acuity by as much as 60%, but the degree of sharpening is graded as a function of deviation of stimulator temperature from normal (neutral) skin temperature; (2) thermal sharpening seems to characterize the body surface since it takes place freely in forearm, forehead, and palm; local differences do, however, become apparent; (3) large thermal sharpening can even occur when one tip of the stimulator is warm, the other cold; and (4) thermal sharpening is easily captured by experiment and is basically the same in magnitude whether assessed by modern forced-choice procedure (controlled criterion) or by the more traditional procedures (uncontrolled criterion) used for more than a century before the advent of signal detection theory. Various arguments are put forth here and elsewhere to suggest that both thermal intensification of pressure sensation and thermal sharpening of gap perception result from direct thermal stimulation of mechanoreceptors and/or polymodal nociceptor networks; neither phenomenon yields readily to a “cognitive” interpretation.     

V - Channel magnification enabled by hybrid optimization algorithm: Enhancement of video super resolution

Although being a really active area of research, television super-resolution remains a difficult problem. Additionally, it is noted that the blur motion and computational crisis hinder the enhancement. As a result, the goal of this research is to present a brand-new smart SR framework for the camera shot. To create High Resolution (HR) videos, first frames in RGB format are converted to HSV and then the V-channel is enhanced. In order to create enriched video frames, a high - dimension grid with enhanced pixel intensity is then created. This paper introduces a particular progression to enable this: Motion estimation, Cubic Spline Interpolation, and Deblurring or Sharpening are the three methods. By carefully adjusting the parameters, the cubic spline interpolation is improved during operation. A brand-new hybrid technique dubbed Lion with Particle Swarm Velocity Update (LPSO-VU), which combines the principles of the Lion Algorithm (LA) and Particle Swarm Optimization (PSO) algorithms, is presented for this optimal tuning purpose. Finally, using the BRISQUE, SDME, and ESSIM metrics, the adequacy of the method is contrasted to other traditional models, and its superiority is demonstrated. Accordingly, the analysis shows that the suggested LPSO-VU model for video frame 1 is 16.6%, 25.56%, 26.2%, 26.2%, and 27.2% superior to the previous systems like PSO, GWO, WOA, ROA, MF-ROA, and LA, respectively, in terms of BRISQUE.     

Activity-driven sharpening of the retinotectal projection in goldfish: Development under stroboscopic illumination prevents sharpening

Blocking or synchronizing activity during regeneration of the retinotectal projection prevents both the sharpening of the retinotopic map recorded on tectum and the refinement of the structure of individual arbors within the plane of the map, and this refinement is triggered by N-methyl-d-aspartate (NMDA) receptors. We tested whether activity-driven refinement also occurs during development of the projection in larval and young adult goldfish. Shortly after hatching, larval goldfish were placed into tanks within light-tight chambers illuminated by a xenon strobe at 1 Hz for 14 h of each daily cycle. Fish were reared for 1.5–2 years, until large enough to record in our retinotectal mapping apparatus (6 cm length). Age- and size-matched controls had normal maps with multiunit receptive fields (MURFs) recorded at each tectal point of 10.8° (0.16 S.E.M., n = 5), whereas the strobe-reared fish had only roughly retino-topic maps with much enlarged MURFs averaging 26.7° (1.41 S.E.M., n = 5). This enlargement represents an abnormal convergence onto each tectal point, as the maps failed to sharpen during development. The arbors of individual retinal axons were stained with horseradish peroxidase (HRP) in larval fish and in adult strobereared and control fish. They were drawn with camera lucida from tectal whole mounts, and analyzed for spatial extent in the plane of the retinotopic map, order of branching, number of branch endings, depth of termination, and caliber of the parent axon. Arbors from larval fish (1–2 weeks) were small (approximately 50 × 40 μm) with less than 10 branches, occupied a single strata, and could not be separated into different classes by caliber of axon. The 87 arbors stained in control adult fish (6 cm long) were much like previously examined adult arbors, with those from fine, medium, and coarse axons averaging 115, 166, and 194 μm in extent, respectively, and having 17–24 branch endings. The 110 arbors from 12 strobe-reared fish were often abnormal. Although the fasciculation was normal, the extrafascicular routes were abnormal with reversing turns. The axons often had branches along their course, and these branches were scattered across a wider extent, rather than forming a distinct cluster. In contrast, neither the number of branches nor the depths of termination was significantly changed in any group. The coarse caliber arbors were most abnormal, being 64% longer and 30% wider than controls. The fine caliber arbors were also significantly larger by about 20%, but the medium caliber arbors were not enlarged. The enlarged arbors partially account for the unsharpened electrophysiological maps. Together the results show that during development, as well as during regeneration, the retinotectal map is subject to an activity-driven sharpening process. © 1993 John Wiley & Sons, Inc.     

Up-regulation of protein kinase C in regenerating optic nerve fibers of goldfish: Immunohistochemistry and kinase activity assay

Protein kinase C (PKC) activation has been associated with synaptic plasticity in many projections, and manipulating PKC in the retinotectal projection strongly affects the activity-driven sharpening of the retinotopic map. This study examined levels of PKC in the regenerating retinotectal projection via immunostaining and assay of activity. A polyclonal antibody to the conserved C2 (Ca²⁺ binding) domain of classical PKC isozymes (anti-panPKC) recognized a single band at 79–80 kD on Western blots of goldfish brain. It stained one class of retinal bipolar cells and the ganglion cells in normal retina, as shown previously. Strong staining was not present in the optic fiber layer of retina or in optic nerve, optic tract, or terminal zone in tectum, with the exception of a single fascicle of optic nerve fibers that by their location and by L1 (E587) staining were identified as those arising from newly added ganglion cells at the retinal margin. Normal tectal sections showed dark staining of a subclass of type XIV neuron with somas at the top of the periventricular layer and an apical dendrite ascending to stratum opticum. In regenerating retina, swollen ganglion cells stained darkly and stained axons were seen in the optic fiber layer. In regenerating optic nerve (2–11 weeks postcrush), all fascicles of optic fibers stained darkly for both PKC and L1(E587). At 5 weeks postcrush, PKC staining could also be seen in the medial and lateral optic tracts and stratum opticum at the front half of the tectum and very lightly over the terminal zones. PKC activity was measured in homogenized tissues dissected from a series of fish with unilateral nerve crush from 1 to 5 weeks previously. Activity levels stimulated by phorbols and Ca²⁺ were measured by phosphorylation of a specific peptide and referred to levels measured in the opposite control side. Regeneration did not increase overall PKC activity in retina or tectum, but in optic nerve there was an 80% rise after the first week. The increased activity verifies that the increased staining in nerve represented an up-regulation of functional PKC during nerve regeneration. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 315–324, 1998     

Presynaptic protein kinase C controls maturation and branch dynamics of developing retinotectal arbors: Possible role in activity‐driven sharpening

Visual activity refines developing retinotectal maps and shapes individual retinal arbors via an NMDA receptor‐dependent mechanism. As retinal axons grow into tectum, they slow markedly and emit many transient side branches behind the tip, assuming a “bottlebrush” morphology. Some branches are stabilized and branch further, giving rise to a compact arbor. The dynamic rate of branch addition and deletion is increased twofold when MK801 is used to block NMDA receptors, as if this prevents release of a stabilizing signal such as arachidonic acid (AA) from the postsynaptic neuron. In optic tract, AA mediates NCAM and L1 stimulation of axon growth by activating presynaptic protein kinase C (PKC) to phosphorylate GAP‐43 and stabilize F‐actin, and, if present in tectum, this growth control pathway could be modulated by postsynaptic activation. To test for the effects on arbor morphology of blocking PKC or AA release, we examined DiO‐labeled retinal axons of larval zebrafish with time‐lapse videomicroscopy. Bath application of the selective PKC inhibitor bisindolylmaleimide from 2 or 3 days onward doubled the rate at which side branches were added and deleted, as seen with MK801, and also prevented maturation of the arbor so that it retained a “bottlebrush” morphology. In order to selectively block the PKC being transported to retinal terminals, we injected the irreversible inhibitor calphostin C into the eye from which the ganglion cells were labeled, and this produced both effects seen with bath application. In contrast, there were no effects of control injections, which included Ringers into the same eye and the same dose into the opposite eye (actually much closer to the tectum of interest), to rule out the possibility that the inhibitor leaked from the eye to act on tectal cells. For comparison, we examined arbors treated with the NMDA blocker MK801 at half‐hour time‐lapse intervals, and detected the twofold rise in rates of branch addition and deletion previously reported in Xenopus larvae, but not the structural effect seen with the PKC inhibitors. In addition, we could produce both effects seen with PKC inhibitors by using RHC80267 to block AA release from DAG lipase, indicating that AA is the main drive for PKC activation. Thus, the results show a distinct role of AA and presynaptic PKC in both maturation of arbor structure and in the dynamic control of branching. The effects on branch dynamics were present regardless of the level of maturity of arbor structure. The fact that they mimicked those of MK801 suggests that presynaptic PKC may be involved in the NMDA receptor‐driven stabilization of developing retinal arbors. © 2003 Wiley Periodicals, Inc. J Neurobiol 58: 328–340, 2004