The mechanisms that regulate synapse formation and maintenance are incompletely understood. In particular, relatively few inhibitors of synapse formation have been identified. Receptor protein tyrosine phosphatase σ (RPTPσ), a transmembrane tyrosine phosphatase, is widely expressed by neurons in developing and mature mammalian brain, and functions as a receptor for chondroitin sulfate proteoglycans that inhibits axon regeneration following injury. Here we address RPTPσ function in the mature brain. We demonstrate increased axon collateral branching in the hippocampus of RPTPσ null mice during normal aging or following chemically-induced seizure, indicating that RPTPσ maintains neural circuitry by inhibiting axonal branching. Previous studies demonstrated a role for pre-synaptic RPTPσ promoting synaptic differentiation during development; however, subcellular fractionation revealed enrichment of RPTPσ in post-synaptic densities. We report that neurons lacking RPTPσ have an increased density of pre-synaptic varicosities in vitro and increased dendritic spine density and length in vivo. RPTPσ knockouts exhibit an increased frequency of miniature excitatory post-synaptic currents, and greater paired-pulse facilitation, consistent with increased synapse density but reduced synaptic efficiency. Furthermore, RPTPσ nulls exhibit reduced long-term potentiation and enhanced novel object recognition memory. We conclude that RPTPσ limits synapse number and regulates synapse structure and function in the mature CNS. © 2012 The Authors Journal of Neurochemistry © 2012 International Society for Neurochemistry.

The presence of a widespread endocannabinoid (eCB) system within the nervous system, including the retina, has been demonstrated in recent years. Expression patterns of the cannabinoid receptor type 1 (CB1R) and enzyme fatty acid amide hydrolase (FAAH) are available for rodents, but data for humans and monkeys are scarce. We, therefore, thoroughly examined the distribution pattern of CB1R and FAAH throughout the retina of the vervet monkey (Chlorocebus sabeus) using confocal microscopy. Our results demonstrate that CB1R and FAAH are expressed throughout the retina, from the foveal pit to the far periphery. CB1R and FAAH are present in the photoreceptor, outer plexiform, inner nuclear, inner plexiform, and retinal ganglion cell layers (PRL, OPL, INL, IPL, and RGCL, respectively). More specifically, in PRL, CB1R and FAAH are preferentially expressed in cones of the central retina. In OPL, these two components of the eCB system are concentrated not only in the cone pedicles but also in rod spherules with, however, a less intense staining pattern. Triple-labeling immunofluorescence revealed that both cone and rod bipolar cells express CB1R and FAAH. Heavy staining is detected in RGC somas and axons. Neither CB1R nor FAAH are found in the retinal glia, the Müller cells. These data indicate that the eCB system is present throughout the primate retina and is ideally positioned to modulate central and peripheral retinal functions.

The endocannabinoid (eCB) system is thought to participate in developmental processes in the CNS. The rodent retina represents a valuable model to study CNS development because it contains well-identified cell types with established developmental timelines. The distribution of cannabinoid receptor type 1 (CB1R) was recently revealed in the developing retina; however, the expression patterns of other elements of this system remain unknown. In this study, we investigated the expression pattern of the degradative enzyme fatty acid amide hydrolase (FAAH), a key regulator of the eCB system, in the rat retina during postnatal development. To identify the cells expressing the enzyme, co-stainings were carried out for FAAH and retinal cell type markers. FAAH was expressed at postnatal day (P) 1 in ganglion and cholinergic amacrine cells. In the course of development, it appeared in cones, horizontal, and bipolar cells. For most cell types (horizontal, cholinergic amacrine cells, and cone bipolar cells), FAAH was transiently expressed, suggesting an important redistribution of the enzyme during postnatal development and thus a potential role of the eCB system in developmental processes. Our results also indicated that, in the adult retina, FAAH is expressed in cones, rod bipolar cells, and some retinal ganglion cells. The presence of FAAH in adult animals supports the hypothesis that the eCB system is involved in retinal functions. Overall these results indicate that, as shown in other structures of the brain, the eCB system could play an instrumental role in the development and function of the retina.

Cannabinoid receptor type 1 (CB1R) participates in developmental processes in the central nervous system (CNS). The rodent retina represents an interesting and valuable model for studying CNS development, because it contains well-identified cell types with clearly established and distinct developmental timelines. Very little is known about the distribution or function of CB1R in the developing retina. In this study, we investigated the expression pattern of CB1R in the rat retina during all stages of postnatal development. Western blots were performed on retinal tissue at different time points between P1 and adulthood. In order to identify the cells expressing the receptor and the age at which this expression started, immunohistochemical co-staining was carried out for CB1R and markers of the different cell types comprising the retina. CB1R was already present at P1 in various cell types, i.e., ganglion, amacrine, horizontal, and mitotic cells. In the course of development, it appeared in cone photoreceptors and bipolar cells. For some cell types (bipolar, Müller, and some amacrine cells), CB1R was transiently expressed, suggesting a potential role of this receptor in developmental processes, such as migration, morphological changes, sub-identity acquisition, and patterned retinal spontaneous activity. Our results also indicated that CB1R is largely expressed in the adult retina (cone photoreceptors and horizontal, most amacrine, and retinal ganglion cells), and may therefore contribute to retinal functions. Overall these results indicate that, as shown in other structures of the brain, CB1R could play an instrumental role in the development and function of the retina.

Endocannabinoids (eCBs) are retrograde neurotransmitters that modulate the function of many types of synapses. The presence of eCBs, their CB1 receptor (CB1R), and metabolizing enzymes at embryonic and early postnatal periods have been linked to developmental processes such as neuronal proliferation, differentiation, and migration, axon guidance, and synaptogenesis. Here, we demonstrate the presence of a functional eCB system in the developing visual system and the role of CB1R during axon growth and retinothalamic development. Pharmacological treatment of retinal explants and primary cortical neuron cultures with ACEA, a selective CB1R agonist, induced a collapse of the growth cone (GC). Furthermore the application of AM251, a CB1R inverse agonist, to the neuronal cultures increased the surface area of GC. In vivo, intraocular injection of ACEA diminished retinal projection growth, while AM251 promoted growth and caused aberrant projections. In addition, compared with their wild-type littermates, CB1R-deficient adult mice revealed a lower level of eye-specific segregation of retinal projections in the dorsal lateral geniculate nucleus. Finally, we found that pharmacological modulation of CB1R affected the trafficking of Deleted in colorectal cancer (DCC) receptor to the plasma membrane in a PKA-dependent manner. Moreover, pharmacological inhibition or genetic inactivation of DCC abolished the CB1R-induced reorganization of the GC. Overall, these findings establish a mechanism by which the CB1R influences GC behavior and nervous system development in concerted action with DCC.

ABSTRACT Individuals with color vision deficiency have difficulties in differentiating colour in their daily activities. Through certain coloured filters, dichromats may report an improvement of their capacity to differentiate colors, but it is not known if this is achieved by means of a chromatic mechanism. The present study attempts to explain the mechanism by which a coloured filter can produce a beneficial effect in dichromatic visual perception and what is the nature of this improvement. Four male protanopes and four normal trichromats (two males and two females) participated in the present study. We evaluated the effect of the red filter (with a spectral transmittance similar to that of the X-Chrom filter) on the detection thresholds for monochromatic light stimuli from 420 to 660 nm in 20 nm steps. The increment spectral sensitivity functions were measured for 1.2 degrees diameter test flashes presented for 300 ms on a 60-cd m(-2) illuminant C background using an optical bench with a monochromator, for both filter and no filter conditions. The capacity to correctly name green, yellow and red for the monochromatic lights of 550, 575 and 625 nm presented for 300 ms on a 60 cd m(-2) illuminant C background screen was also evaluated with and without the red filter. The spectral sensitivity data suggest that, the use of a red filter improves the protanope's capacity to detect long wavelength light stimuli. The results on the colors naming procedure demonstrate that the red filter modifies colour perception in normal and protanope subjects. In normals, only the red color perception is preserved, and typical colour perception for the green and the yellow is lost. Without the filter, all the protanopes demonstrated a residual colour perception for red and green colours. Through the red filter only red colour perception remains. A red filter does not improve the protanopic red-green perception, but it does improve the ability of the protanope to detect long-wavelength light. This improvement seems to arise by means of the luminance mechanism.

New active particulate polymeric vectors based on branched polyester copolymers of hydroxy-acid and allyl glycidyl ether were developed to target drugs to the inflammatory endothelial cell surface. The hydroxyl and carboxyl derivatives of these polymers allow grafting of ligand molecules on the polyester backbones at different densities. A known potent nonselective selectin ligand was selected and synthesized using a new scheme. This synthesis allowed the grafting of the ligand to the polyester polymers, preserving its binding activity as assessed by docking simulations. Selectin expression on human umbilical cord vascular endothelial cells (HUVEC) was induced with the pro-inflammatory bacterial lipopolysaccharide (LPS) or with the nonselective inhibitor of nitric oxide synthase L-NAME. Strong adhesion of the ligand decorated nanoparticles was evidenced in vitro on activated HUVEC. Binding of nanoparticles bearing ligand molecules could be efficiently inhibited by prior incubation of cells with free ligand, demonstrating that adhesion of the nanoparticles is mediated by specific interaction between the ligand and the selectin receptors. These nanoparticles could be used for specific drug delivery to the activated vascular endothelium, suggesting their application in the treatment of diseases with an inflammatory component such as rheumatoid arthritis and cancer.

The netrin-1 receptor DCC is required for the formation of major axonal projections by embryonic cortical neurons, including the corpus callosum, hippocampal commissure, and cortico-thalamic tracts. The presentation of DCC by axonal growth cones is tightly regulated, but the mechanisms regulating DCC trafficking within neurons are not well understood. Here, we investigated the mechanisms regulating DCC recruitment to the plasma membrane of embryonic cortical neurons. In embryonic spinal commissural neurons, protein kinase A (PKA) activation recruits DCC to the plasma membrane and enhances axon chemoattraction to netrin-1. We demonstrate that PKA activation similarly recruits DCC and increases embryonic cortical neuron axon extension, which, like spinal commissural neurons, respond to netrin-1 as a chemoattractant. We then determined if depolarization might recruit DCC to the plasma membrane. Neither netrin-1 induced axon extension, nor levels of plasma membrane DCC, were altered by depolarizing embryonic spinal commissural neurons with elevated levels of KCl. In contrast, depolarizing embryonic cortical neurons increased the amount of plasma membrane DCC, including at the growth cone, and increased axon outgrowth evoked by netrin-1. Inhibition of PKA, phosphatidylinositol-3-kinase, protein kinase C, or exocytosis blocked the depolarization induced recruitment of DCC and suppressed axon outgrowth. Inhibiting protein synthesis did not affect DCC recruitment, nor were the distributions of trkB or NCAM influenced by depolarization, consistent with selective mobilization of DCC. These findings identify a role for membrane depolarization modulating the response of axons to netrin-1 by regulating DCC recruitment to the plasma membrane.

During development, retinal ganglion cells (RGCs) extend their axons toward their thalamic and mesencephalic targets. Their navigation is largely directed by guidance cues present in their environment. Since cAMP is an important second messenger that mediates the neural response to guidance molecules and its intracellular levels seem to decrease significantly following birth, we tested whether modulation of the cAMP/protein kinase A (PKA) pathway would affect the normal development of RGC axons. At postnatal day 1, hamsters received a unilateral intraocular injection of either 0.9% saline solution, 12 mM of the membrane-permeable cAMP analogue (dibutyryl cAMP; db-cAMP), or 10 microM of the PKA inhibitor KT5720. Intraocular elevation of cAMP significantly accelerated RGC axonal growth while inhibition of PKA activity decreased it. Moreover, when highly purified RGC cultures were treated with forskolin (an activator of adenylate cyclase) or cAMP analogues (db-cAMP and Sp-cAMP), neurite length, growth cone (GC) surface area and GC filopodia number were significantly increased. This indicates that intraocular elevation of cAMP acts directly on RGCs. Since these effects were prevented by PKA inhibitors, it demonstrates that cAMP also exerts its action via the PKA pathway. Taken together, these results suggest that the cAMP/PKA cascade is essential for the normal development of retinothalamic projections.

Therapeutic benefit in glial tumors is often limited due to low permeability of delivery systems across the blood-brain barrier (BBB), drug resistance, and poor penetration into the tumor tissue. In an attempt to overcome these hurdles, polyether-copolyester (PEPE) dendrimers were evaluated as drug carriers for the treatment of gliomas. Dendrimers were conjugated to d-glucosamine as the ligand for enhancing BBB permeability and tumor targeting. The efficacy of methotrexate (MTX)-loaded dendrimers was established against U87 MG and U 343 MGa cells. Permeability of rhodamine-labeled dendrimers and MTX-loaded dendrimers across the in vitro BBB model and their distribution into avascular human glioma tumor spheroids was also studied. Glucosylated dendrimers were found to be endocytosed in significantly higher amounts than nonglucosylated dendrimers by both the cell lines. IC 50 of MTX after loading in dendrimers was lower than that of the free MTX, suggesting that loading MTX in PEPE dendrimers increased its potency. Similar higher activity of MTX-loaded glucosylated and nonglucosylated dendrimers was found in the reduction of tumor spheroid size. These MTX-loaded dendrimers were able to kill even MTX-resistant cells highlighting their ability to overcome MTX resistance. In addition, the amount of MTX-transported across BBB was three to five times more after loading in the dendrimers. Glucosylation further increased the cumulative permeation of dendrimers across BBB and hence increased the amount of MTX available across it. Glucosylated dendrimers distributed through out the avascular tumor spheroids within 6 h, while nonglucosylated dendrimers could do so in 12 h. The results show that glucosamine can be used as an effective ligand not only for targeting glial tumors but also for enhanced permeability across BBB. Thus, glucosylated PEPE dendrimers can serve as potential delivery system for the treatment of gliomas.