Integrins in human T cell function: Transdominant regulation, conformational constraints, and intracellular effectors

Integrin adhesion molecules have both positive and negative potential in the regulation of peripheral blood T cell (PB T cell) activation, yet their mechanism of action in the mediation of human T lymphocyte function remains largely undefined. The goals of this study then were to elucidate integrin signaling mechanisms in PB T cells.^ By ligating $\beta$1 integrins with mAb 18D3, it was demonstrated that costimulation of PB T cell proliferation induced by coimmobilizing antibodies specific for $\beta$1, $\beta$2, and $\beta$7 integrin subfamilies in conjunction with the anti-CD3 mAb OKT3 was inhibited. Costimulation of T cell proliferation induced by non-integrins CD4, CD26, CD28, CD44, CD45RA, or CD45RO was unaffected. Inhibition of costimulation correlated with diminished IL-2 production. In his manner, $\beta$1 integrins could regulate heterologous integrins of the $\beta$2 and $\beta$7 subfamilies in a transdominant fashion. It was also demonstrated that integrin costimulation of T cell activation was acutely sensitive to the structural conformation of $\beta$1 integrins. Using the cyclic hexapeptide CWLDVC (TBC772, which is based on the $\alpha4\beta1$ integrin binding site in fibronectin) in soluble form, it was shown that integrins locked into a conformation displaying a neo-epitope called the ligand induced binding site (LIBS) recognized by mAb 15/7 were inhibited from sending mitogenic signals to T cells. When BSA-conjugated TBC772 was coimmobilized with anti-CD3 mAb OKT3, costimulation of proliferation occurred. This suggested that temporally uncoupling integrin receptor occupancy from receptor crosslinking inhibited $\beta$1 integrin signaling mechanisms. When subsets of PB T cells were examined to determine those initially activated by integrins within 6 hours of activation, costimulation induced intracellular accumulation of IL-2 predominantly in the CD4$\sp+$ and CD45RO$\sp+$ T cell subsets. This was similar to a number of PB T cell costimulatory molecules including CD26, CD43, CD44. Only CD28 costimulated IL-2 production from both CD45RA$\sp+$ and CD45RO$\sp+$ subpopulations.^ The GTPase Rho has been implicated in regulating integrin mediated stress fiber formation and anchorage dependent growth in fibroblasts, so studies were initiated to determine if Rho played a role in integrin dependent T cell function. In order to perform this, a technique based on scrape-loading was developed to incorporate macromolecules into PB T cells that maintained their functional activity. With this technique, C3 exoenzyme from Clostridium botulinum was incorporated into PB T cells. C3 ADP-ribosylates Rho proteins on Asn$\sp{41},$ which is in close proximity to the Rho effector domain, rendering it inactive. It was demonstrated that functional Rho is not required for basal or upregulated PB T cell adhesion to $\beta$1 integrin substrates, however PB T cell homotypic aggregation induced by PMA, which is an event mediated predominantly by the integrin $\rm\alpha L\beta2,$ was delayed. PB T cells lacking Rho function displayed altered cell morphology on $\beta$1 integrin ligands, producing stellate, dendritic-like pseudopodia. Rho activity was also found to be required for integrin dependent costimulation of proliferation. When intracellular accumulation of IL-2 was measured, inactivation of Rho prevented both integrin and CD28 costimulatory activity. Rho was identified to lie upstream of signals mediating PKC activation and Ca$\sp{++}$ fluxes, as PMA and ionomycin activation of PB T cells was unaffected by the inactivation of Rho. ^

Signals underlying the induction and expression of long-term, injury-related plasticity in sensory neurons of Aplysia

An important goal in the study of long-term memory is to understand the signals that induce and maintain the underlying neural alterations. In Aplysia, long-term sensitization of defensive reflexes has been examined in depth as a simple model of memory. Extensive studies of sensory neurons (SNs) in Aplysia have led to a cellular and molecular model of long-term memory that has greatly influenced memory research. According to this model, induction of long-term memory in Aplysia depends upon serotonin (5-HT) release and subsequent activation of the cAMP-PKA pathway in SNs. The evidence supporting this model mainly came from studies of long-term synaptic facilitation (LTF) using dissociated (and therefore axotomized) cells growing in culture. However, studies in more intact preparations have produced complex and discrepant results. Because these SNs function as nociceptors, and display similar alterations (long-term hyperexcitability [LTH], LTF, and growth) in models of memory and nerve injury, this study examined the roles of 5-HT and the cAMP-PKA pathway in the induction and expression of long-term, injury-related LTH and LTF in Aplysia SNs. ^ The results presented here suggest that 5-HT is not a primary signal for inducing LTH (and perhaps LTF) in Aplysia SNs. Prolonged treatment with 5-HT failed to induce LTH of Aplysia SNs in either ganglia or dissociated-cell preparations. Treatment with a 5-HT antagonist, methiothepin, during noxious nerve stimulation failed to reduce 24 hr LTH. Furthermore, while 5-HT can induce LTF of SN synapses, this LTF appears to be an indirect effect of 5-HT on other cells. When neural activity was suppressed by elevating divalent cations or by using tetrodotoxin (TTX), 5-HT failed to induce LTF. Unlike LTF, LTH of the SNs could not be produced, even when 5-HT treatment occurred in normal artificial sea water (ASW), suggesting that LTH and LTF are likely to depend on different signals for induction. However, methiothepin reduced the later expression of LTH induced by nerve stimulation, suggesting that 5-HT contributes to the maintenance of LTH in Aplysia SNs.n of somata from the ganglion (which axotomizes SNs) or crushing peripheral n. ^ In summary, this study found that 5-HT and the cAMP-PKA pathway are not involved in the induction of long-term, injury-related LTH of Aplysia SNs, but persistent release of 5-HT and persistent PKA activity contribute to the maintenance of LTH induced by injury. (Abstract shortened by UMI.)^

Integrin signaling in the regulation of lymphocyte morphology

In normal lymphocytes an “inside-out” signal up-regulating integrin adhesion is followed by a ligand mediated “outside-in” signal for cell spreading. Although PKC mediates both events, distinct roles were found for different PLCs. The inhibition of phosphatidylinositol specific PLC decreased both cell adhesion and spreading on fibronectin in T cell receptor/CD28 activated peripheral blood T cells. However, inhibition of phosphatidylcholine specific PLC only blocked cell spreading and did not affect adhesion, indicating that “inside-out” signaling for the integrin α4β1 proceeds through phosphatidylinositol specific PLC and PKC, while the “outside-in” signal utilizes phosphatidylcholine specific PLC and PKC. Furthermore, β1 integrin chain mediated morphological changes in the T lymphocytic cell line HPB-ALL directly paralleled PKA activation, treatment of these cells with an inhibitory anti-β1 antibody blocked PKA activation and cell spreading, and this inhibition could be overcome by activating adenylate cyclase. Furthermore, inhibition of PKA was found to decrease the overall strength of cell adhesion or cellular avidity without affecting individual receptor affinity for soluble ligand. ^ When HPB-ALL cells interact with immobilized FN, two separate morphological phenotypes can be induced. Some cells flattened their cell body into a triangular shape and begin to migrate, while others extended a pseudopod from their stationary cell body. This second morphology recapitulates the shape changes observed during transendothelial migration. During these morphological changes, α4β1 integrins are internalized into endocytic vesicles that ultimately accumulate at the juncture between the cell body and an extending pseudopod. From this juncture, they are rapidly transported down the length of the pseudopod to its most distal end. ^ In addition to an accumulation of integrin containing vesicles, the pseudopod base was found to have increased amounts of the small GTPase RhoA and active PKA. The inhibition of PKA or RhoA resulted in lymphocytes with similar aberrant stellate morphologies. Furthermore, inhibition of PKA blocked the α4β1 mediated phosphorylation of RhoA. The co-localization of active PKA, RhoA and integrin containing endocytic vesicles indicates that integrin triggering can cause the rapid redistribution and activation of key signaling intermediates and raises the possibility that regulation of lymphocyte morphology by PKA and RhoA is through adhesion receptor recycling. ^

Fatal contact shot to the chest caused by the gas jet from a muzzle-loading pistol discharging only black powder and no bullet: case study and experimental simulation of the wounding effect.

In modern medico-legal literature, only a small number of publications deal with fatal injuries from black powder guns. Most of them focus on the morphological features such as intense soot soiling, blast tattooing and burn effects in close-range shots or describe the wound ballistics of spherical lead bullets. Another kind of "unusual" and potentially lethal weapons are handguns destined for firing only blank cartridges such as starter and alarm pistols. The dangerousness of these guns is restricted to very close and contact range shots and results from the gas jet produced by the deflagration of the propellant. The present paper reports on a suicide committed with a muzzle-loading percussion pistol cal. 45. An unusually large stellate entrance wound was located in the precordial region, accompanied by an imprint mark from the ramrod and a faint greenish discoloration (apparently due to the formation of sulfhemoglobin). Autopsy revealed an oversized powder cavity, multiple fractures of the anterior thoracic wall as well as ruptures of the heart, the aorta, the left hepatic lobe and the diaphragm. In total, the zone of mechanical destruction had a diameter of approx. 15 cm. As there was no exit wound and no bullet lodged in the body, the injury was caused exclusively by the inrushing combustion gases of the propellant (black powder) comparable with the gas jet of a blank cartridge gun. In contact shots to ballistic gelatine using the suicide's pistol loaded with black powder but no projectile, the formation of a nearly spherical cavity could be demonstrated by means of a high-speed camera. The extent of the temporary cavity after firing with 5 g of black powder roughly corresponded to the zone of destruction found in the suicide's body.

Ubiquitylation of voltage-gated sodium channels.

Ion channel proteins are regulated by different types of posttranslational modifications. The focus of this review is the regulation of voltage-gated sodium channels (Navs) upon their ubiquitylation. The amiloride-sensitive epithelial sodium channel (ENaC) was the first ion channel shown to be regulated upon ubiquitylation. This modification results from the binding of ubiquitin ligase from the Nedd4 family to a protein-protein interaction domain, known as the PY motif, in the ENaC subunits. Many of the Navs have similar PY motifs, which have been demonstrated to be targets of Nedd4-dependent ubiquitylation, tagging them for internalization from the cell surface. The role of Nedd4-dependent regulation of the Nav membrane density in physiology and disease remains poorly understood. Two recent studies have provided evidence that Nedd4-2 is downregulated in dorsal root ganglion (DRG) neurons in both rat and mouse models of nerve injury-induced neuropathic pain. Using two different mouse models, one with a specific knockout of Nedd4-2 in sensory neurons and another where Nedd4-2 was overexpressed with the use of viral vectors, it was demonstrated that the neuropathy-linked neuronal hyperexcitability was the result of Nav1.7 and Nav1.8 overexpression due to Nedd4-2 downregulation. These studies provided the first in vivo evidence of the role of Nedd4-2-dependent regulation of Nav channels in a disease state. This ubiquitylation pathway may be involved in the development of symptoms and diseases linked to Nav-dependent hyperexcitability, such as pain, cardiac arrhythmias, epilepsy, migraine, and myotonias.

Retinal layer measurements after successful macula-off retinal detachment repair using optical coherence tomography

PURPOSE Optical coherence tomography (OCT) was used to analyze the thickness of various retinal layers of patients following successful macula-off retinal detachment (RD) repair. METHODS Optical coherence tomography scans of patients after successful macula-off RD repair were reanalyzed with a subsegmentation algorithm to measure various retinal layers. Regression analysis was performed to correlate time after surgery with changes in layer thickness. In addition, patients were divided in two groups. Group 1 had a follow-up period after surgery of up to 7 weeks (range, 21-49 days). In group 2, the follow-up period was >8 weeks (range, 60-438 days). Findings were compared to a group of age-matched healthy controls. RESULTS Correlation analysis showed a significant positive correlation between inner nuclear-outer plexiform layer (INL-OPL) thickness and time after surgery (P=0.0212; r2=0.1551). Similar results were found for the ellipsoid zone-retinal pigment epithelium complex (EZ-RPE) thickness (P=0.005; r2=0.2215). Ganglion cell-inner plexiform layer thickness (GCL-IPL) was negatively correlated with time after surgery (P=0.0064; r2=0.2101). For group comparison, the retinal nerve fiber layer in both groups was thicker compared to controls. The GCL-IPL showed significant thinning in group 2. The outer nuclear layer was significantly thinner in groups 1 and 2 compared to controls. The EZ-RPE complex was significantly thinner in groups 1 and 2 compared to controls. In addition, values in group 1 were significantly thinner than in group 2. CONCLUSIONS Optical coherence tomography retinal layer thickness measurements after successful macular-off RD repair revealed time-dependent thickness changes. Inner nuclear-outer plexiform layer thickness and EZ-RPE thickness was positively correlated with time after surgery. Ganglion cell-inner plexiform layer thickness was negatively correlated with time after surgery.

Clinical manifestation of Fuchs uveitis syndrome in childhood

PURPOSE The aim of this study was to describe clinical signs and complications of Fuchs uveitis syndrome (FUS) with onset in childhood. METHODS Ophthalmologic findings and complications in patients with FUS becoming manifest before the age of 16 years were analyzed in a retrospective study at a tertiary referral uveitis center. Inclusion criteria were the presence of pathognomonic FUS findings at any time point and exclusion of any systemic immune-mediated or infectious disease. RESULTS A total of 23 patients (male = 16, female = 7) with juvenile FUS (unilateral n = 20, bilateral n = 3 patients) were included in the study. Mean ages at uveitis and FUS diagnosis were 12.0 ± 4.2 and 22.7 ± 10.7 years, respectively. In six patients, inflammation was noted at age ≤ 7 years. The following inflammatory signs were observed in a total of 26 eyes: ≤ 1+ anterior chamber cell grade (n = 26), vitreous cells (n = 24), fine keratic precipitates (KPs; n = 23), stellate KPs (n = 11), mutton-fat KPs (n = 23), diffuse (n = 24) or inferior (n = 8) distribution of KPs, Koeppe nodules (n = 10), and iris heterochromia (n = 14). A representative subgroup of patients (n = 5) is shown who presented with non-specific clinical signs in the beginning and in whom typical FUS signs became manifest only at a later stage. Secondary complications such as cataract (n = 19), ocular hypertension (n = 3), or glaucomatous disc damage (n = 2) were found after a mean uveitis duration of 11.6, 19.5, and 20.3 years, respectively. CONCLUSION FUS may begin in early childhood, and the characteristic findings may not be present at onset of disease. The diagnosis is often delayed for years, occasionally with the consequence of overtreatment with anti-inflammatory drugs.

MeV ion beam lithography of biocompatible halogenated Parylenes using aperture masks

Parylenes are poly(p-xylylene) polymers that are widely used as moisture barriers and in biomedicine because of their good biocompatibility. We have investigated MeV ion beam lithography using 16O+ ions for writing defined patterns in Parylene-C, which is evaluated as a coating material for the Cochlear Implant (CI) electrode array, a neuroprosthesis to treat some forms of deafness. Parylene-C and -F on silicon and glass substrates as well as 50 μm thick PTFE were irradiated to different fluences (1×1013-1×10161×1013-1×1016 1 MeV 16O+ ions cm−2) through aperture masks under high vacuum and a low pressure (<10−3 mbar) oxygen atmosphere. Biocompatibility of the irradiated and unirradiated surfaces was tested by cell-counting to determine the proliferation of murine spiral ganglion cells. The results reveal that an oxygen ion beam can be used to pattern Parylene-C and -F without using a liquid solvent developer in a similar manner to PTFE but with a ∼25× smaller removal rate. Biocompatibility tests showed no difference in cell adhesion between irradiated and unirradiated areas or ion fluence dependence. Coating the Parylene surface with an adhesion-promoting protein mixture had a much greater effect on cell proliferation.

Variable phenotypic expressivity in inbred retinal degeneration mouse lines: A comparative study of C3H/HeOu and FVB/N rd1 mice

PURPOSE Recent advances in optogenetics and gene therapy have led to promising new treatment strategies for blindness caused by retinal photoreceptor loss. Preclinical studies often rely on the retinal degeneration 1 (rd1 or Pde6b(rd1)) retinitis pigmentosa (RP) mouse model. The rd1 founder mutation is present in more than 100 actively used mouse lines. Since secondary genetic traits are well-known to modify the phenotypic progression of photoreceptor degeneration in animal models and human patients with RP, negligence of the genetic background in the rd1 mouse model is unwarranted. Moreover, the success of various potential therapies, including optogenetic gene therapy and prosthetic implants, depends on the progress of retinal degeneration, which might differ between rd1 mice. To examine the prospect of phenotypic expressivity in the rd1 mouse model, we compared the progress of retinal degeneration in two common rd1 lines, C3H/HeOu and FVB/N. METHODS We followed retinal degeneration over 24 weeks in FVB/N, C3H/HeOu, and congenic Pde6b(+) seeing mouse lines, using a range of experimental techniques including extracellular recordings from retinal ganglion cells, PCR quantification of cone opsin and Pde6b transcripts, in vivo flash electroretinogram (ERG), and behavioral optokinetic reflex (OKR) recordings. RESULTS We demonstrated a substantial difference in the speed of retinal degeneration and accompanying loss of visual function between the two rd1 lines. Photoreceptor degeneration and loss of vision were faster with an earlier onset in the FVB/N mice compared to C3H/HeOu mice, whereas the performance of the Pde6b(+) mice did not differ significantly in any of the tests. By postnatal week 4, the FVB/N mice expressed significantly less cone opsin and Pde6b mRNA and had neither ERG nor OKR responses. At 12 weeks of age, the retinal ganglion cells of the FVB/N mice had lost all light responses. In contrast, 4-week-old C3H/HeOu mice still had ERG and OKR responses, and we still recorded light responses from C3H/HeOu retinal ganglion cells until the age of 24 weeks. These results show that genetic background plays an important role in the rd1 mouse pathology. CONCLUSIONS Analogous to human RP, the mouse genetic background strongly influences the rd1 phenotype. Thus, different rd1 mouse lines may follow different timelines of retinal degeneration, making exact knowledge of genetic background imperative in all studies that use rd1 models.

Differentiation of Diabetic Macular Edema From Pseudophakic Cystoid Macular Edema by Spectral-Domain Optical Coherence Tomography.

PURPOSE: To differentiate diabetic macular edema (DME) from pseudophakic cystoid macular edema (PCME) based solely on spectral-domain optical coherence tomography (SD-OCT). METHODS: This cross-sectional study included 134 participants: 49 with PCME, 60 with DME, and 25 with diabetic retinopathy (DR) and ME after cataract surgery. First, two unmasked experts classified the 25 DR patients after cataract surgery as either DME, PCME, or mixed-pattern based on SD-OCT and color-fundus photography. Then all 134 patients were divided into two datasets and graded by two masked readers according to a standardized reading-protocol. Accuracy of the masked readers to differentiate the diseases based on SD-OCT parameters was tested. Parallel to the masked readers, a computer-based algorithm was established using support vector machine (SVM) classifiers to automatically differentiate disease entities. RESULTS: The masked readers assigned 92.5% SD-OCT images to the correct clinical diagnose. The classifier-accuracy trained and tested on dataset 1 was 95.8%. The classifier-accuracy trained on dataset 1 and tested on dataset 2 to differentiate PCME from DME was 90.2%. The classifier-accuracy trained and tested on dataset 2 to differentiate all three diseases was 85.5%. In particular, higher central-retinal thickness/retinal-volume ratio, absence of an epiretinal-membrane, and solely inner nuclear layer (INL)-cysts indicated PCME, whereas higher outer nuclear layer (ONL)/INL ratio, the absence of subretinal fluid, presence of hard exudates, microaneurysms, and ganglion cell layer and/or retinal nerve fiber layer cysts strongly favored DME in this model. CONCLUSIONS: Based on the evaluation of SD-OCT, PCME can be differentiated from DME by masked reader evaluation, and by automated analysis, even in DR patients with ME after cataract surgery. The automated classifier may help to independently differentiate these two disease entities and is made publicly available.

Treatment of optic neuritis with erythropoietin (TONE): a randomised, double-blind, placebo-controlled trial-study protocol.

INTRODUCTION Optic neuritis leads to degeneration of retinal ganglion cells whose axons form the optic nerve. The standard treatment is a methylprednisolone pulse therapy. This treatment slightly shortens the time of recovery but does not prevent neurodegeneration and persistent visual impairment. In a phase II trial performed in preparation of this study, we have shown that erythropoietin protects global retinal nerve fibre layer thickness (RNFLT-G) in acute optic neuritis; however, the preparatory trial was not powered to show effects on visual function. METHODS AND ANALYSIS Treatment of Optic Neuritis with Erythropoietin (TONE) is a national, randomised, double-blind, placebo-controlled, multicentre trial with two parallel arms. The primary objective is to determine the efficacy of erythropoietin compared to placebo given add-on to methylprednisolone as assessed by measurements of RNFLT-G and low-contrast visual acuity in the affected eye 6 months after randomisation. Inclusion criteria are a first episode of optic neuritis with decreased visual acuity to ≤0.5 (decimal system) and an onset of symptoms within 10 days prior to inclusion. The most important exclusion criteria are history of optic neuritis or multiple sclerosis or any ocular disease (affected or non-affected eye), significant hyperopia, myopia or astigmatism, elevated blood pressure, thrombotic events or malignancy. After randomisation, patients either receive 33 000 international units human recombinant erythropoietin intravenously for 3 consecutive days or placebo (0.9% saline) administered intravenously. With an estimated power of 80%, the calculated sample size is 100 patients. The trial started in September 2014 with a planned recruitment period of 30 months. ETHICS AND DISSEMINATION TONE has been approved by the Central Ethics Commission in Freiburg (194/14) and the German Federal Institute for Drugs and Medical Devices (61-3910-4039831). It complies with the Declaration of Helsinki, local laws and ICH-GCP. TRIAL REGISTRATION NUMBER NCT01962571.

The function of math5 and myocilin in mammalian eye development and disease

Complex molecular events underlie vertebrate eye development and disease. The eye is composed of two major tissue types: the anterior and posterior segments. During development, the retinal progenitor cells differentiate into six neuronal and one non-neuronal cell types. These cell types later organize into the distinct laminar structure of the mature retina which occupies the posterior segment. In the developed anterior segment, both the ciliary body and trabecular meshwork regulate intraocular pressure created by the aqueous humor. The disruption in intraocular pressure can lead to a blinding condition called glaucoma. To characterize molecular mechanisms governing retinal development and glaucoma, two separate mouse knockout lines carrying mutations in math5 and myocilin were subjected to a series of in vivo analyses. ^ Math5 is a murine homologue of Drosophila atonal , a bHLH proneural gene essential for the formation of photoreceptor cells. The expression of math5 coincides with the onset of retinal ganglion cell differentiation. The targeted deletion of mouse math5 revealed that a null mutation inhibits the formation of a majority of the retinal ganglion cells. The mutation also interferes with the normal development of other retinal cell types such as amacrine, bipolar and photoreceptor cells. These results suggest that math5 is a proneural gene responsible for differentiation of retinal ganglion cells and may also have a role in normal development of other neuronal cell types within the retina. ^ Myocilin has two unique protein coding regions bearing homology to non-muscle myosin of Dictyostelium discoideum and to olfactomedin, an extracellular matrix molecule first described in the olfactory epithelium of the bullfrog. Recently, autosomal dominant forms of myocilin mutations have been found in individuals with primary open-angle glaucoma. The genetic linkage to glaucoma suggests a role of myocilin in normal intraocular pressure and ocular function. However, the analysis of mice heterozygous and homozygous for a targeted null mutation in myocilin indicates that it is dispensable for normal intraocular pressure or ocular function. Additionally, the lack of a discernable phenotype in both heterozygous and null mice suggests that haploinsufficiency is not a critical mechanism for MYOC-associated glaucoma in humans. Instead, disease-causing mutations likely act by gain of function. ^ In summary, these studies provide novel insights into the embryonic development of the vertebrate retina, and also begin to uncover the molecular mechanisms responsible for the pathogenesis of glaucoma. ^

Function of histaminergic retinopetal axons in rat and primate retinas

Mammalian retinas receive input from histaminergic neurons in the posterior hypothalamus. These neurons are most active during the waking state of the animal, but their role in retinal information processing is not known. To determine the function of these retinopetal axons, their targets in the rat and monkey retina were identified. Using antibodies to three histamine receptors, HR1, HR2, and HR3, the immunolabeling was analyzed by confocal and electron microscopy. These experiments showed that mammalian retinas possess histamine receptors. In macaques and baboons, diurnal species, HR3 receptors were found at the apex of ON-bipolar cell dendrites in cone pedicles and rod spherules, sclerad to the other neurotransmitter receptors that have been localized there. In addition, HR1 histamine receptors were localized to large puncta in the inner plexiform layer, a subset of ganglion cells and retinal blood vessels. In rats, a nocturnal species, the localization of histamine receptors in the retina was markedly different. Most HR1 receptors were localized to dopaminergic amacrine cells and on elements in the rod spherule. To determine how histaminergic retinopetal axons contribute to retinal information processing, responses of retinal ganglion cells to histamine were analyzed. The effects of histamine on the maintained and light-evoked activity of retinal ganglion cells were analyzed. In monkeys, histamine and the HR3 agonist, methylhistamine, increased or decreased the maintained activity of most ganglion cells, but a few did not respond. The responses of a subset of ganglion cells to light stimuli were decreased by histamine, a finding suggesting that histaminergic retinopetal axons contribute to light adaptation during the day. In rats, histamine nearly always increased the maintained activity and produced both increases and decreases in the light responses. The effects of histamine on maintained activity of ganglion cells in the rat can be partially attributed to HR1-mediated changes in the activity of dopaminergic amacrine cells, at night. Together, these experiments provide the first indication of the function of retinopetal axons in mammalian retinas. ^

Diversity of neuronal connexins in the mammalian retina

Many neurons in the mammalian retina are electrically coupled by intercellular channels or gap junctions, which are assembled from a family of proteins called connexins. Numerous studies indicate that gap junctions differ in properties such as conductance and tracer permeability. For example, A-type horizontal cell gap junctions are permeable to Lucifer Yellow, but B-type horizontal cell gap junctions are not. This suggests the two cell types express different connexins. My hypothesis is that multiple neuronal connexins are expressed in the mammalian retina in a cell type specific manner. Immunohistochemical techniques and confocal microscopy were used to localize certain connexins within well-defined neuronal circuits. The results of this study can be summarized as follows: AII amacrine cells, which receive direct input from rod bipolar cells, are well-coupled to neighboring AIIs. In addition, AII amacrine cells also form gap junctions with ON cone bipolar cells. This is a complex heterocellular network. In both rabbit and primate retina, connexin36 occurs at dendritic crossings in the AII matrix as well as between AIIs and ON cone bipolar cells. Coupling in the AII network is thought to reduce noise in the rod pathway while AII/bipolar gap junctions are required for the transmission of rod signals to ON ganglion cells. In the outer plexiform layer, connexin36 forms gap junctions between cones and between rods and cones via cone telodendria. Cone to cone coupling is thought to reduce noise and is partly color selective. Rod to cone coupling forms an alternative rod pathway thought to operate at intermediate light intensity. A-type horizontal cells in the rabbit retina are strongly coupled via massive low resistance gap junctions composed from Cx50. Coupling dramatically extends the receptive field of horizontal cells and the modulation of coupling is thought to change the strength of the feedback signal from horizontal cells to cones. Finally, there are other coupled networks, such as B-type horizontal cells and S1/S2 amacrine cells, which do not use either connexin36 or Cx50. These results confirm the hypothesis that multiple neuronal connexins are expressed in the mammalian retina and these connexins are localized to particular retinal circuits. ^

Mouse retinal development: Role of cell adhesion and mechanism of gene regulation

Cell differentiation and pattern formation are fundamental processes in animal development that are under intense investigation. The mouse retina is a good model to study these processes because it has seven distinct cell types, and three well-laminated nuclear layers that form during embryonic and postnatal life. β-catenin functions as both the nuclear effector for the canonical Wnt pathway and a cell adhesion molecule, and is required for the development of various organs. To study the function of β-catenin in retinal development, I used a Cre-loxP system to conditionally ablate β-catenin in the developing retina. Deletion of β-catenin led to disrupted laminar structure but did not affect the differentiation of any of the seven cell types. Eliminating β-catenin did not reduce progenitor cell proliferation, although enhanced apoptosis was observed. Further analysis showed that disruption of cell adhesion was the major cause of the observed patterning defects. Overexpression of β-catenin during retinal development also disrupted the normal retinal lamination and caused a transdifferentiation of neurons into pigmented cells. The results indicate that β-catenin functions as a cell adhesion molecule but not as a Wnt pathway component during retinal neurogenesis, and is essential for lamination but not cell differentiation. The results further imply that retinal lamination and cell differentiation are genetically separable processes. ^ Sonic hedgehog (shh) is expressed in retinal ganglion cells under the control of transcription factor Pou4f2 during retinal development. Previous studies identified a phylogenetically conserved region in the first intron of shh containing a Pou4f2 binding site. Transgenic reporter mice in which reporter gene expression was driven by this region showed that this element can direct gene expression specifically in the retina, but expression was not limited to the ganglion cells. From these data I hypothesized that this element is required for shh expression in the retina but is not sufficient for specific ganglion cell expression. To further test this hypothesis, I created a conditional allele by flanking this region with two loxP sites. Lines carrying this allele will be crossed with retinal-specific Cre lines to remove this element in the retina. My hypothesis predicts that alteration in shh expression and subsequent retinal defects will occur in the retinas of these mice. ^