Upregulation of epidermal gap junctional proteins in patients with venous disease.

BACKGROUND: Leg ulceration is a feared complication of venous insufficiency. It is not known whether varicose veins predispose skin to poor wound healing. The expression pattern of gap junctional protein connexin, a known marker of poor wound healing, was investigated across various stages of venous disease. METHODS: Patients undergoing intervention for varicose veins were assessed according to the Clinical Etiologic Anatomic Pathophysiologic (CEAP) classification of varicose veins. Paired 4-mm punch biopsies were taken from above the ankle (pathological) and above the knee (control). Tissues were stained with haematoxylin and eosin, and for connexin 43, connexin 30 and connexin 26. RESULTS: Forty-eight paired biopsies were taken (12 each for CEAP class C0, C2, C4 and C6). The pathological skin showed progressive epithelial hyperthickening, an increase in the number and depth of rete ridges, increased inflammation and loss of dermal architecture with disease progression from C4 onwards. The overall absolute connexin expression and mean connexin expression per cell in the pathological skin similarly increased across the CEAP classes from as early as C2. Increasing levels of connexin in control skin were also noted, indicating progression of the disease proximally. Connexin 43 expression showed the strongest positive correlation between pathological and control skin. CONCLUSION: Connexins were overexpressed in patients with simple varicose veins, with a stepwise increased expression through venous eczema to ulceration. Connexin 43 is a potential biomarker for venous disease. This finding suggests that varicose veins predispose skin to poor wound healing. Surgical relevance The overexpression of connexins, a family of gap junctional proteins, is known to cause poor healing in venous leg ulceration. It is not known whether there is any association with superficial venous disease. Here, connexin proteins were overexpressed in patients with uncomplicated varicose veins, before histological skin changes. Connexin could be a biomarker of venous disease progression.

Abnormal connexin expression in human chronic wounds.

BACKGROUND: Regulated alteration of connexin expression has been shown to be integral to acute wound repair. Downregulation of the gap-junction protein connexin 43 at the wound edge has been correlated with keratinocyte and fibroblast migration, while abnormal overexpression of connexin 43 significantly perturbs healing, as shown in the streptozotocin diabetic rodent impaired healing model. OBJECTIVES: To examine the protein expression levels of connexin 43, in addition to connexins 26 and 30, in a variety of human chronic wounds. METHODS: Wound-edge punch biopsies and a matched control from the arm were taken from a cohort of patients with venous leg, diabetic foot or pressure ulcers. Wound connexin expression in each patient was compared with that in a matched, nonwounded arm punch. Tissue was sectioned, stained and imaged by confocal microscopy using identical parameters per patient to permit quantification. RESULTS: Epidermal connexin 43, connexin 26 and connexin 30, and dermal connexin 43 were discovered to be strikingly upregulated in every ulcer from all three wound types, pointing to connexin upregulation as a common feature between chronic wounds. CONCLUSIONS: This result supports efforts to target connexin 43 to promote cell migration and wound healing in chronic ulcers.

Fibroblast growth factor 4 directs gap junction expression in the mesenchyme of the vertebrate limb Bud.

Pattern in the developing limb depends on signaling by polarizing region mesenchyme cells, which are located at the posterior margin of the bud tip. Here we address the underlying cellular mechanisms. We show in the intact bud that connexin 43 (Cx43) and Cx32 gap junctions are at higher density between distal posterior mesenchyme cells at the tip of the bud than between either distal anterior or proximal mesenchyme cells. These gradients disappear when the apical ectodermal ridge (AER) is removed. Fibroblast growth factor 4 (FGF4) produced by posterior AER cells controls signaling by polarizing cells. We find that FGF4 doubles gap junction density and substantially improves functional coupling between cultured posterior mesenchyme cells. FGF4 has no effect on cultured anterior mesenchyme, suggesting that any effects of FGF4 on responding anterior mesenchyme cells are not mediated by a change in gap junction density or functional communication through gap junctions. In condensing mesenchyme cells, connexin expression is not affected by FGF4. We show that posterior mesenchyme cells maintained in FGF4 under conditions that increase functional coupling maintain polarizing activity at in vivo levels. Without FGF4, polarizing activity is reduced and the signaling mechanism changes. We conclude that FGF4 regulation of cell-cell communication and polarizing signaling are intimately connected.

Antisense down regulation of connexin31.1 reduces apoptosis and increases thickness of human and animal corneal epithelia.

The roles of the gap junction protein connexin31.1 (Cx31.1) are poorly understood, especially as the protein appears to form non-functional channels. Cx31.1 specific antisense oligodeoxynucleotides (ODNs) were designed to evaluate its roles in a corneal epithelium model. Expression of Cx31.1 in corneal epithelium extends from the suprabasal layers of polyhedral wing cells through to the flat squamous cells of superficial layers which are shed into the tear film. Deoxyribozymes (Dzs) were tested for cleavage efficacy using in vitro transcribed Cx31.1 mRNA. Cleavage results showed a putative tertiary structure for Cx31.1 mRNA with one region appearing to have a higher potential for antisense targeting. Application of antisense ODNs designed to this region caused Cx31.1 knockdown in rat and human corneal organotypic culture models, leading to a reduction in apoptosis and a thickening of the corneal epithelium (p=0.0045). Cx31.1 appears to play a role in triggering cell death; knocking it down may provide a novel approach for tissue repair and engineering.

Increased connexin43 gap junction protein in hamster cardiomyocytes during cold acclimatization and hibernation.

OBJECTIVE: The physiology of hibernation is characterized by dramatic reductions of heart rate, respiration, metabolism, blood pressure and body temperature and by resistance to ventricular fibrillation. Gap junctions in the heart provide low resistance pathways, facilitating electrical and metabolic coupling between cardiac muscle cells for coordinated action of the heart and tissue homeostasis. The conductance of these junctions, and therefore their function, is likely to be affected by the physiological changes that take place during hibernation. Our objective was to quantitate gap junction protein levels in cold acclimatization, hibernation and arousal. METHODS: We have used specific antibodies to connexins 43 and 40, in combination with confocal microscopy, to quantitatively analyze the expression of connexin protein in hamster (Mesocricetus auratus) left ventricles in four animal groups: normal controls at euthermy, cold controls (cold-exposed animals that did not undergo hibernation), hibernating animals and animals aroused from hibernation for 2 h. RESULTS: Connexin40 immunostaining was not detected in ventricular cardiomyocytes in any animal group but connexin43 was found in all groups. Connexin43 expression was significantly enhanced in hibernation and cold control ventricular cardiomyocytes. Total plaque area, numerical density and plaque size were higher in the cold controls and hibernating hamsters compared to normal controls and animals aroused from hibernation. CONCLUSION: It is possible that the increased size and number of connexin43 gap junction plaques in the cold controls may represent a compensatory response in order to maintain sufficient gap junction communication during physiological conditions that would reduce conductance. These changes may represent a mechanism by which the hamster avoids ventricular fibrillation during hibernation and arousal.

Regular mosaics of large displaced and non-displaced ganglion cells in the retina of a cichlid fish.

Large retinal ganglion cells in the tilapid cichlid fish Oreochromis spilurus (standard length 15-54 mm) were filled with horseradish peroxidase and studied in flatmounts. Three types, with distinct patterns of dendritic stratification, formed spatially independent, nonrandom mosaics. One type (about 0.3% of all ganglion cells) resembled the outer (off) alpha cells of mammals. They were very large, with thick primary dendrites and large, sparsely branched planar trees in the outer part of the inner plexiform layer (IPL). About 300 were arrayed regularly across each retina, their exact number and spacing depending on its size. Their somata were often displaced into the IPL, even where neighbours in the mosaic were orthotopic. Another type (0.8%) resembled the inner (on) alpha cells of mammals. These had slightly smaller somata that were never displaced and smaller trees in the middle layers of the IPL. About 800 were arrayed uniformly and regularly across each retina. A rarer type (0.06-0.08%) had two planar trees: one forming a coarse mosaic in the outer part of the inner plexiform layer (co-planar with the trees of outer alpha-like cells) and another in the outer plexiform layer. These "biplexiform" cells were smaller and rounder than alpha-like cells and always displaced. The dendrites were finer and less tapered. Cells in which we could identify an outer plexiform tree failed to cover the retina completely, but were nonrandomly distributed. We draw three main conclusions: (1) some nonmammalian vertebrates have separate inner and outer mosaics of large ganglion cells like those of mammals, (2) the vertical displacement of ganglion cell somata can vary widely within a single mosaic and may thus be functionally irrelevant, and (3) biplexiform ganglion cells exist in fish but differ in morphology from the biplexiform types described in some other vertebrates.

Independent mosaics of large inner- and outer-stratified ganglion cells in the goldfish retina.

Goldfish retinal ganglion cells were filled with horseradish peroxidase and studied in flatmounts. Two regular mosaics of large neurons with many of the properties of mammalian alpha ganglion cells were found, differing from each other in spacing, size, and dendritic stratification. The existence of biplexiform ganglion cells with additional dendrites in the outer plexiform layer was also confirmed. One of the two alpha-like mosaics consisted of giant ganglion cells with thick primary dendrites and large, sparsely branched dendritic trees in the outer sublamina of the inner plexiform layer (IPL). In fish 55-65 mm long, about 300 formed a tessellated array across each retina. Their somata (mean area 277 +/- 6 microns 2) were displaced to varying degrees into the IPL, neighbours in the mosaic often occupying different levels. Their dendrites ramified in one stratum near the inner nuclear layer, at a mean depth of 70.8 +/- 0.5% of the IPL. The other alpha-like mosaic comprised about 900 large ganglion cells, with slightly smaller somata (mean area 193 +/- 4 microns 2) in the ganglion cell layer. Most of their dendrites lay in a narrow stratum at 41.9 +/- 0.5% of the depth of the IPL. However, deviations (usually into more vitread strata) were common, which was not true for similar cells in the distantly related cichlid fish Oreochromis. Measurements of nearest neighbour distance (NND) for 4 outer and 4 inner mosaics showed that they were at least as regular as the alpha cell mosaics of mammals: the ratio of the mean NND to the standard deviation ranged from 4.03 for the least regular outer mosaic to 6.47 for the most regular inner mosaic. The wide phylogenetic distribution of these paired, regular mosaics points to a fundamental role in vision. The presence of some variability in dendritic stratification even within the exceptionally regular inner-stratified mosaic suggests that classifications based entirely on the detailed morphology of individual neurons may not always correlate well with their primary functional roles. Where possible, neuronal morphology and spatial distribution should be studied together.

Changes in goldfish retinal ganglion cells during axonal regeneration.

Recent work suggests that mammalian retinal ganglion cells may become more like developing ganglion cells in form while regenerating through a peripheral nerve graft. We have injected Lucifer Yellow into regenerating ganglion cells of goldfish to look for similar changes. Within three weeks of injury, we saw dye-coupling to nearby cells, which is a common developmental feature in many species. Dendrites and axons, which in most mature ganglion cells are smooth, became varicose and hairy, like those examined in mammalian development. Secondary axons arose later, not only as side-branches of the primary axon but also from the soma, as in mammalian development and regeneration. Since, in fish, these responses are clearly an intrinsic part of functional regeneration, their equivalence in fish and mammals strengthens the view that a similar regenerative competence may exist in the retinal ganglion cells of all vertebrates.

Role of gap junctions in the development of the preimplantation mouse embryo.

We have taken several approaches to study the role of gap junctional communication during preimplantation mouse development. Firstly, the normal expression pattern of gap junctions has been characterized using immunostaining in conjunction with laser scanning confocal microscopy. Changes in junctional distribution have been correlated with developmental events. We have gone on to study development and junctional organization in mice which naturally exhibit reduced cell to cell communication (DDK syndrome), and in normal mice in which gap junction permeability has been artificially manipulated. Furthermore, anti-peptide antibodies have been tested for their ability to block gap junction communication and for the effects of such a block on subsequent development. Collectively, the results demonstrate that gap junctional communication plays an important role in the maintenance of compaction and the differentiation of an organized epithelium within an embryo, features which are vital for preimplantation development to progress successfully.

Gap junctions in the vertebrate retina.

The vertebrate retina is a highly laminated assemblage of specialized neuronal types, many of which are coupled by gap junctions. With one interesting exception, gap junctions are not directly responsible for the 'vertical' transmission of visual information from photoreceptors through bipolar and ganglion cells to the brain. Instead, they mediate 'lateral' connections, coupling neurons of a single type or subtype into an extended, regular array or mosaic in the plane of the retina. Such mosaics have been studied by several microscopic techniques, but new evidence for their coupled nature has recently been obtained by intracellular injection of biotinylated tracers, which can pass through gap junctional assemblies that do not pass Lucifer Yellow. This evidence adds momentum to an existing paradigm shift towards a population-based view of the retina, which can now be envisaged both as an array of semi-autonomous vertical processing modules, each extending right through the retina, and as a multi-layered stack of interacting planar mosaics, bearing some resemblance to a set of interleaved neural networks. Junctional conductance across mosaics of horizontal cells is known to be controlled dynamically with a circadian rhythm, and other dynamically-regulated conductance changes are also likely to make important contributions to signal processing. The retina is an excellent system in which to study such changes because many aspects of its structure and function are already well understood. In this review, we summarize the microscopic appearance, coupling properties and functions of gap junctions for each cell type of the neural retina, the regulatory properties that could be provided by selective expression of different connexin proteins, and the evidence for gap junctional coupling in retina development.

Tensile strain increased COX-2 expression and PGE2 release leading to weakening of the human amniotic membrane.

INTRODUCTION: There is evidence that premature rupture of the fetal membrane at term/preterm is a result of stretch and tissue weakening due to enhanced prostaglandin E2 (PGE2) production. However, the effect of tensile strain on inflammatory mediators and the stretch sensitive protein connexin-43 (Cx43) has not been examined. We determined whether the inflammatory environment influenced tissue composition and response of the tissue to tensile strain. METHODS: Human amniotic membranes isolated from the cervix (CAM) or placenta regions (PAM) were examined by second harmonic generation to identify collagen orientation and subjected to tensile testing to failure. In separate experiments, specimens were subjected to cyclic tensile strain (2%, 1 Hz) for 24 h. Specimens were examined for Cx43 by immunofluorescence confocal microscopy and expression of COX-2 and Cx43 by RT-qPCR. PGE2, collagen, elastin and glycosaminoglycan (GAG) levels were analysed by biochemical assay. RESULTS: Values for tensile strength were significantly higher in PAM than CAM with mechanical parameters dependent on collagen orientation. Gene expression for Cx43 and COX-2 was enhanced by tensile strain leading to increased PGE2 release and GAG levels in PAM and CAM when compared to unstrained controls. In contrast, collagen and elastin content was reduced by tensile strain in PAM and CAM. DISCUSSION: Fibre orientation has a significant effect on amniotic strength. Tensile strain increased Cx43/COX-2 expression and PGE2 release resulting in tissue softening mediated by enhanced GAG levels and a reduction in collagen/elastin content. CONCLUSION: A combination of inflammatory and mechanical factors may disrupt amniotic membrane biomechanics and matrix composition.

The pathogenesis of tendon microdamage in athletes: the horse as a natural model for basic cellular research.

The equine superficial digital flexor tendon (SDFT) is a frequently injured structure that is functionally and clinically equivalent to the human Achilles tendon (AT). Both act as critical energy-storage systems during high-speed locomotion and can accumulate exercise- and age-related microdamage that predisposes to rupture during normal activity. Significant advances in understanding of the biology and pathology of exercise-induced tendon injury have occurred through comparative studies of equine digital tendons with varying functions and injury susceptibilities. Due to the limitations of in-vivo work, determination of the mechanisms by which tendon cells contribute to and/or actively participate in the pathogenesis of microdamage requires detailed cell culture modelling. The phenotypes induced must ultimately be mapped back to the tendon tissue environment. The biology of tendon cells and their matrix, and the pathological changes occurring in the context of early injury in both horses and people are reviewed, with a particular focus on the use of various tendon cell and tissue culture systems to model these events.

Cost-effectiveness of denosumab in the treatment of postmenopausal osteoporosis in Canada.

OBJECTIVE: Denosumab is a novel biologic agent approved in Canada for treatment of post-menopausal osteoporosis (PMO) in women at high risk for fracture or who have failed or are intolerant to other osteoporosis therapies. This study estimated cost-effectiveness of denosumab vs usual care from the perspective of the Ontario public payer. METHODS: A previously published PMO Markov cohort model was adapted for Canada to estimate cost-effectiveness of denosumab. The primary analysis included women with demographic characteristics similar to those from the pivotal phase III denosumab PMO trial (FREEDOM; age 72 years, femoral neck BMD T-score -2.16 SD, vertebral fracture prevalence 23.6%). Three additional scenario sub-groups were examined including women: (1) at high fracture risk, defined in FREEDOM as having at least two of three risk factors (age 70+; T-score ≤ -3.0 SD at lumbar spine, total hip, or femoral neck; prevalent vertebral fracture); (2) age 75+; and (3) intolerant or contraindicated to oral bisphosphonates (BPs). Analyses were conducted over a lifetime horizon comparing denosumab to usual care ('no therapy', alendronate, risedronate, or raloxifene [sub-group 3 only]). The analysis considered treatment-specific persistence and post-discontinuation residual efficacy, as well as treatment-specific adverse events. Both deterministic and probabilistic sensitivity analyses were conducted. RESULTS: The multi-therapy comparisons resulted in incremental cost-effectiveness ratios for denosumab vs alendronate of $60,266 (2010 CDN$) (primary analysis) and $27,287 per quality-adjusted life year gained for scenario sub-group 1. Denosumab dominated all therapies in the remaining scenarios. LIMITATIONS: Key limitations include a lack of long-term, real-world, Canadian data on persistence with denosumab as well as an absence of head-to-head clinical data, leaving one to rely on meta-analyses based on trials comparing treatment to placebo. CONCLUSIONS: Denosumab may be cost-effective compared to oral PMO treatments for women at high risk of fractures and those who are intolerant and/or contraindicated to oral BPs.

Optical delineation of human malignant melanoma using second harmonic imaging of collagen.

Skin cancer incidence has increased exponentially over the last three decades. In 2008 skin cancer caused 2280 deaths in the UK, with 2067 due to malignant melanoma. Early diagnosis can prevent mortality, however, conventional treatment requires multiple procedures and increasing treatment times. Second harmonic generation (SHG) imaging could offer diagnosis and demarcation of melanoma borders non-invasively at presentation thereby short-cutting the excision biopsy stage. To test the efficacy and accuracy of SHG imaging of collagen in skin and to delineate the borders of skin cancers, unstained human melanoma biopsy sections were imaged using SHG microscopy. Comparisons with sister sections, stained with H&E or Melan-A were made for correlation of invasion borders. Fresh ex vivo normal human and rat skin was imaged through its whole thickness using SHG to demonstrate this technique is transferable to in vivo tissues. SHG imaging demonstrated detailed collagen distribution in normal skin, with total absence of SHG signal (fibrillar collagen) within the melanoma-invaded tissue. The presence or absence of signal changes dramatically at the borders of the melanoma, accurately demarcating the edges that strongly correlated with H&E and Melan-A defined borders (p<0.002). SHG imaging of ex vivo human and rat skin demonstrated collagen architecture could be imaged through the full thickness of the skin. We propose that SHG imaging could be used for diagnosis and accurate demarcation of melanoma borders on presentation and therefore potentially reduce mortality rates.

Maturational alterations in gap junction expression and associated collagen synthesis in response to tendon function.

Energy-storing tendons including the equine superficial digital flexor tendon (SDFT) contribute to energetic efficiency of locomotion at high-speed gaits, but consequently operate close to their physiological strain limits. Significant evidence of exercise-induced microdamage has been found in the SDFT which appears not to exhibit functional adaptation; the degenerative changes have not been repaired by the tendon fibroblasts (tenocytes), and are proposed to accumulate and predispose the tendon to rupture during normal athletic activity. The anatomically opposing common digital extensor tendon (CDET) functions only to position the digit, experiencing significantly lower levels of strain and is rarely damaged by exercise. A number of studies have indicated that tenocytes in the adult SDFT are less active in collagen synthesis and turnover than those in the immature SDFT or the CDET. Gap junction intercellular communication (GJIC) is known to be necessary for strain-induced collagen synthesis by tenocytes. We postulate therefore that expression of GJ proteins connexin 43 and 32 (Cx43; Cx32), GJIC and associated collagen expression levels are high in the SDFT and CDET of immature horses, when the SDFT in particular grows significantly in cross-sectional area, but reduce significantly during maturation in the energy-storing tendon only. The hypothesis was tested using tissue from the SDFT and CDET of foetuses, foals, and young adult Thoroughbred horses. Cellularity and the total area of both Cx43 and Cx32 plaques/mm(2) of tissue reduced significantly with maturation in each tendon. However, the total Cx43 plaque area per tenocyte significantly increased in the adult CDET. Evidence of recent collagen synthesis in the form of levels of neutral salt-soluble collagen, and collagen type I mRNA was significantly less in the adult compared with the immature SDFT; procollagen type I amino-propeptide (PINP) and procollagen type III amino-propeptide (PIIINP) levels per mm(2) of tissue and PINP expression per tenocyte also decreased with maturation in the SDFT. In the CDET PINP and PIIINP expression per tenocyte increased in the adult, and exceeded those in the adult SDFT. The level of PINP per mm(2) was greater in the adult CDET than in the SDFT despite the higher cellularity of the latter tendon. In the adult SDFT, levels of PIIINP were greater than those of PINP, suggesting relatively greater synthesis of a weaker form of collagen previously associated with microdamage. Tenocytes in monolayers showed differences in Cx43 and Cx32 expression compared with those in tissue, however there were age- and tendon-specific phenotypic differences, with a longer time for 50% recovery of fluorescence after photobleaching in adult SDFT cells compared with those from the CDET and immature SDFT. As cellularity reduces following growth in the SDFT, a failure of the remaining tenocytes to show a compensatory increase in GJ expression and collagen synthesis may explain why cell populations are not able to respond to exercise and to repair microdamage in some adult athletes. Enhancing GJIC in mature energy-storing tendons could provide a strategy to increase the cellular synthetic and reparative capacity.

Limiting burn extension by transient inhibition of Connexin43 expression at the site of injury.

Extension of a burn wound over the first 24h following injury is recognised clinically, and leads to diagnostic and therapeutic dilemmas. In the central nervous system, a similar spread of damage, beyond the initial injury, can occur via the spread of death signals from injured cells to their healthy neighbours via Connexin43 (Cx43) gap junction channels. In the skin, Cx43 is expressed in the basal epidermis and in fibroblasts and dermal appendages. We have used Cx43 specific antisense oligodeoxynucleotide approach to transiently down-regulate Cx43 protein in the early stages of partial thickness cutaneous burn wound healing. Antisense ODNs reduce the spread of tissue damage and neutrophil infiltration around the wound following injury. Epithelial cell proliferation is increased and the rate of wound closure is accelerated, compared to controls. Resultant scarring is smaller with less granulation tissue and more dermal appendages than controls. These findings suggest that Cx43 antisense treatment speeds partial thickness burn wound healing and reduces scarring. We suggest that this approach may provide an effective adjunct to managing partial thickness burn wounds.

Connexin alpha1 and cell proliferation in the developing chick retina.

During the formation of the eye, high levels of connexin alpha1 (connexin 43) are expressed within the tissues of the cornea, lens, and neural retina. In order to determine whether connexin alpha1 plays a role in the regulation of cell proliferation we have used a novel antisense technique to reduce its expression early in development (embryonic days 2-4). Application of Pluronic gel, containing antisense oligodeoxynucleotides (ODNs) to connexin alpha1, to one eye of early chick embryos results in a rapid and significant reduction of alpha1 protein which lasts for 24-48 h. Embryos grown for 48 h, after ODN application to one eye, showed a marked reduction in the diameter of the treated, compared to that of the contralateral untreated, eye. Sections cut from the treated eyes showed that the retina was also reduced in size. TUNEL labeling and staining with propidium iodide showed that apoptosis within the retinae of both treated and untreated eyes was rare and thus that the reduction in the area of the retina brought about by antisense ODNs directed at connexin alpha1 was unlikely to be the result of increased cell death. However, the number of mitotic figures in the ventricular zone of the antisense-treated retinae revealed by propidium iodide staining was significantly reduced (P < 0.0001) to 53 +/- 3.5% (n = 5) of that in the contralateral untreated control eyes. Embryos in which one eye was sham operated, treated with pluronic gel, or treated with sense ODN showed no significant changes in eye size or in the number of mitotic figures within the neural retina. These results point to a role for connexin alpha1-mediated gap-junctional communication in controlling the early wave of neurogenesis in the chick retina.

Retinotopic refinement of the regenerating goldfish optic tract is not linked to activity-dependent refinement of the retinotectal map.

The cut optic nerve of a goldfish can regenerate, restoring an orderly projection from the retina to the optic tectum. At first, regenerating axons make transient connections, many of them in inappropriate tectal locations. Later, their arrangement is gradually refined into an accurate retinotectal map by a process that depends on afferent activity. On their way to the tectum, many regenerating axons make erroneous choices between the two arms (brachia) of the optic tract. However, since they commonly possess divergent collateral branches, a secondary refinement of the brachial pattern can occur by selective collateral elimination. How or why a particular collateral is lost is not known, but we have previously suggested that sibling branches might compete to form stable tectal synapses, implying that there might be a causal link between refinement of the brachial pattern and refinement of the retinotectal map. In this paper, we have tested directly for such a link, blocking map refinement with tetrodotoxin (TTX) or stroboscopic light, verifying the effectiveness of the block and measuring the extent of brachial refinement by standard methods in experimental and control fish. Both TTX and stroboscopic light reliably prevented map refinement, their results being indistinguishable. However, neither had even the slightest detrimental effect on brachial refinement, either 42 days or 70 days after nerve cut. Evidently, neither activity nor a sharp retinotectal projection is necessary for brachial refinement. Theory and experiment both dictate that the basic projection pattern be controlled by a mechanism (such as chemoaffinity) that is independent of activity, and it would seem that selective collateral loss must depend on the same mechanism.