BAR domains as sensors of membrane curvature:the amphiphysin BAR structure

The BAR (Bin/amphiphysin/Rvs) domain is the most conserved feature in amphiphysins from yeast to human and is also found in endophilins and nadrins. We solved the structure of the Drosophila amphiphysin BAR domain. It is a crescent-shaped dimer that binds preferentially to highly curved negatively charged membranes. With its N-terminal amphipathic helix and BAR domain (N-BAR), amphiphysin can drive membrane curvature in vitro and in vivo. The structure is similar to that of arfaptin2, which we find also binds and tubulates membranes. From this, we predict that BAR domains are in many protein families, including sorting nexins, centaurins, and oligophrenins. The universal and minimal BAR domain is a dimerization, membrane-binding, and curvature-sensing module.

Eps15 and Dap160 control synaptic vesicle membrane retrieval and synapse development

Epidermal growth factor receptor pathway substrate clone 15 (Eps15) is a protein implicated in endocytosis, endosomal protein sorting, and cytoskeletal organization. Its role is, however, still unclear, because of reasons including limitations of dominant-negative experiments and apparent redundancy with other endocytic proteins. We generated Drosophila eps15-null mutants and show that Eps15 is required for proper synaptic bouton development and normal levels of synaptic vesicle (SV) endocytosis. Consistent with a role in SV endocytosis, Eps15 moves from the center of synaptic boutons to the periphery in response to synaptic activity. The endocytic protein, Dap160/intersectin, is a major binding partner of Eps15, and eps15 mutants phenotypically resemble dap160 mutants. Analyses of eps15 dap160 double mutants suggest that Eps15 functions in concert with Dap160 during SV endocytosis. Based on these data, we hypothesize that Eps15 and Dap160 promote the efficiency of endocytosis from the plasma membrane by maintaining high concentrations of multiple endocytic proteins, including dynamin, at synapses.

The synapsin cycle:a view from the synaptic endocytic zone

Although the synapsin phosphoproteins were discovered more than 30 years ago and are known to play important roles in neurotransmitter release and synaptogenesis, a complete picture of their functions within the nerve terminal is lacking. It has been shown that these proteins play an important role in the clustering of synaptic vesicles (SVs) at active zones and function as modulators of synaptic strength by acting at both pre- and postdocking levels. Recent studies have demonstrated that synapsins migrate to the endocytic zone of central synapses during neurotransmitter release, which suggests that there are additional functions for these proteins in SV recycling.

Differential efficiency of the endocytic machinery in tonic and phasic synapses

Efficient synaptic vesicle membrane recycling is one of the key factors required to sustain neurotransmission. We investigated potential differences in the compensatory endocytic machineries in two glutamatergic synapses with phasic and tonic patterns of activity in the lamprey spinal cord. Post-embedding immunocytochemistry demonstrated that proteins involved in synaptic vesicle recycling, including dynamin, intersectin, and synapsin, occur at higher levels (labeling per vesicle) in tonic dorsal column synapses than in phasic reticulospinal synapses. Synaptic vesicle protein 2 occurred at similar levels in the two types of synapse. After challenging the synapses with high potassium stimulation for 30 min the vesicle pool in the tonic synapse was maintained at a normal level, while that in the phasic synapse was partly depleted along with expansion of the plasma membrane and accumulation of clathrin-coated intermediates at the periactive zone. Thus, our results indicate that an increased efficiency of the endocytic machinery in a synapse may be one of the factors underlying the ability to sustain neurotransmission at high rates.

Amphiphysin is a component of clathrin coats formed during synaptic vesicle recycling at the lamprey giant synapse

Amphiphysin is a protein enriched at mammalian synapses thought to function as a clathrin accessory factor in synaptic vesicle endocytosis. Here we examine the involvement of amphiphysin in synaptic vesicle recycling at the giant synapse in the lamprey. We show that amphiphysin resides in the synaptic vesicle cluster at rest and relocates to sites of endocytosis during synaptic activity. It accumulates at coated pits where its SH3 domain, but not its central clathrin/AP-2-binding (CLAP) region, is accessible for antibody binding. Microinjection of antibodies specifically directed against the CLAP region inhibited recycling of synaptic vesicles and caused accumulation of clathrin-coated intermediates with distorted morphology, including flat patches of coated presynaptic membrane. Our data provide evidence for an activity-dependent redistribution of amphiphysin in intact nerve terminals and show that amphiphysin is a component of presynaptic clathrin-coated intermediates formed during synaptic vesicle recycling.

Colocalization of synapsin and actin during synaptic vesicle recycling

It has been hypothesized that in the mature nerve terminal, interactions between synapsin and actin regulate the clustering of synaptic vesicles and the availability of vesicles for release during synaptic activity. Here, we have used immunogold electron microscopy to examine the subcellular localization of actin and synapsin in the giant synapse in lamprey at different states of synaptic activity. In agreement with earlier observations, in synapses at rest, synapsin immunoreactivity was preferentially localized to a portion of the vesicle cluster distal to the active zone. During synaptic activity, however, synapsin was detected in the pool of vesicles proximal to the active zone. In addition, actin and synapsin were found colocalized in a dynamic filamentous cytomatrix at the sites of synaptic vesicle recycling, endocytic zones. Synapsin immunolabeling was not associated with clathrin-coated intermediates but was found on vesicles that appeared to be recycling back to the cluster. Disruption of synapsin function by microinjection of antisynapsin antibodies resulted in a prominent reduction of the cytomatrix at endocytic zones of active synapses. Our data suggest that in addition to its known function in clustering of vesicles in the reserve pool, synapsin migrates from the synaptic vesicle cluster and participates in the organization of the actin-rich cytomatrix in the endocytic zone during synaptic activity.

Measurement of Local Sodium Ion Levels near Micellar Surfaces with Fluorescent PET (Photoinduced Electron Transfer) Sensors

Na+ near membranes controls our nerve signals, besides several other crucial bioprocesses. We demonstrate that fluorescent PET (photoinduced electron transfer) sensor molecules target Na+ in nanospaces near micellar membranes with excellent discrimination against H+. They find that Na+ near anionic micelles is concentrated by factors of upto 160. Sensor molecules which are not held tight to the micelle surface find a Na+ amplification factor of 8 only. These findings are strengthened by the employment of control compounds whose PET processes are permanently ‘on’ or permanently ‘off’.

True navigation in migrating gulls requires intact olfactory nerves

During migratory journeys, birds may become displaced from their normal migratory route. Experimental evidence has shown that adult birds can correct for such displacements and return to their goal. However, the nature of the cues used by migratory birds to perform long distance navigation is still debated. In this experiment we subjected adult lesser black-backed gulls migrating from their Finnish/Russian breeding grounds (from >60°N) to Africa (to < 5°N) to sensory manipulation, to determine the sensory systems required for navigation. We translocated birds westward (1080 km) or eastward (885 km) to simulate natural navigational challenges. When translocated westwards and outside their migratory corridor birds with olfactory nerve section kept a clear directional preference (southerly) but were unable to compensate for the displacement, while intact birds and gulls with the ophthalmic branch of the trigeminal nerve sectioned oriented towards their population-specific migratory corridor. Thus, air-borne olfactory information seems to be important for migrating gulls to navigate successfully in some circumstances.

Sustained intraocular VEGF neutralization results in retinal neurodegeneration in the Ins2Akita diabetic mouse

Current therapies that target vascular endothelial growth factor (VEGF) have become a mainstream therapy for the management of diabetic macular oedema. The treatment involves monthly repeated intravitreal injections of VEGF inhibitors. VEGF is an important growth factor for many retinal cells, including different types of neurons. In this study, we investigated the adverse effect of multiple intravitreal anti-VEGF injections (200 ng/μl/eye anti-mouse VEGF164, once every 2 weeks totalling 5-6 injections) to retinal neurons in Ins2(Akita) diabetic mice. Funduscopic examination revealed the development of cotton wool spot-like lesions in anti-VEGF treated Ins2(Akita) mice after 5 injections. Histological investigation showed focal swellings of retinal nerve fibres with neurofilament disruption. Furthermore, anti-VEGF-treated Ins2(Akita) mice exhibited impaired electroretinographic responses, characterized by reduced scotopic a- and b-wave and oscillatory potentials. Immunofluorescent staining revealed impairment of photoreceptors, disruptions of synaptic structures and loss of amacrine and retinal ganglion cells in anti-VEGF treated Ins2(Akita) mice. Anti-VEGF-treated WT mice also presented mild amacrine and ganglion cell death, but no overt abnormalities in photoreceptors and synaptic structures. At the vascular level, exacerbated albumin leakage was observed in anti-VEGF injected diabetic mice. Our results suggest that sustained intraocular VEGF neutralization induces retinal neurodegeneration and vascular damage in the diabetic eye.

Autonomic modification of intestinal smooth muscle contractility

Intestinal smooth muscle contracts rhythmically in the absence of nerve and hormonal stimulation because of the activity of pacemaker cells between and within the muscle layers. This means that the autonomic nervous system modifies rather than initiates intestinal contractions. The practical described here gives students an opportunity to observe this spontaneous activity and its modification by agents associated with parasympathetic and sympathetic nerve activity. A section of the rabbit small intestine is suspended in an organ bath, and the use of a pressure transducer and data-acquisition software allows the measurement of tension generated by the smooth muscle of intestinal walls. The application of the parasympathetic neurotransmitter ACh at varying concentrations allows students to observe an increase in intestinal smooth muscle tone with increasing concentrations of this muscarinic receptor agonist. Construction of a concentration-effect curve allows students to calculate an EC50 value for ACh and consider some basic concepts surrounding receptor occupancy and activation. Application of the hormone epinephrine to the precontracted intestine allows students to observe the inhibitory effects associated with sympathetic nerve activation. Introduction of the drug atropine to the preparation before a maximal concentration of ACh is applied allows students to observe the inhibitory effect of a competitive antagonist on the physiological response to a receptor agonist. The final experiment involves the observation of the depolarizing effect of K+ on smooth muscle. Students are also invited to consider why the drugs atropine, codeine, loperamide, and botulinum toxin have medicinal uses in the management of gastrointestinal problems.

Low-grade malignant Triton tumor of the oral cavity:a case report

Malignant Triton tumor (MTT) is a malignant peripheral nerve sheath tumor showing rhabdomyoblastic differentiation. It is considered a high-grade neoplasm with poor outcome. This report describes an MTT appearing in the oral cavity. On histologic examination the encapsulated lesion was composed of interlacing fascicles of spindle cells and scattered, large, strap-like pleomorphic cells with abundant eosinophilic cytoplasm. No cross striations were seen. Examination of levels through the tissue showed a total of only 4 normal mitoses and no necrosis. Immunohistochemistry demonstrated diffuse S100 positivity in the spindle cells. The large pleomorphic cells were weakly positive for alpha-sarcomeric actin and myoglobin, although variably but strongly positive for desmin. Management involved a small en bloc resection of the maxilla. After 33 months there was no sign of recurrence or distant metastasis. It was concluded that low-grade variants of MTT occur that do not have an aggressive clinical course.

Visual outcomes of cataract surgery performed by supervised novice surgeons during training in rural China

To assess the outcomes of cataract surgery performed by novice surgeons during training in a rural programme. Design: Retrospective study. Participants: Three hundred thirty-four patients operated by two trainees under supervision at rural Chinese county hospitals. Methods: Two trainees performed surgeries under supervision. Visual acuity, refraction and examinations were carried out 3 months postoperatively. Main Outcome Measures: Postoperative uncorrected visual acuity, pinhole visual acuity, causes of visual impairment (postoperative uncorrected visual acuity<6/18) Results: Among 518 operated patients, 426 (82.2%) could be contacted and 334 (64.4% of operated patients) completed the examinations. The mean age was 74.1±8.8 years and 62.9% were women. Postoperative uncorrected visual acuity was available in 372 eyes. Among them, uncorrected visual acuity was ≥6/18 in 278 eyes (74.7%) and <6/60 in 60 eyes (16.1%), and 323 eyes (86.8%) had pinhole visual acuity≥6/18 and 38 eyes (10.2%) had pinhole visual acuity<6/60. Main causes of visual impairment were uncorrected refractive error (63.9%) and comorbid eye disease (24.5%). Comorbid eye diseases associated with pinhole visual acuity<6/60 (n=23, 6.2%) included glaucoma, other optic nerve atrophy, vitreous haemorrhage and retinal detachment. Conclusions: The findings suggest that hands-on training remains safe and effective even when not implemented in centralized training centres. Further refinement of the training protocol, providing postoperative refractive services and more accurate preoperative intraocular lens calculations, can help optimize outcomes. © 2012 The Authors Clinical and Experimental Ophthalmology © 2012 Royal Australian and New Zealand College of Ophthalmologists.

An in vitro model of nociceptors in human trigeminal nerves

Background: The oro-facial region is densely innervated by the trigeminal nerve, which when stimulated can induce noxious pain sensation and contribute to neurogenic inflammation in local tissues. Recent research on the expression of specialised ion channels on the trigeminal nerve has highlighted the need to undertake more extensive studies on ion channel expression/functionality with the aim of elucidating their role in pain sensations. A major family of such ion channels is the transient receptor potential (TRP) channels which are activated by a wide variety of thermal, mechanical or chemical stimuli and merit investigation as possible druggable targets for future analgesics.
Objective: Study of TRP channel expression and regulation in oro-facial tissues is hindered by the fact that the cell bodies of neurons innervating these tissues are located in the trigeminal ganglion. Using dental pulp stem cells differentiated towards peripheral neuronal equivalents (PNEs), we sought to determine TRP channel expression, functionality and potential modulation by cytokines in this novel model.
Method: Dental pulp stem cells (DPSCs) were grown on substrate-coated tissue culture plates and differentiated towards a neuronal phenotype using neuronal induction media. Quantitative polymerase chain reaction (qPCR) was performed on PNEs +/-cytokine treatment. Ion channel functionality was investigated using whole cell patch clamping.
Result: qPCR analysis showed that PNEs expressed the TRP channels TRPA1, TRPV1, TRPV4 and TRPM8. TRPA1 was the most abundantly expressed TRP channel studied whereas TRPM8 was lowly expressed. TRP channel expression was shown to be regulated by treatment with inflammatory cytokines. Patch clamp studies using specific agonists and antagonists for TRPA1 and TRPV1 showed these channels were functional.
Conclusion: PNEs differentiated from DPSCs provide a suitable model for TRP channel expression, regulation, and sensistisation in oro-facial tissues. This human neuronal model has potential for use in pre-clinical studies of novel analgesics.

Immunocytochemical Localization of Substance P Receptors (NK-1 Receptors) in Human Gingival Tissue

Substance P (SP) is a member of the structurally related family of neuropeptides known as the tachykinins. In addition to neurotransmitter roles, the tachykinins are also known to modulate local inflammation which depends on signalling between the neuropeptide molecules and target cells and tissues. SP mediates its effects through a specific receptor, known as the substance P receptor or the neurokinin 1 (NK-1) receptor. The NK-1 receptor is a G-protein associated integral membrane protein and although it has been studied in a wide range of tissues, to date there has been no published data on the localisation of the NK-1 receptor in human gingival tissue. Objective: The aim of this study was to examine the distribution of the NK-1 receptor in human gingival tissue using immunocytochemistry. Method: Gingival tissue was obtained from patients undergoing periodontal surgery. Tissue was fixed in paraformaldehyde and embedded in wax for sectioning. Sections were dewaxed in xylene and then rehydrated in alcohols and phosphate buffered saline. Rehydrated sections were probed with rabbit polyclonal antibody to human NK-1 receptor which was subsequently detected using anti-rabbit horseradish peroxidase conjugate and diaminobenzidine as substrate. Results: Immunocytochemistry revealed that the NK-1 receptor was distributed along nerve fibres and blood vessel endothelial cells, suggesting these areas are main targets for the actions of SP via the NK-1 receptor. Conclusion: This is the first immunocytochemical report of NK-1 receptors in human gingival tissue and provides evidence for possible NK-1 mediated biological effects of SP in human gingival tissue from periodontitis patients.

Age, gender, biometry, refractive error, and the anterior chamber angle among Alaskan Eskimos.

BACKGROUND:

The prevalence of angle-closure glaucoma (ACG) is greater for Eskimos/Inuit than it is for any other ethnic group in the world. Although it has been suggested that this prevalence may be due to a population tendency toward shallower anterior chamber angles, available evidence for other populations such as Chinese with high rates of ACG has not consistently demonstrated such a tendency.

METHODS:

A reticule, slit-lamp, and standard Goldmann one-mirror goniolens were used to make measurements in the anterior chamber (AC) angle according to a previously reported protocol for biometric gonioscopy (BG) (Ophthalmology 1999;106:2161-7). Measurements were made in all four quadrants of one eye among 133 phakic Alaskan Eskimos aged 40 years and older. Automatic refraction, dilated examination of the anterior segment and optic nerve, and A-scan measurements of AC depth, lens thickness, and axial length were also carried out for all subjects.

RESULTS:

Both central and peripheral AC measurements for the Eskimo subjects were significantly lower than those previously reported by us for Chinese, blacks, and whites under the identical protocol. Eskimos also seemed to have somewhat more hyperopia. There were no differences in biometric measurements between men and women in this Eskimo population. Angle measurements by BG seemed to decline more rapidly over life among Eskimos and Chinese than blacks or whites. Although there was a significant apparent decrease in AC depth, increase in lens thickness, and increase in hyperopia with age among Eskimos, all of these trends seemed to reverse in the seventh decade and beyond.

CONCLUSIONS:

Eskimos do seem to have shallower ACs than do other racial groups. Measurements of the AC angle seem to decline more rapidly over life among Eskimos than among blacks or whites, a phenomenon also observed by us among Chinese, another group with high ACG prevalence. This apparent more rapid decline may be due to a cohort effect with higher prevalence of myopia and resulting wider angles among younger Eskimos and Chinese.