RESUMO
PIEZO1 and PIEZO2 are mechanosensitive ion channels that regulate many important physiological processes including vascular blood flow, touch, and proprioception. As the eye is subject to mechanical stress and is highly perfused, these channels may play important roles in ocular function and intraocular pressure regulation. PIEZO channel expression in the eye has not been well defined, in part due to difficulties in validating available antibodies against PIEZO1 and PIEZO2 in ocular tissues. It is also unclear if PIEZO1 and PIEZO2 are differentially expressed. To address these questions, we used single-molecule fluorescence in situ hybridization (smFISH) together with transgenic reporter mice expressing PIEZO fusion proteins under the control of their endogenous promoters to compare the expression and localization of PIEZO1 and PIEZO2 in mouse ocular tissues relevant to glaucoma. We detected both PIEZO1 and PIEZO2 expression in the trabecular meshwork, ciliary body, and in the ganglion cell layer (GCL) of the retina. Piezo1 mRNA was more abundantly expressed than Piezo2 mRNA in these ocular tissues. Piezo1 but not Piezo2 mRNA was detected in the inner nuclear layer and outer nuclear layer of the retina. Our results suggest that PIEZO1 and PIEZO2 are differentially expressed and may have distinct roles as mechanosensors in glaucoma-relevant ocular tissues.
Assuntos
Glaucoma , Canais Iônicos , Animais , Camundongos , Glaucoma/genética , Hibridização in Situ Fluorescente , Canais Iônicos/metabolismo , Mecanotransdução Celular , Camundongos Transgênicos , RNA Mensageiro/genéticaRESUMO
In Drosophila, just as in vertebrates, changes in external temperature are encoded by bidirectional opponent thermoreceptor cells: some cells are excited by warming and inhibited by cooling, whereas others are excited by cooling and inhibited by warming. The central circuits that process these signals are not understood. In Drosophila, a specific brain region receives input from thermoreceptor cells. Here we show that distinct genetically identified projection neurons (PNs) in this brain region are excited by cooling, warming, or both. The PNs excited by cooling receive mainly feed-forward excitation from cool thermoreceptors. In contrast, the PNs excited by warming ('warm-PNs') receive both excitation from warm thermoreceptors and crossover inhibition from cool thermoreceptors through inhibitory interneurons. Notably, this crossover inhibition elicits warming-evoked excitation, because warming suppresses tonic activity in cool thermoreceptors. This in turn disinhibits warm-PNs and sums with feed-forward excitation evoked by warming. Crossover inhibition could cancel non-thermal activity (noise) that is positively correlated among warm and cool thermoreceptor cells, while reinforcing thermal activity which is anti-correlated. Our results show how central circuits can combine signals from bidirectional opponent neurons to construct sensitive and robust neural codes.
Assuntos
Encéfalo/citologia , Encéfalo/fisiologia , Drosophila melanogaster/fisiologia , Temperatura , Termorreceptores/fisiologia , Sensação Térmica/fisiologia , Animais , Drosophila melanogaster/citologia , Feminino , Interneurônios/fisiologiaRESUMO
Glutamatergic neurons are abundant in the Drosophila central nervous system, but their physiological effects are largely unknown. In this study, we investigated the effects of glutamate in the Drosophila antennal lobe, the first relay in the olfactory system and a model circuit for understanding olfactory processing. In the antennal lobe, one-third of local neurons are glutamatergic. Using in vivo whole-cell patch clamp recordings, we found that many glutamatergic local neurons are broadly tuned to odors. Iontophoresed glutamate hyperpolarizes all major cell types in the antennal lobe, and this effect is blocked by picrotoxin or by transgenic RNAi-mediated knockdown of the GluClα gene, which encodes a glutamate-gated chloride channel. Moreover, antennal lobe neurons are inhibited by selective activation of glutamatergic local neurons using a nonnative genetically encoded cation channel. Finally, transgenic knockdown of GluClα in principal neurons disinhibits the odor responses of these neurons. Thus, glutamate acts as an inhibitory neurotransmitter in the antennal lobe, broadly similar to the role of GABA in this circuit. However, because glutamate release is concentrated between glomeruli, whereas GABA release is concentrated within glomeruli, these neurotransmitters may act on different spatial and temporal scales. Thus, the existence of two parallel inhibitory transmitter systems may increase the range and flexibility of synaptic inhibition.
Assuntos
Canais de Cloreto/fisiologia , Drosophila melanogaster/fisiologia , Gânglios dos Invertebrados/fisiologia , Ácido Glutâmico/fisiologia , Inibição Neural/fisiologia , Olfato/fisiologia , Potenciais de Ação/fisiologia , Animais , Canais de Cloreto/genética , Drosophila melanogaster/genética , Feminino , Gânglios dos Invertebrados/citologia , Interneurônios/fisiologia , Ativação do Canal Iônico/fisiologia , Iontoforese , Neurotransmissores/fisiologia , Odorantes , Neurônios Receptores Olfatórios/fisiologia , Técnicas de Patch-Clamp , RNA Interferente Pequeno/genética , Proteínas Vesiculares de Transporte de Glutamato/fisiologia , Ácido gama-Aminobutírico/fisiologiaAssuntos
Blefaroptose/etiologia , Gelo , Miastenia Gravis/diagnóstico , Idoso , Humanos , Masculino , Miastenia Gravis/complicaçõesRESUMO
Purpose: To describe the use of an amniotic membrane graft (AMG) with fibrin sealant to address an overfiltering trabeculectomy flap encountered intraoperatively. Observations: A 35-year-old female with severe primary open angle glaucoma underwent trabeculectomy with mitomycin C due to uncontrolled intraocular pressure (IOP). Intraoperatively, the elastic nature of the scleral flap led to overfiltration, causing persistent anterior chamber shallowing despite numerous sutures. To decrease but not completely shut down aqueous outflow through the trabeculectomy flap, we utilized AMG and fibrin sealant to stabilize the flap. Postoperatively, the patient had a formed anterior chamber, elevated bleb and significantly reduced IOP, without the need for additional glaucoma medications. Conclusions and importance: Amniotic membrane grafts (AMG) with fibrin sealant may help regulate aqueous flow efflux, maintain anterior chamber stability, and mitigate the risk of postoperative hypotony in trabeculectomy surgery. AMG was chosen in this setting given its anti-inflammatory, anti-fibrotic properties, as well as its optically clear nature to allow for post-operative visualization of the flap. AMG allows for early postoperative stabilization of the scleral flap without complete obstruction, and may be useful in patients at risk of early postoperative hypotony.
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Purpose: Verteporfin is a benzoporphyrin derivative which is Food and Drug Administration-approved for treatment of choroidal neovascularization in conjunction with photodynamic therapy. It has been shown to prevent fibrosis and scar formation in several organs and represents a promising novel antifibrotic agent for glaucoma surgery. The goal of this study is to determine the effect of verteporfin on wound healing after glaucoma filtration surgery. Design: Preclinical study using a rabbit model of glaucoma filtration surgery. Subjects: Eight New Zealand white rabbits underwent glaucoma filtration surgery in both eyes. Methods: Eyes were randomized into 4 study groups to receive a postoperative subconjunctival injection of 1 mg/mL verteporfin (n = 4), 0.4 mg/mL mitomycin C (MMC; n = 4), 0.4 mg/mL MMC + 1 mg/mL verteporfin (n = 4), or balanced salt solution (BSS) control (n = 4). Bleb survival, vascularity, and morphology were graded using a standard scale over a 30-day period, and intraocular pressure (IOP) was monitored. At 30 days postoperative or surgical failure, histology was performed to evaluate for inflammation, local toxicity, and scarring. Main Outcome Measures: The primary outcome measure was bleb survival. Secondary outcome measures were IOP, bleb morphology, and bleb histology. Results: Compared to BSS control blebs, verteporfin-treated blebs demonstrated a trend toward increased surgical survival (mean 9.8 vs. 7.3 days, log rank P = 0.08). Mitomycin C-treated blebs survived significantly longer than verteporfin-treated blebs (log rank P = 0.009), with all but 1 MMC-treated bleb still surviving at postoperative day 30. There were no significant differences in survival between blebs treated with combination verteporfin + MMC and MMC alone. Mitomycin C-treated blebs were less vascular than verteporfin-treated blebs (mean vascularity score 0.3 ± 0.5 for MMC vs. 1.0 ± 0.0 for verteporfin, P < 0.01). Bleb histology did not reveal any significant toxicity in verteporfin-treated eyes. There were no significant differences in inflammation or scarring across groups. Conclusions: Although verteporfin remained inferior to MMC with regard to surgical survival, there was a trend toward increased survival compared with BSS control and it had an excellent safety profile. Further studies with variations in verteporfin dosage and/or application frequency are needed to assess whether this may be a useful adjunct to glaucoma surgery. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.
RESUMO
PURPOSE: To provide 4-year data on the efficacy and safety of gonioscopy-assisted transluminal trabeculotomy (GATT) in patients with open-angle glaucoma. DESIGN: Retrospective case series. PARTICIPANTS: Eyes of patients > 18 years of age who underwent GATT by a single surgeon at Wills Eye Hospital with at least 36 months follow-up. METHODS: Postoperative changes in outcome measures including intraocular pressure (IOP), medication use and visual acuity were recorded. Failure was defined as IOP > 21 mmHg or less than 20% reduction below baseline at any postoperative visit after 3 months or need for further glaucoma surgery. MAIN OUTCOME MEASURES: Main outcome measures were failure rate, IOP, number of glaucoma medications, and visual acuity at 4 years. RESULTS: Fifty-nine patients (74 eyes), age 57.1 ± 18.5 years (37.8% female) underwent the GATT procedure. Average follow-up was 47.0 ± 6.7 months (range 35.6-76.5 months). Mean IOP was 27.0 ± 10.0 mmHg preoperatively and 14.8 ± 6.5 mmHg at 4 years (45% IOP decrease; P < 0.01). Mean number of medications decreased from 3.2 ± 1.0 preoperatively to 2.3 ± 1.0 at 4 years (P < 0.01). The cumulative failure rate at 4 years was 53.9%, and the cumulative reoperation rate was 42.0%. No significant differences between patients with primary open-angle glaucoma and other types of glaucoma were found. CONCLUSIONS: Gonioscopy-assisted transluminal trabeculotomy can be a safe and effective conjunctival-sparing surgery for treating various forms of open-angle glaucoma at 4 years. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.
Assuntos
Glaucoma de Ângulo Aberto , Trabeculectomia , Humanos , Feminino , Pré-Escolar , Adulto , Pessoa de Meia-Idade , Idoso , Masculino , Glaucoma de Ângulo Aberto/cirurgia , Gonioscopia , Estudos Retrospectivos , Resultado do Tratamento , SeguimentosRESUMO
Although glaucoma is a disease modulated by eye pressure, the mechanisms of pressure sensing in the eye are not well understood. Here, we investigated associations between mechanosensitive ion channel gene variants and primary open-angle glaucoma (POAG). Common (minor allele frequency > 5%) single nucleotide polymorphisms located within the genomic regions of 20 mechanosensitive ion channel genes in the K2P, TMEM63, PIEZO and TRP channel families were assessed using genotype data from the NEIGHBORHOOD consortium of 3853 cases and 33,480 controls. Rare (minor allele frequency < 1%) coding variants were assessed using exome array genotyping data for 2606 cases and 2606 controls. Association with POAG was analyzed using logistic regression adjusting for age and sex. Two rare PIEZO1 coding variants with protective effects were identified in the NEIGHBOR dataset: R1527H, (OR 0.17, P = 0.0018) and a variant that alters a canonical splice donor site, g.16-88737727-C-G Hg38 (OR 0.38, P = 0.02). Both variants showed similar effects in the UK Biobank and the R1527H also in the FinnGen database. Several common variants also reached study-specific thresholds for association in the NEIGHBORHOOD dataset. These results identify novel variants in several mechanosensitive channel genes that show associations with POAG, suggesting that these channels may be potential therapeutic targets.
Assuntos
Glaucoma de Ângulo Aberto , Humanos , Glaucoma de Ângulo Aberto/genética , Genótipo , Bases de Dados Factuais , Exoma , Canais Iônicos/genéticaRESUMO
PURPOSE: To investigate the association between stereoacuity and the presence of central visual field defects (CVFDs) due to glaucoma. DESIGN: A prospective, cross-sectional cohort study. PARTICIPANTS: Participants with early-to-moderate glaucoma with a visual acuity better than 20/40, less than a 2-line difference in visual acuity between eyes, and 2 reliable Humphrey visual fields (VFs) (24-2 SITA standard) with mean deviation (MD) in the worse eye better than - 12 dB. METHODS: Stereoacuity was measured using the Titmus stereo test. Participants with a significant field defect (P < 0.005) in any 1 of the central 4 points in the 24-2 SITA standard total deviation map in either eye were classified as having a CVFD. Vision-related quality of life (VR-QOL) was measured using 25-item National Eye Institute Visual Function Questionnaire scores. Logistic regression was used to determine the associations between the level of stereoacuity and age, sex, race, glaucoma type, presence of CVFDs, visual acuity, contrast sensitivity, and VF MD. MAIN OUTCOME MEASURES: Stereoacuity in the CVFD and non-CVFD groups. RESULTS: Sixty-five participants met the inclusion criteria. The mean age of the participants was 64.3 ± 8.0 years, and 64.6% were women. The median stereoacuity was 60 arc seconds (interquartile range [IQR], 40-120 arc seconds). Forty-two (65%) patients had CVFDs, and 23 (35%) patients did not. The median stereoacuity of the CVFD group was worse than that of the non-CVFD group (60 arc seconds [IQR, 50-140 arc seconds] vs. 40 arc seconds [IQR, 40-80 arc seconds], respectively; P = 0.001). The non-CVFD group had a higher percentage of participants with normal stereopsis than the non-CVFD group (61% vs. 21%, respectively; P = 0.001). A multivariable analysis found that the presence of CVFDs was associated with worse stereopsis levels (odds ratio, 4.49; P = 0.021). The CVFD group had a lower Visual Functioning Questionnaire-25 (VFQ-25) composite score (84.0 vs. 91.4; P = 0.004) and lower VFQ-25 subscale scores for general vision, near activities, and mental health (P < 0.05). CONCLUSIONS: Central visual field defects were associated with increased odds of poor stereoacuity in patients with early-to-moderate glaucomatous VF loss. Specifically, patients without CVFDs are more likely to have normal stereopsis and higher VR-QOL than those with CVFDs. Patients with CVFDs should be counseled regarding how depth perception difficulties may affect daily living. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.
Assuntos
Glaucoma , Campos Visuais , Humanos , Feminino , Pessoa de Meia-Idade , Idoso , Masculino , Qualidade de Vida/psicologia , Estudos Transversais , Estudos Prospectivos , Escotoma , Transtornos da Visão/psicologia , Percepção de ProfundidadeRESUMO
A 62-year-old woman with mild myopia presented to her local optometrist for a routine examination and was found to have intraocular pressure (IOP) of 30 mm Hg in both eyes and cupped nerves. She had a family history of glaucoma in her father. She was started on latanoprost in both eyes and was referred for a glaucoma evaluation. On initial evaluation, her IOP was 25 mm Hg in the right eye and 26 mm Hg in the left eye. Central corneal thickness measured 592 µm in the right eye and 581 µm in the left eye. Her angles were open to gonioscopy without any peripheral anterior synechia. She had 1+ nuclear sclerosis with a corrected distance visual acuity (CDVA) of 20/25 in the right eye and 20/30- in the left eye and uncorrected near visual acuity of J1+ in each eye. Her nerves were 0.85 mm in the right eye and 0.75 mm in the left eye. Optical coherence tomography (OCT) showed retinal nerve fiber layer thinning and a dense superior arcuate scotoma into fixation in her right eye, and superior and inferior arcuate scotomas in her left eye (Figures 1 and 2JOURNAL/jcrs/04.03/02158034-202307000-00019/figure1/v/2023-06-26T195222Z/r/image-tiffJOURNAL/jcrs/04.03/02158034-202307000-00019/figure2/v/2023-06-26T195222Z/r/image-tiff, Supplemental Figures 1 and 2, available at http://links.lww.com/JRS/A882 and http://links.lww.com/JRS/A883). She was successively trialed on fixed combination brimonidine-timolol, dorzolamide, and netarsudil, in addition to her latanoprost, but her IOP remained in the mid- to upper 20s in both eyes. The addition of acetazolamide lowered the pressure to 19 mm Hg in both eyes, but she tolerated it poorly. Methazolamide was also attempted with similar side effects. We elected to perform left eye cataract surgery combined with 360-degree viscocanaloplasty and insertion of a Hydrus microstent (Alcon Laboratories, Inc.). Surgery was uncomplicated with IOP of 16 mm Hg on postoperative day 1 with no glaucoma medications. However, by postoperative week 3, IOP returned to 27 mm Hg, and despite restarting latanoprost-netarsudil and finishing her steroid taper, IOP remained at 27 mm Hg by postoperative week 6. Brimonidine-timolol was added back to her left eye regimen and at postoperative week 8, IOP had elevated to 45 mm Hg. Maximizing her therapy with the addition of topical dorzolamide and oral methazolamide brought her IOP back down to 30 mm Hg. At that point, the decision was made to proceed with trabeculectomy of the left eye. The trabeculectomy was uneventful. However, postoperative attempts to augment filtration were rendered less successful by extremely thick Tenon layer. At her most recent follow-up the pressure in the left eye was mid-teens with brimonidine-timolol and dorzolamide. Her right eye IOP is in the upper 20s on maximum topical therapy. Knowing her postoperative course in the left eye, how would you manage the right eye? In addition to currently available options, would you consider a supraciliary shunt such as the MINIject (iSTAR) if such a device were U.S. Food and Drug Administration (FDA)-approved?
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Glaucoma de Ângulo Aberto , Glaucoma , Humanos , Estados Unidos , Feminino , Adolescente , Pessoa de Meia-Idade , Glaucoma de Ângulo Aberto/tratamento farmacológico , Glaucoma de Ângulo Aberto/cirurgia , Latanoprosta/uso terapêutico , Metazolamida , Timolol/uso terapêutico , Resultado do TratamentoRESUMO
Normal-tension glaucoma is a form of optic nerve degeneration that is characterized by loss of retinal ganglion cells independent of eye pressure elevation. In this issue of the JCI, Pan et al. report the discovery in a Japanese family of a mutation in the METTL23 gene, which encodes a histone arginine methyltransferase that causes normal-pressure glaucoma in haploinsufficiency. Inherited as an autosomal dominant condition, METTL23 deficiency revealed an important function in the regulation of pS2 and the downstream NF-κB signaling pathway, which has previously been linked to glaucomatous optic nerve degeneration. These findings are the first direct link between defective epigenetic regulatory machinery and genetic forms of optic nerve degeneration.
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Metilação de DNA , Glaucoma , Humanos , Glaucoma/metabolismo , Células Ganglionares da Retina/metabolismo , Degeneração Neural/patologia , Epigênese Genética , Pressão IntraocularRESUMO
We describe here a case report of a novel technique for tube erosion repair, which modifies and utilizes the commercially available tube extender (Model TE). The modification of the tube extender makes the commercially available tube extender more compact and is useful in cases where conjunctival mobility and space are limited. This debulking of the tube extender may reduce the risk of future tube exposure and dellen formation.
Assuntos
Corpos Estranhos no Olho/cirurgia , Implantes para Drenagem de Glaucoma , Glaucoma de Ângulo Aberto/cirurgia , Falha de Prótese , Idoso de 80 Anos ou mais , Feminino , Humanos , Pressão Intraocular/fisiologia , Implantação de Prótese , Retalhos Cirúrgicos , Técnicas de SuturaRESUMO
PRECIS: In open-angle glaucoma, when neuroretinal rim tissue measured by volumetric optical coherence tomography (OCT) scans is below a third of the normal value, visual field (VF) damage becomes detectable. PURPOSE: To determine the amount of neuroretinal rim tissue thickness below which VF damage becomes detectable. METHODS: In a retrospective cross-sectional study, 1 eye per subject (of 57 healthy and 100 open-angle glaucoma patients) at an academic institution had eye examinations, VF testing, spectral-domain OCT retinal nerve fiber layer (RNFL) thickness measurements, and optic nerve volumetric scans. Using custom algorithms, the minimum distance band (MDB) neuroretinal rim thickness was calculated from optic nerve scans. "Broken-stick" regression was performed for estimating both the MDB and RNFL thickness tipping-point thresholds, below which were associated with initial VF defects in the decibel scale. The slopes for the structure-function relationship above and below the thresholds were computed. Smoothing curves of the MDB and RNFL thickness covariates were evaluated to examine the consistency of the independently identified tipping-point pairs. RESULTS: Plots of VF total deviation against MDB thickness revealed plateaus of VF total deviation unrelated to MDB thickness. Below the thresholds, VF total deviation decreased with MDB thickness, with the associated slopes significantly greater than those above the thresholds (P<0.014). Below 31% of global MDB thickness, and 36.8% and 43.6% of superior and inferior MDB thickness, VF damage becomes detectable. The MDB and RNFL tipping points were in good accordance with the correlation of the MDB and RNFL thickness covariates. CONCLUSIONS: When neuroretinal rim tissue, characterized by MDB thickness in OCT, is below a third of the normal value, VF damage in the decibel scale becomes detectable.
Assuntos
Glaucoma de Ângulo Aberto/diagnóstico , Fibras Nervosas/patologia , Disco Óptico/patologia , Doenças do Nervo Óptico/diagnóstico , Células Ganglionares da Retina/patologia , Transtornos da Visão/diagnóstico , Campos Visuais/fisiologia , Adulto , Idoso , Estudos Transversais , Feminino , Glaucoma de Ângulo Aberto/fisiopatologia , Humanos , Imageamento Tridimensional , Pressão Intraocular/fisiologia , Masculino , Pessoa de Meia-Idade , Modelos Teóricos , Disco Óptico/diagnóstico por imagem , Doenças do Nervo Óptico/fisiopatologia , Estudos Retrospectivos , Tomografia de Coerência Óptica/métodos , Transtornos da Visão/fisiopatologia , Testes de Campo VisualRESUMO
Nacre, the crown jewel of natural materials, has been extensively studied owing to its remarkable physical properties for over 160 years. Yet, the precise structural features governing its extraordinary strength and its growth mechanism remain elusive. In this paper, we present a series of observations pertaining to the red abalone (Haliotis rufescens) shell's organic-inorganic interface, organic interlayer morphology and properties, large-area crystal domain orientations and nacre growth. In particular, we describe unique lateral nano-growths and paired screw dislocations in the aragonite layers, and demonstrate that the organic material sandwiched between aragonite platelets consists of multiple organic layers of varying nano-mechanical resilience. Based on these novel observations and analysis, we propose a spiral growth model that accounts for both [001] vertical propagation via helices that surround numerous screw dislocation cores and simultaneous 010 lateral growth of aragonite sheet structure. These new findings may aid in creating novel organic-inorganic micro/nano composites through synthetic or biomineralization pathways.
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Carbonato de Cálcio/química , Gastrópodes/anatomia & histologia , Animais , Calcificação Fisiológica , Carbonato de Cálcio/metabolismo , Gastrópodes/metabolismo , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Difração de Raios XRESUMO
A key tool in neuroscience is the ability to transiently inactivate specific neurons on timescales of milliseconds to minutes. In Drosophila, there are two available techniques for accomplishing this (shibire(ts) and halorhodopsin [1-3]), but both have shortcomings [4-9]. Here we describe a complementary technique using a native histamine-gated chloride channel (Ort). Ort is the receptor at the first synapse in the visual system. It forms large-conductance homomeric channels that desensitize only modestly in response to ligand [10]. Many regions of the CNS are devoid of histaminergic neurons [11, 12], raising the possibility that Ort could be used to artificially inactivate specific neurons in these regions. To test this idea, we performed in vivo whole-cell recordings from antennal lobe neurons misexpressing Ort. In these neurons, histamine produced a rapid and reversible drop in input resistance, clamping the membrane potential below spike threshold and virtually abolishing spontaneous and odor-evoked activity. Every neuron type in this brain region could be inactivated in this manner. Neurons that did not misexpress Ort showed negligible responses to histamine. Ort also performed favorably in comparison to the available alternative effector transgenes. Thus, Ort misexpression is a useful tool for probing functional connectivity among Drosophila neurons.
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Canais de Cloreto/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Regulação da Expressão Gênica , Histamina/metabolismo , Técnicas de Patch-Clamp/métodos , Animais , Encéfalo/fisiologia , Canais de Cloreto/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Feminino , Histamina/genética , Canais Iônicos de Abertura Ativada por Ligante/genética , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Masculino , Neurônios/fisiologiaRESUMO
Alpha-herpesviruses, including herpes simplex virus and pseudorabies virus (PRV), infect the peripheral nervous system (PNS) of their hosts. Here, we describe an in vitro method for studying neuron-to-cell spread of infection as well as viral transport in axons. The method centers on a novel microfluidic chamber system that directs growth of axons into a fluidically isolated environment. The system uses substantially smaller amounts of virus inoculum and media than previous chamber systems and yet offers the flexibility of applying multiple virology and cell biology assays including live-cell optical imaging. Using PRV infection of cultured PNS neurons, we demonstrate that the microfluidic chamber recapitulates all known facets of neuron-to-cell spread demonstrated in animals and other compartmented cell systems.