Name: ex vivo culturing of whole, developing drosophila brains
Uploaded: 2012-07-27T13:00:00
Description: Watch this Scientific Journal Video about Ex vivo Culturing of Whole, Developing Drosophila Brains at JoVE.com

نود أن نشكر جميع أعضاء مختبرنا لمناقشاتهم، وتقديم المشورة المفيدة والتعليقات على المخطوطة. ونحن أيضا نود أن نشكر بشكل خاص مارتا كوخ وحسن باسم لاقتراح ممتاز أن ننتظر حتى 1.5 ساعة APF قبل تشريح أدمغة للزراعة في إعادة عرض التجارب. كما نشكر مركز التصوير NHGRI لاستخدام مجهر متحد البؤر بهم. وأيد هذا العمل من قبل برنامج العلوم العصبية الأساسية لبرنامج بحوث ضمن الجدران NINDS، المعاهد الوطنية للصحة (Z01 NS003106).

أ. إعداد وسائل الإعلام <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تعد وسائل الإعلام كاملة بواسطة فلتر تعقيم شنايدر ذبابة الفاكهة وسائل الإعلام تستكمل مع مصل بقري جنيني 10٪ و 1٪ البنسلين / الستربتومايسين عقار. </li><li style=";text-align:right;direction:rtl"> وينبغي إعداد وسائل ا...

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+time-lapse+imaging+and+dominant+negative+receptors+to+dissect+the+steroid+receptor+control+of+neuronal+remodeling+in+Drosophila.">Use of time-lapse imaging and dominant negative receptors to dissect the steroid receptor control of neuronal remodeling in Drosophila.</a> Development. 133, 275-288 (2006).</li><li>Williamson, W. R., Hiesinger, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Developing+and+Adult+Drosophila+Brains+and+Retinae+for+Live+Imaging.">Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging.</a> J. Vis. Exp. (37), e1936 (2010).</li></ol>

<html> التلاعب الوراثي المزمن في بنية الأنسجة وظيفة، بما في ذلك التحولات والتعبير عن الجينات المحورة، وعادة ما تنتج مجموعة من الآثار المباشرة وغير المباشرة في وقت متأخر العواقب التي يمكن أن يكون من الصعب جدا فصل. ويمكن استكمال الطرق الوراثية مع علم الصيدلة تسهيل استجوا...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم الكاشف </td><td> شركة </td><td> فهرس العدد </td><td> نهائي تركيز </td></tr><tr><td> شنايدر ذبابة الفاكهة وسائل الإعلام </td><td> إينفيتروجن </td><td> 21720-024 </td><td></td></tr><tr><td> الأبقار مصل الجنين (حرارة المعطل) </td><td> إينفيتروجن </td><td> 10082-147 </td><td> 10٪ </td></tr><tr><td> البنسلين / الستربتومايسين عقار </td><td> إينفيتروجن </td><td> 15140-122 </td><td> 1٪ </td></tr><tr><td> الانسولين </td><td> إينفيتروجن </td><td> 12585-014 </td><td> 10μg/ml </td></tr><tr><td> إكديسون </td><td> سيغما الدريخ </td><td> H5142 </td><td> 2μg/ml </td></tr><tr><td> Roscovitine </td><td> سيغما الدريخ </td><td> R7772 </td><td> 100μM </td></tr><tr><td> Olomoucine </td><td> سيغما الدريخ </td><td> O0886 </td><td> 100μM </td></tr><tr><td> طبيعي عنزة المصل </td><td> إينفيتروجن </td><td> 50062Z </td><td> 1٪ </td></tr><tr><td> ألبومين المصل البقري </td><td> فيشر العلمية </td><td> M9501 </td><td> 1٪ </td></tr><tr><td> الفورمالديهايد </td><td> سيغما الدريخ </td><td> D4551 </td><td> 4٪ </td></tr><tr><td> تريتون-X 100 </td><td> فيشر العلمية </td><td> BP151 </td><td> 0.5٪ </td></tr><tr><td> التخزين المؤقت فوسفات المالحة </td><td> إينفيتروجن </td><td> 70011-044 </td><td> 1X </td></tr><tr><td> Vectashield </td><td> ناقلات مختبرات </td><td> H1000 </TD&gt; <td></td></tr><tr><td> إدراجات Millicell خلية الثقافة </td><td> ميليبور </td><td> PI8P01250 </td><td></td></tr><tr><td> Multidish، 4 جيد </td><td> الحرارية العلمية </td><td> 176740 </td><td></td></tr><tr><td> دومون # 5 الملقط </td><td> أدوات العلم غرامة </td><td> 11251-20 </td><td></td></tr><tr><td> مجهر الشرائح </td><td> فيشر العلمية </td><td> 12-544-7 </td><td></td></tr><tr><td> تغطية زجاج </td><td> فيشر العلمية </td><td> 12-548-5M </td><td></td></tr></tbody></table>

ex vivo culturing of whole, developing drosophila brains

ونحن تصف طريقة لاستزراع الأحياء السابقين من أدمغة ذبابة الفاكهة بأكملها. ويمكن استخدام هذا بمثابة الطباق إلى التلاعب الوراثي المزمن عن التحقيق في بيولوجيا الخلايا، وتطوير الهياكل المركزية في المخ من خلال السماح التدخلات الدوائية الحادة والتصوير الحي من العمليات الخلوية. وقد أظهرت كمثال على هذه التقنية، والعمل من قبل مختبر لدينا (1) التي مقصورة غير المعترف بها من قبل التحت خلوية تقع بين حجرات محواري وsomatodendritic من محاور عصبية في الدماغ المركزي ذبابة الفاكهة. وقد تبين في تطوير هذه المقصورة، ويشار إلى هذا الجزء محور عصبي الأولي (AIS) 2، وراثيا تعتمد على كيناز الخلايا العصبية الخاصة التي تعتمد على cyclin، Cdk5. نعرض هنا أن السابقين معاملة المجراة من عقل ذبابة الفاكهة البرية من نوع اليرقات مع roscovitine Cdk5 محددة مثبطات الدوائية وolomoucine 3 يسبب تغيرات حادة في منظمة الأكتين، وفي توطين Fasciclin بروتين على سطح الخلية 2، والتي تحاكي التغيرات ينظر في المسوخ التي تفتقر Cdk5 نشاط وراثيا. ويرد المثال الثاني من تقنية فيفو ثقافة السابقين لإعادة عرض للاتصالات من هيئة فطر الجنينية (MB) الخلايا العصبية جاما خلال التحول من يرقة إلى الكبار. هيئة فطر هي مركز التعلم والذاكرة الشمية في الذبابة 4، وهذه الخلايا العصبية غاما تقليم فروعها محواري وشجيري خلال تطوير العذراء ومن ثم إعادة تمديد فروع في timepoint في وقت لاحق لتأسيس نمط تعصيب الكبار 5. وقد تبين من تشذيب هذه الخلايا العصبية لجماعة الإخوان المسلمين أن يحدث عن طريق انحطاط المحلية لفروع neurite 6، من خلال الآلية التي يتم تشغيلها بواسطة إكديسون، وهو هرمون الستيرويد، ويتصرف بناء على مستقبلات إكديسون B1 7، والتي تعتمد على نشاط ubiquitin-proteasome نظام 6. يمكن أن أسلوبنا في زراعة المجراة سابقا باستخدام البريد لاستجواب مزيد من آلية إعادة عرض التنموية. وجدنا أن في الإعداد فيفو الثقافة السابق، الخلايا العصبية غاما من جماعة الإخوان المسلمين خصت عملية التقليم التنموية مع دورة الوقت مماثلة لتلك التي في الجسم الحي. كان من الضروري، مع ذلك، إلى الانتظار حتى 1.5 ساعة بعد تشكيل puparium قبل explanting النسيج حتى يتسنى للخلايا لارتكاب لا رجعة فيه إلى التحول؛ تشريح الحيوانات في بداية pupariation أدى إلى تحول ضئيلة أو معدومة في الثقافة. وهكذا، بعد إجراء التعديلات المناسبة، يمكن تطبيق ثقافة المجراة سابقا نهج لدراسة ديناميكية، فضلا عن الجوانب حالة مستقرة في علم الأحياء في الدماغ المركزي.

Watch this Scientific Journal Video about Ex vivo Culturing of Whole, Developing Drosophila Brains at JoVE.com

Ex vivo Culturing of Whole, Developing Drosophila Brains

وصف هذه المقالة على الطريقة التي يمكن لأحد أن يقلد<em&gt; في الجسم الحي</em&gt; تطوير<em&gt; ذبابة الفاكهة</em&gt; هيئة فطر في<em&gt; خارج الحي</em&gt; نظام الثقافة.

السابقين التثقيف المجراة من الجامعة، تطوير الأمخاخ ذبابة الفاكهة

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for ...

ex-vivo-culturing-of-whole-developing-drosophila-brains

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JoVE Journal

Neuroscience

Ex vivo Culturing of Whole, Developing Drosophila Brains

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

Electroporation في الجسم الحي من الشبكية ماوس النامية

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

فيفو السابقين علم البصريات الوراثي تشريح الدوائر الخوف في الدماغ شرائح

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

وبسيطة خطوة واحدة بروتوكول تشريح لجبل لالجامع إعداد الكبار<em&gt; ذبابة الفاكهة</em&gt; أدمغة

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

الرحم السابقين انهانسر والثقافات أورجانوتيبيك شريحة من أدمغة الفأر الجنينية للعيش-التصوير لترحيل إينتيرنيورونس جابايرجيك

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model animal for endocrine research, Drosophila melanogaster, which has sophisticated genetic tools and genome information, is being increasingly used. This article describes a method for the calcium imaging of Drosophila brain explants. This method enables the detection of the direct signaling of a hormone to the brain. It is well known that many peptide hormones act through G-protein-coupled receptors (GPCRs), whose activation causes an increase in the intracellular Ca2+concentration. Neural activation also elevates intracellular Ca2+ levels, from both Ca2+ influx and the release of Ca2+ stored in the endoplasmic reticulum (ER). A calcium sensor, GCaMP, can monitor these Ca2+ changes. In this method, GCaMP is expressed in the neurons of interest, and the GCaMP-expressing larval brain is dissected and cultured ex vivo. The test peptide is then applied to the brain explant, and the fluorescent changes in GCaMP are detected using a spinning disc confocal microscope equipped with a CCD camera. Using this method, any water-soluble molecule can be tested, and various cellular events associated with neural activation can be imaged using the appropriate fluorescent indicators. Moreover, by modifying the imaging chamber, this method can be used to image other Drosophila organs or the organs of other animals.

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

This paper describes a protocol for ex vivo calcium imaging of the Drosophila brain. In this method, natural or synthetic compounds can be applied to the buffer to test their ability to activate particular neurons in the brain.

السابقين فيفو تصوير الكالسيوم لتصور استجابات الدماغ للإشارات الغدد الصماء في المورفولوجية

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

التحليل الأيضي اليرقات melanogaster المورفولوجية و "العقول الكبار"

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...

محاذاة الألياف الشبكية OCT الماوس مع الصور البؤرية

Alignment of Visible-Light Optical Coherence Tomography Fibergrams with Confocal Images of the Same Mouse Retina

In recent years, in vivo retinal imaging, which provides non-invasive, real-time, and longitudinal information about biological systems and processes, has been increasingly applied to obtain an objective assessment of neural damage in eye diseases. Ex vivo confocal imaging of the same retina is often necessary to validate the in vivo findings especially in animal research. In this study, we demonstrated a method for aligning an ex vivo confocal image of the mouse retina with its in vivo images. A new clinical-ready imaging technology called visible light optical coherence tomography fibergraphy (vis-OCTF) was applied to acquire in vivo images of the mouse retina. We then performed the confocal imaging of the same retina as the &#34;gold standard&#34; to validate the in vivo vis-OCTF images. This study not only enables further investigation of the molecular and cellular mechanisms but also establishes a foundation for a sensitive and objective evaluation of neural damage in vivo.

تسليط الضوء على المؤلف: التطورات في تصوير الشبكية في الجسم الحي وخارج الجسم الحي لتحسين تشخيص وعلاج الجلوكوما 

The present protocol outlines the steps for aligning in vivo visible-light optical coherence tomography fibergraphy (vis-OCTF) images with ex vivo confocal images of the same mouse retina for the purpose of verifying the observed retinal ganglion cell axon bundle morphology in the in vivo images.

محاذاة الألياف المقطعية للتماسك البصري للضوء المرئي مع الصور متحدة البؤر لشبكية العين للماوس نفسه

In recent years, in vivo retinal imaging, which provides non-invasive, real-time, and longitudinal information about biological systems and processes, has ...

Preparation for Ex Vivo Color Fundus Photography 

To begin the ex vivo color fundus photography, set the high-definition multimedia interface camera to the manual ISO function. Arrange two light sources perpendicular to each other and turn the epi-illumination light sources to full power. At the dissecting microscope, insert the one-millimeter ruby bead into the posterior pole.Next, insert the posterior pole of the eye globe into a 30-milliliter quartz crucible filled with buffer. When the tissue sinks, insert a bracing between the eye and the wall of the crucible. Place the globe in the crucible onto the stereomicroscope stage to observe the interior ocular fundus through the microscope using the lowest magnification.Orient the eye by identifying the tissue mark at the 12 o'clock position, the optic nerve head, and the fovea five degrees below the optic nerve head.