We are grateful to Jochen Zeil, Paul Cooper and Birgit Greiner for sharing their knowledge in insect anatomy and for demonstrating several of the techniques we have described here. We are grateful to the talented and supportive staff at the Centre for Advanced Microscopy at ANU and The Microscopy Unit at MQU. This work was supported by a graduate scholarship to FRE and grants from the Australian Research Council (DE120100019, FT140100221, DP150101172).

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The authors declare no competing interests.

The suite of methods outlined above allow for an effective investigation into the optical system of ants and other insects. These techniques inform our understanding of sampling resolution, optical sensitivity, and potential polarization sensitivity of the eye being studied. This knowledge provides an important foundation for physiological and behavioral investigation into their visual capabilities. Furthermore, while the methods detailed here have focused on ant visual systems, these techniques can be used on other insects, albeit with slight modifications in the protocol (e.g., increasing duration of fixation and infiltration in thicker tissues). Slightly modified protocols have been used to characterize the visual systems of a variety of insects including cicadas22, flies14, bees23, wasps24, butterflies25, and moths26. Although most of the techniques outlined here have been in use for some time, this article takes the opportunity to bring them together in the context of studying the ant&#39;s optical system and compare alternative techniques and describe common pitfalls.
There are many imaging techniques currently available that have overlapping applications and it can be difficult to assess which technique is appropriate for the task at hand. A relevant example here is choosing a technique for overview imaging. The external morphology of the head and eye and relative positioning of the optical system on the head can be done using SEM or photomicrography. The strengths and weaknesses of these techniques have been reviewed27, however, there are some special considerations when imaging eyes. When imaging the relative positioning and size of the eyes, both techniques have their advantages and disadvantages. SEM images lack color information and hence where pigmentation is relevant photomicrography is better. However, SEM images can illustrate fine structures such as inter-ommatidial hairs and facet boundaries in greater detail and even reveal surface features not visible under photomicrography techniques (e.g., ocellar lenses, surface sculpturing of compound eye lenses). SEM is a versatile technique when it comes to exploratory imaging and identifying features of interest because it can operate on a large range of specimen sizes while still retaining very high resolution throughout this range. However, it is not as widely accessible as a dissection microscope and requires a higher level of expertise. There is often no single way of obtaining the information one requires. In such a scenario, it is useful to consider what is available and where it is most important to invest resources.
Nail-polish replicas of the cornea have proven to be most useful in obtaining the most accurate measure of facet numbers and facet diameters. This has now been used in a variety of insects11,22,28,29. While the quality of the images acquired from an SEM is far superior, the curvature of the eye prevents accurate measurements of the whole facet array. Mapping the facet size and facet distribution should also be feasible from scans acquired from micro-computed tomography5.
In both the LM and TEM techniques, it often is difficult to know whether the sample has been prepared and processed well until the final stage of imaging. To avoid complications, it is important to establish good practices such as maintaining clean working spaces and tools, preparing fresh solutions regularly, and thoroughly filtering water. Contaminants that are invisible to the naked eye can ruin EM samples. For this reason, it can be useful to wipe down surfaces and instruments using a solvent, such as ethanol or acetone, and a non-lint producing wipe. This is most relevant when sectioning, staining EM sections, and when preparing SEM samples. Similarly, distilled water sources can present problems and introduce contaminants so it is always best to check filters, change them regularly, and always use freshly filtered water (do not store). Most fixatives, stains, and embedding materials cannot be stored indefinitely and it is important to label all solutions with the date of preparation. It is important to take a systematic approach and set aside enough time to carry out protocols without interruptions.
Adapting techniques to different species is always a matter of trial and error. When working within Formicidae, the main differences lie in the size of the animal and the muscle mass within the head. Ants with more musculature in their head will typically take longer to fix. With very large ants, it is best to remove the mandibular muscles, trachea, and mandibular glands, while ensuring minimum interference with the neural tissue. In small ants and those with few mandibular muscles, it is possible to achieve adequate fixation by just removing the mandibles and exposing the clypeal region. In these cases, small holes using minutiae pins can be made on the head to improve fixation.
It is important to note that environmental conditions can also affect preparations. Hot and humid environments (especially field stations in the tropics) can prove to be a challenge during the infiltration stage. Warm conditions can lead resins to partially polymerize prematurely resulting in the unused resin becoming increasingly more viscous. In this case, the best option is to store the resin in small, single use, containers in the fridge or freezer. Cooling fixatives can be helpful to counter faster tissue decay in warm conditions. However, cooled solutions will disperse more slowly which means that treatment times should be extended to ensure proper penetration.
With these cautions in mind, investigation into the optical system of ants and other insects can prove very rewarding.&#160;Studying the visual system allows us to estimate the size of visual fields, interommatidial angles, optical sensitivity, and sampling resolutions. Understanding the anatomy of the eye informs our understanding and interpretation of animal behavior. For example, anatomy allows us to make predictions on the visual capabilities of animals such as whether they are diurnal or nocturnal, which may not have been previously documented. Given the current knowledge about the visual system of handful of ants, we hope our methods will inspire biologists and myrmecologists to investigate the compound eye and ocelli in ants to further our understanding.

Vision is an important sensory modality for most animals. Vision is especially crucial in the context of navigation for pinpointing goals, establishing and adhering to routes, and obtaining compass information1,2. Insects detect visual information using a pair of compound eyes and, in some cases, one to three dorsally-placed simple eyes called ocelli3,4,5.
The eyes of ants are of particular interest because, while ants are wonderfully diverse, they conserve some key characteristics across species. Despite dramatic variation in anatomy, size, and ecology, the vast majority of species are eusocial and live in colonies; as a result, different species face similar visual challenges in terms of navigating back and forth between a central place and resources. Across ants the same basic eye bauplan can be observed in animals ranging from 0.5-26 mm in body length, from exclusively diurnal to strictly nocturnal species, and from slow walking subterranean to leaping visual predators6,7,8,9,10. All of these staggering differences in ecology and behavior give rise to innumerable permutations of the same basic eye structures to suit different environments, lifestyles, and body-sizes11,12. As a consequence, studying the visual ecology of ants provides a veritable treasure trove of possibilities to the determined investigator.
Understanding the visual system of insects is essential in gaining an insight into their behavioral capabilities. This is apparent from integrative studies which nicely combine anatomy with ecology and behavior to a great success in a few insect groups (e.g., references13,14,15,16,17). Though the field of ant navigation and ant behavior in general has been quite successful, very little emphasis has been placed on ant vision outside of a few selected species. Here, we will elaborate on the techniques involved in investigating eye design of ants. While we will focus on ants, these techniques can be applied, with slight modifications, to other insects, too.

<table><tbody><tr><td>Ant</td><td>Myrmecia midas</td><td></td><td></td></tr><tr><td>Stereomicroscope</td><td>Leica M205 FA</td><td></td><td></td></tr><tr><td>Sputter coater</td><td>Pro Sci Tech</td><td></td><td></td></tr><tr><td>Ethanol</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>Petri dish</td><td>ProSciTech</td><td></td><td></td></tr><tr><td>Dissecting microscope</td><td>Leica MZ6</td><td></td><td></td></tr><tr><td>Insect Pin</td><td>ProSciTech</td><td></td><td></td></tr><tr><td>Colourless nail polish</td><td>Non branded: from any cosmetic store</td><td></td><td></td></tr><tr><td>Glass slide</td><td>ProSciTech</td><td></td><td></td></tr><tr><td>Razor blade</td><td>ProSciTech</td><td></td><td></td></tr><tr><td>Foreceps</td><td>ProSciTech</td><td></td><td></td></tr><tr><td>Cover slip</td><td>ProSciTech</td><td></td><td></td></tr><tr><td>Compound microscope</td><td>Leica DM5000 B</td><td></td><td></td></tr><tr><td>Glutaraldehyde</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>Paraformalydehyde</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>Potassium Chloride (KCl)</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>di-Sodium Hydrogen phosphate (Na2HPO4)</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>Potassium di-Hydrogen Phosphate (KH2PO4)</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>Osmium tetroxide</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>Acetone</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>Araldite Epoxy Resin</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>Pasteur pipette</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>Toluidie Blue</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>Hotplate</td><td>Riechert HK120</td><td></td><td></td></tr></tbody></table>

techniques for investigating the anatomy of the ant visual system

This article outlines a suite of techniques in light microscopy (LM) and electron microscopy (EM) which can be used to study the internal and external eye anatomy of insects. These include traditional histological techniques optimized for work on ant eyes and adapted to work in concert with other techniques such as transmission electron microscopy (TEM) and scanning electron microscopy (SEM). These techniques, although vastly useful, can be difficult for the novice microscopist, so great emphasis has been placed in this article on troubleshooting and optimization for different specimens. We provide information on imaging techniques for the entire specimen (photo-microscopy and SEM) and discuss their advantages and disadvantages. We highlight the technique used in determining lens diameters for the entire eye and discuss new techniques for improvement. Lastly, we discuss techniques involved in preparing samples for LM and TEM, sectioning, staining, and imaging these samples. We discuss the hurdles that one might come across when preparing samples and how best to navigate around them.

The methods described here enable detailed study of the simple and compound eyes of ants. Imaging the dorsal view of the head using Z-stack photomicrography techniques allows one to obtain an overview of the layout of the visual system (Figure 1). This is good preparation for dissections and to determine the required sectioning angle. This technique is also useful for taking measurements such as head width, eye length, and ocellar lens diameters. SEM imaging also gives detailed overview images but additionally allows acquisition of high magnification and high resolution images. Particular regions of interest in the eye can be examined in detail and variations in lens shape can be identified (Figure 2). SEM images are especially useful for resolving ants with small eyes and ocelli. The cornea replicas provide information on the shape, size, and number of lenses in each eye (Figure 3). Semi-thin sections imaged using LM techniques allow investigation of the gross internal anatomy of the eye (Figure 4 and Figure 5); this includes the thickness of the lens, diameter of the crystalline cone, presence of a crystalline cone tract, shape, width, and length of the rhabdom, mapping the dorsal rim area, and location of the primary and secondary pigment cells. This technique can be nicely complemented by ultra-thin sections imaged using TEM, which allows for determining the ultrastructure especially, the microvillar orientation (Figure 4) and the quantifying smaller structures (e.g., width of the constricted crystalline cone tract, Figure 5).
<img alt="Ant caste comparison diagram: worker, alate female, alate male head views for entomology research." class="xfigimg" src="/files/ftp_upload/56339/56339fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: Z-stack photomicrographs of the three castes of the Australian sugar ant, Camponotus consobrinus. This provides an overview of the layout of the visual system in all three castes. Adapted from reference20. Scale bar = 1 mm. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56339/56339fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Insect head and eye morphology, SEM images, compound eyes structure, close-up detailed comparison." class="xfigimg" src="/files/ftp_upload/56339/56339fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Scanning electron micrographs of the ant visual system demonstrating the imaging capabilities of this technique. Top row shows different eye positions and eye sizes in: (A) Myrmecia nigriceps; (B) Opisthopsis pictus; and (C) Amblyopone australis (note the very small eyes, white arrow). Images acquired at high magnification showing: (D) the three simple eyes in workers of Myrmecia nigriceps; different sized compound eye in (E) Rhytidoponera metallica (note the different shaped ommatidia in different regions of the compound eye in yellow), (F) Amblyopone australis, (G) Myrmecia pyriformis, (H) Orectognathus clarki, and (I) Pheidole species. Scale bars = 1 mm (A-C), 100 µm (D-H), 10 µm (I). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56339/56339fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Microscope images of plant root cross-section, cellular structure diagram, vascular tissue analysis." class="xfigimg" src="/files/ftp_upload/56339/56339fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: Cornea replicas of ant eye and ocelli. (A) Replica of the compound eye of a worker of Myrmecia nigriceps. The convex replica was flattened by making incisions.  The inset indicates posterior (p), anterior (a), and dorsal (d), ventral (v) axes. (B) Replica of the ocelli of worker of Myrmecia tarsata. Scale bars = 0.5 mm (A), 10 µm (B). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56339/56339fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Histology micrographs displaying insect photoreceptor cells, cellular orientation, and structural detail." class="xfigimg" src="/files/ftp_upload/56339/56339fig4.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4: LM and EM images of rhabdom cross-sections. (A) Cross-section of distal rhabdoms in Myrmecia nigriceps stained in toluidine blue can be used to distinguish rhabdoms that are circular or rectangular in shape. Transmission electron micrographs show: (B) multiple orientations of microvilli in the circular rhabdom and (C) microvilli oriented in two opposite directions in the rectangular shaped rhabdom. (D) Using light microscopy, the long axis of the rectangular rhabdoms are mapped to show a fan-like organization in the dorsal region of the eye in a queen of Camponotus consobrinus; inset indicates posterior (p), anterior (a), and lateral (l), medial (m) axes. Panel D adapted from reference20. Scale bars = 10 µm (A), 1 µm (B-C), 100 µm (D). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56339/56339fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Histological and electron microscopy comparison of sensory cell structure; cellular anatomy." class="xfigimg" src="/files/ftp_upload/56339/56339fig5v2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 5"> 
Figure 5: LM and EM images of an ommatidium in a light-adapted eye of Myrmecia tarsata. (A) Longitudinal section of an ommatidium showing the cornea (C), crystalline cone (CC), cone tract (ct), rhabdom (Rh) and primary pigment cells (PPC). (B) Dashed rectangular box in panel A from a different section viewed under a TEM to quantify the narrow width of the cone tract. Adapted from reference21. Scale bars = 10 µm. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56339/56339fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Plant cell structure analysis, microscope images; cell wall, tissue section, cellular patterns observed." class="xfigimg" src="/files/ftp_upload/56339/56339fig6.jpg" data-altupdatedat="1747911167000" data-alt="Figure 6"> 
Figure 6: Common problems with semi-thin and ultra-thin sections (fixation and infiltration, cutting and staining). (A) Poor fixation of tissue due to inadequate penetration (arrow) in a semi-thin section of Pheidole species; (B) ripping during sectioning in Iridomyrmex calvus (semi-thin); (C) perfect staining (left) and over staining (right) with toluidine blue in Myrmecia croslandi; (D) pigment (circle) and tissue ripping during sectioning (arrows) due to poor matching of the resin and tissue density (resin too soft). Folding of the section (asterisks), can happen when collecting sections from the knife boat; (E) poor contrast due to insufficient staining (compare to inset), lead citrate crystals (white arrows) from exposure to CO2, and vertical knife mark (black arrows); (F) holes in the tissue (white arrows) caused by poor fixation in a Melophorus hirsutus compound eye; (G) resin too soft, leads to chitter when sectioning seen as vertical ripples in the section; (H) section too thick (~100 nm) resulted in dark image with poor contrast, contaminated distilled water lead to bacteria and particulate matter scattered throughout the section (white arrows) in Pheidole species. Scale bar = 25 µm (A-B), 10 µm (C-H). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56339/56339fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Techniques for Investigating the Anatomy of the Ant Visual System at JoVE.com

Techniques for Investigating the Anatomy of the Ant Visual System

This article outlines a suite of techniques in light and electron microscopy to study the internal and external eye anatomy of insects. These include several traditional techniques optimized for work on ant eyes, detailed troubleshooting, and suggestions for optimization for different specimens and regions of interest.

This article outlines a suite of techniques in light microscopy (LM) and electron microscopy (EM) which can be used to study the internal and external eye ...

techniques-for-investigating-the-anatomy-of-the-ant-visual-system

Nanyang Technological University

Research

JoVE Journal

Environment

13.2K Views.  Australian National University. Dit artikel beschrijft een reeks van technieken in licht- en elektronenmicroscopie te bestuderen van de interne en externe oog anatomie van insecten. Het gaat hierbij om verschillende traditionele technieken geoptimaliseerd voor werk op mier ogen, gedetailleerde probleemoplossing, en suggesties voor optimalisatie voor verschillende exemplaren en regio's van belang.

Video: Techniques for Investigating the Anatomy of the Ant Visual System

Visual Classical Conditioning in Wood Ants

Several species of insects have become model systems for studying learning and memory formation. Although many studies focus on freely moving animals, studies implementing classical conditioning paradigms with harnessed insects have been important for investigating the exact cues that individuals learn and the neural mechanisms underlying learning and memory formation. Here we present a protocol for evoking visual associative learning in wood ants through classical conditioning. In this paradigm, ants are harnessed and presented with a visual cue (a blue cardboard), the conditional stimulus (CS), paired with an appetitive sugar reward, the unconditional stimulus (US). Ants perform a Maxilla-Labium Extension Reflex (MaLER), the unconditional response (UR), which can be used as a readout for learning. Training consists of 10 trials, separated by a 5-minute intertrial interval (ITI). Ants are also tested for memory retention 10 minutes or 1 hour after training. This protocol has the potential to allow researchers to analyze, in a precise and controlled manner, the details of visual memory formation and the neural basis of learning and memory formation in wood ants.

We present a protocol for the classical conditioning of harnessed ants that permits researchers to study visual learning and memory formation at a level of analysis not possible with freely moving individuals.

Several species of insects have become model systems for studying learning and memory formation. Although many studies focus on freely moving animals, ...

Behavior

The Gateway to the Brain: Dissecting the Primate Eye

The visual system in humans is considered the gateway to the world and plays a principal role in the plethora of sensory, perceptual and cognitive processes. It is therefore not surprising that quality of vision is tied to quality of life . Despite widespread clinical and basic research surrounding the causes of visual disorders, many forms of visual impairments, such as retinitis pigmentosa and macular degeneration, lack effective treatments. Non-human primates have the closest general features of eye development to that of humans. Not only do they have a similar vascular anatomy, but amongst other mammals, primates have the unique characteristic of having a region in the temporal retina specialized for high visual acuity, the fovea1. Here we describe a general technique for dissecting the primate retina to provide tissue for retinal histology, immunohistochemistry, laser capture microdissection, as well as light and electron microscopy. With the extended use of the non-human primate as a translational model, our hope is that improved understanding of the retina will provide insights into effective approaches towards attenuating or reversing the negative impact of visual disorders on the quality of life of affected individuals.

The non-human primate is an important translational species for our understanding of development and aging. The anatomical organization of the primate retina may provide important insights into normal and pathological conditions in humans.

The visual system in humans is considered the  gateway  to the world and plays a principal role in the plethora of sensory, perceptual and cognitive ...

Biology

Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees (Apis mellifera L.)

Honey bees (Apis mellifera L.) are eusocial insects and well known for their complex division of labor and associative learning capability1, 2. The worker bees spend the first half of their life inside the dark hive, where they are nursing the larvae or building the regular hexagonal combs for food (e.g. pollen or nectar) and brood3. The antennae are extraordinary multisensory feelers and play a pivotal role in various tactile mediated tasks4, including hive building5 and pattern recognition6. Later in life, each single bee leaves the hive to forage for food. Then a bee has to learn to discriminate profitable food sources, memorize their location, and communicate it to its nest mates7. Bees use different floral signals like colors or odors7, 8, but also tactile cues from the petal surface9 to form multisensory memories of the food source. Under laboratory conditions, bees can be trained in an appetitive learning paradigm to discriminate tactile object features, such as edges or grooves with their antennae10, 11, 12, 13. This learning paradigm is closely related to the classical olfactory conditioning of the proboscis extension response (PER) in harnessed bees14. The advantage of the tactile learning paradigm in the laboratory is the possibility of combining behavioral experiments on learning with various physiological measurements, including the analysis of the antennal movement pattern.

Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees Apis mellifera L.

In this protocol we show how to condition harnessed honey bees to tactile stimuli and introduce a 2D motion capture technique for analyzing the kinematics of fine-scale antennal sampling pattern.

Honey bees (Apis mellifera L.) are eusocial insects and well known for their complex division of labor and associative learning capability1, 2. The worker ...

Neuroscience

Dissection, Immunohistochemistry and Mounting of Larval and Adult Drosophila Brains for Optic Lobe Visualization

The Drosophila optic lobe, comprised of four neuropils: the lamina, medulla, lobula and lobula plate, is an excellent model system for exploring the developmental mechanisms that generate neural diversity and drive circuit assembly. Given its complex three-dimensional organization, analysis of the optic lobe requires that one understand how its adult neuropils and larval progenitors are positioned relative to each other and the central brain. Here, we describe a protocol for the dissection, immunostaining and mounting of larval and adult brains for optic lobe imaging. Special emphasis is placed on the relationship between mounting orientation and the spatial organization of the optic lobe. We describe three mounting strategies in the larva (anterior, posterior and lateral) and three in the adult (anterior, posterior and horizontal), each of which provide an ideal imaging angle for a distinct optic lobe structure.

Dissection, Immunohistochemistry and Mounting of Larval and Adult Drosophila Brains for Optic Lobe Visualization

This protocol describes three steps to prepare larval and adult Drosophila optic lobes for imaging: 1) brain dissections, 2) immunohistochemistry and 3) mounting. Emphasis is placed on step 3, as distinct mounting orientations are required to visualize specific optic lobe structures.&#160;

The Drosophila optic lobe, comprised of four neuropils: the lamina, medulla, lobula and lobula plate, is an excellent model system for exploring the ...

Developmental Biology

An Explant System for Time-Lapse Imaging Studies of Olfactory Circuit Assembly in Drosophila

~Neurons are precisely interconnected to form circuits essential for the proper function of the brain. The Drosophila olfactory system provides an excellent model to investigate this process since 50 types of olfactory receptor neurons (ORNs) from the antennae and maxillary palps project their axons to 50 identifiable glomeruli in the antennal lobe and form synaptic connections with dendrites from 50 types of second-order projection neurons (PNs). Previous studies mainly focused on identifying important molecules that regulate the precise targeting in the olfactory circuit using fixed tissues. Here, an antennae-brain explant system that recapitulates key developmental milestones of olfactory circuit assembly in culture is described. Through dissecting the external cuticle and cleaning opaque fat bodies covering the developing pupal brain, high quality images of single neurons from live brains can be collected using two-photon microscopy. This allows time-lapse imaging of single ORN axon targeting from live tissue. This approach will help reveal important cell biological contexts and functions of previously identified important genes and identify mechanisms underpinning the dynamic process of circuit assembly.

An Explant System for Time-Lapse Imaging Studies of Olfactory Circuit Assembly in Drosophila

This protocol describes the dissection procedure, culture condition, and live imaging of an antennae-brain explant system for the study of the olfactory circuit assembly.

~Neurons are precisely interconnected to form circuits essential for the proper function of the brain. The Drosophila olfactory system provides an ...

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at the synaptic level. The Active Zone (AZ) is the region at the presynaptic membrane that mediates neurotransmitter release and is composed of a dense collection of scaffold proteins. AZs of Drosophila melanogaster (Drosophila) photoreceptors undergo molecular remodeling after prolonged exposure to natural ambient light. Thus the level of neuronal activity can rearrange the molecular composition of the AZ and contribute to the regulation of the functional output.
Starting from the light exposure set-up preparation to the immunohistochemistry, this protocol details how to quantify the number, the spatial distribution, and the delocalization level of synaptic molecules at AZs in Drosophila photoreceptors. Using image analysis software, clusters of the GFP-fused AZ component Bruchpilot were identified for each R8 photoreceptor (R8) axon terminal. Detected Bruchpilot spots were automatically assigned to individual R8 axons. To calculate the distribution of spot frequency along the axon, we implemented a customized software plugin. Each axon&#39;s start-point and end-point were manually defined and the position of each Bruchpilot spot was projected onto the connecting line between start and end-point. Besides the number of Bruchpilot clusters, we also quantified the delocalization level of Bruchpilot-GFP within the clusters. These measurements reflect in detail the spatially resolved synaptic dynamics in a single neuron under different environmental conditions to stimuli.

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Here we show how to quantify the number and spatial distribution of synaptic active zones in Drosophila melanogaster&#160;photoreceptors, highlighted with a genetically encoded molecular marker, and their modulation after prolonged exposure to light.

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

Analyzing Dendritic Morphology in Columns and Layers

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and columns to facilitate brain wiring during development and information processing in developed animals. Postsynaptic neurons elaborate dendrites in type-specific patterns in specific layers to synapse with appropriate presynaptic terminals. The fly medulla neuropil is composed of 10 layers and about 750 columns; each column is innervated by dendrites of over 38 types of medulla neurons, which match with the axonal terminals of some 7 types of afferents in a type-specific fashion. This report details the procedures to image and analyze dendrites of medulla neurons. The workflow includes three sections: (i) the dual-view imaging section combines two confocal image stacks collected at orthogonal orientations into a high-resolution 3D image of dendrites; (ii) the dendrite tracing and registration section traces dendritic arbors in 3D and registers dendritic traces to the reference column array; (iii) the dendritic analysis section analyzes dendritic patterns with respect to columns and layers, including layer-specific termination and planar projection direction of dendritic arbors, and derives estimates of dendritic branching and termination frequencies. The protocols utilize custom plugins built on the open-source MIPAV (Medical Imaging Processing, Analysis, and Visualization) platform and custom toolboxes in the matrix laboratory language. Together, these protocols provide a complete workflow to analyze the dendritic routing of Drosophila medulla neurons in layers and columns, to identify cell types, and to determine defects in mutants.

Here, we show how to analyze dendritic routing of Drosophila medulla neurons in columns and layers. The workflow includes a dual-view imaging technique to improve the image quality and computational tools for tracing, registering dendritic arbors to the reference column array and for analyzing the dendritic structures in 3D space.

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and ...

Olfactory Experience-Induced Synaptic Remodeling in Drosophila's Juvenile Brain

Experience-Dependent Remodeling of Juvenile Brain Olfactory Sensory Neuron Synaptic Connectivity in an Early-Life Critical Period

Early-life olfactory sensory experience induces dramatic synaptic glomeruli remodeling in the Drosophila juvenile brain, which is experientially dose-dependent, temporally restricted, and transiently reversible only in a short, well-defined critical period. The directionality of brain circuit synaptic connectivity remodeling is determined by the specific odorant acting on the respondent receptor class of olfactory sensory neurons. In general, each neuron class expresses only a single odorant receptor and innervates a single olfactory synaptic glomerulus. In the Drosophila genetic model, the full array of olfactory glomeruli has been precisely mapped by odorant responsiveness and behavioral output. Ethyl butyrate (EB) odorant activates Or42a receptor neurons innervating the VM7 glomerulus. During the early-life critical period, EB experience drives dose-dependent synapse elimination in the Or42a olfactory sensory neurons. Timed periods of dosed EB odorant exposure allow investigation of experience-dependent circuit connectivity pruning in juvenile brain. Confocal microscopy imaging of antennal lobe synaptic glomeruli is done with Or42a receptor-driven transgenic markers that provide quantification of synapse number and innervation volume. The sophisticated Drosophila genetic toolkit enables the systematic dissection of the cellular and molecular mechanisms mediating brain circuit remodeling.

Author Spotlight: Exploring Glial Influence in Experience-Dependent Synaptic Pruning During Critical Periods

We describe here methods for inducing and analyzing olfactory experience-dependent remodeling of antennal lobe synaptic glomeruli in the Drosophila juvenile brain.

Early-life olfactory sensory experience induces dramatic synaptic glomeruli remodeling in the Drosophila juvenile brain, which is experientially ...

Photodiode-Based Optical Imaging for Recording Network Dynamics with Single-Neuron Resolution in Non-Transgenic Invertebrates

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light represents a revolutionary advance for studies of the neural basis of behavior. However, a downside of this development is its tendency to focus investigators on a very small number of &#8220;designer&#8221; organisms (e.g., C. elegans and Drosophila), potentially negatively impacting the pursuit of comparative studies across many species, which is needed for identifying general principles of network function. The present article illustrates how optical recording with voltage-sensitive dyes in the brains of non-transgenic gastropod species can be used to rapidly (i.e., within the time course of single experiments) reveal features of the functional organization of their neural networks with single-cell resolution. We outline in detail the dissection, staining, and recording methods used by our laboratory to obtain action potential traces from dozens to ~150 neurons during behaviorally relevant motor programs in the CNS of multiple gastropod species, including one new to neuroscience &#8211; the nudibranch Berghia stephanieae. Imaging is performed with absorbance voltage-sensitive dyes and a 464-element photodiode array that samples at 1,600 frames/second, fast enough to capture all action potentials generated by the recorded neurons. Multiple several-minute recordings can be obtained per preparation with little to no signal bleaching or phototoxicity. The raw optical data collected through the methods described can subsequently be analyzed through a variety of illustrated methods. Our optical recording approach can be readily used to probe network activity in a variety of non-transgenic species, making it well suited for comparative studies of how brains generate behavior.

This protocol presents a method for imaging neuronal population activity with single-cell resolution in non-transgenic invertebrate species using absorbance voltage-sensitive dyes and a photodiode array. This approach enables a rapid workflow, wherein imaging and analysis can be pursued over the course of a single day.

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light ...

Automated Charting of the Visual Space of Housefly Compound Eyes

This paper describes the automatic measurement of the spatial organization of the visual axes of insect compound eyes, which consist of several thousands of visual units called ommatidia. Each ommatidium samples the optical information from a small solid angle, with an approximate Gaussian-distributed sensitivity (half-width on the order of 1&#730;) centered around a visual axis. Together, the ommatidia gather the visual information from a nearly panoramic field of view. The spatial distribution of the visual axes thus determines the eye's spatial resolution. Knowledge of the optical organization of a compound eye and its visual acuity is crucial for quantitative studies of neural processing of the visual information. Here we present an automated procedure for mapping a compound eye's visual axes, using an intrinsic, in vivo optical phenomenon, the pseudopupil, and the pupil mechanism of the photoreceptor cells. We outline the optomechanical setup for scanning insect eyes and use experimental results obtained from a housefly, Musca domestica, to illustrate the steps in the measurement procedure.

The protocol here describes the measurement of the spatial organization of the visual axes of housefly eyes, mapped by an automatic device, using the pseudopupil phenomenon and the pupil mechanism of the photoreceptor cells.

This paper describes the automatic measurement of the spatial organization of the visual axes of insect compound eyes, which consist of several thousands ...

Techniques for Investigating the Anatomy of the Ant Visual System

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