Name: ex vivo calcium imaging for visualizing brain responses to endocrine signaling in drosophila
Uploaded: 2018-06-02T14:30:00
Description: Watch this Scientific Journal Video about Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila at JoVE.com

This work was supported by Grants-in-Aid for Scientific Research (15K07147, 17K07419) from JSPS (to Hiroshi Ishimoto, Hiroko Sano), an Inamori Foundation Research Grant (to HI), and the Program of the Joint Usage/Research Center for Developmental Medicine, at the Institute of Molecular Embryology and Genetics, Kumamoto University (to HS). We thank Dr. Azusa Kamikouchi and Mr. Daichi Yamada for their help in using the imaging system.

1. Preparation of Larval Brain Explants
<ol>
	<li>Make an imaging chamber. 
	NOTE: Stable recording requires that the brain explants be tethered. Here, a low-cost, hand-made imaging chamber is used in the Ca2+ imaging system.
	<ol>
		<li>Using forceps, scratch the bottom center of a plastic culture dish (35 mm x 10 mm) to create a dent for the ventral ganglion of the larval brain making mounting easier (step 1.3, Figure 1).</li>
		<li>With a toothpick, place a small drop of...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+trisphosphate+and+calcium+signalling.">Inositol trisphosphate and calcium signalling.</a> Nature. 361 (6410), 315-325 (1993).</li><li>Nakai, J., Ohkura, M., Imoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high+signal-to-noise+Ca(2+)+probe+composed+of+a+single+green+fluorescent+protein.">A high signal-to-noise Ca(2+) probe composed of a single green fluorescent protein.</a> Nature Biotechnology. 19 (2), 137-141 (2001).</li><li>Rulifson, E. J., Kim, S. 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The Ca2+ imaging method described here is a useful system for testing the function of hormones in the brain. In vivo, the brain receives hormones from various endocrine organs. In addition, the brain constantly perceives ascending sensory information, which causes spontaneous neuronal activation. This ex vivo system eliminates such noise and yields a better signal/noise ratio than in vivo imaging. In this system, molecules can be tested singly or in combination. In addition to peptid...

Organ-to-organ communication is an evolutionarily conserved strategy for maintaining homeostasis to cope with environmental changes. In humans, a variety of hormones arereleased from the endocrine glands into the circulation. Many of these hormones target the hypothalamus of the brain, which regulates metabolic processes and fundamental behaviors such as feeding1,2. Many hormones have been discovered using mammalian models. However, the mechanisms of their action, especially the interorgan networks in which they participate, remain largely unclear.
Drosophila melanogaster has emerged as a useful model for studying organ-to-organ communication. In insects, many physiological processes are controlled by hormones. Early studies focusing on growth and metamorphosis used large insects. In these studies, the removal or transplantation of specific organs predicted the existence of inter-organ signaling molecules; later, Juvenile hormone (JH), Prothoracicotropic hormone (PTTH), and Ecdysone were biochemically purified3,4,5. A large family of intercellular signaling peptides is thought to be involved in various physiological events during the insect life cycle6,7. Most of these peptides act on G-protein-coupled receptors (GPCRs), although the specific GPCRs were initially difficult to identify using conventional approaches. The publication of the Drosophila whole genome sequence8&#160;was the breakthrough that enabled the identification of Drosophila bioactive peptides based on their homology to those found in other insects. In addition, the receptors for several peptides were identified from GPCRs predicted in the genome using cell-culture-based GPCR-ligand binding assays. Next, expression analyses predicted the organ-to-organ pathways elicited by these peptides and receptors. Notably, many of the putative peptide receptors are expressed in the brain, suggesting that the brain is a major target of peptide hormones9. Moreover, the advanced genetic tools in Drosophila have contributed to the identification of physiological roles of peptide-GPCR combinations. For instance, the GAL4 and LexA-based binary transcriptional systems enable gene knockdown or overexpression in a spatially and temporally controlled manner. GAL4, a transcription factor identified in yeast, binds to a specific cis-regulatory sequence called Upstream Activation Sequence (UAS). In the GAL4/UAS system, the driver line provides tissue-specific or stage-specific GAL4 expression and the responder line carries UAS upstream of the gene of interest or the construct to drive shRNA expression. LexA/LexAop system is based on a similar mechanism. Phenotypic analyses of tissue-specific peptide-knockdown and tissue-specific GPCR-knockdown animals can reveal information about the peptide-GPCR signaling&#39;s mode and site of action. However, the conclusions that can be reached solely by genetic data are limited. On the other hand, once the putative target of a particular peptide hormone is narrowed down to a tissue or cell type, ex vivo calcium imaging in organ explants can be used to elucidate the organ-to-organ communication mediated by peptide-GPCR signaling. Upon the activation of a Gq-coupled GPCR, the intracellular Ca2+ concentration is increased due to the release of Ca2+ from the ER10. In the brain, neural activation also elevates the intracellular Ca2+ levels. Such Ca2+ increases can be detected by a calcium sensor, GCaMP, which undergoes conformational changes in the presence of Ca2+ resulting in fluorescence emission11.
In this article, a calcium imaging method using Drosophila brain explants is described. To test the ability of a peptide to activate specific neurons, a test peptide is applied to the GCaMP-expressing brain explant, and fluorescence changes are monitored by confocal microscopy. The involvement of the GPCR is then confirmed by performing the same assay using a mutant brain lacking the GPCR. This combination of imaging and genetics provides precise information about organ-to-organ communication by peptides-GPCRs. Possible modifications and applications of this protocol are also discussed.

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	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">Tungsten rod</td>
			<td style="width:327px;">A-M systems, Sequim, WA, USA</td>
			<td style="width:165px;">717000</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Blu Tack</td>
			<td>Bostik, Paris, France</td>
			<td>3049100</td>
			<td>putty-like reusable adhesives</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Watch glass, square, 1 5/8 in</td>
			<td>Carolina Biological Supply Company, Burlington, NC, USA</td>
			<td>742300</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PBS</td>
			<td>TAKARA Bio Inc., Kusatsu, Shiga, Japan</td>
			<td>T900</td>
			<td>Phosphated buffered salts</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">CCHa2</td>
			<td>SCRUM Inc., Tokyo, Japan</td>
			<td></td>
			<td>Custum-synthesized peptide</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ghrerin</td>
			<td>Peptide institute Inc., Osaka, Japan</td>
			<td>4372-s</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Nociceptin</td>
			<td>Peptide institute Inc., Osaka, Japan</td>
			<td>4313-v</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Axio Imager A2</td>
			<td>Carl Zeiss, Oberkochen, Germany</td>
			<td>Axio Imager A2</td>
			<td>fluorescence microscope</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Objective W N-Achroplan 20x/0.5 M27</td>
			<td>Carl Zeiss, Oberkochen, Germany</td>
			<td>420957-9900-000</td>
			<td>water-immersion objective lens</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">P-725 PIFOC objective scanner with long travel range</td>
			<td>Physik Instrumente GmbH &#38; Co. KG, Karlsruhe, Germany</td>
			<td>N/A</td>
			<td>piezoelectric-activated lens mover</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Confocal Scanner Unit CSU-W1</td>
			<td>Yokogawa Electric Corporation, Tokyo, Japan</td>
			<td>CSU-W1</td>
			<td>spinning disc confocal head</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">ImagEM C9100-13</td>
			<td>Hamamatsu Photonics, Sizuoka, Japan</td>
			<td>C9100-13</td>
			<td>EM-CCD camera</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">OBIS 488 nm LS 60 mW</td>
			<td>Coherent, Santa Clara, CA, USA</td>
			<td>1178770</td>
			<td>488-nm laser</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">488/568/647 nm Yokogawa dichroic beamsplitter</td>
			<td>Semrock, Rochester, NY, USA</td>
			<td>Di01-T405/488/568/647-13x15x0.5</td>
			<td>dichroic beam splitter</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">528/38 nm BrightLine single-band bandpass filter</td>
			<td>Semrock, Rochester, NY, USA</td>
			<td>FF01-528/38-25</td>
			<td>emission filter</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">&#181;Manager</td>
			<td>Open Imaging, Inc.</td>
			<td>N/A</td>
			<td>https://micro-manager.org</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">ImageJ</td>
			<td>U. S. National Institutes of Health</td>
			<td>N/A</td>
			<td>https://imagej.nih.gov/ij/</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">TurboReg</td>
			<td>Philippe Th&#233;venaz, Biomediccal Imaging Group, Swiss Federal Institute of Technology Lausanne</td>
			<td>N/A</td>
			<td>http://bigwww.epfl.ch/thevenaz/turboreg/</td>
		</tr>
	</tbody>
</table>

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.

Using this protocol, the activation of brain insulin-producing cells (IPCs) by the peptide hormone, CCHa2, was examined. IPCs of the wild-type brain were labeled with GCaMP6s using a combination of dilp2-GAL412 (a gift from Dr. Rulifson) and UAS-GCaMP6s13 (a gift from Dr. Kim). Larval brain explants were treated with a synthetic CCHa2 peptide, and the fluorescence from GCaMP6s was recorded in real time. In this experiment, ...

Watch this Scientific Journal Video about Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila at JoVE.com

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.

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 ...

This method can help answer key questions in the endocrinology field, as well as the neuroscience field, such as our research to review brains responses to endocrine signals. The main advantage of this technique is that you can examine direct effects of endocrine signals on the brain separated from other tissues. To begin this procedure, use forceps to scratch the bottom center of a plastic culture dish to create a dent for easier mounting of the ventral ganglion of the larval brain after dissection.With a toothpick, place a small drop of superglue on either side of the dent, and attach a tungsten rod to the glue. Then, using a small piece of putty-like reusable adhesive, make a circular wall surrounding the dent. To dissect the larval brain, 90 to 120 hours after egg laying collect larvae and wash them at least three times with distilled water to remove adherent food debris.Next, place a larva on a square one and a half inch watch glass filled with ice cold PBS. Use a pair of forceps to gently grab the middle part of the larva. Use another pair of forceps to grab and pull the mouth hooks gently to separate the anterior part of the larva containing the brain from the rest of the body.Then, hold the anterior tip with the forceps and turn the larva inside out. Remove the extraneous tissues attached to the brain, such as imaginal discs, fat bodies, and ring gland. Gently separate the brain from the mouth parts.Afterwards, apply 200 microliters of PBS to the inside of the adhesive ring prepared earlier in the imaging chamber. Gently suck the dissected brain with PBS into a Pasteur pipette, and transfer it to the imaging chamber. In the imaging chamber, grab the muscle fibers extending from the ventral ganglia and insert the brain gently into the dent underneath the tungsten wire.Then, pull the tungsten wire slightly up to place the brain at the correct position for imaging. To acquire calcium fluorescence images, place the imaging chamber containing the brain explant under the microscope. Lower the objective lens until it touches the PBS, and under bright field illumination position the brain and bring it into focus.Switch to fluorescent light and adjust the focus on the GCaMP success labeled cells. Start the acquisition at 250 ms/frame, at a resolution of 512 x 512 pixels in water cooled mode. Then, adjust the exposure time to obtain the fluorescence values within the CCD camera dynamic range, but not lower than 1000 arbitrary units with 16 bit images.Once the imaging parameters are determined, take the images for one minute before peptide administration to detect baseline signal intensities. Subsequently, apply the test peptide directly, by pipetting 100 microliters of the prepared peptide solution into the larval bath. Record the GCaMP success emission for a few minutes.To analyze the data, open the analysis software and use the first frame which was before peptide application as a reference image. Then, select Plugins, TurboReg. Choose the serial image file as the Source, and the reference image as the Target.Next, check Rigid Body and Accurate for the processing method and quality, respectively. Subsequently, click Batch to start image processing. Select multiple regions of interest by using Analyze, Tools, ROI Manager, in image tray.Then, measure the pixel intensity by clicking More, Multi Measure, OK, in the ROI Manager window. In this experiment, Wild type brain was exposed to CCHa2, Ghrelin, and Nociceptin, whereas CCHa2 Receptor mutant brain was exposed to CCHa2. GCaMP6 signals in insulin producing cells were detected by confocal microscopy at 250 ms/frame, and here are the still images of selected time points.The ratio of the change of ROI intensity to baseline signal intensity at each time point was plotted. ROIs where set on cell bodies that were detected in the same focal plane. The solid lines indicate the mean of five to 10 samples, and the dotted lines mark upper and lower limits of the standard error of the mean.The shaded areas indicate the variation of the signals in the experiments. Once mastered, this technique can be done in one hour, if it is preformed properly. While attempting this procedure, it's important to remember to prepare the brain sample quickly, to avoid damage to it.This procedure can be combined with genetics in order to answer additional questions like on receptor specificity. The system used in this protocol is easy to prepare and reusable. Thus, this protocol will be useful for, like in screening.After its development, this technique paved the way for researchers in the field of endocrinology and neuroscience to explore hormonal networks that control brain function in Drosophila.

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Research

JoVE Journal

Neuroscience

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory bulb1-5. Olfactory information in the external world is transmitted to the antennal lobe by olfactory sensory neurons (OSNs), which segregate to distinct regions of neuropil called glomeruli according to the specific olfactory receptor they express. Here, OSN axons synapse with both local interneurons (LNs), whose processes can innervate many different glomeruli, and projection neurons (PNs), which convey olfactory information to higher olfactory brain regions.
Optical imaging of the activity of OSNs, LNs and PNs in the antennal lobe - traditionally using synthetic calcium indicators (e.g. calcium green, FURA-2) or voltage-sensitive dyes (e.g. RH414) - has long been an important technique to understand how olfactory stimuli are represented as spatial and temporal patterns of glomerular activity in many species of insects6-10. Development of genetically-encoded neural activity reporters, such as the fluorescent calcium indicators G-CaMP11,12 and Cameleon13, the bioluminescent calcium indicator GFP-aequorin14,15, or a reporter of synaptic transmission, synapto-pHluorin16 has made the olfactory system of the fruitfly, Drosophila melanogaster, particularly accessible to neurophysiological imaging, complementing its comprehensively-described molecular, electrophysiological and neuroanatomical properties2,4,17. These reporters can be selectively expressed via binary transcriptional control systems (e.g. GAL4/UAS18, LexA/LexAop19,20, Q system21) in defined populations of neurons within the olfactory circuitry to dissect with high spatial and temporal resolution how odor-evoked neural activity is represented, modulated and transformed22-24.
Here we describe the preparation and analysis methods to measure odor-evoked responses in the Drosophila antennal lobe using G-CaMP25-27. The animal preparation is minimally invasive and can be adapted to imaging using wide-field fluorescence, confocal and two-photon microscopes.

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

We describe an established technique to measure and analyze odor-evoked calcium responses in the antennal lobe of living Drosophila melanogaster.

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory ...

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the precise molecular steps as well as the activity of specific neurons in multi-functional nuclei of brain areas such as the hypothalamus remain. This problem includes identification of the molecular components involved in the regulation of various neurohormone signal transduction cascades. Elevations of intracellular Ca2+ play an important role in regulating the sensitivity of neurons, both at the level of signal transduction and at synaptic sites.
New tools have emerged to help identify neurons in the myriad of brain neurons by expressing green fluorescent protein (GFP) under the control of a particular promoter. To monitor both spatially and temporally stimulus-induced Ca2+ responses in GFP-tagged neurons, a non-green fluorescent Ca2+ indicator dye needs to be used. In addition, confocal microscopy is a favorite method of imaging individual neurons in tissue slices due to its ability to visualize neurons in distinct planes of depth within the tissue and to limit out-of-focus fluorescence. The ratiometric Ca2+ indicator fura-2 has been used in combination with GFP-tagged neurons1. However, the dye is excited by ultraviolet (UV) light. The cost of the laser and the limited optical penetration depth of UV light hindered its use in many laboratories. Moreover, GFP fluorescence may interfere with the fura-2 signals2. Therefore, we decided to use a red fluorescent Ca2+ indicator dye. The huge Stokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength. We had previously good results using fura-red in combination with GFP-tagged olfactory neurons3. The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons4. Fura-red based Ca2+ imaging was also successfully combined with GFP-tagged pancreatic &beta;-cells and GFP-tagged receptors expressed in HEK cells5,6. A little quirk of fura-red is that its fluorescence intensity at 650 nm decreases once the indicator binds calcium7. Therefore, the fluorescence of resting neurons with low Ca2+ concentration has relatively high intensity. It should be noted, that other red Ca2+-indicator dyes exist or are currently being developed, that might give better or improved results in different neurons and brain areas.

In this protocol, we update recent progress in imaging Ca2+ signals of GFP-tagged neurons in brain tissue slices using a red fluorescent Ca2+ indicator dye.

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca2+-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and&#160;&#8212; with the addition of its cofactor coelenterazine &#8212; emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca2+-transients and Ca2+-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca2+-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Here we present a novel Ca2+-imaging approach using a bioluminescent reporter. This approach uses a fused construct GFP-aequorin which binds to Ca2+ and emits light, eliminating the need for light excitation. Significantly this method permits long continuous imaging, access to deep brain structures and high temporal resolution.

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of individual cells. Since an increase of intracellular calcium (Ca2+) in the cytosol often occurs because of the electrical activity, or in response to a myriad of other stimuli, this process can be monitored by the imaging of cells loaded with fluorescent calcium-sensitive dyes.&#160; However, it is difficult to image this response in an individual cell type within whole tissue because these dyes are taken up by all cell types within the tissue. In contrast, genetically encoded calcium indicators (GECIs) can be expressed by an individual cell type and fluoresce in response to an increase of intracellular Ca2+, thus permitting the imaging of Ca2+ signaling in entire populations of individual cell types. Here, we apply the use of the GECIs GCaMP3/6 to the mouse neuromuscular junction, a tripartite synapse between motor neurons, skeletal muscle, and terminal/perisynaptic Schwann cells. We demonstrate the utility of this technique in classic ex vivo tissue preparations. Using an optical splitter, we perform dual-wavelength imaging of dynamic Ca2+ signals and a static label of the neuromuscular junction (NMJ) in an approach that could be easily adapted to monitor two cell-specific GECI or genetically encoded voltage indicators (GEVI) simultaneously. Finally, we discuss the routines used to capture spatial maps of fluorescence intensity. Together, these optical, transgenic, and analytic techniques can be employed to study the biological activity of distinct cell subpopulations at the NMJ in a wide variety of contexts.

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

Here we present a protocol to image calcium signaling in populations of individual cell types at the murine neuromuscular junction.

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of ...

Preparing Adult Drosophila melanogaster for Whole Brain Imaging during Behavior and Stimuli Responses

We present a method developed specifically to image the whole Drosophila brain during ongoing behavior such as walking. Head fixation and dissection are optimized to minimize their impact on behavior. This is first achieved by using a holder that minimizes movement hindrances. The back of the fly&#8217;s head is glued to this holder at an angle that allows optical access to the whole brain while retaining the fly&#8217;s ability to walk, groom, smell, taste and see. The back of the head is dissected to remove tissues in the optical path and muscles responsible for head movement artefacts. The fly brain can subsequently be imaged to record brain activity, for instance using calcium or voltage indicators, during specific behaviors such as walking or grooming, and in response to different stimuli. Once the challenging dissection, which requires considerable practice, has been mastered, this technique allows to record rich data sets relating whole brain activity to behavior and stimulus responses.

Preparing Adult Drosophila melanogaster for Whole Brain Imaging during Behavior and Stimuli Responses

We present a method specifically tailored to image the whole brain of adult Drosophila during behavior and in response to stimuli. The head is positioned to allow optical access to the whole brain, while the fly can move its legs and the antennae, the tip of the proboscis, and the eyes can receive sensory stimuli.

We present a method developed specifically to image the whole Drosophila brain during ongoing behavior such as walking. Head fixation and dissection are ...

In vivo Calcium Imaging of Mouse Geniculate Ganglion Neuron Responses to Taste Stimuli

Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using calcium as a proxy for neuronal activity, these techniques provide a way to monitor the responses of individual cells within large neuronal ensembles to a variety of stimuli in real time. We, and others, have applied these techniques to image the responses of individual geniculate ganglion neurons to taste stimuli applied to the tongues of live anesthetized mice. The geniculate ganglion is comprised of the cell bodies of gustatory neurons innervating the anterior tongue and palate as well as some somatosensory neurons innervating the pinna of the ear. Imaging the taste-evoked responses of individual geniculate ganglion neurons with GCaMP has provided important information about the tuning profiles of these neurons in wild-type mice as well as a way to detect peripheral taste miswiring phenotypes in genetically manipulated mice. Here we demonstrate the surgical procedure to expose the geniculate ganglion, GCaMP fluorescence image acquisition, initial steps for data analysis, and troubleshooting. This technique can be used with transgenically encoded GCaMP, or with AAV-mediated GCaMP expression, and can be modified to image particular genetic subsets of interest (i.e., Cre-mediated GCaMP expression). Overall, in vivo calcium imaging of geniculate ganglion neurons is a powerful technique for monitoring the activity of peripheral gustatory neurons and provides complementary information to more traditional whole-nerve chorda tympani recordings or taste behavior assays.

Here we present how to expose the geniculate ganglion of a live, anesthetized laboratory mouse and how to use calcium imaging to measure the responses of ensembles of these neurons to taste stimuli, allowing for multiple trials with different stimulants. This allows for in depth comparisons of which neurons respond to which tastants.

Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to correlate this with local hemodynamics. This protocol uses transgenic mice that have been prepared with a chronic cranial window and express the genetically encoded calcium indicator, RCaMP1.07, under the &#945;-smooth muscle actin promoter to specifically label mural cells, such as vascular smooth muscle cells and ensheathing pericytes. Steps are outlined on how to prepare a tail vein catheter for intravenous injection of fluorescent dyes to trace blood flow, as well as how to measure brain pericyte calcium and local blood vessel hemodynamics (diameter, red blood cell velocity, etc.) by two photon microscopy in vivo through the cranial window in ketamine/xylazine anesthetized mice. Finally, details are provided for the analysis of calcium fluctuations and blood flow movies via the image processing algorithms developed by Barrett et al. 2018, with an emphasis on how these processes can be adapted to other cellular imaging data.

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

This protocol presents steps to acquire and analyze fluorescent calcium images from brain ensheathing pericytes and blood flow data from nearby blood vessels in anesthetized mice. These techniques are useful for studies of mural cell physiology and can be adapted to investigate calcium transients in any cell type.

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to ...

Two-Photon and Wide-Field Calcium Imaging in the Superior Colliculus

Calcium Imaging in Mouse Superior Colliculus

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the emergence of the cerebral cortex. It receives direct inputs from ~30 types of retinal ganglion cells (RGCs), with each encoding a specific visual feature. It remains elusive whether the SC simply inherits retinal features or if additional and potentially de novo processing occurs in the SC. To reveal the neural coding of visual information in the SC, we provide here a detailed protocol to optically record visual responses with two complementary methods in awake mice. One method uses two-photon microscopy to image calcium activity at single-cell resolution without ablating the overlaying cortex, while the other uses wide-field microscopy to image the whole SC of a mutant mouse whose cortex is largely undeveloped. This protocol details these two methods, including animal preparation, viral injection, headplate implantation, plug implantation, data acquisition, and data analysis. The representative results show that the two-photon calcium imaging reveals visually evoked neuronal responses at single-cell resolution, and the wide-field calcium imaging reveals&#160;neural activity across the entire SC. By combining these two methods, one can reveal the neural coding in the SC at different scales, and such combination can also be applied to other brain regions.

Author Spotlight: Unveiling Neural Coding and Mechanisms of Visual Processing in the Superior Colliculus 

This protocol details the procedure for imaging calcium responses in the superior colliculus (SC) of awake mice, including imaging single-neuron activity with two-photon microscopy while leaving the cortex intact in wild-type mice, and imaging the entire SC with wide-field microscopy in partial-cortex mutant mice.

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the ...

Monitoring Epileptiform Events in &lt;em&gt;Drosophila&lt;/em&gt; via Calcium Imaging

Ex Vivo Calcium Imaging for Drosophila Model of Epilepsy

Epilepsy is a neurological disorder characterized by recurrent seizures, partially correlated with genetic origin, affecting over 70 million individuals worldwide. Despite the clinical importance of epilepsy, the functional analysis of neural activity in the central nervous system is still to be developed. Recent advancements in imaging technology, in combination with stable expression of genetically encoded calcium indicators, such as GCaMP6, have revolutionized the study of epilepsy at both brain-wide and single-cell resolution levels. Drosophila melanogaster has emerged as a tool for investigating the molecular and cellular mechanisms underlying epilepsy due to its sophisticated molecular genetics and behavioral assays. In this study, we present a novel and efficient protocol for ex vivo calcium imaging in GCaMP6-expressing adult Drosophila to monitor epileptiform activities. The whole brain is prepared from cac, a well-known epilepsy gene, knockdown flies for calcium imaging with a confocal microscope to identify the neural activity as a follow-up to the bang-sensitive seizure-like behavior assay. The cac knockdown flies showed a higher rate of seizure-like behavior and abnormal calcium activities, including more large spikes and fewer small spikes than wild-type flies. The calcium activities were correlated to seizure-like behavior. This methodology serves as an efficient methodology in screening the pathogenic genes for epilepsy and exploring the potential mechanism of epilepsy at the cellular level.

Author Spotlight: Insights into the Techniques and Findings of Recent Advancements in Epilepsy Research 

Here, we present a protocol for ex vivo calcium imaging in GCaMP6-expressing adult Drosophila to monitor epileptiform activities. The protocol provides a valuable tool for investigating ictal events in adult Drosophila through ex vivo calcium imaging, allowing for exploration of the potential mechanisms of epilepsy at the cellular levels.

Epilepsy is a neurological disorder characterized by recurrent seizures, partially correlated with genetic origin, affecting over 70 million individuals ...