Name: Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices (Video) | JoVE Journal
Uploaded: 2012-08-24T13:00:00.000+00:00
Duration: 9 min 14 s
Description: Learn calcium imaging in GFP-tagged hypothalamic neurons using red fluorescent indicator dye and confocal microscopy. This method enables spatial and temporal analysis of stimulus-induced calcium signaling in mouse brain slices.

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We thank our colleagues who participated in the work summarized here. This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 894), the DFG Schwerpunktprogramm 1392 'Integrative analysis of olfaction' and by the Volkswagen Foundation (TLZ). TLZ is a Lichtenberg Professor of the Volkswagen Foundation.

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No conflicts of interest declared.

A major question in neuroscience is to understand how the brain processes social information. A predominant source of information necessary for social recognition is encoded by olfactory or pheromonal signals. The detection of these signals by neuronal populations in the nose and the recognition of the signals in the brain, especially the hypothalamus, play a key role in many social processes and influence hormones and other neuroendocrine factors13-16. An essential obstacle of analyzing neuronal responses in ...

There was a typo in the abstract of <a href="http://www.jove.com/video/4213">Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices</a>.

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

<table><tbody><tr><td>Agar</td><td>Sigma</td><td>A1296</td><td></td></tr><tr><td>Fura-red/AM</td><td>Invitrogen</td><td>F-3021</td><td></td></tr><tr><td>Pluronic F-127</td><td>Sigma</td><td>P2443</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Fisher Scientific</td><td>BP231</td><td></td></tr><tr><td>Vibrating-Blade Microtome Hyrax V 50</td><td>Zeiss</td><td>9770170</td><td></td></tr><tr><td>Cooling Device CU 65 for Microtome Hyrax V 50</td><td>Zeiss</td><td>9920120</td><td></td></tr><tr><td>O&lt;sub&gt;2&lt;/sub&gt;/CO&lt;sub&gt;2&lt;/sub&gt; Incubator, CB210-UL</td><td>Binder</td><td>0019389</td><td></td></tr><tr><td>Super glue, Loctite 406TM</td><td>Henckel</td><td>142580</td><td></td></tr><tr><td>Double spatulas, spoon shape</td><td>Bochem</td><td>3182</td><td></td></tr><tr><td>Microspoon spatulas, spoon shape</td><td>Bochem</td><td>3344</td><td></td></tr><tr><td>Spring Scissors, Moria-Vannas-Wolff - 7mm Blades</td><td>Fine Science Tools</td><td>15370-52</td><td></td></tr><tr><td>Spring Scissors, Vannas - 3mm Blades</td><td>Fine Science Tools</td><td>15000-00</td><td></td></tr><tr><td>Wagner Scissors</td><td>Fine Science Tools</td><td>14071-12</td><td></td></tr><tr><td>Medical Forceps, Dumont 7b</td><td>Fine Science Tools</td><td>11270-20</td><td></td></tr><tr><td>Large Rectangular Open Bath Chamber (RC-27)</td><td>Warner Instruments</td><td>64-0238</td><td></td></tr><tr><td>Confocal Microscope BioRad Radiance 2100</td><td>Zeiss</td><td>n.a.</td><td></td></tr></tbody></table>

imaging calcium responses in gfp-tagged neurons of hypothalamic mouse brain slices

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 ...

The overall goal of this procedure is to monitor stimulus induced calcium responses, both in GFP tagged neurons, spatially, and temporally in brain slices using a non-green fluorescent calcium indicator dye. This is accomplished by first extracting the brain of a transgenic mouse expressing GFP in specific cells. The second step is to prepare tissue slices with a microtome.Next, the calcium indicator dye loading solution is pipetted onto the slices to enable the dye to be taken up by the brain cells. The final step is to perform a timestamp recording of fluorescent cells using confocal microscopy. Ultimately, calcium responses in GFP tagged neurons are shown using multicolor calcium imaging analysis.The main advantage of this technique over existing methods of confocal microscopy is that we can image calcium in green fluorescent GFP tech neurons using a redshifted fluorescent calcium indicator die. The huge stroke shift of the fewer red permits us to do multicolor analysis of the red fluorescence in combination with the green GFP using a single excitation wavelengths. We first had the idea for this method when we were confronted with the problem of monitoring calcium signals in neurons that we only could identify using a genetic fluorescent tech, which was the green fluorescence protein.I will now demonstrate a various critical steps of this procedure. Begin the procedure by pouring the heated agar solution into a square Petri dish to a height of one centimeter after cooling and hardening the 4%agar gel is cut into blocks of one cubic centimeter and placed in a bottle for storage. After decapitating the mouse cut the scalp with a single edge razor blade in the central sagittal direction from the frontal bone to the external occipital protuberance.Subsequently further open the cut scalp first in the rostral lateral direction. Then in the ventral direction. Then make two lateral and one rostral cut.In the foramen magnum with small spring scissor carefully cleave off the cranium. At this moment, the occipital inter parietal and parietal bones should be detached. If the dura matter is still present, carefully remove it with a pair of forceps to prevent damage to the brain.Next, remove the squamal bone, the anterior ethmoidal and the frontal foramen. Now the brain can be easily removed with an inverted micro spoon spatula and the cranial nerves can be severed. The brain should land on its ventral side on a cut resistant surface with the ventral surface of the brain down.Use a single edge razor blade to remove the cerebellum. This straight cut surface will be the basis for mounting the brain on a plate to enable slicing of coronal brain sections. Next, glue the brain with the cut surface on the base plate of the microtome using a small amount of the fast curing high performance cyanoacrylate adhesive super glue.Subsequently glue a one cubic centimeter agar gel block at the ventral side of the brain onto the base plate of the microtome to support and fix the brain while slicing. After the bond has dried, put the plate into the bath of the microtome containing six degrees Celsius cold oxygenated extracellular solution. Then cut the brain into slices of 300 microns thickness with low slicing velocity, the coronal brain slices are collected until the whole brain has been sectioned.Now prepare a 20%onic F1 27 in DMSO solution by adding DMSO solution to onic F1 27 powder. Next, dissolve it by directly sonic the solution for two minutes. After that, add five microliters of 20%onic F1 27 solution to a tube of 50 micrograms of cell permeable furred am.Mix the solution with a pipette. Then add 45 microliters of extracellular solution to the mixture and vortex it. Briefly, add an additional 325 microliters of extracellular solution and sonicate the tube for another three minutes.After sonication, add 1.156 milliliters of oxygenated extracellular solution to obtain the final calcium indicator D loading solution. Transfer the coronal brain slices to a six well cell culture plate filled with oxygenated extracellular solution up to six slices per well. Then aspirate the oxygenated extracellular solution from the chambers being careful not to damage the brain slices.Next pipette 750 microliters of the calcium indicator D loading solution directly to each. Well, the brain slices should be covered by the solution containing rore am. After that, incubate the slices in the oxygen carbon dioxide cell culture incubator for 45 to 60 minutes at 37 degrees Celsius.At the end of the incubation, replace the calcium indicator DI loading solution with fresh oxygenated extracellular solution to prevent overloading the cells. To monitor the stimulus induced changes in the fluorescent signal, transfer one of the fure red loaded brain slices to a recording chamber and position it as desired. After mounting the chamber on the confocal microscope setup, perfuse the slice for 10 minutes with oxygenated extracellular solution to remove any surplus of extracellular calcium indicator dye.After exiting the recording chamber, the PERFUSE eight is collected in a vacuum bottle. Then look at the slice through the microscope at low magnification. Make a note of the orientation of the slice and find the area of interest in the slice in our case the hypothalamic area in the brain.Afterward, change to high magnification. Find the cell of interest in the slice by collecting GFP images and simultaneously checking the fluorescence intensity of the URA red signal. Adjust the laser power to a value that allows measurements in the change of URA red fluorescence intensity, and prevents bleaching of the two fluorophores.Next, start to acquire images at the rates between 0.5 to two hertz to collect the fure and GFP signals. This figure shows the confocal image of the GN, RHR Tau GFP neurons in a coronal brain slice. This is the relatively uniform fluorescence signal observed after loading the brain slice with fure am and this is the merged image showing the neurons loaded with the calcium indicator dye.Here are examples of the somatic stimulus induced calcium responses from individual GFP tagged neurons in different hypothalamic brain areas. The distinct calcium signals between the GN RHR expressing neurons of different hypothalamic nuclei can be compared using the area under the curve as an estimate for the total change in calcium in a given cell during the same period Once mastered. This technique can be done in less than 90 minutes, including the preparation of the calcium indicator dye, the loading and the incubation of the slices if it is performed properly.

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The vomeronasal organ (VNO) detects intraspecies chemical signals that convey social and reproductive information. We have performed Ca2+ imaging experiments using transgenic mice expressing G-CaMP2 in VNO tissue. This approach allows us to analyze the complicated response patterns of the vomeronasal neurons to large numbers of pheromone stimuli.

Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor

Source: Bancroft, E. A., et al. Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons. J. Vis. Exp. (2020) This video demonstrates the real-time visualization of calcium influx in ventral midbrain neurons derived from mouse embryos. Using a viral vector to express a calcium indicator, the neurons are imaged under a confocal microscope to track changes in fluorescence intensity during spontaneous and neurotransmitter-induced calcium activity.

Visualization of Calcium Influx in Ventral Midbrain Neurons Derived from Mouse Embryos

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

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.

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

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.

In vivo Calcium Imaging in Mouse Inferior Olive

This protocol describes the surgical exposure of the dorsal root ganglion (DRG) followed by GCaMP3 (genetically-encoded Ca2+ indicator; Green Fluorescent Protein-Calmodulin-M13 Protein 3) Ca2+ imaging of the neuronal ensembles using Pirt-GCaMP3 mice while applying a variety of stimuli to the ipsilateral hind paw.

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

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.

Calcium Imaging in Mouse Superior Colliculus

The present protocol outlines in vivo calcium imaging for measuring the responses of ensembles of lumbar-6 DRG neurons to somatic and visceral stimuli. Thorough comparisons can be made among neurons responding to different stimuli. This protocol is valuable for investigating mechanisms of visceral pain and somatic stimulation, such as acupuncture.

In Vivo Calcium Imaging of Dorsal Root Ganglia Neurons' Response to Somatic and Visceral Stimuli

The dentate gyrus of the hippocampus carries out essential and distinct functions in learning and memory. This protocol describes a set of robust and efficient procedures for in vivo calcium imaging of granule cells in the dentate gyrus in freely moving mice.

In Vivo Calcium Imaging of Granule Cells in the Dentate Gyrus of Hippocampus in Mice

This protocol describes in vivo GCaMP calcium imaging of intact trigeminal ganglion (TG) neurons. This description includes TG exposure surgery, in vivo confocal microscopy imaging of TG neurons, application of somatic stimuli during TG neurons microscopic imaging, and analysis of in vivo GCaMP calcium imaging data.

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Trigeminal Ganglia

Learn calcium imaging in GFP-tagged hypothalamic neurons using red fluorescent indicator dye and confocal microscopy. This method enables spatial and temporal analysis of stimulus-induced calcium signaling in mouse brain slices.

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

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