Department of Physiology, School of Medicine, University of Saarland, Homburg, Germany
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Schauer, C., Leinders-Zufall, T. Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices. J. Vis. Exp. (66), e4213, doi:10.3791/4213 (2012).
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 β-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.
1. Preparation of Solution and Agarose Gel
|Name of reagent||Abbreviation||Mol. weight||Conc.||Company||Cat. N°|
|Sodium chloride||NaCl||58.44 g/mol||120 mM||VWR||27810|
|Sodium hydrogen carbonate||NaHCO3||84.01 g/mol||25 mM||Merck||106329|
|Potassium chloride||KCl||74.55 g/mol||5 mM||Merck||104936|
|BES*||C6H15NO5S||213.25 g/mol||5 mM||Sigma||14853|
|Magnesium sulfate, anhydrous||MgSO4||120.37 g/mol||1 mM||Sigma||M7506|
|Calcium chloride dihydrate||CaCl*2H2O||147.02 g/mol||1 mM||Merck||102382|
|D(+)-Glucose monohydrate||C6H12O8*H2O||198.17 g/mol||10 mM||Merck||108342|
* N,N-Bis(2-hydroxyethyl)-2-aminoethansulfonic acid
2. Dissection of the Mouse Brain
Please make sure that all animal experimental procedures are performed in accordance with the guidelines established by the animal welfare committees of the respective institutions.
3. Slicing Coronal Hypothalamic Sections of the Mouse Brain
4. Preparation of the Ca2+ Indicator Dye Loading Solution
A critical step in loading neurons remains often the health of the cells which depends on the amount of damage induced by and the speed of the dissection procedure. Another essential step seems to be the use of fresh Pluronic F-127 solution (see 4.1). It is being recommended to make this solution in the laboratory and not to use a premade solution from a vendor. Depending on the temperature, the humidity and the shelf lifetime of the Pluronic F-127 solution, we noted degradation of olfactory and brain neurons during the Ca2+ loading procedure.
5. Microscopy and Analysis
In this protocol, the fluorescence intensity of GFP, which identifies the cell of interest, and of the Ca2+ indicator dye will be measured simultaneously in brain slices. Thus, the confocal microscope should be equipped with the correct laser, filters and two photomultiplier tubes to collect the two emission signals. GFP and the change in fluorescence intensity of fura-red can be measured using a single excitation wavelength of 488 nm. Emission fluorescence from the fluorophores can be collected using a 522/DF35 nm filter for GFP and a long-pass filter for wavelengths greater than 600 nm for fura-red.
Ca2+ signals can be presented as arbitrary fluorescence units or as values (ΔF/F) of the relative change in fluorescence intensity (ΔF) normalized to the baseline fluorescence (F). This procedure results in a negative deflection when the intracellular Ca2+ concentration increases using fura-red as the Ca2+ indicator dye. To ease the interpretation of the results, we recommend multiplying the ΔF/F values with -1 to obtain positive fluorescence signals to display a rise in Ca2+ (Figure 4C).
To compare results between neurons the amplitude and frequency of the Ca2+ signals are usually analyzed. Yet, some Ca2+ signals do not occur with a regular period or comparable amplitudes. Some signals might be strongly influenced by stochastic processes within the cell. Thus, to quantify the total change in Ca2+ in a given cell and to enable comparison of Ca2+ responses between neurons in different brain regions, analysis of the area-under-the-curve (AUC) is more appropriate. This measure for the amount of Ca2+ encompasses any initial Ca2+ transient, second phases and sustained elevated Ca2+ responses and oscillations. In this case care should be taken to analyze the same time period to enable the comparison between neurons.
6. Representative Results
To start characterizing gonadotropin releasing hormone receptor (GnRHR) expressing neurons in the hypothalamus we made use of transgenic mice that express GFP after Cre-mediated excision in GnRHR-expressing neurons4,11. GFP fluorescent neurons were identified in various brain areas, including the hypothalamus. To investigate the physiological properties of these GnRHR neurons, we first recorded Ca2+ signals in hypothalamic slices using a confocal microscope. First, we obtained coronal brain slices from these mice using the above-described protocol. Figure 1 illustrates the necessary tools, material and steps for excising a mouse brain. The coronal hypothalamic brain slices were cut (Figure 2) and then loaded according to the steps in point 4 of the protocol. Single brain slice of the appropriate area are placed in a recording chamber, secured with a harp (Figure 3) and then imaged using a confocal microscope (see steps 5.1-5.9). Figure 4 shows an example of two individual coronal brain slices identifying single GnRHR-τGFP cell bodies, the fluorescence at rest after loading the brain slice with fura-red/AM and the merged confocal image indicating that the GFP neuron had taken up fura-red sufficiently to enable investigation of stimulus-induced Ca2+ signals in these cells. Using our protocol we initially tested whether GnRHR neurons utilize similar Ca2+ signals for stimulus detection in different areas of the hypothalamus in response to direct activation with GnRH (Figure 4E). However, these signals differed in their waveform depending on stimulus strength and brain area4. To quantify the change in the dynamics of the Ca2+ responses, the area-under-the-curve (AUC) can be calculated as a measure for the increase in intracellular Ca2+ (Figure 4E)4. Studies are currently underway to investigate the molecular basis underlying the Ca2+ waves and oscillations, their dependence on sex and hormonal status of the animal, and whether they can be modulated by other natural stimuli.
Figure 1. Tools, material and steps for excising a mouse brain. A. Tools and materials used for brain dissection: 1, Loctite 406 superglue; 2, micro spoon spatula; 3, single edge razor blade; 4, small and medium spring scissors; 5, blunt forceps; 6, scissors; 7, Petri dish containing agar gel block; 8, base plate for mounting brain into microtome. B-F. Images of some steps being described in point 2 of the protocol. B. Photograph of a mouse head indicating the cutting position of the scalp (red line) and arrows (orange) indicating the direction the skin should be pulled away from the bone (see step 2.4). C. Photograph of the mouse head after the skin is pulled away showing the bone structures (see step 2.4). Cutting direction of the scissor and the direction for breaking open the cranium with the blunt forceps is indicated with either the black dashed arrows or grey broad arrows, respectively. D. Photograph of the mouse brain after eliminating the various bone structures (see step 2.5 - 2.7). E. Photograph of the removal of the brain still connected to the skull via the cranial nerves. F. Photograph of a relatively undamaged mouse brain.
Figure 2. Slicing of coronal hypothalamic sections of the mouse brain. A. Photograph indicating the position of the single edge razor blade for eliminating the cerebellum (see step 3.1). B. Position of agar gel block in relation to the brain glued onto the baseplate of the microtome (see step 3.2). C. Cutting of coronal brain slice (note here the location of brain and gel block positions in regard to the cutting blade of the microtome; see step. 3.4). d, dorsal; v, ventral.
Figure 3. Brain slice positioned in recording chamber. A,B. Overview (A) and higher magnification (B) of a Warner Instruments RC-27 open bath recording chamber giving broad access to the hypothalamic areas of the coronal brain slice (see step 5.1). C. U-shaped metal harp containing parallel arrays of nylon threads which will hold the slice in position in the recording chamber (see step 5.2).
Figure 4. Ca2+ signals in τGFP neurons of mouse hypothalamic brain slices. A-D. Identification of a GFP neuron and simultaneous acquisition of the fura-red fluorescence in coronal mouse brain slices. A. Confocal image of a coronal brain slice identifying GnRHR-τGFP neurons (green). B. Relatively uniform fluorescence signal (red) of the brain area shown in A observed after loading the brain slice with fura-red/AM. C. Merged image showing the neurons depicted in A loaded with the Ca2+ indicator dye (yellowish). Boundaries of GFP neuron somata are indicated in dashed white lines, whereas examples of two non-GFP somata (arrows) are indicated in grey lines. D. Example of a GFP and non-GFP neuron with higher amounts of red fluorescence compared to the background. E. Examples of somatic stimulus-induced Ca2+ responses from individual GFP-tagged neurons in different hypothalamic brain areas (Pe, periventricular nucleus; DM, dorsomedial hypothalamus; Arc, arcuate nucleus). Area-under-the-curve (AUC) is depicted by the red area. The distinct Ca2+ signals between GnRHR-expressing neurons from different hypothalamic nuclei can be compared using the AUC as an estimate for the total change in Ca2+ in a given cell during the same period. F. Schematic drawings and diagram indicating the location of the GFP-tagged neurons analyzed in A-E. Upper panel: location of a coronal brain section containing hypothalamic brain regions (red line). Middle and lower panel: Schematic drawing of a brain slice (middle) and magnification of its red boxed area (lower panel) indicating with red dots the approximate position of the recorded GFP-tagged neurons from the Pe, DM and Arc shown in E; black area in lower panel scheme: 3rd ventricle. Lower two diagrams are adapted from Paxinos and Franklin12. Lower left corner number indicates the distance (mm) from Bregma.
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 brain areas like the hypothalamus containing multifunctional nuclei with multiple neurons is the identification of specific neurons of interest.
Many transgenic mouse lines have been developed, in which mainly GFP helps to identify neurons. Unfortunately, the fluorescent property of GFP complicates measurements using Ca2+ indicator dyes such as fluo-3 and fluo-417. We therefore started to investigate GFP-tagged neurons using a red-shifted Ca2+ indicator dye, like fura-red3,4. Fura-red based Ca2+ imaging was previously combined with GFP-tagged pancreatic β-cells and GFP-tagged receptors expressed in HEK cells5,6. Like fura-2, some cross talk between fura-red and GFP has been reported2. Yet, using high quality emission filter sets with specific bandwidths and dichromatic beamsplitter filters can limit the crosstalk/bleed-through between the dyes to some extent. It is wise to confirm the settings of a confocal microscope by measuring both channels (green and red fluorescence) at the same time using (1) brain slices with GFP-tagged neurons, but not loaded with the Ca2+ indicator dye, as well as (2) brain slices without GFP-tagged neurons but loaded with the red-shifted Ca2+ indicator. Using the same settings on the confocal microscope as with a regular experiment, the investigator can then note if any fluorescence is detected in the disparate channel, which should be avoided.
Some investigators use rhod-2 or recommend the use of X-rhod1 as an alternative red-shifted dye in combination with GFP-tagged cells2,18,19. However, rhod AM dyes seem to have a tendency to concentrate in mitochondria20 and have in many neurons low labeling efficiency17. Due to the need of improved red-shifted Ca2+-indicators, investigators and companies are currently developing new probes with hopefully superior performance21.
No conflicts of interest declared.
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.
|Dimethyl sulfoxide||Fisher Scientific||BP231|
|Vibrating-Blade Microtome Hyrax V 50||Zeiss||9770170|
|Cooling Device CU 65 for Microtome Hyrax V 50||Zeiss||9920120|
|O2/CO2 Incubator, CB210-UL||Binder||0019389|
|Super glue, Loctite 406TM||Henckel||142580|
|Double spatulas, spoon shape||Bochem||3182|
|Microspoon spatulas, spoon shape||Bochem||3344|
|Spring Scissors, Moria-Vannas-Wolff - 7mm Blades||Fine Science Tools||15370-52|
|Spring Scissors, Vannas - 3mm Blades||Fine Science Tools||15000-00|
|Wagner Scissors||Fine Science Tools||14071-12|
|Medical Forceps, Dumont 7b||Fine Science Tools||11270-20|
|Large Rectangular Open Bath Chamber (RC-27)||Warner Instruments||64-0238|
|Confocal Microscope BioRad Radiance 2100||Zeiss||n.a.|
Formal Correction: Erratum: Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices
Posted by JoVE Editors on 10/01/2012. Citeable Link.
There was a typo in the abstract of Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices. 'Stokes' was spelt with an 'r' at publication. This has been corrected to
The huge Stokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength.
The huge Strokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength.