This study was supported by the project of National Key R&#38;D Program of China (Project Code no. 2019YFC1709103; no. 2018YFC1707804) and National Natural Science Foundation of China (Project Code no. 81774211; no. 81774432; no. 81801561).

This study was approved by the Ethics Committee of the Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences (reference number D2018-09-29-1). All procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (National Academy Press, Washington, D.C., 1996). Twelve adult Sprague-Dawley male rats (weight 220 &#177; 20 g) were used in this study. Animals [license number SCXK (JING) 2017-0005] were provided by the National Institutes for Food and Drug Control.
1. Innervation of rat cranial dura mater
<ol>
	<li>Perfusions
	<ol>
		<li>Intraperitoneally inject an overdose of tribromoethanol solution (250 mg/kg) to the rat to induce euthanasia.</li>
		<li>Once the breath stops, transcardially perfuse with 100 mL of 0.9% normal saline followed by 250-300 mL of 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4).</li>
		<li>After perfusion, remove the head skin and open the skull to expose the dura mater and dorsal side of the brain. Then dissect out the cranial dura mater along the brainstem to the olfactory bulb in the whole-mount pattern (Figure 1). Perform post-fixation in 4% paraformaldehyde for 2 h, and then cryoprotect in 25% sucrose in 0.1 M PB for more than 24 h at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Fluorescence immunohistochemistry for CGRP and phalloidin labeling 
	NOTE: A combination of fluorescent staining of CGRP and phalloidin was applied to reveal the spatial correlation of dural nerve fibers and blood vessels in the rat cranial dura mater in the whole-mount pattern.
	<ol>
		<li>Rinse the cranial dura mater in 0.1 M PB for about 1 min.</li>
		<li>Incubate the dura mater in a blocking solution containing 3% normal donkey serum and 0.5% Triton X-100 in 0.1 M PB for 30 min.</li>
		<li>Transfer the dura mater into mouse anti-CGRP antibody (1:1000) in 0.1 M PB containing 1% normal donkey serum and 0.5% Triton X-100 overnight at 4 &#176;C11.</li>
		<li>Wash the dura mater three times in 0.1 M PB the following day.</li>
		<li>Incubate the dura mater in a mixed solution of donkey anti-mouse Alexa Fluor 488 secondary antibody (1:500) and phalloidin 568 (1:1000) in 0.1 M PB containing 1% normal donkey serum and 0.5% Triton X-100 for 1.5 h at room temperature (26 &#176;C).</li>
		<li>Wash the dura mater three times in 0.1 M PB.</li>
		<li>Trim the edges and mount it on microscope slides (see Table of Materials).</li>
		<li>Put on coverslips with 50% glycerin before observation.</li>
	</ol>
	</li>
	<li>Observation and recording
	<ol>
		<li>Observe the fluorescent samples under a fluorescent microscope or a confocal imaging system.</li>
		<li>Take the images of the whole-mount dura mater by a fluorescent microscope equipped with a digital camera (4x, NA: 0.13), and use an exposure time of 500 ms. The image mosaics of the dura mater were completed with a software of fluorescent microscope (see Table of Materials).</li>
		<li>Take images of the CGRP-immunoreactive nerve fibers and phalloidin-labeled blood vessels in the dura mater using a confocal microscope. The excitation and emission wavelengths were 488 nm (green) and 594 nm (red). The confocal pinhole is 110 (20x) and 105 &#181;m (40x). The resolution of image capture is 640 x 640 pixels.</li>
		<li>Capture 20 images in 2 &#181;m frames of from each 40 &#181;m thick section and perform single in-focus image integration with a confocal image processing associated software system for 3D analysis as follows: Set Start Focal Plane | Set End focal plane | Set step size | Choose Depth Pattern | Image Capture | Z series.</li>
		<li>Use a photo editing software to adjust the brightness and contrast of images to optimize the visualization. Pay attention to not remove any data from the images.</li>
	</ol>
	</li>
</ol>
2. Retrograde tracing study with FG
<ol>
	<li>Surgical procedures
	<ol>
		<li>Determine the coordinate area of interest in rat cranial dura mater.</li>
		<li>Prepare a 10 &#181;L micro-syringe and test it with liquid paraffin.</li>
		<li>Anesthetize the rats with tribromoethanol solution (150 mg/kg) via intraperitoneal injection. Check for the depth in anesthesia by the lack of response to toe pinch.</li>
		<li>Shave the rat&#39;s head with an electric razor.</li>
		<li>Put blunt ear bars to the rat and place it on the stereotaxic device. Then put the mouth holder and apply ophthalmic ointment on the eyes.</li>
		<li>Clean the surgical site of the head skin using 10% povidone iodine followed by 75% ethanol.</li>
		<li>Make an incision along the midline of the scalp.</li>
		<li>Bluntly remove the periosteum and muscle tissues away from the skull using sterile cotton-tipped applicators (Figure 1A).</li>
		<li>Drill a small hole (~5-7 mm) using a burr drill with a round-tip bit (#106) on the left parietal and temporal bones above the MMA12, and make sure that the cranial dura mater was kept intact (Figure 1B).</li>
		<li>Build a bank around the hole with dental silicate cement to limit the spread of the tracer (Figure 1C).</li>
		<li>Add 2 &#181;L of 2% FG into the hole around MMA with a 10 &#181;L micro-syringe (Figure 1D).</li>
		<li>Cover the hole with a small piece of hemostatic sponge.</li>
		<li>Put a piece of paraffin film on the hole and seal the edges with bone wax to prevent the leakage of tracer and to avoid contaminating to the surrounding tissues.</li>
		<li>Suture the wound with sterile thread.</li>
		<li>Keep the rats in a warm area until they have fully recovered.</li>
		<li>Return the rats back to their cages and add an antibiotic and analgesic into the drinking water.</li>
	</ol>
	</li>
	<li>Perfusions and sections
	<ol>
		<li>After 7 survival days, perfuse these rats as mentioned above in the procedures in section 1.1.</li>
		<li>Dissect out the TG and C1-4 DRGs, then post-fix and cryoprotect them as above mentioned in section 1.1.3 (Figure 1F).</li>
		<li>Cut the TG and DRGs at the thickness of 30 &#181;m on a cryostat microtome system in the sagittal direction and mounted on silane-coated glass slides.</li>
	</ol>
	</li>
	<li>Double immunofluorescences for FG- and CGRP-labeling in the TG and DRGs 
	NOTE: Although the FG-labeling can be directly observed with UV illumination under mercury lamp without additional staining13,14,15,16, the labeled neurons with FG were further examined in TG and cervical DRGs using double immunofluorescences with FG and CGRP for revealing the origins of dural CGRP-immunoreactive nerve fibers in the TG and DRGs.
	<ol>
		<li>Circle the sections with the histochemical pen.</li>
		<li>Incubate the sections for 30 min in a blocking solution containing 3% normal donkey serum and 0.5% Triton X-100 in 0.1 M PB.</li>
		<li>Transfer the samples into the solution of rabbit anti-fluorogold (1:1000) and mouse anti-CGRP antibody (1:1000) in 0.1 M PB containing 1% normal donkey serum and 0.5% Triton X-100 overnight at 4 &#176;C.</li>
		<li>Wash the sections three times in 0.1 M PB the following day.</li>
		<li>Incubate in a mixed solution of donkey anti-rabbit Alexa Fluor 594 (1:500) and donkey anti-mouse Alexa Fluor 488 (1:500) secondary antibody in 0.1 M PB containing 1% normal donkey serum and 0.5% Triton X-100 for 1.5 h at room temperature.</li>
		<li>Wash and apply coverslips to the sections as the procedures in steps 1.2.6 and 1.2.8.</li>
	</ol>
	</li>
	<li>Observation and recording
	<ol>
		<li>Take images of the FG-labeled neurons in TG and DRGs under UV illumination by fluorescent microscope equipped with a digital camera.</li>
		<li>Capture images of the FG- and CGRP-labeled neurons in TG and DRGs under a fluorescent microscope equipped with a digital camera.</li>
		<li>Use the editing software to adjust the brightness and contrast of images and to add labels in the pictures.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

In this study, we have successfully demonstrated the distribution and the origin of CGRP-immunoreactive nerve fibers in the cranial dura mater using immunofluorescence, 3D reconstruction and neural tracing approaches with CGRP antibody and FG neural tracer, providing the histological and chemical evidences to better understand the dural neurovascular network.
As it was known, CGRP plays a critical role in the pathogenesis of migraine4,17. It was shown that increased CGRP can lead to vasodilatation and neurogenic inflammation to cause the peripheral and central sensitization along the trigeminal pathway4,18. CGRP-immunoreactive nerve fibers belong to the unmyelinated peptidergic sensory axons responsible for the transportation of nociceptive signals19. Being consistent with previous studies, here, we clearly demonstrated the distribution of the CGRP-immunoreactive nerve fibers in the cranial dura mater and traced their origin from small- and medium-diameter sensory neurons in the TG and DRGs. These cellular structures could be the sources for synthesizing and releasing CGRP. On the other hand, phalloidin is a specific probe for filamentous actin (F-actin) that is abundant in the smooth muscular and endothelial cells. As a proper candidate, phalloidin was used for labeling the vascular structures and connective tissues10,20,21. Our recent study has shown that, in contrast to alpha smooth muscle actin and CD31, phalloidin is more reliable and sensitive for staining dural arterioles, and is the optimal to combine with CGRP for demonstrating the cranial neurovascular network in detail, which has been published eslewhere10,21.
Neural tract tracing technique is an important tool to investigate the neural origin and termination. In the present study, FG was used for retrograde tracing the origin of CGRP-immunoreactive nerve fibers in the cranial dura mater. Since the cranial dura mater is a thin membrane, the tracer cannot be applied conveniently by the way of injection. Instead, FG was directly added onto the region around MMA in the cranial dura mater according to the method that had been introduced previously22,23, but care must be taken to keep the dura mater intact. Besides, the effort was made to prevent the leakage of FG to the adjacent tissues. Because the FG-labeling can be directly observed under UV illumination of mercury lamp24, by this approach, we checked the site of FG application; it was found that besides the neural propagation, FG was limited in the region circled with dental silicate cement without contaminating the surrounding tissues. The other advantage is that FG-labeled neurons can also be stained in the expected color using immunofluorescence with FG antibody, making it more convenient to be used together with other biomarkers14,15,16. Through this study, we proved that FG not only fits for retrograde tracing the dural innervation but is also a proper candidate to combine with CGRP for determining the chemical characteristics of FG-labeled neurons.
It should be noted here that present methods are preferably used for young animals. At the early stage of rat, the cranial dura mater is more transparent in the whole-mount style. This feature makes it more convenient to visualize the neurovascular structure of the cranial dura mater in a 3D pattern without further transparent treating.
In summary, the present study provides a valuable approach to effectively explore the innervation of the cranial dura mater from the sensory neurons in the TG and cervical DRGs, especially the subtype of small- and medium-diameter sensory neurons with CGRP-immunoreactive expression. From the perspective of methodology, it may provide a valuable reference for further investigating the other kinds of nerve fibers in the cranial dura mater, as well as their origins.

The cranial dura mater is the outermost layer of meninges to protect the brain and contains plentiful blood vessels and different kinds of nerve fibers1,2. Many studies have shown that sensitized cranial dura mater may be the key factor leading to the occurrence of headaches, involving the abnormal vasodilation and innervation3,4,5. Thus, the knowledge of neurovascular structure in the cranial dura mater is important for understanding the pathogenesis of headaches, especially for migraine.
Although the dura innervation has been previously studied with the conventional immunohistochemistry, the spatial correlation of nerve fibers and blood vessels in the cranial dura mater were less studied6,7,8,9. In order to reveal the dural neurovascular structure in more detail, calcitonin gene-related peptide (CGRP) and phalloidin were selected as the markers for respectively staining the dural nerve fibers and blood vessels in the whole-mount cranial dura mater with immunofluorescence and fluorescent histochemistry10. It may be an optimal choice to obtain a three-dimensional (3D) view of neurovascular structure. Additionally, fluorogold (FG) was applied on the area around middle meningeal artery (MMA) in the cranial dura mater to determine the origin of CGRP-immunoreactive nerve fibers, and traced to the trigeminal ganglion (TG) and cervical (C) dorsal root ganglia (DRGs), while the FG-labeled neurons were further examined together with CGRP using immunofluorescence.
The aim of this study was to provide an effective tool for investigating the neurovascular structure in the cranial dura mater for the CGRP-immunoreactive innervation and its origin. By taking the advantage of the transparent whole-mount dura mater and combining the immunofluorescence, retrograde tracing, confocal techniques, and 3D reconstruction, we expected to present a novel 3D view of the neurovascular structure in the cranial dura mater. These methodological approaches may be further served for exploring the pathogenesis of different headaches.

<table><tbody><tr><td>Alexa Fluor 488 donkey anti-mouse IgG (H+L)</td><td>Invitrogen by Thermo Fisher Scientific</td><td>A21202</td><td>Protect from light; RRID: AB_141607</td></tr><tr><td>Brain stereotaxis instrument</td><td>Narishige</td><td>SR-50</td><td></td></tr><tr><td>CellSens Dimension</td><td>Olympus</td><td>Version 1.1</td><td>Software of fluorescent microscope</td></tr><tr><td>Confocal imaging system</td><td>Olympus</td><td>FV1200</td><td></td></tr><tr><td>Fluorogold (FG)</td><td>Fluorochrome</td><td>52-9400</td><td>Protect from light</td></tr><tr><td>Fluorescent imaging system</td><td>Olympus</td><td>BX53</td><td></td></tr><tr><td>Freezing microtome</td><td>Thermo</td><td>Microm International&amp;nbsp;GmbH</td><td></td></tr><tr><td>Olympus FV10-ASW 4.2a</td><td>Olympus</td><td>Version 4.2</td><td>Confocal image processing software system</td></tr><tr><td>Micro Drill</td><td>Saeyang Microtech</td><td>Marathon-N7</td><td></td></tr><tr><td>Mouse anti-CGRP</td><td>Abcam</td><td>ab81887</td><td>RRID: AB_1658411</td></tr><tr><td>Normal donkey serum</td><td>Jackson ImmunoResearch</td><td>017-000-121</td><td></td></tr><tr><td>Phalloidin 568</td><td>Molecular&amp;nbsp;Probes</td><td>A12380</td><td>Protect from light</td></tr><tr><td>Photoshop and&amp;nbsp; Illustration</td><td>Adobe</td><td>CS6</td><td>Photo editing software</td></tr><tr><td>Rabbit anti- Fluorogold</td><td>Abcam</td><td>ab153</td><td>RRID: AB_90738</td></tr><tr><td>Sprague Dawley</td><td>National Institutes for Food and Drug Control</td><td>SCXK (JING) 2014-0013</td><td></td></tr><tr><td>Superfrost plus microscope slides</td><td>Thermo</td><td>#4951PLUS-001</td><td>25x75x1mm</td></tr></tbody></table>

visualizing the calcitonin gene-related peptide immunoreactive innervation of the rat cranial dura mater with immunofluorescence and neural tracing

The aim of this study was to examine the distribution and origin of the calcitonin gene-related peptide (CGRP)-immunoreactive sensory nerve fibers of the cranial dura mater using immunofluorescence, three-dimensional (3D) reconstruction and retrograde tracing technique. Here, the nerve fibers and blood vessels were stained using immunofluorescence and histochemistry techniques with CGRP and fluorescent phalloidin, respectively. The spatial correlation of dural CGRP-immuoreactive nerve fibers and blood vessels were demonstrated by 3D reconstruction. Meanwhile, the origin of the CGRP-immunoreactive nerve fibers were detected by neural tracing technique with fluorogold (FG) from the area around middle meningeal artery (MMA) in the cranial dura mater to the trigeminal ganglion (TG) and cervical (C) dorsal root ganglia (DRGs). In addition, the chemical characteristics of FG-labeled neurons in the TG and DRGs were also examined together with CGRP using double immunofluorescences. Taking advantage of the transparent whole-mount sample and 3D reconstruction, it was shown that CGRP-immunoreactive nerve fibers and phalloidin-labeled arterioles run together or separately forming a dural neurovascular network in a 3D view, while the FG-labeled neurons were found in the ophthalmic, maxillary, and mandibular branches of TG, as well as the C2-3 DRGs ipsilateral to the side of tracer application in which some of FG-labeled neurons presented with CGRP-immunoreactive expression. With these approaches, we demonstrated the distributional characteristics of CGRP-immunoreactive nerve fibers around the blood vessels in the cranial dura mater, as well as the origin of these nerve fibers from TG and DRGs. From the perspective of methodology, it may provide a valuable reference for understanding the complicated neurovascular structure of the cranial dura mater under the physiological or pathological condition.

Neurovascular structure of the cranial dura mater 
After immunofluorescent and fluorescent histochemical staining with CGRP and phalloidin, CGRP-immunoreactive nerve fibers and phalloidin-labeled dural arterioles and connective tissues were clearly demonstrated throughout the whole-mount cranial dura mater in a 3D pattern (Figure 2C,D,E,F). It was shown that both thick and thin CGRP-immunoreactive nerve fibers run in parallel to the dural arterioles, around the vascular wall, or between the blood vessels (Figure 2D,E,F). By taking advantages of the 3D reconstruction, the morphology of dural arterioles and the spatial correlation of the dural CGRP-immunoreactive nerve fibers and arterioles could be clearly demonstrated from different perspectives.
Retrograde-labeled neurons in the TG and DRGs 
Seven days after FG application on the region of MMA in the rat cranial dura mater (Figure 3A), the FG-labeled neurons were detected in TG and cervical DRGs on the ipsilateral side of the tracer application, which was directly observed under the UV illumination of the fluorescent microscopy (Figure 3B,C). FG-labeled neurons were found in all the three branches of TG with higher concentration on the ophthalmic (V1) and maxillary (V2) divisions, and with less on the mandibular division (V3) (Figure 3B). Meanwhile, some of the FG-labeled neurons were also observed in the C2-3 DRGs (Figure 3C).
In addition, double immunofluorescences were also performed with FG and CGRP on the sections of TG and cervical DRGs. According to the diameter of CGRP-immunoreactive neurons in TG and DRGs, most of them were primarily found in the small- and medium-diameter sensory neurons (&#60;50 &#181;m). Some of these type of CGRP-immunoreactive neurons were also labeled with FG in the TG and C2-3 DRGs, indicating that CGRP-immunoreactive nerve fibers in the cranial dura mater originated from these subpopulation of sensory neurons in the TG and DRGs (Figure 4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61742/61742fig01.jpg" /> 
Figure 1: Photographs of main experimental views in the present study. (A) An incision along the midline of the scalp. (B) A hole above the cranial dura mater showing the middle meningeal artery (MMA). (C) A small bank around the hole circled with dental silicate cement for the application of tracer onto the cranial dura mater. (D) Application of fluorogold (FG) into the hole with micro-syringe. (E) The transparent whole-mount dura mater. (F) The outside view of the trigeminal ganglion (TG). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61742/61742fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61742/61742fig02.jpg" /> 
Figure 2: Correlation between calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers and phalloidin (Pha)-labeled arterioles on the whole-mount cranial dura mater. (A) The transparent whole-mount dura mater. (B) The whole-mount dura mater was flattened and mounted on the slide. (C) Distribution of CGRP-immunoreactive nerve fibers and Pha-labeled blood vessels along the middle meningeal artery (MMA). (D,E) The magnified photos from the same areas of d&#160;and e&#160;in panel C. (F) The magnified and adjusted images from panel E with the frame in a 3D pattern. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61742/61742fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61742/61742fig03.jpg" /> 
Figure 3: Distribution of fluorogold (FG)-labeled neurons in the trigeminal ganglion (TG) and cervical (C) dorsal root ganglion (DRG) under UV illumination. (A) The region of dura mater with FG application. (B) Distribution of FG-labeled neurons in the ophthalmic (V1), maxillary (V2), and mandibular (V3) branches of the TG. (C) Distribution of FG-labeled neurons distributed in the C2 DRG. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61742/61742fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61742/61742fig04.jpg" /> 
Figure 4: The representative photographs showing the labeled sensory neurons in trigeminal ganglion (TG) and cervical dorsal root ganglion (DRG) by using double immunofluorescences with fluorogold (FG) and calcitonin gene-related peptide (CGRP). (A,B) The labeled neurons with FG, CGRP, and both FG and CGRP were demonstrated in red, green, and yellow, respectively in TG (A) and DRG (B). (A1-B1), (A2-B2): Panels A and B were separately showed with FG-labeling (A1, B1) and CGRP-labeling (A2, B2).&#160;<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61742/61742fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Visualizing the Calcitonin Gene-Related Peptide Immunoreactive Innervation of the Rat Cranial Dura Mater with Immunofluorescence and Neural Tracing at JoVE.co

Visualizing the Calcitonin Gene-Related Peptide Immunoreactive Innervation of the Rat Cranial Dura Mater with Immunofluorescence and Neural Tracing

Here we present a protocol to visualize spatial correlation of calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers and blood vessels in the cranial dura mater using immunofluorescence and fluorescent histochemistry with CGRP and phalloidin, respectively. In addition, the origin of these nerve fibers was retrograde traced with a fluorescent neural tracer.

The aim of this study was to examine the distribution and origin of the calcitonin gene-related peptide (CGRP)-immunoreactive sensory nerve fibers of the ...

visualizing-calcitonin-gene-related-peptide-immunoreactive

Universidad San Sebastian

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Neuroscience

3.9K Views.  China Academy of Chinese Medical Sciences. Here we present a protocol to visualize spatial correlation of calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers and blood vessels in the cranial dura mater using immunofluorescence and fluorescent histochemistry with CGRP and phalloidin, respectively. In addition, the origin of these nerve fibers was retrograde traced with a fluorescent neural tracer.

Video: Visualizing the Calcitonin Gene-Related Peptide Immunoreactive Innervation of the Rat Cranial Dura Mater with Immunofluorescence and Neural Tracing

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. Since then, the technique was developed further and currently there are many different ways to prepare organotypic cultures. The method presented here was adapted from the one described by Stoppini et al. for the preparation of the slices and from Gogolla et al. for the staining procedure 3,4.
A unique feature of this technique is that it allows you to study different parts of the brain such as hippocampus or cerebellum in their original structure, providing a big advantage over dissociated cultures in which all the cellular organization and neuronal networks are disrupted. In the case of the cerebellum it is even more advantageous because it allows the study of Purkinje cells, extremely difficult to obtain as dissociated primary culture. This method can be used to study certain developmental features of the cerebellum in vitro, as well as for electrophysiological and pharmacological experiments in both wild type and mutant mice.
The method described here was designed to study the effect of apoptotic stimuli such as Fas ligand in the developing cerebellum, using TUNEL staining to measure apoptotic cell death. If TUNEL staining is combined with cell type specific markers, such as Calbindin for Purkinje cells, it is possible to evaluate cell death in a cell population specific manner. The Calbindin staining also serves the purpose of evaluating the quality of the cerebellar cultures.

This method describes the generation of organotypic cerebellar cultures and the effect of certain apoptotic stimuli on the viability of different cerebellar cell types.

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. ...

Investigating the Neural Mechanisms of Aware and Unaware Fear Memory with fMRI

Pavlovian fear conditioning is often used in combination with functional magnetic resonance imaging (fMRI) in humans to investigate the neural substrates of associative learning 1-5. In these studies, it is important to provide behavioral evidence of conditioning to verify that differences in brain activity are learning-related and correlated with human behavior. 
Fear conditioning studies often monitor autonomic responses (e.g. skin conductance response; SCR) as an index of learning and memory 6-8. In addition, other behavioral measures can provide valuable information about the learning process and/or other cognitive functions that influence conditioning. For example, the impact unconditioned stimulus (UCS) expectancies have on the expression of the conditioned response (CR) and unconditioned response (UCR) has been a topic of interest in several recent studies 9-14. SCR and UCS expectancy measures have recently been used in conjunction with fMRI to investigate the neural substrates of aware and unaware fear learning and memory processes 15. Although these cognitive processes can be evaluated to some degree following the conditioning session, post-conditioning assessments cannot measure expectations on a trial-to-trial basis and are susceptible to interference and forgetting, as well as other factors that may distort results 16,17 .
Monitoring autonomic and behavioral responses simultaneously with fMRI provides a mechanism by which the neural substrates that mediate complex relationships between cognitive processes and behavioral/autonomic responses can be assessed. However, monitoring autonomic and behavioral responses in the MRI environment poses a number of practical problems. Specifically, 1) standard behavioral and physiological monitoring equipment is constructed of ferrous material that cannot be safely used near the MRI scanner, 2) when this equipment is placed outside of the MRI scanning chamber, the cables projecting to the subject can carry RF noise that produces artifacts in brain images, 3) artifacts can be produced within the skin conductance signal by switching gradients during scanning, 4) the fMRI signal produced by the motor demands of behavioral responses may need to be distinguished from activity related to the cognitive processes of interest. Each of these issues can be resolved with modifications to the setup of physiological monitoring equipment and additional data analysis procedures. Here we present a methodology to simultaneously monitor autonomic and behavioral responses during fMRI, and demonstrate the use of these methods to investigate aware and unaware memory processes during fear conditioning.

A methodology to investigate the neural mechanisms that support aware and unaware memory processes during fear conditioning is described. This method monitors blood oxygen level dependent (BOLD) functional magnetic resonance imaging, skin conductance response, and unconditioned stimulus expectancy during Pavlovian fear conditioning to assess the neural correlates of distinct memory processes.

Pavlovian fear conditioning is often used in combination with functional magnetic resonance imaging (fMRI) in humans to investigate the neural substrates ...

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the &#947;-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the &#947;-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca2+ traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb.

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose.

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

Dissection of Pelvic Autonomic Ganglia and Associated Nerves in Male and Female Rats

The bilateral major pelvic ganglia (MPG; synonym, pelvic ganglia) are the primary source of postganglionic sympathetic and parasympathetic neurons innervating pelvic organs of rodents; the functionally equivalent structure in humans is the inferior hypogastric plexus. The major pelvic ganglia also provide the route by which lumbar and sacral sensory axons reach the pelvic organs. These complex, mixed ganglia can prove challenging to identify and dissect for further experimental study of normal autonomic mechanisms or to establish preclinical models of disease, injury or visceral pain. Here we describe a protocol to access and visualize these ganglia and their associated nerve tracts. We provide this protocol with schematics for both male and female rats, as the ganglion size and landmarks for identification differ between sexes. The protocol describes removal of the ganglion for in vitro studies, but this method can be integrated into a surgical recovery protocol for experimental interventions (e.g., nerve crush, nerve resection) or for mapping neuronal circuits (e.g., by microinjection of neural tracers). We also demonstrate the primary structures of the ganglion and its associated nerves immediately following dissection and following immunohistochemical staining.

The major pelvic ganglia contain parasympathetic and sympathetic neurons that innervate pelvic organs. Here we describe a dissection method and provide schematics for identification of these ganglia and their associated nerves. These methods can be applied to experimental manipulation of these ganglia in vivo or removal post-mortem for further study.

The bilateral major pelvic ganglia (MPG; synonym, pelvic ganglia) are the primary source of postganglionic sympathetic and parasympathetic neurons ...

Ex Vivo Release of Calcitonin Gene-Related Peptide from the Trigeminovascular System in Rodents

Calcitonin gene-related peptide (CGRP) was first discovered in the 1980s as a splice variant from the calcitonin gene. Since its discovery, its role in migraine pathophysiology has been well established, first by its potent vasodilator properties and subsequently by its presence and function as a neurotransmitter in the sensory trigeminovascular system. The migraine-provoking ability of CGRP gave support to the pharma industry to develop monoclonal antibodies and antagonists inhibiting the effect of CGRP. A new treatment paradigm has proven effective in the prophylactic treatment of migraine. One of the useful tools to further understand migraine mechanisms is the ex vivo model of CGRP release from the trigeminovascular system. It is a relatively simple method that can be used with various pharmacological tools to achieve know-how to further develop new effective migraine treatments. The present protocol describes a CGRP release model and the technique to quantify the effect of pharmacological agents on the amount of CGRP released from the trigeminovascular system in rodents. A procedure describing the experimental approach from euthanasia to the measurement of protein levels is provided. The essential isolation of the trigeminal ganglion and the trigeminal nucleus caudalis from both mice and rats and the preparation of rat dura mater are described in detail. Furthermore, representative results from both species (rats and mice) are presented. The technique is a key tool to investigate the molecular mechanisms involved in migraine pathophysiology by using various pharmacological compounds and genetically modified animals.

Ex Vivo Release of Calcitonin Gene-Related Peptide from the Trigeminovascular System in Rodents

The present protocol describes the ex vivo calcitonin gene-related peptide (CGRP) release model and the strategy to quantify the effect of pharmacological agents on the amount of CGRP released from the trigeminovascular system in rodents.

Calcitonin gene-related peptide (CGRP) was first discovered in the 1980s as a splice variant from the calcitonin gene. Since its discovery, its role in ...

Demonstrating Hairy and Glabrous Skin Innervation in a 3D Pattern Using Multiple Fluorescent Staining and Tissue Clearing Approaches

Skin innervation is an important part of the peripheral nervous system. Although the study of the cutaneous nerve fibers has progressed rapidly, most of the understanding of their distributional and chemical characteristics comes from conventional histochemical and immunohistochemical staining on thin tissue sections. With the development of the tissue clearing technique, it has become possible to view the cutaneous nerve fibers on thicker tissue sections. The present protocol describes multiple fluorescent staining on tissue sections at a thickness of 300 &#181;m from the plantar and dorsal skin of rat hindfoot, the two typical hairy and glabrous skin sites. Here, the calcitonin gene-related peptide labels the sensory nerve fibers, while phalloidin and lymphatic vessel endothelial hyaluronan receptor 1 label the blood and lymphatic vessels, respectively. Under a confocal microscope, the labeled sensory nerve fibers were followed completely at a longer distance, running in bundles in the deep cutaneous layer and freestyle in the superficial layer. These nerve fibers ran in parallel to or surrounded the blood vessels, and lymphatic vessels formed a three-dimensional (3D) network in the hairy and glabrous skin. The current protocol provides a more effective approach to studying skin innervation than the existing conventional methods from the methodology perspective.

The thickness of tissue sections limited the morphological study of the skin innervation. The present protocol describes a unique tissue clearing technique to visualize cutaneous nerve fibers in thick 300 &#181;m tissue sections under confocal microscopy.

Skin innervation is an important part of the peripheral nervous system. Although the study of the cutaneous nerve fibers has progressed rapidly, most of ...

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

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the cytoplasm or the release of intracellular Ca2+ stores. Pirt-GCaMP3-based Ca2+ imaging of primary sensory neurons of the dorsal root ganglion (DRG) in mice offers the advantage of simultaneous measurement of a large number of cells. Up to 1,800 neurons can be monitored, allowing neuronal networks and somatosensory processes to be studied as an ensemble in their normal physiological context at a populational level in vivo. The large number of neurons monitored allows the detection of activity patterns that would be challenging to detect using other methods. Stimuli can be applied to the mouse hindpaw, allowing the direct effects of stimuli on the DRG neuron ensemble to be studied. The number of neurons producing Ca2+ transients as well as the amplitude of Ca2+ transients indicates sensitivity to specific sensory modalities. The diameter of neurons provides evidence of activated fiber types (non-noxious mechano vs. noxious pain fibers, A&#946;, A&#948;, and C fibers). Neurons expressing specific receptors can be genetically labeled with td-Tomato and specific Cre recombinases together with Pirt-GCaMP. Therefore, Pirt-GCaMP3 Ca2+ imaging of DRG provides a powerful tool and model for the analysis of specific sensory modalities and neuron subtypes acting as an ensemble at the populational level to study pain, itch, touch, and other somatosensory signals.

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

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.

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the ...

In Vivo Visualization of the Rat Trigeminal Ganglion With Calcium Indicators

In-Vivo Calcium Imaging of Sensory Neurons in the Rat Trigeminal Ganglion

Genetically encoded calcium indicators (GECIs) enable imaging techniques to monitor changes in intracellular calcium in targeted cell populations. Their large signal-to-noise ratio makes GECIs a powerful tool for detecting stimulus-evoked activity in sensory neurons. GECIs facilitate population-level analysis of stimulus encoding with the number of neurons that can be studied simultaneously. This population encoding is most appropriately done in vivo. Dorsal root ganglia (DRG), which house the soma of sensory neurons innervating somatic and visceral structures below the neck, are used most extensively for in vivo imaging because these structures are accessed relatively easily. More recently, this technique was used in mice to study sensory neurons in the trigeminal ganglion (TG) that innervate oral and craniofacial structures. There are many reasons to study TG in addition to DRG, including the long list of pain syndromes specific to oral and craniofacial structures that appear to reflect changes in sensory neuron activity, such as trigeminal neuralgia. Mice are used most extensively in the study of DRG and TG neurons because of the availability of genetic tools. However, with differences in size, ease of handling, and potentially important species differences, there are reasons to study rat rather than mouse TG neurons. Thus, we developed an approach for imaging rat TG neurons in vivo. We injected neonatal pups (p2) intraperitoneally with an AAV encoding GCaMP6s, resulting in &gt;90% infection of both TG and DRG neurons. TG was visualized in the adult following craniotomy and decortication, and changes in GCaMP6s fluorescence were monitored in TG neurons following stimulation of mandibular and maxillary regions of the face. We confirmed that increases in fluorescence were stimulus-evoked with peripheral nerve block. While this approach has many potential uses, we are using it to characterize the subpopulation(s) of TG neurons changed following peripheral nerve injury.

Author Spotlight: Exploring Peripheral Mechanisms of Neuropathic Pain in Trigeminal Nerve Injury 

Genetically encoded calcium indicators (GECI) enable a robust, population-level analysis of sensory neuron signaling. Here, we have developed a novel approach that allows for in vivo GECI visualization of rat trigeminal ganglia neuron activity.

Genetically encoded calcium indicators (GECIs) enable imaging techniques to monitor changes in intracellular calcium in targeted cell populations. Their ...

Advanced Imaging of Dorsal Root Ganglion Neurons for Sensory Modality Research

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

A technique is described for surgically exposing the dorsal root ganglion (DRG) of the lumbar-6 in a live, anesthetized laboratory mouse, along with the protocol for in vivo calcium imaging of the exposed DRG in response to various visceral and somatic stimuli. Pirt-GCaMP6s mice or C57BL6 mice intrathecally injected with AAV viruses packaged with GCaMP6s were utilized to capture Ca2+ transients. The amplitude of these transients indicates sensitivity to specific sensory modalities. Afferent fibers originate from internal organs, with primary neuronal cell bodies in spinal or vagal ganglia. Studies on visceral nociception and acupuncture analgesia can potentially be conducted on primary sensory neurons using advanced imaging technologies like in vivo calcium imaging, allowing for the recording of neuronal activity ensembles in the intact animal during stimulation or intervention. The responses of DRG neuron ensembles to somatic and visceral stimuli applied to their corresponding receptive fields were recorded. This technique illustrates how neuronal populations react to various types of somatic and visceral stimuli. It is possible to comprehensively compare neuronal ensemble responses to different stimuli, which is a particularly valuable approach in research on visceral pain and segmental mechanisms of somatic stimulation, such as acupuncture.

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.

A technique is described for surgically exposing the dorsal root ganglion (DRG) of the lumbar-6 in a live, anesthetized laboratory mouse, along with the ...

Calcium Imaging of Thoracic Segment T1 DRG for Cardiopulmonary Sensory Neurons

In Vivo Thoracic Dorsal Root Ganglia (DRG) Calcium Imaging and ECG Recording for Studying Peripheral Nerve Stimulation

The dorsal root ganglia (DRG), housing primary sensory neurons, transmit somatosensory and visceral afferent inputs to the dorsal horn of the spinal cord. They play a pivotal role in both physiological and pathological states, including neuropathic and visceral pain. In vivo calcium imaging of DRG enables real-time observation of calcium transients in single units or neuron ensembles. Accumulating evidence indicates that DRG neuronal activities induced by somatic stimulation significantly affect autonomic and visceral functions. While lumbar DRG calcium imaging has been extensively studied, thoracic segment DRG calcium imaging has been less explored due to surgical exposure and stereotaxic fixation challenges. Here, we utilized in vivo calcium imaging at the thoracic1 dorsal root ganglion (T1-DRG) to investigate changes in neuronal activity resulting from somatic stimulations of the forelimb. This approach is crucial for understanding the somato-cardiac reflex triggered by peripheral nerve stimulations (PENS), such as acupuncture. Notably, synchronization of cardiac function was observed and measured by electrocardiogram (ECG), with T-DRG neuronal activities, potentially establishing a novel paradigm for somato-visceral reflex in the thoracic segments.

This study presents a surgical manipulation to expose the T-DRG in anesthetized mice for in vivo calcium imaging, along with synchronous ECG recordings. This method represents a cutting-edge tool for studying peripheral electric nerve stimulation and thoracic visceral organ inputs, as well as their interactions at the primary sensory level.

The dorsal root ganglia (DRG), housing primary sensory neurons, transmit somatosensory and visceral afferent inputs to the dorsal horn of the spinal cord. ...

Visualizing the Calcitonin Gene-Related Peptide Immunoreactive Innervation of the Rat Cranial Dura Mater with Immunofluorescence and Neural Tracing

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Chapters in this video

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Investigating the Neural Mechanisms of Aware and Unaware Fear Memory with fMRI

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Dissection of Pelvic Autonomic Ganglia and Associated Nerves in Male and Female Rats

Ex Vivo Release of Calcitonin Gene-Related Peptide from the Trigeminovascular System in Rodents

Demonstrating Hairy and Glabrous Skin Innervation in a 3D Pattern Using Multiple Fluorescent Staining and Tissue Clearing Approaches

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

In-Vivo Calcium Imaging of Sensory Neurons in the Rat Trigeminal Ganglion

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

In Vivo Thoracic Dorsal Root Ganglia (DRG) Calcium Imaging and ECG Recording for Studying Peripheral Nerve Stimulation