This work was supported by Ochanomizu University, KAKENHI grants (18K14859, 20K15895), JST FOREST Program, Grant Number JPMJFR204G, Research Foundation for Opto-Science and Technology, Kao Research Council for the Study of Healthcare Science, The Japan Association for Chemical Innovation, and TERUMO life science foundation. The author thanks Dr. Takashi Tsuboi, Kazuki Harada, and Akiyo Natsubori for&#160;their supervision of the experiments. The author also thanks members of the laboratory for their support.

All experimental protocols were approved by the Institutional Animal Care and Use Committee of Ochanomizu University, Japan (animal study protocols 22017). All animal experiments were performed according to the guidelines for animal experimentation of Ochanomizu University that conform with the Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions (Ministry of Education, Culture, Sports, Science and Technology, Japan). Efforts were taken to minimize the number of animals used. This study was carried out in compliance with the ARRIVE guidelines.
1. Preparation of transgenic mice
<ol>
	<li>To follow this experiment, use adult male and female BAC GLT-1-G-CaMP7 #817 transgenic mouse line, namely G7NG817 mice24,25 (older than 8 weeks). The background strain of G7NG817 mice25 is C57BL/6J.</li>
	<li>House the mice under a 12 h /12 h light/dark cycle and raise them in groups of up to five mice.</li>
</ol>
2. Catheter creation
<ol>
	<li>Use scissors to adjust the silicon tube (inner diameter 0.5 mm, outer diameter 1.0 mm) to a precise length of 7 cm (Figure 1A-a). 
	NOTE: Prior to the procedures, scissors and silicon tube were sterilized.</li>
	<li>Adhere diminutive plastic beads (inner diameter 3 mm, outer diameter 5 mm) to the tailored tube, ensuring they are situated 3 mm from its terminus (Figure 1A-b). Affix the beads securely using cyanoacrylate glue (medical or standard variants are acceptable).</li>
	<li>Excise a 23 G needle&#39;s apex and subsequently cut 1.5 cm from the needle tip (Figure 1A-c).</li>
	<li>Introduce the sectioned 23 G needle from step 2.3 into the silicon tube&#39;s extremity, opposite to where the beads were affixed (Figure 1A-d).</li>
</ol>
3. Injector creation
<ol>
	<li>Use sterile pliers to excise 1 cm from the needle tip (Figure 1B-a). 
	NOTE: Ensure the orifice at the severed end retains its integrity and does not get deformed, as any distortion could obstruct the injection solution&#39;s flow.</li>
	<li>Couple the adapted 23 G injection needle from step 3.1.1 to a 2.5 mL syringe (Figure 1B-b).</li>
	<li>Segment a silicon tube to the requisite length (a 15 cm silicon tube in this experiment) and sheath it over the 23 G injection needle (Figure 1B-c). 
	NOTE: The volume of solution dispensed will fluctuate based on the tubing&#39;s length. For instance, with a 15 cm tube, approximately 50 &#181;L of solution will linger post experiment.</li>
	<li>Sever the 23 G injection needle 1.5 cm from the needle tip.</li>
	<li>Join the sectioned portion from step 3.4 with the silicon catheter fashioned in step 3.3 (Figure 1B-d). 
	&#8203;NOTE: Ensure the orifices created in steps 3.1 and 3.4 remain undistorted post incision. If rectification is needed, employ pliers to restore its circular configuration.</li>
</ol>
4. Gastric localization
<ol>
	<li>Administer anesthesia to the mouse via 3.0% isoflurane inhalation in a specified chamber. Apply ophthalmic ointment to prevent eye dryness.</li>
	<li>Delicately transfer the anesthetized mouse from the inhalation chamber to the surgical table upon verifying the mouse&#39;s anesthetized state. Provide thermal support throughout the procedure.</li>
	<li>Lay the mouse supine, aligning its mouth proximate to the inhalation apparatus, and modulate the isoflurane concentration from 3.0% to 2.0%. 
	NOTE: Disengage the inferior segment of the isoflurane mask from its superior counterpart and anchor the separated mask (upper segment) to the surgical table using adhesive tape.</li>
	<li>Supplement the mouse&#39;s oral cavity, forelimbs, and hindlimbs to the surgical table using adhesive tape. 
	NOTE: By fixing the mouse to the table, surgical errors due to movement can be prevented even if the mouse is anesthetized.</li>
	<li>Remove the mouse&#39;s hair from the upper left abdomen (150% of the area around the surgical site) by using depilatory cream. Apply a local analgesic topically on the surgery site and wait for 5 min before proceeding.</li>
	<li>Apply a local anesthetic gel to the abdominal skin and wait for 5 to 10 min. Then, disinfect the surgical area 3 times with alternating rounds of iodophor and 70% ethanol. Confirm the depth of anesthesia via a toe-pinch and cover the animal with sterile surgical drapes. Make a skin incision roughly 1.5 cm in length (Figure 1C-b), situated 1 cm to the right of the abdominal median and 5 mm below the xiphoid process (Figure 1C-c). Then, create a 1.5 cm incision in the abdominal wall at the same location as the initial skin incision. 
	NOTE: Exercise caution to avoid inflicting harm to the underlying liver.</li>
	<li>Delicately reposition the left hepatic lobe laterally using blunt-ended forceps, revealing the stomach beneath.</li>
</ol>
5. Catheter insertion
<ol>
	<li>Elevate the stomach and cautiously extricate it through the incision. 
	NOTE: Exercise utmost care to preserve the integrity of adjacent organs, notably the liver, and refrain from exerting undue force on the stomach.</li>
	<li>Use scissors to create a diminutive perforation (approximately 1.5mm in diameter) in the pyloric antrum.</li>
	<li>Introduce the catheter&#39;s terminus into the perforation, ensuring the bead is in direct contact with the pyloric antrum. 
	NOTE: The catheter is disinfected with a 0.1 to 0.5% chlorhexidine gluconate solution prior to insertion.</li>
	<li>Adhere the catheter&#39;s bead to the stomach employing medical-grade cyanoacrylate glue. 
	NOTE: Ensure the stomach remains unattached to surrounding organs.</li>
	<li>Verify the firm attachment of the catheter to the stomach, then delicately reposition the stomach to its innate locale beneath the left hepatic lobe.</li>
	<li>Stitch the abdominal wall, permitting the catheter to extend externally, using a polydioxanone (PDS) suture. After that, close the skin incision in a manner analogous to the abdominal closure.</li>
	<li>Gently reposition the mouse in a sanitized cage. 
	NOTE: Postoperative mice should be singularly housed. Apply a local analgesic topically on the wound closure and follow analgesic plans as recommeneded by the local institutional guidelines.</li>
	<li>Allow a minimum recovery period of 48 h in a standard environment. 
	NOTE: One end of the catheter inserted into the mouse&#39;s stomach is closed with a 23G needle with a sealed tip to prevent the entry of foreign substances and to avoid biting. Since the catheter protruding from the abdomen is short, it prevents the mouse from dragging it through the bedding, which could cause it to open or become entangled.</li>
</ol>
6. Preparation for in vivo transcranial Ca2+ imaging
<ol>
	<li>Administer anesthesia to the mouse using isoflurane (induction at 2%; maintenance between 0.8% and 1.0%).</li>
	<li>Secure the mouse onto a stereotaxic platform using auxiliary ear bars to mitigate the effects of pulsation and respiration. Subsequently, position the mouse under a fluorescence stereo microscope.</li>
	<li>Employ a wideband blue fluorescence filter set (with excitation at 460-490 nm and emission at 520 nm) in conjunction with a mercury light source.</li>
	<li>Capture the images using a camera and process them using the designated imaging software.</li>
	<li>Carefully remove the hair from the scalp using an electric shaver or hair removal cream. 
	NOTE: If using a razor, ensure that it is thoroughly disinfected and be careful not to cut the skin.</li>
	<li>Disinfect the surface of the scalp using a cotton swab or similar tool with a solution of chlorhexidine gluconate diluted to 0.1-0.5%. Next, disinfect with alcohol and repeat this alternating rounds 3x.</li>
	<li>Apply a local anesthetic gel and wait for 5-10 min.</li>
	<li>Prepare to apply dental acrylic cement.
	<ol>
		<li>Use a sterile scalpel blade to cut the scalp. Either completely remove the scalp or make a straight cut from the back of the head to the forehead.</li>
		<li>Use clips to expose the skull by extending any excess skin.</li>
		<li>Use a cotton swab to remove the connective tissue of the periosteum.</li>
		<li>Immediately apply the acrylic cement after removing the periosteum. Act quickly to prevent the skull surface from becoming opaque due to evaporation from the exposed bone.</li>
		<li>Wait for about 5 min for the cement to dry.</li>
	</ol>
	</li>
</ol>
7. Transcranial Ca2+ imaging with intraduodenal glucose administration
<ol>
	<li>Equilibrate both the experimental solution and saline to ambient temperature. Subject the mice to a dietary restriction for a specified duration (e.g., 2 h).</li>
	<li>Purge any residual content within the mouse-side catheter using ~0.03 mL of saline before initiating the injection.</li>
	<li>Aspirate the experimental solution (for instance, a 10% glucose solution prepared in 300 &#181;L of water, complemented with an additional 300 &#181;L of water) into the syringe, then interface it to the catheter. Ascertain the optimal dosage of the solution tailored to the specific experimental objective. 
	NOTE: During this phase, exercise caution to prevent undue strain on the catheter, which might jeopardize the mouse&#39;s internal anatomy. Endeavor to manipulate the mouse-side tubing with utmost gentleness. Additionally, ensure the syringe remains devoid of air post solution aspiration, as entrapped air and bubbles could induce gastrointestinal distension.</li>
	<li>Assess the mouse&#39;s physiological state and activate the imaging software&#39;s recording function by clicking on the recording process button of the software application.</li>
	<li>Capture images at a resolution of 512 x 512 pixels, 16 bit depth, and a frame rate of 10 Hz. 
	NOTE: The recording parameters can be adjusted to align with specific experimental requirements.</li>
	<li>Acquire spontaneous data (e.g., spanning 50 s), followed by the gradual infusion of the experimental solution at a rate tailored to the experiment (here, 0.035 mL/s). 
	NOTE: At the end of the experiment, the mouse will be euthanized promptly by cervical dislocation under isoflurane anesthesia.</li>
</ol>
8. Image data processing and analysis
<ol>
	<li>Bin image data into 64 x 64 pixels.</li>
	<li>Determine hand-drawn regions of interest (ROIs) using ImageJ. Refer to the mouse brain atlas to designate ROIs as the frontal area (M2), the somatosensory area including the Barrel (Somato), the occipital area (Visual), and the retrosplenial region (RSC).</li>
	<li>Use the MATLAB function ReadImageJROI to extract the coordinates corresponding to the ROIs. Access the MATLAB function through the provided link: https://jp.mathworks.com/matlabcentral/fileexchange/32479-readimagejroi</li>
	<li>Compute the average rate of fluorescence intensity change within the ROI using MATLAB.
	<ol>
		<li>Define the fluorescence intensity change rate (&#916;F/F) with equation (1): 
		<img alt="Equation 1" src="/files/ftp_upload/65902/65902eq01.jpg" style="margin: auto;" />&#160; &#160; &#160;(1) 
		Where Ft represents the fluorescence intensity value at a specific time and the average intensity value from the start of recording until 50 s into the injection is denoted as F0.</li>
	</ol>
	</li>
	<li>Account for the delay in the injection solution reaching the organ. Use the average intensity value spanning the three seconds post injection as a baseline. Target waveforms surpassing the baseline intensity value + 1 SD for analysis if they occur 3 s or later post injection.</li>
</ol>

Studying Gut-Brain Interaction via Transcranial Calcium Imaging in Mice

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The authors have no conflicts of interest to disclose.

This article presents how to attach a catheter to the gut and observe transcranial cortex wide Ca2+ signals with transgenic mice that express a genetically encoded Ca2+ indicator. In this experiment, we did not get the imaging data that use a secondary wavelength of the light source. This is because the transgenic mice we employed in this study exhibit a very high fluorescence intensity change rate (~several tens of percent) in response to sensory stimuli due to the properties of the Ca2+ indicator protein (G-CaMP7) employed. Hence, we thought that contamination from hemodynamics was negligible. Furthermore, the neural responses we focused on occurred rapidly within a few seconds, so we considered it unnecessary to account for subsequently generated local functional hyperemia. This catheter implantation method is able to combine with other genetically modified mice to observe neuronal activity in other brain regions and on different timescales. However, if other genetically modified mice are used, the control experiment described above should be performed.
This method of attaching the catheter to the gut is suitable for single-dose injection at low cost and is easy and less invasive than the method proposed earlier29. Traditionally, absorbent cotton is applied to the digestive organ and sutured. However, suturing can impair post-operative recovery in mice. Therefore, we adopted the method of attaching. The reason that we chose plastic beads is they adhered better to the silicone with the cyanoacrylate glue we use.
It has traditionally been postulated that the brain perceives glucose based on the escalation in plasma blood glucose levels and the concomitant hormone release. Consequently, the brain requires several minutes to hours to detect glucose following its absorption from the gut4. Some research has suggested that gut sensing controls mice&#39;s taste preference in addition to oral sweet taste reception1,3,17,22. As an illustrative example, researchers have provided evidence that intragastric (IG) glucose-paired flavored non-nutritive solution and IG nonmetabolizable &#945;-methyl-d-glucopyranoside (MDG)-paired flavored non-nutritive solution can elicit preference for each flavored non-nutritive solution compared to the IG water-paired group. As intraperitoneal glucose injection did not stimulate non-nutritive solution intake30, it was anticipated that gut nutrient sensing would play a crucial role in the food choices of the mice1,19,22.
A recent study has revealed the presence of an electrically excitable cell in the gut, referred to as a &#34;Neuropod cell,&#34; which employs molecular receptors to detect and transduce sensory stimuli onto a vagus nerve via a glutamatergic synapse. While previous studies have primarily focused on the role of GLP-1 in controlling mice&#39;s food feeding, recent investigations have demonstrated that vagus nerve activation by neuropod cells modulates mouse appetite. The vagus nerve subsequently activates the Nucleus Tractus Solitarius, leading to the subsequent activation of dopaminergic neurons19. However, it is unknown if dopaminergic neuron activation also activates the cortex. To categorize cerebral responses following intragastric glucose infusion, we refined the catheter attachment technique for imaging purposes. When employing this technology, it is imperative to exercise caution to prevent organ damage, with particular regard to the liver, which is susceptible to injury during the manipulation required to ascertain the stomach&#39;s location.
Our findings indicate that intragastric glucose injection activates the prefrontal cortex within seconds. However, intragastric glucose administration initiates multiple signaling pathways simultaneously, such as hormonal routes, the vagus nerve, and the spinal afferent nerve. These signals are processed in the brain stem on different timescales. Therefore, future studies should explore how these intricate signals are processed and integrated within the brain.
Moreover, the BAC GLT-1-G-CaMP7 transgenic mouse line we employed in the present study expresses the Ca2+ sensor, G-CaMP7, in both neurons and astrocytes. In principle, the prefrontal Ca2+ elevation obtained by transcranial cortex wide Ca2+ imaging is derived from neurons and/or astrocytes. However, since astrocytes express dopamine receptors if dopamine signaling is involved, it suggests the potential activation of astrocytic IP3/Ca2+ signaling. Since astrocytic IP3/Ca2+ signaling regulates synaptic plasticity, it may also play an important role in changes in sucrose preference and alterations in feeding behavior after glucose reception. Consequently, imaging at cellular resolution using two-photon microscopy would be required to identify whether the response observed in the prefrontal cortex following glucose administration originated from neurons or astrocytes in future studies. Another effective method involves observing cell-specific Ca2+ indicator expression in mice using adeno-associated virus (AAV) through fiber photometry. Furthermore, it is necessary to verify, using electrophysiological techniques, whether the glucose stimulus to the intestine genuinely stimulated the vagus nerve. If neuropod cells are transmitting signals to the vagus nerve by receiving glucose, we anticipate no response from the vagus nerve when an SGLT-1 inhibitor is administered. Finally, validation using various dopamine receptor antagonists would be necessary to confirm whether the vagus nerve projections to the Nucleus Tractus Solitarii (NTS) activated dopaminergic neurons, increasing prefrontal cortex Ca2+ signals. Overall, this system enables a comprehensive investigation of the rapid neural transmission effects from the gut to the brain. It presents a highly promising methodology that could potentially elucidate mechanisms, such as how stress alters sucrose preference regarding gut-brain interactions.

Primarily, gut sensing of glucose occurs via the sweet taste receptors (Tas1r2, Tas1r3) and the sodium-glucose cotransporter 1 (SGLT-1) located in enteroendocrine cells within the intestinal lumen1,2,3,4,5,6. The sensation of glucose in the brain is a process that typically spans from minutes to hours after its absorption from the gut, an occurrence principally based on increased levels of plasma blood sugar and the release of hormones7,8,9,10,11 (e.g., glucagon-like peptide-1 [GLP-1], Peptide YY (PYY), and glucose-dependent insulinotropic polypeptide [GIP]). They reach the arcuate nucleus of the hypothalamus (ARC), where they bind pro-opiomelanocortin (POMC) neurons and agouti-related protein (AgRP) neurons by blood flow and vagus nerve (parasympathetic nerve)12,13,14. The change in the activity of these neurons leads to control of metabolism and feeding behavior15,16.
Recent literature increasingly highlights the significance of gut-brain neural signaling following glucose absorption. This complex pathway can be broadly categorized into two primary mechanisms. The first involves the activation of spinal afferents, ultimately influencing AgRP neuronal activity in the ARC. The second mechanism entails stimulation of the vagus nerve through connections with epithelial cells. Given the multifaceted ways gut epithelial cells can activate the vagus nerve13, its role is considered versatile.
A significant proportion of gut vagus and spinal afferent nerve activities are associated with feeding behaviors17,18,19. However, emerging evidence suggests that certain vagus nerve fibers, projecting to &#34;neuropod cells,&#34; are implicated in glucose preference. Neuropod cells, a subset of enteroendocrine cells, express SGLT-1 on their luminal side and convey sensory stimuli to a vagus nerve via glutamatergic synapses2,20,21,22. Activation of the vagus nerve triggers dopaminergic neurons in the ventral tegmental area (VTA) within seconds19. Notably, inhibiting vagal activation, particularly in the duodenum, using a glutamate antagonist reduces sucrose preference in mice1. This underscores the critical role of neuropod cell activation in modulating food choice behavior post-glucose intake2.
Despite these advancements, a thorough understanding of the rapid intestinal glucose sensing mechanisms and their effect on cortical activities remains a complex enigma. Though brain activation controlled by AgRP neurons does not include the cerebral cortex23, it is known that dopaminergic neurons in the VTA project to the cerebral cortex. However, whether rapid VTA dopaminergic neuronal activation after glucose sensing truly activates the cerebral cortex is unknown. To elucidate this mechanism, we investigated the potential for intragastric glucose administration to rapidly influence cortical Ca2+ dynamics in transgenic mice expressing a genetically encoded calcium indicator.
This paper presents a low-cost and less invasive method to comprehensively understand the changes in cortical activity following intragastric glucose administration using cortex wide Ca2+ imaging in mice Recently, the transcranial cortex wide Ca2+ imaging technique through an intact skull, using transgenic mice expressing genetically encoded calcium indicators, has gained popularity24. Notably, the BAC GLT-1-G-CaMP7 #817 transgenic mouse line (also known as G7NG817 mouse) employed in this study expresses the Ca2+ sensor, G-CaMP7, in neurons and astrocytes25. Due to its high expression density in the cerebral cortex, this transgenic mouse line is especially suitable for transcranial cortex wide Ca2+ imaging using a standard epifluorescence microscope.

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real-time analysis of gut-brain neural communication: cortex wide calcium dynamics in response to intestinal glucose stimulation

Communication between the gastrointestinal tract and the brain after nutrient absorption plays an essential role in food preference, metabolism, and feeding behaviors. Particularly concerning specific nutrients, many studies have elucidated that the assimilation of glucose within gut epithelial cells instigates the activation of many signaling molecules. Hormones such as glucagon-like peptide-1 are renowned as quintessential signaling mediators. Since hormones predominantly influence the brain through circulatory pathways, they slowly modulate brain activity.
However, recent studies have shown two expeditious gut-brain pathways facilitated by the autonomic nervous system. One operates via the spinal afferent neural pathway, while the vagus nerve mediates the other. Consequently, brain responses following glucose assimilation in the gastrointestinal tract are complicated. Moreover, as intestinal stimulation finally induces diverse cortical activities, including sensory, nociceptive, reward, and motor responses, it is necessary to employ methodologies that facilitate the visualization of localized brain circuits and pan-cortical activities to comprehend gut-brain neural transmission fully. Some studies have indicated precipitous alterations in calcium ion (Ca2+) concentrations within the hypothalamus and ventral tegmental area independently through different pathways after intestinal stimulation. However, whether there are changes in cerebral cortex activity has not been known.
To observe cerebral cortex activity after intragastric glucose injection, we developed an imaging technique for real-time visualization of cortex wide Ca2+ dynamics through a fully intact skull, using transgenic mice expressing genetically encoded Ca2+ indicators. This study presents a comprehensive protocol for a technique designed to monitor intestinal stimulation-induced transcranial cortex wide Ca2+ imaging following intragastric glucose injection via an implanted catheter. The preliminary data suggest that administering glucose solution into the gut activates the frontal cortex, which remains unresponsive to water administration.

Transcranial cortex wide Ca2+ imaging with an isoflurane-anesthetized G7NG817 mouse 
We created the catheter following the procedure illustrated in Figure 1. After carefully identifying the position of the stomach, we attached the catheter to that location (Figure 1). After allowing sufficient recovery time following catheter insertion (approximately 48 h), we measured spontaneous neural activity under mild isoflurane anesthesia (0.8-1.0%) to establish the baseline fluorescence intensity changes for subsequent intestinal stimulation observations. As a result, we confirmed the random Ca2+ oscillation patterns that spanned the entire cortex (Figure 2A). Figure 2 provides representative examples of spontaneous brain activity, with the catheters inserted beforehand. We defined the time of the first observable Ca2+ transient peak in the M2 region following the injection as 0.0 s. The pseudo color representation displayed the peak of this Ca2+ transient as the maximum value, with the mean value plus one standard deviation (1 SD) as the minimum value. It was clear that the left and right hemispheres displayed synchronous activity in all regions.
Next, we established ROIs to characterize the waves within each functional area. As shown in Figure 2B, we presented the temporal changes in fluorescence intensity rate for each ROI (Figure 2B). These observed surface Ca2+ oscillations followed a burst suppression pattern, alternating between periods of low activity and high-amplitude fluorescence changes. This activity pattern aligns with previous findings that simultaneously measured cortical surface EEG and Ca2+ dynamics26.
Despite global or regional surface Ca2+ fluctuations between suppression and burst states, the specific cortical region initiating the calcium surge can differ. As previously reported, when a signal stimulates a local cortical region, the area is expected to activate, subsequently driving a global shift in the cortical state27,28.
Changes in cortex Ca2+ dynamics after intragastric glucose injection 
Next, to elucidate how glucose or water administration influences cortical activity, we monitored cortex wide Ca2+ dynamics during and after the injection. As a result, we found alterations in cortical Ca2+ dynamics following intragastric glucose injection. In contrast to the spontaneous activity illustrated in diverse regions of cortical activation, we observed immediate activation in the prefrontal area or the secondary motor cortex (M2) upon direct gut injection of glucose via the catheter, an effect not induced by intragastric water injection (Figure 3A-C). Moreover, we observed that these transient, prominent Ca2+ events tended to occur continuously over approximately 10 s following intragastric glucose injection. Such persistent phenomena were not apparent when water was administered (Figure 3D-F).
The Ca2+ events in each ROI were characterized by calculating the ratio of fluorescence intensity change ratio, using the auditory cortex (Reference) as a reference, at the first peak occurring within 4-8 s after the completion of glucose administration. As a result, a significant change was observed when glucose was injected after comparing the response in the M2 region using the auditory cortex as a reference before and after injection.
In contrast, no such change was observed when water was injected (Figure 3G, water N = 7: before vs. injection, 2.38 &#177; 2.53 vs. 2.05 &#177; 1.84, p &#62; 0.05; glucose N = 7: before vs. injection, 2.48 &#177; 0.97 vs. 3.76 &#177; 2.76, p &#60; 0.01, Wilcoxon signed-rank test). Next, defining the activation levels in each cortical region by the ratio of the value of after injection to before injection, a significant difference was observed only in the M2 region (Figure 3H, M2: 0.92 &#177; 0.89 vs. 1.30 &#177; 0.32, p = 0.006; Somato: 0.95 &#177; 0.076 vs. 1.04 &#177; 0.13, p = 0.27; Visual: 0.92 &#177; 0.3 vs. 1.00 &#177; 0.06, p = 0.89; RSC: 0.97 &#177; 0.27 vs. 1.10 &#177; 0.27, p = 0.60, t-test).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65902/65902fig01.jpg" /> 
Figure 1: Overview of the method. (A) Catheter creation: (a) Cut the silicon tube. (b) Affix a plastic bead to the silicon tube using medical cyanoacrylate glue. (c) Cut the 23 G needle 1.5 cm from the tip. (d) Attach the cut end of the needle to the silicon tube. (B) Injector assembly: (a) Trim a 23 G injection needle by 1 cm. (b) Connect the trimmed injection needle to a 2.5 mL syringe. (c) Sheath the cut section of the injection needle with a silicon tube. (d) Cut the 23 G needle 1.5 cm from the tip. (e) Attach the cut end of the needle to the silicon tube. (C) Surgical procedure: (a) Depilate the surgical area. (b) Incise the skin and the abdominal wall to a length of approximately 1.5 cm. (c) Create a small perforation (around 1.5 mm in diameter) in the pyloric antrum using scissors and insert the catheter (silicon tube). Secure the catheter to the pyloric antrum using medical cyanoacrylate glue. (d) Stitch the abdominal wall and skin using 5/0 silk suture material and needle. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65902/65902fig01large.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/65902/65902fig02.jpg" /> 
Figure 2: Spontaneous cortex wide Ca2+ oscillations in G7NG817 mice with an attached catheter. (A) A representative example of cortex wide Ca2+ oscillations displayed at 0.2 s intervals. Changes in fluorescence intensity are represented with a pseudo color overlay. The time window encircled by the dashed line in B shows the first appearing Ca2+ transient, with its peak set as time 0. The pseudo color representation uses the peak of that Ca2+ transient as the maximum value and the mean +1 SD as the minimum value. (B) Time series changes in fluorescence intensity for each ROI in the example. Line colors correspond to different cortical areas: red represents M2; magenta represents the somatosensory area (Somato); blue represents the visual area (Visual); and green represents the retro splenial region. Salmon colored strip shows the injection time. (C) A single peak within the dashed outline is isolated and displayed in an enlarged view. Abbreviations: SD = standard deviation; ROI = region of interest; RSC = retro splenial region. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65902/65902fig02large.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/65902/65902fig03.jpg" /> 
Figure 3: Cortex wide Ca2+ response during/after glucose or water injection. (A) A representative example of glucose administration displayed at 0.2 s intervals. Changes in fluorescence intensity are represented with a pseudo color overlay. The time window encircled by the dashed line in B shows the first appearing Ca2+ transient after the injection, with its peak set as time 0. The pseudo color representation is based on the peak of the first Ca2+ transient after injection as the maximum value and the mean +1 SD as the minimum value. (B) Time series changes of fluorescence intensity for each ROI in the example. Line colors correspond to different cortical areas: red represents M2, magenta represents the somatosensory area (Somato), blue represents the visual area (Visual), and green represents the retro splenial region. (C) A single peak within the dashed outline is isolated and displayed in an enlarged view. Analyze target calcium wave is the earliest wave that appeared within 4-8 s after injection. (D) A representative example of water injection as glucose control data. (E) Time series changes in fluorescence intensity for each ROI in the example. Line colors are as in B. (F) A single peak within the dashed outline is isolated and displayed in an enlarged view. Analyze target calcium wave is the earliest wave that appeared within 4-8 s after injection. (G) Comparison of M2 spontaneous activity (before) and response after injection (injected). The average fluorescence intensity changes over 50 s before injection, and the peak response in the M2 region between 4 s and 8 s after injection was calculated, using the auditory cortex as a reference. (H) Comparison of activation levels in each cortical region when water or glucose was administered. The ratio of the value of after injection to before injection, as determined in G, was calculated for each region. N.S. represents non-significant (p &#62; 0.05). ** &#60;0.01. Error bars are defined as the standard error of the mean. Abbreviations: SD = standard deviation; ROI = region of interest; RSC = retro splenial region. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65902/65902fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Real-time Analysis of Gut-brain Neural Communication: Cortex wide Calcium Dynamics in Response to Intestinal Glucose Stimulation at JoVE.com

Real-time Analysis of Gut-brain Neural Communication: Cortex wide Calcium Dynamics in Response to Intestinal Glucose Stimulation

Gut-brain communication, facilitated by the vagus nerve, is crucial for communication between the gastrointestinal endocrine system and the brain. However, whether intragastric glucose injection can change cortical activity is still not understood. Here, we offer a comprehensive protocol to observe changes in cortical activity after glucose injection into the duodenum.

Communication between the gastrointestinal tract and the brain after nutrient absorption plays an essential role in food preference, metabolism, and ...

real-time-analysis-gut-brain-neural-communication-cortex-wide-calcium

Nanyang Technological University

Research

JoVE Journal

Neuroscience

585 Views.  Ochanomizu University. Ons onderzoek onthult neurale communicatie tussen de darmen en de hersenen, met name de rol ervan bij voedselvoorkeur. Experimenten waarbij de nervus vagus betrokken was, zijn vaak als een enkele bundel behandeld, maar recent onderzoek heeft aangetoond dat het eigenschappen en orgaanspecificiteit selecteert. Dus ik denk dat het onderzoeken van zijn statistieken, weet je, in de toekomst de aandacht zal trekken.Deze methode voor het bevestigen van een ka...

Video: Real-time analyse van neurale communicatie tussen darmen en hersenen: cortexbrede calciumdynamiek als reactie op intestinale glucosestimulatie

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Simultaneous Calcium Imaging and Glucose Stimulation in Living Zebrafish to Investigate In Vivo &#946;-Cell Function

The pancreatic &#946;-cells sustain systemic glucose homeostasis by producing and secreting insulin according to the blood glucose levels. Defects in &#946;-cell function are associated with hyperglycemia that can lead to diabetes. During the process of insulin secretion, &#946;-cells experience an influx of Ca2+. Thus, imaging the glucose-stimulated Ca2+ influx using genetically encoded calcium indicators (GECIs) provides an avenue to studying &#946;-cell function. Previously, studies showed that isolated zebrafish islets expressing GCaMP6s exhibit significant Ca2+ activity upon stimulation with defined glucose concentrations. However, it is paramount to study how &#946;-cells respond to glucose not in isolation, but in their native environment, where they are systemically connected, vascularized, and densely innervated. To this end, the study leveraged the optical transparency of the zebrafish larvae at early stages of development to illuminate &#946;-cell activity in vivo. Here, a detailed protocol for Ca2+ imaging and glucose stimulation to investigate &#946;-cell function in vivo is presented. This technique allows to monitor the coordinated Ca2+ dynamics in &#946;-cells with single-cell resolution. Additionally, this method can be applied to work with any injectable solution such as small molecules or peptides. Altogether, the protocol illustrates the potential of the zebrafish model to investigate islet coordination in vivo and to characterize how environmental and genetic components might affect &#946;-cell function.

Here, the study presents a protocol for calcium imaging and glucose stimulation of the pancreatic &#946;-cells of the zebrafish in vivo.

The pancreatic &#946;-cells sustain systemic glucose homeostasis by producing and secreting insulin according to the blood glucose levels. Defects in ...

Developmental Biology

In Situ Ca2+ Imaging of the Enteric Nervous System

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ganglionated plexuses housed in the gut wall. Enteric neurons and enteric glia are the only cell types within the enteric ganglia. The activity of enteric neurons and glia is responsible for coordinating intestinal functions. This protocol describes methods for observing the activity of neurons and glia within the intact ENS by imaging intracellular calcium (Ca2+) transients with fluorescent indicator dyes. Our technical discussion focuses on methods for Ca2+ imaging in whole-mount preparations of the myenteric plexus from the rodent bowel. Bulk loading of ENS whole-mounts with a high-affinity Ca2+ indicator such as Fluo-4 permits measurements of Ca2+ responses in individual neurons or glial cells. These responses can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Ca2+ responses in cells of the ENS are recorded using a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Fluorescence measurements obtained using Ca2+ imaging in whole-mount preparations offer a straightforward means of characterizing the mechanisms and potential functional consequences of Ca2+ responses in enteric neurons and glial cells.

In Situ Ca2+ Imaging of the Enteric Nervous System

The enteric nervous system (ENS) is a network of neurons and glia located in the gut wall that controls intestinal reflexes. This protocol describes methods for recording the activity of enteric neurons and glia in live preparations of ENS using Ca2+ imaging.

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ...

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 ...

Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central nervous system. Changes in ENS function are a major cause of GI symptoms and disease and may contribute to GI symptoms reported in neuropsychiatric disorders including autism. It is well established that isolated colon segments generate spontaneous, rhythmic contractions known as Colonic Migrating Motor Complexes (CMMCs). A procedure to analyze the enteric neural regulation of CMMCs in ex vivo preparations of mouse colon is described. The colon is dissected from the animal and flushed to remove fecal content prior to being cannulated in an organ bath. Data is acquired via a video camera positioned above the organ bath and converted to high-resolution spatiotemporal maps via an in-house software package. Using this technique, baseline contractile patterns and pharmacological effects on ENS function in colon segments can be compared over 3-4 hr. In addition, propagation length and speed of CMMCs can be recorded as well as changes in gut diameter and contraction frequency. This technique is useful for characterizing gastrointestinal motility patterns in transgenic mouse models (and in other species including rat and guinea pig). In this way, pharmacologically induced changes in CMMCs are recorded in wild type mice and in the Neuroligin-3R451C mouse model of autism. Furthermore, this technique can be applied to other regions of the GI tract including the duodenum, jejunum and ileum and at different developmental ages in mice.

This article describes a video imaging technique and high-resolution spatiotemporal mapping to identify changes in the neural regulation of colonic motility in adult mice. Subtle effects on gastrointestinal (GI) function can be detected using this approach in isolated tissue preparations to advance our understanding of GI disease.

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central ...

Spatiotemporal Analysis of Microglial Ca2+ Activity at Single-Cell Resolution

Microglia are the sole resident immune cells in the central nervous system. Their morphology is highly plastic, changing depending on their activity. Under homeostatic conditions, microglia possess a highly ramified morphology. This facilitates their monitoring of the surrounding environment through the continuous extending and retracting of their processes. During brain injury and inflammation, however, microglia become activated and undergo dramatic morphological changes, retracting their ramified processes and swelling their cell body. This facilitates activities such as migration and phagocytosis, which microglia undertake to navigate the brain environment to a less pathological state.
This close relationship between microglial morphology and changes in their activity have enabled considerable insights into various microglial functions. However, such morphological and activity changes are themselves phenomena that can result from any number of intracellular signaling pathways. Moreover, the time-lag between stimulus and response, as well as the highly compartmentalized morphology of microglia, make it difficult to isolate the causative mechanisms that underpin function. To solve this problem, we developed a genetically modified mouse line in which a highly sensitive fluorescent Ca2+-indicator protein is specifically expressed in microglia.
After describing methods for in vivo microglial Ca2+ imaging, this paper presents a structured analysis approach that classifies this Ca2+ activity to rationally defined subcellular regions, thus ensuring that the spatial and temporal dimensions of the encoded information are meaningfully extracted. We believe that this approach will provide a detailed understanding of the intracellular signaling rules that govern the diverse array of microglial activities associated with both higher brain functions and pathological conditions.

Spatiotemporal Analysis of Microglial Ca2+ Activity at Single-Cell Resolution

In this paper, we describe a protocol for in vivo imaging of microglial Ca2+ activity and subsequent analysis of its spatiotemporal dynamics. This method enables thorough characterization of how microglia respond to changes in the brain environment, appropriately capturing the fine spatiotemporal scales at which such events occur.

Microglia are the sole resident immune cells in the central nervous system. Their morphology is highly plastic, changing depending on their activity. ...

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 ...

Automated Multimodal Stimulation and Simultaneous Neuronal Recording from Multiple Small Organisms

Fluorescent genetically encoded calcium indicators have contributed greatly to our understanding of neural dynamics from the level of individual neurons to entire brain circuits. However, neural responses may vary due to prior experience, internal states, or stochastic factors, thus generating the need for methods that can assess neural function across many individuals at once. Whereas most recording techniques examine a single animal at a time, we describe the use of wide-field microscopy to scale up neuronal recordings to dozens of Caenorhabditis elegans or other sub-millimeter-scale organisms at once. Open-source hardware and software allow great flexibility in programming fully automated experiments that control the intensity and timing of various stimulus types, including chemical, optical, mechanical, thermal, and electromagnetic stimuli. In particular, microfluidic flow devices provide precise, repeatable, and quantitative control of chemosensory stimuli with sub-second time resolution. The NeuroTracker semi-automated data analysis pipeline then extracts individual and population-wide neural responses to uncover functional changes in neural excitability and dynamics. This paper presents examples of measuring neuronal adaptation, temporal inhibition, and stimulus crosstalk. These techniques increase the precision and repeatability of stimulation, allow the exploration of population variability, and are generalizable to other dynamic fluorescent signals in small biosystems from cells and organoids to whole organisms and plants.

We present a method for the flexible chemical and multimodal stimulation and recording of simultaneous neural activity from many Caenorhabditis elegans worms. This method uses microfluidics, open-source hardware and software, and supervised automated data analysis to enable the measurement of neuronal phenomena such as adaptation, temporal inhibition, and stimulus crosstalk.

Fluorescent genetically encoded calcium indicators have contributed greatly to our understanding of neural dynamics from the level of individual neurons ...

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires careful and in-depth analyses and quantification to capture as much information about the underlying events as possible. Spatial, temporal and intensity parameters intrinsic to Ca2+ signals such as frequency, duration, propagation, velocity and amplitude may provide some biological information required for intracellular signalling. High-resolution Ca2+ imaging typically results in the acquisition of large data files that are time consuming to process in terms of translating the imaging information into quantifiable data, and this process can be susceptible to human error and bias. Analysis of Ca2+ signals from cells in situ typically relies on simple intensity measurements from arbitrarily selected regions of interest (ROI) within a field of view (FOV). This approach ignores much of the important signaling information contained in the FOV. Thus, in order to maximize recovery of information from such high-resolution recordings obtained with Ca2+dyes or optogenetic Ca2+ imaging, appropriate spatial and temporal analysis of the Ca2+ signals is required. The protocols outlined in this paper will describe how a high volume of data can be obtained from Ca2+ imaging recordings to facilitate more complete analysis and quantification of Ca2+ signals recorded from cells using a combination of spatiotemporal map (STM)-based analysis and particle-based analysis. The protocols also describe how different patterns of Ca2+ signaling observed in different cell populations in situ can be analyzed appropriately. For illustration, the method will examine Ca2+ signaling in a specialized population of cells in the small intestine, interstitial cells of Cajal (ICC), using GECIs.

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Genetically encoded Ca2+ indicators (GECIs) have radically changed how in situ Ca2+ imaging is performed. To maximize data recovery from such recordings, appropriate analysis of Ca2+ signals is required. The protocols in this paper facilitate the quantification of Ca2+ signals recorded in situ using spatiotemporal mapping and particle-based analysis.

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires ...

Biology

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from &#946;-cells), glucagon (&#945;-cells) and somatostatin (&#948;-cells). &#946;-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, &#945;-cells and &#948;-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest.
Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of &#946;-cells is minimized.
Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase.
Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.

Here, we present a protocol for imaging and quantifying calcium dynamics in heterogeneous cell populations, such as pancreatic islet cells. Fluorescent reporters are delivered into the peripheral layer of cells within the islet, which is then immobilized and imaged, and per-cell analysis of the dynamics of fluorescence intensity is performed.

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells ...

Real-time Analysis of Gut-brain Neural Communication: Cortex wide Calcium Dynamics in Response to Intestinal Glucose Stimulation

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