Name: intracerebroventricular delivery of gut-derived microbial metabolites in freely moving mice
Uploaded: 2022-06-02T14:15:00
Description: Watch this Scientific Journal Video about Intracerebroventricular Delivery of Gut-Derived Microbial Metabolites in Freely Moving Mice at JoVE.com

We acknowledge the Laboratory Animal Center staff at National Cheng Kung University (NCKU) for caring for the animals. This work was supported by the scholarship from Prof. Kun-Yen Huang Education Fund of CHENG-HSING Medical Foundation to C.-W.L.; the funds from the Ministry of Science and Technology (MOST) in Taiwan: (Undergraduate Research MOST 109-2813-C-006-095-B) to T.-H.Y.; (MOST 107-2320-B-006-072-MY3; 109-2314-B-006-046; 110-2314-B-006-114; 110-2320-B-006-018-MY3) to W.-L.W.; and the Higher Education Sprout Project, Ministry of Education to the Headquarters of University Advancement at NCKU to W.-L.W.

All the experimental protocols and the animals&#39; care were approved by the National Cheng Kung University (NCKU) Institutional Animal Care and Use Committee (IACUC).
1. Preparation for the experimental animal
<ol>
	<li>Obtain 6-8-week-old wild-type C57BL/6JNarl male mice from a vendor.</li>
	<li>House the mice in a standard mouse cage with standard mouse chow and sterilized water ad libitum. 
	&#8203;NOTE: The housing conditions for the Laboratory Animal Cen...

Gut-derived metabolites have been associated with brain-mediated diseases without much precise mechanism, partially due to their multiple binding sites in the body6,12,24. Previous reports indicated that SCFAs could serve as ligands for G protein-coupled receptors, epigenetic regulators, and sources for energy production at multiple sites in the body6,12. To bypass the c...

The human gastrointestinal tract harbors diverse microorganisms impacting the host1,2,3. These gut bacteria can secrete gut-derived metabolites during their utilization of dietary components consumed by the host4,5. Interestingly, the gut metabolites not metabolized in the periphery can be transported to other organs via circulation6. Of note, these secreted metabolites can serve as mediators for the gut-brain axis, defined as the bidirectional communication between the central nervous system and the gut7. Previous studies have shown that gut-derived metabolites can modulate complex behavior and emotion in animals8,9,10,11.
Short-chain fatty acids (SCFAs) are the main metabolites produced by gut microbiota during the fermentation of dietary fiber and indigestible carbohydrates6. Acetate, propionate, and butyrate are the most abundant SCFAs in the gut12. SCFAs serve as the energy source for cells in the gastrointestinal tract. Unmetabolized SCFAs in the gut can be transported to the brain through the portal vein, thus modulating brain and behavior6,12. Previous studies have suggested that SCFAs might play a critical role in neuropsychiatric disorders6,12. For example, intraperitoneal injection of butyrate in BTBR T+ Itpr3tf/J (BTBR) mice, an animal model of autism spectrum disorder (ASD), rescued their social deficits13. Antibiotic-treated rats receiving microbiota from depressive subjects showed an increase in anxiety-like behaviors and fecal SCFAs14. Clinically, alterations in fecal SCFAs levels were observed in people with ASD compared to typically developing controls15,16. People with depression have lower fecal SCFAs levels than healthy subjects17,18. These studies suggested that SCFAs can alter behavior in animals and humans through various routes.
Microbial metabolites exert diverse effects on multiple sites in the body, impacting host physiology and behaviors4,19, including the gastrointestinal tract, vagus nerve, and sympathetic nerve. It is difficult to pinpoint the precise role of gut-derived metabolites in the brain when administering the metabolites via peripheral routes. This paper presents a video-based protocol to investigate the effects of gut-derived metabolites in the brain of a freely moving mouse (Figure 1). We showed that SCFAs could be acutely given through the guide cannula during behavioral tests. The type, volume, and infusion rate of metabolites can be modified depending on the purpose. The site of cannulization can be adjusted to explore the impact of gut metabolites in a specific brain region. We aim to provide scientists with a method to explore the potential impact of gut-derived microbial metabolites on the brain and behavior.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Advil Liqui-Gels Solubilized Ibuprofen A2:D41</td>
			<td>Pfizer</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 donkey anti-rabbit</td>
			<td>ThermoFisher Scientific</td>
			<td>A-21206</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Fluorescent Gold (rabbit polyclonal)</td>
			<td>Millipore</td>
			<td>AB153-I</td>
			<td></td>
		</tr>
		<tr>
			<td>Bottle Top Vacuum Filter, 500 mL, 0.22 &#956;m, PES, Sterile</td>
			<td>NEST</td>
			<td>121921LA01</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C1016</td>
			<td>ACSF: 0.14 g/L</td>
		</tr>
		<tr>
			<td>Chlorhexidine scrub 2%</td>
			<td>Phoenix</td>
			<td>NDC 57319-611-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Chlorhexidine solution</td>
			<td>Phoenix</td>
			<td>NDC 57319-599-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Commercial dummy</td>
			<td>RWD Life Science</td>
			<td>62004</td>
			<td>Single_OD 0.20 mm/ M3.5/G = 0.5 mm</td>
		</tr>
		<tr>
			<td>Commercial guide cannul</td>
			<td>RWD Life Science</td>
			<td>62104</td>
			<td>Single_OD 0.41 mm-27G/ M3.5/C = 2.5 mm&#160;</td>
		</tr>
		<tr>
			<td>Commercial injector</td>
			<td>RWD Life Science</td>
			<td>62204</td>
			<td>Single_OD 0.21 mm-33G/ Mates with M3.5/C = 3.5 mm/G = 0.5 mm</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td>ACSF: 0.61 g/L</td>
		</tr>
		<tr>
			<td>Dental acrylic</td>
			<td>HYGENIC</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixing screws</td>
			<td>RWD Life Science</td>
			<td>62521</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoroshield mounting medium with DAPI</td>
			<td>Abcam</td>
			<td>AB104139</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>ThermoFisher Scientific</td>
			<td>16050130</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin syringes</td>
			<td>BBraun</td>
			<td>XG-LBB-9151133S-1BX</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>Isoflurane&#160;</td>
			<td>Panion &#38; BF biotech</td>
			<td>DG-4900-250D</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P3911</td>
			<td>ACSF: 0.19 g/L</td>
		</tr>
		<tr>
			<td>Ketoprofen&#160;</td>
			<td>Swiss Pharmaceutical</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Lidocaine&#160;</td>
			<td>AstraZeneca</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Low melting point agarose</td>
			<td>Invitrogen</td>
			<td>16520</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td>ACSF: 0.19 g/L</td>
		</tr>
		<tr>
			<td>Microscope cover slips</td>
			<td>MARIENFELD</td>
			<td>101242</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>ThermoFisher Scientific</td>
			<td>4951PLUS-001E</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral oil light, white NF</td>
			<td>Macron Fine Chemicals</td>
			<td>MA-6358-04</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td>ACSF: 7.46 g/L</td>
		</tr>
		<tr>
			<td>NaH2PO4&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S8282</td>
			<td>ACSF: 0.18 g/L</td>
		</tr>
		<tr>
			<td>NaHCO3&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td>ACSF: 1.76 g/L</td>
		</tr>
		<tr>
			<td>n-butyl cyanoacrylate adhesive (tissue adhesive glue)</td>
			<td>3M</td>
			<td>1469SB</td>
			<td>3M Vetbond</td>
		</tr>
		<tr>
			<td>Neural tracer&#160;</td>
			<td>Santa Cruz</td>
			<td>SC-358883</td>
			<td>FluoroGold</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene tube</td>
			<td>RWD Life Science</td>
			<td>62329</td>
			<td>OD 1.50, I.D 0.50 mm and OD 1.09, I.D 0.38 mm</td>
		</tr>
		<tr>
			<td>Puralube Vet (eye) Ointment</td>
			<td>Dechra&#160;</td>
			<td>12920060</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium acetate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S2889</td>
			<td>SCFAs: 13.5 mM</td>
		</tr>
		<tr>
			<td>Sodium azide&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium butyrate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>B5887</td>
			<td>SCFAs: 8 mM</td>
		</tr>
		<tr>
			<td>Sodium propionate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P1880</td>
			<td>SCFAs: 5.18 mM</td>
		</tr>
		<tr>
			<td>Stainless guide cannula</td>
			<td>Chun Ta stainless steel enterprise CO., LTD.</td>
			<td>n/a</td>
			<td>OD 0.63 mm; Local vendor</td>
		</tr>
		<tr>
			<td>Stainless injector</td>
			<td>Chun Ta stainless steel enterprise CO., LTD.</td>
			<td>n/a</td>
			<td>OD 0.3 mm; dummy is made from injector; local vendor</td>
		</tr>
		<tr>
			<td>Superglue</td>
			<td>Krazy Glue</td>
			<td>KG94548R</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Merck</td>
			<td>1.08603.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cannula holder</td>
			<td>RWD Life Science</td>
			<td>B485-68217</td>
			<td></td>
		</tr>
		<tr>
			<td>Ceiling camera</td>
			<td>FOSCAM</td>
			<td>R2</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital stereotaxic instruments</td>
			<td>Stoelting</td>
			<td>51730D</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>INNOVIEW</td>
			<td>SEM-HT/TW</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Bead Sterilizer</td>
			<td>RWD Life Science</td>
			<td>RS1501</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Stoelting</td>
			<td>53800M</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica microscope&#160;</td>
			<td>Leica</td>
			<td>DM2500</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Dissecting Forceps</td>
			<td>ROBOZ</td>
			<td>RS-5136</td>
			<td>Serrated, Slight Curve; Extra Delicate; 0.5mm Tip Width; 4&#34; Length&#160;</td>
		</tr>
		<tr>
			<td>Micro Dissecting Scissors</td>
			<td>ROBOZ</td>
			<td>RS-5918</td>
			<td>4.5&#34; Angled Sharp</td>
		</tr>
		<tr>
			<td>Microinjection controller</td>
			<td>World Precision Instruments (WPI)</td>
			<td>MICRO2T</td>
			<td>SMARTouch Controller</td>
		</tr>
		<tr>
			<td>Microinjection syringe pump</td>
			<td>World Precision Instruments (WPI)</td>
			<td>UMP3T-1</td>
			<td>UltraMicroPump3&#160;&#160;</td>
		</tr>
		<tr>
			<td>Microliter syringe</td>
			<td>Hamilton</td>
			<td>80014</td>
			<td>10 &#181;L</td>
		</tr>
		<tr>
			<td>Optical Fiber Cold Light with double Fiber</td>
			<td>Step</td>
			<td>LGY-150</td>
			<td>Local vendor</td>
		</tr>
		<tr>
			<td>Pet trimmer</td>
			<td>WAHL</td>
			<td>09962-2018</td>
			<td></td>
		</tr>
		<tr>
			<td>Vaporiser for Isoflurane</td>
			<td>Step</td>
			<td>AS-01</td>
			<td>Local vendor</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica</td>
			<td>VT1000S</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Animal behavior video tracking software</td>
			<td>Noldus</td>
			<td>EthoVision</td>
			<td>Version: 15.0.1416</td>
		</tr>
		<tr>
			<td>Leica Application Suite X software</td>
			<td>Leica</td>
			<td>LASX</td>
			<td>Version: 3.7.2.22383</td>
		</tr>
	</tbody>
</table>

intracerebroventricular delivery of gut-derived microbial metabolites in freely moving mice

The impact of gut microbiota and their metabolites on host physiology and behavior has been extensively investigated in this decade. Numerous studies have revealed that gut microbiota-derived metabolites modulate brain-mediated physiological functions through intricate gut-brain pathways in the host. Short-chain fatty acids (SCFAs) are the major bacteria-derived metabolites produced during dietary fiber fermentation by the gut microbiome. Secreted SCFAs from the gut can act at multiple sites in the periphery, affecting the immune, endocrine, and neural responses due to the vast distribution of SCFAs receptors. Therefore, it is challenging to differentiate the central and peripheral effects of SCFAs through oral and intraperitoneal administration of SCFAs. This paper presents a video-based method to interrogate the functional role of SCFAs in the brain via a guide cannula in freely moving mice. The amount and type of SCFAs in the brain can be adjusted by controlling the infusion volume and rate. This method can provide scientists with a way to appreciate the role of gut-derived metabolites in the brain.

The mouse was infused with SCFAs 1 week after recovery from the guide cannula implantation to evaluate locomotor activity in a novel cage. The mouse was placed in a novel cage and infused with 2,100 nL of SCFAs or ACSF in the first 5 min (delivery rate of 7 nL/s) into the brain through the commercial guide cannula implanted in the lateral ventricle of the brain. The locomotor activity in a novel cage was recorded for an additional 30 min after infusion. No difference was observed in the locomotor activity in the novel ca...

Watch this Scientific Journal Video about Intracerebroventricular Delivery of Gut-Derived Microbial Metabolites in Freely Moving Mice at JoVE.com

Intracerebroventricular Delivery of Gut-Derived Microbial Metabolites in Freely Moving Mice

Gut-derived microbial metabolites have multifaceted effects leading to complex behavior in animals. We aim to provide a step-by-step method to delineate the effects of gut-derived microbial metabolites in the brain via intracerebroventricular delivery via a guide cannula.

The impact of gut microbiota and their metabolites on host physiology and behavior has been extensively investigated in this decade. Numerous studies have ...

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