Method Article

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

DOI:

10.3791/63972

⸱

June 2nd, 2022

 ,  , 
1Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, 2Department of Physiology, College of Medicine, National Cheng Kung University

* These authors contributed equally

In This Article

Summary

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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.

Abstract

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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.

Introduction

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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.

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Protocol

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All the experimental protocols and the animals' care were approved by the National Cheng Kung University (NCKU) Institutional Animal Care and Use Committee (IACUC).

1. Preparation for the experimental animal

  1. Obtain 6-8-week-old wild-type C57BL/6JNarl male mice from a vendor.
  2. House the mice in a standard mouse cage with standard mouse chow and sterilized water ad libitum.
    ​NOTE: The housing conditions for the Laboratory Animal Center of NCKU are 22 ± 1 °C temperature, 55% ± 10% humidity, and a 13 h/11 h light/dark cycle.

2. Stereotaxic surgery

  1. Prepare and sterilize the stereotaxic instrument, surgical instruments, and related items.
    NOTE: All items that will directly contact the surgical site should be sterilized to avoid infection.
  2. Anesthetize the mouse by placing it in the Plexiglas cage with 1%-5% isoflurane in oxygen.
    NOTE: Closely observe the mouse to ensure the breathing rate is maintained at around one breath per second.
  3. Take the mouse out of the anesthesia chamber. Shave the surgical site (mouse head) with a pet trimmer. Place the mouse onto the stereotaxic frame by fixing the mouse incisors on the incisor bar in the stereotaxic incisor holder. Cover the nose with the nosecone mask.
  4. Anesthetize the mouse with 1%-2.5% isoflurane in oxygen throughout the stereotaxic surgery. Evaluate the nociception of the mouse via the toe pinch reflex and ensure a constant breathing rate before incising the surgical site.
  5. Place a heating pad (37.0 °C) underneath the mouse on the stereotaxic frame to maintain body temperature during surgery. Alternatively, maintain the core temperature with the aid of a rectal thermal probe connecting the programmed warmer and the heating pad.
  6. Remove the trimmed fur on the head using adhesive tape. Inject the mouse with analgesic Ketoprofen (5 mg/kg) subcutaneously to relieve pain. Apply eye ointment to avoid dry eyes.
  7. Insert the pointed ear bar in the ear canal to fix the head.
  8. Center the head by adjusting the scale of the ear bar.
  9. Tighten the nose clamp on the incisor holder to avoid vertical movement. Press the head gently to check that the head is fixed and avoid the head becoming loose during subsequent surgery.
  10. Disinfect the scalp with three alternating scrubs of chlorhexidine using a cotton swab. Start each scrub from the middle toward the outer side (from the most disinfected central area to the least disinfected area).
    NOTE: The usage of scrubs from the middle to the outside could help scientists minimize infection and contamination from the fur. The outer area is close to the unshaved head region, which still has a great amount of fur and is not easy to disinfect thoroughly.
  11. Incise the scalp in an anterior/posterior manner (<1 cm) using a surgical blade. Open the incision and wipe the skull with a cotton swab held by microdissecting forceps.
    NOTE: The microdissecting forceps, surgical blade, and cotton swabs must be sterilized by autoclaving before surgery. During the surgical process, sterilize all the surgical equipment using a glass bead sterilizer (150 °C) for at least 5 s before and after each animal. Prepare a sterilized beaker to hold the surgical blade and forceps during the surgical process and between animals.
  12. Identify the Bregma and Lambda on the skull. Use the Bregma as the reference to locate the region of interest.
    1. Optional: Mount the stereotaxic drill on the stereotaxic drill holder, and use the tip of the drill to point Bregma as the reference. Sterilize the stereotaxic drill using the glass bead sterilizer (150 °C) for at least 5 s.
  13. Calibrate and align the flat skull horizontal plane in left/right and anterior/posterior planes by Bregma/Lambda.
    ​NOTE: If not in a correct horizontal plane, remount the mouse on the stereotaxic instrument.

3. Commercial customized guide cannula implantation

  1. Identify and label the position of the right lateral ventricle, based on the stereotaxic coordinates: Distance to Bregma, anterior/posterior (A/P): 0.26 mm, medial/lateral (M/L): -1.0 mm, dorsal/ventral (D/V): -2.0 mm20.
    NOTE: The coordinates can be modified based on the region of interest. The coordinates for the lateral ventricle were based on adult C57BL/6J mice with a weight range of 26-30 g. If younger mice are used, refer to the discussion.
  2. Drill a hole (diameter = 1.5 mm) through the skull at the labeled site using a stereotaxic drill for the implantation of a commercial guide cannula.
  3. Drill two to four more holes (diameter = 1.5 mm) on the skull using a stereotaxic drill for the mounting of stainless steel screws.
    NOTE: Sterilize the stereotaxic drill using a glass bead sterilizer (150 °C) for at least 5 s before and after each animal.
  4. Wipe the bone scraps and stop bleeding with a cotton swab.
  5. Wipe the skull with Lidocaine (1 mg/kg) using a cotton swab for local anesthetic, antipruritic, and pain-relieving effects. If bleeding does not stop after drilling, gently place a cotton swab on the hole for hemostasis.
  6. Mount two to four stainless screws on the holes to provide anchors for dental acrylic.
  7. Place the commercial guide cannula on the stereotaxic cannula holder and disinfect with the glass bead sterilizer (150 °C).
    NOTE: The commercial guide cannula, commercial dummy, and commercial injector (Figure 2A) were customized with the specifications shown in the Table of Materials.
  8. Move the stereotaxic cannula holder to the hole drilled for the lateral ventricle and slowly insert the commercial guide cannula into the hole until the desired depth (2.5 mm).
    NOTE: The dorsal/ventral coordinate can be defined by setting the tip of the commercial guide cannula as the reference when the tip is barely inserted into the hole.
  9. Apply 10 µL of n-butyl cyanoacrylate adhesive (tissue adhesive glue) to fix the commercial guide cannula in the drilled hole and wait for 3-4 min. Release the commercial guide cannula gently from the stereotaxic cannula holder and move the holder away.
  10. Apply the dental acrylic to the incised scalp to fix the commercial guide cannula and wait for at least 5 min. Implant the commercial dummy into the commercial guide cannula to avoid cannula clogging by blood or body fluid (Figure 2B).
  11. Release the ear bar from the ear canal and remove the mouse from the stereotaxic frame.
  12. Place the mouse into a new cage with a heating pad underneath for recovery from anesthesia and continuously observe until the mouse fully awakens.
    ​NOTE: Do not leave the animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. An animal that has undergone surgery should not return to the company of other animals until fully recovered.
  13. Return the mouse to single housing or group housing, depending on the institutional IACUC protocol and the experimental design. For group housing, ensure that fewer mice are housed in a cage to minimize unwanted injuries or the detachment of the cannula.
  14. Administer Ibuprofen (0.2 mg/mL) in the drinking water for at least 3 days for postoperative care, and monitor twice a day for signs of pain and distress for at least 3 days.
    1. During postoperative care, apply roxithromycin ointment around the skin to prevent inflammation and infection in the mice.
    2. Keep monitoring the animal's state and give a timely intraperitoneal injection of 5% glucose and/or 0.9% sodium chloride to provide enough energy.
    3. If the state of pain, distress, or infection steadily deteriorates, euthanize the mouse by CO2 inhalation.
  15. Wait for 1 week post operation for the mouse to be ready for the intracerebroventricular delivery of SCFAs and behavioral testing.

4. Preparation of SCFAs

  1. Dissolve the sodium acetate, sodium butyrate, and sodium propionate in artificial cerebrospinal fluid (ACSF) (see the Table of Materials).
  2. Ensure the chemicals are fully dissolved, then adjust the pH to 7.4, and filter the SCFAs mixture through a 0.22 μm filter for sterilization.

5. Set up the infusion system for intracerebroventricular delivery of SCFAs during behavioral testing

  1. Mount a ceiling camera to record the behavior. Connect the camera with a computer to control the video recording software (Figure 3).
  2. Fill a 10 μL syringe with distilled water.
    NOTE: Avoid air bubbles in the microliter syringe.
  3. Connect a microinjection pump with the microinjection controller.
  4. Mount the microliter syringe on the microinjection pump. To install the syringe, press the button to loosen the clamp and install the syringe onto the corresponding position. Close the clamp and tighten the plunger-retaining screw on the microinjection pump (Figure 4A).
  5. Insert the commercial injector into the polyethylene tube (Figure 2A).
  6. Hang the polyethylene tube on the ceiling camera above the testing arena.
  7. Fill the polyethylene tube with distilled water using an insulin syringe. Connect the microliter syringe to the hanging polyethylene tube.
    ​NOTE: Ensure the polyethylene tube is long enough to allow the mouse to move freely throughout the testing arena.

6. System settings of the microinjection controller

  1. Turn on the microinjection controller and press Display All Channels to access the Command screen (Figure 4C). Press Configuration and set Volume Target to 9,800 nL with Delivery Rate to 100 nL/s. Infuse 9,800 nL of distilled water from the polyethylene tube connected to the microliter syringe (press Direction to switch to the Infuse mode and press RUN) (see red squares in the Command screen of Figure 4C).
  2. Press Configuration and set Volume Target to 3,000 nL with Delivery Rate to 100 nL/s. Withdraw 3,000 nL of mineral oil (press Direction to switch to the Withdraw mode and press RUN) (see red squares in the Command screen of Figure 4C).
    NOTE: A clear oil-water phase separation should be observed on the polyethylene tube.
  3. Disassemble the polyethylene tube from the microliter syringe needle. Spit out 3,000 nL of distilled water from the microliter syringe needle (press Direction to switch to the Infuse mode and press RUN).
  4. Insert the microliter syringe back into the polyethylene tube. Press Configuration and set Volume Target to 9,500 nL with Delivery Rate to 100 nL/s. Withdraw 9,500 nL of SCFAs (press Direction to switch to the Withdraw mode and press RUN). Label the oil-SCFAs phase to validate whether the SCFAs are successfully infused.
  5. Press Configuration and set the desired Volume Target with Delivery Rate to 7 nL/s. Press Direction to switch to the Infuse mode (see red squares in the Command screen of Figure 4C).
    NOTE: Determine the volume based on the infusion time. For example, if the infusion time is 3 min for the delivery rate of 7 nL/s, target volume = 1,260 nL.
  6. Press RUN to infuse the microliter syringe forward until the liquid comes out at the front end of the commercial injector before inserting the injector into the cannula for SCFAs injection.

7. Infusion of SCFAs into lateral ventricle through the commercial guide cannula in freely moving mouse

  1. Anesthetize the mouse by placing it in the Plexiglas cage with 1%-5% isoflurane in oxygen.
    NOTE: Closely observe the mouse to ensure the breathing rate is maintained at around one breath per second.
  2. Scruff the mouse and insert the commercial injector into the commercial guide cannula (Figure 4B).
    NOTE: If the commercial guide cannula is plugged by blood or body fluid, gently unclog it with tweezers.
  3. Allow the mouse to recover from anesthesia for 15 min in a cage prior to behavioral testing.
  4. For the basic locomotion test, place the mouse in a novel cage and allow it to freely explore for 35 min. Infuse SCFAs using a Delivery Rate of 7 nL/s for a target volume of 2,100 nL in the first 5 min (press Direction to the Infuse mode and press RUN).
    NOTE: The locomotion in the novel cage can be analyzed using animal behavior video tracking software21,22.
  5. Anesthetize the mouse (repeating step 7.1) and remove the commercial injector from the commercial guide cannula.
    ​NOTE: The mouse can be repeatedly injected with different controls/metabolites after giving the appropriate length of time to wash out the previous injection. As long as the cannula is fixed on the mouse head, the mouse can be repeatedly tested with different metabolites.

8. Restoration of the microinjection system

  1. Disassemble the polyethylene tube from the microliter syringe.
  2. Inject air into the tube using the insulin syringe to discard the distilled water in the polyethylene tube. Empty the microliter syringe.
  3. Press Reset Pos on the Configuration screen to open the Syringe Stop Definition screen (Figure 4C).
  4. Press Withdraw until a beeping sound occurs to reset the microinjection pump to the fully withdrawn position (Figure 4C).
  5. Return to the Command screen and turn off the microinjection controller if there is an **END REACHED** sign on this screen (Figure 4C).

9. Optional: Validation of intracerebroventricular injection by neural tracer

  1. Infuse 2,100 nL of the neural tracer with the Delivery Rate of 7 nL/s to verify the infusion site.
    NOTE: Leave the injector in the guide cannula for 5 min to prevent backflow.
  2. Anesthetize the mouse by an overdose of isoflurane (5%) 30 min after the infusion of neural tracer.
  3. Check the breathing rate and tail/paw pinch reflex in the anesthetized mouse.
    NOTE: Mice must be unresponsive before the next step.
  4. Make a 4-5 cm incision through the skin, muscle, and abdominal wall below the rib cage.
  5. Slightly move the liver away from the diaphragm carefully.
  6. Incise the diaphragm to expose the heart of the mouse.
  7. Perfuse the mouse through the heart with phosphate-buffered saline (PBS) and ice-cold 4% paraformaldehyde in PBS.
  8. Decapitate the mouse and dissect the brain carefully with microdissecting forceps and microdissecting scissors to take out the whole brain23. Place the brain samples in ice-cold 4% paraformaldehyde in PBS for 3-4 days and wash them 3 x 5 min with PBS.
  9. Cut the brain into two parts in the mouse brain slice holder at the eighth cut of the mouse brain slice holder (1 mm/section) from the anterior to posterior direction. Place the brain in the embedding mold and embed the brain samples in low-melting point agarose (4% in PBS).
  10. Glue the brain embedded in agarose onto the stage of the vibratome using superglue. Section the brain coronally into 50 µm brain slices using the vibratome.
  11. Incubate the brain slices in the antibody targeting the neural tracer diluted in blocking buffer (1:1,000 dilution) overnight at room temperature.
    NOTE: The blocking buffer contained 10% horse serum, 0.1% Triton X-100, and 0.02% sodium azide.
  12. Wash the slices 3 x 5 min with PBST (PBS with 0.1% triton X-100).
  13. Incubate the brain slices in fluorescence-dye conjugated secondary antibody diluted in blocking buffer (1:500 dilution) for 2 h at room temperature.
  14. Wash the slices 3 x 5 min with PBS.
  15. Mount the brain slices on the slide with a mounting medium containing 4',6-diamidino-2-phenylindole (DAPI).
  16. Cover the slide with a microscope coverslip.
  17. Apply nail polish on the slide edge to avoid leakage of the mounting medium.
  18. After overnight incubation at room temperature, protected from light, image the fluorescence signal in the infusion site using a fluorescence microscope.

10. Optional: Infusion of metabolites through a customized stainless steel guide cannula in the lateral ventricle in mice

  1. Follow protocol sections 1-8 and replace the commercial guide cannula with a stainless steel guide cannula to infuse chemicals through a stainless steel injector in the mouse.
    NOTE: The pros and cons of the different commercial and stainless steel cannulas are elaborated upon in the discussion section.
  2. Perform the surgical protocol in the same way as described in protocol sections 2 and 3, except remember to replace the commercial guide cannula with a stainless steel guide cannula (Figure 5B).
    NOTE: The stainless steel guide cannula, stainless steel dummy, and stainless steel injector (Figure 5A) were customized with the specifications shown in the Table of Materials. Insert the customized stainless steel guide cannula into the hole until the desired depth (2.0 mm).
  3. Infuse SCFAs via the stainless steel guide cannula using the microinjection system consisting of the microinjection controller and microinjection pump (Figure 4B) (same as protocol sections 4-7). For the stainless steel dummy of the stainless steel guide cannula, bend one side of the stainless steel injector gently until the tip of the other side is 1 mm longer than the stainless steel guide cannula.
  4. Infuse 2,100 nL of the neural tracer through the stainless steel guide cannula into the lateral ventricle in mice (same as protocol section 9).
  5. Collect the sample for 30 min after neural tracer infusion (same as protocol section 9).
  6. Perform image acquisition in the neural tracer infused-brain slices (same as protocol section 9).

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Results

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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...

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Discussion

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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...

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Disclosures

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The authors have no conflicts of interest related to this work.

Acknowledgements

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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.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Material
Advil Liqui-Gels Solubilized Ibuprofen A2:D41Pfizern/a
Alexa Fluor 488 donkey anti-rabbitThermoFisher ScientificA-21206
Anti-Fluorescent Gold (rabbit polyclonal)MilliporeAB153-I
Bottle Top Vacuum Filter, 500 mL, 0.22 μm, PES, SterileNEST121921LA01
CaCl2 Sigma-AldrichC1016ACSF: 0.14 g/L
Chlorhexidine scrub 2%PhoenixNDC 57319-611-09
Chlorhexidine solutionPhoenixNDC 57319-599-09
Commercial dummyRWD Life Science62004Single_OD 0.20 mm/ M3.5/G = 0.5 mm
Commercial guide cannulRWD Life Science62104Single_OD 0.41 mm-27G/ M3.5/C = 2.5 mm 
Commercial injectorRWD Life Science62204Single_OD 0.21 mm-33G/ Mates with M3.5/C = 3.5 mm/G = 0.5 mm
D-(+)-GlucoseSigma-AldrichG8270ACSF: 0.61 g/L
Dental acrylicHYGENICn/a
Fixing screwsRWD Life Science62521
Fluoroshield mounting medium with DAPIAbcamAB104139
Horse serumThermoFisher Scientific16050130
Insulin syringesBBraunXG-LBB-9151133S-1BX1 mL
Isoflurane Panion & BF biotechDG-4900-250D
KCl Sigma-AldrichP3911ACSF: 0.19 g/L
Ketoprofen Swiss Pharmaceuticaln/a
Lidocaine AstraZenecan/a
Low melting point agaroseInvitrogen16520
MgCl2 Sigma-AldrichM8266ACSF: 0.19 g/L
Microscope cover slipsMARIENFELD101242
Microscope slidesThermoFisher Scientific4951PLUS-001E
Mineral oil light, white NFMacron Fine ChemicalsMA-6358-04
NaCl Sigma-AldrichS9888ACSF: 7.46 g/L
NaH2PO4 Sigma-AldrichS8282ACSF: 0.18 g/L
NaHCO3 Sigma-AldrichS5761ACSF: 1.76 g/L
n-butyl cyanoacrylate adhesive (tissue adhesive glue)3M1469SB3M Vetbond
Neural tracer Santa CruzSC-358883FluoroGold
ParaformaldehydeSigma-AldrichP6148
Polyethylene tubeRWD Life Science62329OD 1.50, I.D 0.50 mm and OD 1.09, I.D 0.38 mm
Puralube Vet (eye) OintmentDechra 12920060
Sodium acetate Sigma-AldrichS2889SCFAs: 13.5 mM
Sodium azide Sigma-AldrichS2002
Sodium butyrate Sigma-AldrichB5887SCFAs: 8 mM
Sodium propionate Sigma-AldrichP1880SCFAs: 5.18 mM
Stainless guide cannulaChun Ta stainless steel enterprise CO., LTD.n/aOD 0.63 mm; Local vendor
Stainless injectorChun Ta stainless steel enterprise CO., LTD.n/aOD 0.3 mm; dummy is made from injector; local vendor
SuperglueKrazy GlueKG94548R
Triton X-100Merck1.08603.1000
Equipment
Cannula holderRWD Life ScienceB485-68217
Ceiling cameraFOSCAMR2
Digital stereotaxic instrumentsStoelting51730D
Dissecting microscopeINNOVIEWSEM-HT/TW
Glass Bead SterilizerRWD Life ScienceRS1501
Heating padStoelting53800M
Leica microscope LeicaDM2500
Micro Dissecting ForcepsROBOZRS-5136Serrated, Slight Curve; Extra Delicate; 0.5mm Tip Width; 4" Length 
Micro Dissecting ScissorsROBOZRS-59184.5" Angled Sharp
Microinjection controllerWorld Precision Instruments (WPI)MICRO2TSMARTouch Controller
Microinjection syringe pumpWorld Precision Instruments (WPI)UMP3T-1UltraMicroPump3  
Microliter syringeHamilton8001410 µL
Optical Fiber Cold Light with double FiberStepLGY-150Local vendor
Pet trimmerWAHL09962-2018
Vaporiser for IsofluraneStepAS-01Local vendor
VibratomeLeicaVT1000S
Software
Animal behavior video tracking softwareNoldusEthoVisionVersion: 15.0.1416
Leica Application Suite X softwareLeicaLASXVersion: 3.7.2.22383

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Gut MicrobiotaMicrobial MetabolitesShort Chain Fatty AcidsIntracerebroventricular DeliveryGuide CannulaFreely Moving MiceStereotaxic SurgeryGut Brain AxisBehavioral TestingFluorescence Microscopy

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