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.

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

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 ...

intracerebroventricular-delivery-gut-derived-microbial-metabolites

Research

JoVE Journal

Neuroscience

3.1K Views.  National Cheng Kung University. 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.

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

High-density EEG Recordings of the Freely Moving Mice using Polyimide-based Microelectrode

Electroencephalogram (EEG) indicates the averaged electrical activity of the neuronal populations on a large-scale level. It is widely utilized as a noninvasive brain monitoring tool in cognitive neuroscience as well as a diagnostic tool for epilepsy and sleep disorders in neurology. However, the underlying mechanism of EEG rhythm generation is still under the veil. Recently introduced polyimide-based microelectrode (PBM-array) for high resolution mouse EEG1 is one of the trials to answer the neurophysiological questions on EEG signals based on a rich genetic resource that the mouse model contains for the analysis of complex EEG generation process. This application of nanofabricated PBM-array to mouse skull is an efficient tool for collecting large-scale brain activity of transgenic mice and accommodates to identify the neural correlates to certain EEG rhythms in conjunction with behavior. However its ultra-thin thickness and bifurcated structure cause a trouble in handling and implantation of PBM-array. In the presented video, the preparation and surgery steps for the implantation of PBM-array on a mouse skull are described step by step. Handling and surgery tips to help researchers succeed in implantation are also provided.

In this article, we described the surgery procedure and handling tips for implantation of ultra-thin polyimide-based microelectrode array (PBM-array) on the mouse skull for acquisition of high-density encephalography (EEG) in a mouse model.

Electroencephalogram (EEG) indicates the averaged electrical activity of the neuronal populations on a large-scale level. It is widely utilized as a ...

Recording Single Neurons' Action Potentials from Freely Moving Pigeons Across Three Stages of Learning

While the subject of learning has attracted immense interest from both behavioral and neural scientists, only relatively few investigators have observed single-neuron activity while animals are acquiring an operantly conditioned response, or when that response is extinguished. But even in these cases, observation periods usually encompass only a single stage of learning, i.e. acquisition or extinction, but not both (exceptions include protocols employing reversal learning; see Bingman&#160;et al.1 for an example). However, acquisition and extinction entail different learning mechanisms and are therefore expected to be accompanied by different types and/or loci of neural plasticity.
Accordingly, we developed a behavioral paradigm which institutes three stages of learning in a single behavioral session and which is well suited for the simultaneous recording of single neurons&#39; action potentials. Animals are trained on a single-interval forced choice task which requires mapping each of two possible choice responses to the presentation of different novel visual stimuli (acquisition). After having reached a predefined performance criterion, one of the two choice responses is no longer reinforced (extinction). Following a certain decrement in performance level, correct responses are reinforced again (reacquisition). By using a new set of stimuli in every session, animals can undergo the acquisition-extinction-reacquisition process repeatedly. Because all three stages of learning occur in a single behavioral session, the paradigm is ideal for the simultaneous observation of the spiking output of multiple single neurons. We use pigeons as model systems, but the task can easily be adapted to any other species capable of conditioned discrimination learning.

Learning new stimulus-response associations engages a wide range of neural processes which are ultimately reflected in changing spike output of individual neurons. Here we describe a behavioral protocol allowing for the continuous registration of single-neuron activity while animals acquire, extinguish, and reacquire a conditioned response within a single experimental session.

While the subject of learning has attracted immense interest from both behavioral and neural scientists, only relatively few investigators have observed ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

Intracerebroventricular Injection of Amyloid-&#946; Peptides in Normal Mice to Acutely Induce Alzheimer-like Cognitive Deficits

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, progressive accumulation of A&#946; oligomers and plaques is observed. Such A&#946; abnormalities are believed to block long-term potentiation, impair synaptic function, and induce cognitive deficits. Clinical and experimental evidences have revealed that the acute increase of A&#946; levels in the brain allows development of Alzheimer-like phenotypes. Hence, a detailed protocol describing how to acutely generate an AD mouse model via the intracerebroventricular (ICV) injection of A&#946; is necessary in many cases. In this protocol, the steps of the experiment with an A&#946;-injected mouse are included, from the preparation of peptides to the testing of behavioral abnormalities. The process of preparing the tools and animal subjects before the injection, of injecting the A&#946; into the mouse brain via ICV injection, and of assessing the degree of cognitive impairment are easily explained throughout the protocol, with an emphasis on tips for effective ICV injection of A&#946;. By mimicking certain aspects of AD with a designated injection of A&#946;, researchers can bypass the aging process and focus on the downstream pathology of A&#946; abnormalities.

The amyloid-&#946; (A&#946;)-injected animal model enables the administration of a defined quantity and species of A&#946; fragments and reduces individual differences within each study group. This protocol describes the intracerebroventricular (ICV) injection of A&#946; without stereotactic instruments, enabling the production of Alzheimer-like behavioral abnormalities in normal mice.

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, ...

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such as epilepsy and neurodegenerative disorders. However, it is technically difficult to obtain EEG recordings in neonates as it requires specialized handling and great care. Here, we present a novel method to record EEG in neonatal rat pups (P8-P15). We designed a simple and reliable electrode using computer pin loci; it can be easily implanted into the skull of a rat pup to record high-quality EEG signals in the normal and epileptic brain. Pups were given an intraperitoneal (i.p.) injection of the neurotoxin kainic acid (KA) to induce epileptic seizures. The surgical implantation performed in this procedure is less expensive than other EEG procedures for neonates. This method allows one to record high-quality and stable EEG signals for more than 1 week. Furthermore, this procedure can also be applied to adult rats and mice to study epilepsy or other neurological disorders.

Here, we introduce a novel technique designed to record electroencephalography (EEG) in freely moving neonatal epileptic pups and describe its procedures, features, and applications. This method allows one to record EEG for more than 1 week.

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such ...

Intracerebroventricular Treatment with Resiniferatoxin and Pain Tests in Mice

The transient receptor potential vanilloid type 1 (TRPV1), a thermosensitive cation channel, is known to trigger pain in the peripheral nerves. In addition to its peripheral function, its involvement in brain functions has also been suggested. Resiniferatoxin (RTX), an ultrapotent TRPV1 agonist, has been known to induce long-term desensitization of TRPV1, and this desensitization has been an alternative approach for investigating the physiological relevance of TRPV1-expressing cells. Here we describe a protocol for intracerebroventricular (i.c.v.) treatment with RTX in mice. Procedures are described for testing nociception to peripheral TRPV1 stimulation (RTX test) and mechanical stimulation (tail pressure test) then follow. Although the nociceptive responses of mice that had been administered RTX i.c.v. were comparable to those of the control groups, RTX-i.c.v.-administered mice were insensitive to the analgesic effect of acetaminophen, suggesting that i.c.v. RTX treatment can induce supraspinal-selective TRPV1 desensitization. This mouse model can be used as a convenient experimental system for studying the role of TRPV1 in brain/supraspinal function. These techniques can also be applied to studies of the central actions of other drugs.

The transient receptor potential vanilloid type 1 (TRPV1) in the supraspinal region has been suggested to play some roles in the brain function. Described here is a protocol for intracerebroventricular injection of resiniferatoxin for supraspinal TRPV1 desensitization in mice. Procedures for some pain tests are also presented.

The transient receptor potential vanilloid type 1 (TRPV1), a thermosensitive cation channel, is known to trigger pain in the peripheral nerves. In ...

Noninvasive EEG Recordings from Freely Moving Piglets

The method allows the recording of high-quality electroencephalograms (EEGs) from freely moving piglets directly in the pigpen. We use a one-channel telemetric electroencephalography system in combination with standard self-adhesive hydrogel electrodes. The piglets are calmed down without the use of sedatives. After their release into the pigpen, the piglets behave normally&#8212;they drink and sleep in the same cycle as their siblings. Their sleep phases are used for the EEG recordings.

Here, we present a protocol to record telemetric electroencephalograms (EEGs) from freely moving piglets directly in the pigpen without the use of a sedative, making it possible to record typical EEG patterns during non-REM sleep, like spindle bursts.

The method allows the recording of high-quality electroencephalograms (EEGs) from freely moving piglets directly in the pigpen. We use a one-channel ...

Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain interstitial space with the behavior and/or with the specific outcome of a pathology (e.g., seizures for epilepsy). When studying epilepsy, the microdialysis technique is often combined with short-term or even long-term video-electroencephalography (EEG) monitoring to assess spontaneous seizure frequency, severity, progression and clustering. The combined microdialysis-EEG is based on the use of several methods and instruments. Here, we performed in vivo microdialysis and continuous video-EEG recording to monitor glutamate and aspartate outflow over time, in different phases of the natural history of epilepsy in a rat model. This combined approach allows the pairing of changes in the neurotransmitter release with specific stages of the disease development and progression. The amino acid concentration in the dialysate was determined by liquid chromatography. Here, we describe the methods and outline the principal precautionary measures one should take during in vivo microdialysis-EEG, with particular attention to the stereotaxic surgery, basal and high potassium stimulation during microdialysis, depth electrode EEG recording and high-performance liquid chromatography analysis of aspartate and glutamate in the dialysate. This approach may be adapted to test a variety of drug or disease induced changes of the physiological concentrations of aspartate and glutamate in the brain. Depending on the availability of an appropriate analytical assay, it may be further used to test different soluble molecules when employing EEG recording at the same time.

Here, we describe a method for in vivo microdialysis to analyze aspartate and glutamate release in the ventral hippocampus of epileptic and non-epileptic rats, in combination with EEG recordings. Extracellular concentrations of aspartate and glutamate may be correlated with the different phases of the disease.

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain ...

Long-term Sensory Conflict in Freely Behaving Mice

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning in mice. By permanently wearing a device fixed on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages. Therefore, this protocol readily enables the study of the visual system and multisensory interactions over an extended timeframe that would not be accessible otherwise. In addition to lowering the experimental costs of long-term sensory learning in naturally behaving mice, this approach accommodates the combination of in vivo and in vitro experiments. In the reported example, video-oculography is performed to quantify the vestibulo-ocular reflex (VOR) and optokinetic reflex (OKR) before and after learning. Mice exposed to this long-term sensory conflict between visual and vestibular inputs presented a strong VOR gain decrease but exhibited few OKR changes. Detailed steps of device assembly, animal care, and reflex measurements are hereby reported.

The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning. By permanently wearing a fixed device on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages.

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for ...

Hybrid Microdrive System with Recoverable Opto-Silicon Probe and Tetrode for Dual-Site High Density Recording in Freely Moving Mice

Multi-regional neural recordings can provide crucial information to understanding fine-timescale interactions between multiple brain regions. However, conventional microdrive designs often only allow use of one type of electrode to record from single or multiple regions, limiting the yield of single-unit or depth profile recordings. It also often limits the ability to combine electrode recordings with optogenetic tools to target pathway and/or cell type specific activity. Presented here is a hybrid microdrive array for freely moving mice to optimize yield and a description of its fabrication and reuse of the microdrive array. The current design employs nine tetrodes and one opto-silicon probe implanted in two different brain areas simultaneously in freely moving mice. The tetrodes and the opto-silicon probe are independently adjustable along the dorsoventral axis in the brain to maximize the yield of unit and oscillatory activities. This microdrive array also incorporates a set-up for light, mediating optogenetic manipulation to investigate the regional- or cell type-specific responses and functions of long-range neural circuits. In addition, the opto-silicon probe can be safely recovered and reused after each experiment. Because the microdrive array consists of 3D-printed parts, the design of microdrives can be easily modified to accommodate various settings. First described is the design of the microdrive array and how to attach the optical fiber to a silicon probe for optogenetics experiments, followed by fabrication of the tetrode bundle and implantation of the array into a mouse brain. The recording of local field potentials and unit spiking combined with optogenetic stimulation also demonstrate feasibility of the microdrive array system in freely moving mice.

This protocol describes the construction of a hybrid microdrive array that allows implantation of nine independently adjustable tetrodes and one adjustable opto-silicon probe in two brain regions in freely moving mice. Also demonstrated is a method for safely recovering and reusing the opto-silicon probe for multiple purposes.

Multi-regional neural recordings can provide crucial information to understanding fine-timescale interactions between multiple brain regions. However, ...