This work was supported by the National Natural Science Foundation of China (82104533), the Science &#38; Technology Department of Sichuan Province (2021YJ0175), and the China Postdoctoral Science Foundation (2020M683273). The authors would like to thank Mr. Yuncheng Hong, a senior equipment engineer at Tri-Angels D&#38;H Trading Pte. Ltd. (Singapore) for providing technical services for the microdialysis technique.

The experimental protocol was conducted in accordance with the requirements of the Use of Laboratory Animals and Institutional Animal Care and Use Committee at Chengdu University of Traditional Chinese Medicine (Record number: 2021-11). Male Sprague Dawley (SD) rats (280 &#177; 20 g, 6-8 weeks old) were used for the present study.
1. Brain microdialysis probe implantation surgery
<ol>
	<li>Use 3% and 1.5% isoflurane for the induction and maintenance of rat anesthesia, respectively, using an animal anesthesia system in an air-oxygen mixture at 0.6 L/min. Make sure that the rats are deeply anesthetized without both a pain reflex and a corneal reflex. Use veterinary ointment on the eyes to prevent dryness while under anesthesia.</li>
	<li>Remove the fur on the anesthetized rat&#39;s skull with an electric shaver in the preparation area. Then, fix the anesthetized rat on a stereotaxic brain locator. Disinfect the surgical site before the operation by applying povidone-iodine and ethanol 3x to the region of surgery with a sterile cotton ball. Apply&#160;bupivacaine topically for local analgesia. NOTE: The entire process of microdialysis-based HECF sample acquisition is illustrated in Figure 1.</li>
	<li>Make a 1.5 cm craniofacial incision down the middle with surgical scissors and remove the periosteum using surgical scissors and ophthalmic forceps.</li>
	<li>Consider the bregma as the basal position and pierce the endocranium to drill a 2 mm aperture at the anteroposterior (AP) position (-2 mm), the mediolateral (ML) position (-3.5 mm), and the dorsoventral (DV) position (-3.5 mm) (the hippocampal CA1 region) using a cranial drill.</li>
	<li>Fix the catheter stylet on the gripper of a stereotaxic brain locator and adjust the position of the microdialysis casing at the AP (-2 mm), ML (-3.5 mm), and DV (0 mm) positions. Adjust the DV value of the brain stereo locator, and implant the microdialysis casing into the CA1 region at a depth of 3.5 mm. 
	NOTE: Maintain the temperature of the animal at 37 &#176;C during the operation using an animal temperature maintainer.</li>
	<li>Drill three more apertures with a 2 mm diameter such that the three apertures form a triangle in which the probe aperture is centrally located. Implant screws into the apertures at a depth of 1 mm.</li>
	<li>Fix the probe catheter with dental cement and use a 4-0 surgical suture to close the skin. See Figure 2 for the placement of the probe.</li>
	<li>Place the rat in cages for 7 days to recover. Locally infiltrate bupivacaine (1.5 mg/kg) once daily post-surgery.&#160;Provide food and water ad libitum. Use sodium hyaluronate eye drops 3x a day to prevent dryness after the operation. 
	&#8203;NOTE: Perform all the procedures in a sterile surgical room. Do not leave the animal unattended until it has regained sufficient consciousness to maintain sternal recumbency under a 37 &#176;C condition. Do not return the animal that has undergone surgery to the company of other animals until fully recovered.</li>
</ol>
2. Microdialysis system connection and probe check
<ol>
	<li>Connect the microdialysis pump, microsyringe, awake activity device, and cryogenic sample collector according to the manufacturer&#39;s instructions. Install the microsyringe with ACSF on the microdialysis pump and set the microdialysis pump to a rate of 1 &#181;L/min to discharge the air in the pipeline.</li>
	<li>Connect the pipeline and brain microdialysis probe (membrane: PAES; membrane length: 4 mm; membrane OD: 0.5 mm; cut-off: 20 kDa; shaft length: 14 mm). Operate the microdialysis pump at a rate of 1 &#181;L/min to inject ACSF into the probe until the surface of the probe is slightly moist. Immerse the probe in heparin sodium injection solution for subsequent use. 
	&#8203;NOTE: If a big stream of the ACSF falls from the semipermeable membrane of the probe, as seen with the naked eye under gravity, replace the probe with a new one.</li>
</ol>
3. Collection of HECF from the awake rat
<ol>
	<li>Insert the brain microdialysis probe into the probe catheter and place the rat in a chamber (height: 360 mm; diameter: 400 mm) with padding to make sure the rats are free to move around.</li>
	<li>Connect the pipeline, microsyringe pump, and brain microdialysis probe. Harness the rat via the hole at the top of the rat restraint device and the stainless-steel swivels.</li>
	<li>Turn on the multi-channel swivel controller to avoid intertwining the microdialysis pipelines during the free movement of the rat. Turn on the microsyringe pump and pump the ACSF at a rate of 1 &#181;L/min. Collect HECF periodically after a 60 min equilibration of the microdialysis HECF collection system.</li>
	<li>Ensure that the flow rate of the HECF samples in the refrigerated fraction collector is consistent with ACSF infusion. Collect 20 &#181;L of HECF and automatically change to the next sampling tube. Take care to check whether the probe membrane is damaged when inserting the probe.</li>
</ol>
4. Measurement of the osmotic pressure for the HECF
<ol>
	<li>Turn on the osmometer and log in to the detection system. Click on the Cal button on the touch screen and click on the Res button on the page to clear the previous calibration memory.</li>
	<li>Install a 1.5 mL tube containing 100 &#181;L of pure water without bubbles on the measuring head. Pull the measuring head to the bottom of the cold hydrazine container.</li>
	<li>Enter the sample number 0 on the touch screen and confirm to test. Quickly dip the diode needle into the sample tube, and then quickly pull it out to induce crystallization of the sample at a temperature of -6.2 &#176;C.</li>
	<li>Wait for the screen to display: Push measure head up and click on Cal and Cal 0 in turn to calibrate. Perform measurements with a 300 mOsm calibration solution and measure the osmotic pressure of the HECF samples as described above. 
	&#8203;NOTE: Wipe the measuring head with a soft paper towel after calibration or measurement. The HECF samples with no bubbles should be mixed well.</li>
</ol>
5. Maintenance of the microdialysis system and devices after sampling
<ol>
	<li>Take out the brain microdialysis probe from the probe catheter after sampling termination. Immerse the probe in deionized water and lavage with deionized water for 12 h to remove stranded salt depositions from the pipeline and the probe.</li>
	<li>Remove the probe to place into a 0.05% trypsin solution at 4 &#176;C. Dry the pipelines in an air-dry oven at 25 &#176;C and store them at room temperature. 
	&#8203;NOTE: The microdialysis probes are expensive and this step can increase the reusability of the probes. Proteins that adhere to the probe surface can be digested by trypsin solution to prevent the probe membrane from being blocked by proteins, and trypsin had no effect on the probe material.</li>
</ol>
6. Animal treatment after sampling
<ol>
	<li>After sampling, painlessly euthanize the rats by making them inhale 1.5% isoflurane, followed by an overdose of 5% isoflurane in accordance with animal ethics.</li>
</ol>

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The authors have nothing to disclose.

The pathogenesis of CNS diseases is still not fully understood, which hinders the development of new therapies and drugs. Studies have shown that most CNS diseases are closely related to hippocampal lesions20,21,22. The proposed brain microdialysis technique can target specific regions of the brain, especially the hippocampus, which makes it stand out from the traditional approach of collecting HECF. Probes are placed in the CA1...

Central nervous system (CNS) diseases with high morbidities, such as neurodegenerative disease, traumatic brain injury, high-altitude hypoxia-induced brain injury, and ischemic stroke, are crucial causes of the growing mortality worldwide1,2,3. Real-time monitoring of cytokines and protein changes in specific brain regions contributes to the diagnostic accuracy of CNS diseases and brain pharmacokinetic studies after medication. Traditional scientific research uses brain tissue homogenate or a manual collection of animal interstitial brain fluid for the detection of specific substances and for pharmacokinetic studies. However, this has some shortcomings, such as a limited sample size, the inability to dynamically observe the changes of indicators, and uneven sampling quality4,5,6. Cerebrospinal fluid, an interstitial fluid, protects the brain and spinal cord from mechanical damage. Its composition is different from that of the serum due to the existence of the blood-brain barrier (BBB)7. Direct analysis of cerebrospinal fluid samples is more conducive to disclosing the mechanism of CNS lesions and drug discovery. Inevitably, the cerebrospinal fluid samples, manually obtained directly from the cisterna magna and cerebral ventricles through a syringe, have disadvantages of blood contamination, a random chance of sample collection, uncertainty of quantity, and almost no multiple retrieval possibility8,9. More notably, conventional interstitial brain fluid sampling methods cannot obtain samples from damaged brain regions, which hinders the exploration of the pathogenesis of CNS diseases in specific brain regions and the efficacy evaluation of targeted ethnomedicine therapies9,10.
Brain microdialysis is a technique for sampling interstitial brain fluid in awake animals11. The microdialysis system imitates vascular permeability with the help of a probe implanted in the brain. The microdialysis probe is armed with a semi-permeable membrane and is implanted in specific brain regions. After perfusion with isotonic artificial cerebrospinal fluid (ACSF), the dialyzed interstitial brain fluid can be favorably collected with the benefits of small sample sizes, continuous sampling, and dynamic observation12,13. In terms of location, brain microdialysis probes can be selectively implanted into brain structures or cranial cisterns of interest14. An observation of abnormal levels of an endogenous substance in the hippocampal extracellular fluid (HECF) suggests the occurrences of CNS diseases or the pathogenesis of disease. Several studies have shown that the biomarkers for CNS diseases, such as D-amino acids in schizophrenia, &#946;-amyloid and tau proteins in Alzheimer&#39;s disease, neurofilament light chains in traumatic brain injury, and ubiquitin carboxy-terminal hydrolase L1s in hypoxic ischemia encephalopathy, can be analyzed in cerebrospinal fluid15,16,17. A chemical analysis method based on the brain microdialysis sampling technique can be used to monitor dynamic changes of exogenous compounds such as active ingredients of ethnomedicine, which diffuse and distribute in specific brain regions14.
This article presents the specific process of dynamic HECF acquisition in awake rats and measures its osmotic pressure to ensure sample quality.

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	<tbody>
		<tr>
			<td>&#160;Air-drying oven</td>
			<td>Suzhou Great Electronic Equipment Co., Ltd</td>
			<td>GHG-9240A</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal anesthesia system</td>
			<td>Rayward Life Technology Co., Ltd</td>
			<td>R500IE</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal temperature maintainer</td>
			<td>Rayward Life Technology Co., Ltd</td>
			<td>69020</td>
			<td></td>
		</tr>
		<tr>
			<td>Artificial cerebrospinal fluid</td>
			<td>Beijing leagene biotech. Co., Ltd</td>
			<td>CZ0522</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain microdialysis probe</td>
			<td>&#160;CMA Microdialysis AB</td>
			<td>T56518</td>
			<td></td>
		</tr>
		<tr>
			<td>Catheter</td>
			<td>&#160;CMA Microdialysis AB</td>
			<td>T56518</td>
			<td></td>
		</tr>
		<tr>
			<td>Covance infusion harness</td>
			<td>Instech Laboratories, Inc.</td>
			<td>CIH95</td>
			<td></td>
		</tr>
		<tr>
			<td>Denture base resins</td>
			<td>Shanghai Eryi Zhang Jiang Biomaterials Co., Ltd</td>
			<td>190732</td>
			<td></td>
		</tr>
		<tr>
			<td>Electric cranial drill</td>
			<td>Rayward Life Technology Co., Ltd</td>
			<td>78001</td>
			<td></td>
		</tr>
		<tr>
			<td>Electric shaver</td>
			<td>Rayward Life Technology Co., Ltd</td>
			<td>CP-5200</td>
			<td></td>
		</tr>
		<tr>
			<td>Free movement tank for animals</td>
			<td>&#160;CMA Microdialysis AB</td>
			<td>CMA120</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin sodium injection</td>
			<td>Chengdu Haitong Pharmaceutical Co., Ltd</td>
			<td>H51021208</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodophor</td>
			<td>Sichuan Lekang Pharmaceutical Accessories Co., Ltd</td>
			<td>202201</td>
			<td></td>
		</tr>
		<tr>
			<td>Isofluran</td>
			<td>Rayward Life Technology Co., Ltd</td>
			<td>R510-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Microdialysis catheter stylet</td>
			<td>&#160;CMA Microdialysis AB</td>
			<td>8011205</td>
			<td></td>
		</tr>
		<tr>
			<td>Microdialysis collection tube</td>
			<td>&#160;CMA Microdialysis AB</td>
			<td>7431100</td>
			<td></td>
		</tr>
		<tr>
			<td>Microdialysis collector</td>
			<td>&#160;CMA Microdialysis AB</td>
			<td>CMA4004</td>
			<td></td>
		</tr>
		<tr>
			<td>Microdialysis fep tubing</td>
			<td>&#160;CMA Microdialysis AB</td>
			<td>3409501</td>
			<td></td>
		</tr>
		<tr>
			<td>Microdialysis in vitro stand</td>
			<td>&#160;CMA Microdialysis AB</td>
			<td>CMA130</td>
			<td></td>
		</tr>
		<tr>
			<td>Microdialysis microinjection pump</td>
			<td>&#160;CMA Microdialysis AB</td>
			<td>788130</td>
			<td></td>
		</tr>
		<tr>
			<td>Microdialysis syringe (1.0 mL)</td>
			<td>&#160;CMA Microdialysis AB</td>
			<td>8309020</td>
			<td></td>
		</tr>
		<tr>
			<td>Microdialysis tubing adapter</td>
			<td>&#160;CMA Microdialysis AB</td>
			<td>3409500</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-absorbable surgical sutures</td>
			<td>Shanghai Tianqing Biological Materials Co., Ltd</td>
			<td>S19004</td>
			<td></td>
		</tr>
		<tr>
			<td>Ophthalmic forceps</td>
			<td>Rayward Life Technology Co., Ltd</td>
			<td>F12016-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmometer</td>
			<td>L&#246;ser</td>
			<td>OM 807</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hyaluronate eye drops</td>
			<td>URSAPHARM Arzneimittel GmbH</td>
			<td>H20150150</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotaxie apparatus</td>
			<td>Rayward Life Technology Co., Ltd</td>
			<td>68025</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Rayward Life Technology Co., Ltd</td>
			<td>S14014-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Shanghai Bingyu Fluid technology Co., Ltd</td>
			<td>BY-103</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe needle</td>
			<td>&#160;CMA Microdialysis AB</td>
			<td>T56518</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin solution</td>
			<td>Boster 
			Biological Technology, Ltd.</td>
			<td>PYG0107</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic cleaner</td>
			<td>Guangdong Goote Ultrasonic Co., Ltd</td>
			<td>KMH1-240W8101</td>
			<td></td>
		</tr>
	</tbody>
</table>

real-time dynamic collection of hippocampal extracellular fluid from conscious rats using a microdialysis system

A variety of central nervous system (CNS) diseases are associated with changes in the composition of hippocampal extracellular fluid (HECF). However, difficulty in obtaining HECF in real time from conscious rats has long restricted the evaluation of CNS disease progression and the effectiveness of ethnomedicine therapy. Encouragingly, a brain microdialysis technique can be used for continuous sampling with the advantages of dynamic observation, quantitative analysis, and a small sampling size. This allows the monitoring of changes in the extracellular fluid content for compounds from traditional herbs and their metabolites in the brain of living animals. The aim of this study was thus to accurately implant a cerebrospinal fluid microdialysis probe into the hippocampal region of Sprague Dawley (SD) rats with a three-dimensional brain stereotaxic apparatus, cutting off molecular weights greater than 20 kDa. The high-quality HECF was then obtained from conscious rats using a microdialysis sampling control system with an adjustable sampling rate from 2.87 nL/min - 2.98 mL/min. In conclusion, our protocol provides an efficient, rapid, and dynamic method to obtain HECF in awake rats with the help of microdialysis technology, which provides us with unlimited possibilities to further explore the pathogenesis of CNS-related diseases and evaluate drug efficacy.

Following the above experimental protocol and the sampling parameters set in Table 1, water-like, colorless, and transparent rat HECF was obtained at the set sampling rate (Figure 1K). The osmotic pressure of the obtained rat HECF was 290-310 mOsm/L, which can indirectly ensure the quality of samples18,19.
<img alt="Figure 1" class="xfigimg" src="...

Watch this Scientific Journal Video about Real-Time Dynamic Collection of Hippocampal Extracellular Fluid from Conscious Rats Using a Microdialysis System at JoVE.com

Real-Time Dynamic Collection of Hippocampal Extracellular Fluid from Conscious Rats Using a Microdialysis System

The protocol here provides a detailed real-time dynamic sampling of extracellular fluid from the hippocampus of awake rats using a microdialysis system.

A variety of central nervous system (CNS) diseases are associated with changes in the composition of hippocampal extracellular fluid (HECF). However, ...

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JoVE Journal

Neuroscience

2.3K Views.  Chengdu University of Traditional Chinese Medicine. The protocol here provides a detailed real-time dynamic sampling of extracellular fluid from the hippocampus of awake rats using a microdialysis system.

Video: Real-Time Dynamic Collection of Hippocampal Extracellular Fluid from Conscious Rats Using a Microdialysis System

Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol. However, ethanol is typically self-administered orally, rather than intravenously like many other drugs of abuse. The pharmacokinetics of orally administered drugs are more complex than intravenously administered drugs. Because understanding the relationship between the pharmacological and behavioral effects of ethanol requires knowledge of the time course of ethanol reaching the brain during and after drinking, we use in vivo microdialysis and gas chromatography with flame ionization detection to monitor brain dialysate ethanol concentrations over time. 
Combined microdialysis-behavioral experiments involve the use of several techniques. In this article, stereotaxic surgery, behavioral training and microdialysis, which can be adapted to test a multitude of self-administration and neurochemical centered hypotheses, are included only to illustrate how they relate to the subsequent phases of sample collection and dialysate ethanol analysis. Dialysate ethanol concentration analysis via gas chromatography with flame-ionization detection, which is specific to ethanol studies, is described in detail. Data produced by these methods reveal the pattern of ethanol reaching the brain during the self-administration procedure, and when paired with neurochemical analysis of the same dialysate samples, allows conclusions to be made regarding the pharmacological and behavioral effects of ethanol.

A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol.  ...

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance imaging. However, use of such methods in animal models requires anesthesia and hence both alters the brain state and prevents behavioral measures. An alternative method is the use of in vivo microdialysis to take continuous measurement of brain extracellular fluid concentrations of glucose, lactate, and related metabolites in awake, unrestrained animals. This technique is especially useful when combined with tasks designed to rely on specific brain regions and/or acute pharmacological manipulation; for example, hippocampal measurements during a spatial working memory task (spontaneous alternation) show a dip in extracellular glucose and rise in lactate that are suggestive of enhanced glycolysis1-3,4-5, and intrahippocampal insulin administration both improves memory and increases hippocampal glycolysis6. Substances such as insulin can be delivered to the hippocampus via the same microdialysis probe used to measure metabolites. The use of spontaneous alternation as a measure of hippocampal function is designed to avoid any confound from stressful motivators (e.g. footshock), restraint, or rewards (e.g. food), all of which can alter both task performance and metabolism; this task also provides a measure of motor activity that permits control for nonspecific effects of treatment. Combined, these methods permit direct measurement of the neurochemical and metabolic variables regulating behavior.

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Modulation of hippocampally-dependent spatial working memory by direct intrahippocampal microinjection, accompanied and followed by in vivo microdialysis for metabolites in conscious, behaving animals.

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance ...

Isolation of Cerebrospinal Fluid from Rodent Embryos for use with Dissected Cerebral Cortical Explants

The CSF is a complex fluid with a dynamically varying proteome throughout development and in adulthood. During embryonic development, the nascent CSF differentiates from the amniotic fluid upon closure of the anterior neural tube. CSF volume then increases over subsequent days as the neuroepithelial progenitor cells lining the ventricles and the choroid plexus generate CSF. The embryonic CSF contacts the apical, ventricular surface of the neural stem cells of the developing brain and spinal cord. CSF provides crucial fluid pressure for the expansion of the developing brain and distributes important growth promoting factors to neural progenitor cells in a temporally-specific manner. To investigate the function of the CSF, it is important to isolate pure samples of embryonic CSF without contamination from blood or the developing telencephalic tissue. Here, we describe a technique to isolate relatively pure samples of ventricular embryonic CSF that can be used for a wide range of experimental assays including mass spectrometry, protein electrophoresis, and cell and primary explant culture. We demonstrate how to dissect and culture cortical explants on porous polycarbonate membranes in order to grow developing cortical tissue with reduced volumes of media or CSF. With this method, experiments can be performed using CSF from varying ages or conditions to investigate the biological activity of the CSF proteome on target cells.

The ventricular cerebrospinal fluid (CSF) bathes the neuroepithelial and cerebral cortical progenitor cells during early brain development in the embryo. Here we describe the method developed to isolate ventricular CSF from rodent embryos of different ages in order to investigate its biological function. In addition, we demonstrate our cerebral cortical explant dissection and culture technique that allows for explant growth with minimal volumes of culture medium or CSF.

The CSF is a complex fluid with a dynamically varying proteome throughout development and in adulthood. During embryonic development, the nascent CSF ...

Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are increasingly viewed as an early feature of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD) and Huntington&#8217;s disease (HD). As yet unclear is the mechanism(s) by which axonal injury is induced. We reported the development of a novel technique to produce biologically active, monobiotinylated BDNF (mBtBDNF) that can be used to trace axonal transport of BDNF. Quantum dot-labeled BDNF (QD-BDNF) was produced by conjugating quantum dot 655 to mBtBDNF. A microfluidic device was used to isolate axons from neuron cell bodies. Addition of QD-BDNF to the axonal compartment allowed live imaging of BDNF transport in axons. We demonstrated that QD-BDNF moved essentially exclusively retrogradely, with very few pauses, at a moving velocity of around 1.06 &#956;m/sec. This system can be used to investigate mechanisms of disrupted axonal function in AD or HD, as well as other degenerative disorders.

Axonal transport of BDNF, a neurotrophic factor, is critical for the survival and function of several neuronal populations. Some degenerative disorders are marked by disruption of axonal structure and function. We demonstrated the techniques used to examine live trafficking of QD-BDNF in microfluidic chambers using primary neurons.

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are ...

Real-time Electrophysiology: Using Closed-loop Protocols to Probe Neuronal Dynamics and Beyond

Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where the stimulus applied depends in real-time on the response of the system. Recent applications range from the implementation of virtual reality systems for studying motor responses both in mice1 and in zebrafish2, to control of seizures following cortical stroke using optogenetics3. A key advantage of closed-loop techniques resides in the capability of probing higher dimensional properties that are not directly accessible or that depend on multiple variables, such as neuronal excitability4 and reliability, while at the same time maximizing the experimental throughput. In this contribution and in the context of cellular electrophysiology, we describe how to apply a variety of closed-loop protocols to the study of the response properties of pyramidal cortical neurons, recorded intracellularly with the patch clamp technique in acute brain slices from the somatosensory cortex of juvenile rats. As no commercially available or open source software provides all the features required for efficiently performing the experiments described here, a new software toolbox called LCG5 was developed, whose modular structure maximizes reuse of computer code and facilitates the implementation of novel experimental paradigms. Stimulation waveforms are specified using a compact meta-description and full experimental protocols are described in text-based configuration files. Additionally, LCG has a command-line interface that is suited for repetition of trials and automation of experimental protocols.

Closed-loop protocols are becoming increasingly widespread in modern day electrophysiology. We present a simple, versatile and inexpensive way to perform complex electrophysiological protocols in cortical pyramidal neurons in vitro, using a desktop computer and a digital acquisition board.

Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where ...

Custom-made Microdialysis Probe Design

Microdialysis is a commonly used technique in neuroscience research. Therefore commercial probes are in great demand to monitor physiological, pharmacological and pathological changes in cerebrospinal fluid. Unfortunately, commercial probes are expensive for research groups in public institutions. In this work, a probe assembly is explained in detail to build a reliable, concentric, custom-made microdialysis probe for less than $10. The microdialysis probe consists of a polysulfone membrane with a molecular cut-off of 30 kDa. Probe in vitro recoveries of substances with different molecular weight (in the range of 100-1,600 Da) and different physicochemical properties are compared. The probe yields an in vitro recovery of approximately 20% for the small compounds glucose, lactate, acetylcholine and ATP. In vitro recoveries for neuropeptides with a molecular weight between 1,000-1,600 Da amount to 2-6%. Thus, while the higher molecular weight of the neuropeptides lowered in vitro recovery values, dialysis of compounds in the lower range (up to 500 Da) of molecular weights has no great impact on the in vitro recovery rate. The present method allows utilization of a dialysis membrane with other cut-off value and membrane material. Therefore, this custom-made probe assembly has the advantage of sufficient flexibility to dialyze substances in a broad molecular weight range. Here, we introduce a microdialysis probe with an exchange length of 2 mm, which is applicable for microdialysis in mouse and rat brain regions. However, dimensions of the probe can easily be adapted for larger exchange lengths to be used in larger animals.

Microdialysis is a commonly used technique in neuroscience research. Therefore commercial probes are in great demand.In this work a probe assembly is explained in detail to build a reliable, concentric, custom-made microdialysis probe for less than $10.

Microdialysis is a commonly used technique in neuroscience research. Therefore commercial probes are in great demand to monitor physiological, ...

A Protocol for the Administration of Real-Time fMRI Neurofeedback Training

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control neuroplastic processes and correct abnormal function, or even shift functions from damaged tissue to physiologically healthy brain regions, hold the potential to dramatically improve overall health. Of the current neuroplastic interventions in development, neurofeedback training (NFT) from functional Magnetic Resonance Imaging (fMRI) has the advantages of being completely non-invasive, non-pharmacologic, and spatially localized to target brain regions, as well as having no known side effects. Furthermore, NFT techniques, initially developed using fMRI, can often be translated to exercises that can be performed outside of the scanner without the aid of medical professionals or sophisticated medical equipment. In fMRI NFT, the fMRI signal is measured from specific regions of the brain, processed, and presented to the participant in real-time. Through training, self-directed mental processing techniques, that regulate this signal and its underlying neurophysiologic correlates, are developed. FMRI NFT has been used to train volitional control over a wide range of brain regions with implications for several different cognitive, behavioral, and motor systems. Additionally, fMRI NFT has shown promise in a broad range of applications such as the treatment of neurologic disorders and the augmentation of baseline human performance. In this article, we present an fMRI NFT protocol developed at our institution for modulation of both healthy and abnormal brain function, as well as examples of using the method to target both cognitive and auditory regions of the brain.

The ability to induce and/or control neural plasticity may be critical in future treatments for neurologic disorders and the recovery from brain injury. In this paper, we present a protocol on the use of neurofeedback training with functional magnetic resonance imaging to modulate human brain function.

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control ...

Real-time Iontophoresis with Tetramethylammonium to Quantify Volume Fraction and Tortuosity of Brain Extracellular Space

This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the extracellular space (ECS) of the living brain. The ECS surrounds all brain cells and contains both interstitial fluid and extracellular matrix. The transport of many substances required for brain activity, including neurotransmitters, hormones, and nutrients, occurs by diffusion through the ECS. Changes in the volume and geometry of this space occur during normal brain processes, like sleep, and pathological conditions, like ischemia. However, the structure and regulation of brain ECS, particularly in diseased states, remains largely unexplored. The RTI method measures two physical parameters of living brain: volume fraction and tortuosity. Volume fraction is the proportion of tissue volume occupied by ECS. Tortuosity is a measure of the relative hindrance a substance encounters when diffusing through a brain region as compared to a medium with no obstructions. In RTI, an inert molecule is pulsed from a source microelectrode into the brain ECS. As molecules diffuse away from this source, the changing concentration of the ion is measured over time using an ion-selective microelectrode positioned roughly 100 &#181;m away. From the resulting diffusion curve, both volume fraction and tortuosity can be calculated. This technique has been used in brain slices from multiple species (including humans) and in vivo to study acute and chronic changes to ECS. Unlike other methods, RTI can be used to examine both reversible and irreversible changes to the brain ECS in real time.

This protocol describes real-time iontophoresis, a method that measures physical parameters of the extracellular space (ECS) of living brains. The diffusion of an inert molecule released into the ECS is used to calculate the ECS volume fraction and tortuosity. It is ideal for studying acute reversible changes to brain ECS.

This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the ...

Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. To efficiently and accurately deorphanize ORs, we combine the use of a heterologous cell line to express mammalian ORs and a genetically modified biosensor plasmid to measure cAMP production downstream of OR activation in real time. This assay can be used to screen odorants against ORs and vice versa. Positive odorant-receptor interactions from the screens can be subsequently confirmed by testing against various odor concentrations, generating concentration-response curves. Here we used this method to perform a high-throughput screening of an odorous compound against a human OR library expressed in Hana3A cells and confirmed that the positively-responding receptor is the cognate receptor for the compound of interest. We found this high-throughput detection method to be efficient and reliable in assessing OR activation and our data provide an example of its potential use in OR functional studies.

Characterizing the function of odorant receptors serves an indispensable part in the deorphanization process. We describe a method to measure the activation of odorant receptors in real time using a cAMP assay.

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. ...

An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice

The cerebrospinal fluid (CSF) is a valuable body fluid for analysis in neuroscience research. It is one of the fluids in closest contact with the central nervous system and thus, can be used to analyze the diseased state of the brain or spinal cord without directly accessing these tissues. However, in mice it is difficult to obtain from the cisterna magna due to its closeness to blood vessels, which often contaminate samples. The area for CSF collection in mice is also difficult to dissect to and often only small samples are obtained (maximum of 5-7 &#181;L or less). This protocol describes in detail a technique that improves on current methods of collection to minimize contamination from blood and allow for the abundant collection of CSF (on average 10-15 &#181;L can be collected). This technique can be used with other dissection methods for tissue collection from mice, as it does not impact any tissues during CSF extraction. Thus, the brain and spinal cord are not affected with this technique and remain intact. With greater CSF sample collection and purity, more analyses can be used with this fluid to further aid neuroscience research and better understand diseases affecting the brain and spinal cord.

This protocol describes an improved technique for the abundant collection of cerebrospinal fluid (CSF) with no contamination from blood. With greater sample collection and purity, more analyses can be performed using CSF to further our understanding of diseases that affect the brain and spinal cord.

The cerebrospinal fluid (CSF) is a valuable body fluid for analysis in neuroscience research. It is one of the fluids in closest contact with the central ...