This work was supported by the Nature Science Foundation grant of Hubei Province, China [Grant No. 2018CFB761].

1. Husbandry
<ol>
	<li>Use 12 week old Kunming mice. 
	NOTE: We used mice (n = 40, 20 males, 37&#8211;46 g, mean 42.38 &#177; 2.39 g) from the Experimental Animals Center of&#160;Tongji&#160;Medical&#160;College.</li>
	<li>House the mice under standard conditions with free access to food and drinking water. Keep two mice in a 530 cm2 cage with wood shaving bedding.</li>
	<li>Maintain a room temperature between 21&#8211;23 &#176;C.</li>
	<li>Feed mice with a normal salt diet (0.3% NaCl) throughout the study.</li>
</ol>
2. Blood sample collection
<ol>
	<li>Prepare the following equipment: vacuum tube (1 mL), butterfly needle, eyeglass magnifier, and plastic restraining holder. Place them on a sterile surface (Figure 1).</li>
	<li>Place a mouse in a plastic restraining holder and wash its tail with warm water (20&#8211;30 &#176;C). Wipe the tail with 70% ethanol-saturated cotton balls to expand the vein. Select the right or left tail vein for blood sampling. Grasp the lower portion of the tail gently and keep the tail straight during blood sample collection.</li>
	<li>Collect the blood. If comparing methods, collect blood in two groups: the &#8220;experimental&#8221; group using the procedure we have developed, presented below, and &#8220;conventional&#8221; group using a conventional method, also presented below.
	<ol>
		<li>Experimental collection:
		<ol>
			<li>Wear an eyeglass magnifier to improve the viewing for puncture of the tail vein. Insert the 22 G butterfly needle tip into one of the lateral tail veins at a position approximately half the distance distally from the tip of the tail at an angel approximately 10&#176;, moving towards the base of the tail for multiple samples.</li>
			<li>Insert the rubber end of the butterfly needle into the vacuum blood collection tube to collect blood (Figure 2). 
			NOTE: If blood stops flowing out during blood collection, the needle angle should be quickly adjusted. To avoid blood coagulation in the needle, another puncture position should be selected if blood stops flowing out after 15 s.</li>
		</ol>
		</li>
		<li>Conventional method:
		<ol>
			<li>Insert the needle connected to a syringe into one of the lateral veins approximately one-third of the distance distally from the tip of the tail.</li>
			<li>When blood appears in the hub, pull back the plunger slowly to collect blood (Figure 3)7. 
			NOTE: To further elucidate the effects of varying experience with blood collection, a beginner and expert were chosen to collect blood samples using experimental and conventional methods simultaneously.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>After blood collection, remove the needle and press the puncture point to stop bleeding. Then, release the mouse from the plastic restraining holder and return the mouse to its cage. 
	NOTE: It has been reported that up to 10% of total blood volume can be safely removed from a healthy animal at 2 week intervals8, so about 175 &#181;L of blood was collected each time in accordance with ethical principles.</li>
	<li>Use tubes with EDTA to collect plasma and use tubes without anticoagulants to collect serum. Gently invert the tube several times and put them on ice vertically.</li>
	<li>Centrifuge the blood sample collection tubes in a refrigerated centrifuge at 1,000 x g for 10 min to separate plasma and serum. 
	NOTE: Successful blood collection is defined as obtaining a volume of 175 &#181;L each time. No more than three punctures should be attempted, and a failed blood collection is defined as a total blood volume of less than 175 &#181;L after the third puncture. The sampling duration is defined as the time from the tail vein puncture to removal of the needle after blood collection.</li>
	<li>Collect blood twice at intervals of 2 weeks8.</li>
</ol>
3. Statistical analysis
<ol>
	<li>Use commercially available statistical software for analysis. Present data as mean value &#177; standard deviation, using p &#60; 0.05 as the cutoff for statistical significance.</li>
</ol>

<ol>
	<li>Grouzmann, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+sampling+methodology+is+crucial+for+precise+measurement+of+plasma+catecholamines+concentrations+in+mice.">Blood sampling methodology is crucial for precise measurement of plasma catecholamines concentrations in mice.</a> European Journal of Physiology. 447 (2), 254-258 (2003).</li><li>Heimann, M., Roth, D. R., Ledieu, D., Pfister, R., Classen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sublingual+and+submandibular+blood+collection+in+mice:+a+comparison+of+effects+on+body+weight,+food+consumption+and+tissue+damage.">Sublingual and submandibular blood collection in mice: a comparison of effects on body weight, food consumption and tissue damage.</a> Lab Animal. 44 (4), 352-358 (2010).</li><li>Heimann, M., Kasermann, H. P., Pfister, R., Roth, D. R., Burki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+collection+from+the+sublingual+vein+in+mice+and+hamsters:+a+suitable+alternative+to+retrobulbar+technique+that+provides+large+volumes+and+minimizes+tissue+damage.">Blood collection from the sublingual vein in mice and hamsters: a suitable alternative to retrobulbar technique that provides large volumes and minimizes tissue damage.</a> Lab Animal. 43 (3), 255-260 (2009).</li><li>Hoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+blood+collection+in+the+mouse.">Methods of blood collection in the mouse.</a> Lab Animal. 29 (10), 47-53 (2000).</li><li>Zou, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeated+Blood+Collection+from+Tail+Vein+of+Non-Anesthetized+Rats+with+a+Vacuum+Blood+Collection+System.">Repeated Blood Collection from Tail Vein of Non-Anesthetized Rats with a Vacuum Blood Collection System.</a> Journal of Visualized Experiments. (130), (2017).</li><li>Kilkenny, C., Altman, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+bioscience+research+reporting:+ARRIVE-ing+at+a+solution.">Improving bioscience research reporting: ARRIVE-ing at a solution.</a> Lab Animal. 44 (4), 377-378 (2010).</li><li>Parasuraman, S., Raveendran, R., Kesavan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+sample+collection+in+small+laboratory+animals.">Blood sample collection in small laboratory animals.</a> Journal of Pharmacology and Pharmacotherapeutics. 1 (2), 87-93 (2010).</li><li>Diehl, K. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+good+practice+guide+to+the+administration+of+substances+and+removal+of+blood,+including+routes+and+volumes.">A good practice guide to the administration of substances and removal of blood, including routes and volumes.</a> Journal of Applied Toxicology. 21 (1), 15-23 (2001).</li><li>Wollowitz, A., Bijur, P. E., Esses, D., John Gallagher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+butterfly+needles+to+draw+blood+is+independently+associated+with+marked+reduction+in+hemolysis+compared+to+intravenous+catheter.">Use of butterfly needles to draw blood is independently associated with marked reduction in hemolysis compared to intravenous catheter.</a> Academic Emergency Medicine. 20 (11), 1151-1155 (2013).</li><li>Eder, J. M., Cutter, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+device+for+collecting+cord+blood.">A new device for collecting cord blood.</a> Obstetrics and Gynecology. 86 (5), 850-852 (1995).</li></ol>

The authors have nothing to disclose.

The present study describes an easy-to-learn blood collection method in mice that is superior to the conventional techniques. First, the method can be easily mastered with a high success rate. Second, it is based on the vacuum negative pressure principle and allows for continuous drawing of blood with a reduced risk of direct blood exposure, which also reduces the chance of contamination and hemolysis9. Third, this method is feasible for frequent sampling of blood with adequate volumes from mice over a short period of time for various research purposes. Moreover, the procedure inflicts only minimal injury upon mice, and blood collection can be performed without the use of anesthetics; thus, the influence of the stress response and anesthetics on blood samples can be avoided.
The tail vein is a superior location for blood sampling according to the approved protocol7. However, it is not always easy to obtain sufficient blood volume from thin tail veins with low blood flows. In this case, the skin is usually cut open and vein is penetrated by a lancet, or the end of the tail is removed quickly by a razor.
This protocol aims to improve the methodology of blood collection from mice using the vacuum blood collection system, which requires a vacuum blood sample collection tube, butterfly needle, and eyeglass magnifier. This vacuum blood sampling system is usually used for collecting blood samples from patients in daily clinical practice10. With the help of an eyeglass magnifier, the perfect puncture point of a tail vein is easier to locate. When the tip of a needle is inserted into the tail vein, blood will automatically flow into the vacuum tube due to negative pressure. After withdrawing the needle from the tail vein, the blood that is blocked in the catheter will flow into the collecting vacuum tube due to the vacuum.
The following tips are important for successful application of the method. First, the body weight of each mouse should be 40 g or higher to decrease difficulty in puncturing and obtaining enough blood. Second, in the case of failed blood collection, experimenters should try to withdraw the needle slowly until blood continues to flow out. Third, it is vital to extend the tail to avoid any movement during blood sampling. Holding the needle gently can help to keep the needle tip in the vein as the mouse tail moves. Fourth, if blood stops flowing out during blood collection, the needle angle should be adjusted in a timely manner. In order to avoid blood coagulation in the needle, another puncture position should be selected if blood stops flowing out after 15 s. Finally, the cooperation of two operators is recommended while using this technique to collect blood from mice.&#160;
In short, the adopted vacuum blood collection method for use in mice is safe, feasible, and easy to practice. This method enables frequent sampling of blood with adequate blood volumes from non-anesthetized mice.

Blood sampling from animals involved in experiments is a basic research technique. There are some available techniques for blood collection from mice, including tail snips and puncturing of the heart, retro-orbital plexus, jugular vein, caudal vein, and vena cava. Ideally, blood should be collected in a minimally invasive manner, with minimal impact on the animal&#8217;s health. However, the most commonly used techniques often inflict stress upon animals and can impact research outcomes1. Blood collection from the retro-orbital plexus can be used to obtain enough blood volume from mice2. However, it can result in severe tissue damage and does not allow for obtaining blood repeatedly in short time intervals3.
The caudal vein is a superior location for blood collection, which inflicts minimal injury upon mice. However, the tail veins of mice are thin, and it is sometimes difficult to obtain enough blood through the conventional puncture technique. In some cases, repeated punctures are required to obtain the desired blood volume. Anesthesia is also commonly recommended to facilitate blood sampling from the tail veins of mice.&#160; Moreover, a scalpel, straight edge razor, or sharp scissors may be needed to remove the ends of the tails to obtain the required blood samples4. We have previously reported successful blood collection from the tail veins of non-anaesthetized rats by the vacuum blood sample collection system, which reduced the risk of blood contamination and avoided the need for repeated punctures5. This study reports a similar blood collection method in non-anesthetized mice.

<table border="0" cellpadding="0" cellspacing="0" style="width:585px;" width="583">
	<tbody>
		<tr height="78">
			<td height="78" style="height:78px;width:166px;">Double-pointed needle</td>
			<td style="width:135px;">Shanghai Kang Nong medical instrument co., LTD, China</td>
			<td style="width:129px;">20163151718</td>
			<td style="width:154px;">22G (0.7 mm x 25 mm)</td>
		</tr>
		<tr height="78">
			<td height="78" style="height:78px;width:166px;">Eyeglass magnifier</td>
			<td style="width:135px;">Vergroberung</td>
			<td style="width:129px;"></td>
			<td style="width:154px;">1.5x</td>
		</tr>
		<tr height="78">
			<td height="78" style="height:78px;width:166px;">Normal salt diet for mice</td>
			<td style="width:135px;"></td>
			<td style="width:129px;"></td>
			<td style="width:154px;">Mice received a normal salt diet (0.3% NaCl) throughout the study.</td>
		</tr>
		<tr height="78">
			<td height="78" style="height:78px;width:166px;">Plastic holder</td>
			<td style="width:135px;">Shanghai Kang Nong medical instrument co., LTD, China</td>
			<td style="width:129px;"></td>
			<td style="width:154px;">35-45 g rat hoder</td>
		</tr>
		<tr height="78">
			<td height="78" style="height:78px;width:166px;">SPSS software for statistical analysis</td>
			<td style="width:135px;">SPSS Inc, Chicago; USA</td>
			<td style="width:129px;">Version 17.0.</td>
			<td style="width:154px;"></td>
		</tr>
		<tr height="90">
			<td height="90" style="height:91px;width:166px;">Syringe</td>
			<td style="width:135px;">Shandong wego Medical polymer products co. LTD., China</td>
			<td style="width:129px;">20160911A</td>
			<td style="width:154px;">1 mL (Matching needle size: 0.45&#215;16RW LB)</td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:166px;">Vacuum blood collection system</td>
			<td style="width:135px;">Wuhan Zhi Yuan, medical science and technology co., LTD, China</td>
			<td style="width:129px;">20171222</td>
			<td style="width:154px;">1 mL (&#934;12.4&#215;L75)</td>
		</tr>
	</tbody>
</table>

modified blood collection from tail veins of non-anesthetized mice with a vacuum blood collection system and eyeglass magnifier

Blood sample collection is the basis of experimental animal research. It is of importance to obtain adequate blood samples for various research purposes. The tail veins of mice are small, and it is sometimes difficult to obtain the required blood volume by conventional puncture methods. This study investigates the superiority of repeated blood sample collection from tail veins of mice through use of a vacuum blood collection system and eyeglass magnifier (experimental group) compared to conventional blood sampling methods (conventional group), performed by beginners and experts, respectively. With the help of an eyeglass magnifier, a butterfly needle tip is inserted into the tail vein of each mouse in the experimental group. When the vein is penetrated successfully, a blood sample is collected in the vacuum collection tube by insertion of the rubber end of a butterfly needle into the vacuum blood collection tube. The plunger is then used to collect blood without the help of the eyeglass magnifier in the conventional group. Success rates of blood sample collection by the beginners and experts were shown to be 70% vs. 100% (p &#60; 0.01) in the experimental group and 35% vs. 85% (p &#60; 0.01) in the conventional group. For both beginners and experts, puncture times required for obtaining required blood sample were significantly lower in the experimental group compared to the conventional group (2.40 &#177; 0.75 vs. 2.90 &#177; 0.31, p &#60; 0.05; 1.15 &#177; 0.37 vs. 1.55 &#177; 0.76, p &#60; 0.05). In conclusion, the presented blood sampling technique is feasible and easy to practice and enables frequent sampling of adequate blood volumes from non-anesthetized mice.&#160;

Body mass, blood collection volumes, and sampling durations of the two groups&#160; 
Blood samples were collected from 20 mice (10 males) twice at 2 week intervals in each group. The mean body mass of mice was similar between the experimental and conventional groups for beginners and experts, respectively (42.40 g &#177; 1.42 g vs. 42.65 g &#177; 1.14 g, p &#62; 0.05; 42.55 g &#177; 2.91 g vs. 43.20 g &#177; 2.69 g, p &#62; 0.05). Collected blood volumes and sampling durations were similar between the two groups in experts (184.25 &#181;L &#177; 11.95 &#181;L vs. 171.75 &#181;L &#177; 25.61 &#181;L, p &#62; 0.05; 1.85 min &#177; 0.68 min vs. 2.17 min &#177; 0.80 min, p &#62; 0.05). However, higher collected blood volumes and shorter sampling durations were seen in the experimental group compared to the conventional group in beginners (172.00 &#181;L &#177; 15.17 &#181;L vs. 148.50 &#181;L &#177; 30.22 &#181;L, p &#60; 0.01; 3.11 min &#177; 0.44 min vs. 4.08 min &#177; 0.61 min, p &#60; 0.01). Compared to beginners, experts collected higher blood volumes and showed lower sampling times using experimental and conventional methods (184.25 &#181;L &#177; 11.95 &#181;L vs. 172.00 &#181;L &#177; 15.17 &#181;L, p &#60; 0.01; 171.75 &#181;L &#177; 25.61 &#181;L vs. 148.50 &#181;L &#177; 30.22 &#181;L, p &#60; 0.05; 1.85 min &#177; 0.68 min vs. 3.11 min &#177; 0.44 min, p &#60; 0.01; 2.17 min &#177; 0.80 min vs. 4.08 min &#177; 0.61 min, p &#60; 0.05) (Table 1).&#160;
Success rates and puncture times of the two groups&#160; 
The comparison of success rates between beginners and experts was 70% (14/20) vs. 100% (20/20) (p &#60; 0.01) in the experimental group and 35% (7/20) vs. 85% (17/20) (p &#60; 0.01) in the conventional group. Higher success rates were also seen in the experimental group compared to the conventional group in beginners [70% (14/20) vs. 35% (7/20), p &#60; 0.05]. In both beginners and experts, the number of punctures was significantly lower in the experimental group compared to the conventional group (2.40 &#177; 0.75 vs. 2.90 &#177; 0.31, p &#60; 0.05; 1.15 &#177; 0.37 vs. 1.55 &#177; 0.76, p &#60; 0.05). Compared to beginners, lower puncture times for experts were observed using experimental and conventional methods. (1.15 &#177; 0.37 vs. 2.40 &#177; 0.75, p &#60; 0.01; 1.55 &#177; 0.76 vs. 2.90 &#177; 0.31, p &#60; 0.01) (Table 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59136/59136fig1.jpg" /> 
Figure 1: Equipment. Shown are 1 mL vacuum blood collection tubes and a 22 G butterfly needle (left), an eyeglass magnifier (middle), and a plastic restraining holder (right). <a href="https://www.jove.com/files/ftp_upload/59136/59136fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59136/59136fig2.jpg" /> 
Figure 2: Successful blood collection in the experimental group. <a href="https://www.jove.com/files/ftp_upload/59136/59136fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59136/59136fig3.jpg" /> 
Figure 3: Successful blood collection in the conventional group. <a href="https://www.jove.com/files/ftp_upload/59136/59136fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="2">Experimental group</td>
			<td colspan="2">Conventional group</td>
		</tr>
		<tr>
			<td></td>
			<td>beginner</td>
			<td>expert</td>
			<td>beginner</td>
			<td>expert</td>
		</tr>
		<tr>
			<td>Body mass (g)</td>
			<td>42.4&#177;1.42</td>
			<td>42.55&#177;2.91</td>
			<td>42.65&#177;1.14</td>
			<td>43.20&#177;2.69</td>
		</tr>
		<tr>
			<td>Collected blood volume (&#181;L)</td>
			<td>172.00&#177;15.17</td>
			<td>184.25&#177;11.95##</td>
			<td>148.50&#177;30.22**</td>
			<td>171.75&#177;25.61#</td>
		</tr>
		<tr>
			<td>Sampling duration (min)</td>
			<td>3.11&#177;0.44</td>
			<td>1.85&#177;0.68##</td>
			<td>4.08&#177;0.61**</td>
			<td>2.17&#177;0.80##</td>
		</tr>
		<tr>
			<td>Blood collection times</td>
			<td>20</td>
			<td>20</td>
			<td>20</td>
			<td>20</td>
		</tr>
		<tr>
			<td>Average number of punctures&#160;</td>
			<td>2.40&#177;0.75</td>
			<td>1.15&#177;0.37##</td>
			<td>2.90&#177;0.31*</td>
			<td>1.55&#177;0.76##*</td>
		</tr>
		<tr>
			<td>One time puncture</td>
			<td>3</td>
			<td>17</td>
			<td>0</td>
			<td>12</td>
		</tr>
		<tr>
			<td>Two times puncture&#160;</td>
			<td>6</td>
			<td>3</td>
			<td>2</td>
			<td>5</td>
		</tr>
		<tr>
			<td>Three times puncture&#160;</td>
			<td>5</td>
			<td>0</td>
			<td>5</td>
			<td>0</td>
		</tr>
		<tr>
			<td>Failed</td>
			<td>6</td>
			<td>0</td>
			<td>13</td>
			<td>3</td>
		</tr>
		<tr>
			<td>Success rate</td>
			<td>70%</td>
			<td>100%##</td>
			<td>35%*</td>
			<td>85%##</td>
		</tr>
	</tbody>
</table>
Table 1: Comparison of results between the experimental and conventional groups. *p &#60; 0.05, **p &#60; 0.01, experimental method vs. conventional method. #p &#60; 0.05, ##p &#60; 0.01, beginner vs. expert.

Watch this Scientific Journal Video about Modified Blood Collection from Tail Veins of Non-anesthetized Mice with a Vacuum Blood Collection System and Eyeglass Magnifier at JoVE.com

Modified Blood Collection from Tail Veins of Non-anesthetized Mice with a Vacuum Blood Collection System and Eyeglass Magnifier

This study reports blood sampling from tail vein in mice using a vacuum extraction tube system with eyeglass magnifier. Our method is easy to practice and could be used for repeat blood sampling in mice.

Blood sample collection is the basis of experimental animal research. It is of importance to obtain adequate blood samples for various research purposes. ...

modified-blood-collection-from-tail-veins-non-anesthetized-mice-with

Research

JoVE Journal

Behavior

17.6K Views.  Huazhong University of Science and Technology. This study reports blood sampling from tail vein in mice using a vacuum extraction tube system with eyeglass magnifier. Our method is easy to practice and could be used for repeat blood sampling in mice.

Video: Modified Blood Collection from Tail Veins of Non-anesthetized Mice with a Vacuum Blood Collection System and Eyeglass Magnifier

Method for Simultaneous fMRI/EEG Data Collection during a Focused Attention Suggestion for Differential Thermal Sensation

In the present work, we demonstrate a method for concurrent collection of EEG/fMRI data. In our setup, EEG data are collected using a high-density 256-channel sensor net. The EEG amplifier itself is contained in a field isolation containment system (FICS), and MRI clock signals are synchronized with EEG data collection for subsequent MR artifact characterization and removal. We demonstrate this method first for resting state data collection. Thereafter, we demonstrate a protocol for EEG/fMRI data recording, while subjects listen to a tape asking them to visualize that their left hand is immersed in a cold-water bath and referred to, here, as the cold glove paradigm. Thermal differentials between each hand are measured throughout EEG/fMRI data collection using an MR compatible temperature sensor that we developed for this purpose. We collect cold glove EEG/fMRI data along with simultaneous differential hand temperature measurements both before and after hypnotic induction. Between pre and post sessions, single modality EEG data are collected during the hypnotic induction and depth assessment process. Our representative results demonstrate that significant changes in the EEG power spectrum can be measured during hypnotic induction, and that hand temperature changes during the cold glove paradigm can be detected rapidly using our MR compatible differential thermometry device.

We present a protocol for concurrent collection of EEG/fMRI data, and synchronized MR clock signal recording. We demonstrate this method using a unique paradigm whereby subjects receive &#x2018;cold glove&#x2019; instructions during scanning, and EEG/fMRI data are recorded along with hand temperature measurements both before and after hypnotic induction.

In the present work, we demonstrate a method for concurrent collection of EEG/fMRI data. In our setup, EEG data are collected using a high-density ...

Construction of Microdrive Arrays for Chronic Neural Recordings in Awake Behaving Mice

State-of-the-art electrophysiological recordings from the brains of freely behaving animals allow researchers to simultaneously examine local field potentials (LFPs) from populations of neurons and action potentials from individual cells, as the animal engages in experimentally relevant tasks. Chronically implanted microdrives allow for brain recordings to last over periods of several weeks. Miniaturized drives and lightweight components allow for these long-term recordings to occur in small mammals, such as mice. By using tetrodes, which consist of tightly braided bundles of four electrodes in which each wire has a diameter of 12.5 &mu;m, it is possible to isolate physiologically active neurons in superficial brain regions such as the cerebral cortex, dorsal hippocampus, and subiculum, as well as deeper regions such as the striatum and the amygdala. Moreover, this technique insures stable, high-fidelity neural recordings as the animal is challenged with a variety of behavioral tasks. This manuscript describes several techniques that have been optimized to record from the mouse brain. First, we show how to fabricate tetrodes, load them into driveable tubes, and gold-plate their tips in order to reduce their impedance from M&Omega; to K&Omega; range. Second, we show how to construct a custom microdrive assembly for carrying and moving the tetrodes vertically, with the use of inexpensive materials. Third, we show the steps for assembling a commercially available microdrive (Neuralynx VersaDrive) that is designed to carry independently movable tetrodes. Finally, we present representative results of local field potentials and single-unit signals obtained in the dorsal subiculum of mice. These techniques can be easily modified to accommodate different types of electrode arrays and recording schemes in the mouse brain.

The design and assembly of microdrives for in vivo electrophysiological recordings of brain signals from the mouse is described. By attaching microelectrode bundles to sturdy driveable carriers, these techniques allow for long-term and stable neural recordings. The lightweight design allows for unrestricted behavioral performance by the animal following drive implantation.

State-of-the-art electrophysiological recordings from the brains of freely behaving animals allow researchers to simultaneously examine local field ...

Contextual and Cued Fear Conditioning Test Using a Video Analyzing System in Mice

The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association between environmental cues and aversive experiences. In this test, mice are placed into a conditioning chamber and are given parings of a conditioned stimulus (an auditory cue) and an aversive unconditioned stimulus (an electric footshock). After a delay time, the mice are exposed to the same conditioning chamber and a differently shaped chamber with presentation of the auditory cue. Freezing behavior during the test is measured as an index of fear memory. To analyze the behavior automatically, we have developed a video analyzing system using the ImageFZ application software program, which is available as a free download at http://www.mouse-phenotype.org/. Here, to show the details of our protocol, we demonstrate our procedure for the contextual and cued fear conditioning test in C57BL/6J mice using the ImageFZ system. In addition, we validated our protocol and the video analyzing system performance by comparing freezing time measured by the ImageFZ system or a photobeam-based computer measurement system with that scored by a human observer. As shown in our representative results, the data obtained by ImageFZ were similar to those analyzed by a human observer, indicating that the behavioral analysis using the ImageFZ system is highly reliable. The present movie article provides detailed information regarding the test procedures and will promote understanding of the experimental situation.

This article presents a protocol for a contextual and cued fear conditioning test using a video analyzing system to assess fear learning and memory in mice.

The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association ...

Assessment of Murine Exercise Endurance Without the Use of a Shock Grid: An Alternative to Forced Exercise

Using laboratory mouse models, the molecular pathways responsible for the metabolic benefits of endurance exercise are beginning to be defined. The most common method for assessing exercise endurance in mice utilizes forced running on a motorized treadmill equipped with a shock grid. Animals who quit running are pushed by the moving treadmill belt onto a grid that delivers an electric foot shock; to escape the negative stimulus, the mice return to running on the belt. However, avoidance behavior and psychological stress due to use of a shock apparatus can interfere with quantitation of running endurance, as well as confound measurements of postexercise serum hormone and cytokine levels. Here, we demonstrate and validate a refined method to measure running endurance in na&#239;ve C57BL/6 laboratory mice on a motorized treadmill without utilizing a shock grid. When mice are preacclimated to the treadmill, they run voluntarily with gait speeds specific to each mouse. Use of the shock grid is replaced by gentle encouragement by a human operator using a tongue depressor, coupled with sensitivity to the voluntary willingness to run on the part of the mouse. Clear endpoints for quantifying running time-to-exhaustion for each mouse are defined and reflected in behavioral signs of exhaustion such as splayed posture and labored breathing. This method is a humane refinement which also decreases the confounding effects of stress on experimental parameters.

A method to assess exercise endurance in laboratory mice without the use of a shock grid is demonstrated. This method is a humane refinement that can decrease the confounding effects of stress on experimental parameters.

Using laboratory mouse models, the molecular pathways responsible for the metabolic benefits of endurance exercise are beginning to be defined. The most ...

The Attentional Set Shifting Task: A Measure of Cognitive Flexibility in Mice

Cognitive impairment, particularly involving dysfunction of circuitry within the prefrontal cortex (PFC), represents a core feature of many neuropsychiatric and neurodevelopmental disorders, including depression, post-traumatic stress disorder, schizophrenia and autism spectrum disorder. Deficits in cognitive function also represent the most difficult symptom domain&#160;to successfully treat, as serotonin reuptake inhibitors and tricyclic antidepressants have only modest effects. Functional neuroimaging studies and postmortem analysis of human brain tissue implicate the PFC as being a primary region of dysregulation in patients with these disorders. However, preclinical behavioral assays used to assess these deficits in mouse models which can be readily manipulated genetically and could provide the basis for studies of new treatment avenues have been underutilized. Here we describe the adaptation of a behavioral assay, the attentional set shifting task (AST), to be performed in mice to assess prefrontal cortex mediated cognitive deficits. The neural circuits underlying behavior during the AST are highly conserved across humans, nonhuman primates and rodents, providing excellent face, construct and predictive validity.

The goal of this protocol is to perform a behavioral assay such as the attentional set shifting task (AST) to assess prefrontal cortex-mediated cognitive flexibility in mice.

Cognitive impairment, particularly involving dysfunction of circuitry within the prefrontal cortex (PFC), represents a core feature of many ...

The Modified Hole Board - Measuring Behavior, Cognition and Social Interaction in Mice and Rats

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure multiple dimensions of unconditioned behavior in small laboratory mammals (e.g., mice, rats, tree shrews and small primates). This paradigm is a valuable alternative for the use of a behavioral test battery, since a broad behavioral spectrum of an animal&#8217;s behavioral profile can be investigated in one single test.
The apparatus consists of a box, representing the &#8216;protected&#8217; area, separated from a group compartment. A board, on which small cylinders are staggered in three lines, is placed in the center of the box, representing the &#8216;unprotected&#8217; area of the set-up. The cognitive abilities of the animals can be measured by baiting some cylinders on the board and measuring the working and reference memory. Other unconditioned behavior, such as activity-related-, anxiety-related- and social behavior, can be observed using this paradigm. Behavioral flexibility and the ability to habituate to a novel environment can additionally be observed by subjecting the animals to multiple trials in the mHB, revealing insight into the animals&#8217; adaptive capacities.
Due to testing order effects in a behavioral test battery, na&#239;ve animals should be used for each individual experiment. By testing multiple behavioral dimensions in a single paradigm and thereby circumventing this issue, the number of experimental animals used is reduced. Furthermore, by avoiding social isolation during testing and without the need to food deprive the animals, the mHB represents a behavioral test system, inducing if any, very low amount of stress.

This protocol describes the modified hole board, which is a behavioral test set-up that comprises the characteristics of an open field and a traditional hole board. This set-up enables the differential analysis of unconditioned behavior of small laboratory mammals as well as the analysis of cognitive abilities.

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure ...

Protocol for Data Collection and Analysis Applied to Automated Facial Expression Analysis Technology and Temporal Analysis for Sensory Evaluation

We demonstrate a method for capturing emotional response to beverages and liquefied foods in a sensory evaluation laboratory using automated facial expression analysis (AFEA) software. Additionally, we demonstrate a method for extracting relevant emotional data output and plotting the emotional response of a population over a specified time frame. By time pairing each participant's treatment response to a control stimulus (baseline), the overall emotional response over time and across multiple participants can be quantified. AFEA is a prospective analytical tool for assessing unbiased response to food and beverages. At present, most research has mainly focused on beverages. Methodologies and analyses have not yet been standardized for the application of AFEA to beverages and foods; however, a consistent standard methodology is needed. Optimizing video capture procedures and resulting video quality aids in a successful collection of emotional response to foods. Furthermore, the methodology of data analysis is novel for extracting the pertinent data relevant to the emotional response. The combinations of video capture optimization and data analysis will aid in standardizing the protocol for automated facial expression analysis and interpretation of emotional response data.

A protocol for capturing and statistically analyzing emotional response of a population to beverages and liquefied foods in a sensory evaluation laboratory using automated facial expression analysis software is described.

We demonstrate a method for capturing emotional response to beverages and liquefied foods in a sensory evaluation laboratory using automated facial ...

The Treadmill Fatigue Test: A Simple, High-throughput Assay of Fatigue-like Behavior for the Mouse

Fatigue is a prominent symptom in many diseases and disorders and reduces quality of life for many people. The lack of clear pathogenesis and failure of current interventions to adequately treat fatigue in all patients leaves a need for new treatment options. Despite the therapeutic need and importance of preclinical research in helping identify promising novel treatments, few preclinical assays of fatigue are available. Moreover, the most common preclinical assay used to assess fatigue-like behavior, voluntary wheel running, is not suitable for use with some strains of mice, may not be sensitive to drugs that reduce fatigue, and has relatively low throughput. The current protocol describes a novel, non-voluntary preclinical assay of fatigue-like behavior, the treadmill fatigue test, and provides evidence of its efficacy in detecting fatigue-like behavior in mice treated with a chemotherapy drug known to cause fatigue in humans and fatigue-like behavior in animals. This assay may be a beneficial alternative to wheel running, as fatigue-like behavior and potential interventions can be assessed in a greater number of mice over a shorter time frame, thus permitting faster discovery of new therapeutic options.

Fatigue is a common, undertreated and frequently poorly-understood symptom in many diseases and disorders. New preclinical assays of fatigue may help to improve current understanding and future treatment of fatigue. To that end, the current protocol provides a novel means of measuring fatigue-like behavior in the mouse.

Fatigue is a prominent symptom in many diseases and disorders and reduces quality of life for many people. The lack of clear pathogenesis and failure of ...

A Method for Manipulating Blood Glucose and Measuring Resulting Changes in Cognitive Accessibility of Target Stimuli

Much research in social psychology has investigated the impact of bodily energy need on cognition and decision-making. As such, blood glucose, the body's primary energy source, has been of special interest to researchers for years. Fluctuations in blood glucose have been linked to a variety of changes in cognitive and behavioral processes, such as self-control, political attitudes, and eating behavior. To help meet growing interest in the links between bodily energy need and these processes, this manuscript offers a simple methodology to experimentally manipulate blood glucose using a fasting procedure followed by administration of a sugar-sweetened, unsweetened, or artificially-sweetened beverage. This is followed by presentation of a method for measuring resulting changes in implicit cognition using a lexical decision-task. In this task, participants are asked to identify whether strings of letters are words or non-words and response latencies are recorded. Sample results from a recent publication are presented as an example of the applications for the experimental manipulation of blood glucose and the lexical decision task measures.

The authors introduce a method for manipulating blood glucose and measuring resulting changes in cognitive accessibility of target words using a lexical decision task.

Much research in social psychology has investigated the impact of bodily energy need on cognition and decision-making.  As such, blood glucose, the body's ...

Short Session High Intensity Interval Training and Treadmill Assessment in Aged Mice

High intensity interval training (HIIT) is emerging as a therapeutic approach to prevent, delay, or ameliorate frailty. In particular short session HIIT, with regimens less than or equal to 10 min is of particular interest as several human studies feature routines as short as a few minutes a couple times a week. However, there is a paucity of animal studies that model the impacts of short session HIIT. Here, we describe a methodology for an individually tailored and progressive short session HIIT regimen of 10 min given 3 days a week for aged mice using an inclined treadmill. Our methodology also includes protocols for treadmill assessment. Mice are initially acclimatized to the treadmill and then given baseline flat and uphill treadmill assessments. Exercise sessions begin with a 3 min warm-up, then three intervals of 1 min at a fast pace, followed by 1 min at an active recovery pace. Following these intervals, the mice are given a final segment that starts at the fast pace and accelerates for 1 min. The HIIT protocol is individually tailored as the speed and intensity for each mouse are determined based upon initial anaerobic assessment scores. Additionally, we detail the conditions for increasing or decreasing the intensity for individual mice depending on performance. Finally, intensity is increased for all mice every two weeks. We previously reported in this protocol enhanced physical performance in aged male mice and here show it also increases treadmill performance in aged female mice. Advantages of our protocol include low administration time (about 15 min per 6 mice, 3 days a week), strategy for individualizing for mice to better model prescribed exercise, and a modular design that allows for the addition or removal of the number and length of intervals to titrate exercise benefits.

Short session (&#8804;10 min) high intensity interval training (HIIT) is emerging as an alternative to longer exercise modalities, yet the shorter variants are rarely modeled in animal studies. Here, we describe a 10 min, 3 day a week, uphill treadmill HIIT protocol that enhances physical performance in male and female aged mice.

High intensity interval training (HIIT) is emerging as a therapeutic approach to prevent, delay, or ameliorate frailty. In particular short session HIIT, ...