This research was supported by a grant from the Russian Science Foundation (No. 23-75-30001).

All procedures were performed in accordance with the &#34;Guide for the Care and Use of Laboratory Animals&#34;, Directive 2010/63/EU on the Protection of Animals Used for Scientific Purposes, and the guidelines from the Ministry of Science and High Education of the Russian Federation (N&#186; 742 from 13.11.1984), which have been approved by the Bioethics Commission of the Saratov State University (Protocol No. 7, 22.09.2022).
1. Hardware assembly
<ol>
	<li>Cut a piece of foil textolite that is 1.5 mm thick to the dimensions of 1.5 mm x 2 mm. This part will be referred to as the light-emitting diode (LED) printed circuit board (PCB) (Figure 1C).</li>
	<li>Solder an LED to a PCB, as shown in Figure 1.</li>
	<li>Solder two stranded copper wires to the pins of an LED and then cover them with a sleeve.</li>
	<li>Print out the 3D model of the frame (Figure 1A); place an LED (Figure 1B) and magnets (Figure 1D) on the frame; place a washer (Figure 1E) on the mouse&#39;s head.</li>
	<li>To assemble the circuit (Figure 2), follow these steps.
	<ol>
		<li>First, connect the resistor (Figure 2; R1) between an LED anode and 5 V port on the Arduino.</li>
		<li>Next, connect an LED cathode to the metal-oxide-semiconductor field-effect transistor (MOSFET) (Figure 2; Q1) drain. Then, connect the MOSFET source to the ground.</li>
		<li>Connect the pull-down resistor (Figure 2; R2) between the MOSFET gate and the ground. Finally, connect the MOSFET gate to pin 3 on the Arduino.</li>
	</ol>
	</li>
	<li>Connect a liquid crystal display (LCD) keypad shield to the Arduino.</li>
	<li>Print out the 3D models of the case, the cover plate, and the buttons. Insert the Arduino board with LCD keypad shield, MOSFET, and LED connector into the case, as shown in Figure 3.</li>
</ol>
2. Software guide (Figure 4)
<ol>
	<li>Download the Arduino sketch (.ino file) and open it via the Arduino integrated development environment (IDE) (Supplementary Coding File 1).</li>
	<li>Select the correct communication (COM) port and flash the firmware.</li>
	<li>The interface includes two columns. Use the buttons Left and Right to navigate between these columns. The selection indicator is located to the left of the column.</li>
	<li>The left-hand column on the screen is the pulse width modulation (PWM) duty cycle selection field. Use the Up and Down buttons to adjust the duty cycle. Achieve the 10/20/30 Dj/cm2 dose of PBM with a corresponding 2%/4%/6% PWM duty cycle in a 17 min session.</li>
	<li>The right-hand column on the screen is the RUN/Off field. To select this column, press the Right button on the keypad and then press Select. When the process is running, the activity indicator will blink, and the text on the RUN button will change to OFF. 
	NOTE. Mice are prepared for the experiments over a period of 10 days, including implantation of EEG electrodes, implantation of a chronic catheter into the right lateral ventricle for injection of fluorescent A&#946;, and placement of a plate for PBM.</li>
</ol>
3. Implantation of an EEG recording system (Figure 5)
<ol>
	<li>Weigh the mouse and anesthetize it with a mixture of Ketamine and Xylazine (100 mg/kg; 10 mg/kg, respectively) by intramuscular injection into the thigh. Administer Xylazine as pre-operative analgesia at a concentration of 20 mg/mL at a dose of 1.5 mL/kg.</li>
	<li>When the rear foot withdrawal reflexes and tail pinch response cease, place the mouse in a stereotaxic frame over a heating pad (Figure 5A).</li>
	<li>Apply ophthalmic ointment to the eyelids to prevent drying of the eyeballs during surgery. Repeat this procedure whenever necessary.</li>
	<li>Shave the head in the area from the nasal bones to the occipital bones using a shaving machine and disinfect the exposed skin with alternating rounds of chlorhexidine and alcohol 3 times each.</li>
	<li>Using straight dissecting scissors, cut off the scalp, hold it with micro forceps, clean the skull from fascia, and dry it with cotton swabs. If necessary, use the hemostatic agents.</li>
	<li>Using a drill with a diameter of 1.3 mm, make 2 holes in the temporal bones on each side along the coordinates: AP = -1 mm for the first pair of screws and AP = -3 mm for the second pair of screws.</li>
	<li>Place the EEG screws with wire leads in alcohol for 15 min. Afterward, place the EEG screws in the saline solution.</li>
	<li>Place four silver-plated screws with electrodes into the holes to a depth of 1 mm (Figure 5B).</li>
	<li>Fix the screws on the surface of the skull using dental acrylic so that the electrodes extending from them are located towards the animal&#39;s nose (Figure 5C). Allow the dental acrylic to harden for 15 min.</li>
	<li>Attach an EEG recording sensor to the animal&#39;s nose using dental acrylic. Allow the dental acrylic to harden for 30 min (Figure 5D).</li>
	<li>Place the EMG electrodes on the back of the orbicularis oculi muscle using curved tweezers and fix them with the dental acrylic. Allow the dental acrylic to harden for 15 min (Figure 5E).</li>
	<li>Connect the EEG electrodes to the silver-plated recess of the sensor and solder them using a soldering station. Afterward, fix the EEG electrodes using the dental acrylic (Figure 5F).</li>
	<li>After surgery, place the mouse on a heating pad to maintain body temperature until the animal fully recovers from anesthesia.</li>
	<li>Afterward, put the mouse in an individual home cage with free access to food and water with ibuprofen (40 mg/kg in 200 mL of water) for analgesia after surgery for 10 days. 
	NOTE: Each animal was kept in an individual cage so that the mice could not deform the EEG registration system. Ibuprofen was supplied with water to prevent stress in animals.</li>
</ol>
4. Implantation of a plate for PBM
<ol>
	<li>Weigh the mouse and anesthetize it with a mixture of Ketamine and Xylazine (100 mg/kg; 10 mg/kg, respectively) by intramuscular injection into the thigh 7 days after implantation of the EEG recording system. Administer Xylazine as pre-operative analgesia at a concentration of 20 mg/mL at a dose of 1.5 mL/kg.</li>
	<li>When the rear foot withdrawal reflexes and tail pinch response cease, fix the mouse in a stereotactic system.</li>
	<li>Apply ophthalmic ointment to the eyelids to prevent drying of the eyeballs during surgery. Repeat this procedure whenever necessary.</li>
	<li>Fix a metal plate with a diameter of 5 mm on the occipital bone of the skull using dental acrylic and Dumont forceps. Allow the dental acrylic to harden for 15 min (Figure 6).</li>
</ol>
5. Preparation of a chronic catheter
<ol>
	<li>Mark on the insulin needle a segment 2 cm from the side of the beveled end.</li>
	<li>Fix the insulin needle in the needle holder from the side of the beveled tip to the marked segment.</li>
	<li>Place a 2 cm-PE-10 polyethylene catheter over the entire remaining length of the needle.</li>
	<li>Fill the catheter with a saline solution and cover it with a plastic cap to seal it.</li>
</ol>
6. Implantation of a chronic catheter into the right lateral ventricle
<ol>
	<li>After implantation of the plate for PBM, make a trepanation hole at the coordinates AP = -0.5 mm and ML = 1.2 mm, with a diameter of 1.5 mm, using a drill.</li>
	<li>Place the PE-10 polyethylene catheter in a stereotactic holder and insert it into the mouse skull (DV = 2 mm). Afterward, fix it using the dental acrylic. Allow the dental acrylic to harden for 15 min (Figure 7).</li>
	<li>After surgery, place the mouse on a heating pad to maintain body temperature until the animal fully recovers from anesthesia.</li>
	<li>Afterward, put the mouse in an individual home cage with free access to food and water with ibuprofen (40 mg/kg in 200 mL of water) for analgesia after surgery for 10 days. 
	NOTE: Each animal was kept in an individual cage so that the mice could not deform the EEG registration system. Ibuprofen was supplied with water to prevent stress in animals. Apply antibacterial ointment to the exposed areas of the skull and soft tissues during the rehabilitation period (7 days) to prevent them from drying out.</li>
</ol>
7. tPBM under EEG control of NREM sleep
<ol>
	<li>Connect any commercial EEG recording system to the connector on the mouse&#39;s head and set the requirement value of the PWM duty cycle.</li>
	<li>Monitor the EEG signal and wait for delta rhythm activity. If NREM sleep is seen, initiate the PBM process and stop the process if NREM sleep transitions to rapid eye movement (REM) sleep or wakefulness. The dose increases with each interaction until it reaches the required value.</li>
	<li>When the required PBM dose is obtained, the session is over.</li>
</ol>
8. Confocal imaging of lymphatic removal of A&#946; from mouse brain
<ol>
	<li>Connect a 10 cm catheter to an insulin needle.</li>
	<li>Using a Hamilton syringe with a 29 G needle, prepare an infusion of fluorescent beta-amyloid (FA&#946;) in a volume of 5 &#181;L into the catheter. Let the catheter remain on a Hamilton syringe.</li>
	<li>Fix the mouse&#39;s hand and connect the catheter through a needle to the implanted chronic catheter.</li>
	<li>Connect the catheter to a microinjector. Afterward, place the mouse in an individual box;</li>
	<li>Select the injection rate 0.1 &#181;L/min in the microinjector menu, and press the Start button.</li>
	<li>Make injection of FA&#946; into the right lateral ventricle.</li>
	<li>After FA&#946; administration, make PBM using an LED for 61 min following the algorithm: 17 min - light and 5 min - pause over 61 min.</li>
	<li>After PBM, intravenously inject any tracer for labeling the cerebral vessels via the tail.</li>
	<li>After injection, euthanize mice using the CO2 euthanasia chamber.</li>
	<li>Using sharp, straight scissors, make a small transverse incision in the skin along the trachea, holding the skin with straight non-sharp tweezers.</li>
	<li>Make a longitudinal incision along the entire length of the neck using straight scissors.</li>
	<li>Using curved tweezers, take up the salivary glands and carefully separate them from the connective tissue. Place a wound retractor on the open section of the incision and fix it in such a way as to push back the surrounding tissues.</li>
	<li>Examine the area between the trachea and the cleidomastoid muscle using two curved tweezers from both sides of the neck.</li>
	<li>When the deep cervical lymph node (dcLN) is detected on each side, take off dcLNs using straight tweezers with blunt ends and cut it from the connective tissue.</li>
	<li>Place dcLNs in a Petri dish with a saline solution and cover them with horizontally oriented cover glass (25 mm &#215; 50 mm &#215; 0.17 mm).</li>
	<li>Use any commercial confocal microscope to obtain images of whole dcLNs.</li>
</ol>
9. Analysis of A&#946; in the lysates of brain tissues
<ol>
	<li>Prepare the samples for the assay.
	<ol>
		<li>Euthanize mice using the CO2 euthanasia chamber.</li>
		<li>Decapitate the mouse, remove the skin from its head, and remove the muscles from the skull.</li>
		<li>Make two incisions with sharp, straight scissors from the great occipital foramen to the auditory canal.</li>
		<li>Using straight tweezers, separate the ventral part of the skull, the occipital bone, and bones forming the middle ear cavities.</li>
		<li>Using tweezers, separate the brain from the parietal and frontal bones.</li>
		<li>Using straight scissors, remove the upper jaw and cut off the olfactory bulbs.</li>
		<li>Place the brain in the physiological solution.</li>
		<li>Rinse the brain in cold phosphate-buffered saline to remove excess blood thoroughly and weigh before homogenization.</li>
		<li>Prepare a lysing buffer pH 7.2 containing 1.5 mm KH2PO4, 8 mm Na2HPO4, 3 mm KCl, 137 mm NaCl and 0.1% Tween20, 10 mM EDTA with a freshly prepared protease inhibitory mixture.</li>
		<li>Homogenize the brain in fresh lysis buffer (1 mL of lysis buffer for 200-500 mg tissue sample) with a glass homogenizer on ice.</li>
		<li>Sonicate a resulting suspension with an ultrasonic cell disruptor till the solution is clarified.</li>
		<li>Centrifuge the homogenates at 10,000 &#215; g for 5 min.</li>
		<li>Collect the supernatant using a single channel mechanical pipette (100-1000 &#181;L) and assay immediately or aliquot and store at &#8804;-20 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare the following materials: microplate reader with 450 nm &#177; 10 nm filter; microcentrifuge tubes; single or multi-channel pipettes with high precision and disposable tips; absorbent paper for blotting the microplate; container for wash solution; 0.01 mol/L (or 1x) phosphate buffered saline (PBS); and deionized or distilled water.</li>
	<li>Prepare the reagents.
	<ol>
		<li>Bring the components of the kit and samples to room temperature (RT; 18-25 &#176;C) before use.</li>
		<li>Reconstitute the standard with the 1.0 mL of standard diluent, keep for 10 min at RT, and shake gently (not to foam). The standard stock solution is 300 pg/mL. Prepare 5 tubes containing a volume of 0.6 mL of standard diluent and make a triple dilution series.</li>
		<li>Set up 5 points (300 pg/mL, 100 pg/mL, 33.33 pg/mL, 11.11 pg/mL, and 3.70 pg/mL) of the diluted standard, and the last tubes with as blank containing only the standard diluent (0 pg/mL).</li>
		<li>Quickly spin down the stock solutions of Detection reagent A and Detection reagent B prior to use. Dilute them 100-fold with Assay Diluent A and B to prepare the working concentration.</li>
		<li>Dilute 20 mL of the concentrated wash solution (30x) with 580 mL of deionized or distilled water to make 600 mL of wash solution (1x).</li>
		<li>Aspirate the needed dosage of the solution with sterilized tips, and do not dump the residual solution into the vial again.</li>
	</ol>
	</li>
	<li>Perform the assay.
	<ol>
		<li>Determine wells for a diluted standard, blank, and sample.</li>
		<li>Prepare 5 wells for standard points and one well for blank.
		<ol>
			<li>Add 50 &#181;L each of standard, blank, and sample dilutions into the corresponding wells, respectively. Then, add 50 &#181;L of Detection reagent A to each well immediately.</li>
			<li>Shake the plate gently (a microplate shaker is recommended) and cover it with a plate sealer. Incubate the plate at 37 &#176;C for 1 h. Detection reagent A may appear cloudy. Warm the solution to RT and mix gently until it appears uniform.</li>
		</ol>
		</li>
		<li>Aspirate the solution and wash each well with 350 &#181;L of 1x wash solution with the help of a squirt bottle, multi-channel pipette, manifold dispenser, or auto washer. Leave the plate undisturbed for 1-2 min. Snap the plate onto absorbent paper to completely remove the remaining liquid from all wells. Repeat this procedure 3 times.</li>
		<li>After the last wash, aspirate or decant any remaining wash buffer. Ensure complete removal of the washing solution by inverting the plate and blotting it against absorbent paper.</li>
		<li>Add 100 &#181;L of Detection reagent B working solution to each well and incubate the plate for 30 min at 37 &#176;C after covering it with the plate sealer.</li>
		<li>Repeat the aspiration/washing steps for a total of 5 min.</li>
		<li>Add 90 &#181;L of substrate solution to each well and cover the plate with a new plate sealer. Incubate for 10-20 min at 37 &#176;C (Do not exceed 30 min) protected from light. After adding the substrate solution, the liquid will turn blue.</li>
		<li>Add 50 &#181;L of a stop solution to each well to terminate the reaction. Adding the stop solution will turn the liquid yellow. Tap the plate on its side to mix the liquid. If the color change is inconsistent, gently tap the plate to ensure thorough mixing.</li>
		<li>Ensure complete removal of water and fingerprint on the bottom of the plate and no bubble formation on the liquid surface. Then, read the plate in a microplate reader at 450 nm immediately.</li>
	</ol>
	</li>
	<li>Calculate the results.
	<ol>
		<li>Determine the average of the duplicate readings for each standard, control, and sample. Plot a standard curve with the log of A&#946; 1-42 concentration on the y-axis and absorbance on the x-axis.</li>
		<li>Draw a best-fit curve through the points, which can be determined by regression analysis.</li>
		<li>If diluted samples were used, multiply the concentration obtained from the standard curve by the dilution factor. 
		NOTE: For ELISA, a kit for determining A&#946; 1-42 was used in this study.</li>
	</ol>
	</li>
</ol>

Enhancing Brain Drainage During Sleep with Transcranial Photobiomodulation

The authors have nothing to disclose.

MLVs are an important target for the development of innovative technologies for modulation of the brain&#39;s drainage and removal of cellular debris and wastes from the brain, especially in aged subjects whose MLV function declines1,22. In a homeostatic state, deep sleep is associated with the natural activation of brain tissue cleansing13,14. Therefore, it is obvious to expect that stimulation of MLVs during deep sleep will be more effective than during wakefulness15,16. The new non-invasive and portable technology of tPBM under EEG control of sleep for stimulation of lymphatic removal of soluble A&#946; from the brain of aged male mice is presented here.
The small size of the photo platform (7 mm x 11 mm) and its light weight (1 g) allow it to be firmly and securely mounted on the heads of mice, maintaining their natural motor activity. This also eliminates the need to use anesthesia during tPBM. The tPBM during deep sleep can be performed using any commercial device for an EEG control of sleep stages. Thus, our technology allows performing the study in a home cage, preserving the most natural conditions for animals.
In the first step, an LED wavelength of 1050 nm was randomly selected for the study of the effective dose of photo-effects on lymphatic removal of fluorescent A&#946; from the brain to dcLNs in awake adult (2-3 month old) mice. The choice of this wavelength is related to the previous data on effective stimulation of MLVs with a 1267 nm laser8,10,27,28,29,30,31,32,33,34. This wavelength promotes the direct generation of the singlet oxygen in small quantities in brain tissue and in its meninges, which is one of the mechanisms underlying the PBM of MLVs8,28,34. However, the biological effects of both a 1267 nm laser and an LED 1050 nm are related to the generation of singlet oxygen excitation35. Indeed, the bands of a 1267 nm &#177; 20 nm laser and a 1065 nm &#177; 15 nm LED are related to formation of the singlet oxygen from the ground state of an oxygen molecule. A band of a 1267 nm laser pumps an oxygen molecule directly into the first excited singlet state, while a band of 1065 nm LED corresponds to additional vibrational energy, which decays very fast, releasing heat to the environment. Moreover, water absorption in the 1065 nm wavelength is tenfold lesser than in the 1267 nm wavelength, which makes it more favorable for biological research35. Furthermore, a 1050 nm LED is commercially available in the market and much cheaper compared with the rare and expensive 1267 nm laser. Note that the emission band of a used 1050 nm LED in this study was 40 nm wide, and it only partially overlaps with a singlet oxygen band. However, we demonstrated the significant effect of tPBM on the lymphatic removal of fluorescent A&#946; from the brain to dcLNs using a 1050 nm LED (30 J/cm2). The light dose 30 J/cm2 was determined to be the most effective compared to the light doses 10 J/cm2 and 20 J/cm2 based on the higher intensity of a fluorescent signal from A&#946; in dcLNs. Thus, a 1050 nm LED can be very useful for the application of the tPBM technique to humans, which is consistent with the common trend to use LEDs instead of lasers for PBM36,37.
In the second step, the research aimed to answer the question of which light wavelengths are most effective for lymphatic elimination of fluorescent A&#946; from the brain of adult awake mice. This step of study was performed using a portable photo platform with different light wavelengths (880 nm, 1050 nm, 1300 nm) and modes (1050 nm in continuous and pulse regimes) with the same dose (30 J/cm2). These wavelengths were chosen due to the widespread use in clinical practice for tPBM (880 nm) and new expensive LEDs (1300 nm), which could potentially be clinically significant. In addition, the pulsed and continuous modes only for the 1050 nm LED as a light resource were compared. These data clearly demonstrate that only a 1050 nm LED in pulse mode vs. other wavelengths and the 1050 nm in continuous mode causes a significant lymphatic removal of A&#946; from the brain to dcLNs. These results are consistent with the findings of other researchers, who also indicate the advantage of using a pulsed PBM to effectively achieve biological effects38,39,40.
In the final step, when the effective wavelength, dose and mode were selected, the study of the effects of a 1050 nm LED in pulse mode on the level of soluble A&#946; in the brain of aged mice before and after the 10 day-course of tPBM under an EEG control of NREM and wakefulness was carried out. The immunoassay analysis revealed that the soluble A&#946; content in the brain was significantly higher in aged mice (16-18 months old) compared with adult mice (2-3 months old). It is interesting to note that the 10-day course of tPBM during deep sleep, but not during awake, effectively reduced the soluble A&#946; level in the brains of aged mice to the level of soluble A&#946; in the brains of adult mice. An increase in the A&#946; content in brain tissue of healthy mice and rats with age has also been noted in other studies25,26, which may be associated with an age-related decrease in the MLV functions leading to ineffective cleansing of brain tissue from this toxic protein1,22. The more effective tPBM of removal of soluble A&#946; from brain tissue in sleeping mice compared to awake ones can be explained by the natural activation of brain tissue drainage during sleep. The MLV morphology changes significantly with age, leading to reduced lymphatic removal of metabolites from the aged brain22. However, tPBM during sleep helps restore the lymphatic removal of soluble A&#946; from the brain tissue of aging mice to the level of adult animals.
Considering the scattering of light energy when passing through the skull, tPBM limits its effects only on MLVs without penetrating deep into the brain tissues. However, despite the fact that the lymphatic vessels have not yet been found directly in the brain tissues of humans and animals, growing evidence is emerging indicating the presence of the cerebral lymphatic system41,42,43,44. This explains the results showing the removal of toxins (blood and A&#946;) from the deep structures of the brain (the ventricular system, the hippocampus) to MLVs and further to the periphery (dcLNs)5,7. Over the course of a century, knowledge has accumulated, indicating a close connection between the brain&#39;s drainage and the peripheral lymphatic system45. Even in the absence of a generally recognized lymphatic network in the central nervous system, there are facts indicating lymphatic removal of macromolecules from the deep parts of the brain to the periphery1,2,3,4,5,6,7&#160;This also explains why tPBM, acting only on MLVs, stimulates the clearance of blood and A&#946; from the brain5,7.
In summary, this study presents a portable technology of tPBM under EEG control of sleep designed to photo-stimulate the removal of toxins using the example of soluble A&#946; from the brain of aged mice with the ability to compare the therapeutic effectiveness of different optical resources. The technology can be used in the natural condition of a home cage without anesthesia, maintaining the natural motor activity of mice. These data open up new prospects for the development of non-invasive and clinically promising photo-technologies for correcting age-related changes in the MLV functions and brain&#39;s drainage processes and for the effective cleansing of brain tissues from metabolites and toxins. The proposed technology for photostimulation of the brain&#39;s drainage and lymphatic removal of toxins from the brain has limited effects on MLVs due to the scattering of light energy when passing through the skull. Therefore, the technology can be used to develop new methods for treating brain diseases associated with MLV dysfunction. The most pronounced stimulating photo-effects on MLVs are observed in deep (NREM) sleep, which requires the use of PBM under EEG control. This creates certain difficulties (technical, programming) for simultaneous control of the onset of deep sleep and the supply of light exposure.

Meningeal lymphatic vessels (MLVs) play an important role in the removal of toxins and metabolites from brain tissues1,2,3. Damage of MLVs in various brain diseases, including tumors, traumatic brain injuries, hemorrhages, and neurodegenerative processes, is accompanied by a decrease in the MLV functions leading to the progression of these pathologies1,2,3,4,5,6. Therefore, the development of methods for the stimulation of MLVs opens new horizons in the emergence of effective technologies for the treatment of brain diseases. Recently, non-invasive technology for effective transcranial photobiomodulation (tPBM) has been proposed to stimulate MLVs and remove toxins such as blood and A&#946; from the brain5,7,8,9,10,11,12. It is interesting to note that deep sleep is a natural factor for the activation of lymphatic drainage processes in the brain13,14. Based on this fact, it is logical to assume that the tPBM of MLVs during sleep may have more effective therapeutic effects than during wakefulness9,11,12,15. However, there are currently no commercial technologies for tPBM during sleep16. In addition, animal experiments to study the therapeutic effects of tPBM are performed under anesthesia, which is required to accurately deliver light to the brain. However, anesthesia significantly affects the brain&#39;s drainage, which reduces the quality of research results17.
A&#946; is a metabolic product of normal neural activity18. As it was established in cultured rat cortical neurons, A&#946; is released from them at high rates into the extracellular space (2-4 molecules/neuron/s for A&#946;)19. There is evidence that the dissolved form of A&#946;, located in the extracellular and perivascular spaces, is most toxic to neurons and synapses20. The soluble A&#946; is rapidly cleared from the human brain during 1-2.5 h21. MLVs are the tunnels for removal of the soluble A&#946; from the brain1,7 that declines with age, leading to the accumulation of A&#946; in the aged brain1,22. There is evidence that extracellular abnormalities of A&#946; levels in the brain correlate with cognitive performance in aging and are associated with the development of Alzheimer&#39;s disease (AD)23,24. Therefore, aged and old rodents are considered alternatives to transgenic models for the study of amyloidosis, including AD25,26.
This study presents an original and portable tPBM technology under electroencephalographic (EEG) control of deep or non-rapid eye movement (NREM) sleep in non-anesthetized male C57BL/6 mice of different ages to stimulate lymphatic clearance of A&#946; from the brain into the peripheral lymphatic system (the deep cervical lymph nodes, dcLNs).

<table><tbody><tr><td>0.1% Tween20</td><td>Helicon,&amp;nbsp; Russia</td><td>SB-G2009-100ML</td><td></td></tr><tr><td>Catheter</td><td>Scientific Commodities Inc., USA</td><td>PE-10, 0.28 mm ID &amp;times; 0.61 mm OD</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; chamber</td><td>Binder, Germany</td><td>CB-S 170</td><td></td></tr><tr><td>Confocal microscop</td><td>Nikon, Japan</td><td>A1R MP</td><td></td></tr><tr><td>Dental acrylic</td><td>Zermack, Poland-Russia</td><td>Villacryl S, V130V4Z05</td><td></td></tr><tr><td>Drill</td><td>Foredom, Russia</td><td>SR W-0016</td><td></td></tr><tr><td>Dumont forceps</td><td>Stoelting, USA</td><td>52100-07</td><td></td></tr><tr><td>Evans Blue dye</td><td>Sigma-Aldrich, St. Louis, MO, USA</td><td>206334</td><td></td></tr><tr><td>Hamilton</td><td>Hamilton Bonaduz AG, Switzerland</td><td>29 G needle</td><td></td></tr><tr><td>Ibuprofen</td><td>Sintez OJSC, Russia</td><td>N/A</td><td>&amp;nbsp;Analgesic drug</td></tr><tr><td>Insulin needle</td><td>INSUPEN, Italy</td><td>31 G, 0.25 mm x 6 mm</td><td></td></tr><tr><td>Levomekol antibacterial ointment</td><td>Nizhpharm</td><td>D06C </td><td>For external use at a dose of 40 mg/g, 1 time per day</td></tr><tr><td>Micro forceps</td><td>Stoelting, USA</td><td>52102-02P</td><td></td></tr><tr><td>Microcentrifuge</td><td>Gyrozen, South Korea</td><td>GZ-1312</td><td></td></tr><tr><td>Microinjector</td><td>Stoelting, USA</td><td>53311</td><td></td></tr><tr><td>Non-sharp tweezer</td><td>Stoelting, USA</td><td>52108-83P</td><td></td></tr><tr><td>PINNACLE system</td><td>Pinnacle Technology, USA</td><td>8400-K3-SL</td><td>System for recording EEG (2 channels) and EMG (1 channel) of mice</td></tr><tr><td>Shaving machine</td><td>Braun</td><td>Series 3310s</td><td></td></tr><tr><td>Single and multi-channel pipettes</td><td>Eppendorf, Austria</td><td>Epp 3120 000.020, Epp 3122 000.019</td><td></td></tr><tr><td>Sodium chloride</td><td>Kraspharma, Russia</td><td>N/A</td><td></td></tr><tr><td>Soldering station</td><td>AOYUE, China</td><td>N/A</td><td></td></tr><tr><td>Stereotaxic frame</td><td>Stoelting, USA</td><td>51500</td><td></td></tr><tr><td>Straight dissecting scissors</td><td>Stoelting, USA</td><td>52132-10P</td><td></td></tr><tr><td>Tetracycline</td><td>JSC Tatkhimfarmpreparaty, Russia</td><td>N/A</td><td>Eye ointment</td></tr><tr><td>Tweezer</td><td>Stoelting, USA</td><td>52100-03</td><td></td></tr><tr><td>Ultrasonic cell disrupter</td><td>Biobase, China</td><td>USD-500</td><td></td></tr><tr><td>Wound retractor</td><td>Stoelting, USA</td><td>52125</td><td></td></tr><tr><td>Xylanit</td><td>Nita-Farm, Russia</td><td>N/A</td><td>Muscle relaxant</td></tr><tr><td>Zoletil 100</td><td>Virbac Sante Animale, France</td><td>N/A</td><td>General anesthesia</td></tr></tbody></table>

photobiomodulation under electroencephalographic controls of sleep for stimulation of lymphatic removal of toxins from mouse brain

The meningeal lymphatic vessels (MLVs) play an important role in the removal of toxins from the brain. The development of innovative technologies for the stimulation of MLV functions is a promising direction in the progress of the treatment of various brain diseases associated with MLV abnormalities, including Alzheimer&#39;s and Parkinson&#39;s diseases, brain tumors, traumatic brain injuries, and intracranial hemorrhages. Sleep is a natural state when the brain&#39;s drainage processes are most active. Therefore, stimulation of the brain&#39;s drainage and MLVs during sleep may have the most pronounced therapeutic effects. However, such commercial technologies do not currently exist.
This study presents a new portable technology of transcranial photobiomodulation (tPBM) under electroencephalographic (EEG) control of sleep designed to photo-stimulate removal of toxins (e.g., soluble amyloid beta (A&#946;)) from the brain of aged BALB/c mice with the ability to compare the therapeutic effectiveness of different optical resources. The technology can be used in the natural condition of a home cage without anesthesia, maintaining the motor activity of mice. These data open up new prospects for developing non-invasive and clinically promising photo-technologies for the correction of age-related changes in the MLV functions and brain&#39;s drainage processes and for effectively cleansing brain tissues from metabolites and toxins. This technology is intended both for preclinical studies of the functions of the sleeping brain and for developing clinically relevant treatments for sleep-related brain diseases.

In the first step, the study has focused on establishing the effective light dose (a 1050 nm LED) for stimulation of lymphatic removal of fluorescent A&#946; from the brain to dcLNs in awake adult (2-3 month old, 26-29 g) male BALB/c mice. The light doses were selected randomly as 10 J/cm2, 20 J/cm2, and 30 J/cm2 based on our previous studies of tPBM effects on the removal of different dyes and the red blood cells from the brain7,8,9,10,11,12. The results of confocal imaging presented in Figure 8A-E clearly demonstrate the dose-related effects of tPBM on lymphatic removal of fluorescent A&#946; from the brain to dcLNs. The light dose 30 J/cm2 was determined to be the most effective compared to the light doses 10 J/cm2 and 20 J/cm2 due to a higher intensity of a fluorescent signal from A&#946; in dcLNs.
In the second step, the research was aimed at finding the wavelengths that are most effective for lymphatic elimination of fluorescent A&#946; from the brain of adult (2-3 month old, 26-29 g) awake BALB/c male mice using the portable photo platforms with the different light wavelengths (880 nm, 1050 nm, 1300 nm) and modes (1050 nm in continues and pulse regimes) with a same dose (30 J/cm2). Figure 9A-E shows the confocal imaging results of dcLNs in the tested groups. The findings revealed that the light wavelength 1050 nm in pulse mode vs. other tested wavelengths (880 nm and 1300 nm) and the 1050 nm wavelength in continuous regime caused significantly higher removal of fluorescent A&#946; from the brain to dcLNs.
In the final step, the effects of a 1050 nm LED in pulse mode on the level of soluble A&#946; in the brain of aged mice before and after the 10 day course of tPBM under an EEG control of NREM and wakefulness were studied. The immunoassay analysis revealed that the soluble A&#946; content in the brain was significantly higher in aged male BALB/c mice (16-18 months old, 30-33 g) compared with adult male BALB/c mice (2-3 months old, 26-29 g). The 10 day course of tPBM during deep sleep, but not while awake, effectively reduced the soluble A&#946; level in the brains of aged mice to the level of soluble A&#946; in the brains of adult mice (Figure 10).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67035/67035fig01.jpg" /> 
Figure 1: Hardware assembly process. (A) 3D printed frame, (B) LED, (C) LED printed circuit board, (D) magnets, (E) metallic washer. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67035/67035fig01large.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/67035/67035fig02.jpg" /> 
Figure 2: LED control circuit. R1 - current regulation resistor, R2 - pull-down resistor for the MOSFET gate, Q1 - n-channel MOSFET. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67035/67035fig02large.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/67035/67035fig03.jpg" /> 
Figure 3: Case 3D view. (A) 3D printed cover plate, (B) 3D printed buttons, (C) LCD keypad shield for Arduino, (D) Arduino, (E) 3D printed case, (F) LED connector, (G) Channel MOSFET. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67035/67035fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/67035/67035fig04.jpg" /> 
Figure 4: Software. (A) Select button, (B) Left button, (C) Up/Down button, (D) Right button, (E) Selection indicator, (F) PWM duty cycle selection field, (G) RUN/OFF field. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67035/67035fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/67035/67035fig05.jpg" /> 
Figure 5: Implantation of an EEG recording system. (A) Skull preparation; (B) Implantation of screws; (C) Fixation of screws with the dental acrylic; (D) Fixation of the EEG recording sensor; (E) Implantation and fixation of the EMG electrodes on the back of the orbicularis oculi muscle; (F) The soldered electrodes to the silver-plated recess of the sensor. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67035/67035fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/67035/67035fig06.jpg" /> 
Figure 6: Fixation of a plate for PBM on the occipital bone of the skull. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67035/67035fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/67035/67035fig07.jpg" /> 
Figure 7: Implantation of a chronic catheter into the right lateral ventricle. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67035/67035fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/67035/67035fig08.jpg" /> 
Figure 8: The effects of different LED 1050 nm doses on lymphatic removal of fluorescent A&#946; from the brain to dcLNs. (A-D) Representative images of dcLNs from the (A) control group and the PBM group with (B) 10 J/cm2, (C) 20 J/cm2, and (D) 30 J/cm2; (E) Quantitative analysis of the accumulation of fluorescent A&#946; in dcLNs from the tested groups (n = 8, one-way ANOVA with Duncan post hoc test, **p &#60; 0.01, *p &#60; 0.05). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67035/67035fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/67035/67035fig09.jpg" /> 
Figure 9: The effects of different LED wavelengths on lymphatic removal of fluorescent A&#946; from the brain to dcLNs. (A-D) Representative images of dcLNs from the PBM group with impulse mode 1050 nm (ImpMode_1050, A), 880 nm (ImpMode_880, B), 1300 nm (ImpMode_1300, C) and continuous mode 1050 nm (ContMode_1050 nm, D); (E) Quantitative analysis of accumulation of fluorescent A&#946; in dcLNs from the tested groups (n = 8, one-way ANOVA with Duncan post hoc test, **p &#60; 0.01). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67035/67035fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/67035/67035fig10.jpg" /> 
Figure 10: The PBM effects (LED 1050 nm in impulse mode, 30 J/cm2) during NREM and awake state on the level of soluble A&#946; (pg/mL) in mouse brains of adult and aged mice. A one-way ANOVA with Duncan post hoc test, **p &#60; 0.01, *p &#60; 0.05. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67035/67035fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Coding File 1: lcd1key.ino <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67035/lcd1Key.zip" target="_blank">Please click here to download this File.</a>

Author Spotlight: Developing Photo-Stimulation Technology for Toxin Removal in Sleep-Related Brain Diseases

This study presents an original and portable tPBM technology under electroencephalographic (EEG) control of deep or non-rapid eye movement (NREM) sleep in non-anesthetized male C57BL/6 mice of different ages to stimulate lymphatic clearance of A&#946; from the brain into the peripheral lymphatic system (the deep cervical lymph nodes, dcLNs).

Photobiomodulation Under Electroencephalographic Controls of Sleep for Stimulation of Lymphatic Removal of Toxins from Mouse Brain

The meningeal lymphatic vessels (MLVs) play an important role in the removal of toxins from the brain. The development of innovative technologies for the ...

photobiomodulation-under-electroencephalographic-controls-sleep-for

Nanyang Technological University

Research

JoVE Journal

Neuroscience

619 Views.  Saratov State University. In de studie stellen we een nieuwe manier van technologie voor voor fotostimulatie van veel kiembestrijdende vaten en hersendrainage die slaap en controle doen. Slaap is een natuurlijke staat van activering van de drainage en de reinigende functie van de hersenen, die wordt verminderd bij grote aantallen ziekten van het CZS. Het is noodzakelijk om technologieën te creëren voor de behandeling van hersenziekten tijdens de slaap om de reiniging van de weefsels...

Video: Auteur in de schijnwerpers: ontwikkeling van fotostimulatietechnologie voor het verwijderen van toxines bij slaapgerelateerde hersenziekten

Combining Transcranial Magnetic Stimulation and fMRI to Examine the Default Mode Network

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful rest."1,2,3 It is thought the default mode network corresponds to self-referential or "internal mentation".2,3
It has been hypothesized that, in humans, activity within the default mode network is correlated with certain pathologies (for instance, hyper-activation has been linked to schizophrenia 4,5,6 and autism spectrum disorders 7 whilst hypo-activation of the network has been linked to Alzheimer's and other neurodegenerative diseases 8). As such, noninvasive modulation of this network may represent a potential therapeutic intervention for a number of neurological and psychiatric pathologies linked to abnormal network activation. One possible tool to effect this modulation is Transcranial Magnetic Stimulation: a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.9
In order to explore the default mode network's propensity towards and tolerance of modulation, we will be combining TMS (to the left inferior parietal lobe) with functional magnetic resonance imaging (fMRI). Through this article, we will examine the protocol and considerations necessary to successfully combine these two neuroscientific tools.

In this article, we examine the methodology and considerations relevant to the combination of TMS and fMRI to examine the effects of brain stimulation on the default network.

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful ...

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS display abnormalities in the expression of FMR1 - a protein required for typical, healthy neural development.3 Recent data has suggested that the loss of this protein can cause the cortex to be hyperexcitable thereby affecting overall patterns of neural plasticity.4,5
In addition, Fragile X shows a strong comorbidity with autism: in fact, 30% of children with FXS are diagnosed with autism, and 2 - 5% of autistic children suffer from FXS.6
Transcranial Magnetic Stimulation (a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses 7,8) represents a unique method of exploring plasticity and the manifestations of FXS within affected individuals. More specifically, Theta-Burst Stimulation (TBS), a specific stimulatory protocol shown to modulate cortical plasticity for a duration up to 30 minutes after stimulation cessation in healthy populations, has already proven an efficacious tool in the exploration of abnormal plasticity.9,10 
Recent studies have shown the effects of TBS last considerably longer in individuals on the autistic spectrum - up to 90 minutes.11 This extended effect-duration suggests an underlying abnormality in the brain's natural plasticity state in autistic individuals - similar to the hyperexcitability induced by Fragile X Syndrome.
In this experiment, utilizing single-pulse motor-evoked potentials (MEPs) as our benchmark, we will explore the effects of both intermittent and continuous TBS on cortical plasticity in individuals suffering from FXS and individuals on the Autistic Spectrum.

In this article, we examine the effects of Theta-Burst TMS stimulation on cortical plasticity in individuals suffering from Fragile X syndrome and individuals on the autistic spectrum.

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

State-Dependency Effects on TMS:  A Look at Motive Phosphene Behavior

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.1,2 Within the field of TMS, the term state dependency refers to the initial, baseline condition of the particular neural region targeted for stimulation. As can be inferred, the effects of TMS can (and do) vary according to this primary susceptibility and responsiveness of the targeted cortical area.3,4,5

In this experiment, we will examine this concept of state dependency through the elicitation and subjective experience of motive phosphenes. Phosphenes are visually perceived flashes of small lights triggered by electromagnetic pulses to the visual cortex. These small lights can assume varied characteristics depending upon which type of visual cortex is being stimulated. In this particular study, we will be targeting motive phosphenes as elicited through the stimulation of V1/V2 and the V5/MT+ complex visual regions.6

In this article, we examine the effects of visually relevant state dependency on TMS induced motive phosphenic presentations.

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate ...

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3
In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4
The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053).
In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

Manipulation of Epileptiform Electrocorticograms (ECoGs) and Sleep in Rats and Mice by Acupuncture

Ancient Chinese literature has documented that acupuncture possesses efficient therapeutic effects on epilepsy and insomnia. There is, however, little research to reveal the possible mechanisms behind these effects. To investigate the effect of acupuncture on epilepsy and sleep, several issues need to be addressed. The first is to identify the acupoints, which correspond between humans, rats, and mice. Furthermore, the depth of insertion of the acupuncture needle, the degree of needle twist in manual needle acupuncture, and the stimulation parameters for electroacupuncture (EA) need to be determined. To evaluate the effects of acupuncture on epilepsy and sleep, a feasible model of epilepsy in rodents is required. We administer pilocarpine into the left central nucleus of the amygdala (CeA) to simulate focal temporal lobe epilepsy (TLE) in rats. Intraperitoneal (IP) injection of pilocarpine induces generalized epilepsy and status epilepticus (SE) in rats. Five IP injections of pentylenetetrazol (PTZ) with a one-day interval between each injection successfully induces spontaneous generalized epilepsy in mice. Recordings of electrocorticograms (ECoGs), electromyograms (EMGs), brain temperature, and locomotor activity are used for sleep analysis in rats, while ECoGs, EMGs, and locomotor activity are employed for sleep analysis in mice. ECoG electrodes are implanted into the frontal, parietal, and contralateral occipital cortices, and a thermistor is implanted above the cerebral cortex by stereotactic surgery. EMG electrodes are implanted into the neck muscles, and an infrared detector determines locomotor activity. The criteria for categorizing vigilance stages, including wakefulness, rapid eye movement (REM) sleep, and non-REM (NREM) sleep are based on information from ECoGs, EMGs, brain temperature, and locomotor activity. Detailed classification criteria are stated in the text.

Manipulation of Epileptiform Electrocorticograms ECoGs and Sleep in Rats and Mice by Acupuncture

This paper demonstrates the performance of acupuncture, epilepsy models, and the analysis of sleep in rodents. The acupuncture procedure and the identification of acupoints are described. Pilocarpine or pentylenetetrazol (PTZ) is used to induce epilepsy. Electrocorticogram (ECoG), electromyogram (EMG), brain temperature, and locomotor activity recordings are employed for sleep analysis.

Ancient Chinese literature has documented that acupuncture possesses efficient therapeutic effects on epilepsy and insomnia. There is, however, little ...

Behavior

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of the breadth of molecular regulation. Newer approaches such as high-resolution RNA sequencing (RNA-Seq)1 provides new and expansive information about tissue- or state-specific expression such as relative transcript levels, alternative splicing, and micro RNAs2-4. Prospects for employing the RNA-Seq method in comparative whole transcriptome profiling5 within discrete tissues or between phenotypically distinct groups of individuals affords new avenues for elucidating molecular mechanisms involved in both normal and abnormal physiological states. Recently, whole transcriptome profiling has been performed on human brain tissue, identifying gene expression differences associated with disease progression6. However, the use of next-generation sequencing has yet to be more widely integrated into mammalian studies.
Gene expression studies in mouse models have reported distinct profiles within various brain nuclei using laser capture microscopy (LCM) for sample excision7,8. While LCM affords sample collection with single-cell and discrete brain region precision, the relatively low total RNA yields from the LCM approach can be prohibitive to RNA-Seq and other profiling approaches in mouse brain tissues and may require sub-optimal sample amplification steps. Here, a protocol is presented for microdissection and total RNA extraction from discrete mouse brain regions. Set-diameter tissue corers are used to isolate 13 tissues from 750-&mu;m serial coronal sections of an individual mouse brain. Tissue micropunch samples are immediately frozen and archived. Total RNA is obtained from the samples using magnetic bead-enabled total RNA isolation technology. Resulting RNA samples have adequate yield and quality for use in downstream expression profiling. This microdissection strategy provides a viable option to existing sample collection strategies for obtaining total RNA from discrete brain regions, opening possibilities for new gene expression discoveries.

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling  |

RNA expression profiling of discrete mouse brain regions requires a precise and repeatable tissue collection strategy. A protocol that uses both coronal brain sectioning and tissue corer-assisted microdissection is described here. The yield and quality of total RNA obtained from the resulting samples confirms the utility of the outlined method.

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of ...

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.
	One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.
	Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.
	We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

Fiber-optic Implantation for Chronic Optogenetic Stimulation of Brain Tissue

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard for analyzing patterns of synaptic connectivity, but paired electrophysiological recordings can be both cumbersome and experimentally limiting. The development of optogenetics has introduced an elegant method to stimulate neurons and circuits, both in vitro1 and in vivo2,3. By exploiting cell-type specific promoter activity to drive opsin expression in discrete neuronal populations, one can precisely stimulate genetically defined neuronal subtypes in distinct circuits4-6. Well described methods to stimulate neurons, including electrical stimulation and/or pharmacological manipulations, are often cell-type indiscriminate, invasive, and can damage surrounding tissues. These limitations could alter normal synaptic function and/or circuit behavior. In addition, due to the nature of the manipulation, the current methods are often acute and terminal. Optogenetics affords the ability to stimulate neurons in a relatively innocuous manner, and in genetically targeted neurons. The majority of studies involving in vivo optogenetics currently use a optical fiber guided through an implanted cannula6,7; however, limitations of this method include damaged brain tissue with repeated insertion of an optical fiber, and potential breakage of the fiber inside the cannula. Given the burgeoning field of optogenetics, a more reliable method of chronic stimulation is necessary to facilitate long-term studies with minimal collateral tissue damage. Here we provide our modified protocol as a video article to complement the method effectively and elegantly described in Sparta et al.8 for the fabrication of a fiber optic implant and its permanent fixation onto the cranium of anesthetized mice, as well as the assembly of the fiber optic coupler connecting the implant to a light source. The implant, connected with optical fibers to a solid-state laser, allows for an efficient method to chronically photostimulate functional neuronal circuitry with less tissue damage9 using small, detachable, tethers. Permanent fixation of the fiber optic implants provides consistent, long-term in vivo optogenetic studies of neuronal circuits in awake, behaving mice10 with minimal tissue damage.

The development of optogenetics now provides the means to precisely stimulate genetically defined neurons and circuits, both in vitro and in vivo. Here we describe the assembly and implantation of a fiber optic for chronic photostimulation of brain tissue.

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard ...

Optogenetic Manipulation of Neural Circuits During Monitoring Sleep/wakefulness States in Mice

In recent years, optogenetics has been widely used in many fields of neuroscientific research. In many cases, an opsin, such as channel rhodopsin 2 (ChR2), is expressed by a virus vector in a particular type of neuronal cells in various Cre-driver mice. Activation of these opsins is triggered by application of light pulses which are delivered by laser or LED through optic cables, and the effect of activation is observed with very high time resolution. Experimenters are able to acutely stimulate neurons while monitoring behavior or another physiological outcome in mice. Optogenetics can enable useful strategies to evaluate function of neuronal circuits in the regulation of sleep/wakefulness states in mice. Here we describe a technique for examining the effect of optogenetic manipulation of neurons with a specific chemical identity during electroencephalogram (EEG) and electromyogram (EMG) monitoring to evaluate the sleep stage of mice. As an example, we describe manipulation of GABAergic neurons in the bed nucleus of the stria terminalis (BNST). Acute optogenetic excitation of these neurons triggers a rapid transition to wakefulness when applied during NREM sleep. Optogenetic manipulation along with EEG/EMG recording can be applied to decipher the neuronal circuits that regulate sleep/wakefulness states.

Here, we describe methods of optogenetic manipulation of particular types of neurons during monitoring of sleep/wakefulness states in mice, presenting our recent work on the bed nucleus of the stria terminalis as an example.

In recent years, optogenetics has been widely used in many fields of neuroscientific research. In many cases, an opsin, such as channel rhodopsin 2 ...

A Protocol for Transcranial Photobiomodulation Therapy in Mice

Transcranial photobiomodulation is a potential innovative noninvasive therapeutic approach for improving brain bioenergetics, brain function in a wide range of neurological and psychiatric disorders, and memory enhancement in age-related cognitive decline and neurodegenerative diseases. We describe a laboratory protocol for transcranial photobiomodulation therapy (PBMT) in mice. Aged BALB/c mice (18 months old) are treated with a 660 nm laser transcranially, once daily for 2 weeks. Laser transmittance data shows that approximately 1% of the incident red light on the scalp reaches a 1 mm depth from the cortical surface, penetrating the dorsal hippocampus. Treatment outcomes are assessed by two methods: a Barnes maze test, which is a hippocampus-dependent spatial learning and memory task evaluation, and measuring hippocampal ATP levels, which is used as a bioenergetics index. The results from the Barnes task show an enhancement of the spatial memory in laser-treated aged mice when compared with age-matched controls. Biochemical analysis after laser treatment indicates increased hippocampal ATP levels. We postulate that the enhancement of memory performance is potentially due to an improvement in hippocampal energy metabolism induced by the red laser treatment. The observations in mice could be extended to other animal models since this protocol could potentially be adapted to other species frequently used in translational neuroscience, such as rabbit, cat, dog, or monkey. Transcranial photobiomodulation is a safe and cost-effective modality which may be a promising therapeutic approach in age-related cognitive impairment.

Photobiomodulation therapy is an innovative noninvasive modality for the treatment of a wide range of neurological and psychiatric disorders and can also improve healthy brain function. This protocol includes a step-by-step guide to performing brain photobiomodulation in mice by transcranial light delivery, which can be adapted for use in other laboratory rodents.

Transcranial photobiomodulation is a potential innovative noninvasive therapeutic approach for improving brain bioenergetics, brain function in a wide ...

Photobiomodulation Under Electroencephalographic Controls of Sleep for Stimulation of Lymphatic Removal of Toxins from Mouse Brain

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video