We are grateful for the grants provided by the Ministry of Science and Technology, Taiwan - MOST105-2628-B-038-005-MY3(1-3) and Health and welfare surcharge of tobacco products - MOHW106-TDU-B-212-144001 to S-Y C.

All the protocols and methods used in animal subjects have been approved by Institutional Animal Care and Use Committees (IACUC) of Taipei Medical University (Protocol numbers: LAC-2013-0278; LAC-2015-0397)
1. Primary Neuron Culture
<ol>
	<li>Preparation before culture
	<ol>
		<li>Coat the culture dishes with poly-D-Lysine (Table 1) one day before culture. For a 6-well dish, add 1.5 mL poly-D-Lysine (0.05 mg/mL) into each well. For live-imaging, culture cells on a 12 mm x 12 mm coverslip in a standard coated 6-well plate.</li>
		<li>Remove/recycle the poly-D-lysine and wash the dishes twice with 2 mL ddH2O.</li>
		<li>Prepare the surgical tools: a pair of micro-dissecting scissors, curved-tipped forceps, and standard straight-tipped forceps. Sterilize the surgical tools and keep them in 75% ethanol during surgery.</li>
		<li>Fill 10 cm petri dishes with 20 mL wash medium (Table 1) and keep on ice.</li>
		<li>Fill a 15 mL test tube with 15 mL wash medium and keep the tube on ice.</li>
	</ol>
	</li>
	<li>Brain tissue isolation from different brain regions
	<ol>
		<li>Sacrifice 16-19 day old pregnant female mice with standard euthanization protocols by placing mice in a non-ventilated cage and then intoxicating with CO2. E15.5~E16.5 embryos are recommended for hypothalamic neuronal culture and E16.5~E17.5 are more suitable for hippocampal and cortical neuronal culture.</li>
		<li>Sterilize the platform, dissecting microscope, and surgery tools with 75% ethanol to avoid contamination.</li>
		<li>Cut around the umbilical cord area (dark red area, Figure 1A, 1B dash line) to isolate the pups easily without damaging them (Figure 1C, 1D).</li>
		<li>Remove the head portion of each pup with a pair of scissors (Figure 1E, F), then secure the head portion in place using the fine-tipped straight forceps for the eye region (Figure 1G). Use another fine-tipped curved forceps to remove the outer skin and skull from two sides and peel them off from the anterior to the dorsal direction (Figure 1H, black arrow points to the direction). The brain should be isolated without any damage (Figure 1I). 
		NOTE: It is essential to maintain the integrity of the brain to ensure accuracy when isolating different regions of the brain.</li>
		<li>Keep isolated brains in a clean petri dish with ice-cold wash medium for the following steps.</li>
		<li>Flip the brain and keep the ventral side up. The hypothalamus is a round structure in the middle of the brain (Figure 1J, arrow points to the hypothalamic region). Isolate the hypothalamic tissue with fine-tipped curved forceps. Remove the meninges (which have a yellowish-red color) carefully. 
		CAUTION: Complete removal of meninges is a critical step to avoid fibroblast contamination.</li>
		<li>Hold the olfactory lobe with the sharp forceps and peel off the thin meninges surrounding the whole brain with the curved forceps (Figure 1K, L).</li>
		<li>The hippocampus is a banana-like shape which is embedded in the lower part of the cortex. Flip to the underside of the cortex and separate the hippocampus from the cortex by pulling it to the side with the curved forceps (Figure 1M, N, and O). Be sure to remove all the remaining meninges surrounding the brain tissue.</li>
		<li>When all regions of the brain have been isolated, chop these tissues in the 15 mL tubes containing ice-cold wash medium with the help of the forceps (step 1.1.5). 
		NOTE: Brain tissues can be kept in the ice-cold wash medium for 2-3 h.</li>
	</ol>
	</li>
	<li>Tissue digestion, plating and culture
	<ol>
		<li>Rinse the tissues by inverting the tissue-containing 15 mL tube 2-3 times, and then keep the tube straight to allow the tissues to settle down (1-3 min).</li>
		<li>Remove the upper medium using the glass Pasteur pipette attached to a vacuum system. To wash the tissue, add 15 mL ice cold wash medium and invert the tube 2-3 times. Repeat wash steps 3 times before the next step. 
		CAUTION: Be cautious of the tissues on the bottom when removing the upper medium using a vacuum system.</li>
		<li>After the final wash, remove the wash medium with the help of a 1 mL pipette.</li>
		<li>Incubate tissues with Papain-Trypsin digestion buffer (Table 1) in a 37 &#176;C water bath for 7-14 min. Shake the tubes every two min to ensure all tissues are properly exposed to the digestion buffer. 
		NOTE: Incubation time and the volume of digestion buffer can be adjusted based on the tissue size. For hypothalamic tissue collected from 6-8 pups, 300 &#181;L Papain-Trypsin digestion buffer and 7 min incubation time is recommended. More tissue will require more digestion buffer.</li>
		<li>Neutralize the enzyme&#39;s activity by adding 1 volume of fetal bovine serum. Shake the test tube 3-5 times at room temperature.</li>
		<li>Keep the tube on racks and wait for 1-2 min to allow the tissues to settle down; remove the supernatant carefully with a 1 mL pipette.</li>
		<li>Add 6 mL plating medium (Table 1) into the test tube and pipet the tissue up-and-down 50 times with a 5 mL pipette gently (for a 6-well plate, 1.5 mL plating medium per well is recommended). Most cells will dissociate into single cells after repeated pipetting. 
		NOTE: Try to avoid the formation of bubbles while conducting this step. Most laboratories use flamed glass pasture pipets to dissociate the tissue. A 5 mL pipette with a narrow tip can be a good alternative for this step.</li>
		<li>Keep a tube on the rack and wait for 1-2 min to allow the chunky, un-dissociated tissues to settle down. Take the upper phase containing dissociated cells to a new tube and dilute with plating medium (5x volume plating medium per 1x volume of dissociated cells. For example, add 5 mL plating medium for 1 mL dissociated cells).</li>
		<li>Calculate the cell density using a hemocytometer and seed the required number of cells into the culture plate coated with poly-D-lysine as in step 1.1.1. We recommend 2-4 x 105 cells per well for a 6 well dish/35 mm dish were recommended for neuron imaging studies. A higher density would be needed for protein collection and analysis. 
		NOTE: Do not add too much plating medium, 1-1.5 mL plating medium in one 6-well dish is sufficient for attachment. An excessive amount of medium will prolong the time required for proper cell attachment.</li>
		<li>Wait for 2-3 h and check the seeded neurons under the microscope. Neurons will start to grow neuritis when they attach properly.</li>
		<li>Remove the plating medium and add 2 mL warmed wash medium (37 &#176;C) into the dish to wash off the plating medium. Repeat this step twice.</li>
		<li>Add 2 mL complete culture medium (Table 1) into each well in the 6-well dish.</li>
		<li>Change half of the medium every 3-4 days. Prepare the complete medium freshly each time. Neurons cultured from different mouse brain regions will have different morphology and characteristics (Figure 2).</li>
	</ol>
	</li>
</ol>
2. Transfection of Plasmid DNA into Primary Neuron with Liposome System
CAUTION: For neuron transfection, an endotoxin-free plasmid DNA purification kit (Materials Table) is recommended for DNA preparation. Additional ethanol precipitation can remove excess solvent and enhances DNA concentration.
<ol>
	<li>Liposome-based DNA transfection in primary neurons. 
	NOTE: The transfection time depends on the maturation status required in experiments. The hypothalamic neurons were transfected at DIV 7-10 (DIV, days in vitro).
	<ol>
		<li>DNA: Liposome mixture preparation: Dilute GFP-GLUT4 plasmid DNA16 (2 &#181;g) in a microcentrifuge tube containing 300 &#181;L low serum culture medium. Prepare another microcentrifuge tube with 2 &#181;L liposome in 300 &#181;L low serum culture medium, and incubate for 5 min at room temperature.</li>
		<li>Combine the contents of both tubes and incubate for 20-30 min at room temperature.</li>
		<li>Remove the neuron culture medium from the culture dish. Add 600 &#181;L DNA-liposome mixture into each well and incubate at 37 &#176;C for 4-6 h.</li>
		<li>After 4-6 h, remove DNA-liposome mixture and add 2 mL culture medium.</li>
		<li>Fluorescent signals, such as GFP alone and GLUT4 protein conjugated with GFP tag (Figure 3 and Figure 4), can be observed after 18-72 h.</li>
	</ol>
	</li>
</ol>
3. Live Image Recording
<ol>
	<li>Preparing Cells for Live Imaging
	<ol>
		<li>Culture WT and CCR5-/- hypothalamic neuron cells expressing green fluorescence protein labeled glucose transporter 4 (GFP-GLUT4) on the #1.5 (or 0.17mm thickness) glass coverslip which has been pre-coated with poly-D-Lysine in step 1.1.1 in the 6-well plate.</li>
		<li>Carefully remove the coverslips with neurons using forceps and place it on the glass slides with caution.</li>
		<li>Remove excess medium/liquid with delicate task wipes. Fold the wipes twice, and carefully place them on top of the coverslip. Gently press down the wipes without moving the coverslip. 
		CAUTION: Do not press the coverslip against the glass slide forcefully. The purpose of this step is to ensure that the coverslip does not move/drift during image acquisition caused by buoyant force.</li>
		<li>Place coverslips and slides on the deconvolution microscope stage, with the coverslip facing down, and secured properly. This step is to ensure that the slide position remains constant so users can track the same set of target cells later.</li>
		<li>Observe WT and CCR5-/- hypothalamic neuron cells with a 60x/1.42 NA oil immersion objective lens.</li>
		<li>Treat the selected cell samples with CCL5 (10 ng/mL) or insulin (10 U/mL) for one min. Add 1.5 &#181;L of diluted insulin on the edge of coverslip.</li>
		<li>Visualize and record the selected cell samples immediately. Program the video recording software to record for 30 min.</li>
	</ol>
	</li>
	<li>Deconvolution microscopy and analysis 
	NOTE: This part of the protocol requires the use of a deconvolution microscope and specialized software for analysis.
	<ol>
		<li>Turn on the power of the imaging system, allow microscope stage to initialize properly, and then turn on the LED light source.</li>
		<li>Add immersion oil (refractive index 1.520 for live samples at 37 &#176;C) on a 60x 1.42 NA objective lens. Place the sample slide on the microscope with the coverslip facing toward the objective lens, and secure the slide properly.</li>
		<li>Use bright-field or fluorescence illumination to identify target cells. Adjust the focus until target cells can be clearly observed. Do not move the objective lens outside of the coverslip area to avoid unnecessary scratch marks.</li>
		<li>Identify green fluorescence protein conjugated Glucose Transporter 4 (GFP-GLUT4) by the GFP signal. Identify desired target cells for image acquisition. Selected target cell position can be memorized for future reference (Figure 4).</li>
		<li>Setup proper experimental parameters (including pixel number, excitation wavelength, transmission percentage, exposure time, stack thickness, time interval, and total imaging time) on each target cell. For this experiment, the image pixel number was set at 512 x 512 (it can be set at 1,024 x 1,024 for higher resolution) for GFP signals. The exposure time was set between 0.025 to 0.05 s for every 5 min. Minor lateral x, y, and z adjustments can be controlled by the recommended software (Materials Table).</li>
		<li>Set the exposure parameter to approximately 2,000 to 3,000 counts to achieve maximum pixel intensity. To minimize fluorescence photobleaching, reduce the percentage of excitation light transmission as much as possible while keeping exposure time less than 1 s. Repeat these steps for each additional fluorescence channel(s) and each individual area of interest.</li>
		<li>Set the upper and lower limit of the Z-stack on each target cell. This can be achieved by moving the microscope stage until the top and bottom of the target cell are both slightly out of focus. The users can adjust the resolution of the Z-axis by setting the number of images between the upper and lower limit (which can be done by setting the distance between each image).</li>
		<li>Stacks of images were deconvolved and later analyzed with the help of the respective software &#8211; Velocity from PerkinElmer in this case.</li>
	</ol>
	</li>
</ol>

<ol><li>Levin, B. E., Sherwin, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Peripheral+glucose+homeostasis%3A+does+brain+insulin+matter%3F%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">Peripheral glucose homeostasis: does brain insulin matter?</a> J Clin Invest. 121 (9), 3392-3395 (2011).</li>
<li>Kleinridders, A., Ferris, H. A., Cai, W., Kahn, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Insulin+action+in+brain+regulates+systemic+metabolism+and+brain+function%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">Insulin action in brain regulates systemic metabolism and brain function.</a> Diabetes. 63 (7), 2232-2243 (2014).</li>
<li>Duarte, A. I., Moreira, P. I., Oliveira, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Insulin+in+central+nervous+system%3A+more+than+just+a+peripheral+hormone%5BTitle%5D)+AND+%22J+Aging+Res%22%5BJournal%5D)">Insulin in central nervous system: more than just a peripheral hormone.</a> J Aging Res. 2012, 384017(2012).</li>
<li>Vogt, M. C., Bruning, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CNS+insulin+signaling+in+the+control+of+energy+homeostasis+and+glucose+metabolism+-+from+embryo+to+old+age%5BTitle%5D)+AND+%22Trends+Endocrinol+Metab%22%5BJournal%5D)">CNS insulin signaling in the control of energy homeostasis and glucose metabolism - from embryo to old age.</a> Trends Endocrinol Metab. 24 (2), 76-84 (2013).</li>
<li>Plum, L., Schubert, M., Bruning, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+insulin+receptor+signaling+in+the+brain%5BTitle%5D)+AND+%22Trends+Endocrinol+Metab%22%5BJournal%5D)">The role of insulin receptor signaling in the brain.</a> Trends Endocrinol Metab. 16 (2), 59-65 (2005).</li>
<li>Lin, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dysregulation+of+insulin+receptor+substrate+2+in+beta+cells+and+brain+causes+obesity+and+diabetes%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">Dysregulation of insulin receptor substrate 2 in beta cells and brain causes obesity and diabetes.</a> J Clin Invest. 114 (7), 908-916 (2004).</li>
<li>Werner, E. D., Lee, J., Hansen, L., Yuan, M., Shoelson, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Insulin+resistance+due+to+phosphorylation+of+insulin+receptor+substrate-1+at+serine+302%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Insulin resistance due to phosphorylation of insulin receptor substrate-1 at serine 302.</a> J Biol Chem. 279 (34), 35298-35305 (2004).</li>
<li>Thaler, J. P., Guyenet, S. J., Dorfman, M. D., Wisse, B. E., Schwartz, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hypothalamic+inflammation%3A+marker+or+mechanism+of+obesity+pathogenesis%3F%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">Hypothalamic inflammation: marker or mechanism of obesity pathogenesis?</a> Diabetes. 62 (8), 2629-2634 (2013).</li>
<li>Milanski, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhibition+of+hypothalamic+inflammation+reverses+diet-induced+insulin+resistance+in+the+liver%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">Inhibition of hypothalamic inflammation reverses diet-induced insulin resistance in the liver.</a> Diabetes. 61 (6), 1455-1462 (2012).</li>
<li>Wang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increased+hypothalamic+inflammation+associated+with+the+susceptibility+to+obesity+in+rats+exposed+to+high-fat+diet%5BTitle%5D)+AND+%22Exp+Diabetes+Res%22%5BJournal%5D)">Increased hypothalamic inflammation associated with the susceptibility to obesity in rats exposed to high-fat diet.</a> Exp Diabetes Res. 2012, 847246(2012).</li>
<li>Kitade, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CCR5+plays+a+critical+role+in+obesity-induced+adipose+tissue+inflammation+and+insulin+resistance+by+regulating+both+macrophage+recruitment+and+M1%2FM2+status%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">CCR5 plays a critical role in obesity-induced adipose tissue inflammation and insulin resistance by regulating both macrophage recruitment and M1/M2 status.</a> Diabetes. 61 (7), 1680-1690 (2012).</li>
<li>Kennedy, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Loss+of+CCR5+results+in+glucose+intolerance+in+diet-induced+obese+mice%5BTitle%5D)+AND+%22Am+J+Physiol+Endocrinol+Metab%22%5BJournal%5D)">Loss of CCR5 results in glucose intolerance in diet-induced obese mice.</a> Am J Physiol Endocrinol Metab. 305 (7), E897-E906 (2013).</li>
<li>Chan, O., Burke, J. D., Gao, D. F., Fish, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+chemokine+CCL5+regulates+glucose+uptake+and+AMP+kinase+signaling+in+activated+T+cells+to+facilitate+chemotaxis%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">The chemokine CCL5 regulates glucose uptake and AMP kinase signaling in activated T cells to facilitate chemotaxis.</a> J Biol Chem. 287 (35), 29406-29416 (2012).</li>
<li>Plata-Salaman, C. R., Borkoski, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemokines%2Fintercrines+and+central+regulation+of+feeding%5BTitle%5D)+AND+%22Am+J+Physiol%22%5BJournal%5D)">Chemokines/intercrines and central regulation of feeding.</a> Am J Physiol. 266 (5 Pt 2), R1711-R1715 (1994).</li>
<li>Mayer, C. M., Fick, L. J., Gingerich, S., Belsham, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hypothalamic+cell+lines+to+investigate+neuroendocrine+control+mechanisms%5BTitle%5D)+AND+%22Front+Neuroendocrinol%22%5BJournal%5D)">Hypothalamic cell lines to investigate neuroendocrine control mechanisms.</a> Front Neuroendocrinol. 30 (3), 405-423 (2009).</li>
<li>Lizunov, V. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Insulin+stimulates+fusion%2C+but+not+tethering%2C+of+GLUT4+vesicles+in+skeletal+muscle+of+HA-GLUT4-GFP+transgenic+mice%5BTitle%5D)+AND+%22Am+J+Physiol+Endocrinol+Metab%22%5BJournal%5D)">Insulin stimulates fusion, but not tethering, of GLUT4 vesicles in skeletal muscle of HA-GLUT4-GFP transgenic mice.</a> Am J Physiol Endocrinol Metab. 302 (8), E950-E960 (2012).</li>
<li>Chou, S. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CCL5%2FRANTES+contributes+to+hypothalamic+insulin+signaling+for+systemic+insulin+responsiveness+through+CCR5%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">CCL5/RANTES contributes to hypothalamic insulin signaling for systemic insulin responsiveness through CCR5.</a> Sci Rep. 6, 37659(2016).</li>
</ol>

The authors have nothing to disclose.

The ability to monitor live cells, upon CCL5 or insulin stimulation, is critically important for studying the rapid effect of CCL5 or insulin on GLUT4 movement. In fact, it allows us to visualize the significant difference between WT and CCR5-/- hypothalamic neurons upon insulin stimulation. We have performed the surface labeling of endogenous GLUT4 protein in WT and CCR5-/- hypothalamic neurons at different time points after insulin stimulation17. The labeling of cell surface proteins requires high-specificity antibody with low background. In addition, surface fluorescence quantification can also be challenging and time consuming. Thus, time-lapse recording allows us to be certain that the effect of CCL5 or insulin is a true physiological change based on experimental conditions, rather than a statistical variation. Together with surface labeling of endogenous GLUT4, we provide strong evidence and experiments to demonstrate how CCL5 and CCR5 participate in GLUT4 translocation and insulin signaling.
In modern cell biology and molecular biology studies, many experiments require the utilization of fluorescence microscopy. This technology allows scientists to visualize the spatial relationship between proteins and/or cellular organelles, in addition to movement direction and speed, stimulatory effects, morphological changes, and protein trafficking. However, this technology still has its limitation: when fluorophores are excited, signals emitted from the target protein (or area) can be overwhelmed by background fluorescence. As a result, fluorescence images can appear blurry with expected signals buried deep into background signals. This phenomenon is especially apparent for the observation of membrane-bound proteins.
Total Internal Reflection Fluorescence Microscopy (TIRFM) was developed to overcome this difficulty. It allows scientists to visualize the excitation of selected surface-bound fluorophores without affecting the background fluorophores. It allows scientists to selectively characterize features and events on a very thin surface region such as a plasma membrane. Deconvolution microscopy is a computationally intensive image processing technique that is made possible with the help of technological advancements in recent years. It has been frequently utilized to improve digital fluorescence image resolution. As mentioned previously, when fluorophores are being excited by any type of illumination (such as laser or LED), all fluorophores will emit light signals regardless if they are in focus or not, so the image will always appear blurry. This blurring is caused by a phenomenon called &#34;Point Spread Function&#34; (PSF), as light coming from a small fluorescent source (bright spot) will spread out further and become out of focus (blur). In principle, this event will produce an hourglass-like shaped fluorescent signal, and a fluorescence image can be made up of numerous such light signals. The deconvolution process can reassign all the fluorescence signals to its original bright spot form, and eliminate most of the out-of-focus light to improve image contrast.
In recent years, deconvolution algorithms have generated images with comparable resolution to that of a confocal microscope. Moreover, in comparison with TIRFM, which prevents out-of-focus blur from being detected by a limited excitation region, wide-field microscopy allows all light signals to be detected and reassigns them back to their source through the deconvolution process. Therefore, in practice, deconvolution microscopy has become not only a more efficient image acquisition method, but also a more cost-effective method when compared to TIRF microscopy.

Type 2 diabetes mellitus (T2DM) is a global health crisis. T2DM is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus is the control center which regulates the body&#39;s energy homeostasis, appetite, and circadian rhythms. Most importantly, the hypothalamus also mediates insulin signal responsiveness to regulate systemic metabolism1,2,3,4,5. Disrupting the hypothalamic insulin signaling pathway could induce insulin resistance6,7. The hypothalamus coordinates cellular energy status and secretion of hormones, such as insulin and adipokines (e.g., leptin) from the peripheral tissues, to regulate systemic glucose metabolism, insulin responsiveness, and food intake. Insulin binding to the insulin receptor activates insulin receptor substrate (IRS) proteins, which then activate insulin downstream signaling molecules, such as PI3K (phosphatidylinositol 3-kinase) and AKT (protein kinase B (PKB/AKT)), to induce GLUT4 membrane translocation. Neurons are not the major target for glucose uptake in response to insulin; however, significant levels of GLUT4 expression have been identified in the hypothalamic arcuate nucleus (ARC) region. Therefore, the regulation of GLUT4 in hypothalamic neurons may play an important role in insulin signaling in the brain-peripheral axis.
Many studies have suggested that chronic inflammation and inflammatory chemokines in hypothalamus also play an important role in the development of diabetes and obesity, and inhibition of hypothalamic inflammation can reverse diet-induced insulin resistance8,9,10. Moreover, chemokine-CCL5 (C-C motif ligand 5, also known as RANTES, Regulated-on-Activation-Normal-T-cell-Expressed-and-Secreted) and its receptor CCR5 levels also correlate with the development of T2DM11,12. The roles of CCL5 and CCR5 in insulin function and glucose metabolism remain unclear. One study reported that CCR5 deficiency protected mice from obesity-induced inflammation, macrophage recruitment, and insulin resistance11; in contrast, another study reported that CCR5 deficiency impairs systemic glucose tolerance, as well as adipocyte and muscle insulin signaling12. CCL5 is found to increase glucose uptake in T-cells and to reduce food intake through its action on the hypothalamus13,14, however, both the mechanism of action and the receptors involved are yet to be identified.
It is difficult to study the cellular mechanisms underlying the effect of peripheral tissue inflammation on insulin functioning in hypothalamic neurons. This is due to cellular heterogeneity and neuron circuit feedback regulations. For this reason, an in vitro cell culture model provides a clean model to investigate the effects of the chemokine on hypothalamic insulin signal regulation. Although there are many established immortalized hypothalamic neuronal cell lines for research use, these cell lines expressed different markers, and therefore, represent different types of hypothalamic neurons15. Even though primary hypothalamic cultures can be difficult to maintain, they can provide the most realistic response of hypothalamic neurons upon insulin stimulation, and can also avoid the potential unknown effects which come into play when maintaining cells long-term in culture medium with artificial growth factors.
Herein, we utilize primary hypothalamic neurons from both C57BL/6 wildtype (WT) mouse and CCR5 knockout (CCR5-/-) mouse, and transfect both types of cells with GFP-GLUT4 construct. To investigate the contribution of CCR5 to insulin mediated GLUT4 membrane trafficking, GFP-GLUT4 transfected neurons were treated with insulin or recombinant CCL5. We then characterize the movement of GFP-GLUT4 on the plasma membrane in primary hypothalamic neurons with Deconvolution Microscopy.

<table><tbody><tr><td>DeltaVision deconvolution microscope</td><td>Applied Precision Inc./(GE Healthcare Life Science)&amp;nbsp;</td><td></td><td>equipped with 60x/1.42 NA oil immersion objective lens; used for taking live cell images</td></tr><tr><td>SoftWorX application software</td><td>Applied Precision Inc.</td><td></td><td>used to control microscope and camera</td></tr><tr><td>VoloCITY software</td><td>PerkinElmer</td><td></td><td>used to analyze the images</td></tr><tr><td>Insulin</td><td>Actrapid, Denmark</td><td></td><td></td></tr><tr><td>Liposome</td><td>Invitrogen</td><td>11668019</td><td>Lipofectamine&amp;reg; 2000, used for plasmid DNA transfection</td></tr><tr><td>GFP control plasmid DNA</td><td></td><td></td><td>provided by Professor Samuel Cushman</td></tr><tr><td>GFP-GLUT4 plasmid DNA</td><td></td><td></td><td>provided by Professor Samuel Cushman</td></tr><tr><td>Anti-mouse Alex-488</td><td>Invitrogen</td><td>#A-11001</td><td>Secondary anitbody</td></tr><tr><td>Anti-rabbit IgG -Alex-568</td><td>Invitrogen</td><td>#A-11036</td><td>Secondary anitbody</td></tr><tr><td>CCL5/RANTES</td><td>R&amp;amp;D Systems</td><td>478-MR-025</td><td>10ng/ml</td></tr><tr><td>Kimwipes</td><td>Kimberly-Clark</td><td>#FL42572A</td><td>0.17mm thickness</td></tr><tr><td>12 mm Microscope coverglass</td><td>Deckglaser</td><td>#41001112</td><td>used for immmunstaining study</td></tr><tr><td>micro-dissecting scissors</td><td>Klappenecker</td><td></td><td>Surgical tool</td></tr><tr><td>curved-tipped forceps</td><td>Ideal-tek</td><td></td><td>Surgical tool</td></tr><tr><td>standard straight-tipped forceps</td><td>Ideal-tek</td><td></td><td>Surgical tool</td></tr><tr><td>EndoFree Plasmid Maxi Kit</td><td>QIAGEN</td><td>12362</td><td>Purify Endotoxin free plasmid DNA</td></tr><tr><td>5 ml pipette</td><td>Corning costar&amp;nbsp;</td><td>4487</td><td>dissociate brain tissue</td></tr></tbody></table>

live images of glut4 protein trafficking in mouse primary hypothalamic neurons using deconvolution microscopy

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNF&#945;, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear.
In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

The hypothalamic neurons cultured from mice were further identified by immunostaining with hypothalamic specific protein - pro-opiomelanocortin (POMC) antibody and neuronal marker - microtubule-associated protein 2 (MAP2) (Figure 2A). We confirmed the primary cultured hypothalamic neurons expressed hypothalamic protein POMC. The expression of the CCR5 receptor and CCL5 in hypothalamic neurons were identified with specific antibodies and co-labeled with POMC antibody (Figure 2A, 2B).
After 3 days of culturing, neurons were transfected with GFP DNA (Figure 3) or GFP conjugated GLUT4 (Figure 4). GFP expression can usually be found all over the cell without a specific pattern (Figure 3) but the GLUT4-GFP will express as a punctate-like structure in the cytosol (Figure 4). The transfection kit used in this study is not the most efficient method for neuron transfection; however, it is a less stringent method for better cell survival after transfection, which contributes to better live-cell imaging/recording later. The images of GFP-GLUT4 expressing neurons were taken before time-lapse movies (Figure 4A-B, supplementary video 1, 2) upon insulin stimulation or CCL5 stimulation (Figure 4C, supplementary video 3). The signals of GFP and GFP-GLUT4 are clear and strong in neurons.
Hypothalamic neurons with GLUT4-GFP transfection were further treated with insulin (40 U) to characterize GFP-GLUT4 trafficking. Reprehensive videos of GLUT4-GFP movement upon insulin stimulation in both WT and CCR5-/- hypothalamic neurons are shown as Video 1 and Video 2, respectively.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56409/56409fig1.jpg" /> 
Figure 1: Isolation of tissues from different regions of the mouse brain at the embryonic stage (day 16.5). (A-D) The steps involved in the separation of pups from the placenta. (E, F) The dissection of a pup head from the body. (G-I) The steps involved in the isolation of the whole brain from the skull. The black arrow points in the direction to be pulled while removing the skull using forceps. (J) The isolation of the hypothalamus. The black arrow points to the hypothalamic region between forceps. (K-L) Isolation of the cortex of the mouse brain. The black asterisk indicates the cortical region of the mouse brain and the white arrow points to the separation of the cortex from the whole brain. (M-O) The separation of the hippocampal portion from the cortex. The upper white arrow marks the hippocampal tissue and the lower white arrow marks the cortical tissue. Scale bars = 1 cm (A-F), 200 &#181;m (G-O). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56409/56409fig1large.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/56409/56409fig2.jpg" /> 
Figure 2: Characterization of hypothalamic neuronal marker - POMC and the co-expression of CCL5 and CCR5. (A) Primary cultured hypothalamic neurons were labeled with hypothalamic neuronal marker - POMC (red), the co-expression of CCR5 (green), and neuron marker MAP2 (gray). (B) The CCL5 (green) expression in POMC (red) positive hypothalamic neurons (Adapted from the supplementary data of reference17). Here, DAPI labeled the nucleus with blue color. Scale bars = 50 &#181;m in (A) and (B). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56409/56409fig2large.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/56409/56409fig3.jpg" /> 
Figure 3: GFP protein expression in mouse primary neurons. GFP plasmid DNA transfected into primary cultured neurons after 4 days culture (DIV4) with liposome and expressed for another 3 days (DIV7). (A-B) GFP is expressed in both neuritis and soma. (C-D) Neurons with mock transfection; DAPI labeled the nucleus in (B, D). Scale bars = 100 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56409/56409fig3large.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/56409/56409fig4.jpg" /> 
Figure 4: The snapshots of GFP-GLUT4 in hypothalamic neurons. (A, C) GFP-GLUT4 protein expressed in Wildtype (WT) hypothalamic neurons and (B) CCR5-/- hypothalamic neurons. Neurons were stimulated with insulin (A, B) or CCL5 (C). The arrows point to the GFP-GLUT4 punctate in the neuritis before (-) and after (+) insulin or CCL5 stimulation and asterisks point to the surface GLUT4-GFP before (-) and after (+) CCL5 stimulation in (C). (Figure adapted from reference17). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56409/56409fig4large.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>5x Borade Buffer</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Volume</td>
		</tr>
		<tr>
			<td>Boric Acid</td>
			<td>Sigma-Aldrich</td>
			<td>B6768</td>
			<td>1.55 g</td>
		</tr>
		<tr>
			<td>Borax</td>
			<td>Sigma-Aldrich</td>
			<td>71997</td>
			<td>2.375 g</td>
		</tr>
		<tr>
			<td>ddH2O</td>
			<td></td>
			<td></td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Filtered, Keep at 4 &#176;C</td>
		</tr>
		<tr>
			<td>20x Poly-D-Lysine stock</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Volume</td>
		</tr>
		<tr>
			<td>Poly-D-Lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P6407</td>
			<td>100 mg</td>
		</tr>
		<tr>
			<td>ddH2O</td>
			<td></td>
			<td></td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Filtered, Keep at -20 &#176;C</td>
		</tr>
		<tr>
			<td>1x Poly-D-Lysine</td>
			<td></td>
			<td></td>
			<td>Volume</td>
		</tr>
		<tr>
			<td>20x Poly-D-Lysine</td>
			<td></td>
			<td></td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td>5x Borade Buffer</td>
			<td></td>
			<td></td>
			<td>20 mL</td>
		</tr>
		<tr>
			<td>ddH2O</td>
			<td></td>
			<td></td>
			<td>75 mL</td>
		</tr>
		<tr>
			<td>Total</td>
			<td></td>
			<td></td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Keep at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Wash Medium</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Volume</td>
		</tr>
		<tr>
			<td>DMEM-High glucose</td>
			<td>Gibco</td>
			<td>12800-017</td>
			<td>495 mL</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimyotic</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td>Total</td>
			<td></td>
			<td></td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Keep at 4&#176; C</td>
		</tr>
		<tr>
			<td>Papain-Trypsin digestion buffer:</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Volume/Final Concentration</td>
		</tr>
		<tr>
			<td>Papain (10 mg/mL)</td>
			<td>Sigma-Aldrich</td>
			<td>P4762</td>
			<td>200 &#181;L (2 mg/mL)</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%)</td>
			<td>Gibco</td>
			<td>25200-072</td>
			<td>200 &#181;L (0.05%)</td>
		</tr>
		<tr>
			<td>Wash Medium</td>
			<td></td>
			<td></td>
			<td>600 &#181;L</td>
		</tr>
		<tr>
			<td>Total</td>
			<td></td>
			<td></td>
			<td>1,000 &#181;L</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Keep at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Plating medium:</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Volume</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td>176 mL</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10437-028</td>
			<td>20 mL</td>
		</tr>
		<tr>
			<td>L-glutamate (200 mM)</td>
			<td>Gibco</td>
			<td>25030</td>
			<td>2 mL</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimyotic</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td>2 mL</td>
		</tr>
		<tr>
			<td>Total</td>
			<td></td>
			<td></td>
			<td>200 mL</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Keep at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Complete culture medium</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Volume</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td>95 mL</td>
		</tr>
		<tr>
			<td>N2 supplement (100x)</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>B27 supplement (50x)</td>
			<td>Gibco</td>
			<td>17504-04</td>
			<td>2 mL</td>
		</tr>
		<tr>
			<td>L-glutamate (200 mM)</td>
			<td>Gibco</td>
			<td>25030</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimyotic</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>Total</td>
			<td></td>
			<td></td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Freshly prepared</td>
		</tr>
	</tbody>
</table>
Table 1: Digestion buffer and media composition used in this study.
Supplementary Video 1: Insulin stimulated GFP-GLUT4 movement in WT hypothalamic neurons. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56409/JoVE-Video-1.mov">Please click here to download this file.</a>
Supplementary Video 2: Insulin stimulated GFP-GLUT4 movement in CCR5-/- hypothalamic neurons. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56409/JoVE-Video-2.mov">Please click here to download this file.</a>
Supplementary Video 3: CCL5 stimulated GFP-GLUT4 movement in WT hypothalamic neurons (Video adapted from reference17). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56409/JoVE-Video-3.mov">Please click here to download this file.</a>

Watch this Scientific Journal Video about Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy at JoVE.com

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

This protocol describes a technique for observation of real-time Green Fluorescence Protein (GFP) tagged Glucose Transporter 4 (GLUT4) protein trafficking upon insulin stimulation and characterization of the biological role of CCR5 in the insulin&#8211;GLUT4 signaling pathway with Deconvolution Microscopy.

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

live-images-glut4-protein-trafficking-mouse-primary-hypothalamic

Research

JoVE Journal

Neuroscience

9.6K Views.  Taipei Medical University. Dit protocol wordt een techniek beschreven voor observatie van de real-time groen fluorescentie proteïne (GFP) gelabeld Glucose Transporter 4 (GLUT4) eiwit mensenhandel op insuline stimulatie en karakterisering van de biologische rol van CCR5 in de insuline-GLUT4 signalering traject met deconvolutie microscopie.

Video: Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic 
analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3].  Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons.  One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, 
and maintaining general cell health.  Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons.  A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde.  Our approach to imaging 
GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently 
labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events.  The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.

Live cell imaging is of particular utility when studying the dynamics of organelle trafficking. Here we describe a protocol for live imaging of dense-core vesicles in cultured neurons using wide-field fluorescence microscopy. This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes.

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  ...

Biology

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with increasing connective tissue and decreased neuronal viability that occur with age. Here, we describe a methodology for the dissociation of healthy hypothalamic neurons from adult-aged mice. The ability to study neurons from adult-aged mice allows the use of disease models that manifest at a later age and might be more developmentally accurate for certain studies. Fluorescence imaging of dissociated neurons can be used to study the activity of a population of neurons, as opposed to using electrophysiology to study a single neuron. This is particularly useful when studying a heterogeneous neuronal population in which the desired neuronal type is rare such as for hypothalamic glucose sensing neurons. We utilized membrane potential dye imaging of adult ventromedial hypothalamic neurons to study their responses to changes in extracellular glucose. Glucose sensing neurons are believed to play a role in central regulation of energy balance. The ability to study glucose sensing in adult rodents is particularly useful since the predominance of diseases related to dysfunctional energy balance (e.g.&#160;obesity) increase with age.

The activity of single neurons from adult-aged mice can be studied by dissociating neurons from specific brain regions and using fluorescent membrane potential dye imaging. By testing responses to changes in glucose, this technique can be used to study the glucose sensitivity of adult ventromedial hypothalamic neurons.

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3.
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx due to the abnormal activation of glutamate receptors results in DA excitotoxicity and has been identified as an important mechanism for DA neuron loss. In this study, we isolate, dissociate, and culture midbrain neurons from the mouse ventral mesencephalon (VM) of ED14 mouse embryos. We then infect the long-term primary mouse midbrain cultures with an adeno-associated virus (AAV) expressing a genetically encoded calcium indicator, GCaMP6f under control of the human neuron-specific synapsin promoter, hSyn. Using live confocal imaging, we show that cultured mouse midbrain neurons display spontaneous Ca2+ fluxes detected by AAV-hSyn-GCaMP6f. Bath application of glutamate to midbrain cultures causes abnormal elevations in intracellular Ca2+ within neurons and this is accompanied by caspase-3 activation in DA neurons, as demonstrated by immunostaining. The techniques to identify glutamate-mediated apoptosis in primary mouse DA neurons have important applications for the high content screening of drugs that preserve DA neuron health.

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Here we present a protocol to measure in vitro Ca2+ fluxes in midbrain neurons and their downstream effects on caspase-3 using primary mouse midbrain cultures. This model can be employed to study pathophysiologic changes related to abnormal Ca2+ activity in midbrain neurons, and to screen novel therapeutics for anti-apoptotic properties.

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx ...

Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy

Amyotrophic lateral sclerosis is a neurodegenerative disorder causing progressive muscle weakness and death within 2-5 years following diagnosis. Clinical manifestations include weight loss, dyslipidemia, and hypermetabolism; however, it remains unclear how these relate to motor neuron degeneration. Using a Drosophila model of TDP-43 proteinopathy that recapitulates several features of ALS including cytoplasmic inclusions, locomotor dysfunction, and reduced lifespan, we recently identified broad ranging metabolic deficits. Among these, glycolysis was found to be upregulated and genetic interaction experiments provided evidence for a compensatory neuroprotective mechanism. Indeed, despite upregulation of phosphofructokinase, the rate limiting enzyme in glycolysis, an increase in glycolysis using dietary and genetic manipulations was shown to mitigate locomotor dysfunction and increased lifespan in fly models of TDP-43 proteinopathy. To further investigate the effect on TDP-43 proteinopathy on glycolytic flux in motor neurons, a previously reported genetically encoded, FRET-based sensor, FLII12Pglu-700&#181;&#948;6, was used. This sensor is comprised of a bacterial glucose-sensing domain and cyan and yellow fluorescent proteins as the FRET pair. Upon glucose binding, the sensor undergoes a conformational change allowing FRET to occur. Using FLII12Pglu-700&#181;&#948;6, glucose uptake was found to be significantly increased in motor neurons expressing TDP-43G298S, an ALS causing variant. Here, we show how to measure glucose uptake, ex vivo, in larval ventral nerve cord preparations expressing the glucose sensor FLII12Pglu-700&#181;&#948;6 in the context of TDP-43 proteinopathy. This approach can be used to measure glucose uptake and assess glycolytic flux in different cell types or in the context of various mutations causing ALS and related neurodegenerative disorders.

Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy

Glucose uptake is increased in Drosophila motor neurons affected by TAR DNA binding protein (TDP-43) proteinopathy, as indicated by a FRET-based, genetically encoded glucose sensor.

Amyotrophic lateral sclerosis is a neurodegenerative disorder causing progressive muscle weakness and death within 2-5 years following diagnosis. Clinical ...

Live Imaging of the Mitochondrial Glutathione Redox State in Primary Neurons using a Ratiometric Indicator

Mitochondrial redox homeostasis is important for neuronal viability and function. Although mitochondria contain several redox systems, the highly abundant thiol-disulfide redox buffer glutathione is considered a central player in antioxidant defenses. Therefore, measuring the mitochondrial glutathione redox potential provides useful information about mitochondrial redox status and oxidative stress. Glutaredoxin1-roGFP2 (Grx1-roGFP2) is a genetically encoded, green fluorescent protein (GFP)-based ratiometric indicator of the glutathione redox potential that has two redox-state-sensitive excitation peaks at 400 nm and 490 nm with a single emission peak at 510 nm. This article describes how to perform confocal live microscopy of mitochondria-targeted Grx1-roGFP2 in primary hippocampal and cortical neurons. It describes how to assess steady-state mitochondrial glutathione redox potential (e.g., to compare disease states or long-term treatments) and how to measure redox changes upon acute treatments (using the excitotoxic drug N-methyl-D-aspartate (NMDA) as an example). In addition, the article presents co-imaging of Grx1-roGFP2 and the mitochondrial membrane potential indicator, tetramethylrhodamine, ethyl ester (TMRE), to demonstrate how Grx1-roGPF2 can be multiplexed with additional indicators for multiparametric analyses. This protocol provides a detailed description of how to (i) optimize confocal laser scanning microscope settings, (ii) apply drugs for stimulation followed by sensor calibration with diamide and dithiothreitol, and (iii) analyze data with ImageJ/FIJI.

This article describes a protocol to determine differences in basal redox state and redox responses to acute perturbations in primary hippocampal and cortical neurons using confocal live microscopy. The protocol can be applied to other cell types and microscopes with minimal modifications.

Mitochondrial redox homeostasis is important for neuronal viability and function. Although mitochondria contain several redox systems, the highly abundant ...

Visualizing Proteins in Primary Hippocampal Neurons by Live&#45;Cell Imaging

Using Live&#45;Cell Imaging to Measure the Effects of Pathological Proteins on Axonal Transport in Primary Hippocampal Neurons

Bidirectional transport of cargos along the axon is critical for maintaining functional synapses, neural connectivity, and healthy neurons. Axonal transport is disrupted in multiple neurodegenerative diseases, and projection neurons are particularly vulnerable because of the need to transport cellular materials over long distances and sustain substantial axonal mass. Pathological modifications of several disease-related proteins negatively affect transport, including tau, amyloid-&#946;, &#945;-synuclein, superoxide dismutase, and huntingtin, providing a potential common mechanism by which pathological proteins exert toxicity in disease. Methods to study these toxic mechanisms are necessary to understand neurodegenerative disorders and identify potential therapeutic interventions.
Here, cultured primary rodent hippocampal neurons are co-transfected with multiple plasmids to study the effects of pathological proteins on fast axonal transport using live-cell confocal imaging of fluorescently-tagged cargo proteins. We begin with the harvest, dissociation, and culturing of primary hippocampal neurons from rodents. Then, we co-transfect the neurons with plasmid DNA constructs to express fluorescent-tagged cargo protein and wild-type or mutant tau (used as an exemplar of pathological proteins). Axons are identified in live cells using an antibody that binds an extracellular&#160;domain of neurofascin, an axon initial segment protein, and an axonal region of interest is imaged to measure fluorescent cargo transport.
Using KymoAnalyzer, a freely available ImageJ macro, we extensively characterize the velocity, pause frequency, and directional cargo density of axonal transport, all of which may be affected by the presence of pathological proteins. Through this method, we identify a phenotype of increased cargo pause frequency associated with the expression of pathological tau protein. Additionally, gene-silencing shRNA constructs can be added to the transfection mix to test the role of other proteins in mediating transport disruption. This protocol is easily adaptable for use with other neurodegenerative disease-related proteins and is a reproducible method to study the mechanisms of how those proteins disrupt axonal transport.

Author Spotlight: Understanding the Impact of Pathological Proteins on Axonal Transport in Neurodegenerative Diseases

Here, we demonstrate how to combine transfection of primary hippocampal rodent neurons with live-cell confocal imaging to analyze pathological protein-induced effects on axonal transport and identify mechanistic pathways mediating these effects.

Bidirectional transport of cargos along the axon is critical for maintaining functional synapses, neural connectivity, and healthy neurons. Axonal ...

Confocal Imaging of Neuropeptide Y-pHluorin: A Technique to Visualize Insulin Granule Exocytosis in Intact Murine and Human Islets

Insulin secretion plays a central role in glucose homeostasis under normal physiological conditions as well as in disease. Current approaches to study insulin granule exocytosis either use electrophysiology or microscopy coupled to the expression of fluorescent reporters. However most of these techniques have been optimized for clonal cell lines or require dissociating pancreatic islets. In contrast, the method presented here allows for real time visualization of insulin granule exocytosis in intact pancreatic islets. In this protocol, we first describe the viral infection of isolated pancreatic islets with adenovirus that encodes a pH-sensitive green fluorescent protein (GFP), pHluorin, coupled to neuropeptide Y (NPY). Second, we describe the confocal imaging of islets five days after viral infection and how to monitor the insulin granule secretion. Briefly, the infected islets are placed on a coverslip on an imaging chamber and imaged under an upright laser-scanning confocal microscope while being continuously perfused with extracellular solution containing various stimuli. Confocal images spanning 50 &#181;m of the islet are acquired as time-lapse recordings using a fast-resonant scanner. The fusion of insulin granules with the plasma membrane can be followed over time. This procedure also allows for testing a battery of stimuli in a single experiment, is compatible with both mouse and human islets, and can be combined with various dyes for functional imaging (e.g., membrane potential or cytosolic calcium dyes).

We describe a protocol for visualization of insulin exocytosis in intact islets using pHluorin, a pH-sensitive green fluorescent protein. Isolated islets are infected with adenovirus encoding pHluorin coupled to the vesicle cargo neuropeptide Y. This allows for the detection of insulin granule fusion events by confocal microscopy.

Insulin secretion plays a central role in glucose homeostasis under normal physiological conditions as well as in disease. Current approaches to study ...

Imaging GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

<a href='https://app-jove-com.remotexs.ntu.edu.sg/v/56409/live-images-glut4-protein-trafficking-mouse-primary-hypothalamic'>Source: Changou, C. A., et. al. Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy. J. Vis. Exp. (2017).</a>This video showcases the imaging of insulin-induced GLUT4 translocation in hypothalamic neurons, employing deconvolution microscopy to capture and analyze GFP-GLUT4 trafficking dynamics with high resolution.

Source: Changou, C. A., et. al. Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy. J. Vis. ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

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