Graphical figures were created with BioRender.com. This work was supported by an NIA grant (R01AG060919) and an NSF grant (2030348) to FS.

NOTE: A general view of the experimental procedure is graphically illustrated in Figure 1A. All experiments with mice performed in this study were approved by our institution&#39;s Animal Care and Use Committee (IACUC). We used 3-month-old C57BL6/J mice, which were housed in an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC)-approved vivarium, under the care of a veterinarian. The mice lived in large cages, with a 12 h light-dark cycle, and were fed ad libitum.
1. Conception and pregnancy verification
<ol>
	<li>Place mice of any background and genotype of interest for breeding. Record the date and the weight of the female before conception.</li>
	<li>After 6 h, inspect the female for a plaque with a probe. If the plaque is present, separate the female from the male. If the plaque is not present, keep the female in the cage until the next day, then separate the mice.</li>
	<li>At days 7, 10, and 14 post conception, weigh the female to confirm pregnancy.</li>
</ol>
2. Media, 24-well plate, and material preparation
<ol>
	<li>On the day of cell isolation, place, ready-to-use, poly-D-lysine-coated glass coverslips (see Table of Materials) in a 24-well plate as follows:
	<ol>
		<li>Under a biological hood, sterilize a single package containing 15 coverslips with 70% ethanol and let dry. Open the package and place the coverslips in a 60 mm plate. Shake the plate horizontally to separate the coverslips. Then, invert the plate to pick up single coverslips to be placed in the wells of a 24-well plate.</li>
	</ol>
	</li>
	<li>Wash the coverslips one time with 1.0 mL of sterile Hank&#39;s Balanced Salt Solution (HBSS) for 5 min.</li>
	<li>In the meantime, prepare 20.0 mL of plating media as follows: to 18.31 mL of BME (Basal Medium Eagle, + Earle&#39;s Salts), supplemented with 1.0 mL of heat-inactivated fetal bovine serum (FBS), add 200 &#181;L of sodium pyruvate (from a 100x stock), 200 &#181;L of glutamine (from a 200 nM stock), and 100 &#181;L of penicillin/streptomycin (from a 200x stock).</li>
	<li>Replace the HBSS in the wells with 1.0 mL of plating media and place the plate in the incubator at 37 &#176;C.</li>
	<li>Using a Bunsen burner, fire-polish three Pasteur pipettes in decreasing diameters. Holding the pipet with one hand, insert the tip into the flame and quickly remove it. Repeat the process until the tip smoothens and the diameter is reduced to the desired diameter (assessed by eye).</li>
</ol>
3. Reagent preparation for Neural Tissue Dissociation, following the directions of the Neural Dissociation kit
<ol>
	<li>After warming the Digestion Buffer 1 at room temperature, prepare Enzyme Mix 1 by mixing 50 &#181;L of Enzyme 1 with 1.91 mL of Buffer 1 and Enzyme Mix 2 by mixing 15 &#181;L of Enzyme 2 with 30 &#181;L of Digestion Buffer 2. The mixes are enough to be used for all embryos&#39; brain tissue.</li>
	<li>Prepare 0.5% Bovine Serum Albumin (BSA) in HBSS, for example, 0.25 g in 50.0 mL of HBSS.</li>
</ol>
4. Embryo extraction
<ol>
	<li>Autoclave two straight fine forceps, one curved point forceps, and one pair of fine surgical scissors and sterilize them with 70% ethanol before use. Then, fill the Petri dishes with HBSS.</li>
	<li>Euthanize one E14-E16 pregnant dam in the CO2 chamber and perform cervical dislocation. 
	NOTE: The following steps must be performed under the hood under sterile conditions:</li>
	<li>Sterilize the abdomen with 70% ethanol. Cut open the abdominal cavity from the pubic symphysis to the xiphoid process of the rib cage with surgical scissors and forceps.</li>
	<li>Extract the uterine horn and place it in a 100 mm plate filled with ice-cold HBSS and wash thoroughly.</li>
	<li>Extract and separate all the embryos from the uterus with fine forceps. Quickly decapitate the embryos with fine surgical scissors and/or forceps. Place the heads in the 60 mm Petri dish filled with HBSS.</li>
</ol>
5. Hypothalamus extraction, collection, and tissue dissociation
<ol>
	<li>Place one fine forceps in the eye cavity to hold the brain. Using the other fine forceps, remove the skin and the skull by peeling until the brain is visible. Distinguish the brain from other tissues based on its white appearance. Skin and skull are pink and rich in vasculature.</li>
	<li>Remove the brain from the skull by using the curved point forceps and scoop out the brain from the olfactory bulbs, flipping it upside down.</li>
	<li>Now the cortex is ventral, and the hypothalamus is visible dorsally on the superior surface (Figure 1B). With the curved forceps, remove the layer of meninges and blood vessels until the brain appears white and clear.</li>
	<li>With the curved forceps, separate the hypothalamic area from the rest of the brain.</li>
	<li>Cut the hypothalamus into 3-4 small pieces and with a pipet, transfer the pieces into a 15 mL tube.</li>
	<li>Repeat the steps for the other embryos while the tube is on ice.</li>
	<li>Fill the tube with 6.0 mL of HBSS and let the tissue settle, remove the supernatant, and add Enzyme Mix 1. Gently mix and agitate the tube to prevent the tissue.</li>
	<li>Incubate the tube in a 37 &#176;C water bath for 15 min and agitate the tissue every 5 min to resuspend the tissue.</li>
	<li>After 15 min, add 30 &#956;L of Enzyme mix 2. Dissociate the brain tissue by using the pipette Pasteur with the biggest diameter (&#60;1 mm). Pipet up and down 10x without forming bubbles.</li>
	<li>Incubate for 10 min at 37 &#176;C in the water bath. Gently agitate the tube to resuspend the tissue every 5 min.</li>
	<li>After 10 min, add the remaining 15 &#181;L of Enzyme mix 2. Dissociate the tissue 10x with the other two fire-polished pipettes of decreasing diameter, up and down without forming bubbles.</li>
	<li>To the tube with the dissociated tissue, add immediately 10.0 mL of HBSS-0.5% BSA and centrifuge at 300 &#215; g for 10 min at room temperature.</li>
	<li>Aspirate the supernatant and resuspend the cell pellet in 1.0 mL of HBS-0.5% BSA.</li>
</ol>
6. Cell counting
<ol>
	<li>Dilute the cell suspension 1:5 using HBSS-0.5% BSA.</li>
	<li>Place 10 &#181;L of the diluted cell suspension in a Neubauer counting chamber.</li>
	<li>Under a brightfield microscope, count only the cells that are in the four chamber&#39;s&#160;corner squares. Calculate the average and multiply by 5 &#215; 104. 
	&#8203;NOTE: Make sure there are &#62;106 cells to proceed to cell isolation; the optimal cell number is 107.</li>
</ol>
7. Negative selection
NOTE: Negative selection enables users to obtain a pure primary neuron culture by separating neuronal and non-neuronal cells. Use precooled solutions.
<ol>
	<li>Centrifuge the cell suspension at 300 &#215; g for 3 min (centrifugation can be extended up to 10 min). Gently aspirate the supernatant and resuspend the pellet to a concentration of 107 cells in 80 &#181;L of HBSS-0.5% BSA.</li>
	<li>Add 20 &#181;L of Non-Neuronal Cell Biotin-Antibody Cocktail and incubate for 5 min at 4 &#176;C.</li>
	<li>Wash the cells to remove free antibody with 2.0 mL of HBSS-0.5% BSA and centrifuge at 300 &#215; g for 3 min.</li>
	<li>Gently&#160;aspirate the supernatant and resuspend the pellet in 80 &#181;L of HBSS-0.5% BSA. Add 20 &#181;L of anti-biotin Microbeads, mix thoroughly, and incubate for 10 min at 4 &#176;C.</li>
	<li>Add 0.5 mL of HBSS-0.5% BSA for up to 107 cells and wait until the magnetic column is ready.</li>
</ol>
8. Magnetic separation, negative selection
NOTE: Magnetic separation is a crucial step that allows the separation of the non-neuronal cells from the neuronal cells. The sample containing neuronal and non-neuronal cells is passed through the magnetic field and the non-neuronal cells, which are bound to a biotin-antibody-magnetic bead complex, are trapped in the column (Figure 1C). The free neuronal cells are eluted through the column and are collected in a 15 mL tube.
<ol>
	<li>Prepare the stand (included in the kit) with the separator and the MS column as shown in Figure 1C.</li>
	<li>Open the column and set up the stand only when the cells are ready to be separated.</li>
	<li>Rinse the column with 0.5 mL of HBSS-0.5% BSA. Wait until the solution stops dripping.</li>
	<li>To collect neuronal cells, place a 15 mL tube under the column and pass 0.5 mL of the cell suspension through the column. Collect the eluate in the tube until it stops dripping. To capture residual neuronal cells, wash the column 3 x 0.5 mL of HBSS-0.5% BSA.</li>
	<li>To collect non-neuronal cells, remove the column from the magnet and place it inside a new 15 ml tube. Add 1.0 mL of HBSS--0.5% BSA to the column and use the plunger to collect the magnetically labeled non-neuronal cells.</li>
	<li>Centrifuge the neuronal and non-neuronal cells at 300 &#215; g for 3 min. Gently aspirate the supernatant and resuspend the cells in 1.0 mL of HBSS-0.5% BSA. Count the cells as described previously in section 6.</li>
	<li>If needed, plate the non-neuronal cells in a 24-well plate; otherwise discard them.</li>
</ol>
9. Positive selection
NOTE: Once a pure neuronal cell suspension is obtained, positive selection is carried out to isolate the targeted cells. Cells can be isolated by using a specific biotin-conjugated antibody for a surface antigen. The antibody is recognized by anti-biotin magnetic beads. By flowing the cell&#160;suspension through the column, only the cells of interest are trapped in the magnetic field.
<ol>
	<li>Centrifuge the pure neuronal cell suspension at 300 &#215; g for 3 min. Gently aspirate the supernatant and resuspend the pellet in 80 &#181;L of HBSS-0.5% BSA. Add the specific antibody following the manufacturer&#39;s instructions and incubate at 4 &#176;C for 10 min. 
	NOTE: If cells expressing the LepR are sought, we suggest a Mouse Leptin R Biotinylated Antibody (see Table of Materials) at a concentration of 0.50 &#181;g/106 cells.</li>
	<li>Wash off the excess antibody with 2.0 mL of HBSS-0.5% BSA and centrifuge at 300 &#215; g for 3 min.</li>
	<li>Remove the supernatant, resuspend the pellet in 80 &#181;L of HBSS-0.5% BSA, and add 20 &#181;L of anti-biotin microbeads. Incubate at 4 &#176;C for 10 min.</li>
	<li>For every 107 cells, add 0.5 mL of HBSS-0.5% BSA and wait until the magnetic column is ready.</li>
</ol>
10. Magnetic separation, positive selection
<ol>
	<li>Prepare the stand with the separator and the MS column. Rinse the MS column with 0.5 mL of HBSS-0.5% BSA. Wait until the dripping stops.</li>
	<li>Place a 15 mL tube under the column, pass 0.5 mL of the cell suspension through the column, and collect the eluate comprising the non-specific neuronal cells. To clean the column of residual non-specific neuronal cells, wash with 3 x 0.5 mL of HBSS-0.5% BSA.</li>
	<li>Remove the column from the magnet, place it in a new 15 mL tube, and add 1.0 mL of HBSS-0.5% BSA. Use the plunger to flush out the targeted cells.</li>
	<li>Centrifuge both tubes at 300 &#215; g for 3 min. Gently remove the supernatant and resuspend it in 0.5 mL of plating medium.</li>
	<li>Count the cells as previously described in section 6.</li>
	<li>Plate both targeted cells as the positive control and non-specific cells as the negative control at 120,000-200,000 cells/mm3 density in the 24-well plate previously prepared as described in section 2 and incubate at 37 &#176;C in 5% CO2, 9% O2, and 95% humidity for 12 h.</li>
</ol>
11. Cell culture maintenance
<ol>
	<li>Prepare 20 mL of culture medium with 19.2 mL of Neuronal Culture Medium, 400 &#181;L of B27 supplement (from a 50x stock), 200 &#181;L of glutamine (from a 200 &#181;M stock), and 100 &#181;L of penicillin/streptomycin (from a 200x stock).</li>
	<li>Replace plating media from the 24 well-plate containing neuronal or non-neuronal cells.</li>
	<li>Wash with 2 x 1.0 mL of HBSS.</li>
	<li>Add 1.0 mL of culture media.</li>
	<li>Refresh the media every 2/3 days by replacing 0.5 mL of old media with 0.5 mL of fresh media. 
	&#8203;NOTE: Cells can be maintained in culture and used for up to 21 days in vitro (DIV21).</li>
</ol>
12. Neuron immunofluorescence staining
<ol>
	<li>Twelve hours prior to the staining, prepare a solution consisting of 50/50 methanol and acetone and cool it at -20 &#176;C overnight.</li>
	<li>Wash the neurons in the 24-well plate with 2 x 1.0 mL of 1x phosphate-buffered saline (PBS) for 5 min.</li>
	<li>Replace the PBS solution with 1.0 mL of the 50/50 solution and incubate in ice for 20 min.</li>
	<li>Wash with 1x PBS for 3 x 5 min.</li>
	<li>Block the neurons with 3% BSA in 1x PBS for 1 h at room temperature.</li>
	<li>Prepare the primary antibody solution in 3% BSA in 1x PBS, using the antibody concentration mentioned in the manufacturer&#39;s directions. The antibodies and concentrations used are listed in the Table of Materials.</li>
	<li>Replace the blocking solution with the primary antibody solution and incubate at 4 &#176;C overnight.</li>
	<li>Wash the cells for 3 x 10 min with 1x PBS.</li>
	<li>Prepare the secondary antibody solution with 3% BSA in 1x PBS, using the concentrations of the antibodies following the manufacturer&#39;s&#160;instructions. The secondary antibodies used are listed in the Table of Materials.</li>
	<li>Incubate the cells with the secondary antibody solution at room temperature for 1 h.</li>
	<li>Wash the cells for 3 x 10 min with 1x PBS.</li>
	<li>Leave the neurons in 1x PBS during the mounting procedure. Place a small drop of mounting medium (with or without 4&#39;,6-diamidino-2-phenylindole for nuclear identification) on the microscope slide. Extract one glass coverslip containing neurons with forceps and tap the side of the coverslip on a paper tissue to dry the excess PBS. Flip the coverslip on the mounting medium, making sure the neurons are facing the microscope slides; gently press and remove the excess mounting media with tissue paper.</li>
	<li>The microscope slides are ready to be analyzed with a brightfield or a confocal microscope.</li>
</ol>

Hormonal, Metabolic, and Electrical Regulation of Isolated Hypothalamic Neurons

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The authors declare that they have no conflicts of interest.

Investigating the biochemical and electrical properties of hypothalamic neurons is key to understanding the molecular basis of metabolism, thermoregulation, mood management, feeding behavior, and more. However, the neuronal heterogeneity of the hypothalamus makes this effort challenging, and methods to isolate and study specific hypothalamic sub-populations are needed.
In vivo techniques employ CRE-recombinase, optogenetic, fiber photometry, and calcium imaging. These approaches allow primarily for the study of the electrical properties of hypothalamic neurons, and very few methods are currently available to investigate their non-electrical attributes. The MACS technology developed in this study could provide a technique amenable to isolating specific hypothalamic neuronal sub-populations in vitro, thereby affording targeted treatments and analyses. Neuronal cultures are simpler to manage compared to co-cultures of different neuronal populations. Additionally, pure cultures avoid confounding effects stemming from the presence of glia and microglia. Thus, neurons from the same hypothalamic region and type could be studied in response to specific metabolic and hormonal inputs.
In this protocol, we selected hypothalamic neurons expressing the LepR. Isolated LepR+ cells were cultured to investigate their cellular, morphological, and molecular characteristics which are difficult to study in vivo. The purity of the cultures was 99%, supporting the accuracy of the method. In addition, the LepR+ cells were healthy and viable at DIV7 until DIV21.
This technique, however, has some limitations. E18 or older pure neuron cultures are challenging to maintain. Therefore, the window of extraction is limited to E14-E16. This implies that cellular changes occurring after E16 are missed. For example, the expression of the leptin receptor in ARC neurons increases during the early postnatal period22. The procedure for the isolation must be performed as quickly as possible to reduce cellular stress and death and improve yield. The procedure can take up to 5 h; hence, it is essential to maintain sterile conditions and reduce manipulation to the minimum necessary. The positive selection can lead to low yield due to the low amounts of available tissue, limiting the number of experiments that can be performed with a single preparation. Elevated neuronal death was observed, probably due to low cell density and reduced neuronal connectivity and intraneuronal support.
Moreover, the antibody targeting the antigen of interest must bind to the cell surface to guarantee a correct separation; usually, antibodies used for flow cytometry are suitable for the MACS technique. If the antibody has not been used before in cell separation methods, validation and titration experiments are needed to determine the ideal usage and concentration. The extraction of targeted cells requires a cell surface marker. Here we used a biotinylated antibody but in principle, antibodies conjugated with other molecules, such as FITC (fluorescein isothiocyanate) and PE (purified anti-phycoerythrin), could also be used. MACS technology could also be applied to neurons expressing a fluorophore, such as GFP or another Tag protein, potentially increasing specificity and yield. If a fluorophore is not employed, the alternative would be to confirm the expression of the molecule of interest by immunofluorescence before performing live cell experiments. Future studies will test the validity of these alternatives.
One important aspect that this study did not address concerns the &#34;fidelity&#34; of the sub-neuronal populations. We ascertained that the cultured LepR+ neurons expressed POMC, which is a signature of native ARCPOMC neurons. However, more tests will be necessary to conclude that the LepR+ neuronal cultures recapitulate their native in vivo counterparts. Overall, the MACS neuronal isolation protocol presented here may provide a valid and effective method to study in vitro hypothalamic mechanisms that would be otherwise difficult to investigate in vivo.

The hypothalamus is a multipronged area of the brain that mediates endocrine, autonomic, visceral, and behavioral functions, including feeding, metabolism, sleep, body temperature, social behavior, and sex drive 1,2,3,4,5. Functional heterogeneity is achieved by a synergistic combination of biochemical and electrical mechanisms: hypothalamic neurons fire action potentials and secrete and release hormones and neuropeptides to modulate brain regions and organs of the body. Finally, hypothalamic neurons translate homeostatic messages from the body, responding with long-term and short-term feedback and feedforward regulations6.
The complex neuronal environment of the hypothalamus includes magnocellular endocrine neurons, releasing oxytocin and vasopressin; parvocellular neurons, primarily involved in the systemic hormonal regulation, releasing for instance, thyrotropin-release hormone (TRH), and corticotropin-release hormone (CRH) to the pituitary gland; large peptidergic projection neurons, releasing orexin and melanin-concentrating hormone (MCH); and parvocellular peptidergic neurons of the Arcuate Nucleus (ARC) releasing POMC (proopiomelanocortin) and AgRP (agouti-related protein), named ARCPOMC and ARCAgRP, respectively. Together with secretory cells, other excitatory and inhibitory neurons, including dopaminergic, glutaminergic, and GABAergic neurons 7, are involved in forming intrahypothalamic and extrahypothalamic circuits, thereby creating large-scale coordinated networks of considerable cellular heterogeneity8.
Hypothalamic diversity has been a challenge that researchers have been trying to overcome over the past 50 years. To study this heterogeneity in developing, mature, and aging hypothalami, investigators, on the one hand, have been employing single-cell RNA sequencing to explore neuronal organization, as well as molecular and transcriptomic signatures. This effort has provided an insightful look into the variegated roles of hypothalamic neurons and has addressed connections between cellular identity and its possible role in the physiological system8,9,10. On the other hand, neuronal functions have been investigated by optogenetic manipulations and fiber photometry behavioral approaches, affording a close look at the circuitry structure. In the past two decades, the Cre-recombinase technology has allowed researchers to ontogenetically stimulate or inhibit a targeted group of neurons while observing changes in behaviors and body responses6,11,12.
However, these approaches examine hypothalamic functions from a general perspective without diving deeper into the specific cellular mechanisms or the biological basis for their role within the complex hypothalamic environment. To address this, very few studies have focused on investigating molecular, biochemical, and electrical properties utilizing heterogeneous primary hypothalamic cultures. These studies sought to dissect specific neuronal processes in a complex environment and generated integrative models of physiological mechanisms13,14,15. Nonetheless, non-specific cultures pose significant challenges. For instance, the neurons&#39; physiological connectivity and anatomical distribution are disrupted by plating neurons from different hypothalamic regions that normally would not interact, creating confounding effects. Additionally, each region has different roles and variegated neuronal populations, making it difficult to study simple biological processes.
To address these challenges, in the past decade, new approaches have been implemented to isolate neurons of interest, such as immunopanning, Fluorescent-Activated-Cell-Sorting (FACS), and Magnetic-Activated-Cell-Sorting (MACS). Immunopanning is a strategy employed to purify targeted cells using antibody-coated dishes for a series of non-neuronal (negative) and neuronal (positive) selections. While this technique could, in principle, generate high-yield purified cell cultures, in practice, is mostly used for astrocytes and oligodendrocytes since these cells can resist hours of manipulation16,17. FACS technology is a powerful tool to sort cells based on fluorescent markers and cellular characteristics using flow cytometry18,19,20. However, very few studies used this method to isolate cells for cell culture. The technique is expensive and requires highly skilled personnel to use and maintain; additionally, it is challenging to maintain viable and sterile cells at the end of the sorting procedure21. Overall, MACS appears to be a simple, non-expensive technique to obtain highly pure and viable cultures of hypothalamic primary neurons. The method utilizes magnetic beads linked to the cells via an antibody. This allows the cells to be isolated using the magnetic field of the column.
Here we describe a method based on MACS technology, which is typically used with cortical neurons. This protocol allows to isolate, in principle, viable and highly pure hypothalamic neurons. In this study, we prepare primary cultures of neurons expressing the Leptin Receptor (LepR), such as ARCPOMC and ARCAgRP neurons, that are present only in the Arcuate Nucleus. These neurons respond to leptin, an anorexigenic hormone secreted by the adipose tissue, in biochemical and electrical ways. Therefore, the isolation of this group of neurons in culture allows for the study of their hormonal, metabolic, and electrical properties in vitro.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Embryo extraction</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 curved point forceps</td>
			<td>Fine Science Tools</td>
			<td>11270-20</td>
			<td>Dumont</td>
		</tr>
		<tr>
			<td>1 fine surgical scissor</td>
			<td>Fine Science Tools</td>
			<td>14058-11</td>
			<td>Dumont</td>
		</tr>
		<tr>
			<td>100 mm Petri dish</td>
			<td>Corning</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>2 straight fine forceps</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td>Dumont</td>
		</tr>
		<tr>
			<td>60 mm Petri dish</td>
			<td>Corning</td>
			<td>430196</td>
			<td></td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>Decon Laboratories, INC.</td>
			<td>2801</td>
			<td>Ethanol 190 Proof</td>
		</tr>
		<tr>
			<td>Anti-Biotin MicroBeads 1mL</td>
			<td>Miltenyi Biotec</td>
			<td>130-115-390</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-MAP2 antibody</td>
			<td>Abcam</td>
			<td>ab5392</td>
			<td>1 : 800</td>
		</tr>
		<tr>
			<td>Bench pads</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A9418-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer Y</td>
			<td>Miltenyi Biotec</td>
			<td>130-094-802</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer Z</td>
			<td>Miltenyi Biotec</td>
			<td>130-094-802</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Biotin MicroBeads 1mL</td>
			<td>Miltenyi Biotec</td>
			<td>130-115-390</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A9418-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer Y</td>
			<td>Miltenyi Biotec</td>
			<td>130-094-802</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer Z</td>
			<td>Miltenyi Biotec</td>
			<td>130-094-802</td>
			<td></td>
		</tr>
		<tr>
			<td>Enzyme A</td>
			<td>Miltenyi Biotec</td>
			<td>130-094-802</td>
			<td></td>
		</tr>
		<tr>
			<td>Enzyme P</td>
			<td>Miltenyi Biotec</td>
			<td>130-094-802</td>
			<td></td>
		</tr>
		<tr>
			<td>GG-12-1.5, 12 mm dia.#1.5 thick 100 pc cell culture tested German coverglasses</td>
			<td>Neuvitro Corporation</td>
			<td>GG-12-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco B-27 Supplement 10 mL</td>
			<td>ThermoFisher&#160;</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Basal Medium Eagle (BME) 500 mL&#160;</td>
			<td>ThermoFisher</td>
			<td>21010046</td>
			<td>(+) Earle&#39;s Salts, (-) L-Glutamine&#160;</td>
		</tr>
		<tr>
			<td>Gibco HBBS (1x) Hanks&#39; Balanced Salt Solution&#160; 500 mL</td>
			<td>ThermoFisher</td>
			<td>14025092</td>
			<td>Calcium, Magnesium, No phenol red</td>
		</tr>
		<tr>
			<td>Gibco HI FBS 100 mL</td>
			<td>ThermoFisher</td>
			<td>16140-063</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco L-Glutamine 200 mM (100x)</td>
			<td>ThermoFisher</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Penicilline/Streptomicine&#160;</td>
			<td>ThermoFisher</td>
			<td>15140-122</td>
			<td>10,000 U/mL</td>
		</tr>
		<tr>
			<td>Gibco Sodium Pyruvate (100 mM) 100 mL</td>
			<td>ThermoFisher</td>
			<td>11360070</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniMACS Separator and Starting Kit</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-102</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Leptin R Biotinylated Antibody</td>
			<td>R&#38;D Systems&#160;</td>
			<td>ABAF497</td>
			<td>0.25 &#956;g/106 cells</td>
		</tr>
		<tr>
			<td>MS Column</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td>Neaubeaur-Improved Brightline 100 &#181;m Chamber</td>
			<td>Hausser Scientific&#160;</td>
			<td>3120</td>
			<td></td>
		</tr>
		<tr>
			<td>Neural Tissue Dissociation Kit - Postnatal Neurons</td>
			<td>Miltenyi Biotec</td>
			<td>130-094-802</td>
			<td></td>
		</tr>
		<tr>
			<td>Neuronal Culture Medium 500 mL&#160;</td>
			<td>ThermoFisher</td>
			<td>88283</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-Neuronal Cell Biotin-Antibody Cocktail mouse 1 mL</td>
			<td>Miltenyi Biotec</td>
			<td>130-115-389</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus SZ61 Zoom Stereomicroscope&#160;</td>
			<td>Olympus Life Science</td>
			<td>SZ61/SZ51&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce Primary Neuron Isolation Kit</td>
			<td>ThermoFisher</td>
			<td>88280Y</td>
			<td></td>
		</tr>
		<tr>
			<td>Staining</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-MAP2 antibody</td>
			<td>Abcam</td>
			<td>ab5392</td>
			<td>1 : 800</td>
		</tr>
		<tr>
			<td>Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488</td>
			<td>ThermoFisher</td>
			<td>A32766</td>
			<td>1 : 500</td>
		</tr>
		<tr>
			<td>Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488</td>
			<td>ThermoFisher</td>
			<td>A32790</td>
			<td>1 : 500</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline (DPBS)&#160;</td>
			<td>Sigma Aldrich&#160;</td>
			<td>MFCD00131855</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Chicken IgY (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647</td>
			<td>ThemoFisher</td>
			<td>A32933</td>
			<td>1 : 500</td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 594</td>
			<td>ThermoFisher</td>
			<td>A11037</td>
			<td>1 : 200</td>
		</tr>
		<tr>
			<td>Invitrogen Leptin Receptor Recombinant Rabbit Monoclonal Antibody (JA73-01)</td>
			<td>ThermoFisher</td>
			<td>MA5-32685</td>
			<td>1 : 500</td>
		</tr>
		<tr>
			<td>Mouse Leptin R Biotinylated Antibody</td>
			<td>R&#38;D Systems&#160;</td>
			<td>ABAF497</td>
			<td>1 : 500</td>
		</tr>
		<tr>
			<td>POMC Rabbit mAb&#160;</td>
			<td>Cell Signaling Technology</td>
			<td>D3R1U</td>
			<td>1 : 500</td>
		</tr>
		<tr>
			<td>PSD95 (D74D3) XP Rabbit mAb</td>
			<td>Cell Signaling Technology</td>
			<td>D74D3#3409</td>
			<td>1 : 500</td>
		</tr>
		<tr>
			<td>Streptavidin, Alexa Fluor 594 conjugate</td>
			<td>ThermoFisher</td>
			<td>S11227</td>
			<td>1 : 500</td>
		</tr>
		<tr>
			<td>Synapsin 1 Monoclonal Antibody (7H10G6)</td>
			<td>ThermoFisher</td>
			<td>MA5-31919</td>
			<td>1 : 500</td>
		</tr>
		<tr>
			<td>Vectashield Plus Antifade Mountina Medium with DAPI 10 mL</td>
			<td>Vector Laboratories&#160;</td>
			<td>H-2000</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of targeted hypothalamic neurons for studies of hormonal, metabolic, and electrical regulation

The hypothalamus regulates fundamental metabolic processes by controlling functions as varied as food intake, body temperature, and hormone release. As the functions of the hypothalamus are controlled by specific subsets of neuronal populations, the ability to isolate them provides a major tool for studying metabolic mechanisms. In this regard, the neuronal complexity of the hypothalamus poses exceptional challenges. 
For these reasons, new techniques, such as Magnetic-Activated Cell Sorting (MACS), have been explored. This paper describes a new application of magnetic-activated cell sorting (MACS) using microbead technology to isolate a targeted neuronal population from prenatal mice brains. The technique is simple and guarantees a highly pure and viable primary hypothalamic neuron culture with high reproducibility. The hypothalamus is gently dissociated, neurons are selectively isolated and separated from glial cells, and finally, using a specific antibody for a cell surface marker, the population of interest is selected. 
Once isolated, targeted neurons can be used to investigate their morphological, electrical, and endocrine characteristics and their responses in normal or pathological conditions. Furthermore, given the variegated roles of the hypothalamus in regulating feeding, metabolism, stress, sleep, and motivation, a closer look at targeted and region-specific neurons may provide insight into their tasks in this complex environment.

This paper describes a protocol for the isolation of targeted neurons of the hypothalamus (Figure 1). The scope of the method is to study specific neuronal characteristics in a controlled and isolated context. Thus, mouse embryos were extracted from the pregnant dams at E14-E16. The meninges were removed, and the hypothalamus was isolated from the rest of the brain. The tissue was gently dissociated with two mixtures of enzymes freshly prepared using the referenced dissociation kit. First, non-neuronal cells were separated from neuronal cells-glia, microglia, and neurons were collected in the same single-cell suspension. To this end, non-neuronal cells were labeled using a cocktail of antibodies recognizing non-neuronal surface epitopes. After incubation, the antibody-cell complex was conjugated with magnetic microbeads and subsequently passed through a magnetic column to trap the non-neuronal cells.
This step yielded two cell suspensions, one containing non-neuronal and the other containing neuronal cells. Both suspensions could be plated immediately. Alternatively, the neuronal cell suspension could be further manipulated to separate a neuronal sub-population (targeted suspension) from the rest using the same strategy.&#160;Based on the experiment of interest, the neuronal cells can be plated from 125,000 to 200,000 cells/mm3. The less-dense cultures can be used to analyze neurons at single-cell resolution: from axonal development, synaptic formation, and transmission to electrophysiology. The denser cultures can be used for biochemical analyses, including DNA and RNA extraction, western blot, Southern blot, Northern blot, real-time PCR, and RNA sequencing.
In this study, LepR was targeted to isolate neurons involved in the melanocortin system, such as the ARCPOMC and ARCAgRP neurons. Cells were plated at densities ranging from 120,000 cells/mm3, for LepR+ neurons, to 200,000 cells/mm3 for generic neuronal populations. After 48 h, LepR+ neurons began to form neurites (Figure 2). At DIV4, axonal extensions showed progress, while dendritic processes began to appear. At DIV6, the neurons were sufficiently developed and were therefore ready to be analyzed. Immunofluorescence experiments on LepR+ neurons showed 99% expression of LepR (green, Figure 3A). No glial cells or other non-neuronal cells were observed, confirming the purity of the primary neuronal culture. The neuronal nature of the cells was confirmed by microtubule-associated protein 2 (MAP2) staining, with the identification of axons and dendritic protrusions (Figure 3B). At DIV10, 30% of LepR+ cells expressed POMC (red). This is expected as the majority of LepR+ cells express either POMC or AgRP. Figure 3C,D illustrate co-localization between POMC and LepR signals. Note that the co-localization was prominent at and around the nucleus, as expected.
General cultures containing heterogeneous hypothalamic neuronal populations were used for control. Immunofluorescence demonstrated synaptic connectivity and functionality, as assessed by Synapsin-1 (green) and PSD 95 (red) co-staining (Figure 4A,B). The number of LepR+ neurons present in the general culture was ~5%, a percentage consistent with the notion that the majority of LepR-expressing neurons had been selected during the magnetic separation process (representative LepR+ cells are illustrated in Figure 4C,D). All data generated or analyzed during this study are available at&#160;https://doi-org.remotexs.ntu.edu.sg/10.5061/dryad.cnp5hqc9c.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65674/65674fig01.jpg" /> 
Figure 1: Experimental flow chart and setup. (A) Graphical representation of the experimental procedure.&#160;Go-no-go: &#8805;106 cells are necessary to proceed to cell isolation; the optimal cell number is 107. (B) Representative image of an E16 embryo brain. The hypothalamus and meninges are indicated. (C) MACS setup used for the separation and isolation of targeted cells. Magnetic stand, magnetic separator, and column are indicated. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65674/65674fig01large.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/65674/65674fig02.jpg" /> 
Figure 2: Neuronal culture between DIV2 and DIV6. (A) LepR+ cells obtained from the positive selection show reduced cell density and connectivity, but normal development of neurites (red arrows), axons (blue arrows), and dendrites (green arrows). Cells were plated at a density of 120,000 cells/mm3. Scale bar = 100 &#956;m. (B) Generic hypothalamic neurons, plated at a density of 200,000 cells/mm3, display normal developmental and growth features and connectivity (black arrows). Scale bar = 100 &#956;m. Abbreviations: DIV = days in vitro; LepR = leptin receptor. All data generated or analyzed to construct this figure are available at https://doi-org.remotexs.ntu.edu.sg/10.5061/dryad.cnp5hqc9c. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65674/65674fig02large.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/65674/65674fig03.jpg" /> 
Figure 3: In vivo neurons are recapitulated by cultured LepR+ neurons. (A) Representative images of a culture of neurons expressing the LepR (green; DAPI is blue). Ninety-nine percent of the cells expressed the LepR. Neurons were plated at 120,000 cells/mm3 density. At DIV7, the neurons presented elongated axons, dendritic maturation, and neuronal connectivity (arrows). Scale bar = 40 &#956;m.&#160;(B) Immunofluorescence with&#160;anti-MAP2 was used to confirm the neuronal nature of the cells. Neuronal-specific morphologies such as axons, dendrites, and protrusions are demonstrated (arrows). Scale bar = 40 &#956;m. (C) Magnification of LepR+ cells.&#160;LepR in green and DAPI in blue. Scale bar = 10 &#956;m. (D) Co-staining with LepR (green) and POMC (red). Roughly 30% of LepR+ neurons were co-immunoreactive to POMC, which was detected at the level of the nucleus (red arrows). Scale bar = 10 &#956;m. Abbreviations: DAPI = 4&#39;,6-diamidino-2-phenylindole; MAP2 = microtubule-associated protein 2; POMC = proopiomelanocortin; LepR = leptin receptor. All data generated or analyzed to construct this figure are available at https://doi-org.remotexs.ntu.edu.sg/10.5061/dryad.cnp5hqc9c.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65674/65674fig03large.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/65674/65674fig04.jpg" /> 
Figure 4: In vivo cells are recapitulated by cultured generic hypothalamic neurons. (A) Representative images of generic hypothalamic neuronal culture&#160;stained with Synapsin 1 (green), PSD95 (red), and DAPI (blue). The neurons exhibited well-developed connectivity and synaptic functionality (arrows). Scale bar = 40 &#956;m. (B) Magnification of the&#160;box in (A), showing co-localization of Synapsin 1 (green) and PSD95 (red, arrows). Scale bar = 20 &#956;m.&#160;(C,D) Representative images showing LepR+ cells in a general culture. LepR+ cells (green) accounted for ~5% of the total. Representative LepR+ cells are indicated by arrows. Scale bar = 40 &#956;m.&#160;(E,F) Magnifications of the boxes in (C,D) showing green puncta leptin receptors localized at the soma. Scale bar = 20 &#956;m. All data generated or analyzed to construct this figure are available at&#160;https://doi-org.remotexs.ntu.edu.sg/10.5061/dryad.cnp5hqc9c.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65674/65674fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Hypothalamic Neural Mechanism Insights at JoVE.com

Author Spotlight: Hypothalamic Neural Mechanism Insights 

Here we present a protocol to grow specific hypothalamic cell subtypes in culture. The cells can be selected based on opportune/unique membrane markers and used in many applications, including immunofluorescence, electrophysiological, and biochemical assays.

Isolation of Targeted Hypothalamic Neurons for Studies of Hormonal, Metabolic, and Electrical Regulation

The hypothalamus regulates fundamental metabolic processes by controlling functions as varied as food intake, body temperature, and hormone release. As ...

isolation-targeted-hypothalamic-neurons-for-studies-hormonal

Nanyang Technological University

Research

JoVE Journal

Neuroscience

3.3K Views.  Rutgers University. Here we present a protocol to grow specific hypothalamic cell subtypes in culture. The cells can be selected based on opportune/unique membrane markers and used in many applications, including immunofluorescence, electrophysiological, and biochemical assays.

Video: Author Spotlight: Hypothalamic Neural Mechanism Insights 

Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory  Neurons

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. Caged compounds are molecules made physiologically inactive by a chemical cage that can be broken by a flash of ultraviolet light. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged compounds for the study of olfactory transduction in dissociated mouse olfactory sensory neurons. The process of olfactory transduction (Figure 1) takes place in the cilia of olfactory sensory neurons, where odorant binding to receptors leads to the increase of cAMP that opens cyclic nucleotide-gated (CNG) channels2. Ca entry through CNG channels activates Ca-activated Cl channels. We show how to dissociate neurons from the mouse olfactory epithelium3 and how to activate CNG channels or Ca-activated Cl channels by photolysis of caged cAMP4 or caged Ca5. We use a flash lamp6,7 to apply ultraviolet flashes to the ciliary region to uncage cAMP or Ca while patch-clamp recordings are taken to measure the current in the whole-cell voltage-clamp configuration8-11.

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged cAMP or caged Ca for the study of olfactory transduction in dissociated mouse olfactory sensory neurons.

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. ...

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the precise molecular steps as well as the activity of specific neurons in multi-functional nuclei of brain areas such as the hypothalamus remain. This problem includes identification of the molecular components involved in the regulation of various neurohormone signal transduction cascades. Elevations of intracellular Ca2+ play an important role in regulating the sensitivity of neurons, both at the level of signal transduction and at synaptic sites.
New tools have emerged to help identify neurons in the myriad of brain neurons by expressing green fluorescent protein (GFP) under the control of a particular promoter. To monitor both spatially and temporally stimulus-induced Ca2+ responses in GFP-tagged neurons, a non-green fluorescent Ca2+ indicator dye needs to be used. In addition, confocal microscopy is a favorite method of imaging individual neurons in tissue slices due to its ability to visualize neurons in distinct planes of depth within the tissue and to limit out-of-focus fluorescence. The ratiometric Ca2+ indicator fura-2 has been used in combination with GFP-tagged neurons1. However, the dye is excited by ultraviolet (UV) light. The cost of the laser and the limited optical penetration depth of UV light hindered its use in many laboratories. Moreover, GFP fluorescence may interfere with the fura-2 signals2. Therefore, we decided to use a red fluorescent Ca2+ indicator dye. The huge Stokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength. We had previously good results using fura-red in combination with GFP-tagged olfactory neurons3. The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons4. Fura-red based Ca2+ imaging was also successfully combined with GFP-tagged pancreatic &beta;-cells and GFP-tagged receptors expressed in HEK cells5,6. A little quirk of fura-red is that its fluorescence intensity at 650 nm decreases once the indicator binds calcium7. Therefore, the fluorescence of resting neurons with low Ca2+ concentration has relatively high intensity. It should be noted, that other red Ca2+-indicator dyes exist or are currently being developed, that might give better or improved results in different neurons and brain areas.

In this protocol, we update recent progress in imaging Ca2+ signals of GFP-tagged neurons in brain tissue slices using a red fluorescent Ca2+ indicator dye.

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral delivery or transgenic animals have potential confounds that make interpretation of results from these studies difficult. New radiological tools are emerging, however, that allow one to noninvasively investigate the function of select groups of adult-born neurons through accurate and precise anatomical targeting in small animals. Focal ionizing radiation inhibits the birth and differentiation of new neurons, and allows targeting of specific neural progenitor regions. In order to illuminate the potential functional role that adult hypothalamic neurogenesis plays in the regulation of physiological processes, we developed a noninvasive focal irradiation technique to selectively inhibit the birth of adult-born neurons in the hypothalamic median eminence. We describe a method for Computer tomography-guided focal irradiation (CFIR) delivery to enable precise and accurate anatomical targeting in small animals. CFIR uses three-dimensional volumetric image guidance for localization and targeting of the radiation dose, minimizes radiation exposure to nontargeted brain regions, and allows for conformal dose distribution with sharp beam boundaries. This protocol allows one to ask questions regarding the function of adult-born neurons, but also opens areas to questions in areas of radiobiology, tumor biology, and immunology. These radiological tools will facilitate the translation of discoveries at the bench to the bedside.

The function of adult-born mammalian neurons remains an active area of investigation. Ionizing radiation inhibits the birth of new neurons. Using computer tomography-guided focal irradiation (CFIR), three-dimensional anatomical targeting of specific neural progenitor populations can now be used to assess the functional role of adult neurogenesis.

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result of chemical inputs to its synapses. However, neurons can also be excited by an imposed electric field. In particular, recent clinical applications activate neurons by creating an electric field externally. It is therefore of interest to investigate how the neuron responds to the external field and what causes the action potential. Fortunately, precise and controlled application of an external electric field is possible for embryonic neuronal cells that are excised, dissociated and grown in cultures. This allows the investigation of these questions in a highly reproducible system.
In this paper some of the techniques used for controlled application of external electric field on neuronal cultures are reviewed. The networks can be either one dimensional, i.e. patterned in linear forms or allowed to grow on the whole plane of the substrate, and thus two dimensional. Furthermore, the excitation can be created by the direct application of electric field via electrodes immersed in the fluid (bath electrodes) or by inducing the electric field using the remote creation of magnetic pulses.

Neuronal cultures are a good model for studying emerging brain stimulation techniques via their effect on single neurons or a population of neurons. Presented here are different methods for stimulation of patterned neuronal cultures by an electric field produced directly by bath electrodes or induced by a time-varying magnetic field.

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result ...

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.

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 ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody.
Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 &#176;C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 &#176;C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) &#62; 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...

Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the study of &#945;S aggregation in Parkinson&#39;s Disease (PD). Recently, the development of new animal models through the exogenous inoculation of brain extracts from diseased &#945;S transgenic mice or PD patients has given new hopes to the possibility of creating more adequate cell models of &#945;S aggregation. Unfortunately, when it comes to cells in cultures, administration of raw brain extracts has not proven as successful as in mice and the source of choice of exogenous aggregates is still in vitro preformed &#945;S fibrils.
We have developed a method to induce the formation of intracellular &#945;S inclusions in primary neurons through the exogenous administration of native microsomes-associated &#945;S aggregates, a highly toxic &#945;S species isolated from diseased areas of transgenic mice. This fraction of &#945;S aggregates that is associated with the microsomes vesicles, is efficiently internalized and induces the formation of intracellular inclusions positive for aggregated and phosphorylated &#945;S. Compared to in vitro-preformed fibrils which are made from recombinant &#945;S, our method is faster and guarantees that the pathogenic seeding is made with authentic &#945;S aggregates extracted from diseased animal models of PD, mimicking more closely the type of inclusions obtained in vivo. As a result, availability of tissues rich in &#945;S inclusions is mandatory.
We believe that this method will provide a versatile cell-based model to study the microscopic aspects of &#945;S aggregation and the related cellular pathophysiology in vivo and will be a starting point for the creation of more accurate and sophisticated cell paradigm of PD.

The goal of this protocol is to provide a cell-based system that replicates the formation of alpha-synuclein aggregates in vivo. Intracellular alpha-synuclein inclusions are seeded in primary neurons by the internalization and propagation of exogenous administered native microsomes-associated alpha-synuclein aggregates isolated from diseased alpha-synuclein transgenic mice.

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

Isolation of Targeted Hypothalamic Neurons for Studies of Hormonal, Metabolic, and Electrical Regulation

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Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory Neurons

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures

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

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice