Published: August 4th, 2023
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.
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.
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' 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.
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'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
2. Media, 24-well plate, and material preparation
3. Reagent preparation for Neural Tissue Dissociation, following the directions of the Neural Dissociation kit
4. Embryo extraction
5. Hypothalamus extraction, collection, and tissue dissociation
6. Cell counting
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.
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.
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 suspension through the column, only the cells of interest are trapped in the magnetic field.
10. Magnetic separation, positive selection
11. Cell culture maintenance
12. Neuron immunofluorescence staining
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. 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 https://doi.org/10.5061/dryad.cnp5hqc9c.
Figure 1: Experimental flow chart and setup. (A) Graphical representation of the experimental procedure. Go-no-go: ≥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. Please click here to view a larger version of this figure.
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 μm. (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 μm. Abbreviations: DIV = days in vitro; LepR = leptin receptor. All data generated or analyzed to construct this figure are available at https://doi.org/10.5061/dryad.cnp5hqc9c. Please click here to view a larger version of this figure.
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 μm. (B) Immunofluorescence with 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 μm. (C) Magnification of LepR+ cells. LepR in green and DAPI in blue. Scale bar = 10 μm. (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 μm. Abbreviations: DAPI = 4',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/10.5061/dryad.cnp5hqc9c. Please click here to view a larger version of this figure.
Figure 4: In vivo cells are recapitulated by cultured generic hypothalamic neurons. (A) Representative images of generic hypothalamic neuronal culture stained with Synapsin 1 (green), PSD95 (red), and DAPI (blue). The neurons exhibited well-developed connectivity and synaptic functionality (arrows). Scale bar = 40 μm. (B) Magnification of the box in (A), showing co-localization of Synapsin 1 (green) and PSD95 (red, arrows). Scale bar = 20 μm. (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 μm. (E,F) Magnifications of the boxes in (C,D) showing green puncta leptin receptors localized at the soma. Scale bar = 20 μm. All data generated or analyzed to construct this figure are available at https://doi.org/10.5061/dryad.cnp5hqc9c. Please click here to view a larger version of this figure.
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 "fidelity" 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 authors declare that they have no conflicts of interest.
Graphical figures were created with BioRender.com. This work was supported by an NIA grant (R01AG060919) and an NSF grant (2030348) to FS.
|1 curved point forceps
|Fine Science Tools
|1 fine surgical scissor
|Fine Science Tools
|100 mm Petri dish
|2 straight fine forceps
|Fine Science Tools
|60 mm Petri dish
|Decon Laboratories, INC.
|Ethanol 190 Proof
|Anti-Biotin MicroBeads 1mL
|1 : 800
|Bovine Serum Albumin
|Anti-Biotin MicroBeads 1mL
|Bovine Serum Albumin
|GG-12-1.5, 12 mm dia.#1.5 thick 100 pc cell culture tested German coverglasses
|Gibco B-27 Supplement 10 mL
|Gibco Basal Medium Eagle (BME) 500 mL
|(+) Earle's Salts, (-) L-Glutamine
|Gibco HBBS (1x) Hanks' Balanced Salt Solution 500 mL
|Calcium, Magnesium, No phenol red
|Gibco HI FBS 100 mL
|Gibco L-Glutamine 200 mM (100x)
|Gibco Sodium Pyruvate (100 mM) 100 mL
|MiniMACS Separator and Starting Kit
|Mouse Leptin R Biotinylated Antibody
|0.25 μg/106 cells
|Neaubeaur-Improved Brightline 100 µm Chamber
|Neural Tissue Dissociation Kit - Postnatal Neurons
|Neuronal Culture Medium 500 mL
|Non-Neuronal Cell Biotin-Antibody Cocktail mouse 1 mL
|Olympus SZ61 Zoom Stereomicroscope
|Olympus Life Science
|Pierce Primary Neuron Isolation Kit
|1 : 800
|Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488
|1 : 500
|Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488
|1 : 500
|Dulbecco's Phosphate Buffered Saline (DPBS)
|Goat anti-Chicken IgY (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647
|1 : 500
|Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 594
|1 : 200
|Invitrogen Leptin Receptor Recombinant Rabbit Monoclonal Antibody (JA73-01)
|1 : 500
|Mouse Leptin R Biotinylated Antibody
|1 : 500
|POMC Rabbit mAb
|Cell Signaling Technology
|1 : 500
|PSD95 (D74D3) XP Rabbit mAb
|Cell Signaling Technology
|1 : 500
|Streptavidin, Alexa Fluor 594 conjugate
|1 : 500
|Synapsin 1 Monoclonal Antibody (7H10G6)
|1 : 500
|Vectashield Plus Antifade Mountina Medium with DAPI 10 mL
Copyright © 2024 MyJoVE Corporation. All rights reserved