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.
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 have nothing to disclose.
Graphical figures were created with BioRender.com. This work was supported by an NIA grant (R01AG060919) and an NSF grant (2030348) to FS.
Embryo extraction | |||
1 curved point forceps | Fine Science Tools | 11270-20 | Dumont |
1 fine surgical scissor | Fine Science Tools | 14058-11 | Dumont |
100 mm Petri dish | Corning | 430167 | |
2 straight fine forceps | Fine Science Tools | 11254-20 | Dumont |
60 mm Petri dish | Corning | 430196 | |
70% ethanol | Decon Laboratories, INC. | 2801 | Ethanol 190 Proof |
Anti-Biotin MicroBeads 1mL | Miltenyi Biotec | 130-115-390 | |
Anti-MAP2 antibody | Abcam | ab5392 | 1 : 800 |
Bench pads | |||
Bovine Serum Albumin | Sigma-Aldrich | A9418-50G | |
Buffer Y | Miltenyi Biotec | 130-094-802 | |
Buffer Z | Miltenyi Biotec | 130-094-802 | |
Cell Culture | |||
Anti-Biotin MicroBeads 1mL | Miltenyi Biotec | 130-115-390 | |
Bovine Serum Albumin | Sigma-Aldrich | A9418-50G | |
Buffer Y | Miltenyi Biotec | 130-094-802 | |
Buffer Z | Miltenyi Biotec | 130-094-802 | |
Enzyme A | Miltenyi Biotec | 130-094-802 | |
Enzyme P | Miltenyi Biotec | 130-094-802 | |
GG-12-1.5, 12 mm dia.#1.5 thick 100 pc cell culture tested German coverglasses | Neuvitro Corporation | GG-12-15 | |
Gibco B-27 Supplement 10 mL | ThermoFisher | 17504-044 | |
Gibco Basal Medium Eagle (BME) 500 mL | ThermoFisher | 21010046 | (+) Earle's Salts, (-) L-Glutamine |
Gibco HBBS (1x) Hanks' Balanced Salt Solution 500 mL | ThermoFisher | 14025092 | Calcium, Magnesium, No phenol red |
Gibco HI FBS 100 mL | ThermoFisher | 16140-063 | |
Gibco L-Glutamine 200 mM (100x) | ThermoFisher | 25030-081 | |
Gibco Penicilline/Streptomicine | ThermoFisher | 15140-122 | 10,000 U/mL |
Gibco Sodium Pyruvate (100 mM) 100 mL | ThermoFisher | 11360070 | |
MiniMACS Separator and Starting Kit | Miltenyi Biotec | 130-042-102 | |
Mouse Leptin R Biotinylated Antibody | R&D Systems | ABAF497 | 0.25 μg/106 cells |
MS Column | Miltenyi Biotec | 130-042-201 | |
Neaubeaur-Improved Brightline 100 µm Chamber | Hausser Scientific | 3120 | |
Neural Tissue Dissociation Kit – Postnatal Neurons | Miltenyi Biotec | 130-094-802 | |
Neuronal Culture Medium 500 mL | ThermoFisher | 88283 | |
Non-Neuronal Cell Biotin-Antibody Cocktail mouse 1 mL | Miltenyi Biotec | 130-115-389 | |
Olympus SZ61 Zoom Stereomicroscope | Olympus Life Science | SZ61/SZ51 | |
Pierce Primary Neuron Isolation Kit | ThermoFisher | 88280Y | |
Staining | |||
Anti-MAP2 antibody | Abcam | ab5392 | 1 : 800 |
Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 | ThermoFisher | A32766 | 1 : 500 |
Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 | ThermoFisher | A32790 | 1 : 500 |
Dulbecco's Phosphate Buffered Saline (DPBS) | Sigma Aldrich | MFCD00131855 | |
Goat anti-Chicken IgY (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647 | ThemoFisher | A32933 | 1 : 500 |
Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 | ThermoFisher | A11037 | 1 : 200 |
Invitrogen Leptin Receptor Recombinant Rabbit Monoclonal Antibody (JA73-01) | ThermoFisher | MA5-32685 | 1 : 500 |
Mouse Leptin R Biotinylated Antibody | R&D Systems | ABAF497 | 1 : 500 |
POMC Rabbit mAb | Cell Signaling Technology | D3R1U | 1 : 500 |
PSD95 (D74D3) XP Rabbit mAb | Cell Signaling Technology | D74D3#3409 | 1 : 500 |
Streptavidin, Alexa Fluor 594 conjugate | ThermoFisher | S11227 | 1 : 500 |
Synapsin 1 Monoclonal Antibody (7H10G6) | ThermoFisher | MA5-31919 | 1 : 500 |
Vectashield Plus Antifade Mountina Medium with DAPI 10 mL | Vector Laboratories | H-2000 |