magnetic separation and negative selection of murine neuronal cultures

Regolazione ormonale, metabolica ed elettrica dei neuroni ipotalamici isolati

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.

Autore in primo piano: Approfondimenti sul meccanismo neurale ipotalamico 

Isolamento di neuroni ipotalamici mirati per studi di regolazione ormonale, metabolica ed elettrica

The scope of our research is to study metabolic disorder in relation to hypothalamic dysfunction. We're interested in studying the biochemical and cellular mechanism of targeted primary mammalian hypothalamic neuron, especially the one expressing leptin receptor. The hypothalamus is a complex brain region, involving the maintenance of feeding, sleeping, motivation, and metabolism.And this imply a very diverse neural community. Only few of this population have been study thoroughly, and most of the other needs to further investigated and their cellular mechanism further explained. Hypothalamic neurons and their function and are investigated mainly with electrophysiology, chemogenetics, and optogenetics.And these techniques are very informative in relation to understand animal behavior and brain circuitry. However, very little is known about the cellular and molecular mechanism that drive this behavior. And only by looking deeper at the cellular level, we can address this gaps.

Watch this Scientific Journal Video about Magnetic Separation and Negative Selection of Murine Neuronal Cultures at JoVE.com

Magnetic Separation and Negative Selection of Murine Neuronal Cultures  

Separazione magnetica e selezione negativa di colture neuronali murine

Iniziare centrifugando la sospensione di cellule cerebrali isolate a 300 G per otto minuti. Risospendere il pellet in 80 microlitri di HBSS con soluzione 0,5%BSA. Aggiungere 20 microlitri di cocktail di anticorpi biotina di cellule non neuronali e incubare.Lavare le celle con due millilitri di HBSS con 0,5% di BSA. Quindi risospendere il pellet della centrifuga in 80 microlitri di HBSS con 0,5% di BSA. Ora aggiungi 20 microlitri di microsfere anti-biotina e pipetta per mescolare accuratamente.Dopo l'incubazione a freddo, aggiungere 0,5 millilitri di soluzione HBSS BSA. Installare il supporto prima di risciacquare la colonna con 0,5 millilitri di HBSS con 0,5% di BSA. Quindi posizionare una provetta da 15 millilitri sotto la colonna e far passare 0,5 millilitri di sospensione cellulare attraverso di essa.Eseguire tre lavaggi con 0,5 millilitri di HBSS con soluzione BSA allo 0,5% per catturare le cellule neuronali residue. Rimuovere la colonna dal magnete e posizionarla all'interno di un nuovo tubo da 15 millilitri. Aggiungere un millilitro di HBSS con 0,5% di BSA e utilizzare lo stantuffo per raccogliere le cellule non neuronali marcate magneticamente.Dopo la centrifugazione cellulare, risospendere le cellule in un millilitro di HBSS con una soluzione di BSA allo 0,5%. Contare le cellule su una camera di conteggio Neubauer al microscopio in campo chiaro. I neuroni LepR positivi hanno iniziato a formare neuriti dopo 48 ore.A DIV4 le estensioni assonali hanno mostrato progressi mentre i processi dendritici hanno iniziato a comparire. A DIV6 i neuroni erano sufficientemente sviluppati. Studi di immunofluorescenza hanno dimostrato che non sono state osservate cellule gliali o altre cellule non neuronali.La natura neuronale delle cellule è stata confermata dalla colorazione della proteina due associata ai microtubuli. Al DIV10 il 30% delle cellule LepR positive ha espresso POMC. La connettività e la funzionalità sinaptica sono state osservate in colture generali contenenti popolazioni neuronali ipotalamiche eterogenee.

magnetic-separation-and-negative-selection-of-murine-neuronal-cultures

Research

JoVE Journal

Neuroscience

Magnetic Separation and Negative Selection of Murine Neuronal Cultures

421 Views.  Rutgers University. Iniziare centrifugando la sospensione di cellule cerebrali isolate a 300 G per otto minuti. Risospendere il pellet in 80 microlitri di HBSS con soluzione 0,5%BSA. Aggiungere 20 microlitri di cocktail di anticorpi biotina di cellule non neuronali e incubare.Lavare le celle con due millilitri di HBSS con 0,5% di BSA. Quindi risospendere il pellet della centrifuga in 80 microlitri di HBSS con 0,5% di BSA. Ora aggiungi 20 microlitri di microsfere anti-biotina e pipetta per mescolare accura...

Video: Magnetic Separation and Negative Selection of Murine Neuronal Cultures  

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.

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 ...

Ricerca

Neuroscienze

Murine Embryonal Hypothalamus Extraction, Collection, and Tissue Dissociation

Start by fire-polishing three pasture pipettes using a bunsen burner, creating decreasing diameters. Next, sterilize the abdomen of a euthanized pregnant mouse dam with 70%ethanol. Cut open the abdominal cavity from the pubic synthesis to the xiphoid process of the rib cage.Now, extract the uterine horn from the abdominal cavity and place it in a 100-millimeter plate filled with ice-cold HBSS solution. Extract and separate all embryos from the washed uterus using fine forceps. Quickly decapitate the extracted mice embryos and place the heads in a 60-millimeter Petri dish filled with HBSS.Place a pair of fine forceps in the eye cavity to hold the brain. With another pair, peel the skin and skull until the brain is visible. Scoop the brain out of the olfactory bulbs from the skull and flip it upside down.Observe the cortex on the ventral side and the hypothalamus on the dorsal side. Use curved forceps to remove the layer of meninges and blood vessels until the brain appears white and clear, and separate the hypothalamic area from the rest of the brain. Cut the hypothalamus into three to four small fine pieces, and use a pipette to transfer the pieces into a 15-milliliter tube.Next, fill the tube with six milliliters of HBSS. Then add Enzyme Mix 1 once the tissue has settled and the supernatant removed. Incubate the tube in a 37-degree Celsius water bath for 15 minutes, agitating every five minutes to re-suspend the tissue.Now, add 30 microliters of Enzyme Mix 2 and dissociate the tissue by pipetting up and down 10 times with a large diameter pasture pipette. After an additional 10-minute incubation, add the remaining 15 microliters of Enzyme Mix 2. Utilizing two fire polished pipettes of decreasing diameter, pipette the tissue 10 times.Immediately add 10 milliliters of HBSS with 0.5%BSA to the tube. Then centrifuge the tube at 300 G for 10 minutes at room temperature. Aspirate the supernatant and re-suspend the cell pellet in a one milliliter of HBSS with 0.5%BSA.

Estrazione, raccolta e dissociazione dell'ipotalamo embrionale embrionale murino

Begin by centrifuging the isolated brain cell suspension at 300G for eight minutes. Resuspend the pellet in 80 microliters of HBSS with 0.5%BSA solution. Add 20 microliters of non neuronal cell biotin antibody cocktail and incubate.Wash the cells with two milliliters of HBSS with 0.5%BSA. Then resuspend the centrifuge pellet in 80 microliters of HBSS with 0.5%BSA. Now add 20 microliters of anti biotin microbeads, and pipette to mix thoroughly.After cold incubation add 0.5 milliliters of HBSS BSA solution. Set up the stand before rinsing the column with 0.5 milliliters of HBSS with 0.5%BSA. Next place a 15 milliliter tube under the column and pass 0.5 milliliters of the cell suspension through it.Perform three washes with 0.5 milliliters of HBSS with 0.5%BSA solution to capture residual neuronal cells. Remove the column from the magnet and place it inside a new 15 milliliter tube. Add one milliliter of HBSS with 0.5%BSA and use the plunger to collect magnetically labeled non neuronal cells.After cell centrifugation, resuspend the cells in one milliliter of HBSS with 0.5%BSA solution. Count the cells on a neubauer counting chamber under a brightfield microscope. LepR positive neurons began to form neurites after 48 hours.At DIV4 axonal extensions showed progress while dendritic processes began to appear. At DIV6 the neurons were sufficiently developed. Immunofluorescent studies showed that no glial cells or other non neuronal cells were observed.The neuronal nature of the cells was confirmed by microtubule associated protein two staining. At DIV10 30%of LepR positive cells expressed POMC. Synaptic connectivity and functionality were observed in general cultures containing heterogeneous hypothalamic neuronal populations.

Magnetic Separation and Positive Selection of Murine Neuronal Cultures

Begin by centrifuging the murine-pure neuronal cell suspension at 300 G for eight minutes. Gently aspirate the supernatant and resuspend the pellet in 80 microliters of HBSS with 0.5 BSA solution. After adding the specific antibody, incubate the tubes at four degrees Celsius for 10 minutes.Wash the excess antibody with HBSS-BSA solution and centrifuge. Add 20 microliters of antibiotin microbeads to the pellet, resuspended in HBSS-BSA. Incubate the tube at four degrees Celsius for 10 minutes.Then, add 0.5 milliliters of HBSS with 0.5%BSA solution for every 10 to the seven cells. Rinse the column with 0.5 milliliters of HBSS with 0.5%BSA solution. Once the dripping stops, place a 15-milliliter tube beneath the column and pass 0.5 milliliters of cell suspension through it.Collect the eluate containing nonspecific neuronal cells, then clean the column with three washes of 0.5 milliliters of HBSS with 0.5%BSA. Now remove the column from the magnet and place it in a new 15-milliliter tube. Add one milliliter of HBSS with 0.5%BSA and use the plunger to flush out the targeted cells.After centrifugation and supernatant aspiration, resuspend the pellet in 0.5 milliliters of plating medium. Count the cells in a Neubauer counting chamber under a bright-field microscope. Plate the targeted cells as the positive control and non-specific cells as the negative control in the prepared 24-well plate.Incubate at 37 degrees Celsius for 12 hours. LepR-positive neurons began to form neurites after 48 hours. At DIV4, axonal extensions showed progress, While dendritic processes began to appear.At DIV6, the neurons were sufficiently developed. Immunofluorescent studies showed that no glial cells or other non-neuronal cells were observed. The neuronal nature of the cells was confirmed by microtubule-associated protein 2 staining.At DIV10, 30%of LepR-positive cells expressed POMC. Synaptic connectivity and functionality were observed in general cultures containing heterogeneous hypothalmic neuronal populations.