murine embryonal hypothalamus extraction, collection, and tissue dissociation

Regulação hormonal, metabólica e elétrica de neurônios hipotalâmicos isolados

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.

Autor em destaque: Insights do mecanismo neural hipotalâmico 

Isolamento de neurônios hipotalâmicos direcionados para estudos de regulação hormonal, metabólica e elétrica

Watch this Scientific Journal Video about Murine Embryonal Hypothalamus Extraction, Collection, and Tissue Dissociation at JoVE.com

Extração, Coleta e Dissociação de Tecidos do Hipotálamo Embrionário Murino

Comece polindo três pipetas de pastagem usando um queimador de bunsen, criando diâmetros decrescentes. Em seguida, esterilizar o abdômen de uma mãe de rata grávida eutanasiada com etanol 70%. Abra a cavidade abdominal da síntese púbica para o processo xifoide da caixa torácica.Agora, extraia o corno uterino da cavidade abdominal e coloque-o em uma placa de 100 milímetros preenchida com solução gelada de HBSS. Extrair e separar todos os embriões do útero lavado usando pinças finas. Decapite rapidamente os embriões de camundongos extraídos e coloque as cabeças em uma placa de Petri de 60 milímetros cheia de HBSS.Coloque um par de pinças finas na cavidade ocular para segurar o cérebro. Com outro par, descasque a pele e o crânio até que o cérebro esteja visível. Tire o cérebro dos bulbos olfativos do crânio e vire-o de cabeça para baixo.Observe o córtex no lado ventral e o hipotálamo no lado dorsal. Use pinças curvas para remover a camada de meninges e vasos sanguíneos até que o cérebro pareça branco e claro, e separe a área hipotalâmica do resto do cérebro. Corte o hipotálamo em três a quatro pequenos pedaços finos e use uma pipeta para transferir as peças para um tubo de 15 mililitros.Em seguida, encha o tubo com seis mililitros de HBSS. Em seguida, adicione Enzyme Mix 1 uma vez que o tecido tenha se estabilizado e o sobrenadante removido. Incubar o tubo em banho-maria de 37 graus Celsius por 15 minutos, agitando a cada cinco minutos para ressuspender o tecido.Agora, adicione 30 microlitros de Enzyme Mix 2 e dissociar o tecido pipetando para cima e para baixo 10 vezes com uma pipeta de pastagem de grande diâmetro. Após mais 10 minutos de incubação, adicione os restantes 15 microlitros de Enzyme Mix 2. Utilizando duas pipetas polidas a fogo de diâmetro decrescente, pipetar o tecido 10 vezes.Adicione imediatamente 10 mililitros de HBSS com 0,5% BSA ao tubo. Em seguida, centrifugar o tubo a 300 G por 10 minutos à temperatura ambiente. Aspirar o sobrenadante e ressuspender o pellet de células em um mililitro de HBSS com BSA 0,5%.

murine-embryonal-hypothalamus-extraction-collection-tissue

Research

JoVE Journal

Neuroscience

Murine Embryonal Hypothalamus Extraction, Collection, and Tissue Dissociation

624 visualizações. Comece polindo três pipetas de pastagem usando um queimador de bunsen, criando diâmetros decrescentes. Em seguida, esterilizar o abdômen de uma mãe de rata grávida eutanasiada com etanol 70%. Abra a cavidade abdominal da síntese púbica para o processo xifoide da caixa torácica.Agora, extraia o corno uterino da cavidade abdominal e coloque-o em uma placa de 100 milímetros preenchida com solução gelada de HBSS. Extrair e separar todos os embriões do útero lavado usando pinç...

Vídeo: Extração, Coleta e Dissociação de Tecidos do Hipotálamo Embrionário Murino

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 ...