Agradecemos aos nossos colegas que participaram do trabalho aqui resumidas. Este trabalho foi financiado por doações do Deutsche Forschungsgemeinschaft (SFB 894), &quot;análise integrativa do olfato&quot; o DFG Schwerpunktprogramm 1392 e pela Fundação Volkswagen (TLZ). TLZ é professor Lichtenberg da Fundação Volkswagen.

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+loading+of+Rhod+2+into+mitochondria+shows+mitochondrial+Ca2++transients+during+the+contractile+cycle+in+adult+rabbit+cardiac+myocytes.">Selective loading of Rhod 2 into mitochondria shows mitochondrial Ca2+ transients during the contractile cycle in adult rabbit cardiac myocytes.</a> Biochem Biophys. Res. Commun. 236, 738-742 (1997).</li><li>Meshik, X. A., Hyrc, K. L., Goldberg, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+Asante+Calcium+Red+-+a+novel+ratiometric+indicator+with+long+excitation+wavelength.">Properties of Asante Calcium Red - a novel ratiometric indicator with long excitation wavelength.</a> , (2010).</li></ol>

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Uma questão importante em neurociência é compreender como o cérebro processa a informação social. A principal fonte de informações necessárias para o reconhecimento social é codificada por sinais olfativos ou pheromonal. A detecção destes sinais por populações neuronais no nariz e ao reconhecimento dos sinais no cérebro, especialmente o hipotálamo, desempenham um papel fundamental em muitos processos sociais e de hormonas e de outros factores de influência neuroendócrinos 13-16. Um obstácul...

There was a typo in the abstract of <a href="http://www.jove.com/video/4213">Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices</a>.

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

<table border="1"><tbody><tr><td> Nome </td><td> Companhia </td><td> Gato. N ° </td></tr><tr><td> Agar </td><td> Sigma </td><td> A1296 </td></tr><tr><td> Fura-red/AM </td><td> Invitrogen </td><td> F-3021 </td></tr><tr><td> Pluronic F-127 </td><td> Sigma </td><td> P2443 </td></tr><tr><td> Dimetilsulfóxido </td><td> Fisher Scientific </td><td> BP231 </td></tr><tr><td> Vibratório-Blade Microtome Hyrax V 50 </td><td> Zeiss </td><td> 9770170 </td></tr><tr><td> Dispositivo de arrefecimento CU 65 para Micrótomo Hyrax V 50 </td><td> Zeiss </td><td> 9920120 </td></tr><tr><td> O 2 / CO 2 Incubadora, CB210-UL </td><td> Encadernador </td><td> 0019389 </td></tr><tr><td> Super cola, Loctite 406TM </td><td> Henckel </td><td> 142580 </td></tr><tr><td> Espátulas duplas, forma colher </td><td>Bochem </td><td> 3182 </td></tr><tr><td> Espátulas Microspoon, forma colher </td><td> Bochem </td><td> 3344 </td></tr><tr><td> Tesoura de mola, Moria-Vannas-Wolff - Lâminas 7 milímetros </td><td> Belas Science Tools </td><td> 15370-52 </td></tr><tr><td> Tesoura de mola, Vannas - Lâminas 3 milímetros </td><td> Belas Science Tools </td><td> 15000-00 </td></tr><tr><td> Wagner Tesoura </td><td> Belas Science Tools </td><td> 14071-12 </td></tr><tr><td> Médico Fórceps, 7b Dumont </td><td> Belas Science Tools </td><td> 11270-20 </td></tr><tr><td> Câmara de banho grande retangular Aberto (RC-27) </td><td> Warner Instruments </td><td> 64-0238 </td></tr><tr><td> Radiance BioRad Confocal Microscope 2100 </td><td> Zeiss </td><td> nd </td></tr></tbody></table>

imaging calcium responses in gfp-tagged neurons of hypothalamic mouse brain slices

Apesar de um aumento enorme no nosso conhecimento sobre os mecanismos subjacentes a codificação de informação no cérebro, a questão central sobre os passos precisos moleculares, bem como a actividade de neurónios específicos em multi-funcional núcleos de áreas cerebrais, tais como o hipotálamo permanecem. Este problema abrange a identificação dos componentes moleculares envolvidos na regulação de vários neuro-hormonais cascatas de transdução de sinal. Elevações de Ca 2 + intracelular desempenham um papel importante na regulação da sensibilidade de neurónios, quer ao nível de transdução de sinal e em locais sinápticos. Novas ferramentas surgiram para ajudar a identificar os neurónios a miríade de neurónios do cérebro por expressar a proteína fluorescente verde (GFP) sob o controlo de um promotor particular. Para monitorar tanto espacialmente e temporalmente estímulo induzido Ca 2 + respostas em GFP-tagged neurônios, um não-verde fluorescente Ca 2 + indicador corante needs a ser utilizado. Além disso, a microscopia confocal é um método preferido de neurónios individuais de imagem em fatias de tecido devido à sua capacidade de visualizar os neurónios em planos distintos de profundidade dentro do tecido e para limitar fora de foco fluorescente. O Ca 2 + raciométrica indicador fura-2 tem sido utilizado em combinação com GFP-tagged neurônios 1. No entanto, o corante é excitada por luz ultravioleta (UV). O custo do laser e a profundidade de penetração limitada óptico da luz UV pode prejudicar a sua utilização em muitos laboratórios. Além disso, a GFP fluorescente pode interferir com os sinais de fura-2 2. Por isso, decidimos usar um vermelho fluorescente Ca 2 + corante indicador. A mudança Strokes enorme de fura-vermelho permite multicolor análise da fluorescência vermelha em combinação com GFP utilizando um comprimento de onda de excitação único. Tivemos bons resultados anteriormente usando fura-vermelho em combinação com GFP-tagged neurônios olfativos 3. Os protocolos para os cortes de tecidos olfactivos parecia estar a trabalhar eQually bem em neurônios do hipotálamo 4. Fura-vermelho base de Ca 2 + da imagem foram também combinados com sucesso com GFP-tagged β pancreáticas e células GFP-tagged receptores expressos em células HEK 5,6. Uma peculiaridade pouco de fura-vermelho é que a sua intensidade de fluorescência a 650 nm diminui uma vez que o indicador se liga de cálcio 7. Assim, a fluorescência de neurónios de repouso com baixa concentração de Ca + 2 tem intensidade relativamente alta. Note-se, que os outros vermelhos Ca 2 + indicadoras de corantes existem ou estão sendo desenvolvidos atualmente, que poderia dar melhores resultados ou melhores em neurônios e áreas diferentes do cérebro.

Watch this Scientific Journal Video about Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices at JoVE.com

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

Neste protocolo, atualizar o progresso recente na imagem Ca<sup&gt; 2 +</sup&gt; Sinais de neurónios GFP-tagged em fatias de cérebro de tecido, utilizando um vermelho fluorescente Ca<sup&gt; 2 +</sup&gt; Corante indicador.

Respostas de imagem de cálcio nos neurônios GFP-tagged fatias de cérebro de rato Hipotalâmicos

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

imaging-calcium-responses-gfp-tagged-neurons-hypothalamic-mouse-brain

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JoVE Journal

Neuroscience

17.8K visualizações.  University of Saarland, Homburg, Germany. Neste protocolo, atualizar o progresso recente na imagem Ca 2 + Sinais de neurónios GFP-tagged em fatias de cérebro de tecido, utilizando um vermelho fluorescente Ca 2 + Corante indicador.

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

Dual Somatic Recordings from Gonadotropin-Releasing Hormone (GnRH) Neurons Identified by Green Fluorescent Protein (GFP) in Hypothalamic Slices

Gonadotropin-Releasing Hormone (GnRH) is a small neuropeptide that regulates pituitary release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). These gonadotropins are essential for the regulation of reproductive function. The GnRH-containing neurons are distributed diffusely throughout the hypothalamus and project to the median eminence where they release GnRH from their axon terminals into the hypophysiotropic portal system (1). In the portal capillaries, GnRH travels to the anterior pituitary gland to stimulate release of gonadotropins into systemic circulation. GnRH release is not continuous but rather occurs in episodic pulses. It is well established that the intermittent manner of GnRH release is essential for reproduction (2, 3).
Coordination of activity of multiple GnRH neurons probably underlies GnRH pulses. Total peptide content in GnRH neurons is approximately 1.0 pg/cell (4), of which 30% likely comprises the releasable pool. Levels of GnRH during a pulse (5, 6), suggest multiple GnRH neurons are probably involved in neurosecretion. Likewise, single unit activity extracted from hypothalamic multi-unit recordings during LH release indicates changes in activity of multiple neurons (7). The electrodes with recorded activity during LH pulses are associated with either GnRH somata or fibers (8). Therefore, at least some of this activity arises from GnRH neurons.
The mechanisms that result in synchronized firing in hypothalamic GnRH neurons are unknown. Elucidating the mechanisms that coordinate firing in GnRH neurons is a complex problem. First, the GnRH neurons are relatively few in number. In rodents, there are 800-2500 GnRH neurons. It is not clear that all GnRH neurons are involved in episodic GnRH release. Moreover, GnRH neurons are diffusely distributed (1). This has complicated our understanding of coordination of firing and has made many technical approaches intractable. We have optimized loose cell-attached recordings in current-clamp mode for the direct detection of action potentials and developed a recording approach that allows for simultaneous recordings from pairs of GnRH neurons.

Dual Somatic Recordings from Gonadotropin-Releasing Hormone GnRH Neurons Identified by Green Fluorescent Protein GFP in Hypothalamic Slices

Activity in neuronal systems often requires synchronous action potential discharges from neurons within a specific population. For example, pulses of gonadotropin-releasing hormone (GnRH) likely require coordinated activity between GnRH neurons. We present our methodological approach for reliably obtaining simultaneous electrophysiological recordings from the diffusely distributed GnRH neurons.

Dupla Recordings somática de Hormônio Liberador de Gonadotropina (GnRH) Neurônios Identificados por proteína verde fluorescente (GFP) em fatias Hypothalamic

Gonadotropin-Releasing Hormone (GnRH) is a small neuropeptide that regulates pituitary release of luteinizing hormone (LH) and follicle-stimulating ...

Neurociência

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory bulb1-5. Olfactory information in the external world is transmitted to the antennal lobe by olfactory sensory neurons (OSNs), which segregate to distinct regions of neuropil called glomeruli according to the specific olfactory receptor they express. Here, OSN axons synapse with both local interneurons (LNs), whose processes can innervate many different glomeruli, and projection neurons (PNs), which convey olfactory information to higher olfactory brain regions.
Optical imaging of the activity of OSNs, LNs and PNs in the antennal lobe - traditionally using synthetic calcium indicators (e.g. calcium green, FURA-2) or voltage-sensitive dyes (e.g. RH414) - has long been an important technique to understand how olfactory stimuli are represented as spatial and temporal patterns of glomerular activity in many species of insects6-10. Development of genetically-encoded neural activity reporters, such as the fluorescent calcium indicators G-CaMP11,12 and Cameleon13, the bioluminescent calcium indicator GFP-aequorin14,15, or a reporter of synaptic transmission, synapto-pHluorin16 has made the olfactory system of the fruitfly, Drosophila melanogaster, particularly accessible to neurophysiological imaging, complementing its comprehensively-described molecular, electrophysiological and neuroanatomical properties2,4,17. These reporters can be selectively expressed via binary transcriptional control systems (e.g. GAL4/UAS18, LexA/LexAop19,20, Q system21) in defined populations of neurons within the olfactory circuitry to dissect with high spatial and temporal resolution how odor-evoked neural activity is represented, modulated and transformed22-24.
Here we describe the preparation and analysis methods to measure odor-evoked responses in the Drosophila antennal lobe using G-CaMP25-27. The animal preparation is minimally invasive and can be adapted to imaging using wide-field fluorescence, confocal and two-photon microscopes.

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

We describe an established technique to measure and analyze odor-evoked calcium responses in the antennal lobe of living Drosophila melanogaster.

Imagens de cálcio de odor-provocou respostas no Drosophila Lobo antenal

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory ...

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

Preparação de fatias agudas zona subventricular for Imaging Cálcio

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

Imagem de dois fotões cálcio em dendritos neuronais no cérebro fatias

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

Imagem de cálcio Ratiometric de neurônios individuais se comportando Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model animal for endocrine research, Drosophila melanogaster, which has sophisticated genetic tools and genome information, is being increasingly used. This article describes a method for the calcium imaging of Drosophila brain explants. This method enables the detection of the direct signaling of a hormone to the brain. It is well known that many peptide hormones act through G-protein-coupled receptors (GPCRs), whose activation causes an increase in the intracellular Ca2+concentration. Neural activation also elevates intracellular Ca2+ levels, from both Ca2+ influx and the release of Ca2+ stored in the endoplasmic reticulum (ER). A calcium sensor, GCaMP, can monitor these Ca2+ changes. In this method, GCaMP is expressed in the neurons of interest, and the GCaMP-expressing larval brain is dissected and cultured ex vivo. The test peptide is then applied to the brain explant, and the fluorescent changes in GCaMP are detected using a spinning disc confocal microscope equipped with a CCD camera. Using this method, any water-soluble molecule can be tested, and various cellular events associated with neural activation can be imaged using the appropriate fluorescent indicators. Moreover, by modifying the imaging chamber, this method can be used to image other Drosophila organs or the organs of other animals.

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

This paper describes a protocol for ex vivo calcium imaging of the Drosophila brain. In this method, natural or synthetic compounds can be applied to the buffer to test their ability to activate particular neurons in the brain.

Ex Vivo Imagem de cálcio para visualizar as respostas do cérebro a sinalização endócrina em Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

Rotulagem Balística de Neurônios Piramidais em Fatias Cerebrais e na Cultura Celular Primária

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

In vivo Calcium Imaging of Mouse Geniculate Ganglion Neuron Responses to Taste Stimuli

Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using calcium as a proxy for neuronal activity, these techniques provide a way to monitor the responses of individual cells within large neuronal ensembles to a variety of stimuli in real time. We, and others, have applied these techniques to image the responses of individual geniculate ganglion neurons to taste stimuli applied to the tongues of live anesthetized mice. The geniculate ganglion is comprised of the cell bodies of gustatory neurons innervating the anterior tongue and palate as well as some somatosensory neurons innervating the pinna of the ear. Imaging the taste-evoked responses of individual geniculate ganglion neurons with GCaMP has provided important information about the tuning profiles of these neurons in wild-type mice as well as a way to detect peripheral taste miswiring phenotypes in genetically manipulated mice. Here we demonstrate the surgical procedure to expose the geniculate ganglion, GCaMP fluorescence image acquisition, initial steps for data analysis, and troubleshooting. This technique can be used with transgenically encoded GCaMP, or with AAV-mediated GCaMP expression, and can be modified to image particular genetic subsets of interest (i.e., Cre-mediated GCaMP expression). Overall, in vivo calcium imaging of geniculate ganglion neurons is a powerful technique for monitoring the activity of peripheral gustatory neurons and provides complementary information to more traditional whole-nerve chorda tympani recordings or taste behavior assays.

Here we present how to expose the geniculate ganglion of a live, anesthetized laboratory mouse and how to use calcium imaging to measure the responses of ensembles of these neurons to taste stimuli, allowing for multiple trials with different stimulants. This allows for in depth comparisons of which neurons respond to which tastants.

In vivo Imagem de cálcio do rato Geniculado Gânglio Respostas ao Sabor Estímulo

Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

In vivo Imagem de cálcio em rato de azeitona inferior

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Detection of G Protein-coupled Receptor Expression in Mouse Vagal Afferent Neurons using Multiplex In Situ Hybridization

This study describes a protocol for the multiplex in situ hybridization (ISH) of the mouse jugular-nodose ganglia, with a particular emphasis on detecting the expression of G protein-coupled receptors (GPCRs). Formalin-fixed jugular-nodose ganglia were processed with the RNAscope technology to simultaneously detect the expression of two representative GPCRs (cholecystokinin and ghrelin receptors) in combination with one marker gene of either nodose (paired-like homeobox 2b, Phox2b) or jugular afferent neurons (PR domain zinc finger protein 12, Prdm12). Labeled ganglia were imaged using confocal microscopy to determine the distribution and expression patterns of the aforementioned transcripts. Briefly, Phox2b afferent neurons were found to abundantly express the cholecystokinin receptor (Cck1r) but not the ghrelin receptor (Ghsr). A small subset of Prdm12 afferent neurons was also found to express Ghsr and/or Cck1r. Potential technical caveats in the design, processing, and interpretation of multiplex ISH are discussed. The approach described in this article may help scientists in generating accurate maps of the transcriptional profiles of vagal afferent neurons.

Detection of G Protein-coupled Receptor Expression in Mouse Vagal Afferent Neurons using Multiplex In Situ Hybridization

Multiplex in situ hybridization (ISH) was employed to simultaneously visualize the transcripts for two G protein-coupled receptors and one transcription factor in the entire vagal ganglionic complex of the adult mouse. This protocol could be used to generate accurate maps of the transcriptional profiles of vagal afferent neurons.

Detecção de expressão receptora acoplada à proteína G em neurônios aferentes do mouse vagal usando multiplex in situ hibridização

This study describes a protocol for the multiplex in situ hybridization (ISH) of the mouse jugular-nodose ganglia, with a particular emphasis on detecting ...