Wir danken unseren Kollegen, die in der Arbeit beteiligt hier zusammengefasst. Diese Arbeit wurde durch Fördermittel der Deutschen Forschungsgemeinschaft (SFB 894), der DFG Schwerpunktprogramm 1392 &quot;Integrative Analyse des Geruchssinns&quot; und von der Volkswagen-Stiftung (TLZ) unterstützt. TLZ ist eine Lichtenberg Professor der Volkswagen-Stiftung.

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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>

Keine Interessenskonflikte erklärt.

Eine wichtige Frage in den Neurowissenschaften ist es zu verstehen, wie das Gehirn soziale Informationen verarbeitet. Eine vorherrschende Quelle von Informationen, die für die gesellschaftliche Anerkennung wird durch Geruchs-oder Pheromone Signale kodiert. Der Nachweis dieser Signale durch neuronale Populationen in der Nase und der Erkennung der Signale im Gehirn, insbesondere der Hypothalamus, eine wichtige Rolle spielen in vielen sozialen Prozesse und Einfluss Hormone und andere neuroendokrine Faktoren 13-16.</s...

There was a typo in the abstract of <a href="http://jove.unicartagenaproxy.elogim.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> Name </td><td> Firma </td><td> Cat. Nr. </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> Dimethylsulfoxid </td><td> Fisher Scientific </td><td> BP231 </td></tr><tr><td> Vibrierende-Blade Mikrotom Hyrax V 50 </td><td> Zeiss </td><td> 9770170 </td></tr><tr><td> Kühlgerät CU 65 für Mikrotom Hyrax V 50 </td><td> Zeiss </td><td> 9920120 </td></tr><tr><td> O 2 / CO 2 Inkubator, CB210-UL </td><td> Binder </td><td> 0019389 </td></tr><tr><td> Super Kleber, Loctite 406TM </td><td> Henckel </td><td> 142580 </td></tr><tr><td> Doppelspatel, Löffelform </td><td>Bochem </td><td> 3182 </td></tr><tr><td> Mikrolöffel Spatel, Löffel Form </td><td> Bochem </td><td> 3344 </td></tr><tr><td> Federschere, Moria-Vannas-Wolff - 7mm Blades </td><td> Fine Science Tools </td><td> 15370-52 </td></tr><tr><td> Federschere, Vannas - 3mm Blades </td><td> Fine Science Tools </td><td> 15000-00 </td></tr><tr><td> Wagner Scissors </td><td> Fine Science Tools </td><td> 14071-12 </td></tr><tr><td> Medical Forceps, Dumont 7b </td><td> Fine Science Tools </td><td> 11270-20 </td></tr><tr><td> Große rechteckige Open Bath Chamber (RC-27) </td><td> Warner Instruments </td><td> 64-0238 </td></tr><tr><td> Konfokalen Mikroskop BioRad Radiance 2100 </td><td> Zeiss </td><td> na </td></tr></tbody></table>

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

Trotz einer enormen Steigerung in unserem Wissen über die Mechanismen der Kodierung von Informationen im Gehirn, eine zentrale Frage, die die genauen molekularen Schritte sowie die Aktivität bestimmter Nervenzellen im multifunktionalen Kerne von Hirnarealen wie dem Hypothalamus bleiben. Dieses Problem umfasst die Identifizierung der molekularen Komponenten der Regulation verschiedener Neurohormon Signaltransduktionskaskaden beteiligt. Erhöhungen der intrazellulären Ca 2 + spielen eine wichtige Rolle bei der Regulierung der Empfindlichkeit von Nervenzellen, sowohl auf der Ebene der Signaltransduktion und an synaptischen Websites. Neue Werkzeuge sind entstanden, um zur Ermittlung Neuronen in der Vielzahl von Neuronen des Gehirns durch Expression grün fluoreszierendes Protein (GFP) unter der Kontrolle eines bestimmten Promotors. Sowohl räumlich als auch zeitlich Stimulus-induzierten überwachen Ca 2 +-Antworten in GFP-markierten Neuronen, eine nicht-grünen fluoreszierenden Ca 2 +-Indikatorfarbstoff nEEDS verwendet werden. Darüber hinaus ist ein beliebtes Konfokalmikroskopie Verfahren zum Abbilden einzelner Neuronen in Gewebeschnitten durch seine Fähigkeit, Neuronen in verschiedenen Ebenen der Tiefe innerhalb des Gewebes sichtbar zu machen und außerhalb des Fokus Fluoreszenz begrenzen. Die ratiometrische Ca 2 +-Indikator Fura-2 wurde in Kombination mit Neuronen verwendet ein GFP-markierten. Jedoch wird der Farbstoff durch ultraviolettes (UV) Licht angeregt. Die Kosten für den Laser und die begrenzte optische Eindringtiefe des UV-Lichts verhindert die Verwendung in vielen Laboratorien. Darüber hinaus kann GFP-Fluoreszenz mit den Fura-2 Signale 2 stören. Deshalb haben wir beschlossen, ein rot fluoreszierendes Ca 2 +-Indikator-Farbstoff verwenden. Die riesige Strokes Verschiebung der Fura-Rot erlaubt multicolor Analyse der roten Fluoreszenz in Kombination mit GFP mit einer einzigen Anregungswellenlänge. Wir hatten zuvor gute Ergebnisse mit Fura-Rot in Kombination mit GFP-markierten olfaktorischen Neuronen 3. Die Protokolle für olfaktorischen Gewebeschnitten schien e arbeitenQually auch in Neuronen des Hypothalamus 4. Fura-rot Basis Ca 2 +-Imaging wurde auch erfolgreich mit GFP-markierten Pankreas β-Zellen und GFP-markierten Rezeptoren in HEK-Zellen 5,6 Ausdruck kombiniert. Ein wenig Laune des Fura-Rot ist, dass seine Fluoreszenzintensität bei 650 nm, wenn die Anzeige bindet Calcium 7 abnimmt. Daher weist die Fluoreszenz des ruhenden Neuronen mit niedrigen Ca 2 +-Konzentration relativ hoher Intensität. Es sollte angemerkt werden, dass andere rote Ca 2 +-Indikatorfarbstoffe existieren oder derzeit entwickelt werden, könnte das eine bessere oder verbesserte Ergebnisse in verschiedenen Neuronen und Hirnareale.

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

In diesem Protokoll, aktualisieren wir die jüngsten Fortschritte in der Bildgebung Ca<sup&gt; 2 +</sup&gt; Signale GFP-markierten Neuronen im Gehirn Gewebeschnitte mit einem rot fluoreszierenden Ca<sup&gt; 2 +</sup&gt; Indikatorfarbstoff.

Imaging Calcium Antworten in GFP-markierten Neuronen des Hypothalamus Maus Hirnschnitten

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

17.8K Aufrufe.  University of Saarland, Homburg, Germany. In diesem Protokoll, aktualisieren wir die jüngsten Fortschritte in der Bildgebung Ca 2 + Signale GFP-markierten Neuronen im Gehirn Gewebeschnitte mit einem rot fluoreszierenden Ca 2 + Indikatorfarbstoff.

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

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Vibrodissociation von Neuronen aus Nager Hirnschnitten zu Study Synaptic Transmission and Image präsynaptischen

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Forschung

Neurowissenschaften

Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within the same species 1,2. These intraspecies signals, the pheromones, as well as signals from some predators 3, activate the vomeronasal sensory neurons (VSNs) with high levels of specificity and sensitivity 4. At least three distinct families of G-protein coupled receptors, V1R, V2R and FPR 5-14, are expressed in VNO neurons to mediate the detection of the chemosensory cues. To understand how pheromone information is encoded by the VNO, it is critical to analyze the response profiles of individual VSNs to various stimuli and identify the specific receptors that mediate these responses.
The neuroepithelia of VNO are enclosed in a pair of vomer bones. The semi-blind tubular structure of VNO has one open end (the vomeronasal duct) connecting to the nasal cavity. VSNs extend their dendrites to the lumen part of the VNO, where the pheromone cues are in contact with the receptors expressed at the dendritic knobs. The cell bodies of the VSNs form pseudo-stratified layers with V1R and V2R expressed in the apical and basal layers respectively 6-8. Several techniques have been utilized to monitor responses of VSNs to sensory stimuli 4,12,15-19. Among these techniques, acute slice preparation offers several advantages. First, compared to dissociated VSNs 3,17, slice preparations maintain the neurons in their native morphology and the dendrites of the cells stay relatively intact. Second, the cell bodies of the VSNs are easily accessible in coronal slice of the VNO to allow electrophysiology studies and imaging experiments as compared to whole epithelium and whole-mount preparations 12,20. Third, this method can be combined with molecular cloning techniques to allow receptor identification.
Sensory stimulation elicits strong Ca2+ influx in VSNs that is indicative of receptor activation 4,21. We thus develop transgenic mice that express G-CaMP2 in the olfactory sensory neurons, including the VSNs 15,22. The sensitivity and the genetic nature of the probe greatly facilitate Ca2+ imaging experiments. This method has eliminated the dye loading process used in previous studies 4,21. We also employ a ligand delivery system that enables application of various stimuli to the VNO slices. The combination of the two techniques allows us to monitor multiple neurons simultaneously in response to large numbers of stimuli. Finally, we have established a semi-automated analysis pipeline to assist image processing.

The vomeronasal organ (VNO) detects intraspecies chemical signals that convey social and reproductive information. We have performed Ca2+ imaging experiments using transgenic mice expressing G-CaMP2 in VNO tissue. This approach allows us to analyze the complicated response patterns of the vomeronasal neurons to large numbers of pheromone stimuli.

Imaging neuronalen Antworten in Scheiben Zubereitungen von Vomeronasalorgan Ausdruck eines genetisch kodierte Kalzium-Sensor

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within ...

Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study these two systems and how they interact has been with the use of in vivo preparations. Major advances have been made with acute brain slices containing the thalamocortical and cortico-thalamic pathways in the somatosensory, visual, and auditory systems. With key refinements to our recent modification of the auditory thalamocortical slice1, we are able to more reliably capture the projections between most of the major auditory midbrain and forebrain structures: the inferior colliculus (IC), medial geniculate body (MGB), thalamic reticular nucleus (TRN), and the auditory cortex (AC). With portions of all these connections retained, we are able to answer detailed questions that complement the questions that can be answered with in vivo preparations. The use of flavoprotein autofluorescence imaging enables us to rapidly assess connectivity in any given slice and guide the ensuing experiment. Using this slice in conjunction with recording and imaging techniques, we are now better equipped to understand how information processing occurs at each point in the auditory forebrain as information ascends to the cortex, and the impact of descending cortical modulation. 3-D printing to build slice chamber components permits double-sided perfusion and broad access to networks within the slice and maintains the widespread connections key to fully utilizing this preparation.

Using multiple angles to cut the mouse pup brain, we improve upon a previously-described acute brain slice which captures the connections between most of the major auditory midbrain and forebrain structures.

Modifikation eines Colliculo-thalamokortikalen Maus Hirnschnitt, Einbindung der 3D-Druckkammerkomponenten und Multiskalen Optical Imaging

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

Neuronalen Aktivität im primären somatosensorischen Cortex mit Thy1Imaging-GCaMP6s transgene Mäuse

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

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.

Zwei-Photonen-Kalzium Imaging in neuronalen Dendriten in Hirnschnitten

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

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 Calcium Imaging of Mouse Geniculate Ganglion Neuron Reaktionen auf Geschmacksreize

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 Calcium Imaging in Maus 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 ...

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the cytoplasm or the release of intracellular Ca2+ stores. Pirt-GCaMP3-based Ca2+ imaging of primary sensory neurons of the dorsal root ganglion (DRG) in mice offers the advantage of simultaneous measurement of a large number of cells. Up to 1,800 neurons can be monitored, allowing neuronal networks and somatosensory processes to be studied as an ensemble in their normal physiological context at a populational level in vivo. The large number of neurons monitored allows the detection of activity patterns that would be challenging to detect using other methods. Stimuli can be applied to the mouse hindpaw, allowing the direct effects of stimuli on the DRG neuron ensemble to be studied. The number of neurons producing Ca2+ transients as well as the amplitude of Ca2+ transients indicates sensitivity to specific sensory modalities. The diameter of neurons provides evidence of activated fiber types (non-noxious mechano vs. noxious pain fibers, A&#946;, A&#948;, and C fibers). Neurons expressing specific receptors can be genetically labeled with td-Tomato and specific Cre recombinases together with Pirt-GCaMP. Therefore, Pirt-GCaMP3 Ca2+ imaging of DRG provides a powerful tool and model for the analysis of specific sensory modalities and neuron subtypes acting as an ensemble at the populational level to study pain, itch, touch, and other somatosensory signals.

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

This protocol describes the surgical exposure of the dorsal root ganglion (DRG) followed by GCaMP3 (genetically-encoded Ca2+ indicator; Green Fluorescent Protein-Calmodulin-M13 Protein 3) Ca2+ imaging of the neuronal ensembles using Pirt-GCaMP3 mice while applying a variety of stimuli to the ipsilateral hind paw.

Im lebenden Organismus Calcium-Bildgebung neuronaler Ensembles in Netzwerken primärer sensorischer Neurone in intakten dorsalen Wurzelganglien

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the ...

Zwei-Photonen- und Weitfeld-Kalziumbildgebung im Colliculus superior

Calcium Imaging in Mouse Superior Colliculus

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the emergence of the cerebral cortex. It receives direct inputs from ~30 types of retinal ganglion cells (RGCs), with each encoding a specific visual feature. It remains elusive whether the SC simply inherits retinal features or if additional and potentially de novo processing occurs in the SC. To reveal the neural coding of visual information in the SC, we provide here a detailed protocol to optically record visual responses with two complementary methods in awake mice. One method uses two-photon microscopy to image calcium activity at single-cell resolution without ablating the overlaying cortex, while the other uses wide-field microscopy to image the whole SC of a mutant mouse whose cortex is largely undeveloped. This protocol details these two methods, including animal preparation, viral injection, headplate implantation, plug implantation, data acquisition, and data analysis. The representative results show that the two-photon calcium imaging reveals visually evoked neuronal responses at single-cell resolution, and the wide-field calcium imaging reveals&#160;neural activity across the entire SC. By combining these two methods, one can reveal the neural coding in the SC at different scales, and such combination can also be applied to other brain regions.

Autor im Rampenlicht: Enthüllung der neuronalen Kodierung und der Mechanismen der visuellen Verarbeitung im Colliculus superior 

This protocol details the procedure for imaging calcium responses in the superior colliculus (SC) of awake mice, including imaging single-neuron activity with two-photon microscopy while leaving the cortex intact in wild-type mice, and imaging the entire SC with wide-field microscopy in partial-cortex mutant mice.

Kalzium-Bildgebung beim Colliculus superior der Maus

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the ...

Imaging Calcium Antworten in GFP-markierten Neuronen des Hypothalamus Maus Hirnschnitten

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In vivo Calcium Imaging in Maus Inferior Olive

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Kalzium-Bildgebung beim Colliculus superior der Maus

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