Questo lavoro è stato finanziato dal Consiglio svedese della ricerca, Università di Uppsala e Fredrik e Ingrid Thurings Foundation. ZJ tenuto una borsa di studio post-dottorato da EXODIAB e Sällskapet Svenska för Medicinsk Forskning. Ringraziamo Frank Lee per l&#39;assistenza tecnica.

<ol>
	<li>Pavlov, I., Savtchenko, L. P., Kullmann, D. M., Semyanov, A., Walker, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outwardly+rectifying+tonically+active+GABAA+receptors+in+pyramidal+cells+modulate+neuronal+offset,+not+gain.">Outwardly rectifying tonically active GABAA receptors in pyramidal cells modulate neuronal offset, not gain.</a> J Neurosci. 29, 15341-15350 (2009).</li><li>Chao, H. T., Chen, H., Samaco, R. C., Xue, M., Chahrour, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysfunction+in+GABA+signalling+mediates+autism-like+stereotypies+and+Rett+syndrome+phenotypes.">Dysfunction in GABA signalling mediates autism-like stereotypies and Rett syndrome phenotypes.</a> Nature. 468, 263-269 (2010).</li><li>Clarkson, A. N., Huang, B. S., Macisaac, S. E., Mody, I., Carmichael, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reducing+excessive+GABA-mediated+tonic+inhibition+promotes+functional+recovery+after+stroke.">Reducing excessive GABA-mediated tonic inhibition promotes functional recovery after stroke.</a> Nature. 468, 305-309 (2010).</li><li>Jin, Z., Jin, Y., Mendu, K. M., Degerman, E., Groop, L., Birnir, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+reduces+neuronal+excitability+by+turning+on+GABA(A)+channels+that+generate+tonic+current.">Insulin reduces neuronal excitability by turning on GABA(A) channels that generate tonic current.</a> Plos One. 6, e16188-e16188 (2011).</li><li>Nusser, Z., Mody, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+modulation+of+tonic+and+phasic+inhibitions+in+dentate+gyrus+granule+cells.">Selective modulation of tonic and phasic inhibitions in dentate gyrus granule cells.</a> J Neurophysiol. 87, 2624-2628 (2002).</li><li>Semyanov, A., Walker, M. C., Kullmann, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA+uptake+regulates+cortical+excitability+via+cell+type-specific+tonic+inhibition.">GABA uptake regulates cortical excitability via cell type-specific tonic inhibition.</a> Nat Neurosci. 6, 484-490 (2003).</li><li>Birnir, B., Korpi, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+sub-cellular+location+and+intracellular+neuronal+proteins+on+properties+of+GABA(A)+receptors.">The impact of sub-cellular location and intracellular neuronal proteins on properties of GABA(A) receptors.</a> Curr Pharm Des. 13, 3169-3177 (2007).</li><li>Olsen, R. W., Sieghart, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+Union+of+Pharmacology.+LXX.+Subtypes+of+gamma-aminobutyric+acid(A)+receptors:+classification+on+the+basis+of+subunit+composition,+pharmacology,+and+function.">International Union of Pharmacology. LXX. Subtypes of gamma-aminobutyric acid(A) receptors: classification on the basis of subunit composition, pharmacology, and function.</a> Update. Pharmacol Rev. 60, 243-260 (2008).</li><li>Hamill, O. P., Marty, A., Neher, E., Sakmann, B., Sigworth, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+patch-clamp+techniques+for+high-resolution+current+recording+from+cells+and+cell-free+membrane+patches.">Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.</a> Pflugers Arch. 391, 85-100 (1981).</li><li>Sakmann, B., Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Single-Channel Recording. , (1995).</li><li>Zilberter, Y., Zilberter, T., Bregestovski, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+activity+in+vitro+and+the+in+vivo+reality:+the+role+of+energy+homeostasis.">Neuronal activity in vitro and the in vivo reality: the role of energy homeostasis.</a> Trends Pharmacol Sci. 31, 394-401 (2010).</li><li>Spruston, N., McBain, C., Andersen, P., Morris, R., Amaral, D., Bliss, T., O'keefe, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+properties+of+hippocampal+neurons.">Structural and functional properties of hippocampal neurons.</a> The Hippocampus Book. , (2007).</li><li>Andersen, P., Morris, R., Amaral, D., Bliss, T., O'keefe, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Hippocampus Book. , (2007).</li><li>Eghbali, M., Curmi, J. P., Birnir, B., Gage, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampal+GABA(A)+channel+conductance+increased+by+diazepam.">Hippocampal GABA(A) channel conductance increased by diazepam.</a> Nature. 388, 71-75 (1997).</li><li>Lindquist, C. E., Birnir, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Graded+response+to+GABA+by+native+extrasynaptic+GABA+receptors.">Graded response to GABA by native extrasynaptic GABA receptors.</a> J Neurochem. 97, 1349-1356 (2006).</li><li>Birnir, B., Eghbali, M., Cox, G. B., Gage, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA+concentration+sets+the+conductance+of+delayed+GABAA+channels+in+outside-out+patches+from+rat+hippocampal+neurons.">GABA concentration sets the conductance of delayed GABAA channels in outside-out patches from rat hippocampal neurons.</a> J Membr Biol. 181, 171-183 (2001).</li><li>Birnir, B., Eghbali, M., Everitt, A. B., Gage, P. W. B. i. c. u. c. u. l. l. i. n. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pentobarbital+and+diazepam+modulate+spontaneous+GABA(A)+channels+in+rat+hippocampal+neurons.">pentobarbital and diazepam modulate spontaneous GABA(A) channels in rat hippocampal neurons.</a> Br J Pharmacol. 131, 695-704 (2000).</li><li>Birnir, B., Everitt, A. B., Gage, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+GABAA+channels+in+rat+dentate+gyrus.">Characteristics of GABAA channels in rat dentate gyrus.</a> J Membr Biol. 142, 93-102 (1994).</li></ol>

Nessun conflitto di interessi dichiarati.

L&#39;intera-cella corrente rappresenta l&#39;insieme delle correnti generate da canali attivi nella cella. La farmacologia specifica e cloruro di permeabilità dei canali GABAA permette di identificare le correnti associate a questi canali. La natura transitoria di attivazione di canali sinaptica e l&#39;attivazione tonica dei canali extrasinaptica permette di differenziare facilmente tra queste due popolazioni di canali GABAA espressi in un neurone. Tuttavia, solo con un singolo canale registrazioni si può studiare le proprietà cinetiche delle molecole canale che generano le correnti tonico. A differenza dei canali sinaptici che attivano entro 100 ms, i canali GABAA tonica sono attivati ​​con una latenza nella gamma di minuti dal momento GABA è applicata 4,7,15,16,17,18. Questa attivazione ritardata dei risultati canali che le proprietà del canale molti non possono essere studiate usando cellule intere registrazioni. Al canale singolo livello i diversi sotto-popolazioni di canali GABAA possono essere identificati sulla base di diverse proprietà funzionali e farmacologiche. Così, prendendo un vantaggio di ciò che le diverse configurazioni di patch-clamp hanno da offrire, è possibile studiare in dettaglio la funzione molecolare e farmacologia del GABA-attivati ​​i canali che generano le correnti toniche e sinaptica nei neuroni.

Diverse patch-clamp sistemi hardware e software sono disponibili e tutti funzionano utilizzando gli stessi principi di base. Nel nostro laboratorio usiamo gli amplificatori Axoclamp e il software PClamp (Molecular Devices, USA) per registrare e analizzare i nostri esperimenti. Un altro marchio popolare è amplificatori HEKA e software (HEKA Elektronik, Germania) Microforge: Narishige MF-830 (Giappone); Vibrotome: ad esempio Leica VT1200S (Germania) o Campden Strumenti vibrotome (UK); microscopio: Nikon Eclipse E600FN; Bagnomaria; Micromanipolatori: misura e micromanipolatori Narishige (Giappone); Estrattore: DMZ- universale (Germania) o Narishige PC-10 (Giappone). Elettrodo di vetro: GC150F-15 (Harvard Apparatus, UK).

gaba-activated single-channel and tonic currents in rat brain slices

Il GABA A canali sono presenti in tutti i neuroni e si trovano sia a sinapsi e al di fuori delle sinapsi dove generano correnti fasica e tonica, rispettivamente 4,5,6,7 Il GABA Un canale è un pentamerica GABA-dipendenti canale del cloro. Le subunità del canale sono raggruppati in 8 famiglie (α1-6, β1-3, γ1-3, δ, ε, θ, π e ρ). Due alfa, due beta e uno da 3 subunità ° modulo funzionale del canale 8. Unendo studi di sottotipo specifico GABA-attivati ​​monocanale molecole con studi tra cui tutte le popolazioni del GABA A canali nel neurone diventa possibile comprendere il meccanismo di base di inibizione neuronale e come viene modulato da agenti farmacologici. Usiamo il patch-clamp 9,10 per studiare le proprietà funzionali dei canali GABA A nei neuroni vivi in fettine di ippocampo cervello e registrare il singolo canale e cellule intere correnti. Abbiamo ulteriormente esaminare come i canali sono influenzati da differenti concentrazioni di GABA, altri farmaci e fattori intra ed extracellulare. Per informazioni dettagliate descrizione teorica e pratica del patch-clamp metodo si veda The Single-Channel Registrazioni a cura di B ed E Sakman Neher 10.

Watch this Scientific Journal Video about GABA-activated Single-channel and Tonic Currents in Rat Brain Slices at JoVE.com

GABA-activated Single-channel and Tonic Currents in Rat Brain Slices

Usiamo il patch-clamp per misurare GABA-activated monocanale correnti (GABAA canali, recettori GABA) e le correnti sinaptiche e tonico che generano nei neuroni. L&#39;attivazione dei canali diminuisce l&#39;eccitabilità neuronale nella salute e nella malattia<sup&gt; 1,2,3,4</sup&gt;.

Correnti a singolo canale e Tonico GABA-attivati ​​in fettine di cervello di ratto

The GABAA channels are present in all neurons and are located both at synapses and outside of synapses where they generate phasic and tonic currents, ...

gaba-activated-single-channel-and-tonic-currents-in-rat-brain-slices

Research

JoVE Journal

Neuroscience

25.1K Views.  Uppsala University, Sweden. Usiamo il patch-clamp per misurare GABA-activated monocanale correnti (GABAA canali, recettori GABA) e le correnti sinaptiche e tonico che generano nei neuroni. L'attivazione dei canali diminuisce l'eccitabilità neuronale nella salute e nella malattia 1,2,3,4.

Video: GABA-activated Single-channel and Tonic Currents in Rat Brain Slices

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

Associati a cellule intere registrazioni in organotipiche fettine di ippocampo

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

Ricerca

Neuroscienze

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

Ex Vivo optogenetic dissezione di circuiti paura nel cervello fette

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

Registrazioni Whole-cell patch-clamp nel cervello fette

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

Registrazione di plasticità sinaptica in fettine ippocampali Acute mantenuti in un piccolo volume riciclaggio - aspersione- e sistema di immersione-tipo camera

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

Valutazione di densità di sinapsi nelle fette Hippocampal del cervello del roditore

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

Whole-cell Currents Induced by Puff Application of GABA in Brain Slices

Pharmacological administration is commonly used when conducting whole-cell patch-clamp recording in brain slices. One of the best methods of drug application during electrophysiological recording is the puff technique, which can be used to study the effect of pharmacological reagents on neuronal activities in brain slices. The greatest advantage of puff application is that the drug concentration around the recording site increases rapidly, thus preventing desensitization of membrane receptors. Successful use of puff application involves careful attention to the following elements: the concentration of the drug, the parameters of the puff micropipette, the distance between the tip of a puff micropipette and the neuron recorded, and the duration and pressure driving the puff (pounds per square inch, psi). This article describes a step-by-step procedure for recording whole-cell currents induced by puffing gamma-aminobutyric acid (GABA) onto a neuron of a prefrontal cortical slice. Notably, the same procedure can be applied with minor modifications to other brain areas such as the hippocampus and the striatum, and to different preparations, such as cell cultures.

We describe the puff technique, by which pharmacological reagents can be administered during whole-cell patch-clamp recording, and highlight various aspects of the features that are crucial for its success.

Cellule intere correnti indotte dall'applicazione di Puff di GABA nelle fette del cervello

Pharmacological administration is commonly used when conducting whole-cell patch-clamp recording in brain slices. One of the best methods of drug ...

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.

Imaging del due-fotone calcio nei dendriti neuronali nelle fette del cervello

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

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

Un approccio alternativo per studiare eventi primari nella neurodegenerazione utilizzando fettine di cervello di ratto Ex Vivo

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) is a promising approach when pharmacological treatment fails or neurosurgery is not recommended. Acute brain slices coupled to microelectrode arrays (MEAs) represent a valuable tool to study neuronal network interactions and their modulation by electrical stimulation. As compared to conventional extracellular recording techniques, they provide the added advantages of a greater number of observation points and a known inter-electrode distance, which allow studying the propagation path and speed of electrophysiological signals. However, tissue oxygenation may be greatly impaired during MEA recording, requiring a high perfusion rate, which comes at the cost of decreased signal-to-noise ratio and higher oscillations in the experimental temperature. Electrical stimulation further stresses the brain tissue, making it difficult to pursue prolonged recording/stimulation epochs. Moreover, electrical modulation of brain slice activity needs to target specific structures/pathways within the brain slice, requiring that electrode mapping be easily and quickly performed live during the experiment. Here, we illustrate how to perform the recording and electrical modulation of 4-aminopyridine (4AP)-induced epileptiform activity in rodent brain slices using planar MEAs. We show that the brain tissue obtained from mice outperforms rat brain tissue and is thus better suited for MEA experiments. This protocol guarantees the generation and maintenance of a stable epileptiform pattern that faithfully reproduces the electrophysiological features observed with conventional field potential recording, persists for several hours, and outlasts sustained electrical stimulation for prolonged epochs. Tissue viability throughout the experiment is achieved thanks to the use of a small-volume custom recording chamber allowing for laminar flow and quick solution exchange even at low (1 mL/min) perfusion rates. Quick MEA mapping for real-time monitoring and selection of stimulating electrodes is performed by a custom graphic user interface (GUI).

We illustrate how to perform recording and electrical modulation of 4-aminopyridine-induced epileptiform activity in rodent brain slices using microelectrode arrays. A custom recording chamber maintains tissue viability throughout prolonged experimental sessions. Live electrode mapping and selection of stimulating pairs are performed by a custom graphical user interface.

Registrazione e modulazione di attività Epileptiform in fettine di cervello del roditore accoppiate a matrici di microelettrodi

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

Valutazione dell'induzione della depressione a lungo termine nelle fette di cerebellar adulto

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...

Correnti a singolo canale e Tonico GABA-attivati ​​in fettine di cervello di ratto

Correnti a singolo canale e Tonico GABA-attivati in fettine di cervello di ratto