Supportato da University of Kentucky, Dipartimento di Biologia, Ufficio di studi universitari e il College of Arts &amp; Sciences.

Nessun conflitto di interessi dichiarati.

Potenziale di membrana Già nel 1902, Bernstein aveva a che fare con i temi di un potenziale di riposo nel assone di un calamaro. E &#39;interessante considerare come queste prime idee e osservazioni di Berstein (1902) e Nernst (1888) in seguito influenzato la ricerca in fisiologia della membrana. (Vedi recensione di Malmivuo e Plonsey, 1995, disponibile anche sul www <a href="http://www.bem.fi/book/">http://www.bem.fi/book/</a> ). Ci sono ancora, fino ad oggi, le scoperte in corso sulla funzione dei canali ionici e le proprietà delle membrane biologiche che sono molto importanti per comprendere la fisiologia cellulare, che si riferisce alla funzione di tessuti, organi e sistemi. Il confronto degli effetti sperimentali e teoricamente derivata esterno [K +] sul potenziale di membrana a riposo indica l&#39;influenza di ioni sul potenziale di membrana. Ulteriori esperimenti utilizzando questa stessa preparazione rimangono da eseguire per affrontare le domande fondamentali fisiologico. Alcuni sono stati evidenziati nel 1968 da Atwood e Parnas e devono ancora essere pienamente affrontato. Con le tecniche ottenute in questo esercizio, si può procedere a rispondere a molte domande ancora in altre preparazioni sperimentali così come nelle applicazioni fisiologiche legate alla medicina e alla salute. Abbiamo dimostrato l&#39;utilità di una preparazione invertebrati modello per affrontare questioni fondamentali pertinenti a tutti gli animali. Con le conoscenze acquisite sui gradienti elettrochimici degli ioni in questo esercizio di cui sopra, è ora possibile passare alla eccitabilità delle membrane esaminando la trasmissione sinaptica a preparati neuromuscolari in gamberi. Misura Risposte Synaptic I dati forniti per la prima parte di questo laboratorio, e il film associati, hanno fornito passaggi chiave per la registrazione di potenziali di membrana e indagare la struttura muscolare. Nella seconda parte di questo laboratorio, la dimostrazione di dissezione e di registrazione, la trasmissione sinaptica alla NMJs di unità motorie fasica e tonica ha fornito una esposizione a concetti fondamentali nella fisiologia. L&#39;esposizione ad un circuito neurale, che può in parte può essere usata per spiegare comportamenti associati, nell&#39;animale intatto ha un potenziale non solo per gli studenti per studiare varie questioni di tipo aperto all&#39;interno del loro esercizio di laboratorio, ma anche per la ricerca futura sui circuiti neuronali in un pozzo invertebrati preparazione stabilita (Kennedy et al, 1969;. Antonsen e Edwards, 2003) Queste preparazioni possono essere utilizzati anche per studiare facilitazione sinaptica, depressione e lungo termine plasticità (non indagato in questo studio di laboratorio). Anche all&#39;interno di alcune specie di gamberi, plasticità neuronale dipende dallo stimolo condizioni sperimentali (Mercier e Atwood, 1989;. Cooper et al, 1998) così come la loro ambiente naturale. In che misura la capacità di alterare l&#39;efficacia sinaptica e la dinamica dei muscoli serve l&#39;animale resta ancora da esplorare. Dal gambero si alterano il loro comportamento in relazione alle variazioni stagionali e il ciclo di muta, ci sono relativamente a lungo termine differenze attività nei loro sistemi neuromuscolare. E &#39;stato dimostrato che la fasica terminali del motore nervose dei muscoli artiglio più vicino esporre la morfologia classica fasica durante l&#39;inverno, ma si gonfiano e diventano più varicose per tutta la lunghezza del terminale durante i mesi estivi (Lnenicka 1993; Lnenicka e Zhao, 1991). Alcuni primi studi condotti in laterale gamberi giganti (LG) interneuroni all&#39;interno del cordone nervoso ventrale dimostrato la presenza di giunzioni gap (Johnson, 1924; Watanabe e Grundfest, 1961). E &#39;noto che la CO2 ha un effetto sulla comunicazione elettrica di disaccoppiamento giunzioni gap (Arellano et al, 1990). E &#39;stato recentemente dimostrato che il cordone nervoso e la comunicazione all&#39;interno del sistema nervoso centrale-sensoriale-motorio-muscolare del circuito, come descritto nella presente relazione, è anche sensibile al CO 2 l&#39;esposizione, che indica la presenza di giunzioni gap (Bierbower, 2010; Bierbower e Cooper, 2010) L&#39;attività spontanea della radice 3 ° motore è stato un argomento in quanto il 1960, quando Eckert (1961) ha esaminato se il tonico di tiro statico organo recettore muscolare (MRO) nello stesso segmento o vicini potrebbe spiegare l&#39;unità motoria spontanea. In questi studi precedenti è emerso che l&#39;attività è stata guidata all&#39;interno del cordone nervoso ventrale (VNC) eventualmente da centri superiori (Eckert, 1961; Kennedy e Takeda, 1965a, b;. Strawn et al, 2000). Poiché la presenza di CO 2 smesso l&#39;attività spontanea, si può assumere in qualche parte del disco ai motoneuroni potrebbe esserci giunzioni gap o glutamatergica disco eccitatori. Il NMJs sono bloccati o esibire diminuita sensibilità al glutammato in presenza di CO 2, e possono essere bloccoKED come pure all&#39;interno del sistema nervoso centrale (Bierbower, 2010; Bierbower e Cooper, 2010; vedi anche Badre et al, 2005).. L&#39;azione di diversi neuromodulatori è anche facilmente studiato i vari tipi di NMJs (Cooper e Cooper, 2009; Griffis et al, 2000;. Southard et al, 2000;.. Strawn et al, 2000). Inoltre, varie influenze sono esercitate dai neuromodulatori sul circuito nervoso centrale. È stato suggerito che la 5-HT e neuroni octopaminergic può funzionare come &#39;guadagno-setter&#39; nel alterando la produzione di circuiti neuronali (Ma et al, 1992;. Schneider et al, 1996;. Hörner et al, 1997;. Edwards et al., 2002). Molto lavoro resta da fare prima di poter comprendere appieno gli effetti dei neuromodulatori sulle cellule bersaglio individuali. Dato che neuromodulatori diversi possono lavorare in concerto con gli altri, l&#39;analisi della loro azione mista è uno spazio per la ricerca futura (Djokaj et al., 2001). Inoltre, pochi studi, in particolare nei vertebrati, affrontare gli effetti dei neuromodulatori su percorsi intero che può regolare uno specifico comportamento. In questo senso-SNC-motore preparazione unità si può esaminare l&#39;influenza sia di input sensoriali e neuromodulatori sull&#39;attività dei motoneuroni (Kennedy et al., 1969). Poiché è stato ipotizzato che il 5-HT gioca un ruolo nella regolazione dello stato comportamentale di gamberi, aragoste e granchi (Livingstone et al, 1980;.. Sneddon et al, 2000), diversi tentativi sono stati fatti per determinare la sua concentrazione in il VNC, l&#39;emolinfa, e nei gangli isolati di aragoste (Livingstone et al, 1980;. Harris-Warrick e Kravitz 1984;. Fadool et al, 1988). Tuttavia, c&#39;è stata una notevole variazione nelle misure registrato, precludendo specifiche relazioni dose-risposta, che potrebbe spiegare le azioni comportamentali. Un gambero di fiume con gli artigli tenuto in posizione rialzata e con la coda nascosta sotto il suo addome è stato pensato di esporre una posizione dominante (Livingstone et al., 1980). Lo stato di flessione addominale in gamberi non sembra essere la postura che gambero dominante, all&#39;interno di una coppia, mostra durante le interazioni sociali e, pur mantenendo uno status dominante gerarchico (Listerman et al., 2000). Gamberi sottomesso sarà anche infilare sotto i loro addomi se stessi come si ritirano da un avversario. Rimboccare coda tale è anche visto come una postura di difesa (Listerman et al., 2000). Questi comportamenti sono stati prontamente osservati in campo e in laboratorio (Bovbjerg, 1953, 1956; Bruski e Dunham, 1987; Li et al, 2000;.. Listerman et al, 2000). È interessante notare che le posture del comportamento osservato nel aragoste (Livingstone et al., 1980) sono invertiti per la 5-HT e iniezioni octopamina nel gambero australiano, distruttore Cherax (McRae, 1996). Forse, risposte completamente diverse sarebbe osservata nella preparazione flessore superficiale nel gambero australiano. Inoltre, poiché il dominio è generalmente dimensioni legati tra gamberi, ci si aspetterebbe un sistema di risposta molto rapido in plastica per mutate condizioni sociali (Strawn et al., 2000). La plasticità in risposta a neuromodulatori negli invertebrati è uno spazio aperto di indagine. Wyttenbach, Johnson e Hoy (1999) hanno prodotto dei media digitali e un manuale di laboratorio per sperimentazioni che coinvolgono le varie gamberi muscolare stesso presentati in questo rapporto, oltre ad altre preparazioni di gamberi. Questa è una risorsa eccellente per le esercitazioni degli studenti.

<ol><li> Gambero di fiume (Procambarus clarkii). Atchafalaya biologica Supply Co., Raceland, LA., USA. </li><li> Standard di gamberi salina: Modificato dalla soluzione di Van Harreveld (1936). (In mm) 205 NaCl, 5.3 KCl; 13,5 CaCl 2 2H 2 O; 2,45 MgCl 2 6H 2 O, 5 HEPES e portato a pH 7.4. Serotonina, dopamina, glutammato e sono realizzati in soluzione salina gamberi. Soluzione fissante di Bouin è stato utilizzato direttamente e blu di metilene è fatto in soluzione salina gamberi. Tutti i prodotti chimici sono ottenuti da Sigma Chemical Company (St. Louis, MO). </li><li> Strumenti di dissezione: Fine # 5 pinzette, forbici, lama portalama, spille # 26002-20 insetti (tutti ottenuti da Strumenti Scienze Fine (USA), Inc., 373-G Vintage Park Drive, Foster City, CA 94404-1139) . </li><li> Sylgard a fondo piatto in vetro </li><li> Bicchieri (di tenere soluzioni chimiche) </li><li> I segnali elettrici vengono registrati in linea ad una interfaccia PowerLab 26T a un computer (ADInstruments, Colorado Springs, CO, USA). Noi utilizziamo software standard da ADInstruments nome o Campo Grafico. </li><li> Modello 3000 AC / DC amplificatore per le registrazioni intracellulari così come extracellulare può essere utilizzato. </li><li> Dissezione microscopio con funzione di zoom per le registrazioni intracellulari. Per la registrazione focale su terminali visualizzato un microscopio composto con obiettivi in ​​posizione verticale (4 x e 20X) viene utilizzato. Uno ha bisogno di una fonte di luce Hg. </li><li> Per le registrazioni intracellulari usiamo tubo capillare di vetro (catalogo # 30-31-0 da FHC, Brunswick, ME, 04011, USA). L&#39;elettrodo intracellulare dovrebbe avere una resistenza di 20 a 30 mOhm. Elettrodi intracellulari sono stati riempiti con 3 M KCl. </li><li> Un elettrodo di aspirazione viene utilizzata per registrare i segnali extracellulari. </li><li> Gabbia di Faraday. </li><li> Illuminatore ad alta intensità (sorgente luminosa). </li><li> Micromanipolatore </li></ol>

membrane potentials, synaptic responses, neuronal circuitry, neuromodulation and muscle histology using the crayfish: student laboratory exercises

Lo scopo di questa relazione è quello di contribuire a sviluppare una comprensione degli effetti causati da gradienti ionici attraverso una membrana biologica. Due aspetti che influenzano il potenziale di membrana di una cellula e che affronteremo in questi esperimenti sono: (1) la concentrazione di ioni di K + all&#39;esterno della membrana, e (2) la permeabilità della membrana agli ioni specifici. Il addominale muscoli estensori gamberi sono in raggruppamenti con alcuni tonico essere (lento) e altri fasica (veloce) nel loro fenotipo biochimico e fisiologico, così come nella loro struttura, i motoneuroni che innervano questi muscoli sono corrispondentemente diverse caratteristiche funzionali. Noi usiamo questi muscoli così come il superficiale, il muscolo flessore tonico addominali per dimostrare la proprietà nella trasmissione sinaptica. Inoltre, si introduce un senso-SNC-motoneurone-muscolare circuito per dimostrare l&#39;effetto della stimolazione sensoriale cuticolare così come l&#39;influenza dei neuromodulatori su alcuni aspetti del circuito. Con le tecniche ottenute in questo esercizio, si può cominciare a rispondere a molte domande ancora in altre preparazioni sperimentali così come nelle applicazioni fisiologiche legate alla medicina e alla salute. Abbiamo dimostrato l&#39;utilità di preparati invertebrati modello per affrontare questioni fondamentali pertinenti a tutti gli animali.

Watch this Scientific Journal Video about Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises at JoVE.com

Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises

Gli esperimenti dimostrano un approccio facile per gli studenti di acquisire esperienza nell&#39;esame struttura muscolare, le risposte sinaptiche, gli effetti dei gradienti ionici e permeabilità di potenziali di membrana. Inoltre, un senso-SNC-motorio-muscolare circuito è presentato per mostrare un modo per testare gli effetti dei composti su un circuito neuronale.

Potenziali di membrana, Risposte Synaptic, circuiti neuronali, neuromodulazione e Istologia muscolare Utilizzando la Gamberi: Esercizi di laboratorio per studenti

The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that ...

membrane-potentials-synaptic-responses-neuronal-circuitry

Research

JoVE Journal

Neuroscience

58.4K Views.  University of Kentucky. Gli esperimenti dimostrano un approccio facile per gli studenti di acquisire esperienza nell'esame struttura muscolare, le risposte sinaptiche, gli effetti dei gradienti ionici e permeabilità di potenziali di membrana. Inoltre, un senso-SNC-motorio-muscolare circuito è presentato per mostrare un modo per testare gli effetti dei composti su un circuito neuronale.

Video: Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises

Recording Behavioral Responses to Reflection in Crayfish

Social behavior depends on sensory input from the visual, mechanical and olfactory systems. One important issue concerns the relative roles of each sensory modality in guiding behavior. The role of visual inputs has been examined by isolating visual stimuli from mechanical and chemosensory stimuli. In some studies (Bruski &amp; Dunham, 1987: Delgado-Morales et al., 2004) visual inputs have been removed with blindfolds or low light intensity, and effects of remaining sensory modalities have been elucidated. An alternative approach is to study the effects of visual inputs in the absence of any appropriate mechanical and chemosensory cues. This approach aims to identify the exclusive role of visual inputs. 
We have used two methods to provide visual stimuli to crayfish without providing chemical and mechanical cues. In one method, crayfish are videotaped in an aquarium where half of the walls are covered in mirrors to provide a reflective environment, and the other half are covered in a non-reflective (matte finish) plastic. This gives the crayfish a choice between reflective and non-reflective environments. The reflective environment provides visual cues in the form of reflected images of the crayfish as it moves throughout half of the tank; these visual cues are missing from the non-reflective half of the tank. An alternative method is to videotape the behavior of crayfish in an aquarium separated by a smaller chamber at each end, with a crayfish in one small chamber providing visual cues and an inert object in the opposite small chamber providing visual input from a non-moving, non-crayfish source. 
Our published results indicate that responses of crayfish to the reflective environment depend on socialization and dominance rank. Socialized crayfish spent more time in the reflective environment and exhibited certain behaviors more frequently there than in the non-reflective environment; isolated crayfish showed no such differences. Crayfish that were housed in same-sex pairs developed a social rank of either dominant or subordinate. Responses to reflection differed between dominant and subordinate crayfish (May &amp; Mercier, 2006; May &amp; Mercier, 2007). Dominant crayfish spent more time on the reflective side, entered reflective corners more frequently and spent more time in reflective corners compared to the non-reflective side. Subordinate crayfish walked in reverse more often on the reflective side than on the non-reflective side. Preliminary data suggest similar effects from visual cues provided by a crayfish in a small adjoining chamber (May et al., 2008).

We have developed two methods for studying effects of visual cues on behavior in the absence of tactile and chemical cues. One method involves videotaping responses of crayfish to reflective walls in an aquarium; the other examines effects of visual inputs provided by a live crayfish behind a transparent partition.

Registrazione di risposte comportamentali alla riflessione in gamberi di fiume

Social behavior depends on sensory input from the visual, mechanical and olfactory systems.  One important issue concerns the relative roles of each ...

Ricerca

Neuroscienze

Physiological Recordings of High and Low Output NMJs on the Crayfish Leg Extensor Muscle

We explain in detail how to expose and conduct electrophysiological recordings of synaptic responses for high (phasic) and low (tonic) output motor neurons innervating the extensor muscle in the walking leg of a crayfish. Distinct differences are present in the physiology and morphology of the phasic and tonic nerve terminals. The tonic axon contains many more mitochondria, enabling it to take a vital stain more intensely than the phasic axon. The tonic terminals have varicosities, and the phasic terminal is filiform. The tonic terminals are low in synaptic efficacy but show dramatic facilitated responses. In contrast, the phasic terminals are high in quantal efficacy but show synaptic depression with high frequency stimulation. The quantal output is measured with a focal macropatch electrode placed directly over the visualized nerve terminals. Both phasic and tonic terminals innervate the same muscle fibers, which suggests that inherent differences in the neurons, rather than differential retrograde feedback from the muscle, account for the morphological and physiological differentiation.

This article demonstrates how to conduct electrophysiological recordings of synaptic responses on the extensor muscle in the walking leg of a crayfish and how the nerve terminals are visualized to show the gross morphological differences of high- and low-output nerve terminals.

Le registrazioni fisiologiche NMJs uscita alta e bassa del muscolo estensore gamberi Leg

We explain in detail how to expose and conduct electrophysiological recordings of synaptic responses for high (phasic) and low (tonic) output motor ...

Muscle Receptor Organs in the Crayfish Abdomen: A Student Laboratory Exercise in Proprioception

The primary purpose of this experiment is to demonstrate primary sensory neurons conveying information of joint movements and positions as proprioceptive information for an animal. An additional objective of this experiment is to learn anatomy of the preparation by staining, dissection and viewing of neurons and sensory structures under a dissecting microscope. This is performed by using basic neurophysiological equipment to record the electrical activity from a joint receptor organ and staining techniques. The muscle receptor organ (MRO) system in the crayfish is analogous to the intrafusal muscle spindle in mammals, which aids in serving as a comparative model that is more readily accessible for electrophysiological recordings. In addition, these are identifiable sensory neurons among preparations. The preparation is viable in a minimal saline for hours which is amenable for student laboratory exercises. The MRO is also susceptible to neuromodulation which encourages intriguing questions in the sites of modulatory action and integration of dynamic signals of movements and static position along with a gain that can be changed in the system.

The primary purpose of this experiment is to understand how primary sensory neurons convey information of joint movements and positions as proprioceptive information for an animal. An additional objective of this report is present the anatomy of the preparation by dissection and viewing of neurons under a dissecting microscope.

Receptor Organi muscolare all&#39;addome gamberi: un esercizio di laboratorio per studenti in propriocezione

The primary purpose of this experiment is to demonstrate primary sensory neurons conveying information of joint movements and positions as proprioceptive ...

Physiological Experimentation with the Crayfish Hindgut: A Student Laboratory Exercise

The purpose of the report is to describe dissection techniques for preparing the crayfish hindgut and to demonstrate how to make physiological recordings with a force transducer to monitor the strength of contraction. In addition, we demonstrate how to visually monitor peristaltic activity, which can be used as a bioassay for various peptides, biogenic amines and neurotransmitters. This preparation is amenable to student laboratories in physiology and for demonstrating pharmacological concepts to students. This preparation has been in use for over 100 years, and it still offers much as a model for investigating the generation and regulation of peristaltic rhythms and for describing the mechanisms underlying their modulation. The pharmacological assays and receptor sub-typing that were started over 50 years ago on the hindgut still contribute to research today. This robust preparation is well suited to training students in physiology and pharmacology.

In this report we demonstrate techniques that can be used to investigate the biology of the crayfish hindgut. We show how to dissect a crayfish abdomen and study the associated anatomy, physiology and modulation of activity. The peristaltic activity and strength of contractions are measured using a force transducer.

La sperimentazione con la fisiologica hindgut gamberi: un esercizio di laboratorio per studenti

The purpose of the report is to describe dissection techniques for preparing the crayfish hindgut and to demonstrate how to make physiological recordings ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

Analisi quantitativa di Replenishment pool delle vescicole sinaptiche in colture di neuroni granuli cerebellari utilizzando coloranti FM

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

Diffusione laterale e esocitosi delle proteine ​​di membrana in neuroni in coltura valutata utilizzando Recovery fluorescenza e fluorescenza-loss photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

Veloce micro-ionoforesi di glutammato e GABA: un utile strumento per indagare l&#39;integrazione Synaptic

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Proprioception and Tension Receptors in Crab Limbs: Student Laboratory Exercises

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons responsible for proprioception as they are detecting joint position and movement, and muscle tension. Electrical activity from crustacean proprioceptors and tension receptors is recorded by basic neurophysiological instrumentation, and a transducer is used to simultaneously measure force that is generated by stimulating a motor nerve. In addition, we demonstrate how to stain the neurons for a quick assessment of their anatomical arrangement or for permanent fixation. Staining reveals anatomical organization that is representative of chordotonal organs in most crustaceans. Comparing the tension nerve responses to the proprioceptive responses is an effective teaching tool in determining how these sensory neurons are defined functionally and how the anatomy is correlated to the function. Three staining techniques are presented allowing researchers and instructors to choose a method that is ideal for their laboratory.

Physiological and anatomical techniques are demonstrated to address function and structure for joint proprioceptors and muscle tension receptors in crustacean walking limbs.

Propriocezione e la tensione recettori in Crab Arti: Esercizi di laboratorio per studenti

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons ...

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the pathophysiological basis of these diseases, it is necessary to precisely characterize in which way the processing of information is disturbed between the different neuronal parts of the circuit. Using extracellular in vivo electrophysiological recordings, it is possible to accurately delineate neuronal activity within a neuronal network. The application of this method has several advantages over alternative techniques, e.g., functional magnetic resonance imaging and calcium imaging, as it allows a unique temporal and spatial resolution and does not rely on genetically engineered organisms. However, the use of extracellular in vivo recordings is limited since it is an invasive technique that cannot be universally applied. In this article, a simple and easy to use method is presented with which it is possible to simultaneously record extracellular potentials such as local field potentials and multiunit activity at multiple sites of a network. It is detailed how a precise targeting of subcortical nuclei can be achieved using a combination of stereotactic surgery and online analysis of multi-unit recordings. Thus, it is demonstrated, how a complete network such as the hyperdirect cortico-basal ganglia loop can be studied in anesthetized animals in vivo.

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

In this study, the methodology is presented on how to perform multi-site in vivo electrophysiological recordings from the hyperdirect pathway under urethane anesthesia.

acuto<em&gt; In Vivo</em&gt; Registrazioni elettrofisiologiche dei potenziali del campo locale e dell&#39;attività multi-unità dal percorso iperdirezionale nei ratti anestetizzati

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the ...

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like mouse and rat astrocyte monocultures: Brain cells can be isolated from different brain regions, and after a week of cell culture, non-astrocytic cells are shaken off by placing the culture dishes on a shaker for 6 h in the incubator. The remaining astrocytes are then passaged into new plates with an astrocyte-specific medium (termed NB+H). NB+H contains low concentrations of heparin-binding EGF-like growth factor (HBEGF), which is used in place of serum in medium. After growing in NB+H, AWESAM astrocytes have a stellate morphology and feature fine processes. Moreover, these astrocytes have more in vivo-like gene expression than astrocytes generated by previously published methods. Ca2+ imaging, vesicle dynamics, and other events close to the membrane can thus be studied in the fine astrocytic processes in vitro, e.g., using live cell confocal or TIRF microscopy. Notably, AWESAM astrocytes also exhibit spontaneous Ca2+ signaling similar to astrocytes in vivo.

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM protocol described here is optimal for culturing murine astrocytes in isolation from other brain cells in a fast, simple, and inexpensive manner. AWESAM astrocytes exhibit spontaneous Ca2+ signaling, morphology, and gene expression profiles similar to astrocytes in vivo.

Coltura In Vivo-piace murini astrociti utilizzando il protocollo AWESAM veloce, semplice e poco costoso

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like ...