Este trabajo fue apoyado por la Fundación de Neurociencias de Investigación y subvenciones del Fondo Blasker-Rose-Miah de la Fundación San Diego y el G. Harold y Leila Y. Mathers Fundación Caritativa.

<ol>
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Neurosci. Methods. , (2011).</li><li>Bieszczad, K. M., Weinberger, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+strategy+trumps+motivational+level+in+determining+learning-induced+auditory+cortical+plasticity.">Learning strategy trumps motivational level in determining learning-induced auditory cortical plasticity.</a> Neurobiology of Learning and Memory. 93, 229-239 (2010).</li><li>Sagvolden, T. I. m. p. u. l. s. i. v. e. n. e. s. s., overactivity, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=poorer+sustained+attention+improve+by+chronic+treatment+with+low+doses+of+l-amphetamine+in+an+animal+model+of+Attention-Deficit/Hyperactivity+Disorder+(ADHD).">poorer sustained attention improve by chronic treatment with low doses of l-amphetamine in an animal model of Attention-Deficit/Hyperactivity Disorder (ADHD).</a> Behav. Brain Funct. 7, 6 (2011).</li><li>Sagvolden, T., Johansen, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+Models+of+ADHD.">Rat Models of ADHD.</a> Curr. Top Behav. Neurosci. , (2011).</li><li>Brown, D. C., Nichols, J. A., Thomas, F., Dinh, L., Atzori, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotinic+modulation+of+auditory+attentional+shift+in+the+rat.">Nicotinic modulation of auditory attentional shift in the rat.</a> Behavioural brain research. 210, 273-279 (2010).</li><li>Johnson, K. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+discrimination:+neural+processes+preceding+discrimination+decision.">Sensory discrimination: neural processes preceding discrimination decision.</a> J. Neurophysiol. 43, 1793-1815 (1980).</li><li>Johnson, K. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+discrimination:+decision+process.">Sensory discrimination: decision process.</a> J. Neurophysiol. 43, 1771-1792 (1980).</li><li>Fraser, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+anxiety-+and+locomotion-related+behaviours+in+mice:+a+new+way+of+using+old+tests.">Measuring anxiety- and locomotion-related behaviours in mice: a new way of using old tests.</a> Psychopharmacology (Berl). 211, 99-112 (2010).</li><li>Hui, G. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditioned+tone+control+of+brain+reward+behavior+produces+highly+specific+representational+gain+in+the+primary+auditory+cortex.">Conditioned tone control of brain reward behavior produces highly specific representational gain in the primary auditory cortex.</a> Neurobiology of Learning and Memory. 92, 27-34 (2009).</li><li>Feduccia, A. A., Duvauchelle, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auditory+stimuli+enhance+MDMA-conditioned+reward+and+MDMA-induced+nucleus+accumbens+dopamine,+serotonin+and+locomotor+responses.">Auditory stimuli enhance MDMA-conditioned reward and MDMA-induced nucleus accumbens dopamine, serotonin and locomotor responses.</a> Brain Res. Bull. 77, 189-196 (2008).</li><li>Kudoh, M., Shibuki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sound+sequence+discrimination+learning+motivated+by+reward+requires+dopaminergic+D2+receptor+activation+in+the+rat+auditory+cortex.">Sound sequence discrimination learning motivated by reward requires dopaminergic D2 receptor activation in the rat auditory cortex.</a> Learning &amp; Memory. 13, 698-69 (2006).</li><li>Shiflett, M. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cue-elicited+reward-seeking+requires+extracellular+signal-regulated+kinase+activation+in+the+nucleus+accumbens.">Cue-elicited reward-seeking requires extracellular signal-regulated kinase activation in the nucleus accumbens.</a> J. Neurosci. 28, 1434-1443 (2008).</li></ol>

La producción y el libre acceso de este video-artículo es patrocinado por Med Associates, Inc.

Un aspecto crítico en el diseño de cualquier tarea auditiva de comportamiento es la eliminación de sonidos no deseados que resultan de la dispersión y la deflexión en el entorno de prueba. Control de sonido deficiente puede tener un efecto significativo en el comportamiento se está probando y va a producir resultados erróneos o no se puede interpretar, incluso. La cámara de comportamiento utilizado en el sistema descrito aquí está diseñado específicamente para ser acústicamente transparente a fin de evitar la deflexión del sonido de las paredes de la cámara. En efecto, cuando se mide desde el centro de la cámara, la deflexión sonido era eficaz indetectable (datos no presentados). A pesar de que hemos desarrollado este sistema principalmente para el estudio del sistema auditivo, puede ser fácilmente adaptado por otros investigadores para estudiar otros sistemas sensoriales. Modificaciones se pueden hacer fácilmente en software y hardware para diferentes tareas sin cambiar la configuración global del sistema. El panel de operaciones del sistema modular hace particularmente polivalenciaLe permitiendo que para la adición y / o sustitución de los diferentes dispositivos para las nuevas tareas conductuales. Por ejemplo, las tareas de comportamiento olfativo puede ser implantado mediante la entrega de estímulos olfativos en el dispositivo de empuje nariz. Las cinco opciones de iluminación de pared asoman la nariz con estímulo olfativo de los Asociados MED (ENV-115A-DE) puede ser fácilmente instalado en el panel de control para las complejas tareas olfativas. Además, todos los dispositivos operantes pueden ser fácilmente reemplazados por aquellos que están diseñados para los ratones sin cambiar la configuración del sistema. El control preciso temporal de cada dispositivo operante, así como la grabación de alta resolución de los eventos individuales en un ensayo dado, permiten la manipulación precisa de los dispositivos para personalizar el diseño de las tareas de comportamiento para hacer frente a las diferentes funciones cognitivas del cerebro (ver más abajo). Cuando se combina con la grabación de la actividad neuronal, una rica variedad de temas en el campo de las neurociencias pueden ser estudiados con este sistema. Por ejemplo, en la unauditory tarea se ha descrito anteriormente, las siguientes preguntas relacionadas con las funciones cognitivas del cerebro pueden ser investigados en un solo ensayo: (1). Motivación: Dado que cada proceso se inicia por animal &quot;auto-motivado&quot; nariz-poke acción (Figura 5A-a y Figura 3), la motivación por lo tanto se pueden evaluar cuantitativamente mediante la medición del número total de ensayos realizados por un animal en un determinado período de sesiones, o el número de intentos consecutivos a cabo 6,7. (2). Atención: La clave para la obtención de una recompensa en un ensayo es reconocer correctamente las señales auditivas. En aproximadamente el 25% de las ratas que no podían ser entrenados para realizar la tarea, la falta de atención a las señales auditivas era el factor principal. En contraste, en las ratas que aprendieron la tarea, una pausa momentánea de comportamientos era evidente durante la presentación auditiva señal (véase la Figura 5A-B y la Figura 3). Mediante el uso de este sistema, es así posible a las ratas de pantalla (i) para la atención definición &quot;cit &quot;y (ii) estudiar los mecanismos neurales de la atención cuando se combina con las grabaciones neuronales mientras el animal está atendiendo a las señales auditivas 10.8. (3). Decisión: A partir del reconocimiento de las señales auditivas, el animal tiene que decidir qué dirección tomar para acercarse a la palanca correcta dentro de una ventana de tiempo limitado (Figura 5A-C). Por tanto, es también un paradigma efectivo para el estudio de la toma de decisiones 11,12. (4). Paciencia: El tiempo de extensión de la palanca se puede controlar de tal manera que el animal tiene que esperar a que la palanca después de llegar al punto donde la palanca se extenderá (Figura 5A-d). Al variar la longitud de espera, en la medida de la paciencia de un animal puede ser probado y cuantificado 13. (5). Recompensas: El objetivo final de la tarea es para obtener la recompensa (Figura 5A-e y la Figura 3). Tareas conductuales que utilizan este sistema por lo tanto puedeser fácilmente diseñado para estudiar muchos aspectos de los problemas de decisión recompensa-y la función de los sistemas de valores del cerebro 14-17.

a fully automated and highly versatile system for testing multi-cognitive functions and recording neuronal activities in rodents

Hemos desarrollado un sistema completamente automatizado para la prueba de la conducta operante y la grabación de la actividad neuronal mediante el cual varias funciones cerebrales cognitivas pueden ser investigados en una secuencia única tarea. La característica única de este sistema es un hecho a medida, la cámara acústicamente transparente que elimina muchas de las cuestiones relacionadas con el control auditivo de referencia en la mayoría de cámaras disponibles en el mercado. La facilidad con que los dispositivos operantes se pueden añadir o sustituido hace que este sistema muy versátil, permitiendo la aplicación de una variedad de auditivo, visual y olfativo tareas de comportamiento. La automatización del sistema permite fina temporal (10 ms) el control y la precisión de sellado de tiempo de cada evento en una secuencia de comportamiento prediseñado. Cuando se combina con un sistema de grabación multi-canal de electrofisiología, múltiples funciones cerebrales cognitivas, tales como la motivación, la atención, la toma de decisiones, la paciencia, y las recompensas, se pueden examinar de forma secuencial o de forma independiente.

Watch this Scientific Journal Video about A Fully Automated and Highly Versatile System for Testing Multi-cognitive Functions and Recording Neuronal Activities in Rodents at JoVE.com

A Fully Automated and Highly Versatile System for Testing Multi-cognitive Functions and Recording Neuronal Activities in Rodents

En este informe, presentamos un sistema totalmente automatizado y altamente versátil capaz de probar simultáneamente múltiples comportamientos cognitivos y el registro de las actividades neuronales de los roedores.

Un sistema totalmente automatizado y altamente versátil para las pruebas múltiples funciones cognitivas y el registro de las actividades neuronales en los roedores

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be ...

a-fully-automated-highly-versatile-system-for-testing-multi-cognitive

Research

JoVE Journal

Neuroscience

14.2K vistas.  The Neurosciences Institute, San Diego, CA. En este informe, presentamos un sistema totalmente automatizado y altamente versátil capaz de probar simultáneamente múltiples comportamientos cognitivos y el registro de las actividades neuronales de los roedores.

Video: A Fully Automated and Highly Versatile System for Testing Multi-cognitive Functions and Recording Neuronal Activities in Rodents

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3. Specifically, it affects the ability of neurons to receive inputs from other cells2 and contributes to the propagation of action potentials4,5. The morphology of the neurites also affects how information is processed. The diversity of dendrite morphologies facilitate local and long range signaling and allow individual neurons or groups of neurons to carry out specialized functions within the neuronal network6,7. Alterations in dendrite morphology, including fragmentation of dendrites and changes in branching patterns, have been observed in a number of disease states, including Alzheimer's disease8, schizophrenia9,10, and mental retardation11. The ability to both understand the factors that shape dendrite morphologies and to identify changes in dendrite morphologies is essential in the understanding of nervous system function and dysfunction.
Neurite morphology is often analyzed by Sholl analysis and by counting the number of neurites and the number of branch tips. This analysis is generally applied to dendrites, but it can also be applied to axons. Performing this analysis by hand is both time consuming and inevitably introduces variability due to experimenter bias and inconsistency. The Bonfire program is a semi-automated approach to the analysis of dendrite and axon morphology that builds upon available open-source morphological analysis tools. Our program enables the detection of local changes in dendrite and axon branching behaviors by performing Sholl analysis on subregions of the neuritic arbor. For example, Sholl analysis is performed on both the neuron as a whole as well as on each subset of processes (primary, secondary, terminal, root, etc.) Dendrite and axon patterning is influenced by a number of intracellular and extracellular factors, many acting locally. Thus, the resulting arbor morphology is a result of specific processes acting on specific neurites, making it necessary to perform morphological analysis on a smaller scale in order to observe these local variations12.
The Bonfire program requires the use of two open-source analysis tools, the NeuronJ plugin to ImageJ and NeuronStudio. Neurons are traced in ImageJ, and NeuronStudio is used to define the connectivity between neurites. Bonfire contains a number of custom scripts written in MATLAB (MathWorks) that are used to convert the data into the appropriate format for further analysis, check for user errors, and ultimately perform Sholl analysis. Finally, data are exported into Excel for statistical analysis. A flow chart of the Bonfire program is shown in Figure 1.

We have developed a computer program to analyze neuronal morphology. In combination with two existing open source analysis tools, our program performs Sholl analysis and determines the number of neurites, branch points, and neurite tips. The analyses are performed so that local changes in neurite morphology can be observed.

Automatizado de Análisis de la morfología neuronal Sholl digitalizados en múltiples escalas

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

Investigación

Neurociencias

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. For this protocol the freezing medium used is 10% dimethyl sulfoxide (DMSO) diluted in Hank's Buffered Salt Solution (HBSS). Blocks of cortical tissue are transferred to cryovials containing the freezing medium and slowly frozen at -1&deg;C/min in a rate-controlled freezing container. Post-thaw processing and dissociation of frozen tissue blocks consistently produced neuronal-enriched cultures which exhibited rapid neuritic growth during the first 5 days in culture and significant expansion of the neuronal network within 10 days. Immunocytochemical staining with the astrocytic marker glial fibrillary acidic protein (GFAP) and the neuronal marker beta-tubulin class III, revealed high numbers of neurons and astrocytes in the cultures. Generation of neural precursor cell cultures after tissue block dissociation resulted in rapidly expanding neurospheres, which produced large numbers of neurons and astrocytes under differentiating conditions. This simple cryopreservation protocol allows for the rapid, efficient, and inexpensive preservation of cortical brain tissue blocks, which grants increased flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

Here, we describe a method for efficient cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. This simple protocol provides flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

La criopreservación de tejido cortical Bloques para la generación de cultivos neuronales altamente enriquecido

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly ...

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

Derivación eficiente de los Derechos Humanos progenitores neuronales y neuronas de pluripotentes células madre embrionarias humanas mediante la inducción de moléculas pequeñas

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

Large-scale Recording of Neurons by Movable Silicon Probes in Behaving Rodents

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of neurons and circuits requires methods with the spatial selectivity and temporal resolution appropriate for mechanistic analysis of neural ensembles in the behaving animal, i.e. recording of representatively large samples of isolated single neurons. Ensemble monitoring of neuronal activity has progressed remarkably in the past decade in both small and large-brained animals, including human subjects1-11. Multiple-site recording with silicon-based devices are particularly effective because of their scalability, small volume and geometric design.
Here, we describe methods for recording multiple single neurons and local field potential in behaving rodents, using commercially available micro-machined silicon probes with custom-made accessory components. There are two basic options for interfacing silicon probes to preamplifiers: printed circuit boards and flexible cables. Probe supplying companies (<a href="http://www.neuronexustech.com/" >http://www.neuronexustech.com/</a>; <a href="http://www.sbmicrosystems.com/">http://www.sbmicrosystems.com/</a>; <a href="http://www.acreo.se/">http://www.acreo.se/</a>) usually provide the bonding service and deliver probes bonded to printed circuit boards or flexible cables. Here, we describe the implantation of a 4-shank, 32-site probe attached to flexible polyimide cable, and mounted on a movable microdrive. Each step of the probe preparation, microdrive construction and surgery is illustrated so that the end user can easily replicate the process.

We describe methods for large-scale recording of multiple single units and local field potential in behaving rodents with silicon probes. Drive fabrication, probe attachment to the drive and probe implantation processes are illustrated in sufficient details for easy replication.

A gran escala de grabación de las neuronas por medio de sondas de silicio móviles en comportarse roedores

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

Registro y análisis de los ritmos circadianos en funcionamiento las ruedas Actividad en roedores

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

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.

Fast Micro-iontoforesis de glutamato y GABA: una herramienta útil para investigar la integración sináptica

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

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

La inducción de la plasticidad de astrocítico Receptores por manipulación de Neuronales cocción Precios

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

Simultáneo<em&gt; Ex vivo</em&gt; Pruebas funcionales de dos retinas por<em&gt; In vivo</em&gt; Sistema Electrorretinograma

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

Estimulación eléctrica transcraneal crónica y grabación Intracortical en ratas

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ablation of neurons is an effective method for this purpose when neurons are selectively labeled with fluorescent probes. In the present study, protocols for laser ablating a subpopulation of neurons using a two-photon microscope and testing of its functional and behavioral consequences are described. In this study, prey capture behavior in zebrafish larvae is used as a study model. The pretecto-hypothalamic circuit is known to underlie this visually-driven prey catching behavior. Zebrafish pretectum were laser-ablated, and neuronal activity in the inferior lobe of the hypothalamus (ILH; the target of the pretectal projection) was examined. Prey capture behavior after pretectal ablation was also tested.

Here, we present a protocol to ablate a genetically labeled subpopulation of neurons by a two-photon laser from Zebrafish larvae.

Ablación de una población Neuronal utilizando un láser de dos fotones y su evaluación mediante grabación comportamiento y proyección de imagen de calcio en las larvas de pez cebra

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ...