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1.
J Neurophysiol ; 129(6): 1468-1481, 2023 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-37198134

RESUMEN

Calyx terminals make afferent synapses with type I hair cells in vestibular epithelia and express diverse ionic conductances that influence action potential generation and discharge regularity in vestibular afferent neurons. Here we investigated the expression of hyperpolarization-activated current (Ih) in calyx terminals in central and peripheral zones of mature gerbil crista slices, using whole cell patch-clamp recordings. Slowly activating Ih was present in >80% calyces tested in both zones. Peak Ih and half-activation voltages were not significantly different; however, Ih activated with a faster time course in peripheral compared with central zone calyces. Calyx Ih in both zones was blocked by 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino) pyrimidinium chloride (ZD7288; 100 µM), and the resting membrane potential became more hyperpolarized. In the presence of dibutyryl-cAMP (dB-cAMP), peak Ih was increased, activation kinetics became faster, and the voltage of half-activation was more depolarized compared with control calyces. In current clamp, calyces from both zones showed three different categories of firing: spontaneous firing, phasic firing where a single action potential was evoked after a hyperpolarizing pulse, or a single evoked action potential followed by membrane potential oscillations. In the absence of Ih, the latency to peak of the action potential increased; Ih produces a small depolarizing current that facilitates firing by driving the membrane potential closer to threshold. Immunostaining showed the expression of HCN2 subunits in calyx terminals. We conclude that Ih is found in calyx terminals across the crista and could influence conventional and novel forms of synaptic transmission at the type I hair cell-calyx synapse.NEW & NOTEWORTHY Calyx afferent terminals make synapses with vestibular hair cells and express diverse conductances that impact action potential firing in vestibular primary afferents. Conventional and nonconventional synaptic transmission modes are influenced by hyperpolarization-activated current (Ih), but regional differences were previously unexplored. We show that Ih is present in both central and peripheral calyces of the mammalian crista. Ih produces a small depolarizing resting current that facilitates firing by driving the membrane potential closer to threshold.


Asunto(s)
Células Ciliadas Vestibulares , Vestíbulo del Laberinto , Animales , Células Ciliadas Vestibulares/fisiología , Neuronas Aferentes , Potenciales de Acción/fisiología , Potenciales de la Membrana , Mamíferos
2.
J Neurophysiol ; 114(3): 2053-64, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26203105

RESUMEN

Electrophysiological recordings from brain slices are typically performed in small recording chambers that allow for the superfusion of the tissue with artificial extracellular solution (ECS), while the chamber holding the tissue is mounted in the optical path of a microscope to image neurons in the tissue. ECS itself is inexpensive, and thus superfusion rates and volumes of ECS consumed during an experiment using standard ECS are not critical. However, some experiments require the addition of expensive pharmacological agents or other chemical compounds to the ECS, creating a need to build superfusion systems that operate on small volumes while still delivering appropriate amounts of oxygen and other nutrients to the tissue. We developed a closed circulation tissue chamber for slice recordings that operates with small volumes of bath solution in the range of 1.0 to 2.6 ml and a constant oxygen/carbon dioxide delivery to the solution in the bath. In our chamber, the ECS is oxygenated and recirculated directly in the recording chamber, eliminating the need for tubes and external bottles/containers to recirculate and bubble ECS and greatly reducing the total ECS volume required for superfusion. At the same time, the efficiency of tissue oxygenation and health of the section are comparable to standard superfusion methods. We also determined that the small volume of ECS contains a sufficient amount of nutrients to support the health of a standard brain slice for several hours without concern for either depletion of nutrients or accumulation of waste products.


Asunto(s)
Encéfalo/fisiología , Electrofisiología/instrumentación , Técnicas de Placa-Clamp/instrumentación , Animales , Encéfalo/citología , Electrofisiología/métodos , Gerbillinae , Neuronas/fisiología , Técnicas de Placa-Clamp/métodos
3.
J Neurophysiol ; 112(11): 2901-15, 2014 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-25185813

RESUMEN

Neurons in the medial nucleus of the trapezoid body (MNTB) are well known for their prominent excitatory inputs, mediated by the calyx of Held. Less attention has been paid to the prominent inhibitory inputs that MNTB neurons also receive. Because of their auditory nature, both excitatory and inhibitory synapses are highly active in vivo. These high levels of activity are known to reduce excitatory synaptic currents considerably, such that in vivo synaptic currents produced by the calyx are smaller than typically measured in standard brain slice experiments. The goal of this study was to investigate the properties of the inhibitory inputs in the Mongolian gerbil (Meriones unguiculatus) under activity levels that correspond to those in the intact brain to facilitate a direct comparison between the two inputs. Our results suggest that inhibitory inputs to MNTB are largely mediated by a fast and phasic glycinergic component, and to a lesser degree by a GABAergic component. The glycinergic component can sustain prolonged high levels of activity. Even when challenged with stimulus patterns consisting of thousands of stimuli over tens of minutes, glycinergic inputs to MNTB maintain large conductances and fast decays and even facilitate substantially when the stimulation frequency is increased. The inhibition is mediated by a relatively small number of independent input fibers. The data presented here suggest that inhibitory inputs to MNTB sustain high levels of activity and need to be considered for a full understanding of mechanisms underlying processing of auditory information in MNTB.


Asunto(s)
Potenciales Postsinápticos Excitadores , Glicina/farmacología , Potenciales Postsinápticos Inhibidores , Cuerpo Trapezoide/fisiología , Animales , Vías Auditivas/citología , Vías Auditivas/fisiología , Femenino , Gerbillinae , Masculino , Neuronas/efectos de los fármacos , Neuronas/fisiología , Cuerpo Trapezoide/citología , Ácido gamma-Aminobutírico/farmacología
4.
Eur J Neurosci ; 40(6): 2867-77, 2014 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-24995587

RESUMEN

Calyx of Held giant presynaptic terminals in the auditory brainstem form glutamatergic axosomatic synapses that have advanced to one of the best-studied synaptic connections of the mammalian brain. As the auditory system matures and adjusts to high-fidelity synaptic transmission, the calyx undergoes extensive structural and functional changes - in mice, it is formed at about postnatal day 3 (P3), achieves immature function until hearing onset at about P10 and can be considered mature from P21 onwards. This setting provides a unique opportunity to examine the repertoire of genes driving synaptic structure and function during postnatal maturation. Here, we determined the gene expression profile of globular bushy cells (GBCs), neurons giving rise to the calyx of Held, at different maturational stages (P3, P8, P21). GBCs were retrogradely labelled by stereotaxic injection of fluorescent cholera toxin-B, and their mRNA content was collected by laser microdissection. Microarray profiling, successfully validated with real time quantitative polymerase chain reaction and nCounter approaches, revealed genes regulated during maturation. We found that mostly genes implicated in the general cell biology of the neuron were regulated, while most genes related to synaptic function were regulated around the onset of hearing. Among these, voltage-gated ion channels and calcium-binding proteins were strongly regulated, whereas most genes involved in the synaptic vesicle cycle were only moderately regulated. These results suggest that changes in the expression patterns of ion channels and calcium-binding proteins are a dominant factor in defining key synaptic properties during maturation of the calyx of Held.


Asunto(s)
Tronco Encefálico/crecimiento & desarrollo , Tronco Encefálico/fisiología , Neuronas/fisiología , Sinapsis/fisiología , Animales , Tronco Encefálico/citología , Toxina del Cólera , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Ontología de Genes , Inmunohistoquímica , Captura por Microdisección con Láser , Análisis por Micromatrices , Microscopía Confocal , Datos de Secuencia Molecular , Técnicas de Trazados de Vías Neuroanatómicas , Neuronas/citología , Técnicas de Placa-Clamp , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Sinapsis/genética , Técnicas de Cultivo de Tejidos
5.
Hear Res ; 443: 108962, 2024 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-38295585

RESUMEN

Nestin expression is associated with pluripotency. Growing evidence suggests nestin is involved in hair cell development. The objective of this study was to investigate the morphology and role of nestin-expressing cells residing in the early postnatal murine inner ear. A lineage-tracing nestin reporter mouse line was used to further characterize these cells. Their cochleae and vestibular organs were immunostained and whole-mounted for cell counting. We found Nestin-expressing cells present in low numbers throughout the inner ear. Three morphotypes were observed: bipolar, unipolar, and globular. Mitotic activity was noted in nestin-expressing cells in the cochlea, utricle, saccule, and crista. Nestin-expressing cell characteristics were then observed after hair cell ablation in two mouse models. First, a reporter model demonstrated nestin expression in a significantly higher proportion of hair cells after hair cell ablation than in control cochleae. However, in a lineage tracing nestin reporter mouse, none of the new hair cells which repopulated the organ of Corti after hair cell ablation expressed nestin, nor did the nestin-expressing cells change in morphotype. In conclusion, Nestin-expressing cells were identified in the cochlea and vestibular organs. After hair cell ablation, nestin-expressing cells did not react to the insult. However, a small number of nestin-expressing cells in all inner ear tissues exhibited mitotic activity, supporting progenitor cell potential, though perhaps not involved in hair cell regeneration.


Asunto(s)
Cóclea , Vestíbulo del Laberinto , Animales , Ratones , Cóclea/metabolismo , Células Ciliadas Auditivas/metabolismo , Nestina/genética , Nestina/metabolismo , Sáculo y Utrículo/metabolismo , Vestíbulo del Laberinto/metabolismo
6.
Hear Res ; 404: 108215, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33677192

RESUMEN

Efferent innervation of the inner hair cells changes over time. At an early age in mice, inner hair cells receive efferent feedback, which helps fine-tune tonotopic maps in the brainstem. In adulthood, inner hair cell efferent innervation wanes but increases again in older animals. It is not clear, however, whether age-related inner hair cell efferents increase along the entire range of the cochlear frequencies, or if this increase is restricted to a particular frequency-region, and whether this phenomenon occurs in both sexes. Age-related hearing loss, presbycusis, affects men and women differently. In mice, this difference is also strain specific. In aging black six mice, the auditory brainstem response thresholds increase in females earlier than in males. Here, we study age-related increase of the inner hair cell efferent innervation throughout the cochlea before hearing onset, in one month old and in ten months old and older male and female black six mice. We collected confocal images of immunostained inner hair cell efferents and quantified the labeled terminals in the entire cochlea using a machine learning algorithm. The overall number of the inner hair cell efferents in both sexes did not change significantly between age-groups. The distribution of the inner hair cell efferent innervation did not differ across frequencies in the cochlea. However, in females, inner hair cells received on average up to four times more efferent innervation than in males per each of the frequency regions tested. Sex differences were also found in the oldest age-group tested (≥ 10 months) where on average inner hair cells received six times more efferents in females than in males of matching age. Our findings emphasize the importance of including both sexes in sensorineural hearing loss research.


Asunto(s)
Células Ciliadas Auditivas Internas , Caracteres Sexuales , Envejecimiento , Animales , Cóclea , Femenino , Masculino , Ratones , Neuronas Eferentes , Sinapsis
7.
Stem Cell Res Ther ; 12(1): 352, 2021 06 19.
Artículo en Inglés | MEDLINE | ID: mdl-34147129

RESUMEN

BACKGROUND: Hearing loss affects 25% of the population at ages 60-69 years. Loss of the hair cells of the inner ear commonly underlies deafness and once lost this cell type cannot spontaneously regenerate in higher vertebrates. As a result, there is a need for the development of regenerative strategies to replace hair cells once lost. Stem cell-based therapies are one such strategy and offer promise for cell replacement in a variety of tissues. A number of investigators have previously demonstrated successful implantation, and certain level of regeneration of hair and supporting cells in both avian and mammalian models using rodent pluripotent stem cells. However, the ability of human stem cells to engraft and generate differentiated cell types in the inner ear is not well understood. METHODS: We differentiate human pluripotent stem cells to the pre-placodal stage in vitro then transplant them into the mouse cochlea after selective and complete lesioning of the endogenous population of hair cells. RESULTS: We demonstrate that hair cell ablation prior to transplantation leads to increased engraftment in the auditory sensory epithelium, the organ of Corti, as well as differentiation of transplanted cells into hair and supporting cell immunophenotypes. CONCLUSION: We have demonstrated the feasibility of human stem cell engraftment into an ablated mouse organ of Corti.


Asunto(s)
Células Madre Embrionarias Humanas , Células Madre Pluripotentes , Animales , Diferenciación Celular , Cóclea , Células Ciliadas Auditivas , Humanos , Ratones , Trasplante de Células Madre
8.
J Neurosci Methods ; 347: 108955, 2021 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-32971134

RESUMEN

BACKGROUND: Some experimental approaches in neuroscience research require the precise placement of a recording electrode, pipette or other tool into a specific brain area that can be quite small and/or located deep beneath the surface. This process is typically aided with stereotaxic methods but remains challenging due to a lack of advanced technology to aid the experimenter. Currently, procedures require a significant amount of skill, have a high failure rate, and take up a significant amount of time. NEW METHOD: We developed a next generation robotic stereotaxic platform for small rodents by combining a three-dimensional (3D) skull profiler sub-system and a full six degree-of-freedom (6DOF) robotic platform. The 3D skull profiler is based on structured illumination in which a series of horizontal and vertical line patterns are projected onto an animal skull. These patterns are captured by two two-dimensional (2D) CCD cameras which reconstruct an accurate 3D skull surface profile based on structured illumination and geometrical triangulation. Using the reconstructed 3D profile, the skull can be repositioned using a 6DOF robotic platform to accurately align a surgical tool. RESULTS: The system was evaluated using mechanical measurement techniques, and the accuracy of the platform was demonstrated using agar brain phantoms and animal skulls. Additionally, a small and deep brain nucleus (the medial nucleus of the trapezoid body) were targeted in rodents to confirm the targeting accuracy. CONCLUSIONS: The new stereotaxic system can accomplish "skull-flat" rapidly and precisely and with minimal user intervention, and thus reduces the failure rate of such experiments.


Asunto(s)
Procedimientos Quirúrgicos Robotizados , Animales , Imagenología Tridimensional , Fantasmas de Imagen , Cráneo/diagnóstico por imagen , Cráneo/cirugía , Técnicas Estereotáxicas
9.
Hear Res ; 394: 107934, 2020 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-32204962

RESUMEN

The application of gene therapy is widely expanding in research and continuously improving in preparation for clinical applications. The inner ear is an attractive target for gene therapy for treating environmental and genetic diseases in both the auditory and vestibular systems. With the lack of spontaneous cochlear hair cell replacement, hair cell regeneration in adult mammals is among the most important goals of gene therapy. In addition, correcting gene defects can open up a new era for treating inner ear diseases. The relative isolation and small size of the inner ear dictate local administration routes and carefully calculated small volumes of reagents. In the current review, we will cover effective timing, injection routes and types of vectors for successful gene delivery to specific target cells within the inner ear. Differences between research purposes and clinical applications are also discussed.


Asunto(s)
Oído Interno , Enfermedades del Laberinto , Terapia Genética , Células Ciliadas Auditivas , Humanos , Enfermedades del Laberinto/genética , Enfermedades del Laberinto/terapia , Sistema Vestibular
10.
Otolaryngol Head Neck Surg ; 161(6): 1004-1011, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31570054

RESUMEN

OBJECTIVES: (1) To characterize changes in brainstem neural activity following unilateral deafening in an animal model. (2) To compare brainstem neural activity from unilaterally deafened animals with that of normal-hearing controls. STUDY DESIGN: Prospective controlled animal study. SETTING: Vivarium and animal research facilities. SUBJECTS AND METHODS: The effect of single-sided deafness on brainstem activity was studied in Chinchilla lanigera. Animals were unilaterally deafened via gentamycin injection into the middle ear, which was verified by loss of auditory brainstem responses (ABRs). Animals underwent measurement of ABR and local field potential in the inferior colliculus. RESULTS: Four animals underwent chemical deafening, with 2 normal-hearing animals as controls. ABRs confirmed unilateral loss of auditory function. Deafened animals demonstrated symmetric local field potential responses that were distinctly different than the contralaterally dominated responses of the inferior colliculus seen in normal-hearing animals. CONCLUSION: We successfully developed a model for unilateral deafness to investigate effects of single-sided deafness on brainstem plasticity. This preliminary investigation serves as a foundation for more comprehensive studies that will include cochlear implantation and manipulation of binaural cues, as well as functional behavioral tests.


Asunto(s)
Tronco Encefálico/fisiopatología , Chinchilla , Modelos Animales de Enfermedad , Pérdida Auditiva Unilateral/etiología , Animales , Potenciales Evocados Auditivos del Tronco Encefálico/fisiología
11.
Trends Neurosci ; 41(11): 823-837, 2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-30033182

RESUMEN

Hearing loss in mammals is an irreversible process caused by degeneration of the hair cells of the inner ear. Current therapies for hearing loss include hearing aids and cochlear implants that provide substantial benefits to most patients, but also have several shortcomings. There is great interest in the development of regenerative therapies to treat deafness in the future. Cell-based therapies, based either on adult, multipotent stem, or other types of pluripotent cells, offer promise for generating differentiated cell types to replace lost or damaged hair cells of the inner ear. In this review, we focus on the methods proposed and avenues for research that seem the most promising for stem cell-based auditory sensory cell regeneration, from work collected over the past 15 years.


Asunto(s)
Diferenciación Celular/fisiología , Tratamiento Basado en Trasplante de Células y Tejidos , Sordera/terapia , Pérdida Auditiva/terapia , Animales , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Células Ciliadas Auditivas/metabolismo , Humanos , Regeneración/fisiología
12.
PLoS One ; 11(8): e0160241, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27489949

RESUMEN

Principal neurons in the medial nucleus of the trapezoid body (MNTB) receive strong and temporally precise excitatory input from globular bushy cells in the cochlear nucleus through the calyx of Held. The extremely large synaptic currents produced by the calyx have sometimes led to the view of the MNTB as a simple relay synapse which converts incoming excitation to outgoing inhibition. However, electrophysiological and anatomical studies have shown the additional presence of inhibitory glycinergic currents that are large enough to suppress action potentials in MNTB neurons at least in some cases. The source(s) of glycinergic inhibition to MNTB are not fully understood. One major extrinsic source of glycinergic inhibitory input to MNTB is the ventral nucleus of the trapezoid body. However, it has been suggested that MNTB neurons receive additional inhibitory inputs via intrinsic connections (collaterals of glycinergic projections of MNTB neurons). While several authors have postulated their presence, these collaterals have never been examined in detail. Here we test the hypothesis that collaterals of MNTB principal cells provide glycinergic inhibition to the MNTB. We injected dye into single principal neurons in the MNTB, traced their projections, and immunohistochemically identified their synapses. We found that collaterals terminate within the MNTB and provide an additional source of inhibition to other principal cells, creating an inhibitory microcircuit within the MNTB. Only about a quarter to a third of MNTB neurons receive such collateral inputs. This microcircuit could produce side band inhibition and enhance frequency tuning of MNTB neurons, consistent with physiological observations.


Asunto(s)
Neuronas/metabolismo , Cuerpo Trapezoide/metabolismo , Animales , Femenino , Gerbillinae , Inmunohistoquímica , Lisina/análogos & derivados , Lisina/química , Masculino , Microscopía Fluorescente , Neuronas/patología , Técnicas de Placa-Clamp , Análisis de la Célula Individual , Sinapsis/metabolismo , Proteína 25 Asociada a Sinaptosomas/metabolismo , Cuerpo Trapezoide/citología
13.
J Neurosci Methods ; 134(2): 121-31, 2004 Apr 30.
Artículo en Inglés | MEDLINE | ID: mdl-15003378

RESUMEN

We have recently applied Lindau-Neher's capacitance measurement technique to study vesicle trafficking at the calyx-type synapse in the rat medial nucleus of the trapezoid body (MNTB) in slice conditions. This application made the MNTB synapse an excellent model for the study of exocytosis and endocytosis at conventional active zones. However, the application was only made at calyces that are presumably equivalent to a single-compartment circuit because their passive current transients decayed mono-exponentially. Here, we determined whether the application could be extended to majority of calyces whose passive current transients decayed bi-exponentially. By comparison of calyces with mono- or bi-exponential decay in their passive current transients, we found similar properties in respect to: (1) the capacitance jump induced by trains of action-potential equivalent stimuli, which reflects exocytosis; (2) the size of a releasable vesicle pool; (3) the time course of the decay after the capacitance jump, which reflects endocytosis; and (4) the transient capacitance artifact observed in the presence of Cd(2+) that blocks exocytosis. These similar properties were also obtained from modeling calyces as a single- or two-compartment circuit. Thus, capacitance measurements may be extended to the majority of calyces, which may facilitate the study of rapid vesicle trafficking at conventional active zones.


Asunto(s)
Tronco Encefálico/citología , Capacidad Eléctrica , Neuronas/fisiología , Sinapsis/fisiología , Animales , Animales Recién Nacidos , Axones/metabolismo , Tronco Encefálico/fisiología , Cadmio/farmacología , Simulación por Computador , Potenciales Postsinápticos Excitadores/fisiología , Técnicas In Vitro , Isoquinolinas/metabolismo , Modelos Neurológicos , Neuronas/citología , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp/métodos , Ratas , Ratas Wistar , Sinapsis/clasificación , Sinapsis/efectos de los fármacos , Factores de Tiempo
14.
Artículo en Inglés | MEDLINE | ID: mdl-25120436

RESUMEN

Neurons in the medial nucleus of the trapezoid body (MNTB) receive prominent excitatory input through the calyx of Held, a giant synapse that produces large and fast excitatory currents. MNTB neurons also receive inhibitory glycinergic inputs that are also large and fast, and match the calyceal excitation in terms of synaptic strength. GABAergic inputs provide additional inhibition to MNTB neurons. Inhibitory inputs to MNTB modify spiking of MNTB neurons both in-vitro and in-vivo, underscoring their importance. Surprisingly, the origin of the inhibitory inputs to MNTB has not been shown conclusively. We performed retrograde tracing, anterograde tracing, immunohistochemical experiments, and electrophysiological recordings to address this question. The results support the ventral nucleus of the trapezoid body (VNTB) as at least one major source of glycinergic input to MNTB. VNTB fibers enter the ipsilateral MNTB, travel along MNTB principal neurons and produce several bouton-like presynaptic terminals. Further, the contribution of GABA to the total inhibition declines during development, resulting in only a very minor fraction of GABAergic inhibition in adulthood, which is matched in time by a reduction in expression of a GABA synthetic enzyme in VNTB principal neurons.


Asunto(s)
Red Nerviosa/fisiología , Inhibición Neural/fisiología , Neuronas/fisiología , Sinapsis/fisiología , Cuerpo Trapezoide/anatomía & histología , Factores de Edad , Animales , Animales Recién Nacidos , Toxina del Cólera/metabolismo , Estimulación Eléctrica , Electroporación , Lateralidad Funcional , Glutamato Descarboxilasa/metabolismo , Proteínas de Transporte de Glicina en la Membrana Plasmática/genética , Proteínas de Transporte de Glicina en la Membrana Plasmática/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Compuestos Heterocíclicos con 3 Anillos/metabolismo , Técnicas In Vitro , Ratones , Ratones Transgénicos , Red Nerviosa/crecimiento & desarrollo , Técnicas de Placa-Clamp , Rodaminas , Cuerpo Trapezoide/crecimiento & desarrollo
15.
J Vis Exp ; (71): e4358, 2013 Jan 18.
Artículo en Inglés | MEDLINE | ID: mdl-23354055

RESUMEN

In vivo recordings from single neurons allow an investigator to examine the firing properties of neurons, for example in response to sensory stimuli. Neurons typically receive multiple excitatory and inhibitory afferent and/or efferent inputs that integrate with each other, and the ultimate measured response properties of the neuron are driven by the neural integrations of these inputs. To study information processing in neural systems, it is necessary to understand the various inputs to a neuron or neural system, and the specific properties of these inputs. A powerful and technically relatively simple method to assess the functional role of certain inputs that a given neuron is receiving is to dynamically and reversibly suppress or eliminate these inputs, and measure the changes in the neuron's output caused by this manipulation. This can be accomplished by pharmacologically altering the neuron's immediate environment with piggy-back multibarrel electrodes. These electrodes consist of a single barrel recording electrode and a multibarrel drug electrode that can carry up to 4 different synaptic agonists or antagonists. The pharmacological agents can be applied iontophoretically at desired times during the experiment, allowing for time-controlled delivery and reversible reconfiguration of synaptic inputs. As such, pharmacological manipulation of the microenvironment represents a powerful and unparalleled method to test specific hypotheses about neural circuit function. Here we describe how piggy-back electrodes are manufactured, and how they are used during in vivo experiments. The piggy-back system allows an investigator to combine a single barrel recording electrode of any arbitrary property (resistance, tip size, shape etc) with a multibarrel drug electrode. This is a major advantage over standard multi-electrodes, where all barrels have more or less similar shapes and properties. Multibarrel electrodes were first introduced over 40 years ago, and have undergone a number of design improvements until the piggy-back type was introduced in the 1980s. Here we present a set of important improvements in the laboratory production of piggy-back electrodes that allow for deep brain penetration in intact in vivo animal preparations due to a relatively thin electrode shaft that causes minimal damage. Furthermore these electrodes are characterized by low noise recordings, and have low resistance drug barrels for very effective iontophoresis of the desired pharmacological agents.


Asunto(s)
Electrodos Implantados , Iontoforesis/instrumentación , Neuronas/efectos de los fármacos , Neuronas/fisiología , Animales , Encéfalo/fisiología , Encéfalo/cirugía
16.
PLoS One ; 8(7): e67626, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23874433

RESUMEN

Recently developed optogenetic tools provide powerful approaches to optically excite or inhibit neural activity. In a typical in-vivo experiment, light is delivered to deep nuclei via an implanted optical fiber. Light intensity attenuates with increasing distance from the fiber tip, determining the volume of tissue in which optogenetic proteins can successfully be activated. However, whether and how this volume of effective light intensity varies as a function of brain region or wavelength has not been systematically studied. The goal of this study was to measure and compare how light scatters in different areas of the mouse brain. We delivered different wavelengths of light via optical fibers to acute slices of mouse brainstem, midbrain and forebrain tissue. We measured light intensity as a function of distance from the fiber tip, and used the data to model the spread of light in specific regions of the mouse brain. We found substantial differences in effective attenuation coefficients among different brain areas, which lead to substantial differences in light intensity demands for optogenetic experiments. The use of light of different wavelengths additionally changes how light illuminates a given brain area. We created a brain atlas of effective attenuation coefficients of the adult mouse brain, and integrated our data into an application that can be used to estimate light scattering as well as required light intensity for optogenetic manipulation within a given volume of tissue.


Asunto(s)
Encéfalo/fisiología , Encéfalo/efectos de la radiación , Luz , Estimulación Luminosa , Dispersión de Radiación , Animales , Mapeo Encefálico/métodos , Femenino , Masculino , Ratones , Fibras Ópticas
17.
J Comp Neurol ; 518(7): 1008-29, 2010 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-20127803

RESUMEN

Bassoon and Piccolo contribute to the cytomatrix of active zones (AZ), the sites of neurotransmitter release in nerve terminals. Here, we examined the 3D localization of Bassoon and Piccolo in the rat calyx of Held between postnatal days 9 and 21, the period of hearing onset characterized by pronounced structural and functional changes. Bassoon and Piccolo were identified by immunohistochemistry (IHC) on slices of the brainstem harboring calyces labeled with membrane-anchored green fluorescent protein (mGFP). By using confocal microscopy and 3D reconstructions, we examined the distribution of Bassoon and Piccolo in calyces delineated by mGFP. This allowed us to discriminate calyceal IHC signals from noncalyceal signals located in the spaces between the calyceal stalks, which could mimic a calyx-like distribution. We found that both proteins were arranged in clusters resembling the size of AZs. These clusters were located along the presynaptic membrane facing the principal cell, close to or overlapping with synaptic vesicle (SV) clusters. Only about 60% of Bassoon and Piccolo clusters overlapped, whereas the remaining clusters contained predominantly Bassoon or Piccolo, suggesting differential targeting of these proteins within a single nerve terminal and potentially heterogeneous AZs functional properties. The total number of Bassoon and Piccolo clusters, which may approximate the number of AZs, was 405 +/- 35 at P9 and 601 +/- 45 at P21 (mean +/- SEM, n = 12). Normalized to calyx volume at P9 and P21, the density of clusters was similar, suggesting that the absolute number of clusters, not density, may contribute to the functional maturation associated with hearing onset.


Asunto(s)
Vías Auditivas/crecimiento & desarrollo , Proteínas del Citoesqueleto/metabolismo , Audición/fisiología , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Neuropéptidos/metabolismo , Sinapsis/metabolismo , Animales , Vías Auditivas/citología , Vías Auditivas/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Imagenología Tridimensional/métodos , Inmunohistoquímica , Masculino , Microscopía Confocal , Neuronas/fisiología , Puente/citología , Puente/crecimiento & desarrollo , Puente/metabolismo , Terminales Presinápticos/metabolismo , Ratas , Ratas Sprague-Dawley , Vesículas Sinápticas/metabolismo , Factores de Tiempo
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