RESUMEN
Studying chemosensory processing desires precise chemical cue presentation, behavioral response monitoring, and large-scale neuronal activity recording. Here we present Fish-on-Chips, a set of optofluidic tools for highly-controlled chemical delivery while simultaneously imaging behavioral outputs and whole-brain neuronal activities at cellular resolution in larval zebrafish. These include a fluidics-based swimming arena and an integrated microfluidics-light sheet fluorescence microscopy (µfluidics-LSFM) system, both of which utilize laminar fluid flows to achieve spatiotemporally precise chemical cue presentation. To demonstrate the strengths of the platform, we used the navigation arena to reveal binasal input-dependent behavioral strategies that larval zebrafish adopt to evade cadaverine, a death-associated odor. The µfluidics-LSFM system enables sequential presentation of odor stimuli to individual or both nasal cavities separated by only ~100 µm. This allowed us to uncover brainwide neural representations of cadaverine sensing and binasal input summation in the vertebrate model. Fish-on-Chips is readily generalizable and will empower the investigation of neural coding in the chemical senses.
Asunto(s)
Encéfalo , Pez Cebra , Animales , Pez Cebra/fisiología , Larva , Cadaverina , Encéfalo/fisiología , Microscopía Fluorescente/métodosRESUMEN
Antibodies have diverse applications due to their high reaction specificities but are sensitive to denaturation when a higher working temperature is required. We have developed a simple, highly scalable and generalizable chemical approach for stabilizing off-the-shelf antibodies against thermal and chemical denaturation. We demonstrate that the stabilized antibodies (termed SPEARs) can withstand up to 4 weeks of continuous heating at 55 °C and harsh denaturants, and apply our method to 33 tested antibodies. SPEARs enable flexible applications of thermocycling and denaturants to dynamically modulate their binding kinetics, reaction equilibrium, macromolecular diffusivity and aggregation propensity. In particular, we show that SPEARs permit the use of a thermally facilitated three-dimensional immunolabeling strategy (termed ThICK staining), achieving whole mouse brain immunolabeling within 72 h, as well as nearly fourfold deeper penetration with threefold less antibodies in human brain tissue. With faster deep-tissue immunolabeling and broad compatibility with tissue processing and clearing methods without the need for any specialized equipment, we anticipate the wide applicability of ThICK staining with SPEARs for deep immunostaining.
Asunto(s)
Anticuerpos , Encéfalo , Animales , Anticuerpos/metabolismo , Encéfalo/metabolismo , Humanos , RatonesRESUMEN
Current understanding of human brain development is rudimentary due to suboptimal in vitro and animal models. In particular, how initial cell positions impact subsequent human cortical development is unclear because experimental spatial control of cortical cell arrangement is technically challenging. 3D cell printing provides a rapid customized approach for patterning. However, it has relied on materials that do not represent the extracellular matrix (ECM) of brain tissue. Therefore, in the present work, a lipid-bilayer-supported printing technique is developed to 3D print human cortical cells in the soft, biocompatible ECM, Matrigel. Printed human neural stem cells (hNSCs) show high viability, neural differentiation, and the formation of functional, stimulus-responsive neural networks. By using prepatterned arrangements of neurons and astrocytes, it is found that hNSC process outgrowth and migration into cell-free matrix and into astrocyte-containing matrix are similar in extent. However, astrocytes enhance the later developmental event of axon bundling. Both young and mature neurons migrate into compartments containing astrocytes; in contrast, astrocytes do not migrate into neuronal domains signifying nonreciprocal chemorepulsion. Therefore, precise prepatterning by 3D printing allows the construction of natural and unnatural patterns that yield important insights into human cerebral cortex development.
Asunto(s)
Bioimpresión , Corteza Cerebral/crecimiento & desarrollo , Membrana Dobles de Lípidos/química , Ingeniería de Tejidos , Astrocitos/citología , Astrocitos/metabolismo , Diferenciación Celular , Movimiento Celular , Colágeno/química , Combinación de Medicamentos , Matriz Extracelular/química , Humanos , Laminina/química , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Neuronas/citología , Neuronas/metabolismo , Impresión Tridimensional , Proteoglicanos/química , Andamios del Tejido/químicaRESUMEN
Brain-wide activities revealed by neuroimaging and recording techniques have been used to predict motor and cognitive functions in both human and animal models. However, although studies have shown the existence of micrometer-scale spatial organization of neurons in the motor cortex relevant to motor control, two-photon microscopy (TPM) calcium imaging at cellular resolution has not been fully exploited for the same purpose. Here, we ask if calcium imaging data recorded by TPM in rodent brain can provide enough information to predict features of upcoming movement. We collected calcium imaging signal from rostral forelimb area in layer 2/3 of the motor cortex while mice performed a two-dimensional lever reaching task. Images of average calcium activity collected during motion preparation period and inter-trial interval (ITI) were used to predict the forelimb reach results. The evaluation was based on a deep learning model that had been applied for object recognition. We found that the prediction accuracy for both maximum reaching location and trial outcome based on motion preparation period but not ITI were higher than the probabilities governed by chance. Our study demonstrated that imaging data encompassing information on the spatial organization of functional neuronal clusters in the motor cortex is useful in predicting motor acts even in the absence of detailed dynamics of neural activities.
RESUMEN
The mechanisms underlying the emergence of learned motor skill representation in primary motor cortex (M1) are not well understood. Specifically, how motor representation in the deep output layer 5b (L5b) is shaped by motor learning remains virtually unknown. In rats undergoing motor skill training, we detect a subpopulation of task-recruited L5b neurons that not only become more movement-encoding, but their activities are also more structured and temporally aligned to motor execution with a timescale of refinement in tens-of-milliseconds. Field potentials evoked at L5b in vivo exhibit persistent long-term potentiation (LTP) that parallels motor performance. Intracortical dopamine denervation impairs motor learning, and disrupts the LTP profile as well as the emergent neurodynamical properties of task-recruited L5b neurons. Thus, dopamine-dependent recruitment of L5b neuronal ensembles via synaptic reorganization may allow the motor cortex to generate more temporally structured, movement-encoding output signal from M1 to downstream circuitry that drives increased uniformity and precision of movement during motor learning.
Asunto(s)
Aprendizaje , Corteza Motora/fisiología , Destreza Motora , Animales , Dopamina/metabolismo , Electrofisiología , Potenciación a Largo Plazo , Masculino , Corteza Motora/química , Plasticidad Neuronal , Ratas , Ratas Sprague-DawleyRESUMEN
Much recent discussion about the origin of Parkinsonian symptoms has centered around the idea that they arise with the increase of beta frequency waves in the EEG. This activity may be closely related to an oscillation between subthalamic nucleus (STN) and globus pallidus. Since STN is the target of deep brain stimulation, it had been assumed that its action is on the nucleus itself. By means of simultaneous recordings of the firing activities from populations of neurons and the local field potentials in the motor cortex of freely moving Parkinsonian rats, this study casts doubt on this assumption. Instead, we found evidence that the corrective action is upon the cortex, where stochastic antidromic spikes originating from the STN directly modify the firing probability of the corticofugal projection neurons, destroy the dominance of beta rhythm, and thus restore motor control to the subjects, be they patients or rodents.