RESUMO
Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a number of model organisms and can reliably detect three or more action potentials in short bursts in several systems in vivo. Through protein structure determination, targeted mutagenesis, high-throughput screening, and a battery of in vitro assays, we have increased the dynamic range of GCaMP3 by severalfold, creating a family of "GCaMP5" sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, and in vivo in Caenorhabditis chemosensory neurons, Drosophila larval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2- to 3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combining in vivo imaging with electrophysiology we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3. GCaMP5 allows more sensitive detection of neural activity in vivo and may find widespread applications for cellular imaging in general.
Assuntos
Sinalização do Cálcio , Corantes Fluorescentes/química , Fluorometria/métodos , Proteínas de Fluorescência Verde/química , Neuroimagem/métodos , Neurônios/química , Peptídeos/química , Transmissão Sináptica , Animais , Astrócitos/química , Astrócitos/ultraestrutura , Caenorhabditis elegans , Cristalografia por Raios X , Drosophila melanogaster/crescimento & desenvolvimento , Feminino , Corantes Fluorescentes/análise , Genes Sintéticos , Vetores Genéticos , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/isolamento & purificação , Células HEK293/química , Células HEK293/ultraestrutura , Hipocampo/química , Hipocampo/citologia , Humanos , Larva , Lasers , Camundongos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Junção Neuromuscular/química , Junção Neuromuscular/ultraestrutura , Neurônios/fisiologia , Neurônios/ultraestrutura , Neurópilo/química , Neurópilo/fisiologia , Neurópilo/ultraestrutura , Neurônios Receptores Olfatórios/química , Neurônios Receptores Olfatórios/fisiologia , Neurônios Receptores Olfatórios/ultraestrutura , Peptídeos/análise , Peptídeos/genética , Estimulação Luminosa , Conformação Proteica , Ratos , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Células Bipolares da Retina/química , Células Bipolares da Retina/fisiologia , Células Bipolares da Retina/ultraestrutura , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
In this study, a thulium (Tm:YAP) laser system was developed for brain surgery applications. As the Tm:YAP laser is a continuous-wave laser delivered via silica fibers, it would have great potential for stereotaxic neurosurgery with highest local absorption in the IR region. The laser system developed in this study allowed the user to set the power level, exposure time, and modulation parameters (pulse width and on-off cycles). The Tm:YAP laser beam (200-600 mW, 69-208 W/cm(2)) was delivered from a distance of 2 mm to cortical and subcortical regions of ex-vivo Wistar rat brain tissue samples via a 200-µm-core optical fiber. The system performance, dosimetry study, and ablation characteristics of the Tm:YAP laser were tested at different power levels by maximizing the therapeutic effects and minimizing unwanted thermal side-effects. The coagulation and ablation diameters were measured under microscope. The maximum ablation efficiency (100 × ablation diameter/coagulation diameter) was obtained when the Tm:YAP laser system was operated at 200 mW for 10 s. At this laser dose, the ablation efficiency was found to be 71.4% and 58.7% for cortical and subcortical regions, respectively. The fiber-coupled Tm:YAP laser system in hence proposed for the delivery of photothermal therapies in medical applications.
Assuntos
Encéfalo/cirurgia , Terapia a Laser/métodos , Animais , Encéfalo/patologia , Técnicas In Vitro , Terapia a Laser/instrumentação , Terapia a Laser/estatística & dados numéricos , Ratos , Ratos Wistar , TúlioRESUMO
The Merkel cell-neurite (MCN) complex generates slowly adapting type 1 (SA1) response when mechanically stimulated. Both serotonin (5-HT) and glutamate have been implicated in the generation of normal SA1 responses, but previous studies have been inconclusive as to what their roles are or how synaptic transmission occurs. In this study, excised dorsal skin patches from common water frogs (Rana ridibunda) were stimulated by von Frey hairs during perfusion in a tissue bath, and single-unit spike activity was recorded from SA1 fibres. Serotonin had no significant effect on the SA1 response at low (10 µM) concentration, significantly increased activity in a force-independent manner at 100 µM, but decreased activity with reduced responsiveness to force at 1 mM. Glutamate showed no effect on the responsiveness to force at 100 µM. MDL 72222 (100 µM), an ionotropic 5-HT3 receptor antagonist, completely abolished the responsiveness to force, suggesting that serotonin is released from Merkel cells as a result of mechanical stimulation, and activated 5-HT3 receptors on the neurite. The metabotropic 5-HT2 receptor antagonist, ketanserin, greatly reduced the SA1 fibre's responsiveness to force, as did the non-specific glutamate receptor antagonist, kynurenic acid. This supports a role for serotonin and glutamate as neuromodulators in the MCN complex, possibly by activation and/or inhibition of signalling cascades in the Merkel cell associated with vesicle release. Additionally, it was observed that SA1 responses contained a force-independent component, similar to a dynamic response observed during mechanical vibrations.
