RESUMO
To study the complex synaptic interactions underpinning dendritic information processing in single neurons, experimenters require methods to mimic presynaptic neurotransmitter release at multiple sites with no physiological damage. We show that laser scanning systems built around large-aperture acousto-optic deflectors and high numerical aperture objective lenses provide the sub-millisecond, sub-micron precision necessary to achieve physiological, exogenous synaptic stimulation. Our laser scanning systems can produce the sophisticated spatio-temporal patterns of synaptic input that are necessary to investigate single-neuron dendritic physiology.
Assuntos
Estimulação Acústica/instrumentação , Acústica/instrumentação , Potenciais de Ação/fisiologia , Microscopia Confocal/instrumentação , Neurônios/citologia , Neurônios/fisiologia , Estimulação Luminosa/instrumentação , Animais , Células Cultivadas , Desenho de Equipamento , Análise de Falha de Equipamento , Hipocampo/citologia , Hipocampo/fisiologia , Hipocampo/efeitos da radiação , Luz , Neurônios/efeitos da radiação , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Mice are an excellent model for studying mammalian hearing and transgenic mouse models of human hearing, loss are commonly available. However, the mouse cochlea is substantially smaller than other animal models routinely used to study cochlear physiology. This makes study of their hair cells difficult. We develop a novel methodology to optically image calcium within living hair cells left undisturbed within the excised mouse cochlea. Fresh cochleae are harvested, left intact within their otic capsule bone, and fixed in a recording chamber. The bone overlying the cochlear epithelium is opened and Reissner's membrane is incised. A fluorescent calcium indicator is applied to the preparation. A custom-built upright two-photon microscope was used to image the preparation using 3-D scanning. We are able to image about one third of a cochlear turn simultaneously, in either the apical or basal regions. Within one hour of animal sacrifice, we find that outer hair cells demonstrate increased fluorescence compared with surrounding supporting cells. This methodology is then used to visualize hair cell calcium changes during mechanotransduction over a region of the epithelium. Because the epithelium is left within the cochlea, dissection trauma is minimized and artifactual changes in hair cell physiology are expected to be reduced.
Assuntos
Cálcio/metabolismo , Cóclea/citologia , Células Ciliadas Auditivas Internas/metabolismo , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Estimulação Acústica , Animais , Cálcio/análise , Permeabilidade da Membrana Celular/fisiologia , Cóclea/anatomia & histologia , Fluoresceínas/metabolismo , Células Ciliadas Auditivas Internas/citologia , Camundongos , Projetos de PesquisaRESUMO
The Nikon C1 confocal laser scanning microscope is a relatively inexpensive and user-friendly instrument. We describe a straightforward method to convert the C1 for multiphoton microscopy utilizing direct coupling of a femtosecond near-infrared laser into the scan head and fiber optic transmission of emission light to the three-channel detector box. Our adapted system can be rapidly switched between confocal and multiphoton mode, requires no modification to the original system, and uses only a few custom-made parts. The entire system, including scan mirrors and detector box, remain under the control of the user-friendly Nikon EZ-C1 software without modification.