Abstract
Intelligent behavior and cognitive functions in mammals depend on cortical microcircuits made up of a variety of excitatory and inhibitory cells that form a forest-like complex across six layers. Mechanistic understanding of cortical microcircuits requires both manipulation and monitoring of multiple layers and interactions between them. However, existing techniques are limited as to simultaneous monitoring and stimulation at different depths without damaging a large volume of cortical tissue. Here, we present a relatively simple and versatile method for delivering light to any two cortical layers simultaneously. The method uses a tiny optical probe consisting of two microprisms mounted on a single shaft. We demonstrate the versatility of the probe in three sets of experiments: first, two distinct cortical layers were optogenetically and independently manipulated; second, one layer was stimulated while the activity of another layer was monitored; third, the activity of thalamic axons distributed in two distinct cortical layers was simultaneously monitored in awake mice. Its simple-design, versatility, small-size, and low-cost allow the probe to be applied widely to address important biological questions.
Highlights
Despite over half a century of intense investigation, we still do not understand the role of layers in the complicated microcircuitry of the cortex (Adesnik and Naka, 2018)
To take advantage of the ease and flexibility of the single endoscope approach while allowing the probing of multiple layers, we developed a double-μPeriscope that is capable of optogenetic stimulation, fluorescence imaging or both of two separate cortical layers (Figure 1a, b)
A previous in vitro study showed that combined stimulation of cell body and distal apical dendrites within a close time window induces a large, regenerative, long-lasting calcium plateau potential that evokes a burst somatic action potentials (APs) (Larkum et al, 1999) – a phenomenon referred to as ‘backpropagation-activated calcium spike firing’ or BAC firing
Summary
Despite over half a century of intense investigation, we still do not understand the role of layers in the complicated microcircuitry of the cortex (Adesnik and Naka, 2018). Multichannel electrical recording is the most widely used approach to measure the neural responses to optogenetic stimulation. This approach is preferred over the optical approaches available (e.g., two-p hoton imaging) because of the relative ease of use and lower costs. The problem with this approach, is that extracellular signals represent the summation of the activity of all structures (dendrites, axons, and cell bodies) from all cell types surrounding each electrode; even worse, extracellular activity is usually very complex in vivo, in the awake brain
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