Abstract
Neurons are classified according to action potential firing in response to current injection. While such firing patterns are shaped by the composition and distribution of ion channels, modelling studies suggest that the geometry of dendritic branches also influences temporal firing patterns. Verifying this link is crucial to understanding how neurons transform their inputs to output but has so far been technically challenging. Here, we investigate branching-dependent firing by pruning the dendritic tree of pyramidal neurons. We use a focused ultrafast laser to achieve highly localized and minimally invasive cutting of dendrites, thus keeping the rest of the dendritic tree intact and the neuron functional. We verify successful dendrotomy via two-photon uncaging of neurotransmitters before and after dendrotomy at sites around the cut region and via biocytin staining. Our results show that significantly altering the dendritic arborisation, such as by severing the apical trunk, enhances excitability in layer V cortical pyramidal neurons as predicted by simulations. This method may be applied to the analysis of specific relationships between dendritic structure and neuronal function. The capacity to dynamically manipulate dendritic topology or isolate inputs from various dendritic domains can provide a fresh perspective on the roles they play in shaping neuronal output.
Highlights
Studying the input-output transfer function of neurons is essential to understanding information processing in the brain
We used our previously reported custom-built 2P laser scanning microscope[25,26] to obtain a 3D view of layer V (L5) cortical pyramidal cells (PCs) labelled with Alexa-488 from which we chose the site for dendrotomy
Time series of 2P images were taken of the target site up to two hours after dendrotomy to examine the dendritic structure around the cut
Summary
Studying the input-output transfer function of neurons is essential to understanding information processing in the brain. The exact underlying principle for all these processes is not yet fully understood, it has been demonstrated that using a femtosecond (fs) pulsed laser for surgery allows for cutting of submicron-sized structures[18,19,20] with minimal or negligible damage to surrounding tissue This technique has been applied to the mammalian central nervous system, in vivo to produce vascular disruptions in rat brain parenchyma to model stroke[21] and to dissect dendrites and ablate single spines in the rat cortex[22], and in vitro to sever axons, which have submicron diameters[23,24]
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