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.
Studying the input-output transfer function of neurons is essential to understanding information processing in the brain. Neurons can be classified by electrophysiological means via their distinctive temporal firing patterns of action potentials following the injection of current at the soma1,2,3,4. A neuron’s firing pattern is shaped by the composition and distribution of ion channels in the membrane and by dendritic morphology5,6,7.
Neurons have a functional polarity; that is, they have a uni-directional flow of information. Inputs from other cells arrive at the dendrites while the output from their integration is sent to other neurons through the axon via synapses. Synaptic integration is highly dependent on the timing and location of inputs, the properties of dendrites and the interaction between cellular compartments. Inputs to various dendritic domains, e.g. apical or basal, are hypothesized to play specific roles in generating an action potential. Yet the functional significance of inputs to these dendritic domains remains cryptic8,9,10. To systematically study their functions, the morphological complexity of dendritic structure can be modified by isolating inputs from different domains by physically cutting dendrites (dendrotomy).
Several mechanical techniques11,12,13,14,15 have been used to disconnect neurons from their processes in brain slices. These techniques, however, are invasive and incur collateral damage to the cell and the surrounding tissue. As such, their application has been mostly limited to dissection of the main apical dendrite. The challenge is to realize a tool and establish an efficient opto-electrophysiological protocol that can access dendrites embedded deep within brain tissue and perform highly targeted dendrotomy while keeping the neuron functional and the surrounding neuropile largely intact.
Non-linear multi-photon processes can facilitate highly localized dissection of dendrites within brain tissue. The key is to use a highly penetrating near-infrared (NIR) femtosecond (fs) pulse laser focused to a diffraction-limited focal volume. Cutting is achieved at the focus where a cascade of nonlinear light-tissue interactions such as photochemical processes, plasma formation, tissue expansion and protein denaturation due to high temperature occur16,17. Although 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 structures18,19,20 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 stroke21 and to dissect dendrites and ablate single spines in the rat cortex22, and in vitro to sever axons, which have submicron diameters23,24.
With such precision and flexibility in controlling dissection sites, we sought to cut the dendritic trees of pyramidal neurons and characterize the electrophysiological properties of neurons during and following dendrotomy. We monitor neurons electrophysiologically post dendrotomy and the separation is verified by evoking two-photon (2P) glutamate uncaging responses at identified spines proximal and distal to the site of dendrotomy and by biocytin staining post hoc. The effects of small- and large-extent dendrotomy on neuronal firing characteristics are investigated.
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Go, M. A. et al. Targeted pruning of a neuron’s dendritic tree via femtosecond laser dendrotomy. Sci. Rep. 6, 19078; doi: 10.1038/srep19078 (2016).