In vivo multiphoton nanosurgery on cortical neurons

Abstract

Abstract. Two-photon microscopy has been used to perform high spatial resolution imaging of spine plasticity in the intact neocortex of living mice. Multiphoton absorption has also been used as a tool for the selective disruption of cellular structures in living cells and simple organisms. In this work, we exploit the spatial localization of multiphoton excitation to perform selective lesions on the neuronal processes of cortical neurons in living mice expressing fluorescent proteins. Neurons are irradiated with a focused, controlled dose of femtosecond laser energy delivered through cranial optical windows. The morphological consequences are then characterized with time lapse 3-D two-photon imaging over a period of minutes to days after the procedure. This methodology is applied to dissect single dendrites with submicrometric precision without causing any visible collateral damage to the surrounding neuronal structures. The spatial precision of this method is demonstrated by ablating individual dendritic spines, while sparing the adjacent spines and the structural integrity of the dendrite. The combination of multiphoton nanosurgery and in vivo imaging in mammals represents a promising tool for neurobiology and neuropharmacology research. The nonlinear nature of two-photon fluorescence provides an absorption volume spatially confined to the focal region. The localization of the excitation is maintained even in strongly scattering tissues, allowing deep high-resolution microscopy. In combination with fluorescent protein expression techniques, two-photon microscopy has become an indispensable tool to image cortical plasticity in living mice.1 In parallel to its application in imaging, multiphoton absorption has also been used as a tool for the selective disruption of intracellular structures.2,3 A similar approach has also been applied in vivo, where two-photon imaging and laser-induced lesions have been combined. 4,5 Other groups have taken advantage of multiphoton absorption to ablate or dissect individual neurons. Multiphoton nanosurgery has been performed in worms to study axon regeneration 6 and dissect the role of specific neurons in behavior.7 Nevertheless, the potential of this technique has not been fully explored in the mammalian central nervous system. We demonstrate a method for performing multiphoton nanosurgery in the central nervous system of mice, and discuss the widespread applications in neurobiology research.We exploited the spatial localization and deep penetration of multiphoton excitation to perform selective lesions on the neuronal processes of cortical neurons in mice expressing fluorescent proteins ͑thy1-YFP-H and thy1-GFP-M transgenic lines 8 ͒. Neurons were irradiated with a focused, controlled dose of femtosecond laser energy delivered through a permanent cranial optical window. Transgenic mice aged 3 to 5 months were used in all experiments unless otherwise stated. Mice were deeply anaesthetized with an intraperitoneal injection of ketamine ͑0.13 mg per g body weight͒ and xylazine ͑0....

In vivo single branch axotomy induces GAP-43–dependent sprouting and synaptic remodeling in cerebellar cortex

“…In Vivo Imaging and Laser Axotomy. The basic design of our TPF imaging system has already been described (16). Briefly, a mode-locked Ti:Sapphire laser (Chameleon; Coherent) (120-fs width pulses, 90-MHz repetition rate) was coupled into a custom-made scanning system based on a pair of galvanometric mirrors (VM500; GSI Lumonics).…”

“…The restricted absorption volume and deep penetration of multiphoton excitation (15) can be used as a tool to dissect single neurites in the brain of adult mice in vivo (16). The severed neuron can be imaged by two-photon fluorescence (TPF) microscopy (17,18), so that the reactive plasticity of the injured process can be monitored in optically accessible parts of the adult CNS in vivo.…”

Advanced Optical Techniques to Explore Brain Structure and Function

“…72 Laser nanosurgery has been demonstrated in the mammalian CNS as well. 73,74 The high spatial precision of multi-photon laser nanosurgery allows ablating even individual dendritic spines, while sparing the adjacent spines and the structural integrity of the dendrite (see Fig. 6).…”

Laser Nanosurgery of Cerebellar Axons <em>In Vivo</em>

“…Taking advantage of transgenic mice in which fluorescent proteins are selectively expressed in subpopulations of neurons [18][19][20] , TPF microscopy has been applied to the exploration of synaptic plasticity and axonal elongation during development in vivo 21,22 .The capability of singularly damaged neurons to regrow after injury can be investigated by coupling in vivo monitoring by two-photon imaging with a model of injury specifically targeted to the axon of interest. Multi-photon absorption of femtosecond pulses has been used to disrupt single dendrites or even single spines 5,23 . Moreover, this injury paradigm allows cutting single axonal branches without disrupting the contacting dendrite 6 .…”

“…The investigation of the timelapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo 3,4 . This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex [5][6][7][8][9] . This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo.…”