Optical microscopy methods for understanding learning and memory

2016

The brain is a complex organ consisting of neurons and non-neuronal cells. Communication between neurons takes place via synapses, whose morphological remodeling is thought to be crucial for information processing and storage in the mammalian brain. Recently, this neuro-centric view of synaptic function has evolved, also taking into account the glial processes in close vicinity of the synapse. However, as their structure is well below the spatial resolution of conventional light microscopy, progress in investigating them in a physiological environment, the intact brain, has been impeded. Indeed, little is known on the nanoscale morphological variations of dendritic spines, the interaction with glial processes, and how these affect synaptic transmission in vivo. Here, we aim to visualize the dynamic nano-morphology of dendritic spines in mouse somatosensory cortex in vivo. We implemented super-resolution 2P-STED time-lapse imaging, which allows for high spatial resolution and deep ti...

Proceedings of SPIE - The International Society for Optical Engineering

In combination with fluorescent protein (XFP) expression techniques, two-photon microscopy has become an indispensable tool to image cortical plasticity in living mice. In parallel to its application in imaging, multi-photon absorption has also been used as a tool for the dissection of single neurites with submicrometric precision without causing any visible collateral damage to the surrounding neuronal structures. In this work, multi-photon nanosurgery is applied to dissect single climbing fibers expressing GFP in the cerebellar cortex. The morphological consequences are then characterized with time lapse 3-dimensional two-photon imaging over a period of minutes to days after the procedure. Preliminary investigations show that the laser induced fiber dissection recalls a regenerative process in the fiber itself over a period of days. These results show the possibility of this innovative technique to investigate regenerative processes in adult brain. In parallel with imaging and man...

Frontiers in Ultrafast Optics: Biomedical, Scientific, and Industrial Applications X, 2010

In combination with fluorescent protein (XFP) expression techniques, two-photon microscopy has become an indispensable tool to image cortical plasticity in living mice. In parallel to its application in imaging, multi-photon absorption has also been used as a tool for the dissection of single neurites with submicrometric precision without causing any visible collateral damage to the surrounding neuronal structures. In this work, multi-photon nanosurgery is applied to dissect single climbing fibers expressing GFP in the cerebellar cortex. The morphological consequences are then characterized with time lapse 3-dimensional two-photon imaging over a period of minutes to days after the procedure. Preliminary investigations show that the laser induced fiber dissection recalls a regenerative process in the fiber itself over a period of days. These results show the possibility of this innovative technique to investigate regenerative processes in adult brain.

ENeuro, 2015

Two-photon microscopy in combination with a technique involving the artificial expression of fluorescent protein has enabled the direct observation of dendritic spines in living brains. However, the application of this method to primate brains has been hindered by the lack of appropriate labeling techniques for visualizing dendritic spines. Here, we developed an adeno-associated virus vector-based fluorescent protein expression system for visualizing dendritic spines in vivo in the marmoset neocortex. For the clear visualization of each spine, the expression of reporter fluorescent protein should be both sparse and strong. To fulfill these requirements, we amplified fluorescent signals using the tetracycline transactivator (tTA)-tetracycline-responsive element system and by titrating down the amount of Thy1S promoter-driven tTA for sparse expression. By this method, we were able to visualize dendritic spines in the marmoset cortex by two-photon microscopy in vivo and analyze the turnover of spines in the prefrontal cortex. Our results demonstrated that short spines in the marmoset cortex tend to change more frequently than long spines. The comparison of in vivo samples with fixed samples showed that we did not detect all existing spines by our method. Although we found glial cell proliferation, the damage of tissues caused by window construction was relatively small, judging from the comparison of spine length between samples with or without window construction. Our new labeling technique for two-photon imaging to visualize in vivo dendritic spines of the marmoset neocortex can be applicable to examining circuit reorganization and synaptic plasticity in primates.

T wo-photon laser-scanning fluorescence microscopy (Denk et al. 1990) has made it possible to image neurons over 600 pm deep within a living slice or organism, with submicrometer resolution and in three dimensions (3D) for many hours without photodamage (Potter et al. 1996a). With true 4D microscopy (i.e., 3D with time), it is now feasible to capture neural development and synaptic plasticity in the act of happening, eliminating many uncertainties associated with between-animal comparisons of fixed-tissue specimens. By using pulsed IR laser light to excite fluorescent labels (e.g., dyes, fluorescent proteins, or endogenous fluorophores) that are normally excited by visible light, excitation is restricted to the focal plane, greatly reducing photobleaching and phototoxicity (see Chapter 7). The IR illumination is scattered less than visible light, allowing imaging 2-3 times deeper than with standard confocal microscopy. In addition, because the fluorescent signal emanates only from the focus of the scanning IR laser beam within the specimen, no confocal aperture is necessary to remove the out-of-focus signal. This means that two-photon microscopy has an inherently higher signal-to-noise ratio (SNR) compared with confocal microscopy. Excellent references for two-photon microscopy, and for labeling and imaging living specimens, include Denk et al. (1995) and Terasaki and Dailey (1995). In this chapter, I describe two-photon imaging hardware, pointing out potential pitfalls in microscope construction and operation. I also describe technical considerations for successful two-photon microscopy in two experimental systems: (1) 4D imaging of living neurons transplanted to cultured hippocampal slices from neonatal rats and (2) 4D imaging of dendritic spines within acute hippocampal slices from adult rats. Figure 1 shows the components of the imaging setup.

2001

abstract We wish to understand how neural systems store, recall, and process information. We are using cultured networks of cortical neurons grown on microelectrode arrays as a model system for studying the emergent properties of ensembles of living neurons. We have developed a 2-way communication interface between the cultured network and a computer-generated animal, the Neurally Controlled Animat.

F1000 - Post-publication peer review of the biomedical literature, 2004

Dendritic spines of pyramidal neurons in the cerebral cortex undergo activity-dependent structural remodelling 1-5 that has been proposed to be a cellular basis of learning and memory 6. How structural remodelling supports synaptic plasticity 4,5 , such as long-term potentiation 7 , and whether such plasticity is input-specific at the level of the individual spine has remained unknown. We investigated the structural basis of long-term potentiation using two-photon photolysis of caged glutamate at single spines of hippocampal CA1 pyramidal neurons 8. Here we show that repetitive quantum-like photorelease (uncaging) of glutamate induces a rapid and selective enlargement of stimulated spines that is transient in large mushroom spines but persistent in small spines. Spine enlargement is associated with an increase in AMPA-receptor-mediated currents at the stimulated synapse and is dependent on NMDA receptors, calmodulin and actin polymerization. Long-lasting spine enlargement also requires Ca/calmodulin-dependent protein kinase II. Our results thus indicate that spines individually follow Hebb's postulate for learning. They further suggest that small spines are preferential sites for long-term potentiation induction, whereas large spines might represent physical traces of long-term memory. We investigated the structural plasticity of individually identified spines on proximal apical dendrites of CA1 pyramidal neurons in rat hippocampal slice preparations, using two-photon uncaging (at 720nm) of a caged glutamate compound (MNI (4-methoxy-7-nitroindolinyl)glutamate) 8. Neurons within the hippocampal slices were transfected biolistically with an expression vector for enhanced green fluorescent protein (eGFP) and were imaged with a second two-photon laser at 910 nm. Two-photon uncaging allows the release of glutamate within a small focal volume 8 , so that activation of glutamate receptors is limited to a region within ca. 1 μm of the uncaging beam. The power of the laser was set at ca. 5mW, which induced currents through AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-sensitive receptors with amplitudes in the range of miniature excitatory postsynaptic currents (see Methods). Repetitive stimulation of NMDA (N-methyl-D-aspartate)-sensitive

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.

Real-Time Imaging, 2002

M ultiphoton laser scanning microscopy offers improved axial resolution over confocal microscopy for three-dimensional (3D) imaging of fluorescently labeled cells. It also greatly reduces phototoxicity and photobleaching outside the plane of focus when collecting 3D series of optical sections in living tissue. This permits images to be collected at shorter intervals with less harm to the specimen. To facilitate the transition from confocal to twophoton in vivo imaging, we have compared some of the most commonly used fluorescent vital dyes for their applicability to confocal and two-photon laser scanning microscopy. We find that many of the dyes commonly used for morphological imaging can be used for two-photon excitation microscopy. However, the optimal wavelengths for two-photon excitation cannot always be deduced from the one-photon fluorescence spectra of the fluorophores. As improvements in imaging technology are beginning to make near-real-time in vivo fluorescence imaging more feasible, flexible software that can perform temporal as well as spatial analyses of anatomical data becomes increasingly important. Here we demonstrate how we have used Object-Image, an extension of the popular public-domain NIH Image software, together with a set of custom macros for time-lapse morphometric analysis, to analyze neuronal branch growth and complexity in three dimensions over time.

Proceedings of the National Academy of Sciences, 2012

Multiphoton microscopy is a powerful tool in neuroscience, promising to deliver important data on the spatiotemporal activity within individual neurons as well as in networks of neurons. A major limitation of current technologies is the relatively slow scan rates along the z direction compared to the kHz rates obtainable in the x and y directions. Here, we describe a custom-built microscope system based on an architecture that allows kHz scan rates over hundreds of microns in all three dimensions without introducing aberration. We further demonstrate how this high-speed 3D multiphoton imaging system can be used to study neuronal activity at millisecond resolution at the subcellular as well as the population level.