Advances in light microscopy for neuroscience.

Since the work of Golgi and Cajal, light microscopy has remained a key tool for neuroscientists to observe cellular properties. Ongoing advances have enabled new experimental capabilities using light to inspect the nervous system across multiple spatial scales, including ultrastructural scales finer than the optical diffraction limit. Other progress permits functional imaging at faster speeds, at greater depths in brain tissue, and over larger tissue volumes than previously possible. Portable, miniaturized fluorescence microscopes now allow brain imaging in freely behaving mice. Complementary progress on animal preparations has enabled imaging in head-restrained behaving animals, as well as time-lapse microscopy studies in the brains of live subjects. Mouse genetic approaches permit mosaic and inducible fluorescence-labeling strategies, whereas intrinsic contrast mechanisms allow in vivo imaging of animals and humans without use of exogenous markers. This review surveys such advances and highlights emerging capabilities of particular interest to neuroscientists.

Miniaturized integration of a fluorescence microscope.

The light microscope is traditionally an instrument of substantial size and expense. Its miniaturized integration would enable many new applications based on mass-producible, tiny microscopes. Key prospective usages include brain imaging in behaving animals for relating cellular dynamics to animal behavior. Here we introduce a miniature (1.9 g) integrated fluorescence microscope made from mass-producible parts, including a semiconductor light source and sensor. This device enables high-speed cellular imaging across ∼0.5 mm2 areas in active mice. This capability allowed concurrent tracking of Ca2+ spiking in >200 Purkinje neurons across nine cerebellar microzones. During mouse locomotion, individual microzones exhibited large-scale, synchronized Ca2+ spiking. This is a mesoscopic neural dynamic missed by prior techniques for studying the brain at other length scales. Overall, the integrated microscope is a potentially transformative technology that permits distribution to many animals and enables diverse usages, such as portable diagnostics or microscope arrays for large-scale screens.

High-speed, miniaturized fluorescence microscopy in freely moving mice.

A central goal in biomedicine is to explain organismic behavior in terms of causal cellular processes. However, concurrent observation of mammalian behavior and underlying cellular dynamics has been a longstanding challenge. We describe a miniaturized (1.1 g mass) epifluorescence microscope for cellular-level brain imaging in freely moving mice, and its application to imaging microcirculation and neuronal Ca(2+) dynamics.

Zolpidem reduces hippocampal neuronal activity in freely behaving mice: a large scale calcium imaging study with miniaturized fluorescence microscope.

Therapeutic drugs for cognitive and psychiatric disorders are often characterized by their molecular mechanism of action. Here we demonstrate a new approach to elucidate drug action on large-scale neuronal activity by tracking somatic calcium dynamics in hundreds of CA1 hippocampal neurons of pharmacologically manipulated behaving mice. We used an adeno-associated viral vector to express the calcium sensor GCaMP3 in CA1 pyramidal cells under control of the CaMKII promoter and a miniaturized microscope to observe cellular dynamics. We visualized these dynamics with and without a systemic administration of Zolpidem, a GABAA agonist that is the most commonly prescribed drug for the treatment of insomnia in the United States. Despite growing concerns about the potential adverse effects of Zolpidem on memory and cognition, it remained unclear whether Zolpidem alters neuronal activity in the hippocampus, a brain area critical for cognition and memory. Zolpidem, when delivered at a dose known to induce and prolong sleep, strongly suppressed CA1 calcium signaling. The rate of calcium transients after Zolpidem administration was significantly lower compared to vehicle treatment. To factor out the contribution of changes in locomotor or physiological conditions following Zolpidem treatment, we compared the cellular activity across comparable epochs matched by locomotor and physiological assessments. This analysis revealed significantly depressive effects of Zolpidem regardless of the animal's state. Individual hippocampal CA1 pyramidal cells differed in their responses to Zolpidem with the majority (∼ 65%) significantly decreasing the rate of calcium transients, and a small subset (3%) showing an unexpected and significant increase. By linking molecular mechanisms with the dynamics of neural circuitry and behavioral states, this approach has the potential to contribute substantially to the development of new therapeutics for the treatment of CNS disorders.

Long-term dynamics of CA1 hippocampal place codes.

Using Ca(2+) imaging in freely behaving mice that repeatedly explored a familiar environment, we tracked thousands of CA1 pyramidal cells' place fields over weeks. Place coding was dynamic, as each day the ensemble representation of this environment involved a unique subset of cells. However, cells in the ∼15-25% overlap between any two of these subsets retained the same place fields, which sufficed to preserve an accurate spatial representation across weeks.

In vivo brain imaging using a portable 2.9 g two-photon microscope based on a microelectromechanical systems scanning mirror.

We present a two-photon microscope that is approximately 2.9 g in mass and 2.0 x 1.9 x 1.1 cm(3) in size and based on a microelectromechanical systems (MEMS) laser-scanning mirror. The microscope has a focusing motor and a micro-optical assembly composed of four gradient refractive index lenses and a dichroic microprism. Fluorescence is captured without the detected emissions reflecting off the MEMS mirror, by use of separate optical fibers for fluorescence collection and delivery of ultrashort excitation pulses. Using this microscope we imaged neocortical microvasculature and tracked the flow of erythrocytes in live mice.