Benchtop mesoSPIM: a next-generation open-source light-sheet microscope for cleared samples.

In 2015, we launched the mesoSPIM initiative, an open-source project for making light-sheet microscopy of large cleared tissues more accessible. Meanwhile, the demand for imaging larger samples at higher speed and resolution has increased, requiring major improvements in the capabilities of such microscopes. Here, we introduce the next-generation mesoSPIM ("Benchtop") with a significantly increased field of view, improved resolution, higher throughput, more affordable cost, and simpler assembly compared to the original version. We develop an optical method for testing detection objectives that enables us to select objectives optimal for light-sheet imaging with large-sensor cameras. The improved mesoSPIM achieves high spatial resolution (1.5 µm laterally, 3.3 µm axially) across the entire field of view, magnification up to 20×, and supports sample sizes ranging from sub-mm up to several centimeters while being compatible with multiple clearing techniques. The microscope serves a broad range of applications in neuroscience, developmental biology, pathology, and even physics.

Leptomeningeal collaterals regulate reperfusion in ischemic stroke and rescue the brain from futile recanalization.

Recanalization is the mainstay of ischemic stroke treatment. However, even with timely clot removal, many stroke patients recover poorly. Leptomeningeal collaterals (LMCs) are pial anastomotic vessels with yet-unknown functions. We applied laser speckle imaging, ultrafast ultrasound, and two-photon microscopy in a thrombin-based mouse model of stroke and fibrinolytic treatment to show that LMCs maintain cerebral autoregulation and allow for gradual reperfusion, resulting in small infarcts. In mice with poor LMCs, distal arterial segments collapse, and deleterious hyperemia causes hemorrhage and mortality after recanalization. In silico analyses confirm the relevance of LMCs for preserving perfusion in the ischemic region. Accordingly, in stroke patients with poor collaterals undergoing thrombectomy, rapid reperfusion resulted in hemorrhagic transformation and unfavorable recovery. Thus, we identify LMCs as key components regulating reperfusion and preventing futile recanalization after stroke. Future therapeutic interventions should aim to enhance collateral function, allowing for beneficial reperfusion after stroke.

The Benchtop mesoSPIM: a next-generation open-source light-sheet microscope for large cleared samples.

In 2015, we launched the mesoSPIM initiative (www.mesospim.org), an open-source project for making light-sheet microscopy of large cleared tissues more accessible. Meanwhile, the demand for imaging larger samples at higher speed and resolution has increased, requiring major improvements in the capabilities of light-sheet microscopy. Here, we introduce the next-generation mesoSPIM ("Benchtop") with significantly increased field of view, improved resolution, higher throughput, more affordable cost and simpler assembly compared to the original version. We developed a new method for testing objectives, enabling us to select detection objectives optimal for light-sheet imaging with large-sensor sCMOS cameras. The new mesoSPIM achieves high spatial resolution (1.5 µm laterally, 3.3 µm axially) across the entire field of view, a magnification up to 20x, and supports sample sizes ranging from sub-mm up to several centimetres, while being compatible with multiple clearing techniques. The new microscope serves a broad range of applications in neuroscience, developmental biology, and even physics.

3D Reconstruction of the Clarified Rat Hindbrain Choroid Plexus.

The choroid plexus (CP) acts as a regulated gate between blood and cerebrospinal fluid (CSF). Despite its simple histology (a monostratified cuboidal epithelium overlying a vascularized stroma), this organ has remarkably complex functions several of which involve local interaction with cells located around ventricle walls. Our knowledge of CP structural organization is mainly derived from resin casts, which capture the overall features but only allow reconstruction of the vascular pattern surface, unrelated to the overlying epithelium and only loosely related to ventricular location. Recently, CP single cell atlases are starting to emerge, providing insight on local heterogeneities and interactions. So far, however, few studies have described CP spatial organization at the mesoscale level, because of its fragile nature and deep location within the brain. Here, using an iDISCO-based clearing approach and light-sheet microscopy, we have reconstructed the normal rat hindbrain CP (hCP) macro- and microstructure, using markers for epithelium, arteries, microvasculature, and macrophages, and noted its association with 4th ventricle-related neurovascular structures. The hCP is organized in domains associated to a main vessel (fronds) which carry a variable number of villi; the latter are enclosed by epithelium and may be flat (leaf-like) or rolled up to variable extent. Arteries feeding the hCP emerge from the cerebellar surface, and branch into straight arterioles terminating as small capillary anastomotic networks, which run within a single villus and terminate attaching multiple times to a large tortuous capillary (LTC) which ends into a vein. Venous outflow mostly follows arterial pathways, except for the lateral horizontal segment (LHS) and the caudal sagittal segment. The structure of fronds and villi is related to the microvascular pattern at the hCP surface: when LTCs predominate, leaflike villi are more evident and bulge from the surface; different, corkscrew-like villi are observed in association to arterioles reaching close to the CP surface with spiraling capillaries surrounding them. Both leaf-like and corkscrew-like villi may reach the 4th ventricle floor, making contact points at their tip, where no gap is seen between CP epithelium and ependyma. Contacts usually involve several adjacent villi and may harbor epiplexus macrophages. At the junction between medial (MHS) and lateral (LHS) horizontal segment, arterial supply is connected to the temporal bone subarcuate fossa, and venous outflow drains to a ventral vein which exits through the cochlear nuclei at the Luschka foramen. These vascular connections stabilize the hCP overall structure within the 4th ventricle but make MHS-LHS joint particularly fragile and very easily damaged when removing the brain from the skull. Even in damaged samples, however, CP fronds (or isolated villi) often remain strongly attached to the dorsal cochlear nucleus (DCN) surface; in these fronds, contacts are still present and connecting "bridges" may be seen, suggesting the presence of real molecular contacts rather than mere appositions.

Context-dependent limb movement encoding in neuronal populations of motor cortex.

Neuronal networks of the mammalian motor cortex (M1) are important for dexterous control of limb joints. Yet it remains unclear how encoding of joint movement in M1 depends on varying environmental contexts. Using calcium imaging we measured neuronal activity in layer 2/3 of the M1 forelimb region while mice grasped regularly or irregularly spaced ladder rungs during locomotion. We found that population coding of forelimb joint movements is sparse and varies according to the flexibility demanded from individual joints in the regular and irregular context, even for equivalent grasping actions across conditions. This context-dependence of M1 encoding emerged during task learning, fostering higher precision of grasping actions, but broke apart upon silencing of projections from secondary motor cortex (M2). These findings suggest that M1 exploits information from M2 to adapt encoding of joint movements to the flexibility demands of distinct familiar contexts, thereby increasing the accuracy of motor output.

The mesoSPIM initiative: open-source light-sheet microscopes for imaging cleared tissue.

Light-sheet microscopy is an ideal technique for imaging large cleared samples; however, the community is still lacking instruments capable of producing volumetric images of centimeter-sized cleared samples with near-isotropic resolution within minutes. Here, we introduce the mesoscale selective plane-illumination microscopy initiative, an open-hardware project for building and operating a light-sheet microscope that addresses these challenges and is compatible with any type of cleared or expanded sample ( www.mesospim.org ).

iDISCO+ for the Study of Neuroimmune Architecture of the Rat Auditory Brainstem.

The lower stations of the auditory system display a complex anatomy. The inner ear labyrinth is composed of several interconnecting membranous structures encased in cavities of the temporal bone, and the cerebellopontine angle contains fragile structures such as meningeal folds, the choroid plexus (CP), and highly variable vascular formations. For this reason, most histological studies of the auditory system have either focused on the inner ear or the CNS by physically detaching the temporal bone from the brainstem. However, several studies of neuroimmune interactions have pinpointed the importance of structures such as meninges and CP; in the auditory system, an immune function has also been suggested for inner ear structures such as the endolymphatic duct (ED) and sac. All these structures are thin, fragile, and have complex 3D shapes. In order to study the immune cell populations located on these structures and their relevance to the inner ear and auditory brainstem in health and disease, we obtained a clarified-decalcified preparation of the rat hindbrain still attached to the intact temporal bone. This preparation may be immunolabeled using a clearing protocol (based on iDISCO+) to show location and functional state of immune cells. The observed macrophage distribution suggests the presence of CP-mediated communication pathways between the inner ear and the cochlear nuclei.

Tissue Clearing and Light Sheet Microscopy: Imaging the Unsectioned Adult Zebra Finch Brain at Cellular Resolution.

The inherent complexity of brain tissue, with brain cells intertwining locally and projecting to distant regions, has made three-dimensional visualization of intact brains a highly desirable but challenging task in neuroscience. The natural opaqueness of tissue has traditionally limited researchers to techniques short of single cell resolution such as computer tomography or magnetic resonance imaging. By contrast, techniques with single-cell resolution required mechanical slicing into thin sections, which entails tissue distortions that severely hinder accurate reconstruction of large volumes. Recent developments in tissue clearing and light sheet microscopy have made it possible to investigate large volumes at micrometer resolution. The value of tissue clearing has been shown in a variety of tissue types and animal models. However, its potential for examining the songbird brain remains unexplored. Songbirds are an established model system for the study of vocal learning and sensorimotor control. They share with humans the capacity to adapt vocalizations based on auditory input. Song learning and production are controlled in songbirds by the song system, which forms a network of interconnected discrete brain nuclei. Here, we use the CUBIC and iDISCO+ protocols for clearing adult songbird brain tissue. Combined with light sheet imaging, we show the potential of tissue clearing for the investigation of connectivity between song nuclei, as well as for neuroanatomy and brain vasculature studies.

Multiphoton in vivo imaging with a femtosecond semiconductor disk laser.

We use an ultrafast diode-pumped semiconductor disk laser (SDL) to demonstrate several applications in multiphoton microscopy. The ultrafast SDL is based on an optically pumped Vertical External Cavity Surface Emitting Laser (VECSEL) passively mode-locked with a semiconductor saturable absorber mirror (SESAM) and generates 170-fs pulses at a center wavelength of 1027 nm with a repetition rate of 1.63 GHz. We demonstrate the suitability of this laser for structural and functional multiphoton in vivo imaging in both Drosophila larvae and mice for a variety of fluorophores (including mKate2, tdTomato, Texas Red, OGB-1, and R-CaMP1.07) and for endogenous second-harmonic generation in muscle cell sarcomeres. We can demonstrate equivalent signal levels compared to a standard 80-MHz Ti:Sapphire laser when we increase the average power by a factor of 4.5 as predicted by theory. In addition, we compare the bleaching properties of both laser systems in fixed Drosophila larvae and find similar bleaching kinetics despite the large difference in pulse repetition rates. Our results highlight the great potential of ultrafast diode-pumped SDLs for creating a cost-efficient and compact alternative light source compared to standard Ti:Sapphire lasers for multiphoton imaging.

An R-CaMP1.07 reporter mouse for cell-type-specific expression of a sensitive red fluorescent calcium indicator.

Genetically encoded calcium indicators (GECIs) enable imaging of in vivo brain cell activity with high sensitivity and specificity. In contrast to viral infection or in utero electroporation, indicator expression in transgenic reporter lines is induced noninvasively, reliably, and homogenously. Recently, Cre/tTA-dependent reporter mice were introduced, which provide high-level expression of green fluorescent GECIs in a cell-type-specific and inducible manner when crossed with Cre and tTA driver mice. Here, we generated and characterized the first red-shifted GECI reporter line of this type using R-CaMP1.07, a red fluorescent indicator that is efficiently two-photon excited above 1000 nm. By crossing the new R-CaMP1.07 reporter line to Cre lines driving layer-specific expression in neocortex we demonstrate its high fidelity for reporting action potential firing in vivo, long-term stability over months, and versatile use for functional imaging of excitatory neurons across all cortical layers, especially in the previously difficult to access layers 4 and 6.

Early synaptic deficits in the APP/PS1 mouse model of Alzheimer's disease involve neuronal adenosine A2A receptors.

Synaptic plasticity in the autoassociative network of recurrent connections among hippocampal CA3 pyramidal cells is thought to enable the storage of episodic memory. Impaired episodic memory is an early manifestation of cognitive deficits in Alzheimer's disease (AD). In the APP/PS1 mouse model of AD amyloidosis, we show that associative long-term synaptic potentiation (LTP) is abolished in CA3 pyramidal cells at an early stage. This is caused by activation of upregulated neuronal adenosine A2A receptors (A2AR) rather than by dysregulation of NMDAR signalling or altered dendritic spine morphology. Neutralization of A2AR by acute pharmacological inhibition, or downregulation driven by shRNA interference in a single postsynaptic neuron restore associative CA3 LTP. Accordingly, treatment with A2AR antagonists reverts one-trial memory deficits. These results provide mechanistic support to encourage testing the therapeutic efficacy of A2AR antagonists in early AD patients.

Cellular distribution of the NMDA-receptor activated synapto-nuclear messenger Jacob in the rat brain.

In previous work, we found that the protein messenger Jacob is involved in N-methyl-D-aspartate receptor (NMDAR) signaling to the nucleus and cAMP response element-binding protein (CREB) mediated gene expression in hippocampal primary neurons. Particularly, extrasynaptic NMDAR activation drives Jacob efficiently into the nucleus where it then induces gene expression that promotes neurodegeneration. However, the protein also translocates to the nucleus in CA1 neurons after Schaffer collateral long-term potentiation (LTP) but not long-term depression (LTD), suggesting that Jacob might be involved in hippocampal and LTP-dependent learning and memory processes. Not much is known about the cellular and subcellular distribution of the protein in brain. In this paper, we provide an overview of the expression of Jacob in rat brain with special emphasis on the hippocampus. We show that Jacob is abundant in hippocampal pyramidal neurons and interneurons but absent from astrocytes and microglia. Interestingly, we found that Jacob is also present in mossy fiber axons. Double immunofluorescence confocal laser scans with presynaptic markers demonstrate that Jacob is indeed found at excitatory but not inhibitory presynaptic sites. Accordingly, we found no substantial co-localization of Jacob with a postsynaptic marker of inhibitory synapses, gephyrin. In contrast, almost all postsynaptic density protein 95 (PSD-95) positive excitatory postsynaptic sites also exhibited strong Jacob-immunofluorescence. Taken together, these data support a synaptic and nuclear role of Jacob that implicates long-distance NMDAR signaling to the nucleus in excitatory neurons.

Encoding and transducing the synaptic or extrasynaptic origin of NMDA receptor signals to the nucleus.

The activation of N-methyl-D-aspartate-receptors (NMDARs) in synapses provides plasticity and cell survival signals, whereas NMDARs residing in the neuronal membrane outside synapses trigger neurodegeneration. At present, it is unclear how these opposing signals are transduced to and discriminated by the nucleus. In this study, we demonstrate that Jacob is a protein messenger that encodes the origin of synaptic versus extrasynaptic NMDAR signals and delivers them to the nucleus. Exclusively synaptic, but not extrasynaptic, NMDAR activation induces phosphorylation of Jacob at serine-180 by ERK1/2. Long-distance trafficking of Jacob from synaptic, but not extrasynaptic, sites depends on ERK activity, and association with fragments of the intermediate filament α-internexin hinders dephosphorylation of the Jacob/ERK complex during nuclear transit. In the nucleus, the phosphorylation state of Jacob determines whether it induces cell death or promotes cell survival and enhances synaptic plasticity.

Two-photon excitation STED microscopy in two colors in acute brain slices.

Many cellular structures and organelles are too small to be properly resolved by conventional light microscopy. This is particularly true for dendritic spines and glial processes, which are very small, dynamic, and embedded in dense tissue, making it difficult to image them under realistic experimental conditions. Two-photon microscopy is currently the method of choice for imaging in thick living tissue preparations, both in acute brain slices and in vivo. However, the spatial resolution of a two-photon microscope, which is limited to ~350 nm by the diffraction of light, is not sufficient for resolving many important details of neural morphology, such as the width of spine necks or thin glial processes. Recently developed superresolution approaches, such as stimulated emission depletion microscopy, have set new standards of optical resolution in imaging living tissue. However, the important goal of superresolution imaging with significant subdiffraction resolution has not yet been accomplished in acute brain slices. To overcome this limitation, we have developed a new microscope based on two-photon excitation and pulsed stimulated emission depletion microscopy, which provides unprecedented spatial resolution and excellent experimental access in acute brain slices using a long-working distance objective. The new microscope improves on the spatial resolution of a regular two-photon microscope by a factor of four to six, and it is compatible with time-lapse and simultaneous two-color superresolution imaging in living cells. We demonstrate the potential of this nanoscopy approach for brain slice physiology by imaging the morphology of dendritic spines and microglial cells well below the surface of acute brain slices.

Nuclear translocation of jacob in hippocampal neurons after stimuli inducing long-term potentiation but not long-term depression.

BACKGROUND: In recent years a number of potential synapto-nuclear protein messengers have been characterized that are thought to be involved in plasticity-related gene expression, and that have the capacity of importin- mediated and activity-dependent nuclear import. However, there is a surprising paucity of data showing the nuclear import of such proteins in cellular models of learning and memory. Only recently it was found that the transcription factor cyclic AMP response element binding protein 2 (CREB2) transits to the nucleus during long-term depression (LTD), but not during long-term potentiation (LTP) of synaptic transmission in hippocampal primary neurons. Jacob is another messenger that couples NMDA-receptor-activity to nuclear gene expression. We therefore aimed to study whether Jacob accumulates in the nucleus in physiological relevant models of activity-dependent synaptic plasticity. METHODOLOGY/PRINCIPAL FINDINGS: We have analyzed the dynamics of Jacob's nuclear import following induction of NMDA-receptor dependent LTP or LTD at Schaffer collateral-CA1 synapses in rat hippocampal slices. Using time-lapse imaging of neurons expressing a Jacob-Green-Fluorescent-Protein we found that Jacob rapidly translocates from dendrites to the nucleus already during the tetanization period of LTP, but not after induction of LTD. Immunocytochemical stainings confirmed the nuclear accumulation of endogenous Jacob in comparison to apical dendrites after induction of LTP but not LTD. Complementary findings were obtained after induction of NMDA-receptor dependent chemical LTP and LTD in hippocampal primary neurons. However, in accordance with previous studies, high concentrations of NMDA and glycine as well as specific activation of extrasynaptic NMDA-receptors resembling pathological conditions induce an even more profound increase of nuclear Jacob levels. CONCLUSIONS/SIGNIFICANCE: Taken together, these findings suggest that the two major forms of NMDA-receptor dependent synaptic plasticity, LTP and LTD, elicit the transition of different synapto-nuclear messengers albeit in both cases importin-mediated retrograde transport and NMDA-receptor activation is required.