Hans-Ulrich Dodt

The Endocannabinoid System Controls Key Epileptogenic Circuits in the Hippocampus

Balanced control of neuronal activity is central in maintaining function and viability of neuronal circuits. The endocannabinoid system tightly controls neuronal excitability. Here, we show that endocannabinoids directly target hippocampal glutamatergic neurons to provide protection against acute epileptiform seizures in mice. Functional CB1 cannabinoid receptors are present on glutamatergic terminals of the hippocampal formation, colocalizing with vesicular glutamate transporter 1 (VGluT1). Conditional deletion of the CB1 gene either in cortical glutamatergic neurons or in forebrain GABAergic neurons, as well as virally induced deletion of the CB1 gene in the hippocampus, demonstrate that the presence of CB1 receptors in glutamatergic hippocampal neurons is both necessary and sufficient to provide substantial endogenous protection against kainic acid (KA)-induced seizures. The direct endocannabinoid-mediated control of hippocampal glutamatergic neurotransmission may constitute a promising therapeutic target for the treatment of disorders associated with excessive excitatory neuronal activity.

Three-dimensional imaging of solvent-cleared organs using 3DISCO.

The examination of tissue histology by light microscopy is a fundamental tool for investigating the structure and function of organs under normal and disease states. Many current techniques for tissue sectioning, imaging and analysis are time-consuming, and they present major limitations for 3D tissue reconstruction. The introduction of methods to achieve the optical clearing and subsequent light-sheet laser scanning of entire transparent organs without sectioning represents a major advance in the field. We recently developed a highly reproducible and versatile clearing procedure called 3D imaging of solvent-cleared organs, or 3DISCO, which is applicable to diverse tissues including brain, spinal cord, immune organs and tumors. Here we describe a detailed protocol for performing 3DISCO and present its application to various microscopy techniques, including example results from various mouse tissues. The tissue clearing takes as little as 3 h, and imaging can be completed in ∼45 min. 3DISCO is a powerful technique that offers 3D histological views of tissues in a fraction of the time and labor required to complete standard histology studies.

Visualizing unstained neurons in living brain slices by infrared DIC-videomicroscopy.

With a combination of infrared illumination, differential interference contrast (DIC) and image intensification by video, unstained living neurons have been visualized up to a depth of 50-100 microns in 300-microns thick brain slices. Recording and application pipettes could be placed under visual guidance. When applied to slices from rat neocortex and hippocampus, pyramidal and non-pyramidal neurons could be differentiated with medium magnification (20x-40x objective). Local neuronal clusters and bundling of apical dendrites of pyramidal cells were visible. The use of an objective with high numerical aperture (63x, N.A. 1.4) allowed the visualization of structures in the submicron range in neocortex and hippocampus. In combination with electrophysiological recordings, infrared DIC-videomicroscopy should facilitate the search for morphological changes underlying neuronal plasticity and the characterization of neuronal networks.

Cannabinoid Receptor Type 1 Located on Presynaptic Terminals of Principal Neurons in the Forebrain Controls Glutamatergic Synaptic Transmission

It is widely accepted that cannabinoids regulate GABA release by activation of cannabinoid receptor type 1 (CB1). Results obtained from a variety of brain regions consistently indicate that cannabinoid agonists can also reduce glutamatergic synaptic transmission. However, there are still conflicting data concerning the role of CB1 in cannabinoid-induced inhibition of glutamatergic transmission in cortical areas. Here, we provide direct evidence that activation of CB1 on terminals of principal neurons controls excitatory synaptic responses in the forebrain. In slices of the basolateral amygdala, the CA1 region of the hippocampus, and the primary somatosensory cortex of wild-type mice, application of the CB1 agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN55,212-2; WIN) (5 μm) reduced evoked excitatory postsynaptic responses. In contrast, in slices obtained from conditional mouse mutants lacking CB1 in all principal forebrain neurons but not in GABAergic interneurons (CB1f/f;CaMKIIαCre), WIN no longer affected glutamatergic synaptic transmission in any of the brain regions tested. Compatible with a presynaptic mechanism, WIN did not change the sensitivity to focally uncaged l-glutamate. WIN reduced glutamatergic responses in slices obtained from mice lacking CB1 exclusively in GABAergic neurons (CB1f/f;Dlx5/6-Cre), thus excluding the involvement of CB1 expressed on GABAergic neurons in this effect of the drug. The present data strongly indicate that excitatory synaptic transmission in forebrain areas is directly modulated by CB1 expressed on presynaptic axon terminals originating from glutamatergic neurons.

Chemical Clearing and Dehydration of GFP Expressing Mouse Brains

Generally, chemical tissue clearing is performed by a solution consisting of two parts benzyl benzoate and one part benzyl alcohol. However, prolonged exposure to this mixture markedly reduces the fluorescence of GFP expressing specimens, so that one has to compromise between clearing quality and fluorescence preservation. This can be a severe drawback when working with specimens exhibiting low GFP expression rates. Thus, we screened for a substitute and found that dibenzyl ether (phenylmethoxymethylbenzene, CAS 103-50-4) can be applied as a more GFP-friendly clearing medium. Clearing with dibenzyl ether provides improved tissue transparency and strikingly improved fluorescence intensity in GFP expressing mouse brains and other samples as mouse spinal cords, or embryos. Chemical clearing, staining, and embedding of biological samples mostly requires careful foregoing tissue dehydration. The commonly applied tissue dehydration medium is ethanol, which also can markedly impair GFP fluorescence. Screening for a substitute also for ethanol we found that tetrahydrofuran (CAS 109-99-9) is a more GFP-friendly dehydration medium than ethanol, providing better tissue transparency obtained by successive clearing. Combined, tetrahydrofuran and dibenzyl ether allow dehydration and chemical clearing of even delicate samples for UM, confocal microscopy, and other microscopy techniques.

Three-dimensional reconstruction and segmentation of intact Drosophila by ultramicroscopy

Genetic mutants are invaluable for understanding the development, physiology and behaviour of Drosophila. Modern molecular genetic techniques enable the rapid generation of large numbers of different mutants. To phenotype these mutants sophisticated microscopy techniques are required, ideally allowing the 3D-reconstruction of the anatomy of an adult fly from a single scan. Ultramicroscopy enables up to cm fields of view, whilst providing micron resolution. In this paper, we present ultramicroscopy reconstructions of the flight musculature, the nervous system, and the digestive tract of entire, chemically cleared, drosophila in autofluorescent light. The 3D-reconstructions thus obtained verify that the anatomy of a whole fly, including the filigree spatial organization of the direct flight muscles, can be analysed from a single ultramicroscopy reconstruction. The recording procedure, including 3D-reconstruction using standard software, takes no longer than 30 min. Additionally, image segmentation, which would allow for further quantitative analysis, was performed.

NMDA and AMPA receptors on neocortical neurons are differentially distributed

The distribution of glutamate receptor subtypes on the surface of neurons is highly relevant for synaptic activation and signal processing in the neocortex. As a novel approach we have used infra‐red videomicroscopy in combination with photostimulation or microiontophoresis in brain slices of rat neocortex to map the distribution of N‐methyl‐d‐aspartate (NMDA) and α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptors on pyramidal neurons of layer V. Both modes of application revealed a spatially distinct distribution of glutamate receptor subtypes: the soma and the proximal dendrite of neurons are highly sensitive to NMDA, whereas the more distal parts of the dendrite are more sensitive to AMPA. An implication is that NMDA receptors near the soma might regulate the amplification of synaptic signals resulting from AMPA receptor activation on remote dendritic sites.

Light sheet microscopy of living or cleared specimens.

Light sheet microscopy is a versatile imaging technique with a unique combination of capabilities. It provides high imaging speed, high signal-to-noise ratio and low levels of photobleaching and phototoxic effects. These properties are crucial in a wide range of applications in the life sciences, from live imaging of fast dynamic processes in single cells to long-term observation of developmental dynamics in entire large organisms. When combined with tissue clearing methods, light sheet microscopy furthermore allows rapid imaging of large specimens with excellent coverage and high spatial resolution. Even samples up to the size of entire mammalian brains can be efficiently recorded and quantitatively analyzed. Here, we provide an overview of the history of light sheet microscopy, review the development of tissue clearing methods, and discuss recent technical breakthroughs that have the potential to influence the future direction of the field.

Actions of noradrenaline on neocortical neurons in vitro

The effects of noradrenaline (NA) and alpha- and beta-adrenergic agonists on neocortical neurons in rat brain slices were studied by intracellular recording. NA added to the perfusion medium decreased the orthodromically evoked excitatory postsynaptic potential (EPSP) and increased the neuronal excitability on current injection. The decrease of the EPSP by NA was mimicked by alpha-adrenoceptor activation. The beta-adrenergic agonist, isoprenaline, increased the neuronal excitability to current injections similarly to NA. In addition, isoprenaline markedly increased the amplitude of a late component of the EPSP (I-EPSP). The increase of the I-EPSP was long-lasting (up to 90 min). Since this late component of the EPSP is blocked by the NMDA (N-methyl-D-aspartate)-antagonist APV (D-2-amino-5-phosphonovalerate), an enhancement of this excitatory synaptic transient via beta-adrenoceptors may be relevant for the facilitatory effect of NA on neuronal plasticity.

Ultramicroscopy: 3D reconstruction of large microscopical specimens

Ultramicroscopy is a microscopical technique that allows optical sectioning and 3D reconstruction of biological and medical specimens. While in confocal microscopy specimen size is limited to several hundred micrometers at best, using ultramicroscopy even centimeter sized objects like whole mouse embryos can be reconstructed with micrometer resolution. This is possible by using a combination of a clearing procedure and the principle of lightsheet illumination. We present ultramicroscopic 3D reconstructions of whole immunohistochemically labelled mouse embryos and adult Drosophila, giving detailed insight into their anatomy. Its speed and simplicity makes ultramicroscopy ideally suited for high-throughput phenotype screening of transgenic mice and thus will benefit the investigation of disease models.