Fluorescence Microscopy Journal Club

As part of our work in fluorescence microscopy, we regularly come across great research in super resolution, neuroscience and 2-photon imaging. Members of our Fluorescence Journal Club receive brief reviews of select papers, collected below. If you would like to be added to our email list for these reviews, please email us at productinfo@bruker.com.

ASYMMETRIC PACKAGING OF POLYMERASES WITHIN VESICULAR STOMATITIS VIRUS
Jeffery Hodges, Xiaolin Tang, Michael B. Landesman, John B. Ruedas, Anil Ghimire,
Manasa V. Gudheti, Jacques Perrault, Erik M. Jorgensen, Jordan M. Gerton, Saveez Saffarian
Biochemical and Biophysical Research Communications, Volume 440, Issue 2, p271–276, 18 October 2013
doi: 10.1016/j.bbrc.2013.09.064
(view article: http://www.sciencedirect.com/science/article/pii/S0006291X13015453)


The vesicular stomatitis virus (VSV) a prototypical negative sense single-stranded RNA virus commonly used in the study of viral evolution and pathology. The study of viral behavior and structural organization in such viruses as VSV serves as a building block for the broader understanding of retroviral virus families, such as rabies and HIV. Furthermore, VSV has been targeted for its ability to be genetically modified for use in vaccines for Ebola and rabies, as well as its ability to be used in a variety of oncology treatments.

VSV is a bullet-shaped virus approximately 180 nm long by 80 nm wide, and contains multiple copies of L and P polymerase proteins that are used in transcribing and replicating the N-RNA of the virion. The polymerase engages with promoter sites located at the 3’ end of the RNA strand, determined previously by CryoEM, located at the bullet end of the virion. The authors use a combination of sub-diffraction techniques, namely super-resolution optical microscopy and atomic force microscopy (AFM) to determine the location and quantification of these polymerase proteins within the manifold of the virus to determine its spatial relationship to the transcriptional promoter site of the viral genome.

A series of super-resolution optical localization microscopy experiments on the VSV virus were conducted to measure the relative distance of the L and P proteins to the surface of the virus. This was accomplished by a novel combination of recombinant viruses encoding for the fluorescent protein eGFP for use in conventional diffraction-limited imaging to use as a center-of-mass marker for either the L or P protein, coupled with a super-resolution image of the virus membrane to determine the structural manifold of the VSV virus, leading to the conclusion that the placement of the L and P proteins being near the blunt end of the virus.

Verification of this phenomenon was corroborated via AFM in a fluorescent-free assay where the surface stiffness of the VSV virus was directly measured. The increase in the protein load at one end of the virus or the other will directly relate to a differing stiffness value for the virus surface, with the AFM being used as an active force sensor. These finding agreed well with the findings determined from imaging the virions with optical super-resolution techniques.

In conclusion, the authors determine that the L and P proteins, and hence the polymerase for the N-RNA, are packaged asymmetrically within the volume of the VSV virus. This has direct consequences for transcription events after viral entry into a host cell, and the knowledge of the location of the polymerase with respect to the position and layout of the N-RNA can help further the modeling of the replication of these viruses once they infect a host cell. This paper is a nice example of using super-resolution localization microscopy to determine not only spatial location but quantification as well of labeled protein, as well as incorporating another microscopy technique to further validate the optical results.

DELAUNAY ALGORITHM AND PRINCIPAL COMPONENT ANALYSIS FOR 3D VISUALIZATION OF MITOCHONDRIAL DNA NUCLEOIDS BY BIPLANE FPALM/DSTORM
Alán, L., Špaček, T. & Ježek, P. Eur
Biophys J (2016) 45: 443.
doi:10.1007/s00249-016-1114-5
(view article: https://link.springer.com/article/10.1007/s00249-016-1114-5)


Super-resolution localization microscopy determines the spatial location of fluorophores below the diffraction limit of light by isolating single point sources in time and determining their position through statistical fitting of their recorded point-spread functions to either optical theory or to a calibration curve. A localization microscopy data set then consists of a set of localized points in 3D space containing the location of the fluorophores within the biological sample, which are then computationally rendered to create an image containing spatial information below the classical diffraction limit. Due to the nature of the method, the final image is a computer-generated image of localized data points, and not a conventional captured image.

Various methods of image reconstruction can be employed within localization microscopy, such as direct rendering of the localization data of spheroids of size associated with their localization precision, convolving every localization with a Gaussian distribution, and adaptive histogram binning. Another popular method is based upon Delaunay triangulation, where the two-dimensional case the data set of localized points is connected in such a way that no data points lie within the circumcircle of the triangle formed by three connected points. The data set can then be segmented into “pixels” whose size is based upon the local density of localization points. In three dimensions, this is extended to the circumsphere created by a unique polyhedron composed of four localization points, with no localization points in the interior. This can lead to direct assessment of the local density of points in three-dimensional space, and estimations for the volume that the proteins in question occupy.

COMBINING SINGLE RNA SENSITIVE PROBES WITH SUBDIFFRACTION-LIMITED AND LIVE-CELL IMAGING ENABLES THE CHARACTERIZATION OF VIRUS DYNAMICS IN CELLS
Alonas et al. ACS Nano 8: 302-315 (2014)
ACS Nano, 2014, 8 (1), pp 302–315, DOI: 10.1021/nn405998v, Publication Date (Web): December 18, 2013
(view full article: http://pubs.acs.org/doi/abs/10.1021/nn405998v)


Super-resolution imaging has been gaining popularity and use in recent research. This week’s article shows an example of how super-resolution imaging can be used to study the distribution of viral matrix protein (M) and cellular proteins such as F-actin using newly developed labeling techniques, and can be utilized as general methodology for studying RNA viruses such as influenza and Ebola.

This paper presents a novel method for fluorescently labeling genomic RNA (gRNA) of hSRV (human respiratory syncytial virus) virions using multiply labeled tetravalent RNA imaging probes (MTRIPs).

Cells were infected with hRSV and viral RNA was labeled using MTRIPs. The virions were then harvested from the cell, deposited on glass coverslips and subsequently stained for either the viral nucleoprotein (N) or viral fusion protein (F). Filamentous virion morphology was characterized using three dimensional single molecule localization super-resolution microscopy. Super-resolution images showed that the F-protein was distributed evenly along the length of the filaments in contrast to gRNA and N protein which were distributed unevenly. The gRNA and N protein overlapped tightly in co-localized areas whereas the F protein appeared to surround the gRNA. The axial distributions indicated that F protein was located away from the center of the virion when compared to N protein and gRNA.

REMODELING OF THE SARCOMERIC CYTOSKELETON IN CARDIAC VENTRICULAR MYOCYTES DURING HEART FAILURE AND AFTER CARDIAC RESYNCHRONIZATION THERAPY
Lichter et al. Journal of Molecular Cellular Cardiology 72: 186-195 (2014),
DOI: http://dx.doi.org/10.1016/j.yjmcc.2014.03.012, Publication Date (Web): March 20, 2014
(view full article: www.sciencedirect.com/science/article/pii/S0022282814000893


Understanding structural changes at the molecular level in disease models can provide new insights into underlying disease mechanisms. The authors of this week’s paper used single molecule localization super resolution microscopy to study the effect of cardiac failure on the sarcomeric cytoskeleton after cardiac failure.

Canine cardiomyocytes were used as a model system to study structural changes in the sarcomere associated cytoskeletal protein, alpha-actinin, after inducing synchronous or dyssynchronous heart failure (SHF or DHF) and following cardiac resynchronization therapy (CRT). In control cells the organization of alpha-actinin was found to be in a regular, pararell, transverse sheet pattern consistent with the arrangement found in striated muscle. In heart failure and CRT cells there was a reduction in the overall spatial regularity of alpha-actinin and increase in the presence of longitudinal alpha-actinin depositions connecting adjacent parallel alpha-actinin sheets. This decreased regularity was most pronounced in DHF.

SEQUENTIAL COMBINATION THERAPY OF OVARIAN CANCER WITH DEGRADABLE N-(2-HYDROXYPROPYL)METHACRYLAMIDE COPOLYMER PACLITAXEL AND GEMCITABINE CONJUGATES
Rui Zhang, Jiyuan Yang, Monika SIma, Yan Zhou, and Jindřich Kopeček
Proceedings of the National Academy of Sciences 111(33): 12181–12186 (2014)
doi:10.1073/nature.1406233111
(view full article: www.pnas.org/content/111/33/12181.long)


Single Molecule localization super resolution imaging offers the ability to track the distribution of specific molecule types throughout cells and tissues. In this paper, the authors utilized that potential in order to evaluate the intracellular mechanism of delivery of therapeutic agents for cancer treatment.

Biopolymers are being increasingly used as drug carriers in cancer treatment. The authors characterize the effect of second-generation high-molecular weight backbone-degradable HPMA copolymer carriers on A2780 human ovarian carcinoma xenografts. Three dimensional single molecule localization super-resolution imaging was used to elucidate conjugate internalization and drug release by using FITC-P-Cy5 as a model conjugate. Super-resolution imaging showed that FITC-P-Cy5 was internalized via endocytosis and colocalized with lysosomes and late endosomes. FITC and Cy5 signals were colocalized initially. Over time, Cy5 molecules were located at further distances from FITC due to the release of Cy5 from polymer side chains.

The super-resolution images indicate that bond cleavage occurs in the lysosomes and that the functional payload can diffuse into the cytoplasm. Besides shedding light on the internal mechanisms of a particular type of drug delivery system, it’s an excellent example of how single molecule localization super-resolution imaging can be used to track the fate of specific molecule types.

CD169-MEDIATED TRAFFICKING OF HIV TO PLASMA MEMBRANE INVAGINATIONS IN DENDRITIC CELLS ATTENUATES EFFICACY OF ANTI-GP120 BROADLY NEUTRALIZING ANTIBODIES
Hisashi Akiyama, Nora-Guadalupe Pina Ramirez, Manasa V. Gudheti, Suryaram Gummuluru
PLoS Pathogens, Vol 11, Issue 3:e1004751.March 2015
doi: 10.1371/journal.ppat.1004751
(view full article: journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.100475111)


Dendritic cells (DCs) play a crucial role in eliciting adaptive immunity to pathogen infection. Certain pathogens such as the HIV virus have been shown to hijack DCs and use them to spread the virus to T cells, a mechanism known as trans-infection. This is thought to be mediated by the CD169 surface receptors on DCs, which capture the HIV virus and form virus containing compartments (VCCs). Broadly, the goal of this paper is to understand the mechanism of VCC formation and how the HIV virus utilizes VCCs to evade the immune system.

Single molecule 3D super-resolution microscopy was used to study the degree of association of CD169 and HIV-1 particles. The HIV-1 p24 gag protein was labeled with Cy3B and CD169 with Alexa 647. Two color 3D z-stacks were acquired to create a three-dimensional image of CD169 and HIV-1 interaction which showed a close association of the two entities in VCCs at the cell periphery about ~800 nm to 1 µm deep from the cell surface. Electron microscopy images corroborated results obtained with super-resolution microscopy.

In summary, the results indicate that VCCs are surface accessible but have a considerable invagination due to which the captured virus particles are protected from anti-gp120 broadly neutralizing antibodies. The interaction of HIV-1 particles with CD169 receptors could thereby protect the HIV virus from phagocytic degradation and anti-viral neutralization.

Personally, what stood out in the paper for me was how 3D super-resolution microscopy made it possible to obtain a high resolution snapshot of how the HIV-1 particles interact with CD169 receptors. Being able to show that they are co-localized (but not endocytosed) close to the surface of the cell in an invagination (pocket) that protects the virus from being degraded is a crucial piece of the puzzle.

IN VIVO VISUALIZATION OF STROMAL MACROPHAGES VIA LABEL-FREE FLIM-BASED METABOLITE IMAGING
Joseph M. Szulczewski1,2,3, David R. Inman2,3, David Entenberg4, Suzanne M. Ponik2, Julio Aguirre-Ghiso5, James Castracane6, John Condeelis4, Kevin W. Eliceiri3 and Patricia J. Keely1,2,3
Sci Rep. 2016 May 25;6:25086
doi: 10.1038/srep25086
(view article: http://www.nature.com/articles/srep25086)


In the last years, in vivo imaging with two-photon microscopy has been recognized as a very powerful technique mainly used to investigate the physiology and pathology of the brain. However, besides neuroscience, a wide range of other biomedical applications have taken advantage of this outstanding technique to elucidate biological aspects of life sciences, including immunology and cancer research.

Interesting evidence from the literature suggests that the presence of tumor-associated macrophages plays a critical role in the progression of cancer and correlates with a poorer patient prognosis. For this reason, an approach that could allow imaging unstained macrophages in live tissue or fresh biopsies would be of great importance for better diagnosis and targeted therapeutic development.

In this paper, Szulczewski et al. report a novel strategy to non-invasively characterize the cellular metabolism as well as to identify the macrophage population in the intact mammary tumor microenvironment of a mouse, by studying the endogenous fluorescence of metabolic co-factors NADH and FAD multiphoton and fluorescence life time imaging (FLIM)..

In particular, they could discriminate between tumor cells (with high autofluorescent NADH signal intensity) and stromal cells (with high endogenous FAD fluorescence intensity, predominantly phagocytic macrophages). Stromal collagen surrounding the tumor could also be imaged by second harmonic generation.

By taking advantage of Fluorescence Lifetime Imaging Microscopy, the cellular metabolism of the two different cell types has been characterized, showing that the FAD bright cells display a highly glycolytic NADH-FLIM signature, with a significantly shorter NADH average fluorescence lifetime with respect to the tumor cells. These results demonstrate that the glycolytic metabolic heterogeneity between monocytes and tumor cells can be used for imaging contrast.

In conclusion, this paper reports for the first time a novel way to use intravital, live, metabolic imaging in vivo to non-invasively and quantitatively identify distinct cell types within the breast tumor microenvironment, with high spatial and temporal resolution.

The relevance of this new label-free FLIM-based metabolite imaging approach in vivo is linked to the opportunity to be exploited as a readout for clinical diagnostics in the future, as several studies have demonstrated a correlation between NADH-FLIM changes and disease progression in biopsied colon, breast and melanoma tissue.

DISTINCT CONTRIBUTION OF ADULT-BORN HIPPOCAMPAL GRANULE CELLS TO CONTEXT ENCODING
Nathan B. Danielson, Patrick Kaifosh, Jeffrey D. Zaremba, Matthew Lovett-Barron, Joseph Tsai, Christine A. Denny, Elizabeth M. Balough, Alexander R. Goldberg, Liam J. Drew, Rene Hen, Attila Losonczy, and Mazen Kheirbek
Neuron 2016 Apr 6;90(1):101-12
doi: 10.1016/j.neuron.2016.02.0196
(view article: http://www.ncbi.nlm.nih.gov/pubmed/26971949)


Adult-born granule cells have been implicated in cognition and mood; however, it remains unknown how these cells behave in-vivo. The authors used two-photon calcium imaging to monitor the activity of young adult-born neurons in awake behaving mice. The authors found that young adult-born neurons fire at higher rate in vivo but paradoxically exhibit less spatial tuning than their mature counterparts. When presented with different contexts, mature granule cells underwent robust remapping of their spatial representations and the few spatially tuned adult-born cells remapped to a similar degree. The authors next used optogenetic silencing to confirm the direct involvement of adult-born granule cells in context encoding and discrimination, consistent with their proposed role in pattern separation. These results provide the first in vivo characterization of adult-born granule cells and reveal their participation in the encoding of novel information.

PRIMARY CILIA ARE NOT CALCIUM-RESPONSIVE MECHANOSENSORS
M. Delling, A. A. Indzhykulian, X. Liu, Y. Liu, T. Xie, D. P. Corey, and D. E. Clapham.
Nature. 2016 Mar 31; 531(7596): 656–660.
doi:10.1038/nature17426
(view article: http://www.nature.com/nature/journal/v531/n7596/full/nature17426.html)


In this paper, Delling et al question the widely held hypothesis that primary cilia act as Calcium responsive mechanosensors (CaRMS). This hypothesis has been used to explain a large range of biological observations, like left-right axis determination in embryonic development or polycystic kidney disease. The authors developed a transgenic mouse, Arl13b–mCherry–GECO1.2, expressing a ratiometric genetically encoded calcium indicator confined to the primary cilia and were able to demonstrate the complete lack of mechanically induced calcium increases in primary cilia upon mechanical stimulation. They found no CaRMS in osteocyte-like cells, mouse embryonic fibroblasts, or, indeed, any primary cilia examined. They found that in all cases where the calcium concentration in cilia increased, the calcium rise was initiated at other sites in the cell and diffused from the cytoplasm into the cilium.

They explain the previous hypothesis by out of focus data, motion, and light path artifacts in combination with low acquisition rates where calcium originating from the cytoplasm can diffuse into the cilium in less than 200 ms and be mistaken as originating from within the cilium. Temporal resolution is critical. The authors used the Opterra swept-field confocal in combination with a fast camera in order to reach supra video rate acquisitions in 2 colours, and performed the acquisitions in slit mode in order to both increase light delivery to the camera and avoid excessive photobleaching.

These findings have a significant implication on established models based on an incorrect hypothesis and encourage researchers to reinvestigate other mechanisms for regulation of ciliary ion channels.

PROJECTIONS FROM NEOCORTEX MEDIATE TOP-DOWN CONTROL OF MEMORY RETRIEVAL
Priyamvada Rajasethupathy, Sethuraman Sankaran, James H. Marshel, Christina K. Kim,Emily Ferenczi, Soo Yeun Lee, Andre Berndt, Charu Ramakrishnan, Anna Jaffe, Maisie Lo, Conor Liston & Karl Deisseroth
Nature 526, 653–659 (29 October 2015)
doi:10.1038/nature15389
(view full article: www.nature.com/nature/journal/v526/n7575/abs/nature15389.html23)


While great progress in understanding the molecular and physiological mechanisms of memory formation have been made at the synaptic level, little is known as to how populations of individual neurons form networks representing memory.

The authors provide an extensive study identifying a hypothesized, but previously unidentified, monosynaptic connection between the anterior cingulate region of the prefrontal cortex and the CA1 and CA3 regions of the hippocampus. Using a variety of techniques including imaging for anatomical tracing, electrophysiology, optogenetic stimulation in freely moving animals and 2P imaging and optogenetic stimulation of restrained animals in a virtual environment they demonstrate a functional connection between the AC and CA1 and CA3 that appears to play a role on contextual memory formation and retrieval.

Their work provides a guide to suggested approaches for performing a complete study of connectivity between brain regions for the purpose of understanding memory formation and retrieval. Their use of retrograde and anterograde labelling with optogenetic probes provides useful guidance in terms of utilizing this strategy for demonstrating functional connections.

The identification of the AC-CA monosynaptic connection furthers our understanding of memory formation in general, as well as having clinical implications for psychiatric disorders such as post-traumatic stress disorder, schizophrenia and drug addiction.

THE NEURAL REPRESENTATION OF TASTE QUALITY AT THE PERIPHERY
Robert P. J. Barretto, Sarah Gillis-Smith, Jayaram Chandrashekar, David A. Yarmolinsky, Mark J. Schnitzer, Nicholas J. P. Ryba & Charles S. Zuker
Nature 517, 373–376 (15 January 2015) doi:10.1038/nature13873
(view full article:http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4297533/)


Multiphoton microscopy provides intravital imaging of neuronal structure and function in vivo in brain regions that lie within 1 mm of the surface of the brain. Imaging deeper structures is outside the range of standard multiphoton imaging. However, the use of a gradient refractive index microendoscope (GRIN lens), coupled with multiphoton microscopy, allows imaging of deeper structures in the brain.

In this paper the authors describe the use of a GRIN lens to perform calcium imaging of neurons in the geniculate ganglion, a structure lying 4 mm below the brain surface. Their interest in the geniculate ganglion was that as the initial neural station in the gustatory system, it receives inputs from all 5 types of dedicated taste receptor cells.

They were able to monitor neural activity of ensembles of geniculate ganglion neurons in response to individual as well as mixed taste stimuli. Their findings support a model of a direct match between taste receptor cells and ganglion neurons. They also demonstrate a robust imaging preparation for further work exploring molecular markers defining taste and how their stimulus quality is transmitted to the brain.

THE DEVELOPMENT OF CORTICAL CIRCUITS FOR MOTION DISCRIMINTATION
Gordon B Smith, Audrey Sederberg, Yishai M Elyada, Stephen D Van Hooser3, Matthias Kaschube & David Fitzpatrick
Nature Neuroscience 18,252–261(2015)
doi:10.1038/nn.3921
(view full article: www.nature.com/neuro/journal/v18/n2/full/nn.3921.htmll)


Two photon in vivo calcium imaging has become a standard technique for measuring neuronal activity and providing the spatio-temporal information to understand the function of neural networks. Developmental paradigms provide an opportunity to observe changes in neural function that are related to natural experiences.

In this paper from MPI Florida, multiphoton in vivo calcium imaging is used to track development of visual cortex in ferrets. The group found that after eye opening, they were able to observe and measure changes in neuronal response properties that appear to be critical for development of motion discrimination.

SIMULTANEOUS ALL-OPTICAL MANIPULATION AND RECORDING OF NEURAL CIRCUIT ACTIVITY
WITH CELLULAR RESOLUTION IN VIVO

Adam M Packer, Lloyd E Russell, Henry WP Dalgleish, and Michael Häusser
Nature Methods, (2014)
doi:10.1038/nmeth.3217
(view full article: www.nature.com/nmeth/journal/vaop/ncurrent/full/nmeth.3217.html)


Optical stimulation methods have been a staple of neuroscience research for a number of years, having been used extensively to study neuronal signaling in vitro, often at the synaptic level, and in vivo in behaving animals, although not at the cellular level.

This exciting paper out of Michael Häusser’s lab at University College London describes methods utilizing multiphoton microscopy to both optically record and optically stimulate at the cellular level in fields containing hundreds of cells that form neural circuits. A key piece of technology implemented in their instrumental configuration is the use of a spatial-light modulator to create precise patterns of light to simultaneously stimulate specific groups of cells and at the same time record their activity and the activity of neighboring cells.

In an interview the authors describe their methodology as being able to both read and write brain activity, opening up the door for experimental designs that allow conversations with the brains of behaving animals in order to unravel the mysteries of brain activity.

MECHANISMS OF AMPHETAMINE ACTION ILLUMINATED THROUGH OPTICAL MONITORING OF DOPAMINE SYNAPTIC VESCICLES IN DROSOPHILA BRAIN
Zachary Freyberg, Mark S. Sonders, Jenny I. Aguilar, Takato Hiranita, Caline S. Karam, Jorge Flores, Andrea B. Pizzo, Yuchao Zhang, Zachary J. Farino, Audrey Chen, Ciara A. Martin, Theresa A. Kopajtic, Hao Fei, Gang Hu, Yi-Ying Lin, Eugene V. Mosharov, Brian D. McCabe, Robin Freyberg, Kandatege Wimalasena, Ling-Wei Hsin, Dalibor Sames, David E. Krantz, Jonathan L. Katz, David Sulzer & Jonathan A. Javitch
Nature Communications. 2016 Feb 16;7:10652
doi: 10.1038/ncomms10652
(view article: http://www.nature.com/articles/ncomms10652 )


In this paper, two-photon microscopy is utlized in a scientific research context of important sociological impact. Specifically, the authors are investigating the effects of amphetamine, one of the most widely used and abused drugs, on the neuronal microenvironment by following in real time the dynamics of dopamine vesicles in the viable ex vivo Drosophila brain.

In a series of experiments, a fluorescent substrate of the vesicular monoamine transporter (VMAT) and a fluorescent biosensor of the intraluminal vesicular pH are used in order to directly monitor monoamine loading and release from synaptic vesicles, as well as changes in monoamine vesicle pH under amphetamine treatment in WT and transporter mutant Drosophila brains.

By combining different scientific approaches, such as genetic manipulation, optical monitoring through two-photon microscopy and pharmacology, the group was able to demonstrate for the first time the molecular mechanisms at the basis of amphetamine-mediated dopamine extracellular release, which determines the psychomotor stimulation and the behavioral effects already observed in mammals.

Clinically relevant amphetamine concentrations are able to alkalize dopamine vesicle pH and induce content release after its functional active transportation at both the plasma membrane (via DAT) and at the vesicular membrane (via VMAT) levels. Moreover, the mechanism of alkalization was also innovatively identified as an antiport, i.e. net export of H+ ions via VMAT for every amphetamine molecule transported into the vesicle.

In conclusion, the results provide a model for how pharmacologically relevant concentrations of amphetamines increase extracellular dopamine. The results also demonstrate the viability and utility of a novel experimental system for invesitgating the physiology of intact monoaminergic vesicles.

GATING OF HIPPOCAMPAL ACTIVITY, PLASTICITY AND MEMORY BY ENTORHINAL CORTEX LONG-RANGE INHIBITION
Jayeeta Basu, Jeffrey D. Zaremba, Stephanie K Cheung, Frederick L. Hitti, Boris V. Zemelman, Atilla Losonczy, Steven A. Siegelbaum
Science, VOL 351, ISSUE 6269, Jan 8th 2016
(view full article: science.sciencemag.org/content/351/6269/aaa5694.full?ijkey=wGUElGP8cA.BA&keytype=ref&siteid=sci )


The hippocampus and entorhinal cortex (EC) are physically interconnected brain areas. The CA1 cells in the hippocampus integrate direct excitatory input from the EC with indirect excitatory input from the upstream hippocampal CA3 area, and both pathways are implicated in memory storage. Interestingly it was recently found that medial EC additionally sends long-range inhibitory projections (LRIPs) that form synapses on CA1 inhibitory neurons.

Here the authors investigate whether lateral EC also sends inhibitory connection to the CA1 area of the hippocampus and if so how this influences paired EC and hippocampal CA3 inputs thus long-term memory storage. Neurons in the lateral EC in contrast to neurons in medial EC exhibit little spatial selectivity but lateral EC conveys important contextual and object-related information to the hippocampus.

The authors used a customized Bruker two-photon microscope (Ultima) that allowed for calcium-imaging in head fixed mice, photoactivation and integration with electrophysiological instrumentation.

They confirmed the presence of the LRIPs from LEC to the CA1 area. Although the silencing of LRIPs in hippocampus did not prevent memory formation in behavioral tests, it caused mice to show inappropriate fear response to a neutral context and a diminished ability to distinguish a novel object from a familiar object. The long-range projecting interneurons form synapses on interneurons in the CA1. Intracellular recording demonstrated that LRIP suppressed the activity of a subclass of cholecystokinin-expressing interneurons (CCK IN). These interneurons were normally strongly excited by the CA3 input. The LRPI transiently inhibited the activity of CCK IN allowing for enhancing CA3c input to the CA1 that arrives precisely 20 ms after LRPI activation. This disinhibition enabled generation of dendritic spikes in the distal dendrites of the CA1 pyramidal neurons and to induce synaptic plasticity.

The facilitating effect of LRIPs on the CA3 excitation and synaptic plasticity found in this paper is in line with emerging recognition that interneurons can control memory through either direct inhibition of pyramidal cells or disinhibitory circuits (Freund and Gulyas 1997, Lovett-Barron et al. 2014, Wolff et al. 2014). It seems that disinhibition is a conserved circuit mechanism contributing to learning and memory expression and can be linked to most behavioral functions including auditory fear learning (Letzkus et al. 2011) and spatial navigation (Chambers et al. 2003).

MULTIMODAL CHEMOSENSORY CIRCUITS CONTROLLING MALE COURTSHIP IN DROSOPHILA
E. Josephine Clowney, Shinya Iguchi, Jennifer J. Bussell, Elias Scheer, Vanessa Ruta
Neuron, Volume 87, Issue 5, p1036–1049, 2 September 2015
(view full article: www.cell.com/neuron/abstract/S0896-6273(15)00647-9)


The study of innate behaviors is a central tenant of behavioral science and has a rich experimental provenance traced back to classic naturalist observational studies in the early and mid-twentieth century. The authors leverage published knowledge of a subset of neurons previously identified through genetic markers and thought to describe the potential for a complete innate behavior, and utilize an array of modern optical microscopy techniques to provide a functional and anatomical description of the neural circuits responsible for the expression of male courtship in Drosophila.

In a series of elegant experiments utilizing a behavioral assay in conjunction with in vivo 2 photon imaging they demonstrate functional activation of sexualy dimorphic P1 interneurons that correlates with behavioral expression that is ethologically appropriate with respect to presented stimuli (male and female Drosophila of different strains).

Using in vivo photoactivation techniques utilizing channelrhodopsin expression in P1 neurons, they demonstrate that optical activation of P1 neurons results in long lasting release of the expression of behavior related to male courtship in Drosophila.

In a series of in vitro experiments the authors utilize functional 2 photon imaging, 3-D anatomical mapping using 2 photon imaging, photoactivatable fluorescent proteins and 2 photon ablation to functionally and anatomically map the gustatory and olfactory sensory afferents that connect to P1 neurons.

In their conclusion, the authors present an anatomical and functional model showing how feedforward excitatory and inhibitory signaling can interact with P1 neurons to release a behavioral response that is appropriate to external species specific stimuli. In addition to describing the neural basis of a particular innate behavior, their model suggests a general cirguit mechanism for release of innate behaviors in response to appropriate stimuli. Personally I found this paper to be a fun read, as an undergraduate course in Animal Behavior many years ago initiated my own interest in studying the nervous system

TEMPERATURE REPRESENTATION IN THE DROSOPHILA BRAIN
Dominic D. Frank, Genevieve C. Jouandet, Patrick J. Kearney, Lindsey J. Macpherson & Marco Gallio
Nature 519, 358–361 (19 March 2015)
doi:10.1038/nature14284
(view full article: www.nature.com/nature/journal/v519/n7543/full/nature14284.html )


Drosophila has been used as an animal model for well over 100 years, and still remains a popular model, especially given full sequencing of the genome of several species, and a rapid reproductive cycle which facilitates genetic engineering. In addition to being a popular model for developmental biology, Drosophila is also a useful model for studying neural circuits in vivo, providing a simpler nervous system than mammalian models while still exhibiting behavior that can be conditioned.

In “Temperature representation in the Drosophila brain” from Dr. Marco Gallio’s lab at Northwestern University, the group uses two-photon guided conversion of photoactivatable GFP to track the projections of thermosensory neurons, and also perform calcium imaging using UAS.GCaMP6m to measure activity of neurons in brain areas receiving projections.

Their functional imaging showed a dose response relationship between calcium changes and temperature, and they also show correlation between neuronal activity as measured by calcium and a two choice behavioral task.

The authors found some surprises concerning the functional characterization of the projections they studied.

LONG-TERM TWO-PHOTON IMAGING IN AWAKE MACAQUE MONKEY
Ming Li, Fang Liu, Hongfei Jiang, Tai Sing Lee, and Shiming Tang
Neuron. 2017 Feb 8. pii: S0896-6273(17)30051-X
doi: 10.1016/j.neuron.2017.01.027
(view article: http://www.cell.com/neuron/abstract/S0896-6273(17)30051-X)


In this week’s article, Li et al overcome the main technical challenges associated with long-term two-photon imaging in awake behaving monkeys. The combination of a novel design of cranial optical window and stable genetically encoded indicators enables the authors to successfully image neuronal activity in the visual cortex as a response to visual stimuli across months. To date, some technical challenges have made it difficult to achieve long term stable two photon imaging in awake macaque monkeys, equivalent of what can be achieved in rodents.

Li et al overcome these challenges with i). the development of a new cranial window design combining the use of glass coverslip optical window and an artificial dura membrane insulating the window chamber as well as reinforced head restraints to stabilize the animal skull. ii) cortical tissue movement during experiments was addressed by image registration using 2D cross-correlation algorithm. The authors averaged 1000 frames at the middle of the imaging protocol to then use as a reference for image registration and hence movement correction. Finally iii) the use of stable calcium indicators delivered by AAV injection. These enabled the authors to image the same neurons at 100 days interval in awake behaving animal.

In addition, Li et al performed simultaneous electrophysiology studies under 2 photon imaging done with a Bruker Ultima system, using microelectrodes penetrating through micropores drilled through the glass imaging window.

Macaque monkeys are similar to human beings in many aspects of behavior, brain structure and function, making them good animal models for human neurological diseases as well as for visual cognition and other high order cognitive functions. In this article Li et al overcome the limitation associated with stable long-term study of neuronal activity in awake monkeys that will enable fundamental advances in our understanding of higher cognitive function at the level of molecular and neuronal circuits.

VALIDATION OF A NEW GENETICALLY ENCODED VOLTAGE REPORTER
Joseph M. Szulczewski1,2,3, David R. Inman2,3, David Entenberg4, Suzanne M. Ponik2, Julio Aguirre-Ghiso5, James Castracane6, John Condeelis4, Kevin W. Eliceiri3 and Patricia J. Keely1,2,3
Physiol Rep,3 (7), 2015, e12468
doi: 10.14814/phy2.12468
(view article: http://physreports.physiology.org/content/3/7/e12468 )

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One of the ultimate goals of neuroscience is to understand how electrical activity of the brain produces behavior. A substantial amount of current neuroscience research is focused on directly monitoring neural activity through the use of optical methods in awake, behaving animal models.

A substantial amount of the work done to date has relied on the use of monitoring calcium transients in neurons through the use of genetically encoded optical reporters. While providing a robust method to begin unravelling the complexity of neural networks that produce and govern behavior, calcium flux is not a direct measure of electrical events occurring in neurons. Voltage sensors have existed for a number of years, but have historically had issues related to optical sensitivity. In the last few years, genetically encoded optical reporters have provided a new class of molecules more amenable to optical recording techniques.

In this paper, the authors validate a new genetically encoded voltage reporter Voltage Sensitive Fluorescence Protein (VSFP) Butterfly 2.1 in cortical layer L2/3 in the Rasgrf2-2A-dCre;Camk2a-tTA;Ai78 mouse model.

Using in vitro brain slices methods, the authors report that Butterfly 2.1 exhibits excellent signal to noise as an optical reporter with both multiphoton and wide field optical recording techniques. They also find specific and reliable functional expression exclusively in the soma, dendrites, and axons of L2/3 pyramidal neurons. Butterfly 2.1 in L2/3 neurons is capable of monitoring both action potentials and synaptically meditated responses with good spatial and temporal frequency. Their findings suggest that Butterfly 2.1 in cortical layer L2/3 in the Rasgrf2-2A-dCre;Camk2a-tTA;Ai78 mouse model would provide a product preparation for in vivo behavioral studies examining neural activity in pyramidal layers L2/3.

CAGED COMPOUNDS FOR MULTICHROMIC OPTICAL INTERROGATION OF NEURAL SYSTEMS
Amatrudo, J. M., Olson, J. P., Agarwal, H. K. and Ellis-Davies, G. C. R.
European Journal of Neuroscience, 16 January 2015, 41: 5–16.
doi: 10.1111/ejn.12785
(view full article: onlinelibrary.wiley.com/doi/10.1111/ejn.12785/abstract )


Caged compounds have been a standard tool for physiologists for nearly 40 years, allowing precise chemical stimulation of neurons. Initially developed caged compounds were typically activated with UV or near UV radiation. The more recent use of nonlinear lasers for uncaging provides even greater precision as the uncaging event is precisely localized in 3-D.

Historically uncaging experiments used a single compound which activated or inhibited action potentials. Recent developments have produced caged compounds with absorption at longer wavelengths, opening the possibility of multichromatic optical interrogation of neural systems with caged compounds.

The authors offer an excellent review of the current state of caged compounds, as well as describing their work to develop multichromatic pairs of caged compounds for use with nonlinear excitation. They demonstrate the feasibility of non-linear multichromatic uncaging, and provide a methodological guidance for employing the technique.

They make the case that caged compound pairs provide an excellent tool for bi-directional activation studies, with good control of concentration at localization.

IMPERMANENCE OF DENDRITIC SPINES IN LIVE ADULT CA1 HIPPOCAMPUS
Alessio Attardo, James E. Fitzgerald & Mark J. Schnitzer
Nature (2015) doi:10.1038/nature14467
(view full article: www.nature.com/nature/journal/v523/n7562/full/nature14467.html )


Understanding the biological basis of memory is one of the major goals of neuroscience research. A brain region in mammals that appears to play a critical role in episodic memory formation is the hippocampus, which transiently retains information for about 3-4 weeks in mouse models. While neural synapses are thought to be the elements of information storage, to date there has been no direct evidence linking hippocampal synapse formation and persistence with hippocampal dependent memory. .

The authors of this week’s paper used in vivo time lapse multiphoton microscopy to study turnover of basal dendritic spines in the CA1 region of the hippocampus. Turnover of dendritic spines is thought to reflect formation of excitatory synaptic connections, and so offered the opportunity to monitor synaptic changes over a period of time.

In order to access the hippocampus with multiphoton microscopy they employed microendoscopes in order to optically reach the hippocampus. They also employed novel processing and modelling algorithms to differentiate merged spines and to model dendritic turnover.

Their results suggest that dendritic turnover rates in the hippocampus match the course of memory retention in the hippocampus. They also find that the dendritic turnover rate in the hippocampus is markedly different than in the neocortex, an area thought to play a role in long term memory retention. They suggest that employing the techniques they demonstrate in this paper in conjunction with learning paradigms will provide an opportunity to look for direct relationships between synapse formation and learning.

DISTINCT Kv CHANNEL SUBTYPES CONTRIBUTE TO DIFFERENCES IN SPIKE SIGNALING PROPERTIES IN THE AXON INITIAL SEGMENT AND PRESYNAPTIC BOUTONS OF CEREBELLAR INTERNEURONS
Matthew J. M. Rowan, Elizabeth Tranquil, and Jason M. Christie
The Journal of Neuroscience, 7 May 2014, 34(19): 6611-6623
(view full article: www.jneurosci.org/content/34/19/6611.full )


While the distribution of voltage gated K+ channels is thought to provide functional benefits in action potential signaling, little is known about the organization of voltage sensitive K+ channels in compact cell types. The authors utilize multiphoton imaging and visible wavelength uncaging to study voltage sensitive K+ channels in cerebellar stellate interneurons in mice. They utilize a multiphoton microscope operating in point detection mode to measure changes in a voltage sensitive dye in order to precisely localize fluorescence changes at a high sampling frequency, while using a secondary scanner and a visible laser to provide precisely located diffraction limited uncaging of a caged voltage gated K+ channel inhibitor. They found that cerebellar stellate interneurons contain two types of voltage gated K+ channels which are localized differentially to specific regions of axons.

NANOSCALE DISTRIBUTION OF PRESYNAPTIC Ca2+ CHANNELS AND ITS IMPACT ON VESICULAR RELEASE DURING DEVELOPMENT
Yukihiro Nakamura, Harumi Harada, Naomi Kamasawa, Ko Matsui, Jason S. Rothman, Ryuichi Shigemoto, R. Angus Silver, David A. DiGregorio, and Tomoyuki Takahashi
Neuron, (2015), 85, 1–14
(view full article: www.cell.com/neuron/abstract/S0896-6273%2814%2901047-2 )


Multiple types of imaging modalities are often required to unravel a scientific question. The team on this paper used an EM technique (freeze fracture replica immunogold labelling) along with confocal point detection, to study the coupling of voltage gated calcium2+ channels (VGCCs) to presynaptic vesicles.

Their findings provide a clearer picture of the nanoscale topography of VGCCs and vesicles and suggest that a perimeter model of vesicle to VGCC cluster coupling explains synaptic precision and efficacy during development.

RELAXATION OF INTERKINETOCHORE TENSION AFTER SEVERING OF A K-FIBER DEPENDS ON THE LENGTH OF THE K-FIBER STUB
Ana Milas, Iva M Tolić
Matters Select, 2016,
doi: 10.19185/matters.201603000025
(view article: https://sciencematters.io/articles/201603000025)

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During mitosis, the genetic material of the cell will be equally distributed to both daughter cells. Correct segregation of genetic material requires that sister chromatids of each chromosome attach to microtubules extending from the opposite spindle poles. The attachment of microtubules to chromosomes is mediated by kinetochores, protein complexes on the chromosome. Microtubules bound to kinetochores form bundles known as k-fibers, which generate tension on sister kinetochores. Interkinetochore tension is required for passage through the spindle assembly checkpoint and for the subsequent segregation of sister chromatids.

In this article, the authors have used live-cell imaging combined with 2P photoablation to shine light on how, during cell division, the tension forces acting on kinetochores are generated.

The unique design of the Bruker Opterra swept field microscope allows multi-color imaging combined with simultaneous photostimulation. The authors used this technique for photoablation of microtubules. The Opterra system combined to a pulsed infrared laser allowed the authors to perform precise localized nanosurgery of microtubules in living dividing cells. They demonstrated the effect of the nanoablation site on the microtubule relative to the distance from the kinetochore and highlighted the key role of the bridging fiber in the interkinetochore tension.

CALSYNTENIN-1 REGULATES AXON BRANCHING AND ENDOSOMAL TRAFFICKING DURING SENSORY NEURON DEVELOPMENT IN VIVO
Olga Y. Ponomareva, Ian C. Holmen, Aiden J. Sperry, Kevin W. Eliceiri, and Mary C. Hallorani
The Journal of Neuroscience, 9 July 2014, 34(28): 9235-9248
doi: 10.1523/JNEUROSCI.0561-14.2014
(view full article: www.jneurosci.org/content/34/28/9235 )


Precise regulation of axon formation and branching is a critical requirement for development of functional neuronal circuits. While there is a growing body of work describing the role of molecular signals that may influence branching, little is known about the molecular mechanisms regulating compartmentalization of axons from individual neurons.

The authors use a number of confocal imaging techniques to identify Clstn-1 as a critical regulator of axon branching and compartmentalization during development of vertebrate sensory neurons. In particular, using high speed, high resolution imaging in vivo imaging in zebrafish embryos, they find that specific endosomal populations display different dynamics in different neuronal compartments, and that endosome trafficking appears to play an important role in peripheral axon branching. They also show that Clstn-1 regulates endosomal dynamics and endosomal transport from neuronal cell bodies into axons, and conclude that a major function of Clstn-1 in the context of axon branching is regulation of endosomal trafficking.

METABOLIC IMAGING OF HEAD AND NECK CANCE ORGANOIDS
Amy T. Shah, Tiffany M. Heaster, Melissa C. Skala
PLOS ONE, 2017, 12(1):e0170415
DOI: 10.1371/journal.pone.0170415
(view article: http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0170415#sec001)


Drug discovery is a time consuming and expensive process that that requires high throughput screening assays to evaluate drug candidates during early stage development. In the current paper the authors demonstrate the use of optical imaging of cell metabolism in organoids derived from head and neck tumors as a potential model for development of screening assays for oncology therapeutics.

The authors use organoids as a sample as they provide a 3 dimensional matrix, which has been demonstrated to provide a more appropriate in vitro model than cell lines grown in a three dimensional matrix. Optical metabolic imaging included metabolic imaging where label free fluorescence imaging of NAD(P)H and FAD and NAD(P)H and FAD lifetimes were measured with a multiphoton microscope. These optical imaging methods are advantageous as the are label free and also extremely sensitive. Their assays procedures proved to be quite sensitive, as they were able to detect response to therapeutic agents within 1 day, which is a much earlier time point than assays utilizing cell death, cell proliferation and tumor volume. The optical methods employed allowed analysis at the cellular level, and revealed heterogeneity of cell populations.

The current paper suggests that optical imaging methods used with organoid samples could well provide a viable assay for drug discovery for head and neck cancer, and by extension for other oncology disease areas.