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. Sign up to automatically receive the monthly Journal Club via email:


Shaon Basu, Sebastian D. Mackowiak, Henri Niskanen, Dora Knezevic, Vahid Asimi, Stefanie Grosswendt, Hylkje Geertsema, Salaheddine Ali, Ivana Jerkovic, Helge Ewers, Stefan Mundlos, Alexander Meissner, Daniel M. Ibrahim, and Denes Hnisz

(view article: https://doi.org/10.1016/j.cell.2020.04.018)

Genetic disorders occur when a person’s DNA sequence is altered away from the normal sequence. This can result from the mutation of one or multiple genes, environmental factors in conjunction with gene mutations, or chromosomal damage. Many genetic disorders are inherited at birth. Of those, more than 30 are caused by an abnormal expansion of short, repetitive DNA sequence elements. Most of these repeat expansions result in expansions of alanine or glutamine in cellular proteins which are linked to developmental disorders and neurodegenerative diseases, respectively. Previous studies have not focused on the impacts of repeat expansions on protein function but rather on tendencies to form abnormal aggregates, and changes in subcellular localization or proteolytic processing. However, understanding the effects of repeat expansions on the function of impacted proteins is essential for determining how these diseases can potentially be treated.

In this paper from Basu et al. in Cell, the authors investigated repeat expansions occurring in intrinsically disordered regions (IDRs) of transcription factors (TFs) and their effects on the TFs phase separation capacity and ability to form transcriptional condensates. They found that disease-associated repeat expansions in the studied TFs resulted in altered phase separation capacity and ability to co-condense with transcriptional co-activators in comparison to wildtype TFs, resulting in the proposal that changes to TF phase separation and condensate formation may play an important role in human pathologies associated with repeat expansions of TF IDRs. The Vutara 352 imaging platform along with SRX software was utilized for localization, visualization, and statistical analysis of STORM data (stochastic optical reconstruction microscopy). Authors imaged wildtype (HOXD13) and mutated protein (HOXD13 + 7A alleles) and a co-stain of BRD4, a co-activator, to identify that HOXD13-condensates have altered composition in vivo. To observe this, they used co-localization analysis in SRX to determine the Manders overlap coefficients of HOXD13 with BRD4 and HOXD13 +7A with BRD4 and found that wildtype HOXD13 puncta had a higher degree of colocalization with BRD4 than the repeat expansion variant, indicating alteration in condensate composition as a result of the repeat expansions. Super-resolution microscopy was necessary to make this observation as the HOXD13 puncta are less than 100 nm in size, well below the diffraction limit of light.

Peri Kurshan, Sean Merrill, Yongming Dong, Chen Ding, Marc Hammarlund, Jihong Bai, Erik Jorgensen and Kang Shen

(view article: https://doi.org/10.1016/j.neuron.2018.09.007)

The nervous system is formed through cell cell connections, at specialized contact sites called synapses, forming neuronal circuits. How the nervous system correctly builds these connections has remained a mystery, but synaptic adhesion proteins, proteins expressed both pre- and postsynaptically, have been proposed to determine the specificity of connections. In the paper from Kurshan et al. the authors present data that a short form of the synaptic adhesion protein neurexin is required for assembly of presynaptic components even though this isoform lacks the canonical extracellular adhesion domains. Suggesting, that it is not its cell adhesion properties, but a direct function of this protein. They also discovered that neurexin worked in parallel with the Wnt receptor, Frizzled, but that this function was independent of its ligand Wnt and in fact Wnt resulted in reduction in synapses through removal of the Frizzled receptor from the plasma membrane by endocytosis. Together these results show how positive and negative synaptic development factors come together to help wire the nervous system.

The authors use a combination of genetics, confocal microscopy, electrophysiology and superresolution mcroscopy with the Vutara 352; in Caenorhabditis elegans to reveal that a short form of neurexin is required for proper development of synapses. While confocal microscopy was useful in determining whether synapses were present or absent from the nervous system, super resolution microscopy was required to determine the substructure of the synapse in these mutants. The authors performed live super resolution microscopy using the Vutara 352 on C. elegans expressing Skylan-S and Halo-tagged synaptic proteins labeled with the Janelia fluor JF-646 (available for free to academic labs from Luke Lavis at Janelia, HHMI). The authors relied upon the 3D multi-color imaging to be able to image the synapses in a whole animal several microns from the coverslip. Confocal microscopy was not sufficient for this experiment because the average C. elegans synapse is approximately 400 nm across, while the active zone is on the order of 100-200 nms, below the diffraction limit of light. Using the Fourier Ring Correlation program included in the advanced statistics package in the Vutara SRX software the authors were able to determine the overall resolution of their images to be 40 nm laterally and 70 nm axially. Further, by using the DBSCAN algorithm, also included in the SRX software, the authors were able to determine the degree of clustering of each of the synaptic proteins tested. This analysis revealed that neurexin colocalized with calcium channels at the center of the synapse (the active zone, where the signal is propagated between cells), and this colocalization was higher than that of other active zone components suggesting there may be a functional connection between calcium channels and neurexin. Further, in neurexin mutants calcium channels were greatly reduced at the active zone, in both number and in the size of the calcium channel clusters present suggesting that neurexin is required for proper calcium channel clustering at the synapse. This work could only have been accomplished using he 3D capabilities of the Vutara 352 super resolution microscope along with the advanced statistics package included in the Vutara SRX software.

Pengpeng Li, Sean Merrill, Erik Jorgensen and Kang Shen
Neuron. 2016 May 04; 90(3): 564–580.
(view article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5266526/)

Neurons are highly polarized cells, with defined processes containing presynaptic axons and postsynaptic dendritic regions. These poles are defined molecularly with presynaptic proteins trafficked to the axon while postsynaptic proteins are trafficked to dendrites. The clathrin adaptor proteins AP-1 and AP-3 are thought to help in the molecular sorting of transmembrane proteins from the neuronal golgi apparatus, but their mechanism of action remains unclear. In the paper by Li et al. in Neuron the authors discover that pre- and postsynaptic transmembrane proteins are sorted by novel di-leucine motifs on said proteins and that different adaptor proteins with different affinities for the novel dileucine motifs sort these proteins to the correct part of the cell. The sorting appears to happen in the golgi apparatus and the adaptor AP-3 sorts proteins with the axonal dileucine motif to the presynaptic axon, while AP-1 sorts proteins with the dendritic dileucine motif to the postsynaptic dendrite.

The paper uses several imaging modalities combined with powerful Caenorhabditis elegans genetics to tackle the problem of how the adaptor proteins AP-1 and AP-3 help sort proteins in neurons. Using standard confocal imaging the authors could track whether different pre and post synaptic components were sorted normally, but they could not resolve whether the different adaptor proteins were sorting at the same or different sites in the golgi apparatus. To circumvent this problem the authors used the Vutara SR350 microscope to image the adaptor proteins AP-1 and AP-3 in the cell bodies of neurons in live C. elegans. The authors previously imaged AP-1 and AP-3 by diffraction limited microscopy and determined the two adaptor proteins were colocalized. However, in a technical tour de force the authors developed a method to live label C. elegans with cell permeable dyes compatible with superresolution microscopy and then imaged the dyes in the nervous system of a living animal to show that in fact the two proteins were present in different parts of the golgi apparatus. Through tagging AP-1 and AP-3 with orthogonal tags, HALO and SNAPf, and then labeling these tags with the cell permeable dyes JF546 and JF646 (available for free from Luke Lavis) the authors were able to determine that AP-1 and AP-3 were localized to adjacent, but distinct domains in the golgi, suggesting that the initial sorting of cargo to axons or dendrites occurs in different domains of the golgi apparatus.

Guy Nir1^, Irene Farabella2^, Cynthia Pérez Estrada1,3,4 ^, Carl G. Ebeling5^, Brian J. Beliveau1,6,7, Hiroshi M. Sasaki6,7, Soun H. Lee1, Son C. Nguyen1†, Ruth B. McCole1, Shyamtanu Chattoraj1, Jelena Erceg1, Jumana AlHaj Abed1, Nuno M. C. Martins1, Huy Q. Nguyen1, Mohammed Hannan1, Sheikh Russell3, Neva C. Durand3,8, Suhas S.P. Rao3,4,9, Jocelyn Y. Kishi6,7, Paula Soler-Vila2, Michele Di Pierro4, José N. Onuchic4, Steven Callahan10, John Schreiner10, Jeff Stuckey11*, Peng Yin6,7*, Erez Lieberman Aiden3,4,8,12*, Marc A. Marti- Renom2,13,14,15*, C.-ting Wu1,6*

(view article: https://www.biorxiv.org/content/early/2018/07/28/374058)

While progress has been made in understanding the organization and structure of chromosomes in situ, the size as well sequential and structural heterogeneity of chromosomal DNA within chromosomal regions make it a particularly challenging molecule to study. Understanding the organizational structure of chromosomes in situ would greatly benefit in understanding chromosomal function on both normal and pathological conditions.

The authors present a method for 3-D visualization of chromosomal DNA at the super resolution level through the use of OligoSTORM. Using a sequential labelling strategy with single molecule localization microscopy, the authors “walk” along 8.16 megabases (Mbs) of chromosome 19 of primary fibroblasts. The resulting images resolved structures of individual compartments within the chromosomal region imaged.

The authors also obtained Hi-C contact frequency data and integrated it with the localization microscopy data to produce 3-D models at 10 kilobase (kb) resolution.

Unknown patterns of chromosomal organization were observed in their results, as well as significant structural variability. The authors discuss the implications of their findings, and suggest that their methods could allow studies of chromosomes as single, fully integrated units of structure and function.

Philip R Nicovich, Dylan M Owen & Katharina Gaus
Nature Protocols Volume 12, 453–460, February 2017
(view article: https://www.nature.com/nprot/journal/v12/n3/full/nprot.2016.166.html)

The advent of single molecule localization microscopy (SMLM), where individual molecules are serially imaged and their spatial coordinates recorded, allows for the imaging and characterization of structural details in biological samples below the classical diffraction limit. While this technique is capable of creating images with structural detail in the tens of nanometer range, the images generated in SMLM are a computer-generated reconstruction of the localization data. In SMLM, the inherent strength in this imaging modality lies in the quantitative nature of the underlying data. Standard quantitative analysis in other microscopy modalities requires analysis on a conventional image, while SMLM allows for direct statistical analysis on the underlying data.

The field of single molecule localization microscopy is now entering a maturation phase, where the growth and development is happening less within the sphere of novel imaging but rather in the realm of in-depth examination of SMLM data. The comprehensive review paper by Nicovich, Owen, and Gaus aptly cover the rich and diverse area of SMLM quantitative statistical analysis developed by the research field in the past ten years. This paper covers the initial developments of SMLM imaging, and highlights the various analytical domains and the types of experiments and biologically relevant information that can be distracted from this type of imaging modality. This paper covers the most impactful analysis methods currently used within the research community, areas in where they are applicable to extracting biological information, and how the full extent of the data structure of SMLM can be best utilized. This is a comprehensive review paper for a researcher beginning to utilize SMLM for their research, giving numerous examples and methods that can be applied to imaging data to allow direct hypothesis testing in experimental biology.

Pooja Munnilal Tiwari, Daryll Vanover, Kevin E. Lindsay, Swapnil Subhash Bawage, Jonathan L. Kirschman, Sushma Bhosle, Aaron W. Lifland, Chiara Zurla & Philip J. Santangelo
Nature Communications Volume 9, p.946, October 2018

(view article: https://www.nature.com/articles/s41467-018-06508-3)

The lung is a critical prophylaxis target for numerous infectious agents, including human respiratory syncytial virus (RSV) and influenza. Targeted delivery of therapeutics to the organ of interest has the potential to minimize systemic toxicity, anti-antibody immune responses, and reduce the amount of drug required to achieve therapeutic levels. Tiwari et al. develop a modular, synthetic mRNA-based approach to express neutralizing antibodies directly in the lung via to prevent RSV infections in vivo. The research presented demonstrates that GPI-anchored mRNA-expressed antibodies are retained on the plasma membrane of transfected cells and that expressed neutralizing antibodies with and without a membrane anchor can prevent infections in vitro and in vivo. The authors developed a modular toolbox to express synthetic, modified mRNA and prevent viral infections in the lung in a two-step process first by expressing whole palivizumab (secreted, termed sPali) in the lung via synthetic mRNA delivery by intratracheal aerosol. Second, the well-characterized glycosylphosphatidylinositol (GPI) membrane anchor sequence was linked from the decay accelerating factor (DAF) to the palivizumab heavy chain mRNA. Anchored palivizumab was termed aPali; this paper’s hypothesis is that cells transfected with aPali would retain the immunoglobulin on the epithelial surface, increasing its concentration in the lung and improving efficacy.

Tiwari et al. sought to delineate the mechanism by which aPali prevented infection of transfected cells. Conventional spinning disk confocal microscopy does not afford the necessary axial resolution to determine if RSV particles are internalized in aPali transfected cells or merely attached to the plasma membrane. To overcome this limitation of conventional optical microscopy, single molecule localization microscopy was utilized via the dSTORM method to image the spatial distribution of aPali. In transfected cells, the researchers observed aPali at the plasma membrane along the contour of the cell, and at various post-infection timepoints, individual RSV virions of ~100-300 nm were observed between 100 and 200 nm above the aPali-labeled membrane. These results revealed that RSV particles were not internalized over time, demonstrating that the mechanism by which aPali inhibits infection is by preventing fusion and cytosolic uptake of RSV. This level of optical verification of the localization of the RSV virions would not be possible with conventional microscopy methods and is only possible through the use of advanced optical techniques such as single molecule localization microscopy.

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.

Alán, L., Špaček, T. & Ježek, P. Eur
Biophys J (2016) 45: 443.
(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.

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.

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.

Rui Zhang, Jiyuan Yang, Monika SIma, Yan Zhou, and Jindřich Kopeček
Proceedings of the National Academy of Sciences 111(33): 12181–12186 (2014)
(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.

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.

Blundon JA, Roy NC1, Teubner BJW, Yu J, Eom TY, Sample KJ, Pani A, Smeyne RJ, Han SB, Kerekes RA, Rose DC, Hackett TA, Vuppala PK, Freeman BB 3rd, Zakharenko SS
Science 356, 1352-1356 (2017)

(view article: https://www.ncbi.nlm.nih.gov/pubmed/28663494)

Circuits in the auditory cortex are highly susceptible to acoustic influences during an early postnatal critical period. The auditory cortex selectively expands neuronal representations of enriched acoustic stimuli, a process important for human language acquisition. Adults lack this plasticity. Blundon, Roy at al. demonstrate that with pharmacological intervention, the brain’s cortical plasticity in adult mice could be restored to an extent that is normally seen only in juveniles.

Specifically, the researchers exposed mice to repeated stimulation with pure-tone sound for more than 5 days, while simultaneously rendering the A1R (adenosine A1 receptor) signaling thus reducing presynaptic release of glutamate to the cortex. Reduction of neurotransmitter release led to proportional increase in the cortical area. This effect could be achieved only if A1R signaling was suppressed in the auditory thalamus but not in the cortex. To verify that cortical map plasticity could also be detected on individual neurons, the authors measured tone-evoked activity in individual cortical neurons over time, using a genetically encoded sensor of calcium transients. Suppression of A1R signaling in thalamocortical neurons led to an overall shift in proportion of neurons responding preferentially to the target frequency.

The fact that cortical map plasticity could be induced in adult animals by suppressing A1R signaling suggests that this is a mechanism that is related to the demarcation of the critical period for map plasticity.

The findings should spur research into noninvasive therapies to treat conditions relating to perceptual deficits.

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.

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.

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.
(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.

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)
(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.

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.

Gordon B Smith, Audrey Sederberg, Yishai M Elyada, Stephen D Van Hooser3, Matthias Kaschube & David Fitzpatrick
Nature Neuroscience 18,252–261(2015)
(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.


Adam M Packer, Lloyd E Russell, Henry WP Dalgleish, and Michael Häusser
Nature Methods, (2014)
(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.

Bindocci E, Savtchouk I, Liaudet N, Becker D, Carriero G, Volterra A.
Front Cell Neurosci. 2017; 11: 107. Science. 2017 May 19;356(6339). pii: eaai8185.
doi: 10.1126/science.aai8185
(view article: http://science.sciencemag.org/content/356/6339/eaai8185.long)

In this paper, Bindocci et al. shine light on the role of astrocytes in brain physiology focusing on synaptic and vascular functions, and deciphering the astrocyte’s activity through three-dimensional two-photon calcium imaging. Studies so far have monitored only small portions of total astrocytes, contained in a single one-dimensional (1D) line or 2D plane. Given the highly 3D nature of astrocytes and their relations with neighboring vascular and neuronal elements, only a small fraction of the entire cell volume can be imaged in a single 2 photon plane as precisely reported in this study, leading to an underestimate of the activity of the cells that the authors are addressing by the development of a new approach.

This paper details and validates the approach consisting of a full-cell 3D approach to study astrocyte activity and thus biology by combining high speed imaging (AOD and resonant scanner), fast focusing z piezo element and high-sensitivity GaAsPs detectors available on their Bruker Ultima system, as well as new analytical methods to perfom fast volumetric acquisition and analysis of full-cell astrocyte activity. 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.

Their findings highlight the role of astrocytes in synaptic and vascular functions in the brain. In response to neuronal activity, astrocytes show intracellular Ca2+ elevations that result in downstream effects on synaptic transmission and plasticity. Astrocytic Ca2+ elevations also trigger vascular responses that may be involved in the control of cerebral blood flow. They studied Ca2+ dynamics in entire astrocyte volumes, including during axon-astrocyte interactions. In both awake mice and brain slices, they found that Ca2+ activity in an individual astrocyte is scattered throughout the cell, largely compartmented within each region, and preponderantly local within regions. Processes and endfeet displayed frequent fast activity, whereas the soma was infrequently active.

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.

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).

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

Dominic D. Frank, Genevieve C. Jouandet, Patrick J. Kearney, Lindsey J. Macpherson & Marco Gallio
Nature 519, 358–361 (19 March 2015)
(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.

Banala, S., #Arvin, M.C., Bannon, N.M., Jin, X-T., Macklin, J.J., Wang, Y., Peng. C., Zhao, G., Marshall, J.J., Gee, K.G., Wokosin, D.L., Kim. V.J., McIntosh, J.M., Contractor, A., Lester, H.A., Kozorovitskiy, Y., *Drenan, R.M., and *Lavis, L.D. (2018).
Nature Methods 15(5): 347-50
1 Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA Departments of 2 Pharmacology and 3 Physiology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA 4 Department of Neurobiology, Weinberg School of Arts and Sciences, Northwestern University, Evanston, IL, USA 5 Molecular Probes, ThermoFisher, Eugene, OR, USA 6 Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA 7 These authors contributed equally 8 Corresponding authors emails: lavisl@janelia.hhmi.org, drenan@northwestern.edu
(View article: https://docs.wixstatic.com/ugd/4fa92a_0d13ef92c74b43b5bdceaaec78bcb9ef.pdf)

Photostimulation techniques utilizing caged compounds have revolutionized neuroscience and provided a tool that has allowed precise probing of neural circuits and characterization of receptor behavior at the synaptic level. To date the range of receptors accessible through uncaging protocols has been limited due to the small number of ligands available in a caged format.

The authors describe a novel method for caging tertiary amines using an unconventional quaternary amine linkage. They successfully synthesized photoactivatable nicotine (PA-Nic), and demonstrate its use in 1- and 2-photon illumination protocols for photostimulation. Using brain slices they focused primarily on neurons in the Medial Habenula and showed that synthesized PA-Nic was specific to nicotinic acetylcholine receptors (nAChRs), and demonstrated a dose response relationship between light dose and biological response as measured by voltage clamp recording. Utilizing 1-P and 2-P laser point stimulation, they demonstrate spatial specificity, and provide preliminary findings showing the potential for PA-Nic for uncovering spatial mapping questions related to neurons with nAChRs.

The authors also propose the utility of their synthesis methods for developing photoactivatable versions of other compounds containing tertiary amines such as fentanyl and escitalopram.

In summary, this is a groundbreaking piece of work that greatly expands the potential pharmacological tool kit for optopharmacology studies.

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.

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 )

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.

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.

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.

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.

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.

Ana Milas, Iva M Tolić
Matters Select, 2016,
doi: 10.19185/matters.201603000025
(view article: https://sciencematters.io/articles/201603000025)

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.

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.

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.