Material below is adapted from the SfN Short Course Two-Photon Calcium Imaging in the Macaque Monkey, by Kristina J. Nielsen, PhD. Short Courses are daylong scientific trainings on emerging neuroscience topics and research techniques held the day before SfN’s annual meeting.

To learn more about the human brain, researchers often turn to looking at the brains of other primates, like the Rhesus macaque. Similar to the human brain in both organization and function, non-human primate brains can also be evaluated during tasks requiring complex thought, perception, and decision making. Understanding the neural circuitry underlying these processes will provide clues into the human brain, both its normal functioning and disruptions that lead to mental disorders. However, probing these circuits requires advanced techniques.

Two-photon microscopy can investigate neural circuits at both the level of networks and individual synapses. When coupled with calcium imaging, recordings can be made from hundreds of neurons simultaneously or within discrete dendritic spines. Popular calcium dyes like Oregon Green BAPTA-1AM (OGB) have low background, high stability, and a high signal-to-noise ratio that allows single action potentials to be detected.

However, two-photon microscopy in non-human primates presents challenges that prevent it from being widely used. Typically, imaging is done in the horizontal plane, but the size and shape of the non-human primate brain make this difficult; most areas of interest are too lateral or caudal The best option to get around this is to rotate the microscope — a feature becoming more common on two-photon microscopes.

Tilting the microscope is not without its own problems, but special imaging wells and simplified frames to hold the setup in place can be created. Another unique feature of imaging in non-human primates is that the dura must be removed, as it is not transparent.

An additional major consideration is the calcium dye. OGB isn’t fluorescent until it enters the cells, making it hard to track its progress. To ensure the dye isn’t getting clogged or flowing backwards onto the brain, another dye, Sulforhodamine 101 (SR101) can be used in conjunction with OGB. Also, OGB labels both neurons and astrocytes while SR101 specifically labels astrocytes, so a combination can distinguish both cell types. One consideration to keep in mind during analysis is that the high firing rate in the non-human primate brain (and the slow decay of OGB) may make it difficult to detect single spikes. Additionally, saturation may cause differences among groups of neurons to be over or underestimated.

Finally, even in anesthetized animals, breathing and pulse can cause small movements that distort the images. Small craniotomies followed by a layer of agarose and a coverslip clamped to the imaging well can help to prevent distortions from breathing, while problems caused by the pulse can generally be corrected afterwards, during analysis.

The hope for the future is to be able to image non-human primates while they are awake. This will require optimizing the system for genetically encoded calcium indicators (GECIs) to allow long-term, repeated imaging and making more tweaks to the setup to deal with movement. But the potential of two-photon microscopy make refining the technique worth it. Its high resolution coupled with the ability to simultaneously monitor hundreds of neurons promises to yield valuable insight into the circuitry of the primate brain.

Material below is adapted from the SfN Short Course Mapping Brain Circuitry with a Light Microscope, by Pavel Osten MD, PhD and Troy W. Margrie, PhD. Short Courses are daylong scientific trainings on emerging neuroscience topics and research techniques held the day before SfN’s annual meeting.  

Since the days of Camillo Golgi and Santiago Ramón Y Cajal, light microscopes have provided neuroscientists a glimpse into the anatomy of the brain. Advances in the microscopes themselves and methods to stain and trace neurons have revealed various pathways and structures, as well as organization and hierarchies specific to certain brain regions. Three large projects (the Mouse Brain Architecture Project, the Allen Mouse Brain Connectivity Atlas, and the Mouse Connectome Project) are currently attempting to take our knowledge to another level, by mapping all the connections within the brain. Together with new technologies, these projects are helping bring the possibility of a complete map of the mouse brain within reach.

Recent developments of automated light microscopy instruments significantly increase the amount of data collected while reducing the time researchers spend collecting it. One automated approach combines two-photon microscopy with tissue sectioning, such as in serial two-photon tomography. A second approach is light-sheet fluorescence microscopy, which uses chemicals to render mouse brains transparent. CLARITY, a new twist on this method, avoids earlier problems with the clearing chemicals dimming fluorescent signals.

To identify the connections, anterograde tracers, retrograde tracers and viruses are used. Anterograde tracers label projections as they are transported from the cell body towards the axon terminal, while retrograde tracers label as they move backwards, from the terminal towards the cell body. To visualize specific synaptic contacts, viruses that can cross one or more synapses can be used. Viruses can also be targeted to particular cells by combining them with other viruses expressing specific receptors or with Cre-recombinase transgenic mice, enabling mapping within selective brain regions or cell types. Each of the three projects uses a different combination of these strategies to complement each other and allow for cross-validation of their results.

Challenges associated with charting all the connections include the sheer size of the datasets (several terabytes per brain), registering each brain onto a reference atlas, and ensuring standardized analytical methods are used across the different research groups. Additionally, these tracing methods do not allow the function of circuits to be probed, only the structure. But, by combining methods used for mapping connections with single-cell electrophysiology or calcium imaging, questions about functions of certain cells within a network can be addressed. Specific cell types can then be targeted with optogenetics, allowing causality to be tested. Together these techniques will enable the creation of  a complete structure-function map for different behaviors.

Like the connectome of the roundworm C. elegans, a complete mouse brain connectome will reveal the entire brain’s circuitry and provide a starting point for further research. It will allow the various functions of different circuits to be probed, telling us which neurons are responsible for certain behaviors, and help us understand diseases caused by disruptions to these circuits. Light microscopy instruments have come a long way from the days of Golgi and Ramón Y Cajal, and we currently stand on the cusp of a detailed understanding of all the connections, both local and long-range, within the mouse brain.

Material below is adapted from the SfN Short Course Mesoscale Two-Photon Microscopy Engineering a Wide Field of View with Cellular Resolution, by Jeffrey N. Stirman, PhD, and Spencer L. Smith, PhD. Short Courses are daylong scientific trainings on emerging neuroscience topics and research techniques held the day before SfN’s annual meeting.

Mapping activity in the brain requires both a wide lens — to see connections extending across brain regions — and a fine focus — to see the cells forming those connections. Two-photon microscopy coupled with calcium imaging meets the criteria for a fine focus; it can detect activity within individual neurons or within a local population of neurons. But beyond a small window, two-photon excitation lessens and the resolution suffers. This narrows the field of view, making mapping long-range connections impossible. Fortunately, newly described tweaks to both the microscopes and imaging techniques are making “mesoscale” two-photon microscopy possible, allowing wider views than can span brain regions a millimeter or more apart. Recently, this type of imaging has been performed in vivo to map activity in the visual system of mice.

In a laser scanning microscope, the laser beam bounces off two mirrors that rotate back and forth to move the laser’s focus across the specimen. One of the main determinants of the field of view is the size of the angles these mirrors take as the laser scans. Higher scanning angles are required for viewing a larger field, so the first step in mesoscale two-photon microscopy is considering how to accommodate such high scanning angles. Accommodations include using objectives with high numerical apertures (contributing to better resolution) and short focal lengths (allowing wider views).

High scanning angles can also accentuate aberrations, distortions created by the imaging system. This is a problem in two-photon microscopy because a clear focal spot is needed to achieve two-photon excitation, and aberrations can prevent the formation of such a focal point, limiting the usable field. Again, accommodations can be made, such as using a compound lens, creating a custom lens, or correcting distortions with adaptive optics.

Finally, large fields of views can mean scanning takes a long time, a problem for some applications. Different scanning techniques can increase imaging speed. Resonant scanners, which scan at a rate four to twelve times faster than conventional scanners, provide high enough scanning angles and a wide field of view. Acousto-optical deflectors use sound waves to bounce the beam back and forth, improving speed a hundredfold but not necessarily widening the field of view. Arbitrary line scanning does a quick pass to find the cells of interest, and then directs the laser to those cells in particular, cutting down on time wasted scanning regions without active neurons. Additionally, these methods can be combined to use multiple beams to scan separate areas. The more beams, the faster the imaging speed.

Of course, other approaches (such as using two microscopes or quickly repositioning the microscope or sample) could also enable imaging of multiple brain regions. But by making certain accommodations to the microscope and technique, mesoscale two-photon microscopy can be achieved in a novel way. Mesoscale imaging will allow detection of both the cells making connections and the range of the connections, creating an activity map of unprecedented detail and scope.

Material below is adapted from the SfN Short Course Simultaneous Holographic Imaging of Neuronal Circuits in Three Dimensions, by Sean Quirin, PhD, et al. Short Courses are daylong scientific trainings on emerging neuroscience topics and research techniques held the day before SfN’s annual meeting.

Traditional microscopy techniques are limiting in that most only reflect two dimensions of a three-dimensional biological system. Optical imaging, such as two-photon microscopy to measure voltage-sensitive or calcium-sensitive dyes, is minimally invasive and has the resolution to study the individual cells that make up large neuronal networks. However, images from these experiments are captured in two dimensions; to collect data spanning a chunk of tissue, sequential images are scanned one at a time, losing any activity that occurs at the same time in different planes. 

A new technique seeks to solve this problem by simultaneously collecting data in three dimensions. To achieve this, a conventional two-photon microscope first collects structural data (such as the location of cells), and then a hologram is generated to target regions of interest (such as active cells) in three-dimensional space. A spatial light modulator illuminates the chosen cells while a phase mask system simultaneously picks up their fluorescence. With the new method, two neurons firing together can be imaged together, even if they are spread apart.

The capabilities of this system have been demonstrated both in vitro and in vivo. Using this technique in slices from the mouse dentate gyrus, individual cells were identified and imaged — a feat in and of itself in an area so densely packed with cells. No matter the cell’s location in three-dimensional space, calcium signals could be clearly separated and quantified. Thus, the technique could be used to visualize patterns of activity responsible for memory with unprecedented detail and precision.

This technique was also demonstrated in vivo in larval zebrafish. Using the two-photon, holographic approach, calcium dynamics were measured across the brain with enough precision and speed to make a three-dimensional map of synaptic activity, a first in these animals. However, this was possible in larval zebrafish because they are transparent, a convenience not true of most animals. This new imaging system is not yet optimized to image through layers of tissue because the light scatters too much. Imaging techniques are continuing to improve, though, and applying new strategies may remedy this problem soon.

Material below summarizes the article 3D Electron Microscopy Study of Synaptic Organization of the Normal Human Transentorhinal Cortex and Its Possible Alterations in Alzheimer’s Disease, published on June 19, 2019, in eNeuro and authored by M. Domínguez-Álvaro, M. Montero-Crespo, L. Blazquez-Llorca, J. DeFelipe, and L. Alonso-Nanclares.

Highlights

• The majority of synapses are excitatory on spine heads (55%) and have a small disk shape. Nevertheless, a relatively large proportion of excitatory synapses are established on dendritic shafts (37%).

• Excitatory and inhibitory synapses show different preferences for their postsynaptic targets (spines or dendritic shafts).

• In Alzheimer’s disease (AD), morphological synaptic changes and a reduction in the number of synapses targeting spine heads occur.

Screenshot of the software user interface (EspINA). In the main window, the sections are viewed through the xy plane (as obtained by FIB/SEM microscopy). The other two orthogonal planes, yz and xz, are also shown in adjacent windows (on the right). The list of identified synapses is shown on the left. Synapses are colored in green. (From Figure 3 of Dominguez-Álvaro et al. 2019, eNeuro).

Study Question

What is the synaptic organization of the transentorhinal cortex and what are the possible synaptic alterations that occur in Alzheimer’s disease?

How This Research Advances What We Know

Determination of postsynaptic targets, as well as the shape and size of the synaptic junctions, provides critical data about synaptic functionality and circuit organization.

As far as we know, 3D synaptic morphology has never been performed together with the identification of postsynaptic targets in normal human brain samples or in the brain of patients with Alzheimer’s disease (AD). Thus, this study represents the first attempt to unveil the synaptic organization of the neuropil of the human brain at the ultrastructural level.

Thanks to 3D electron microscopy (FIB/SEM technology), it is possible to obtain large numbers of 3D reconstructed synapses. This study provides a large new quantitative ultrastructure dataset of the transentorhinal cortex, including both normal and AD cases.

In addition, the morphological synaptic alterations and changes in the postsynaptic targets found in AD may further understanding of the relationship between alterations of the synaptic circuits and the cognitive deterioration observed in these patients.

Experimental Design or Methodology

Human brain tissue samples from five control cases (with no recorded neurological or psychiatric alterations) and five AD patients (time between death and tissue processing was lower than 4h in all samples) were analyzed using FIB/SEM technology. Using this 3D electron microscopy method, we determined the morphology and postsynaptic target (spines or dendritic shafts) of the 4722 identified synapses in the neuropil of layer II of the human transentorhinal cortex. Moreover, we differentiated between excitatory and inhibitory synapses (based on their differential morphology). For data analysis, we applied appropriated statistical analysis and contingency tables.

Results

First, both in control cases and AD patients, excitatory and inhibitory synapses show different preferences for postsynaptic targets: Excitatory synapses have a significant preference for spine heads, while inhibitory synapses have a significant preference for dendritic shafts.

Second, in relation to the shape of synapses, in control and AD patients, the vast majority of synapses have a disk (macular) shape, followed by synapses with one or more holes (perforated), synapses with indentations in the perimeter (horseshoe-shaped), and synapses with two or more portions (fragmented).

Third, AD patients show an increase in the number of fragmented synapses and a reduction in the number of synapses targeting spine heads.

Interpretation

The large quantitative synaptic ultrastructure dataset of the normal transentorhinal cortex would be useful for comparing with other brain regions when the data become available. The aim would be to try to determine if the same synaptic organization exists in cortical regions that are cytoarchitectonically and functionally different.

Furthermore, this study provides evidence of a reduction in the number of synapses targeting spine heads in AD patients. The loss of spines and, therefore, the loss of synapses and circuits could be related to cognitive decline observed in AD.

Also, we observe morphological synaptic alterations in these patients that could be due to the activation of compensatory mechanisms in response to the synaptic loss that occurs in this cortical region during the progression of the disease. However, it could be possible that this kind of mechanism fails and synaptic activity would fall off.

Visit eNeuro to read the original article and explore other content. Read other summaries of eNeuro and JNeurosci papers in the Neuronline collection SfN Journals: Research Article Summaries.

3D Electron Microscopy Study of Synaptic Organization of the Normal Human Transentorhinal Cortex and Its Possible Alterations in Alzheimer’s Disease. M. Domínguez-Álvaro, M. Montero-Crespo, L. Blazquez-Llorca, J. DeFelipe, and L. Alonso-Nanclares. eNeuro June 2019, 6 (4) 0140–19.2019; DOI: 10.1523/ENEURO.0140-19.2019

About the Contributors

Marta Domínguez-Álvaro

Domínguez-Álvaro is a graduate student at the Polytechnic University of Madrid, in Spain, where she works in the Cajal Laboratory of Cortical Circuits in the Center for Biomedical Technology.

Lidia Alonso-Nanclares, PhD

Alonso-Nanclares is an assistant researcher in the Cajal Laboratory of Cortical Circuits in the Center for Biomedical Technology, at the Polytechnic University of Madrid, in Spain, and the Cajal Institute (CSIC).

Material below summarizes the article 3D Imaging of Axons in Transparent Spinal Cords from Rodents and Nonhuman Primates, published on March 26, 2015, in eNeuro and authored by Cynthia Soderblom, Do-Hun Lee, Abdul Dawood, Melissa Carballosa, Andrea Jimena Santamaria, Francisco D. Benavides, Stanislava Jergova, Robert M. Grumbles, Christine K. Thomas, Kevin K. Park, James David Guest, Vance P. Lemmon, Jae K. Lee, and Pantelis Tsoulfas.

The beginning of this century has seen some major advances in light microscopy, particularly related to neuroscience. These developments in microscopy, coupled with techniques that make tissues transparent, are enabling microscopes to visualize the cellular architecture of whole tissues in 3D with unprecedented detail.

One of these advances in microscopy has been light sheet fluorescent microscopy (LSFM). The underlying method was developed in 1902 by Richard Zsigmondy and Henry Siedentopf to enhance the microscopic resolution for studying colloidal gold. The modern light sheet fluorescence microscopy was first pioneered by Voie and colleagues and originally named orthogonal-plane fluorescence optical sectioning. Arne Voie, David Burns, and Francis Spelman focused a laser beam into a thin sheet to illuminate a fluorescent sample and captured the reflected light using a different objective lens-oriented perpendicular to the plane of illumination (i.e., light sheet).

This is in contrast to confocal laser microscopy, where the laser and the reflected light travel through the same objective lens. Using this method, these authors were able to reconstruct a cleared guinea pig cochlea in 3D. The basic configuration of that instrument lay the foundation for all subsequent versions of LSFM microscopes.

In confocal microscopes, the optical sectioning of the specimen is based on discriminating the out-of-focus reflected light by using a pinhole. However, the excitation light excites all of the fluorophores as it passes though the specimen, which often leads to photobleaching. Furthermore, point-by-point scanning along the entire specimen makes imaging too slow and unwieldy for large specimens that are millimeters in thickness. In addition, there is a drop in image brightness with increasing depth caused by light scattering and absorption. In LSFM, the laser light sheet, typically 2-6  microns, illuminates only one thin plane of the sample surrounding the focal plane of the detection lens, and thus there is no out of focus light. Therefore, the photo-bleaching or photo-damage is much less than conventional laser scanning microscopy including 2-photon systems.

One limitation of LSFMs using single side illumination, especially with large tissue, is that any obstacles (e.g., air bubbles or high concentration of fluorophores) cast shadows along the illumination path, which appear as stripes in the acquired image. A way to get around this problem is to illuminate from two opposing sides and merge the images, such as in the Ultramicroscope. In addition, adjusting the thickness of the laser sheet and the use of multi-view imaging can provide enhanced resolution. Thus, LSFM is ideal for 3D reconstruction of clear specimens and for in vivo imaging of transparent organisms.

Similar to light microscopy, there have been significant advances in tissue-clearing techniques during this century.

The first successful attempt to render fixed anatomical preparations transparent was done by Walter Spalteholtz. He used a solution of benzyl alcohol and methyl salicylate, which was later modified by others to produce Murray’s clear solution. Murray's clear was mostly used to study development of vertebrate embryos.

The first 3D reconstructions of clear embryos were obtained by using optical projection tomography, and later, Hans-Ulrich Dodt’s lab pioneered the use of light sheet microscopy on cleared whole-mount specimens, such as whole mouse brains expressing green fluorescent protein (GFP).

Dodt and Frank Bradke’s laboratories later developed the 3DISCO (3-dimensional imaging of solvent cleared organs) method of imaging rodent brain and spinal cord by combining Ultramicroscopy with tetrahydrofuran-based tissue clearing.

A few years later, Karl Deisseroth’s lab developed CLARITY, which is a hydrogel-based method that also allows antibody penetration for immunohistochemical labeling in whole tissue. CLARITY is also compatible with LSFM, a method termed COLM (clarity-optimized light-sheet microscopy). Both 3DISCO and CLARITY have relied on the use of transgenic mice in which subpopulation of neurons are brightly labeled with GFP.

However, we have developed AAV (adeno-associated virus)-based fluorescent labeling methods that can be used with 3DISCO to image non-transgenic animals such as rats. In addition, the use of viruses allows much better spatiotemporal control over labeling methods as compared to transgenic animals. Therefore, the combination of LSFM with multiple tissue clearing strategies and neuronal labeling methods will greatly aid our understanding of the structure-function relationship of the central nervous system under normal and diseased conditions.

Visit eNeuro to read the original article and explore other content. Read other summaries of JNeurosci and eNeuro papers in the Neuronline collection SfN Journals: Research Article Summaries.

3D Imaging of Axons in Transparent Spinal Cords from Rodents and Nonhuman Primates. Cynthia Soderblom, Do-Hun Lee, Abdul Dawood, Melissa Carballosa, Andrea Jimena Santamaria, Francisco D. Benavides, Stanislava Jergova, Robert M. Grumbles, Christine K. Thomas, Kevin K. Park, James David Guest, Vance P. Lemmon, Jae K. Lee, Pantelis Tsoulfas. eNeuro Mar 2015, 2 (2) DOI: 10.1523/ENEURO.0001-15.2015

About the Contributor

Jae Lee, PhD

Jae Lee is an assistant professor at the Miami Project to Cure Paralysis, University of Miami School of Medicine.

Material below is adapted from the SfN Short Course Acousto-optical Scanning–Based High-Speed 3D Two-Photon Imaging in Vivo, by Balázs Rózsa, MD, PhD, Gergely Szalay, and Gergely Katona. Short Courses are daylong scientific trainings on emerging neuroscience topics and research techniques held the day before SfN’s annual meeting.

Oftentimes when imaging the brain, a wider view is sacrificed for finer detail, or vice versa. Although the ideal imaging technique could resolve both signals propagating within single neurons and signals sent across a whole network of hundreds of neurons, the two views present different challenges. Imaging in single cells must be able to distinguish activity in adjacent compartments (the soma compared to the dendrite, for example) while imaging networks of hundreds of cells must be able to measure simultaneous activity in cells that are hundreds of micrometers apart. Because of this, most current technologies are optimized for just one of the two views. A microscope that could tackle both would need to be fast yet fine-detailed, focused yet far-reaching.

Recently, researchers have created a high-speed, three-dimensional, two-photon system that meets all these requirements. The system uses an acousto-optical deflector (AOD), which diffracts the laser beam with sound waves. The two-photon technology provides depth, enabling exploration deeper into the cortex. All together, the microscope has a millimeter scanning range and a sub-millisecond speed, both far-reaching and fast.

The system has two main modes, a random-access scanning mode (for calcium imaging across hundreds of neurons) and a trajectory scanning mode (for measuring calcium spikes along the length of a dendrite). Four AODs are used (two for the horizontal scanning lens and two for the vertical scanning lens) to compensate for possible drifting of the focal spot. Drift can also be corrected by picking a landmark (such as a particularly bright glial cell), regularly checking its position, and recalibrating the microscope as need be. The special grouping of the four AODs increases the imaging range almost three-fold.

This technology scans large volumes  (around 1 mm³) of tissue, ideal for experiments that require imaging regions of interest that are spread apart, whether they are multiple areas along a single dendrite, within a single cell, or across large networks. Random-access point scanning identifies points of interest to quickly scan in three dimensions, making it a convenient way to image neuronal networks. The frame scanning mode can be used to move or rotate areas in three-dimensions and can help find regions of interest. Trajectory scanning follows multiple neuronal processes simultaneously and can even be done continuously by allowing the focal spot to drift along a neuronal process.

High-speed, three-dimensional, two-photon imaging enables researchers to scan entire neuronal networks or focus on the dynamics of single dendrites. The combination of high speed and high resolution allows activity within dendritic spines to be resolved, while the combination of high speed and extensive volume permits simultaneous recording across networks. Looking ahead, there is potential for improvement to increase the field of view, better correct for artifacts, lower cost, or gain access to deeper cortical areas. For now, though, the advanced system — and the types of data it can generate — is exciting.

Read the latest research on the use of microscopy in neural circuit interrogation research:

• Wide-Area All-Optical Neurophysiology in Acute Brain Slices
• Wide. Fast. Deep: Recent Advances in Multiphoton Microscopy of In Vivo Neuronal Activity
• Analysis of Parvocellular and Magnocellular Visual Pathways in Human Retina
• 3D Electron Microscopy Study of Synaptic Organization of Normal Human Transentorhinal Cortex and Its Possible Alterations in Alzheimer’s Disease
• Fully Affine Invariant Methods for Cross-Session Registration of Calcium Imaging Data