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 summarizes the article Synaptic Connections of Aromatase Circuits in the Medial Amygdala are Sex Specific, published on May 29, 2020, in eNeuro and authored by Addison Billing, Marcelo Henrique Correia, Diane A. Kelly, Geng-Lin Li, and Joseph Bergan.

Highlights

• Aromatase-producing cells in the mouse medial amygdala receive input originating from sensory cells in the vomeronasal organ that express a single class of receptors known to detect chemicals that convey information about the age, sex, and breeding condition of other mice.

• We did not observe sex differences in the intrinsic electrophysiological properties of aromatase-producing cells in the medial amygdala; however, we found a robust anatomical sex difference in the number of synapses that connect the accessory olfactory bulb – and thus the vomeronasal organ - to these cells.

• Males have nearly an order of magnitude more of these synaptic connections than do females, an anatomical difference that could play a role in shaping sex differences in social behaviors mediated by the medial amygdala.

Increased miR-182 Expression in the aIC Reduces Local QR2 mRNA Levels and Improves Novel Taste Learning. Gould, N.L. et al. eNeuro.0067-20.2020 (2020).

Study Question
Are there sex differences in circuit anatomy and/or function that might produce the sex differences observed in the social behaviors mediated by the medial amygdala?

How This Research Advances What We Know
Mice broadcast their sex, age, and reproductive availability to other mice with chemical markers that they detect with sensory cells inside a structure called the vomeronasal organ (VNO) that is embedded in the nasal septum. Signals from these cells are relayed to the medial amygdala (MeA), a brain region that is a hub for social behavior processing, in a two neuron circuit that passes through the accessory olfactory bulb (AOB). The MeA is known to show sex differences when processing scent information about the sex of another mouse, implying that sex differences exist in the anatomy and function of its circuits. We investigated whether these differences could be found in a subset of MeA neurons that produce aromatase, a key enzyme for producing estradiol, a hormone which plays   a critical role in establishing and maintaining sex-specific social behaviors.

Experimental Design/Methodology
We used mice genetically engineered to express red fluorescent protein in neurons that produce aromatase in the brain. This trait let us distinguish these cells from other neuron types inside the medial amygdala and to examine the electrophysiological behavior of neurons that make aromatase versus those that do not in the medial amygdala. We also injected a virus into the medial amygdala that would carry a green fluorescent label from infected aromatase-positive cells across incoming synapses to upstream neurons in their circuits. We then rendered these brains transparent and imaged them with a lightsheet microscope; these data let us reconstruct the paths and connections of all labeled neurons. In both parts of the study, we analyzed data with unpaired t-tests and a mixed model ANOVA.

Results
Our electrophysiological tests of the MeA found little evidence of sex differences in aromatase-producing cells for an array of critical electrophysiological properties including: input resistance, threshold voltage for eliciting an action potential, and the number of action potentials produced at specific current levels. We also found no sex differences for these characteristics in aromatase-negative neurons. Instead, we found that aromatase-producing cells in the MeA produced fewer action potentials in response to the same current levels than aromatase-negative neurons, suggesting that aromatase defines a class of MeA neurons with shared electrophysiological properties.

Our neuron tracing tests showed that the inputs to aromatase-producing MeA neurons from the anterior olfactory bulb (AOB) originate almost entirely from its anterior region; and their dendritic arbors were predominantly associated with glomeruli that receive sensory information from V1R cells in the vomeronasal organ. Males had significantly more of these inputs than females, even after we normalized for the number of neurons initially infected in the MeA.

Interpretation
Aromatase-expressing neurons in the medial amygdala have been implicated as key parts of the pathways that mediate social behaviours in mice; our results show that these cells are in a circuit with sensory cells that respond to chemicals that convey social signals. The number of these connections shows a robust sex difference: on average, each aromatase-expressing neuron in the male MeA gets inputs from almost an order of magnitude more AOB neurons than are seen in the female MeA. We found no evidence of sex differences in the intrinsic electrophysical properties of this subset of MeA neurons, suggesting that the difference in the volume of synapses connecting the AOB to the MeA could help create the sex differences in mouse social behaviors mediated by this region. An exciting future goal will be to understand the functional implications of this sex difference in circuit anatomy.

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.

Muscarinic-Dependent miR-182 and QR2 Expression Regulation in the Anterior Insula Enables Novel Taste Learning. Nathaniel L. Gould, Alina Elkobi, Efrat Edry, Jonathan Daume, and Kobi Rosenblum. eNeuro 26 March 2020, 0067-20.2020; DOI: 10.1523/ENEURO.0067-20.2020

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To understand the function of the brain and how its dysfunction leads to brain diseases, it is essential to have a deep understanding of the cell type composition of the brain, how the cell types are connected with each other, and what their roles are in circuit function.

Join this interactive Meet-the-Expert session to hear Hongkui Zeng discuss her research team’s experience in building multiple platforms and generating large-scale datasets to characterize the transcriptomic, physiological, morphological, and connectional properties of different types of neurons in a standardized manner, toward a multimodal taxonomy of cell types and a description of their wiring diagram for the mouse brain, with a primary focus on the cortex.

These data contribute significantly to the BRAIN Initiative Cell Census Network’s efforts and collectively provide an integrated resource to the neuroscience community to facilitate the investigation of the cellular diversity, circuit composition, and circuit function.

After the talk, Sheena Josselyn will moderate a 30-minute conversation in which you’ll have an opportunity to pose questions through a Q&A box.

After this virtual session, participants at all career stages will be able to:

• Achieve a better understanding of the different approaches of single cell characterization.
• Describe the number and basic properties of cell types in the mouse cortex.
• Know where and how to find the cell type resources

Speakers

Hongkui Zeng, PhD

Hongkui Zeng is the executive director of the structured science division at the Allen Institute for Brain Science, which is working to develop and operate high-throughput pipelines to generate large-scale, public datasets and tools to fuel neuroscience discovery. There, she has also led several research programs or projects, including the Transgenic Technology program, Human Cortex Gene Survey project, Allen Mouse Brain Connectivity Atlas project, and Mouse Cell Types and Connectivity program. Previously, she worked at a biotechnology start-up where she studied the genomic repertoire of the G-protein coupled receptor (GPCR) gene family, and the function of many GPCR genes in behavioral models of CNS diseases. Zeng earned a PhD in molecular and cell biology from Brandeis University and completed postdoctoral training at Massachusetts Institute of Technology.

Sheena Josselyn, PhD

Sheena Josselyn is a senior scientist in the neurosciences and mental health program at The Hospital for Sick Children and professor in psychology and physiology at the Institute of Medical Sciences at the University of Toronto. Sheena’s current research focus studies how information is encoded stored and used in the brain, primarily using mouse models. She received her Bachelor’s Degree at Queen’s University at Kingston and her PhD at the University of Toronto which are both located in Canada. Her previous position being a Research Associate in the Department of Neurobiology at UCLA and prior to that she worked in the Department of Psychiatry at Yale University School of Medicine.

Read the latest research on mapping circuits and networks:

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• Capillary-Based and Stokes-Based Trapping of Serial Sections for Scalable 3D-EM Connectomics
• Functional Connectome Analyses Reveal the Human Olfactory Network Organization
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