Name: Transmission Electron Microscopy as the Visualization Technique for Analysis of Circadian Synaptic Plasticity in the Mouse Barrel Cortex (Video) | JoVE Journal
Uploaded: 2025-08-19T13:30:00.000+00:00
Duration: 12 min 6 s
Description: Learn transmission electron microscopy to analyze circadian synaptic plasticity in the mouse barrel cortex. This protocol reveals diurnal changes in inhibitory and excitatory synapse numbers and dendritic spine morphology.

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This work is supported by grants from the National Science Centre in Poland, NCN OPUS20 nr UMO-2020/39/B/NZ7/03366 to EP and the Jagiellonian University Medical College, nr N41/DBS/001129 to MJ.

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</ol>

The authors have no conflicts of interest to declare.

Here, we presented the methodology used for studying the circadian plasticity of synapses and the reconstruction of dendritic spines in the barrel cortex of mice. To ensure reliable results, the circadian plasticity study should include at least four time points. Our research showed that data from two time points -- one during the rest phase (day) and one during the activity phase (night) -- provided information on the daily differences between the activity phases of animals under LD 12:12 conditions. Total synapse densi...

Circadian rhythms are generated by circadian clocks in almost all processes in an organism. In animals and humans, they have been detected at molecular, cellular, and whole-organism levels, as well as in their behavior. The circadian system of an organism consists of the main circadian clock (pacemaker) and peripheral clocks. All circadian clocks generate circadian oscillations through the cyclic expression of clock genes, which are controlled by their proteins. The molecular mechanism of the clock generates circadian rhythms with a period of ~1 day (longer or shorter than 24 h), but under day/night conditions, the period of endogenous rhythms is synchronized to 24 h. Many circadian rhythms have been detected in the physiology of the nervous system of invertebrates and vertebrates; however, some studies have shown that they are also present in synaptic and neuronal plasticity, that is, changes in the number and structure of synapses and dendrites1,2,3,4.
The mouse somatosensory cortex provides an excellent site for studying synaptic plasticity throughout the day due to its well-defined organization and direct link to animal locomotor activity5,6,7,8,9. In layer IV of the somatosensory cortex, there are distinct neural structures (barrels) that are highly noticeable even in an unstained brain10,11. Their arrangement reflects the morphology and organization of the whiskers on the animal&#39;s snout, allowing the precise mapping of sensory stimuli. The large barrels are organized in five regular rows, each containing between four and seven barrels. The rows of barrels that correspond to large whiskers are marked with capital letters from A to E and represent the whiskers arranged from the eye area downward along the snout. Each barrel consists of two main areas: the hollow, the central part filled with neuropil, and the wall called the side, which is dominated by the bodies of stellate cells. The barrels are separated from each other by areas of lower cell density, called septa. Each barrel receives impulses from a specific whisker located on the contralateral side of the animal&#39;s snout10,12.
The division into specific barrels, arranged in regular rows, allows for quick and easy identification of the selected area10,13. The selection of barrels from row B for analysis of circadian changes is based on studies of activity-dependent plasticity14,15,16,17. Row B is characterized by well-defined somatotopic boundaries and contains only four distinct large barrels, simplifying its location in samples. Due to its central position, row B neuronal activity is correlated with natural mouse behaviors such as environmental exploration and locomotor activity. For counting synapses on dendritic spines and shafts, it is important to select the central parts of the barrels, where cell bodies are sparse12,15,18.
Immunohistochemistry combined with confocal microscopy imaging is a technique that has played a crucial role in clarifying the key mechanisms of synaptic plasticity in mice. While using confocal microscopy provides high-resolution images with reduced background fluorescence and improved cellular structure clarity, it suffers from photobleaching and requires complex sample preparation, which may change the state of biological tissues. Its reliance on fluorescent markers can limit the types of proteins that can be studied simultaneously due to their overlap. Confocal immunofluorescence microscopy provides a means to label specific proteins, their expression levels, and localization associated with synaptic activity by using antibodies to detect target antigens in fixed tissue sections. Although this method offers specificity and flexibility to study various proteins involved in neurotransmission, it is limited by its inability to provide real-time data on dynamic processes due to the fixation step involved. The inconsistency in the antibody binding could also result in unreliable results19,20,21.
The use of electron microscopy instead of immunofluorescence methods with light microscopy allows for significantly higher resolution and greater precision in both localization selection and the distinction of neural structures. Serial transmission electron microscopy (TEM) sections help to visualize the synaptic structure at the ultrastructural level and have the advantage of being used for studying synapse formation or elimination. TEM enables researchers to analyze the physical properties of synapses and changes in the density and structure that are called the plasticity of synapses. Although sample preparation and imaging are labor-intensive, TEM provides a more detailed understanding of changes that happen during learning, memory formation, and processing of sensory information, and during the day, year, and animal age.
Our method aims to investigate the circadian dynamics of synaptic density and morphology by using a minimum four-time-point-based approach combined with the stereological dissector method based on serial TEM images. The stereological dissector method allows for reliable estimation of synapse numbers even when using only a few ultrathin serial sections22,23,24,25,26. The well-defined somatotopic organization of the barrel cortex ensures precise anatomical selection of study locations, while serial TEM allows differentiation of synapse types (excitatory and inhibitory) and their locations (on dendritic spines and shafts). This method provides a valuable tool for researchers investigating diurnal and circadian neuroplasticity, enabling the exploration of how environmental and internal factors influence synaptic dynamics. The individual components of our method enable its application for analysis of neuroplasticity changes in a much broader range of studies, not necessarily limited to circadian-related changes.

<table><tbody><tr><td>Aclar film</td><td>Agar scientific</td><td>AGL4458</td><td></td></tr><tr><td>Blender software&amp;nbsp;</td><td></td><td></td><td>version: v. 2.91.2</td></tr><tr><td>Cacodylic acid, sodium salt trihydrate</td><td>Polysciences</td><td>01131-100</td><td></td></tr><tr><td>Concave slide</td><td>Menzel</td><td>9.161 151</td><td></td></tr><tr><td>Diamond knife&amp;nbsp;</td><td>Diatome</td><td>15-US</td><td>Knife angle: Ultra 45&amp;deg;&amp;nbsp;</td></tr><tr><td>Digital camera&amp;nbsp;</td><td>Nikon&amp;nbsp;</td><td>DXM 1200 F</td><td></td></tr><tr><td>di-Sodium Hydrogen Phosphate Dodecahydrate</td><td>POCH</td><td>799280115</td><td></td></tr><tr><td>Embedding resin</td><td>Polysciences</td><td>08792-1</td><td>Luft formulation&amp;nbsp;</td></tr><tr><td>Ethanol</td><td>Pol-Aura</td><td>PA-11-0004</td><td></td></tr><tr><td>GIMP software</td><td></td><td></td><td>version: v 3.0.4</td></tr><tr><td>Lead citrate</td><td>TAAB</td><td>L018</td><td></td></tr><tr><td>Light microscopy&amp;nbsp;</td><td>Nikon&amp;nbsp;</td><td>Optiphot</td><td></td></tr><tr><td>Osmium tetroxide&amp;nbsp;</td><td>Polysciences</td><td>0223C-10</td><td>Crystalline (99.95%)</td></tr><tr><td>Paintbrush</td><td></td><td></td><td></td></tr><tr><td>Photoshop software</td><td></td><td></td><td>version: CS5</td></tr><tr><td>Potassium ferricyanide</td><td>Sigmaaldrich</td><td>P8131</td><td></td></tr><tr><td>Propylene oxide&amp;nbsp;</td><td>Sigmaaldrich</td><td>82320</td><td>puriss. p.a., &amp;ge;99.5% (GC)</td></tr><tr><td>Single slot grids</td><td>Agar scientific</td><td>AGG2525</td><td>2 x 0.75 mm or 2 x 1 mm</td></tr><tr><td>Sodium chloride&amp;nbsp;</td><td>Chempur</td><td>117941206</td><td></td></tr><tr><td>Sodium dihydrogen phosphate dihydrate</td><td>Chempur</td><td>117991808</td><td></td></tr><tr><td>Stereo microscope</td><td>pzo</td><td></td><td></td></tr><tr><td>Syring filter&amp;nbsp;</td><td>Biosens</td><td>BS25PES045</td><td></td></tr><tr><td>TEM&amp;nbsp;</td><td>Joel</td><td>JEM-2100</td><td></td></tr><tr><td>Ultramicrotome</td><td>Leica</td><td>UC7</td><td></td></tr><tr><td>Uranyl acetate</td><td>Lachema</td><td></td><td></td></tr><tr><td>Vibratome</td><td>Leica</td><td>VT1000 S</td><td></td></tr></tbody></table>

transmission electron microscopy as the visualization technique for analysis of circadian synaptic plasticity in the mouse barrel cortex

Transmission Electron Microscopy as the Visualization Technique for Analysis of Circadian Synaptic Plasticity in the Mouse Barrel Cortex

Examining circadian synaptic plasticity requires housing mice under different lighting conditions (light/dark cycle, LD 12:12, and constant darkness, DD), providing access to running wheels, and sacrificing them at four defined time points within 24 h-at the beginning and middle of the day/subjective day and at the beginning and middle of the night/subjective night. Brains are then properly fixed for transmission electron microscopy (TEM). The barrel cortex, with its precise somatotopic organization, provides an ideal model for such analysis. To obtain the required brain area, the brains are tangentially cut with a vibratome, and then, sections containing the barrel cortex are selected and embedded in Polybed resin. From the prepared blocks containing the selected barrels, consecutive ultrathin sections are cut. Synaptic density, excitatory and inhibitory, is analysed from electron micrographs using the stereological dissector method. Additionally, TEM images are used for 3D reconstructions of dendritic spines. Changes in the shape of dendritic spines indicate remodeling of neurons during the day. The number of excitatory synapses peaks during sleep (day) in mice, while inhibitory synapses peak during their activity phase (in the middle of the night).

To properly apply our method to the analysis of circadian synaptic changes, it is necessary to begin by selecting at least four time points at equal intervals, with two points for each phase of the animals' activity (every 6 h). At these designated time points, the animals are sacrificed, and their brains are collected. This approach allows for the identification of daily or circadian patterns of synaptic plasticity and links them to changes in the animals' locomotor activity (Figure 1). Usi...

The presented protocol describes the use of transmission electron microscopy (TEM) to quantify circadian changes in the mouse barrel cortex, mainly focusing on synapse number and dendritic spine morphology.

Examining circadian synaptic plasticity requires housing mice under different lighting conditions (light/dark cycle, LD 12:12, and constant darkness, DD), ...

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Research

JoVE Journal

Neuroscience

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Source: Villarreal, S., et. al., Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons. J. Vis. Exp. (2017)This video demonstrates the analysis of synaptic vesicle endocytosis in cultured hippocampal neurons through horseradish peroxidase labeling and electron microscopy.

Transmission Electron Microscopic Imaging of Synaptic Vesicle Endocytosis in Mouse Hippocampal Neurons 

Source: Maruoka, H., et. al. Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice. J. Vis. Exp. (2022)     This video shows a two-photon microscopy procedure for imaging neuronal activity and microglial dynamics in an awake mouse during visual stimulation, highlighting calcium transients and cellular interactions.

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in an Awake Mouse

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

This method creates a tangible, familiar environment for the mouse to navigate and explore during microscopic imaging or single-cell electrophysiological recordings, which require firm fixation of the animal’s head.

Flat-floored Air-lifted Platform: A New Method for Combining Behavior with Microscopy or Electrophysiology on Awake Freely Moving Rodents

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

We developed a novel technique in electron microscopy, "flash-and-freeze," that enables the visualization of membrane dynamics with ms temporal resolution. This technique combines the optogenetic stimulation of neurons with high-pressure freezing. Here, we demonstrate the procedures and describe the protocols in detail.

Flash-and-Freeze: A Novel Technique to Capture Membrane Dynamics with Electron Microscopy

This method describes a chronic preparation that allows optical access to the hippocampus of living mice. This preparation can be used to perform longitudinal optical imaging of neuronal structural plasticity and activity-evoked cellular plasticity over a period of several weeks.

Longitudinal Two-Photon Imaging of Dorsal Hippocampal CA1 in Live Mice

Presented here is a protocol for the implantation of a chronic cranial window for the longitudinal imaging of brain cells in awake, head-restrained mice.

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

Here we present a protocol describing viral transduction of discrete brain regions with optogenetic constructs to permit synapse-specific electrophysiological characterization in acute rodent brain slices.

Ex Vivo Optogenetic Interrogation of Long-Range Synaptic Transmission and Plasticity from Medial Prefrontal Cortex to Lateral Entorhinal Cortex

Here, we describe a protocol combining adeno-associated virus injection with cranial window implantation for simultaneous imaging of microglial dynamics and neuronal activity in awake mice.

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

Learn transmission electron microscopy to analyze circadian synaptic plasticity in the mouse barrel cortex. This protocol reveals diurnal changes in inhibitory and excitatory synapse numbers and dendritic spine morphology.

Transmission Electron Microscopy as the Visualization Technique for Analysis of Circadian Synaptic Plasticity in the Mouse Barrel Cortex (Video) | JoVE Journal

Transmission Electron Microscopy as the Visualization Technique for Analysis of Circadian Synaptic Plasticity in the Mouse Barrel Cortex

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