Volume electron microscopy analysis of synapses in primary regions of the human cerebral cortex.

Functional and structural studies investigating macroscopic connectivity in the human cerebral cortex suggest that high-order associative regions exhibit greater connectivity compared to primary ones. However, the synaptic organization of these brain regions remains unexplored. In the present work, we conducted volume electron microscopy to investigate the synaptic organization of the human brain obtained at autopsy. Specifically, we examined layer III of Brodmann areas 17, 3b, and 4, as representative areas of primary visual, somatosensorial, and motor cortex. Additionally, we conducted comparative analyses with our previous datasets of layer III from temporopolar and anterior cingulate associative cortical regions (Brodmann areas 24, 38, and 21). 9,690 synaptic junctions were 3D reconstructed, showing that certain synaptic characteristics are specific to particular regions. The number of synapses per volume, the proportion of the postsynaptic targets, and the synaptic size may distinguish one region from another, regardless of whether they are associative or primary cortex. By contrast, other synaptic characteristics were common to all analyzed regions, such as the proportion of excitatory and inhibitory synapses, their shapes, their spatial distribution, and a higher proportion of synapses located on dendritic spines. The present results provide further insights into the synaptic organization of the human cerebral cortex.

Analysis of Neuronal Morphology by Two-Photon Microscopy.

During the development of mammalian brains, pyramidal neurons in the cerebral cortex form highly organized six layers with different functions. These neurons undergo developmental processes such as axon extension, dendrite outgrowth, and synapse formation. A proper integration of the neuronal connectivity through dynamic changes of dendritic branches and spines is required for learning and memory. Disruption of these crucial developmental processes is associated with many neurodevelopmental and neurodegenerative disorders. To investigate the complex dendritic architecture, several useful staining tools and genetic methods to label neurons have been well established. Monitoring the dynamics of dendritic spine in a single neuron is still a challenging task. Here, we provide a methodology that combines in vivo two-photon brain imaging and in utero electroporation, which sparsely labels cortical neurons with fluorescent proteins. This protocol may help elucidate the dynamics of microstructure and neural complexity in living rodents under normal and disease conditions.

Imaging of Structural Plasticity of Dendritic Spines with Two-Photon Microscopy.

Plasticity of synaptic transmission underlies learning and memory. It is accompanied by changes in the density and size of synapses, collectively called structural plasticity. Therefore, understanding the mechanism of structural plasticity is critical for understanding the mechanism of synaptic plasticity. In this chapter, we describe the procedures and equipment required to image structural plasticity of a single dendritic spine, which hosts excitatory synapses in the central nervous system, and underlying molecular interactions/biochemical reactions using two-photon fluorescence lifetime microscopy (2P-FLIM) in combination with Förster resonance energy transfer (FRET)-based biosensors.

Improved DiOlistic labelling technique for neurons in situ: Detailed visualisation of dendritic spines and concurrent histochemical-detection in fixed tissue.

DiOlistic labelling is a robust, unbiased ballistic method that utilises lipophilic dyes to morphologically label neurons. While its efficacy on freshly dissected tissue specimens is well-documented, applying DiOlistic labelling to stored, fixed brain tissue and its use in polychromatic multi-marker studies poses significant technical challenges. Here, we present an improved, step-by-step protocol for DiOlistic labelling of dendrites and dendritic spines in fixed mouse tissue. Our protocol encompasses the five key stages: Tissue Preparation, Dye Bullet Preparation, DiOlistic Labelling, Confocal Imaging, and Image Analysis. This method ensures reliable and consistent labelling of dendritic spines in fixed mouse tissue, combined with increased throughput of samples and multi-parameter staining and visualisation of tissue, thereby offering a valuable approach for neuroscientific research.

Key morphological features of human pyramidal neurons.

The basic building block of the cerebral cortex, the pyramidal cell, has been shown to be characterized by a markedly different dendritic structure among layers, cortical areas, and species. Functionally, differences in the structure of their dendrites and axons are critical in determining how neurons integrate information. However, within the human cortex, these neurons have not been quantified in detail. In the present work, we performed intracellular injections of Lucifer Yellow and 3D reconstructed over 200 pyramidal neurons, including apical and basal dendritic and local axonal arbors and dendritic spines, from human occipital primary visual area and associative temporal cortex. We found that human pyramidal neurons from temporal cortex were larger, displayed more complex apical and basal structural organization, and had more spines compared to those in primary sensory cortex. Moreover, these human neocortical neurons displayed specific shared and distinct characteristics in comparison to previously published human hippocampal pyramidal neurons. Additionally, we identified distinct morphological features in human neurons that set them apart from mouse neurons. Lastly, we observed certain consistent organizational patterns shared across species. This study emphasizes the existing diversity within pyramidal cell structures across different cortical areas and species, suggesting substantial species-specific variations in their computational properties.

AFM Imaging Reveals MicroRNA-132 to be a Positive Regulator of Synaptic Functions.

The modification of synaptic and neural connections in adults, including the formation and removal of synapses, depends on activity-dependent synaptic and structural plasticity. MicroRNAs (miRNAs) play crucial roles in regulating these changes by targeting specific genes and regulating their expression. The fact that somatic and dendritic activity in neurons often occurs asynchronously highlights the need for spatial and dynamic regulation of protein synthesis in specific milieu and cellular loci. MicroRNAs, which can show distinct patterns of enrichment, help to establish the localized distribution of plasticity-related proteins. The recent study using atomic force microscopy (AFM)-based nanoscale imaging reveals that the abundance of miRNA(miR)-134 is inversely correlated with the functional activity of dendritic spine structures. However, the miRNAs that are selectively upregulated in potentiated synapses, and which can thereby support prospective changes in synaptic efficacy, remain largely unknown. Using AFM force imaging, significant increases in miR-132 in the dendritic regions abutting functionally-active spines is discovered. This study provides evidence for miR-132 as a novel positive miRNA regulator residing in dendritic shafts, and also suggests that activity-dependent miRNAs localized in distinct sub-compartments of neurons play bi-directional roles in controlling synaptic transmission and synaptic plasticity.

Biophysical Modeling of Synaptic Plasticity.

Dendritic spines are small, bulbous compartments that function as postsynaptic sites and undergo intense biochemical and biophysical activity. The role of the myriad signaling pathways that are implicated in synaptic plasticity is well studied. A recent abundance of quantitative experimental data has made the events associated with synaptic plasticity amenable to quantitative biophysical modeling. Spines are also fascinating biophysical computational units because spine geometry, signal transduction, and mechanics work in a complex feedback loop to tune synaptic plasticity. In this sense, ideas from modeling cell motility can inspire us to develop multiscale approaches for predictive modeling of synaptic plasticity. In this article, we review the key steps in postsynaptic plasticity with a specific focus on the impact of spine geometry on signaling, cytoskeleton rearrangement, and membrane mechanics. We summarize the main experimental observations and highlight how theory and computation can aid our understanding of these complex processes.

Neural circuit changes in neurological disorders: Evidence from in vivo two-photon imaging.

Neural circuits, such as synaptic plasticity and neural activity, are critical components of healthy brain function. The consequent dynamic remodeling of neural circuits is an ongoing procedure affecting neuronal activities. Disruption of this essential process results in diseases. Advanced microscopic applications such as two-photon laser scanning microscopy have recently been applied to understand neural circuit changes during disease since it can visualize fine structural and functional cellular activation in living animals. In this review, we have summarized the latest work assessing the dynamic rewiring of postsynaptic dendritic spines and modulation of calcium transients in neurons of the intact living brain, focusing on their potential roles in neurological disorders (e.g. Alzheimer's disease, stroke, and epilepsy). Understanding the fine changes that occurred in the brain during disease is crucial for future clinical intervention developments.

Effects of sleep deprivation of various durations on novelty-related object recognition memory and object location memory in mice.

Sleep is essential for important physiological functions. Impairment of learning and memory function caused by lack of sleep is a common physiological phenomenon of which underlying changes in synaptic plasticity in the hippocampus are not well understood. The possible different effects of sleep deprivation (SD) lasting for various durations on learning and memory function and hippocampal synaptic plasticity are still not completely clear. In this study, we used a modified multiple platform method (MMPM) to induce rapid eye movement SD (REM SD), lasting for 24 h, 48 h, and 72 h, separately. The novel place recognition (NPR) and novel object recognition (NOR) tasks were used to test the novelty-related object recognition memory (ORM) and object location memory (OLM) of mice. Then, hippocampal synaptic plasticity was evaluated after all behavioural experiments. The results showed that REM SD played a key role in OLM but not in ORM. Specifically, 24 h REM SD improved novelty-related OLM, accompanied by a significantly increased hippocampal synaptic plasticity, including gain of dendritic spines, increased expression of hippocampal GluA1, and enhanced long-term potentiation (LTP), whereas 48 h REM SD showed no effect on OLM or the hippocampal synaptic plasticity mentioned above; however, 72 h REM SD impaired novelty-related OLM and weakened hippocampal synaptic plasticity, including serious loss of dendritic spines, decreased expression of hippocampal GluA1, and significantly attenuated LTP. Our results suggest that REM SD of various durations has different effects on both novelty-related OLM and hippocampal synaptic plasticity.

Cirbp-PSD95 axis protects against hypobaric hypoxia-induced aberrant morphology of hippocampal dendritic spines and cognitive deficits.

Hypobaric hypoxia (HH) is a typical characteristic of high altitude environment and causes a spectrum of pathophysiological effects, including headaches, gliovascular dysfunction and cognitive retardation. Here, we sought to understand the mechanisms underlying cognitive deficits under HH exposure. Our results showed that hypobaric hypoxia exposure impaired cognitive function and suppressed dendritic spine density accompanied with increased neck length in both basal and apical hippocampal CA1 region neurons in mice. The expression of PSD95, a vital synaptic scaffolding molecule, is down-regulated by hypobaric hypoxia exposure and post-transcriptionally regulated by cold-inducible RNA-binding protein (Cirbp) through 3'-UTR region binding. PSD95 expressing alleviates hypoxia-induced dendritic spine morphology changes of hippocampal neurons and memory deterioration. Moreover, overexpressed Cirbp in hippocampus rescues HH-induced abnormal expression of PSD95 and attenuates hypoxia-induced dendritic spine injury and cognitive retardation. Thus, our findings reveal a novel mechanism that Cirbp-PSD-95 axis appears to play an essential role in HH-induced cognitive dysfunction in mice.

Unique properties of dually innervated dendritic spines in pyramidal neurons of the somatosensory cortex uncovered by 3D correlative light and electron microscopy.

Pyramidal neurons (PNs) are covered by thousands of dendritic spines receiving excitatory synaptic inputs. The ultrastructure of dendritic spines shapes signal compartmentalization, but ultrastructural diversity is rarely taken into account in computational models of synaptic integration. Here, we developed a 3D correlative light-electron microscopy (3D-CLEM) approach allowing the analysis of specific populations of synapses in genetically defined neuronal types in intact brain circuits. We used it to reconstruct segments of basal dendrites of layer 2/3 PNs of adult mouse somatosensory cortex and quantify spine ultrastructural diversity. We found that 10% of spines were dually innervated and 38% of inhibitory synapses localized to spines. Using our morphometric data to constrain a model of synaptic signal compartmentalization, we assessed the impact of spinous versus dendritic shaft inhibition. Our results indicate that spinous inhibition is locally more efficient than shaft inhibition and that it can decouple voltage and calcium signaling, potentially impacting synaptic plasticity.

Rapid Ultrastructural Changes in the PSD and Surrounding Membrane after Induction of Structural LTP in Single Dendritic Spines.

The structural plasticity of dendritic spines is considered to be an important basis of synaptic plasticity, learning, and memory. Here, we induced input-specific structural LTP (sLTP) in single dendritic spines in organotypic hippocampal slices from mice of either sex and performed ultrastructural analyses of the spines using efficient correlative light and electron microscopy. We observed reorganization of the PSD nanostructure, such as perforation and segmentation, at 2-3, 20, and 120 min after sLTP induction. In addition, PSD and nonsynaptic axon-spine interface (nsASI) membrane expanded unevenly during sLTP. Specifically, the PSD area showed a transient increase at 2-3 min after sLTP induction. The PSD growth was to a degree less than spine volume growth at 2-3 min and 20 min after sLTP induction but became similar at 120 min. On the other hand, the nsASI area showed a profound and lasting expansion, to a degree similar to spine volume growth throughout the process. These rapid ultrastructural changes in PSD and surrounding membrane may contribute to rapid electrophysiological plasticity during sLTP.SIGNIFICANCE STATEMENT To understand the ultrastructural changes during synaptic plasticity, it is desired to efficiently image single dendritic spines that underwent structural plasticity in electron microscopy. We induced structural long-term potentiation (sLTP) in single dendritic spines by two-photon glutamate uncaging. We then identified the same spines at different phases of sLTP and performed ultrastructural analysis by using an efficient correlative light and electron microscopy method. We found that postsynaptic density undergoes dramatic modification in its structural complexity immediately after sLTP induction. Meanwhile, the nonsynaptic axon-spine interface area shows a rapid and sustained increase throughout sLTP. Our results indicate that the uneven modification of synaptic and nonsynaptic postsynaptic membrane might contribute to rapid electrophysiological plasticity during sLTP.

A large-scale nanoscopy and biochemistry analysis of postsynaptic dendritic spines.

Dendritic spines, the postsynaptic compartments of excitatory neurotransmission, have different shapes classified from 'stubby' to 'mushroom-like'. Whereas mushroom spines are essential for adult brain function, stubby spines disappear during brain maturation. It is still unclear whether and how they differ in protein composition. To address this, we combined electron microscopy and quantitative biochemistry with super-resolution microscopy to annotate more than 47,000 spines for more than 100 synaptic targets. Surprisingly, mushroom and stubby spines have similar average protein copy numbers and topologies. However, an analysis of the correlation of each protein to the postsynaptic density mass, used as a marker of synaptic strength, showed substantially more significant results for the mushroom spines. Secretion and trafficking proteins correlated particularly poorly to the strength of stubby spines. This suggests that stubby spines are less likely to adequately respond to dynamic changes in synaptic transmission than mushroom spines, which possibly explains their loss during brain maturation.

Super-resolution STED microscopy in live brain tissue.

STED microscopy is one of several fluorescence microscopy techniques that permit imaging at higher spatial resolution than what the diffraction-limit of light dictates. STED imaging is unique among these super-resolution modalities in being a beam-scanning microscopy technique based on confocal or 2-photon imaging, which provides the advantage of superior optical sectioning in thick samples. Compared to the other super-resolution techniques that are based on widefield microscopy, this makes STED particularly suited for imaging inside live brain tissue, such as in slices or in vivo. Notably, the 50 nm resolution provided by STED microscopy enables analysis of neural morphologies that conventional confocal and 2-photon microscopy approaches cannot resolve, including all-important synaptic structures. Over the course of the last 20 years, STED microscopy has undergone extensive developments towards ever more versatile use, and has facilitated remarkable neurophysiological discoveries. The technique is still not widely adopted for live tissue imaging, even though one of its particular strengths is exactly in resolving the nanoscale dynamics of synaptic structures in brain tissue, as well as in addressing the complex morphologies of glial cells, and revealing the intricate structure of the brain extracellular space. Not least, live tissue STED microscopy has so far hardly been applied in settings of pathophysiology, though also here it shows great promise for providing new insights. This review outlines the technical advantages of STED microscopy for imaging in live brain tissue, and highlights key neurobiological findings brought about by the technique.

Bone mesenchymal stem cells attenuate resiniferatoxin-induced neuralgia via inhibiting TRPA1-PKCδ-P38/MAPK-p-P65 pathway in mice.

Treatment of neuropathic pain (NP) resulting from nerve injury is one of the most complicated and challenging in modern practice. Pharmacological treatments for NP are not fully effectively and novel approaches are requisite. Recently, transplantation of bone mesenchymal stem cells (BMSCs) has represented a promising approach for pain relief and neural repair, but how it produces beneficial effects on resiniferatoxin (RTX) induced nerve injury is still unclear. Here, we identified the BMSCs' analgesic effects and their potential mechanisms of microglial cells activation on RTX induced neuralgia. Immunostaining, biochemical studies demonstrated that microglia rather than astrocyte cells activation involved in RTX induced mechanical hyperalgesia, whereas the GFP-labeled BMSCs alleviated this mechanical hyperalgesia. Moreover, pain-related TRPA1, PKCδ, CaMKIIɑ (Calcium/calmodulin dependent protein kinase II), P38/MAPK (mitogen-activated protein kinase), p-P65 activation and expression in the spinal cord were significantly inhibited after BMSC administration. In addition, BMSCs treated RTX mice displayed a lower density of mushroom dendritic spines. Our research suggested that activation of PKCδ-CaMKIIɑ-P38/MAPK-p-P65 pathway and mushroom dendritic spines abnormal increase in the spinal cord is the main mechanism of RTX induced neuropathic pain, and transplant of BMSCs to the damaged nerve may offer promising approach for neuropathic pain.

Spine dynamics in the brain, mental disorders and artificial neural networks.

In the brain, most synapses are formed on minute protrusions known as dendritic spines. Unlike their artificial intelligence counterparts, spines are not merely tuneable memory elements: they also embody algorithms that implement the brain's ability to learn from experience and cope with new challenges. Importantly, they exhibit structural dynamics that depend on activity, excitatory input and inhibitory input (synaptic plasticity or 'extrinsic' dynamics) and dynamics independent of activity ('intrinsic' dynamics), both of which are subject to neuromodulatory influences and reinforcers such as dopamine. Here we succinctly review extrinsic and intrinsic dynamics, compare these with parallels in machine learning where they exist, describe the importance of intrinsic dynamics for memory management and adaptation, and speculate on how disruption of extrinsic and intrinsic dynamics may give rise to mental disorders. Throughout, we also highlight algorithmic features of spine dynamics that may be relevant to future artificial intelligence developments.

The importance of ultrastructural analysis of memory.

Plasticity of glutamatergic synapses in the hippocampus is believed to underlie learning and memory processes. Surprisingly, very few studies report long-lasting structural changes of synapses induced by behavioral training. It remains, therefore, unclear which synaptic changes in the hippocampus contribute to memory storage. Here, we systematically compare how long-term potentiation of synaptic transmission (LTP) (a primary form of synaptic plasticity and cellular model of memory) and behavioral training affect hippocampal glutamatergic synapses at the ultrastructural level enabled by electron microscopy. The review of the literature indicates that while LTP induces growth of dendritic spines and post-synaptic densities (PSD), that represent postsynaptic part of a glutamatergic synapse, after behavioral training there is transient (< 6 h) synaptogenesis and long-lasting (> 24 h) increase in PSD volume (without a significant change of dendritic spine volume), indicating that training-induced PSD growth may reflect long-term enhancement of synaptic functions. Additionally, formation of multi-innervated spines (MIS), is associated with long-term memory in aged mice and LTP-deficient mutant mice. Since volume of PSD, as well as atypical synapses, can be reliably observed only with electron microscopy, we argue that the ultrastructural level of analysis is required to reveal synaptic changes that are associated with long-term storage of information in the brain.

Three-Dimensional Synaptic Organization of Layer III of the Human Temporal Neocortex.

In the present study, we have used focused ion beam/scanning electron microscopy (FIB/SEM) to perform a study of the synaptic organization of layer III of Brodmann's area 21 in human tissue samples obtained from autopsies and biopsies. We analyzed the synaptic density, 3D spatial distribution, and type (asymmetric/symmetric), as well as the size and shape of each synaptic junction of 4945 synapses that were fully reconstructed in 3D. Significant differences in the mean synaptic density between autopsy and biopsy samples were found (0.49 and 0.66 synapses/µm3, respectively). However, in both types of samples (autopsy and biopsy), the asymmetric:symmetric ratio was similar (93:7) and most asymmetric synapses were established on dendritic spines (75%), while most symmetric synapses were established on dendritic shafts (85%). We also compared several electron microscopy methods and analysis tools to estimate the synaptic density in the same brain tissue. We have shown that FIB/SEM is much more reliable and robust than the majority of the other commonly used EM techniques. The present work constitutes a detailed description of the synaptic organization of cortical layer III. Further studies on the rest of the cortical layers are necessary to better understand the functional organization of this temporal cortical region.

Upregulation of eIF4E, but not other translation initiation factors, in dendritic spines during memory formation.

Local translation can provide a rapid, spatially targeted supply of new proteins in distal dendrites to support synaptic changes that underlie learning. Learning and memory are especially sensitive to manipulations of translational control mechanisms, particularly those that target the initiation step, and translation initiation at synapses could be a means of maintaining synapse specificity during plasticity. Initiation predominantly occurs via recruitment of ribosomes to the 5' mRNA cap by complexes of eukaryotic initiation factors (eIFs), and the interaction between eIF4E and eIF4G1 is a particularly important target of translational control pathways. Pharmacological inhibition of eIF4E-eIF4G1 binding impairs formation of memory for aversive Pavlovian conditioning as well as the accompanying increase in polyribosomes in the heads of dendritic spines in the lateral amygdala (LA). This is consistent with a role for initiation at synapses in memory formation, but whether eIFs are even present near synapses is unknown. To determine whether dendritic spines contain eIFs and whether eIF distribution is affected by learning, we combined immunolabeling with serial section transmission electron microscopy (ssTEM) volume reconstructions of LA dendrites after Pavlovian conditioning. Labeling for eIF4E, eIF4G1, and eIF2α-another key target of regulation-occurred in roughly half of dendritic spines, but learning effects were only found for eIF4E, which was upregulated in the heads of dendritic spines. Our results support the possibility of regulated translation initiation as a means of synapse-specific protein targeting during learning and are consistent with the model of eIF4E availability as a central point of control.

Selective Ablation of BDNF from Microglia Reveals Novel Roles in Self-Renewal and Hippocampal Neurogenesis.

Microglia, the resident immune cells of the CNS, have emerged as key regulators of neural precursor cell activity in the adult brain. However, the microglia-derived factors that mediate these effects remain largely unknown. In the present study, we investigated a role for microglial brain-derived neurotrophic factor (BDNF), a neurotrophic factor with well known effects on neuronal survival and plasticity. Surprisingly, we found that selective genetic ablation of BDNF from microglia increased the production of newborn neurons under both physiological and inflammatory conditions (e.g., LPS-induced infection and traumatic brain injury). Genetic ablation of BDNF from microglia otherwise also interfered with self-renewal/proliferation, reducing their overall density. In conclusion, we identify microglial BDNF as an important factor regulating microglia population dynamics and states, which in turn influences neurogenesis under both homeostatic and pathologic conditions.SIGNIFICANCE STATEMENT (1) Microglial BDNF contributes to self-renewal and density of microglia in the brain. (2) Selective ablation of BDNF in microglia stimulates neural precursor proliferation. (3) Loss of microglial BDNF augments working memory following traumatic brain injury. (4) Benefits of repopulating microglia on brain injury are not mediated via microglial BDNF.