Multisynaptic Boutons Research Articles

Multi-synaptic boutons are a feature of CA1 hippocampal connections in the stratum oriens

Excitatory synapses are typically described as single synaptic boutons (SSBs), where one presynaptic bouton contacts a single postsynaptic spine. Using serial section block-face scanning electron microscopy, we found that this textbook definition of the synapse does not fully apply to the CA1 region of the hippocampus. Roughly half of all excitatory synapses in the stratum oriens involved multi-synaptic boutons (MSBs), where a single presynaptic bouton containing multiple active zones contacted many postsynaptic spines (from 2 to 7) on the basal dendrites of different cells. The fraction of MSBs increased during development (from postnatal day 22 [P22] to P100) and decreased with distance from the soma. Curiously, synaptic properties such as active zone (AZ) or postsynaptic density (PSD) size exhibited less within-MSB variation when compared with neighboring SSBs, features that were confirmed by super-resolution light microscopy. Computer simulations suggest that these properties favor synchronous activity in CA1 networks.

A Method for Identification of Multisynaptic Boutons in Electron Microscopy Image Stack of Mouse Cortex

Recent electron microscopy (EM) imaging techniques make the automatic acquisition of a large number of serial sections from brain samples possible. On the other hand, it has been proven that the multisynaptic bouton (MSB), a structure that consists of one presynaptic bouton and multiple postsynaptic spines, is closely related to sensory deprivation, brain trauma, and learning. Nevertheless, it is still a challenging task to analyze this essential structure from EM images due to factors such as imaging artifacts and the presence of complicated subcellular structures. In this paper, we present an effective way to identify the MSBs on EM images. Using normalized images as training data, two convolutional neural networks (CNNs) are trained to obtain the segmentation of synapses and the probability map of the neuronal membrane, respectively. Then, a series of follow-up operations are employed to obtain rectified segmentation of synapses and segmentation of neurons. By incorporating this information, the MSBs can be reasonably identified. The dataset in this study is an image stack of mouse cortex that contains 178 serial images with a size of 6004 pixels × 5174 pixels and a voxel resolution of 2 nm × 2 nm × 50 nm. The precision and recall on MSB detection are 68.57% and 94.12%, respectively. Experimental results demonstrate that our method is conducive to biologists’ research on MSBs’ properties.

Changes of Synaptic Structures Associated with Learning, Memory and Diseases

Synaptic plasticity is widely believed to be the cellular basis of learning and memory. It is influenced by various factors including development, sensory experiences, and brain disorders. Long-term synaptic plasticity is accompanied by protein synthesis and trafficking, leading to structural changes of the synapse. In this review, we focus on the synaptic structural plasticity, which has mainly been studied with in vivo two-photon laser scanning microscopy. We also discuss how a special type of synapses, the multi-contact synapses (including those formed by multi-synaptic boutons and multi-synaptic spines), are associated with experience and learning.

Estrogen Restores Multisynaptic Boutons in the Dorsolateral Prefrontal Cortex while Promoting Working Memory in Aged Rhesus Monkeys.

Humans and nonhuman primates are vulnerable to age- and menopause- related decline in working memory, a cognitive function reliant on area 46 of the dorsolateral prefrontal cortex (dlPFC). We showed previously that presynaptic mitochondrial number and morphology in monkey dlPFC neurons correlate with working memory performance. The current study tested the hypothesis that the types of synaptic connections these boutons form are altered with aging and menopause in rhesus monkeys and that these metrics may be coupled with mitochondrial measures and working memory. Using serial section electron microscopy, we examined the frequencies and characteristics of nonsynaptic, single-synaptic, and multisynaptic boutons (MSBs) in the dlPFC. In contrast to our previous observations in the monkey hippocampal dentate gyrus, where MSBs comprised ∼40% of boutons, the vast majority of dlPFC boutons were single-synaptic, whereas MSBs constituted a mere 10%. The frequency of MSBs was not altered by normal aging, but decreased by over 50% with surgical menopause induced by ovariectomy in aged monkeys. Cyclic estradiol treatment in aged ovariectomized animals restored MSB frequencies to levels comparable to young and aged premenopausal monkeys. Notably, the frequency of MSBs positively correlated with working memory scores, as measured by the average accuracy on the delayed response (DR) test. Furthermore, MSB incidence positively correlated with the number of healthy straight mitochondria in dlPFC boutons and inversely correlated with the number of pathological donut-shaped mitochondria. Together, our data suggest that MSBs are coupled to cognitive function and mitochondrial health and are sensitive to estrogen. Significance statement: Many aged menopausal individuals experience deficits in working memory, an executive function reliant on recurrent firing of prefrontal cortex (PFC) neurons. However, little is known about the organization of presynaptic inputs to these neurons and how they may be altered with aging and menopause. Multisynaptic boutons (MSBs) were of particular interest, because they form multiple synapses and can enhance coupling between presynaptic and postsynaptic neurons. We found that higher MSB frequency correlated with better working memory performance in rhesus monkeys. Additionally, aged surgically menopausal monkeys experienced a 50% loss of MSBs that was restored with cyclic estradiol treatment. Together, our findings suggest that hormone replacement therapy benefits cognitive aging, in part by retaining complex synaptic organizations in the PFC.

Effects of context-drug learning on synaptic connectivity in the basolateral nucleus of the amygdala in rats.

Context-drug learning produces structural and functional synaptic changes in the circuitry of the basolateral nucleus of the amygdala (BLA). However, how the synaptic changes translated to the neuronal targets was not established. Thus, in the present study, immunohistochemistry with a cell-specific marker and the stereological quantification of synapses was used to determine if context-drug learning increases the number of excitatory and inhibitory/modulatory synapses contacting the gamma-aminobutyric acid (GABA) interneurons and/or the pyramidal neurons in the BLA circuitry. Amphetamine-conditioned place preference increased the number of asymmetric (excitatory) synapses contacting the spines and dendrites of pyramidal neurons and the number of multisynaptic boutons contacting pyramidal neurons and GABA interneurons. Context-drug learning increased asymmetric (excitatory) synapses onto dendrites of GABA interneurons and increased symmetric (inhibitory or modulatory) synapses onto dendrites but not perikarya of these same interneurons. The formation of context-drug associations alters the synaptic connectivity in the BLA circuitry, findings that have important implications for drug-seeking behavior.

Retrieval of contextual memories increases activity-regulated cytoskeleton-associated protein in the amygdala and hippocampus

Activity-regulated cytoskeleton-associated protein (Arc) integrates information from multiple intracellular signaling cascades and, in turn, regulates cytoskeletal proteins involved in structural synaptic modifications. The purposes of the present study were: (1) to determine if the retrieval of contextual memories would induce Arc in hippocampal and amygdalar neurons; (2) use unbiased stereology at the ultrastructural level to quantify synapses contacting Arc-labeled (Arc+) and unlabeled (Arc-) postsynaptic structures in brain regions in which the amount of Arc integrated density (ID) correlated strongly with the degree of amphetamine conditioned place preference (AMPH CPP). The retrieval of contextual memories increased the Arc ID in the dentate gyrus, cornu ammonis (CA)1, and CA3 fields of the hippocampus and the basolateral, lateral, and central nuclei of the amygdala but not the primary auditory cortex, a control region. Stereological quantification of Arc+ and Arc- synapses in the basolateral nucleus of the amygdala (BLA) was undertaken because the strongest relationship between the amount of Arc ID and AMPH CPP was observed in the BLA. The retrieval of contextual memories increased the number and density of asymmetric (presumed excitatory) synapses contacting Arc+ spines and dendrites of BLA neurons, symmetric (presumed inhibitory or modulatory) synapses contacting Arc+ dendrites of BLA neurons, and multisynaptic boutons contacting Arc+ postsynaptic structures. Thus, the retrieval of contextual memories increases Arc in the amygdala and hippocampus, an effect that could be important for approach behavior to a drug-associated context.

Characterization of MSB Synapses in Dissociated Hippocampal Culture with Simultaneous Pre- and Postsynaptic Live Microscopy

Multisynaptic boutons (MSBs) are presynaptic boutons in contact with multiple postsynaptic partners. Although MSB synapses have been studied with static imaging techniques such as electron microscopy (EM), the dynamics of individual MSB synapses have not been directly evaluated. It is known that the number of MSB synapses increases with synaptogenesis and plasticity but the formation, behavior, and fate of individual MSB synapses remains largely unknown. To address this, we developed a means of live imaging MSB synapses to observe them directly over time. With time lapse confocal microscopy of GFP-filled dendrites in contact with VAMP2-DsRed-labeled boutons, we recorded both MSBs and their contacting spines hourly over 15 or more hours. Our live microscopy showed that, compared to spines contacting single synaptic boutons (SSBs), MSB-contacting spines exhibit elevated dynamic behavior. These results are consistent with the idea that MSBs serve as intermediates in synaptic development and plasticity.

The Synaptic Connections between Cortical Areas V1 and V2 in Macaque Monkey

The primary visual cortex (V1) and V2 together form approximately 24% of the total neocortex of the macaque monkey and have each other as their major partners. The major target of the V1 projection to V2 is layer 4, where it forms clusters of boutons, which form asymmetric (excitatory) synapses mainly with dendritic spines (75%). The remainder form synapses with dendritic shafts. The synapses found on spines were often more complex, perforated postsynaptic densities than those found on dendritic shafts. The reciprocal projection from V2 to V1 targeted layers 1, 2/3, and 5 and was formed of axons of different morphologies. One axon type, originating from superficial layer pyramidal cells, had a morphology resembling those of local pyramidal cell collaterals. These axons arborized in layers 1, 2/3, and 5 of V1. Another type of axon, arborizing in layer 1, was slender (0.3 microm), unbranched, unmyelinated, and uniformly covered with boutons terminaux and formed asymmetric synapses mainly with slender spines. Yet a third type of axon also confined to layer 1, was thick (>1 microm), branched, heavily myelinated, and formed separate small clusters of large ( approximately 1 microm) en passant multisynaptic boutons that formed asymmetric synapses mainly with large flat spines. These data show the existence of a reciprocal excitatory loop between V1 and V2 that is formed by different axonal types, each with preferred layers of termination.

Synaptic connection from cortical area V4 to V2 in macaque monkey

The major target of the V4 projection in V2 is layer 1, where it forms a tangential spread of asymmetric (excitatory) synapses. This is characteristic of a "feedback" projection. Some axons formed discrete clusters of bouton terminaux between lengths of myelinated axon, while others were unbranched and formed a continuous distribution of en passant boutons with no intercalated myelin. Minor projections were found in layers 2/3 and 6. Dendritic spines were the most frequently encountered targets of the V4 projection (80% in layer 1 and layer 2/3, 94% in layer 6). The remaining targets were dendritic shafts. In layer 1, 69% of target dendrites (12% of all targets) had characteristics identifying them as smooth (GABAergic) cells. In layer 2/3 and layer 6 virtually all the shaft synapses were on smooth dendrites (86% and 100%, respectively). Multisynaptic boutons were rare (mean 1.1 synapses per bouton). Synapses formed in layer 6 were smaller than those of layer 1 (mean area 0.073 microm(2) vs. 0.117 microm(2)). Synapses formed with spines had a more complex postsynaptic density than those formed with dendritic shafts. With respect to targets and synaptic type and size and morphology of synapses, the feedback projection from V4 to V2 resembles those of feedforward projections. The principal difference between the feedforward and feedback projection is in the lamina location of their terminal boutons. The concentration of the V4 projection on layer 1, where it forms asymmetric synapses mainly with spines, suggests that it excites the distal apical dendrites of pyramidal cells.

Ultrastructural correlates of vesicular docking in the rat dentate gyrus

To determine the extent to which CA1 synapses are typical of those found in other regions of the hippocampal formation, we have carried out a quantitative analysis of synapses in the middle molecular layer of the rat dentate gyrus, reconstructed from serial electron microscopy, and have compared these data with previous observations from CA1. In general, the morphology of synapses in areas CA1 and the dentate agree, other than an increased density of multisynaptic boutons. Thus, it seems that either area may form an equally effective model for the function of individual synapses in the hippocampal formation. In addition, the current study examines presynaptic curvature, which recent mathematical models have suggested may have profound effects on synaptic transmission. When synapses of distinct curvature profiles (i.e., presynaptically concave, convex, and flat) are examined, the average characteristics of these three synapse populations are distinct. In general, concave synapses have a greater number of morphologically docked vesicles, and thus, likely a greater probability of release. This, however, seems to be accounted for by the fact that these synapses are larger—the spatial density of docked vesicles remains identical across these curvature profiles. This study provides crucial data for further modeling of individual synapse function.

Connection from cortical area V2 to V3A in macaque monkey

The V2 projection to V3 A was labeled by pressure microinjecting biotinylated dextran amine (BDA) and Phaseolus vulgaris lectin (PHA-L) into V2 just posterior to the lunate sulcus. Dense terminal labeling in clusters was found in layer 4, with a weaker terminal projection in layer 3. About 3.5--4.1% of the synapses in the densest bouton clusters in layer 4 were made by labeled boutons. All were asymmetric (Gray's type 1) synapses, made by spiny, excitatory neurons. The most frequently encountered synaptic targets were spines (76% in layer 4, 98% in layer 2/3). The remainder of the synaptic targets were dendritic shafts, of which just less than half (44%) had the characteristic ultrastructure of smooth (inhibitory) cells. Multisynaptic boutons were rare (mean synapses per bouton for layer 4 1.2, for layer 2/3 1.1). The mean size of the postsynaptic densities found on spines (0.11 microm(2)) was not significantly different from that for dendrites (0.09 microm(2)). In terms of their type, laminar location, number, and targets, the synapses that formed the V2 projection to V3 A are typical of a major, excitatory, feedforward projection of macaque visual cortex.

Connection from cortical area V2 to MT in macaque monkey.

The extrastriate visual area of the macaque monkey called MT or V5, receives its input from multiple sources. We have previously examined the synaptic connections made by V1 cells that project to MT (Anderson et al., 1998). Here, we provide a similar analysis of the projection from V2 to MT. The major target of the V2 projection in MT is layer 4, where it forms clusters of asymmetric (excitatory) synapses. Unlike the V1 projection, it also forms synapses in layers 1 and 2 and does not form synapses in layer 6. The most frequently encountered targets of boutons labeled from V2 were spines (67% in layer 4; 82% in layer 2/3). Unusually, only 5/12 boutons examined in layer 1 actually formed synapses. Unlike the V1 projection, multisynaptic boutons were rare (mean, 1.1 synapses per bouton vs. 1.7 for the V1 projection). Like the V1 projection, the input to MT from any point in V2 is sparse (contributing approximately 4-6% of the asymmetric synapses in the densest clusters in layer 4). The synapses of the V2 projection were similar in size to those of the V1 projection (0.1 microm(2) vs. 0.09 microm(2)) and both formed more complex postsynaptic densities on spines than on dendritic shafts. The clear differences between the V1 and V2 projection to MT indicate that their functions are complementary rather than completely overlapping.

Innervation of cat visual areas 17 and 18 by physiologically identified X- and Y- type thalamic afferents. I. Arborization patterns and quantitative distribution of postsynaptic elements.

Specific thalamic afferents to visual areas 17 and 18 were physiologically classified as X or Y type and injected with horseradish peroxidase (HRP). The axons were examined under the light microscope and were then processed for correlated electron microscopy. X axons arborized in area 17 and in the border between area 17 and 18. The X axons all formed terminals throughout layer 6, but were heterogeneous in their distribution in layer 4. They either occupied the entire width of sublayers 4A and 4B or were strongly biased toward layer 4A. Y axons also arborized in layers 4 and 6, but in area 17 they did not form boutons in sublamina 4B. Some Y axons projected only to area 18; others branched and arborized in both areas 17 and 18. Only the collaterals of one X axons were found to enter area 18; all the others were restricted to area 17. Y axons formed three to four separate patches of boutons about 300-400 microns in diameter, while all but one X axon formed a single elongated patch. Y axons had thicker main branches (3-4 microns) than X axons (1.5-2.5 microns) at their point of entry to the cortex. The main axon trunks and their medium-calibre collaterals were myelinated, but the preterminal segments were unmyelinated and studded with boutons. Each X or Y axon contacted about seven to ten somata, but Y axons made more contacts per soma (three to six) than did X axons (two to three). In addition to somatic synapses, both X and Y axons formed asymmetric (type 1) synapses on dendritic spines and shafts, with spines forming the most frequent targets (80%). Each Y bouton made, on average, 1.64 synapses in area 17 and 1.79 synapses in area 18, whereas each X bouton made only 1.27 synapses on average. Although there are proportionally fewer Y axons than X axons entering area 17, the Y axons provide as many synapses as the X axons because of their larger arbors and multisynaptic boutons.

Laminar cell counts and geniculo-cortical boutons in area 17 of cat and monkey

Counts have been made of the number of cells in individual laminae of the cortex in area 17 of the macaque monkey and cat, and degenerating terminals of geniculo-cortical axons have been studied with the electron-microscope in laminae IVab and IVc of the cortex of area 17 and lamina IV of area 18 of the visual cortex of the cat. The increase in number of cells through the depth of cortex in area 17 of the monkey compared with the cat would appear to involve about equally all laminae. In lamina IVab of area 17 of the cat the axon terminals of Y-type geniculo-cortical fibres make many more synapses per bouton than those of X-type fibres ending in lamina IVc. The geniculo-cortical axons ending in lamina IV of area 18 also make more multisynaptic boutons than those in lamina IVc of area 17. Thus, the terminals in the cortex of the X and Y functional types of fibre show comparable differences in both the cat and monkey.